(1980). "Immunodeficiency and cryptosporidiosis. Demonstration at the Royal College of Physicians of London." Br Med J 281(6248): 1123-7.
	
(1984). "Cryptosporidiosis among children attending day-care centers--Georgia, Pennsylvania, Michigan, California, New Mexico." MMWR Morb Mortal Wkly Rep 33(42): 599-601.
	
(1989). "Common-source outbreak of giardiasis--New Mexico." MMWR Morb Mortal Wkly Rep 38(23): 405-7.
	
(1994). "Assessment of inadequately filtered public drinking water--Washington, D.C., December 1993." MMWR Morb Mortal Wkly Rep 43(36): 661-9.
	The risk for waterborne infectious diseases increases when filtration and other standard water-treatment measures fail. On December 6, 1993, water-treatment plant operators in the District of Columbia (DC) began to have difficulty maintaining optimal filter effectiveness. On December 7, filter performance worsened, and levels of turbidity (i.e., small suspended particles) exceeded those permitted by U.S. Environmental Protection Agency (EPA) standards. On December 8, DC residents were advised to boil water intended for drinking because of high municipal water turbidity that may have included microbial contaminants. Although adequate chlorination of the DC municipal water was maintained throughout the period of increased turbidity, the parasite Cryptosporidium parvum is highly resistant to chlorination. Because of the increased risk for infection with this organism and other enteric pathogens, the DC Commission of Public Health and CDC conducted four investigations to determine whether excess cases of diarrheal illness occurred because residents drank inadequately filtered water. This report describes the results of these investigations.

(1994). "Cryptosporidium infections associated with swimming pools--Dane County, Wisconsin, 1993." MMWR Morb Mortal Wkly Rep 43(31): 561-3.
	In March and April 1993, an outbreak of cryptosporidiosis in Milwaukee resulted in diarrheal illness in an estimated 403,000 persons (1). Following that outbreak, testing for Cryptosporidium in persons with diarrhea increased substantially in some areas of Wisconsin; by August 1, 1993, three of six clinical laboratories in Dane County were testing routinely for Cryptosporidium as part of ova and parasite examinations. In late August 1993, the Madison Department of Public Health and the Dane County Public Health Division identified two clusters of persons with laboratory-confirmed Cryptosporidium infection in Dane County (approximately 80 miles west of Milwaukee). This report summarizes the outbreak investigations.

(1996). "Foodborne outbreak of diarrheal illness associated with Cryptosporidium parvum--Minnesota, 1995." MMWR Morb Mortal Wkly Rep 45(36): 783-4.
	On September 29, 1995, the Minnesota Department of Health (MDH) received reports of acute gastroenteritis among an estimated 50 attendees of a social event in Blue Earth County on September 16. This report summarizes the epidemiologic and laboratory investigations of the outbreak, which indicate the probable cause for this foodborne outbreak was Cryptosporidium parvum.

(1996). "Outbreak of cryptosporidiosis at a day camp--Florida, July-August 1995." MMWR Morb Mortal Wkly Rep 45(21): 442-4.
	On July 27, 1995, the Alachua County Public Health Unit (ACPHU) in central Florida was notified of an outbreak of gastroenteritis among children and counselors at a day camp on the grounds of a public elementary school. This report summarizes the outbreak investigation, which implicated Cryptosporidium parvum as the causative agent and underscores the role of contaminated water as a vehicle for transmission of this organism.

(1997). "Outbreaks of Escherichia coli O157:H7 infection and cryptosporidiosis associated with drinking unpasteurized apple cider--Connecticut and New York, October 1996." MMWR Morb Mortal Wkly Rep 46(1): 4-8.
	In October 1996, unpasteurized apple cider or juice was associated with three outbreaks of gastrointestinal illness. In the Western United States, an outbreak of Escherichia coli O157:H7 infections associated with unpasteurized commercial apple juice caused illness in 66 persons and one death. In addition, one outbreak of apple cider-related E. coli O157:H7 infections and another of cider-related Cryptosporidium parvum infections occurred in the Northeast. Apple cider is a traditional beverage produced and consumed in the fall. Cider often is manufactured locally at small cider mills where apples are crushed in presses, and the cider frequently is not pasteurized before sale. This report summarizes the clinical and epidemiologic features of the two apple cider-related outbreaks, which suggest that current practices for producing apple cider may not be adequate to prevent microbial contamination.

(1997). "Outbreaks of pseudo-infection with Cyclospora and Cryptosporidium--Florida and New York City, 1995." MMWR Morb Mortal Wkly Rep 46(16): 354-8.
	Efforts to expand the scope of surveillance and diagnostic testing for emerging infectious diseases also may increase the potential for identifying pseudo-outbreaks (i.e., increases in incidence that may result from enhanced surveillance) and outbreaks of pseudo-infection (i.e., clusters of false-positives for infection). This report describes the investigations of outbreaks of pseudo-infection with Cyclospora in Florida and Cryptosporidium in New York City in 1995 after health departments in those jurisdictions had initiated surveillance for these emerging organisms. These investigations emphasize 1) the need for laboratory training in the identification of emerging pathogens and 2) the importance of confirmation by reference laboratories as an early step in the investigation of any apparent outbreak caused by an emerging pathogen.

(1998). "Foodborne outbreak of cryptosporidiosis--Spokane, Washington, 1997." MMWR Morb Mortal Wkly Rep 47(27): 565-7.
	On December 29, 1997, the Spokane Regional Health District received reports of acute gastroenteritis among members of a group attending a dinner banquet catered by a Spokane restaurant on December 18. The illness was characterized by a prolonged (3-9 days) incubation period and diarrhea, which led public health officials to suspect a parasitic cause of the illness. Eight of 10 stool specimens obtained from ill banquet attendees were positive for Cryptosporidium using both modified acid-fast and auramine-rhodamine staining of concentrated specimens. This report summarizes the epidemiologic investigation of the outbreak, which suggests that foodborne transmission occurred through a contaminated ingredient in multiple menu items.

(1998). "Outbreak of cryptosporidiosis associated with a water sprinkler fountain--Minnesota, 1997." MMWR Morb Mortal Wkly Rep 47(40): 856-60.
	Cryptosporidiosis associated with recreational water exposure is becoming recognized more frequently. This report summarizes the investigation of a large outbreak of cryptosporidiosis associated with exposure to a water sprinkler fountain at the Minnesota Zoo. The initial cases were not diagnosed as cryptosporidiosis by the health-care system despite patients seeking care, underscoring the need for increased awareness of cryptosporidiosis and routine laboratory diagnostic practices among health-care providers.

(1999). "False-positive laboratory tests for Cryptosporidium involving an enzyme-linked immunosorbent assay--United States, November 1997-March 1998." MMWR Morb Mortal Wkly Rep 48(1): 4-8.
	From November 1997 through March 1998, the number of positive tests for Cryptosporidium increased in several locations in the United States. Several laboratories (e.g., the New York state laboratory and the Medical Science Laboratories in Wisconsin) retested original stool specimens and could not confirm the original positive test result. Following reports to the manufacturer by the Massachusetts, New York, and Wisconsin state health departments about possibly inaccurate test results, Alexon-Trend (Ramsey, Minnesota) notified its laboratory customers in a March 25, 1998, letter that three lots of its enzyme-linked immunosorbent assay (ELISA) 24 well (catalog number 540-24) ProSpecT Cryptosporidium Microplate Assay (lot numbers 970717,975011, and 980401) and seven lots of its ELISA 96 well (catalog number 540-96) ProSpect Cryptosporidium Microplate Assay (lot numbers 970696, 970775, 970883, 975006, 980402, 980808, and 980809) were subject to a "non-specific reaction between some stool specimens and the microplate assay" (i.e., a false-positive test result) (K. Hood, Alexon-Trend, personal communication, March 25, 1998). Alexon-Trend directed laboratories to discontinue using kits with implicated lot numbers. This report summarizes an analysis of reports of false-positive tests and describes identification of apparent clusters in three states.

(2000). "Outbreak of gastroenteritis associated with an interactive water fountain at a beachside park--Florida, 1999." MMWR Morb Mortal Wkly Rep 49(25): 565-8.
	Since 1989, approximately 170 outbreaks associated with recreational water venues (e.g., swimming pools, waterparks, fountains, hot tubs and spas, lakes, rivers, and oceans) have been reported, with almost half resulting in gastrointestinal illness (1-5). This report summarizes the investigation of an outbreak of gastroenteritis in Florida during 1999. The findings indicated that Shigella sonnei and Cryptosporidium parvum infections caused illness in persons exposed to an "interactive" water fountain at a beachside park.

(2001). "Prevalence of parasites in fecal material from chlorinated swimming pools--United States, 1999." MMWR Morb Mortal Wkly Rep 50(20): 410-2.
	As a result of the 1998 outbreak of infection with the chlorine-sensitive pathogen Escherichia coli O157:H7 at a waterpark in Georgia, many public health departments updated their guidelines for disinfecting pools following a fecal accident. Many of these guidelines recommended treating all fecal accidents as if they contained the highly chlorine-resistant parasite Cryptosporidium parvum, generally resulting in hyperchlorination and pool closures of up to a day. To determine whether fecal accidents commonly contained Cryptosporidium, the prevalence of this parasite and the moderately chlorine sensitive parasite Giardia intestinalis was assessed by asking swimming pool operators throughout the United States to collect formed stools from fecal accidents in their pools. This report summarizes the results of this study and provides recommendations for disinfecting pools following fecal accidents.

(2002). "Manufacturer's recall of rapid assay kits based on false positive Cryptosporidium antigen tests--Wisconsin, 2001-2002." MMWR Morb Mortal Wkly Rep 51(9): 189.
	The Wisconsin Division of Public Health and the Wisconsin State Laboratory of Hygiene (WSLH) reported that a recent cluster of cryptosporidiosis cases in a three-county area in southeastern Wisconsin was the result of false-positive tests. During December 1, 2001-February 1, 2002, approximately 30 cases of cryptosporidiosis were diagnosed at a laboratory in southeastern Wisconsin using the Becton, Dickinson, and Company (Franklin Lakes, New Jersey) ColorPAC Cryptosporidium/Giardia rapid assay (lot number 219370, expiration date 2002-06-05). Seventeen stool specimens, which were collected from 11 patients and tested positive by the rapid assay, were re-evaluated at WSLH. Six of these stool specimens were in EcoFix (Meridian Bioscience Inc., Cincinnati, Ohio), eight were in Cary-Blair transport media, and three were formalin fixed. All 17 specimens tested negative for Cryptosporidium at WSLH using the hot safranin stain and MeriFluor (Meridian Bioscience Inc., Cincinnati, Ohio) Cryptosporidium/Giardia direct fluorescent antibody kit with concentrated specimens.

(2004). "Manufacturer's recall of rapid cartridge assay kits on the basis of false-positive Cryptosporidium antigen tests--Colorado, 2004." MMWR Morb Mortal Wkly Rep 53(9): 198.
	The Colorado Department of Public Health and Environment (CDPHE) has determined that a fourfold increase in the number of reported cryptosporidiosis cases in Colorado during January-February 2004 might be attributed primarily to false-positive test results. Since January 1, 2004, a total of 13 in-state cases and one out-of-state case were reported to CDPHE. During the previous 7 years, an average of three cases were reported during January-February. In eight of 14 patients, rapid testing was performed by using the ImmunoCard STAT! Cryptosporidium/Giardia Rapid Assay (Meridian Bioscience, Inc., Cincinnati, Ohio). This assay is a solid-phase qualitative immunochromatographic assay designated to detect and distinguish between Giardia intestinalis (lamblia) and Cryptosporidium parvum in aqueous extracts of human fecal specimens. Seven of these samples were tested by using lot no. 081093 (expires August 11, 2004). Of the seven samples that tested positive initially for Cryptosporidium with this lot number, four were retested by using other, more specific tests. One patient sample was positive by direct microscopy, one was negative by direct microscopy, and two were negative by direct fluorescent-antibody testing, suggesting that results for three of the four samples were false positive. The results of testing for Giardia intestinalis (lamblia) with these kits are unclear. Several other states have noted increases in the number of reported cryptosporidiosis cases that also might be associated with use of these rapid assays.

(2004). "Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--selected sites, United States, 2003." MMWR Morb Mortal Wkly Rep 53(16): 338-43.
	In the United States, an estimated 76 million persons contract foodborne and other acute diarrheal illnesses each year. CDC's Emerging Infections Program Foodborne Diseases Active Surveillance Network (FoodNet) collects data on diseases caused by enteric pathogens transmitted commonly through food in nine U.S. sites. FoodNet quantifies and monitors the incidence of these infections by conducting active surveillance for laboratory-diagnosed illness. This report describes preliminary surveillance data for 2003 and compares them with 1996-2002 data. The data indicate substantial declines in the incidence of infections caused by Campylobacter, Cryptosporidium parvum, Escherichia coli O157, Salmonella, and Yersinia enterocolitica. These data represent progress toward meeting the 2010 national health objectives of reducing the incidence of foodborne infections (objective nos. 10.1a, 10.1b, and 10.1d). However, increased efforts are needed to reduce further the incidence of foodborne illnesses, particularly among children.

(2005). "Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food--10 sites, United States, 2004." MMWR Morb Mortal Wkly Rep 54(14): 352-6.
	Foodborne illnesses are a substantial health burden in the United States. The Foodborne Diseases Active Surveillance Network (FoodNet) of CDC's Emerging Infections Program collects data from 10 U.S. sites on diseases caused by enteric pathogens transmitted commonly through food. FoodNet quantifies and monitors the incidence of these infections by conducting active, population-based surveillance for laboratory-diagnosed illness. This report describes preliminary surveillance data for 2004 and compares them with baseline data from the period 1996-1998. The 2004 data indicate declines in the incidence of infections caused by Campylobacter, Cryptosporidium, Shiga toxin-producing Escherichia coli (STEC) O157, Listeria, Salmonella, and Yersinia. Declines in Campylobacter and Listeria incidence are approaching national health objectives (objectives 10-1a through 1d); for the first time, the incidence of STEC O157 infections in FoodNet is below the 2010 target. However, further efforts are needed to sustain these declines and to improve prevention of foodborne infections; efforts should be enhanced to reduce pathogens in food animal reservoirs and to prevent contamination of produce.

Abbaszadegan, M., M. S. Huber, et al. (1997). "Detection of viable Giardia cysts by amplification of heat shock-induced mRNA." Appl Environ Microbiol 63(1): 324-8.
	Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.

Abd-El-Wahed, M. M. (1999). "Cryptosporidium infection among sheep in Qalubia Governorate, Egypt." J Egypt Soc Parasitol 29(1): 113-8.
	The present study was carried to explore the incidence of Cryptosporidium infection among lambs in Qalubia governorate as well as to study the ability of three staining techniques developed for rapid identification of Cryptosporidium oocysts in faecal samples of 120 neonatal lambs less than one month old. The stains used in this investigation were modified Zeihl-Neelsen stain (MZN), Safranin-methylene blue (SMB) and Giemsa stain (GS) to detect the recovered oocysts with their incidence variation. The diagnostic value was raised by direct smear and concentration floatation technique. The infection rates with Cryptosporidium oocysts among the investigated faecal samples from neonatal sheep were 82, 58, 36, out of 120 (68.3, 48.3 and 30%) by modified Zeihl-Neelsen stain, Safranin-methylene blue and Giemsa stain respectively. The lambs less than one week had higher infection rate. The relationship between age susceptibility and infection was discussed. The use of modified Zeihl-Neelsen stain was currently recommended procedure with the concentration floatation technique for the accurate diagnosis and rapid identification of Cryptosporidium oocysts.

Abrahamsen, M. S., A. A. Schroeder, et al. (1996). "Differential mRNA display analysis of gene expression in Cryptosporidium parvum-infected HCT-8 cells." J Eukaryot Microbiol 43(5): 80S-81S.
	
Abrahamsen, M. S., T. J. Templeton, et al. (2004). "Complete genome sequence of the apicomplexan, Cryptosporidium parvum." Science 304(5669): 441-5.
	The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

Ahourai, P., A. Ezzi, et al. (1985). "Cryptosporidium spp. in new born lambs in Iran." Trop Anim Health Prod 17(1): 6-8.
	In a study conducted to investigate the causes of the death of new born lambs due to diarrhoea 237 cases were studied. In 16 of these lambs necropsied at four to 10 days old organisms considered to be Cryptosporidia at various stages of its life-cycle were associated with the luminal surface of the epithelium of the intestinal tract. The histopathology and the mechanism of the diarrhoea caused by the parasite and the resulting deaths are discussed.

Aiello, A. E., L. Xiao, et al. (1999). "Microsatellite analysis of the human and bovine genotypes of Cryptosporidium parvum." J Eukaryot Microbiol 46(5): 46S-47S.
	
Akiyoshi, D. E., R. Balakrishnan, et al. (2002). "Molecular characterization of ribonucleotide reductase from Cryptosporidium parvum." DNA Seq 13(3): 167-72.
	The Apicomplexan enteric parasite, Cryptosporidium, infects a broad range of mammals, birds, fish and reptiles. Cryptosporidium parvum, the principal causative agent of human cryptosporidiosis, has emerged as a major contributor to waterborne outbreaks of this disease. The absence of effective drugs against C. parvum has necessitated the search for new drug targets. One attractive class of target enzymes is ribonucleotide reductase, an essential enzyme required for the de novo synthesis of deoxyribonucleotides. We report the cloning and sequencing of the small and large subunits of ribonucleotide reductase from a C. parvum genotype 2 isolate, GCH1. Southern blot analysis showed that these subunits are encoded by single copy genes. Both subunits have been expressed as recombinant proteins in Escherichia coli.

Akiyoshi, D. E., X. Feng, et al. (2002). "Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species." Infect Immun 70(10): 5670-5.
	Cryptosporidium parvum TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and beta-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of C. parvum genotype 1 and is currently used in several research projects including human volunteer studies.

Al-Herrawy, A. Z., S. E. Elowa and E. A. Morsy (2005). "Fate of Cryptosporidium during wastewater treatmnent via constructed wetland systems." International Journal of Environmental Studies 62(3): 293-300.
	
Ali, M. A., A. Z. Al-Herrawy, et al. (2004). "Detection of enteric viruses, Giardia and Cryptosporidium in two different types of drinking water treatment facilities." Water Res 38(18): 3931-9.
	In this study, two types of drinking water treatment facilities (two conventional drinking water treatment plants (DWTPs) and two compact units (Cus)) were compared referring to their production capacity. Water samples were collected from three main points: (a) different water treatment steps (b) washings of sand filters and (c) distribution system at different distances from the water treatment plants. Both viruses and protozoa were concentrated from each water sample by adsorption and accumulation on the same nitrocellulose membrane filters (0.45 microm pore size). Enteroviruses were detected by plaque infectivity assay in BGM cells and HAV, HEV and Norovirus were detected by RT-PCR. Giardia and Cryptosporidium were detected by conventional staining methods and PCR. The results revealed that enterovirus load at the intake ranged between 10-15 PFU/L for the two compact units and between 4.5 and 75 PFU/L for the two conventional DWTPs. The virus load in distribution system of the first type DWTPs at 1 km from the plant was the same as that of the intake. Viruses in the other type of treatment plants CUs at 1, 5 and 7 km, were much reduced. Investigation of raw water sediments of the two DWTPs showed enterovirus counts between 12 and 17.5 PFU/L. Virus count was reduced in sand of filters after washing. Giardia cysts were equally detected by microscopy and PCR in only intake samples of EL-Hawamdia CU (33.3%) and Meet Fares DWTP (50%). Cryptosporidium oocysts were equally detected by microscopy and PCR in intake samples of Abo EL-Nomros CU (100%), EL-Hawamdia CU (66.7%) and Fowa DWTP (50%). At Meet Fares DWTP three positive intake samples for Cryptosporidium were detected by PCR, compared with only two positive samples by microscopy. Giardia cysts and Cryptosporidium oocysts were detected in raw water sediment and sand of filters before washing. Only one sample from Meet Fares DWTP sand of filters after washing was positive for both Giardia and Cryptosporidium. It can be concluded that the poor microbial quality of the water may be due to improper operational skills and management of the various water treatment plants (especially at the two high capacity treatment plants).

Ali, S. A. and D. R. Hill (2003). "Giardia intestinalis." Curr Opin Infect Dis 16(5): 453-60.
	PURPOSE OF REVIEW: Giardia intestinalis (syn. duodenalis or lamblia) is one of the most common intestinal parasites in the world, with an estimated 2.8 x 10(6) infections per year in humans, and it contributes to diarrhea and nutritional deficiencies in children in developing regions. The wide prevalence of Giardia and its unique place in evolutionary biology have led to ongoing research. RECENT FINDINGS: Research into the basic biology of Giardia has highlighted some of its unique properties as an 'early-branching' eukaryote. Although Giardia do not contain mitochondria, they have developed pathways to perform some mitochondrial functions. Investigations into encystation and excystation have identified new gene products that are important in cyst wall formation, and signal transduction events that occur during excystation. The ability to transfect Giardia stably will lead to an improved understanding of its development and metabolism. Molecular typing of G. intestinalis isolates indicates that most animal parasites are not associated with human infection. Insights into immunology have helped define the role of IL-6 in the early control of murine giardiasis, and the contributions of IgA in controlling infection. Further studies of giardiasis in poorly nourished children in developing regions supports an important contributing role of Giardia in stunting and cognitive impairment. Finally, new diagnostic assays using antigen detection are being evaluated and a new agent, nitazoxanide, has been approved in the USA for the treatment of giardiasis and cryptosporidiosis in children. SUMMARY: Research into the biology of Giardia should increase knowledge about protist differentiation and will complement studies in other biological systems. Continued study of the role of Giardia in chronic diarrhea and malnutrition in developing regions will help focus strategies to improve childhood growth and nutrition.

Alvarez-Pellitero, P. and A. Sitja-Bobadilla (2002). "Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species, Sparus aurata L. and Dicentrarchus labrax L." Int J Parasitol 32(8): 1007-21.
	Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.

Alves, M., O. Matos, et al. (2003). "Microsatellite analysis of Cryptosporidium hominis and C. parvum in Portugal: a preliminary study." J Eukaryot Microbiol 50 Suppl: 529-30.
	
Alves, M., L. Xiao, et al. (2006). "Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal." Parasitol Res 99(3): 287-292.
	To investigate the transmission of cryptosporidiosis in Portugal, Cryptosporidium hominis and Cryptosporidium parvum from HIV-infected patients, cattle, and wild ruminants were characterized by sequence analysis of the 60-kDa glycoprotein (GP60) gene. Fourteen subtypes within nine subtype families were identified, and three of the subtype families (If, IIb, and IId) were restricted or largely limited to Portugal. Parasites from cattle from various regions in Portugal and wild ruminants in Lisbon showed limited genetic heterogeneity (only two subtype families). All wild ruminants had the same subtype, which was also the predominant subtype in cattle all over Portugal and was found in nine HIV-infected patients in Lisbon. Two other C. parvum subtypes were only restricted to limited locations. In contrast, human parasites displayed 13 subtypes in nine subtype families, with most of the infections caused by parasites in Ib, IIa, IIc, and IId families. Two of the C. parvum subtype families (IIc and IIb) had only been found in humans. The high overall parasite diversity and high percentage of C. hominis infections attributable to Ib and C. parvum infections to IId represent unique characteristics of Cryptosporidium transmission in humans in Portugal.

Alves, M., L. Xiao, et al. (2005). "Occurrence and molecular characterization of Cryptosporidium spp. in mammals and reptiles at the Lisbon Zoo." Parasitol Res 97(2): 108-12.
	The presence of Cryptosporidium parasites in mammals and reptiles kept at the Lisbon Zoo was investigated. A total of 274 stool samples were collected from 100 mammals and 29 reptiles. The species and genotype of the isolates identified by light microscopy were determined by nested PCR and sequence analysis of a fragment of the small subunit rRNA gene. Cryptosporidium oocysts were found in one black wildebeest (Connochaetes gnou), one Prairie bison (Bison bison bison) and in one Indian star tortoise (Geochelone elegans). The PCR and sequence analysis of these three isolates showed that those excreted by the Prairie bison were Cryptosporidium mouse genotype, those from the black wildebeest were from a new Cryptosporidium genotype and those infecting the Indian star tortoise were Cryptosporidium tortoise genotype. The present work reports a new Cryptosporidium genotype in a black wildebeest and the first finding of the Cryptosporidium mouse genotype in a ruminant.

Alves, M., L. Xiao, et al. (2003). "Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal." J Clin Microbiol 41(6): 2744-7.
	Cryptosporidium parvum and Cryptosporidium hominis isolates from human immunodeficiency virus-infected patients, cattle, and wild ruminants were characterized by PCR and DNA sequencing analysis of the 60-kDa glycoprotein gene. Seven alleles were identified, three corresponding to C. hominis and four corresponding to C. parvum. One new allele was found (IId), and one (IIb) had only been found in Portugal. Isolates from cattle and wild ruminants clustered in two alleles. In contrast, human isolates clustered in seven alleles, showing extensive allelic diversity.

Amahmid, O., S. Asmama, et al. (1999). "The effect of waste water reuse in irrigation on the contamination level of food crops by Giardia cysts and Ascaris eggs." Int J Food Microbiol 49(1-2): 19-26.
	In Marrakech, raw sewage has been used for farming purposes for several decades for many types of crops. This study aimed to determine the contamination level of Giardia cysts and Ascaris eggs for crops designated for human consumption. Collected crops in irrigated fields were turnip, marrow, squash, potatoes, pepper and eggplant. Field trials were also carried out on four crops, coriander, carrots, mint and radish, using three water types for irrigation, i.e. raw waste water, treated waste water (sedimentation and 16 days retention) and fresh water. Giardia cysts were detected at a level of 5.1 cysts/kg in potatoes, while Ascaris eggs were observed in numbers varying between 0.18 eggs/kg in potatoes and 0.27 eggs/kg in turnip. Field trials confirmed that irrigation of crops by raw waste water leads to contamination. Giardia and Ascaris were isolated in coriander at concentrations of 254 cysts/kg and 2.7 eggs/kg, respectively; mint was also highly contaminated with numbers reaching 96 cysts/kg and 4.63 eggs/kg. Carrots and radish were contaminated and respective numbers observed for Giardia were 155 and 59.1 cysts/kg; Ascaris was discovered in numbers of 0.7 and 1.64 eggs/kg, respectively. However, cultures irrigated with treated waste water and fresh water were free from contamination. Cysts and eggs on coriander persisted for a maximum of 8 days.

Amar, C., S. Pedraza-Diaz, et al. (2001). "Extraction and genotyping of Cryptosporidium parvum DNA from fecal smears on glass slides stained conventionally for direct microscope examination." J Clin Microbiol 39(1): 401-3.
	A method was developed for extracting cryptosporidial DNA from stained fecal smears on glass microscope slides. The correct genotype of Cryptosporidium parvum was amplified by PCR from 89 (85%) of 105 smears following conventional staining but not from negative controls. This technique may have applications for analysis of other infectious agents.

Amar, C. F., R. M. Chalmers, et al. (2002). "Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides." Lett Appl Microbiol 35(6): 486-8.
	AIMS: A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides. METHODS AND RESULTS: Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested. CONCLUSIONS: This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.

Amar, C. F., P. H. Dear, et al. (2002). "Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces." J Clin Microbiol 40(2): 446-52.
	An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalis assemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpi gene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpi gene of G. duodenalis assemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalis genotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.

Andrews, C. D., R. Fayer, et al. (1990). "Continuous in vitro cultivation of Sarcocystis cruzi." J Parasitol 76(2): 254-5.
	Sporozoites of Sarcocystis cruzi were inoculated onto monolayer cultures of bovine pulmonary artery endothelial (CPA) cells. Sporozoites entered the cells, formed large and small multinucleate schizonts, and produced large numbers of merozoites. Continuous cultivation from the original sporozoite inoculum has been maintained for more than 1,320 days by subinoculating merozoites onto new cultures of CPA cells. During this time, the capacity to produce both types of schizonts was preserved, and schizogony was the only form of reproduction that was observed.

Appelbee, A. J., L. M. Frederick, et al. (2003). "Prevalence and genotyping of Giardia duodenalis from beef calves in Alberta, Canada." Veterinary Parasitology 112(4): 289-294.
	Giardia infections in domestic cattle has come under increasing scrutiny owing to the potential contamination of surface and ground waters through manure distribution on fields and pasture runoff. The objective of the study was to determine the prevalence and genotypes of Giardia duodenalis in beef calves in major beef cow calf farms in Alberta, Canada. Fecal samples were collected from beef calves aged 2-10 weeks at nine farms in Alberta. Samples were examined for the presence of G. duodenalis cysts by immunofluorescent staining. Giardia cysts were found in 168 of the 495 fecal samples examined, with prevalence ranging from 7 to 60% among farms. Genotypic analysis of positive isolates utilizing PCR and sequencing of a 292 bp fragment of the 16S-rRNA locus, revealed the hoofed livestock genotype in 41 of the 42 isolates. One isolate was identical to the Assemblage A genotype. The results of this study demonstrate that beef calves in this area are primarily infected with the livestock genotype which is thought to be specific to artiodactyl hosts and non-infective to humans. This suggests that the Giardia carried by beef cattle may be a minimal zoonotic threat. (C) 2003 Elsevier Science B.V. All rights reserved. [References: 15] 15

Aramini, J. J., C. Stephen, et al. (1999). "Potential contamination of drinking water with Toxoplasma gondii oocysts." Epidemiol Infect 122(2): 305-15.
	The world's first documented toxoplasmosis outbreak associated with a municipal water supply was recognized in 1995 in Victoria, British Columbia, Canada. It was hypothesized that domestic cat (Felis catus) or cougar (Felis concolor) faeces contaminated a surface water reservoir with Toxoplasma gondii oocysts. An extensive investigation of the Victoria watershed 1 year following the outbreak documented the presence of an endemic T. gondii cycle involving the animals inhabiting the area. Cats and cougars were observed throughout the watershed. Serological evidence of T. gondii infection was demonstrated among domestic cats living in the Victoria area. Cougars were found to shed T. gondii oocysts. Serological evidence of T. gondii infection in deer mice living in the riparian environments of the watershed suggested that T. gondii oocysts were being shed near the water edge. Contamination of Victoria's water supply with T. gondii oocysts potentially occurred during the study period and future waterborne toxoplasmosis outbreaks in this and other communities are possible.

Ares-Mazas, M. E., B. Fernandez-da Ponte, et al. (1999). "Oocysts, IgG levels and immunoblot patterns determined for Cryptosporidium parvum in bovine examined during a visit to a farm (northeastern Spain)." Vet Parasitol 81(3): 185-93.
	Single fecal and serum samples were individually collected from 101 bovines selected at random during a visit to a farm in northeastern Spain (Group I, 26 animals aged 2-36 days; Group II, 34 animals aged 1.5-4.5 months; Group III, 41 animals aged 20-24 months). Testing for the presence of Cryptosporidium parvum oocysts in feces (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) indicated that 26% animals were infected (81% of Group I, 15% of Group II and 0% of Group III). Serological testing (ELISA for detection of specific anti-C. parvum IgG) indicated that 59% animals were seropositive (12% of Group I, 74% of Group II and 78% of Group III). Immunoblotting results indicate that cattle sera recognize C. parvum antigens of widely varying molecular weights and that the number of antigens recognized increases with age. Immunoblots revealed that some of the sera belonging to the Group I reacted with protein fractions between 15 and 20 kDa but none recognized the 21-23 kDa antigen. Only few sera in the Group II recognized the protein fraction between 15 and 20 kDa. The recognition of 21-23 kDa fraction was observed by four sera from uninfected and seropositive animals. Sera from all the seronegative Group II animals recognized few antigens and always with molecular weight greater than 50 kDa. Serum samples from both seropositive and seronegative animals belonging to the Group III recognized antigens with molecular weight ranging 15-20 kDa. Surprisingly, the protein fractions between 21 and 28 kDa reacted with approximately 30% of the sera from seropositive animals and only one of the nine sera from seronegative animals. The recognition of 42-46 kDa antigens increased with the age and only reacted with the sera from uninfected animals.

Armon, R., D. Gold, et al. (2002). "Surface and subsurface irrigation with effluents of different qualities and presence of Cryptosporidium oocysts in soil and on crops." Water Sci Technol 46(3): 115-22.
	A large variety of human pathogens are excreted in wastewater including bacteria, viruses, protozoan cysts and helminth eggs. In raw sewage, human pathogens reach high numbers, thereafter decreasing successively at each treatment step. However, the final effluents still contain a large fraction of these pathogens that may pose a serious public health. Among the various crops irrigated with effluents, vegetables are the most vulnerable to contamination. Vegetables, usually eaten raw (uncooked) or with rich dressings (causing regrowth of some pathogenic bacteria) pose the main threat to humans. The importance of microbiological and parasitological criteria for reused water has been repeatedly emphasized. Some microbiological recommendations based on epidemiological data have been established for untreated wastewater, there is still a need to define the criteria for effluent quality required for unrestricted crop irrigation. This paper presents a field study comparison of two irrigation methods: surface and subsurface of field crops (mainly vineyard) and follow-up of Cryptosporidium oocysts in soil at different depths (0 to 90 cm). Oocysts were isolated at all depths without a clear pattern of distribution (0 to 640 oocysts/g). In addition different vegetables irrigated with different effluent qualities were tested for the presence of Cryptosporidium oocysts and Giardia cysts. The highest prevalence of oocysts was found on zucchini that has a sticky and hairy outer surface (80 to 10,000 oocysts/0.5 kg).

Arrowood, M. J. and K. Donaldson (1996). "Improved purification methods for calf-derived Cryptosporidium parvum oocysts using discontinuous sucrose and cesium chloride gradients." J Eukaryot Microbiol 43(5): 89S.
	
Arrowood, M. J., M. R. Hurd, et al. (1995). "A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry." J Parasitol 81(3): 404-9.
	A flow cytometric method for the quantification of Cryptosporidium parvum oocysts in stool specimens was developed to replace conventional microscopic immunofluorescent assays. Fecal pellets were collected from control (uninfected) severe combined immune-deficient mice, suspended in 2.5% potassium dichromate at a ratio of 400 microliter per pellet, and homogenized by vortexing. Purified oocytes were added to the samples (10(5), 10(4), 10(3), and 10(2)/ml). Aliquots (200 microliters) of the vortexed samples were centrifuged over microscale discontinuous sucrose gradients. The oocyst-containing fractions were collected, washed, and incubated with an oocyst-specific monoclonal antibody (labeled with fluorescein isothiocyanate) for 30 min at 37 degrees C. Sample volumes were adjusted to 600 microliters with phosphate-buffered saline and assayed by using logical gating of forward/side scatter and fluorescence signal on a flow cytometer. Seeded samples showed a linear correlation with the number of oocysts recovered from the gradients. Analyses of stool samples from chronically infected mice demonstrated that the flow cytometry method was approximately 10 times more sensitive than conventional immunofluorescent assays.

Arrowood, M. J. and C. R. Sterling (1987). "Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients." J Parasitol 73(2): 314-9.
	Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The oocyst recovery method utilized 2 sequential discontinuous sucrose gradients followed by 1 Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls.

Arrowood, M. J., C. R. Sterling, et al. (1991). "Immunofluorescent microscopical visualization of trails left by gliding Cryptosporidium parvum sporozoites." J Parasitol 77(2): 315-7.
	Cryptosporidium parvum sporozoites that exhibited gliding motility in vitro were examined by immunofluorescence with anticryptosporidial monoclonal antibodies (Mabs) for surface antigen deposition on poly-L-lysine-coated glass microscope slides. The Mabs that revealed trails are specific for an immunodominant 23-kDa antigen previously localized to the sporozoite surface.

Arrowood, M. J., L. T. Xie, et al. (1994). "In vitro assays of maduramicin activity against Cryptosporidium parvum." J Eukaryot Microbiol 41(5): 23S.
	
Arrowood, M. J., L. T. Xie, et al. (1996). "Disinfection of Cryptosporidium parvum oocysts by pulsed light treatment evaluated in an in vitro cultivation model." J Eukaryot Microbiol 43(5): 88S.
	
Ashford, R. W. (1979). "Occurrence of an undescribed coccidian in man in Papua New Guinea." Ann Trop Med Parasitol 73(5): 497-500.
	
Asmundsson, I. M., S. J. Upton, et al. (2001). "Five new species of Coccidia (Apicomplexa: Eimeriidae) from colubrid snakes of Ecuador." J Parasitol 87(5): 1077-81.
	Fecal samples from 11 colubrid snakes, representing 10 species, collected in Ecuador during October 1994 were examined for coccidian parasites. Feces of 4 individuals, representing 4 host species, contained coccidian oocysts. Three species of Eimeria and 2 species of Isospora were observed and are described here as new. Oocysts of both Eimeria and Isospora were found in the feces of a slug-eating snake, Dipsas vermiculata. Sporulated oocysts of the Eimeria sp. are spheroid to subspheroid, 16.7 by 16.6 microm (14-18 by 14-18 microm) and those of the Isospora sp. are spheroid and 15.0 microm (13-18 microm) in diameter. Imantodes cenchoa, the common bluntheaded treesnake, was infected with a species of Eimeria. These sporulated oocysts are ellipsoid, 23.3 by 16.2 microm (25-21 by 15-17 microm). Sporulated eimerian oocysts from Leptodeira annulata, the southern cat-eyed snake, are subspheroid, 22.5 by 18.8 microm (19-26 by 17-21 microm). Feces of a juvenile Imantodes lentiferus, the bluntheaded vine snake, contained ovoid to ellipsoid isosporan oocysts, which measured 21.6 by 15.0 microm (20-23 by 14-16 microm) when sporulated.

Aspinall, T. V., D. Marlee, et al. (2002). "Prevalence of Toxoplasma gondii in commercial meat products as monitored by polymerase chain reaction--food for thought?" Int J Parasitol 32(9): 1193-9.
	DNA was extracted from 71 meat samples obtained from UK retail outlets. All of these DNA preparations gave the expected polymerase chain reaction products when amplified with primers specific for the species from which the meat originated. A second polymerase chain reaction analysis, using primers specific for the Toxoplasma gondii SAG2 locus, revealed the presence of this parasite in 27 of the meat samples. Restriction analysis and DNA sequencing showed that 21 of the contaminated meats contained parasites genotyped as type I at the SAG2 locus, whilst six of the samples contained parasites of both types I and II. Toxoplasma- positive samples were subjected to further polymerase chain reaction analysis to determine whether any carried an allele of the dihydropteroate synthase gene that has recently been shown to be causally associated with sulfonamide resistance in T. gondii. In all cases, this analysis confirmed that parasites were present in the samples and, additionally, revealed that none of them carried the drug-resistant form of dihydropteroate synthase. These results suggest that a significant proportion of meats commercially available in the UK are contaminated with T. gondii. Although none of the parasites detected in this study carried the sulfonamide-resistance mutation, a simplified procedure for monitoring this situation merits development.

Atwill, E. R., S. M. Camargo, et al. (2001). "Quantitative shedding of two genotypes of Cryptosporidium parvum in California ground squirrels (Spermophilus beecheyi)." Appl Environ Microbiol 67(6): 2840-3.
	Sixteen percent of California ground squirrels (Spermophilus beecheyi) were found to be shedding an average of 53,875 Cryptosporidium parvum oocysts/g of feces. Male squirrels had a higher prevalence and higher intensity of shedding than did female squirrels. The majority of C. parvum isolates matched a bovine-murine genotype, with a few isolates resembling a porcine genotype. Higher intensities of shedding by males may enhance dissemination and genotypic mixing of this protozoa given males' proclivity to disperse to nonnatal colonies.

Atwill, E. R., J. A. Harp, et al. (1998). "Evaluation of periparturient dairy cows and contact surfaces as a reservoir of Cryptosporidium parvum for calfhood infection." Am J Vet Res 59(9): 1116-21.
	OBJECTIVE: To determine whether periparturient cows or contact surfaces to which newborn calves are exposed are reservoirs of Cryptosporidium parvum oocysts. ANIMALS: Periparturient cows and their calves. PROCEDURE: Using direct fluorescent antibody (DFA) and acid-fast (AF) assays, fecal samples taken before and after calving from periparturient cows were tested for C parvum oocysts. Fecal samples from calves were collected every other day from age 7 to 21 days and were tested by use of the AF assay. Topsoil from close-up and maternity pens and scrapings from wooden walls and floors of calf hutches were tested for C parvum oocysts by use of DFA assay. RESULTS: None of the 384 fecal samples obtained 1 to 21 days before or after calving or on the day of calving from 154 periparturient cows contained detectable C parvum oocysts. Despite this lack of detectable periparturient shedding, the period prevalence of calfhood infection was 92% (123/134) from age 7 to 21 days. Soil samples from the close-up and maternity pens where newborn calves spend the first 12 hours of life also were negative for C parvum oocysts. Wood scrap ings from the outer 2 mm of the walls and floors of empty and cleaned calf hutches that were ready to receive calves were C parvum oocyst-positive. CONCLUSIONS: Conditional on sensitivity of DFA, periparturient cows did not appear to shed detectable C parvum oocysts. In contrast, the floors and walls of wooden calf hutches contained detectable C parvum oocysts on the surface.

Atwill, E. R., B. Hoar, et al. (2003). "Improved quantitative estimates of low environmental loading and sporadic periparturient shedding of Cryptosporidium parvum in adult beef cattle." Appl Environ Microbiol 69(8): 4604-10.
	Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding of C. parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a > or = 90% probability oocyst concentrations as low as 3.2 oocysts g of feces(-1), with a 54% probability of detecting just one oocyst g of feces(-1). Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces(-1). The estimated environmental loading rate of C. parvum ranged from 3,900 to 9,200 oocysts cow(-1) day(-1), which is substantially less than a previous estimate of 1.7 x 10(5) oocysts cow(-1) day(-1) (range of 7.7 x 10(4) to 2.3 x 10(5) oocysts cow(-1) day(-1)) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.

Atwill, E. R., L. Hou, et al. (2002). "Transport of Cryptosporidium parvum oocysts through vegetated buffer strips and estimated filtration efficiency." Appl Environ Microbiol 68(11): 5517-27.
	Vegetated buffer strips were evaluated for their ability to remove waterborne Cryptosporidium parvum from surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log(10) reductions for spiked C. parvum oocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm(3). Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborne C. parvum (1- to 2-log(10) reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log(10) reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of <or=20% and a length of >or=3 m should function to remove >or=99.9% of C. parvum oocysts from agricultural runoff generated during events involving mild to moderate precipitation.

Atwill, E. R., E. Johnson, et al. (1999). "Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow-calf herds." Am J Vet Res 60(4): 420-5.
	OBJECTIVE: To evaluate fecal shedding of Cryptosporidium parvum from California cow-calf herds with respect to age, geographic region, temporal effects, and association with watery feces. ANIMALS: Cows and calves from 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Associations between age, geographic region, month of collection, watery feces, and likelihood of shedding C parvum were evaluated. RESULTS: 3.9% of cattle were shedding C parvum oocysts. Prevalence of shedding among calves ranged from 0 to 13%, and was 0.6% among cattle > or = 12 months old. The odds of shedding C parvum among 2-month-old calves were 41 times greater than among cattle > 4 months old. The odds of shedding C parvum among cattle tested in May were 8.7 times greater than among cattle tested during June, July, or August. The odds of infected individuals having watery feces were 3 to 4 times greater than for noninfected individuals, but the etiologic fraction was only 8 to 9%. CONCLUSIONS AND CLINICAL RELEVANCE: Substantial fecal shedding of C parvum by cow-calf herds was limited to calves 1 to 4 months old, with low prevalence detected in older animals. Risk of contamination of watersheds with C parvum was limited to those periods when young calves were in the herd. Although the odds of having watery feces were greater for animals infected with C parvum than for noninfected animals, the low etiologic fraction suggests that most calves with watery feces were not infected with C parvum.

Atwill, E. R., E. M. Johnson, et al. (1999). "Association of herd composition, stocking rate, and duration of calving season with fecal shedding of Cryptosporidium parvum oocysts in beef herds." J Am Vet Med Assoc 215(12): 1833-8.
	OBJECTIVE: To evaluate the association of herd demographics, parturition variables, stocking rate, and rotational grazing practices with the probability of fecal shedding of Cryptosporidium parvum from beef cow-calf herds in California. DESIGN: Cross-sectional study. SAMPLE POPULATION: 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Association between various demographic and management factors and the probability of shedding C parvum were statistically evaluated. RESULTS: Adjusted for age and month of collection of a fecal sample, cattle from herds with a high number of young calves (< or = 2 months old) on the day of sample collection, a high stocking rate (No. of cattle/acre/mo), or a longer calving season were more likely to shed C parvum oocysts, compared with cattle from herds with fewer young calves, a lower stocking rate, or a shorter calving season. Cattle from herds with a higher number of older calves (> 2 months old) on the day of sample collection were less likely to shed C parvum oocysts, compared with cattle from herds with fewer older calves. Using our multivariate model, rotational grazing systems or season of onset of calving were not associated with shedding status for C parvum oocysts. CONCLUSIONS AND CLINICAL RELEVANCE: Reproductive management that would result in a shorter calving season and use of a lower stocking rate for cattle may be associated with reduced risk of C parvum shedding. Intensive rotational grazing systems and time of year for onset of calving season apparently have little effect on reducing prevalence of oocyst shedding.

Atwill, E. R., M. D. Pereira, et al. (2006). "Environmental Load of Cryptosporidium parvum Oocysts from Cattle Manure in Feedlots from the Central and Western United States." J Environ Qual 35(1): 200-6.
	The first step in assessing the risk of water contamination by Cryptosporidium parvum oocysts from feedlot cattle (Bos taurus) production systems is to quantify the number of C. parvum oocysts present in the fecal material deposited by feedlot cattle. Our primary objective for this project was to estimate the daily environmental load of C. parvum oocysts in fecal material deposited by feedlot cattle from across the central and western USA. Our secondary goal was to genotype isolates of C. parvum from feedlot cattle to help facilitate proper identification of mammalian sources of waterborne C. parvum. Based on 5274 fecal samples from 22 feedlots in seven states (California, Washington, Colorado, Oklahoma, Texas, Nebraska, and South Dakota), we estimated a point prevalence of C. parvum of 0.99 to 1.08% in fecal material from feedlot pens from a wide range of climates and a diverse range of feedlot management systems. On average, fresh fecal material from throughout feedlot systems (recent arrivals to nearing slaughter) contained about 1.3 to 3.6 oocysts/g feces, which roughly translates to about 2.8 x 10(4) to 1.4 x 10(5) oocysts/animal perday.

Atwill, E. R., R. A. Sweitzer, et al. (1997). "Prevalence of and associated risk factors for shedding Cryptosporidium parvum oocysts and Giardia cysts within feral pig populations in California." Appl Environ Microbiol 63(10): 3946-9.
	Populations of feral pigs (Sus scrofa) may serve as an environmental reservoir of Cryptosporidium parvum oocysts and Giardia sp. cysts for source water. We conducted a cross-sectional study to determine the prevalence of and associated demographic and environmental risk factors for the shedding of C. parvum oocysts and Giardia sp. cysts. Feral pigs were either live-trapped or dispatched from 10 populations located along the coastal mountains of western California, and fecal samples were obtained for immunofluorescence detection of C. parvum oocysts and Giardia sp. cysts. We found that 12 (5.4%) and 17 (7.6%) of 221 feral pigs were shedding C. parvum oocysts and Giardia sp. cysts, respectively. The pig's sex and body condition and the presence of cattle were not associated with the probability of the shedding of C. parvum oocysts. However, younger pigs (< or = 8 months) and pigs from high-density populations (> 2.0 feral pigs/km2) were significantly more likely to shed oocysts compared to older pigs (> 8 months) and pigs from low-density populations (< or = 1.9 feral pigs/km2). In contrast, none of these demographic and environmental variables were associated with the probability of the shedding of Giardia sp. cysts among feral pigs. These results suggest that given the propensity for feral pigs to focus their activity in riparian areas, feral pigs may serve as a source of protozoal contamination for surface water.

Atwill, E. R., K. W. Tate, et al. (2006). "Efficacy of natural grassland buffers for removal of Cryptosporidium parvum in rangeland runoff." J Food Prot 69(1): 177-84.
	Our goal for this project was to estimate the retention efficiency of natural grassland buffers for Cryptosporidium parvum. Three sets of 16 plots (2.0 by 3.0 m) were established at 5, 20, and 35% slopes. Within each set of 16 plots, residual dry vegetation matter treatments of 225, 560, and 900 kg/ha were implemented, along with a noncut control averaging 4,500 kg/ ha. Buffer width treatments were implemented by placing cattle fecal material containing known loads of C. parvum 0.1, 1.1, or 2.1 m up-slope of the runoff collector. Grassland buffers of 1.1 and 2.1 m generated 3.2- to 8.8-log and 3.6- to 8.8-log retention of C. parvum, respectively, across the range of residual dry vegetation matter, land slope, rainfall, and runoff conditions examined during this project. Buffers with an increased percent land slope exhibited improved the retention efficiencies, whereas buffers experiencing larger maximum annual runoff events exhibited reduced retention efficiencies. Water-quality data from the 0.1-m-wide buffer plots (effectively no buffer) demonstrated that the majority of C. parvum oocysts (98 to 99.999%) were retained in the fecal matrix for the duration of the storm season, irrespective of the presence of a vegetated buffer. In conclusion, these results support the assertion that grassland buffers are an effective method for reducing animal agricultural inputs of waterborne C. parvum into drinking and irrigation water supplies.

Awad-el-Kariem, F. M., D. C. Warhurst, et al. (1994). "Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction." Parasitology 109 ( Pt 1): 19-22.
	The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).

Axelsson-Olsson, D., J. Waldenstrom, et al. (2005). "Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni." Appl Environ Microbiol 71(2): 987-92.
	We showed by a laboratory experiment that four different Campylobacter jejuni strains are able to infect the protozoan Acanthamoeba polyphaga. C. jejuni cells survived for longer periods when cocultured with amoebae than when grown in culture alone. The infecting C. jejuni cells aggregated in amoebic vacuoles, in which they were seen to be actively moving. Furthermore, a resuscitation of bacterial cultures that were previously negative in culturability tests was observed after reinoculation into fresh amoeba cultures. After spontaneous rupture of the amoebae, C. jejuni could be detected by microscopy and culturability tests. Our results indicate that amoebae may serve as a nonvertebrate reservoir for C. jejuni in the environment.

Bajer, A., M. Bednarska, et al. (1997). "Wildlife rodents from different habitats as a reservoir for Cryptosporidium parvum." Acta Parasitologica 42(4): 192-194.
	A variety of domestic and wild animals are considered to be potential sources of cryptosporidiosis in humans. However, despite considerable information available on Cryptosporidium spp. in humans and ruminants, a paucity of information exists for many species of wildlife rodents. The current study reports the prevalence of Cryptosporidium infection among rodent populations from different habitats in northern Poland. In autumn 1996, spring and summer 1997, a total of 287 animals (8 species of rodents from three different habitats: Clethrionomys glareolus and Apodemus flavicollis and A. sylvaticus from forest; A. agrarius, Microtus arvalis and M. oeconomus from field/meadow; Castor fiber and Ondatra zibethicus from semi-aquatic habitat) were examined for Cryptosporidium oocysts. Direct wet smears from unconcentrated faecal specimens were stained using a modified Ziehl-Neelsen method and/or direct, immunofluorescent (MerIFluor Cryptosporidium/Giardia) assay. Faecal examination revealed that 41 of 132 (31%) C. glareolus, 26 of 96 (27%) Apodemus sp., 19 of 28 (68%) Microtus sp., 1 A. agrarius, 2 of 19 (10.5% colonies of C fiber and 5 of 12 (42%) O. zibethicus were infected with Cryptosporidium oocysts. The intensity of infection was generally low, with <5 oocysts/slide film in most of the samples studied. The morphological characteristics and measurements of the oocysts (mean size 4.8 X 4.5 mum) conformed with those of C. parvum. This study indicates, for the first time in Poland, that semi-aquatic rodents, mostly O. zibethicus, and possibly rodents from other habitats may be involved as reservoirs of infection for C. parvum.

Bajer, A., J. M. Behnke, et al. (2000). "The common vole (Microtus arvalis) as a competent host for Cryptosporidium parvum." Acta Parasitologica 45(3): 178.
	
Bajer, A., J. M. Behnke, et al. (1999). "First evidence of Ehrlichia sp in wild Microtus arvalis from Poland." Acta Parasitologica 44(3): 204-205.
	We examined a total of 73 Microtus arvalis, 168 Clethrionomys glareolus and 17 Apodemus flavicollis trapped in the Mazury Lakes district of North-Eastern Poland, in the spring, summer and autumn of 1998. Three M. arvalis, (2 in summer and 1 in autumn) carried Ehrlichia sp. (overall prevalence = 4.1%), whereas infection was not detected in the other rodent species. We hypothesize that Ixodes ricinus (the most common tick in the region) with which the animals were heavily infested, constitutes the likely natural vector for this pathogen and that M. arvalis are its natural reservoir.

Bajer, A., S. Caccio, et al. (2003). "Preliminary molecular characterization of Cryptosporidium parvum isolates of wildlife rodents from Poland." J Parasitol 89(5): 1053-5.
	Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.

Barbee, S. L., D. J. Weber, et al. (1999). "Inactivation of Cryptosporidium parvum oocyst infectivity by disinfection and sterilization processes." Gastrointest Endosc 49(5): 605-11.
	BACKGROUND: Cryptosporidium parvum is a common cause of self-limited gastroenteritis in the normal host but may cause severe disease in immunocompromised persons. Person-to-person transmission has been well documented in households, child care centers, and hospitals. Because contaminated environmental surfaces and medical devices such as endoscopes may play a role in disease transmission, we studied the susceptibility of C parvum to chemical agents commonly used for disinfection and evaluated the efficacy of sterilization processes. METHODS: Seven disinfectants were studied at their use dilution using a suspension test. Antimicrobial activity was assessed with the use of a cell infectivity assay. RESULTS: All sterilization processes tested (steam, ethylene oxide, Sterrad 100) inactivated 3 logs or greater of C parvum. The only liquid disinfectant/sterilant able to inactivate greater than 3 logs of C parvum was 6% and 7.5% hydrogen peroxide. Agents that did not completely inactivate C parvum included hydrogen peroxide at lower concentrations or exposure times, peracetic acid, sodium hypochlorite, a phenolic, a quaternary ammonium compound, 2% glutaraldehyde, and ortho-phthalaldehyde. CONCLUSIONS: Most high-level disinfectants used on endoscopes have limited efficacy against C parvum. However, the infectivity of C parvum on dry surfaces decreases rapidly. Therefore, current cleaning and high-level disinfection guidelines are adequate to prevent nosocomial transmission of C parvum by means of endoscopes.

Barr, S. C., G. F. Jamrosz, et al. (1994). "Use of paromomycin for treatment of cryptosporidiosis in a cat." J Am Vet Med Assoc 205(12): 1742-3.
	Cryptosporidium oocysts were found in the feces of a 6-month-old female cat with persistent diarrhea. The oocysts disappeared from the feces immediately after treatment with paromomycin (165 mg/kg of body weight, PO, for 5 days), and the diarrhea eventually resolved.

Bern, C., Y. Ortega, et al. (2002). "Epidemiologic differences between cyclosporiasis and cryptosporidiosis in Peruvian children." Emerg Infect Dis 8(6): 581-5.
	We compared the epidemiologic characteristics of cyclosporiasis and cryptosporidiosis in data from a cohort study of diarrhea in a periurban community near Lima, Peru. Children had an average of 0.20 episodes of cyclosporiasis/year and 0.22 episodes of cryptosporidiosis/year of follow-up. The incidence of cryptosporidiosis peaked at 0.42 for 1-year-old children and declined to 0.06 episodes/child-year for 5- to 9-year-old children. In contrast, the incidence of cyclosporiasis was fairly constant among 1- to 9-year-old children (0.21 to 0.28 episodes/child-year). Likelihood of diarrhea decreased significantly with each episode of cyclosporiasis; for cryptosporidiosis, this trend was not statistically significant. Both infections were more frequent during the warm season (December to May) than the cooler season (June to November). Cryptosporidiosis was more frequent in children from houses without a latrine or toilet. Cyclosporiasis was associated with ownership of domestic animals, especially birds, guinea pigs, and rabbits.

Berrilli, F., D. Di Cave, et al. (2004). "Genotype characterisation of Giardia duodenalis isolates from domestic and farm animals by SSU-rRNA gene sequencing." Vet Parasitol 122(3): 193-9.
	In order to investigate the genotypes of Giardia duodenalis from domestic and farm animals in Italy, 21 Giardia isolates, 17 from dogs, 1 from cat and 3 from dairy calves, were genetically characterised by SSU-rRNA gene sequencing. Among dogs, 76.5% of isolates showed the dog-specific genotypes (Assemblages C, D and C/D mixed Assemblage) and 23.5% exhibit potential zoonotic genotypes (Assemblage A and A/C mixed Assemblages). The cat isolate belonged to assemblage A, whereas the sequences among the isolates from calves were found to correspond to hoofed-livestock genotype, namely Assemblage E. These findings suggest that infection of humans by zoonotic genotypes from domestic animals could be of low epidemiological significance, although possible. The present study represents the first contribute to the knowledge of G. duodenalis genotypes in domestic and farm animals from Italy.

Bertrand, I., C. Gantzer, et al. (2004). "Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method." J Microbiol Methods 57(1): 41-53.
	The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.

Bertrand, I., C. Gantzer, et al. (2004). "Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method." J Microbiol Methods 57(1): 41-53.
	The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.

Betancourt, W. Q. and J. B. Rose (2004). "Drinking water treatment processes for removal of Cryptosporidium and Giardia." Vet Parasitol 126(1-2): 219-34.
	Major waterborne cryptosporidiosis and giardiasis outbreaks associated with contaminated drinking water have been linked to evidence of suboptimal treatment. Cryptosporidium parvum oocysts are particularly more resistant than Giardia lamblia cysts to removal and inactivation by conventional water treatment (coagulation, sedimentation, filtration and chlorine disinfection); therefore, extensive research has been focused on the optimization of treatment processes and application of new technologies to reduce concentrations of viable/infectious oocysts to a level that prevents disease. The majority of the data on the performance of treatment processes to remove cysts and oocysts from drinking water have been obtained from pilot-tests, with a few studies performed in full-scale conventional water treatment plants. These studies have demonstrated that protozoan cyst removal throughout all stages of the conventional treatment is largely influenced by the effectiveness of coagulation pretreatment, which along with clarification constitutes the first treatment barrier against protozoan breakthrough. Physical removal of waterborne Crytosporidium oocysts and Giardia cysts is ultimately achieved by properly functioning conventional filters, providing that effective pretreatment of the water is applied. Disinfection by chemical or physical methods is finally required to inactivate/remove the infectious life stages of these organisms. The effectiveness of conventional (chlorination) and alternative (chlorine dioxide, ozonation and ultra violet [UV] irradiation) disinfection procedures for inactivation of Cryptosporidium has been the focus of much research due to the recalcitrant nature of waterborne oocysts to disinfectants. This paper provides technical information on conventional and alternative drinking water treatment technologies for removal and inactivation of the protozoan parasites Cryptosporidium and Giardia.

Beuchat, L. R. (1996). "Pathogenic microorganisms associated with fresh produce." Journal of Food Protection 59(2): 204-216.
	The presence of numerous genera of spoilage bacteria, yeasts and molds, and an occasional pathogen on fresh produce has been recognized for many years. Several outbreaks of human gastroenteritis have been linked to the consumption of contaminated fresh vegetables and, to a lesser extent, fruits. Salads containing raw vegetables have been identified as vehicles of traveler's diarrhea, an illness sometimes experienced by visitors to developing countries. Enterotoxigenic Escherichia coli is the most common cause of this illness. Enterohemorrhagic E. coli, specifically serotype O157:H7, has been implicated as the causative agent in an outbreak of gastroenteritis resulting from the consumption of cantaloupes. Outbreaks of salmonellosis in humans have been attributed to consumption of contaminated tomatoes, mustard cress, bean sprouts, cantaloupe, and watermelon. An onion-associated outbreak of Shigella flexneri gastroenteritis has recently been reported in the United States. Outbreaks of human listeriosis have been epidemiologically linked to the consumption of fresh cabbage and lettuce. Gastrointestinal illness caused by the consumption of raw vegetable seed sprouts contaminated by Bacillus cereus has been documented. The ability of Aeromonas hydrophila and Aeromonas sobria to produce several virulence factors has been documented and their fairly common occurrence in water raises concern over public health risks that may be associated with the consumption of salad vegetables, although their role as agents in foodborne illness has not been fully confirmed. Viruses are not likely to grow on contaminated vegetables and fruits but can survive long enough to cause life-threatening illness in humans. An increased per capita consumption of fresh and lightly processed produce in the United States and other countries, coupled with an increase in importation of produce to these countries from regions where standards for growing and handling produce may be compromised, has resulted in heightened interest in outbreaks of human gastroenteritis that may be attributed to contaminated fresh produce, particularly salad vegetables. Likewise methods of handling, processing, packaging, and distribution of fresh produce on a regional or local scale within countries are receiving attention in terms of identifying and controlling microbiological hazards. Hazard analysis critical control point (HACCP) programs are being developed in an effort to minimize the risk of illness associated with consumption of fresh produce. Examples of pathogenic microorganisms associated with fresh produce as well as procedures that can be used to reduce their incidence at the point of consumption are discussed.

Bier, J. W. (1991). "Isolation of parasites on fruits and vegetables." Southeast Asian J Trop Med Public Health 22 Suppl: 144-5.
	The current FDA method to recover parasites from fruits and vegetables is derived from procedures used to isolate parasitic protozoa from water. A 1kg portion of fruit or vegetable is divided into 200 g subportions. The subportions are sequentially processed in a sonic cleaning bath with 1.5 liters of detergent solution (1% sodium dodecyl sulfate, 0.1% Tween 80) and sonicated for 10 minutes. As each subsample is removed, it is thoroughly drained. After this sonic treatment, the wash water is collected in a polypropylene beaker, transferred to 50 ml polypropylene centrifuge tubes and centrifuged for 15 min at 1500 x g. The sediment is consolidated into one tube along with two rinsings of each tube. The final sediment is fixed in 4% formaldehyde for 10 minutes before examination for parasites. Indirect fluorescent antibody is applied to stain the parasites (Giardia spp. and/or Cryptosporidium spp.) by using commercial kits when available. If a large quantity of extraneous matter is contained in the sediment it may be reduced by layering on Sheather's fluid and centrifuging at 1500 x g for 15 minutes. The supernatant is collected and washed twice in distilled water. This procedure is adequate for protozoa and nonoperculate helminth eggs; operculate helminth eggs may be cleaned by extraction with ethyl acetate. When cabbage and lettuce were seeded at 1 organism/g, the rate of recovery for Cryptosporidium parvum with the FDA method was 1%. When cabbage was seeded at 1 egg/g and 10 eggs/g, the average rate of recovery of decorticated eggs of Ascaris sp. or untreated Trichuris sp. was 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Black, E. K., G. R. Finch, et al. (1996). "Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection." FEMS Microbiol Lett 135(2-3): 187-9.
	In vitro excystation, vital dyes (4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), and infectivity in neonatal CD-1 mice were used to assess the viability of Cryptosporidium parvum oocysts after chemical disinfection. In vitro excystation and DAPI/PI staining provided similar estimates of viability in bench-scale experiments, but both of these methods significantly overestimated the viability when compared with infectivity (Pr < or = 0.01). Infectivity was the most reliable measure of the viability of C. parvum oocysts following chemical disinfection.

Blagburn, B. L. and W. L. Current (1983). "Accidental infection of a researcher with human Cryptosporidium." J Infect Dis 148(4): 772-3.
	
Blunt, D. S., N. V. Khramtsov, et al. (1997). "Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule." Clin Diagn Lab Immunol 4(1): 11-3.
	We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.

Blunt, D. S., B. A. Montelone, et al. (1996). "Sequence of the parasitic protozoan, Cryptosporidium parvum, putative protein disulfide isomerase-encoding DNA." Gene 181(1-2): 221-3.
	A composite 1876-bp DNA encoding a putative protein disulfide isomerase (PDI) has been constructed from clones isolated from Cryptosporidium parvum (C. parvum) genomic and cDNA libraries and the nucleotide sequence determined. As predicted from the open reading frame (ORF), the protein product has a predicted molecular size of 54 kDa and a high degree of homology to PDIs from other species.

Bonafonte, M. T., R. M. Lloyd, Jr., et al. (1996). "Cloning and expression of sporozoite and oocyst Cryptosporidium parvum recombinant proteins." J Eukaryot Microbiol 43(5): 83S.
	
Bonafonte, M. T., L. M. Smith, et al. (2000). "A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice." Exp Parasitol 96(1): 32-41.
	In the present study, we focused on a 23-kDa antigen, Cp23, which has been shown to be a major target of humoral immune responses in Cryptosporidium parvum infections and is present in both the sporozoite and merozoite stages. Recombinant Cp23 antigen was shown to stimulate a specific proliferative response by splenocytes and mesenteric lymph node cells from infected interferon gamma knockout BALB/c mice. Cp23 stimulation also induced TNF-alpha, IL-2, and IL-5 mRNA production by spleen cells from infected animals. In contrast, IL-12 mRNA was decreased by Cp23 stimulation compared with unstimulated splenocytes. These data suggest that, as with humoral responses, Cp23 is an important target of cellular immune responses in experimental C. parvum infections. The potential role of this antigen in conferring protective immunity is also discussed.

Bosch-Driessen, L. E., T. T. Berendschot, et al. (2002). "Ocular toxoplasmosis: clinical features and prognosis of 154 patients." Ophthalmology 109(5): 869-78.
	PURPOSE: To ascertain the clinical features, visual outcome, and recurrence rates of ocular toxoplasmosis (OT) in a large series of patients. To determine the efficacy of various treatment strategies and identify the patients at risk of visual loss. DESIGN: Retrospective noncomparative observational case series. PARTICIPANTS: One hundred fifty-four consecutive patients with active lesions of OT (first attack and/or recurrence) were identified in a cohort of 1300 consecutive patients with uveitis. Mean follow-up was 5.8 years. INTERVENTION: A review of the medical records of 154 patients with active OT. MAIN OUTCOME MEASURES: Patients were subdivided according to clinical and laboratory criteria. Numerous variables were compared per patient and group, including age and gender distribution, onset and course of infection, clinical ocular features, laboratory data, therapeutic strategies and their outcomes, number of recurrences, complications, final visual acuity, and features associated with poor visual outcome. RESULTS: Primary retinal lesions were observed in 28% and a combination of active lesions and old retinochoroidal scars in 72% of the patients at first presentation to the ophthalmologist. Mean age at first presentation with an active OT lesion was 29.5 years. Patients with primary OT were older than those with a combination of active lesions and old scars (P < 0.001). Serologic characteristics of the acute phase of systemic infection were found in 11% of the patients. Ocular involvement in these patients was associated with advanced age at onset (P < 0.001) and was characterized by severe intraocular inflammation. Most (82%) of the patients with serologic characteristics of the acute phase of systemic infection had primary lesions (compared with 23% of OT in the chronic phase of systemic infection; P < 0.001). Extensive retinal lesions were more frequently observed during the acute phase of systemic infection (P = 0.02) and in patients with primary OT (P < 0.04). Recurrences, which developed in 79% of all patients followed for more than 5 years, were located predominantly in previously affected eyes (with old scars) in contrast to the sporadic cases of recurrence in the healthy contralateral eye (P < 0.0001). Standard short-term therapeutic modalities had no effect on visual outcome or future recurrence rates. Legal blindness in one or both eyes was confirmed for 24% of the patients. Blindness of both eyes was more frequent in patients with congenital OT (P < 0.001). Risk factors for visual loss included congenital infection, OT manifesting during the acute phase of systemic infection, central location and/or extensive retinal lesions, and the administration of corticosteroids without a shield of antiparasitic drugs. CONCLUSIONS: Legal blindness in at least one eye developed in 24% of the patients with OT. Recurrences, which developed in 79% of the patients with long-term follow-up, were located predominantly in eyes with toxoplasmic scars. Various short-term therapeutic modalities had no effect on visual outcomes or future recurrence rates, with the exception of a poor visual outcome for patients who received corticosteroids without a shield of antiparasitic drugs.

Bosch-Driessen, L. H., S. Karimi, et al. (2000). "Retinal detachment in ocular toxoplasmosis." Ophthalmology 107(1): 36-40.
	PURPOSE: To report on the clinical course and prognosis of retinal breaks and detachment occurring in patients with ocular toxoplasmosis. DESIGN: Retrospective cross-sectional observational study. PARTICIPANTS: One hundred fifty consecutive patients with ocular toxoplasmosis. INTERVENTION: A review of all records of patients with ocular toxoplasmosis who had consulted our department from 1990 through 1997 was performed. MAIN OUTCOME MEASURES: The presence of retinal detachment or breaks and possible risk factors, such as age, myopia, the interval between the last recurrence of inflammation and the onset of retinal detachment, severity of vitritis, previous treatment methods, and the location of the retinal abnormalities, were analyzed. RESULTS: We found a frequency of 6% (9/150) for retinal detachment and an additional 5% (7/150) for retinal breaks among our patients with ocular toxoplasmosis. Attacks of active ocular toxoplasmosis preceding the retinal detachment or retinal breaks were characterized by severe intraocular inflammation. The frequency of myopia in our patients with retinal detachment or retinal breaks was significantly higher than in patients with ocular toxoplasmosis without retinal detachment or retinal breaks. The functional prognosis for the patients with retinal detachment was poor; legal blindness (visual acuity < or = 20/200) resulting from retinal detachment occurred in five of the nine patients. CONCLUSIONS: Careful retinal examination in ocular toxoplasmosis is warranted, especially in patients with myopia and severe intraocular inflammation.

Brandonisio, O. (2006). "Waterborne transmission of Giardia and cryptosporidium." Parassitologia 48(1-2): 91-4.
	Giardia and Cryptosporidium spp. are parasitic protozoa which are frequent etiologic agents of waterborne diseases. This lecture will summarize the main biological and environmental factors involved in the potential risk for waterborne transmission of giardiosis and cryptosporidiosis, which have caused many outbreaks in different geographical areas. In particular, the current epidemiological situation of these parasitoses in Italy will be analysed, on the basis of research carried out on humans and on the environment. Finally, current methods for evaluating the presence of Giardia cysts and Cryptosporidium oocysts in water and new methods for cyst/oocyst removal from drinking water and wastewater will be examined.

Brandonisio, O., F. Portincasa, et al. (2000). "Giardia and Cryptosporidium in water: evaluation of two concentration methods and occurrence in wastewater." Parassitologia 42(3-4): 205-9.
	Giardia and Cryptosporidium are important agents of water-borne parasitic diseases. In this work we have examined the recovery efficiency of two methods for concentrating Giardia cysts and Cryptosporidium oocysts from water: a membrane filtration method and a crossflow filtration method. Results demonstrated a higher recovery efficiency for crossflow filtration method in comparison to the membrane filtration method. In addition, Giardia cysts and Cryptosporidium oocysts concentration was evaluated in wastewater samples submitted to chemical flocculation or chemical flocculation followed by slow sand filtration. Results showed that slow sand filtration was capable of reducing the number of Giardia cysts, but not of Cryptosporidium oocysts in wastewater.

Browning, J. R. and D. G. Ives (1987). "Environmental Health and the Water Distribution System: A Case History of an Outbreak of Giardiasis." Journal of the Institution of Water and Environmental Management Vol. 1: No. 1, p 55-60.
	In July/August 1985, a significant outbreak of giardiasis occurred in part of south Bristol (England). An epidemiological inquiry carried out strongly suggested that the outbreak was water-borne. Although no positive evidence was found to link the outbreak with mains water, the Company immediately accepted its feasibility and cooperated fully with the Health Authority in the ensuing investigation. This paper describes the investigation to ascertain whether the supply system was involved, and considers some of the broader implications of the situation. Neither the source of the infection nor the means of its transmission was ever established despite extensive and detailed investigation. Also highlighted is the difficulty of tracing the cause and course of an assumed microbiological pollution of a distribution system when, unlike a river or source pollution, evidence of it emerges only after the appropriate incubation period. (Author 's abstract)

Bukhari, Z., W. G. Glen, et al. (1999). "Effects of pH on a fluorogenic vital dyes assay (4',6'-diamidino-2-phenyl-indole and propidium iodide) for Cryptosporidium sp. oocysts." Water Research 33(13): 3037-3039.
	The fluorogenic vital dyes assay utilizing 4',6'-diamidino-2-phenyl-indole (DAPI) and propidium iodide (PI) can be used for determining the viability of individual Cryptosporidium parvum oocysts isolated from environmental samples; however this assay can be affected by the pH of the oocyst suspending medium, resulting in formation of fluorescent yellow DAPI crystals at neutral or alkaline pH. Interference from DAPI crystals can lead to oocyst occlusion, making their viability assessment difficult or subjective. When the oocyst suspending medium is rinsed with HBSS adjusted to pH 4, DAPI crystallization is reduced or prevented without affecting oocyst staining characteristics with these two dyes or with fluorescein isothiocyanate conjugated anti-Cryptosporidium monoclonal antibody.

Bukhari, Z., T. M. Hargy, et al. (1999). "Medium-pressure UV for oocyst inactivation." Journal of the American Water Works Association 91(3): 86-94.
	In water treatment as in life, it is unusual to find that a well-aimed pellet gun can be as effective as an elephant gun, but that is just what the authors of this article did. Although ultraviolet (UV) light was known to effectively inactivate Cryptosporidium oocysts, it was thought that high dosages were required. Bukhari and colleagues successfully inactivated oocysts using much lower UV dosages. The key? Finding the right assay to determine whether oocysts had indeed been inactivated. Mouse infectivity assays were used to test the degree of inactivation as well as the reliability of the surrogate assays (vital dyes and maximized in vitro excystation) that had been used previously. In both bench- and demonstration-scale studies, very low exposure to medium-pressure UV light (19 mJ cm super(-2)) resulted in 3.9-log inactivation of oocyst - as determined by mouse infectivity assays. Surrogate assays, on the other hand, substantially underestimated the reduction in oocyst viability. On the basis of the mouse infectivity studies, the authors conclude that UV is a cost-effective, relatively easy-to-use technology that can reduce concentrations of Cryptosporidium oocysts in drinking water.

Bukhari, Z., M. M. Marshall, et al. (2000). "Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts." Appl Environ Microbiol 66(7): 2972-80.
	Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.

Bukhari, Z., R. M. McCuin, et al. (1998). "Immunomagnetic separation of Cryptosporidium parvum from source water samples of various turbidities." Appl Environ Microbiol 64(11): 4495-9.
	Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.

Bukhari, Z. and H. V. Smith (1995). "Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces." J Clin Microbiol 33(10): 2592-5.
	Bovine fecal samples (1 g) negative for Cryptosporidium sp. oocysts were seeded with 7 x 10(4) Cryptosporidium parvum oocysts and purified by either water-ether concentration, sucrose density flotation, or zinc sulfate flotation to evaluate oocyst recovery. The effect of these purification techniques on the viability of recovered oocysts was also evaluated. Significantly higher numbers of seeded oocysts were recovered by water-ether concentration (recovery rate, 46 to 75%) than by sucrose density (24 to 65%) or zinc sulfate (22 to 41%) flotation methods. In addition, water-ether concentration did not exert a significant effect on the viability of the population of oocysts recovered, whereas sucrose density flotation and zinc sulfate flotation selectively concentrated viable oocysts. The water-ether concentration procedure is recommended for use in epidemiological studies in which both oocyst enumeration and viability assessment are required.

Bukhari, Z. and H. V. Smith (1997). "Cryptosporidium parvum: oocyst excretion and viability patterns in experimentally infected lambs." Epidemiol Infect 119(1): 105-8.
	Cryptosporidium parvum infections of domestic animals can have a considerable economic impact and as oocysts are voided in the faeces of infected hosts, environmental contamination with agricultural waste has also become a matter of concern. Since only viable oocysts are potentially infectious, the numbers of oocysts excreted during infection can have important implications for both veterinary and public health. During the course of infection in experimentally infected lambs, oocyst viability was assessed by a fluorogenic vital dyes assay and by a maximized in vitro excystation assay. The excreted oocyst populations contained a higher proportion of viable oocysts 5-11 days post infection (d.p.i.) than later in the infection. Oocyst viability declined consistently 11-15 d.p.i. and coincided with periods when peaks in serum and intestinal anti-Cryptosporidium antibodies have been reported to occur. Infected lambs excreted a mean of 4.8 (standard error [S.E.] +/- 0.4) x 10(9) oocysts per g of faeces, of which half were non-viable and therefore of no significance for disease transmission. This study demonstrates that the numbers of viable oocysts excreted by infected lambs is smaller than previously suspected.

Bukhari, Z., H. V. Smith, et al. (1997). "Occurrence of Cryptosporidium spp oocysts and Giardia spp cysts in sewage influents and effluents from treatment plants in England." HEALTH-RELATED WATER MICROBIOLOGY 1996: 385-390.
	Cryptosporidium parvum and Giardia duodenalis can cause severe diarrhoea in infected individuals and their transmissive stages, oocysts and cysts, are voided in large numbers with the faeces of infected hosts. Contaminated sewage effluents are recognized as a potential source of waterborne (oo)cysts. In this investigation methods optimized for the recovery of both from a range of wastewaters were used to determine the occurrence of these organisms in influents, effluents and sludges from seven sewage treatment works in England. The data indicated the presence of small numbers of oocysts both in sewage influent and effluent samples whereas cysts were detected more frequently and at higher concentrations in both influents and effluents. Whilst sludge samples from 1/5 sites contained oocysts, cysts were detected from all five sites. These investigations indicate that discharge of sewage effluents into a watercourse, which may be used for potable water abstraction, can contaminate that watercourse with potentially infectious oocysts. In addition, the application of sludge to land can be responsible for contaminating watercourses with (oo)cysts following run-off or leaching.

Bull, S., R. Chalmers, et al. (1998). "Cross-reaction of an anti-Cryptosporidium monoclonal antibody with sporocysts of Monocystis species." Vet Parasitol 77(2-3): 195-7.
	The non-specific cross-reaction of a fluorescently labelled anti-Cryptosporidium monoclonal antibody was observed microscopically when testing faecal specimens from small mammals. The reactive particles were identified as sporocysts of the Gregarine family Monocystidae, and indicate that considerable care should be taken so that false positives are not recorded.

Buret, A., N. denHollander, et al. (1990). "Zoonotic potential of giardiasis in domestic ruminants." J Infect Dis 162(1): 231-7.
	This study was conducted to assess the prevalence and zoonotic potential of giardiasis in domestic ruminants. Prevalence of infection was 17.7% in sheep and 10.4% in cattle and was significantly higher in lambs and calves (35.6% and 27.7%, respectively). Naturally infected lambs released cysts intermittently for months. Giardia trophozoites from sheep had typical claw hammer-shaped median bodies and were successfully cultured in TYI-S-33 medium, and cytosolic, cytoskeletal, and membrane fractions exhibited protein profiles similar to human isolates (WB). Immunoblotting showed that sera from infected sheep recognized human Giardia, sera from patients with giardiasis recognized Giardia from sheep, and in both cases recognition involved antigenic proteins of similar molecular weight. Cyst output and clinical signs in ovine infection resemble human disease and the organisms infecting humans and ruminants are morphologically and antigenically similar. It is postulated that domestic ruminants may be a reservoir for human infection and vice versa, thus classifying giardiasis as a zooanthroponotic disease.

Buret, A., D. G. Gall, et al. (1990). "Intestinal protozoa and epithelial cell kinetics, structure and function." Parasitol Today 6(12): 375-80.
	Intestinal protozoa are not only common enteric pathogens in the tropics but also the high incidence of infection among immunocompromised patients in northern countries has evoked an increased interest in these parasites. Although enteric protozoa are a major cause of diarrhea and malabsorption in humans and other animals, the pathophysiology of gut disturbances caused by them remains poorly understood. Clinical signs related to enteric protozoan disease commonly involve malabsorption, diarrhea, weight loss or retarded weight gain and anorexua. Since these infections are most prevalent and most severe in the young, this may translate into considerable illness among children and significant loss to the agricultural economy where domestic animals are prone to infection. In this review we describe the effects of intestinal protozoan diseases on the structure, kinetics and function of absorptive intestinal cells and other epithelial cells, and correlate morphological injury with physiological alterations in the parasitized gut. Some of the interactions between immune responses and pathophysiology will be discussed, but in-depth discussion of intestinal immunity has recently been undertaken by other authors.

Buret, A., D. G. Gall, et al. (1990). "Effects of murine giardiasis on growth, intestinal morphology , and disaccharidase activity." J Parasitol 76(3): 403-9.
	The aim of this study was to assess the effects of Giardia muris on host growth and food intake, small intestinal morphometrics, mucosal enzyme activities, and brush border ultrastructure. Weanling mice infected with 1,000 G. muris cysts were compared to control and pair-fed sham-treated animals. Infection with G. muris resulted in decreased food intake and retarded growth. In infected animals, villus atrophy was observed in the duodenum throughout the study period and in the jejunum on days 8 and 50. On day 30, whereas jejunal architecture returned to normal in infected animals, malnourished pair-fed animals exhibited a compensatory increase in villus height. Sucrase and maltase were depressed in infected animals on days 2-24. On day 8 jejunal disaccharidases in pair-fed animals were also decreased but to a lesser extent than in infected animals. On day 24, disaccharidase values for control and infected mice were similar, whereas values in pair-fed animals were increased. On day 8, jejunal microvilli were shorter in infected animals than in control and pair-fed animals. This brush border injury was present throughout the jejunum and was also observed in pair-fed animals, but to a lesser extent. These findings suggest that G. muris retards growth in weanling mice, results in small intestinal injury, and interferes with the compensatory response to malnutrition of the infected host. Villus atrophy and brush border enzyme deficiencies associated with the disease mainly occur in the duodenum and jejunum, where trophozoites are most numerous. In infected and in pair-fed animals, the decrease in jejunal disaccharidase activities correlated with a diffuse shortening of brush border microvilli.

Burg, J. L., C. M. Grover, et al. (1989). "Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction." J Clin Microbiol 27(8): 1787-92.
	We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

Burkhalter, R. S., C. A. Smith, et al. (1998). "The signature 10-hydroxy stearic acid thought to correlate with infectivity in oocysts of Cryptosporidium species is an artifact." Lipids 33(8): 829-33.
	Heating or freezing leads to loss in infectivity of oocysts of Cryptosporidium parvum toward neonatal BALB/c mice and is reflected in the profile of the polar lipid fatty acids. Upon loss of infectivity, the ratio of polar lipid to neutral lipid fatty acid decreased and the relative proportions of 18:1n-9 also decreased; proportions of 18:2n-6 and 20:5n-6 increased, whereas the proportions of 16:0 remained constant with freezing. During these investigations, a novel fatty acid, 10-OH 18:0, was discovered in the glycolipid fraction. The identification of a fatty acid unique to species of Cryptosporidium was thought to provide a specific biomarker for this organism. Cryptosporidium also demonstrated fluctuations in absolute quantities of 10-OH 18:0 with events that lead to loss of infectivity. This led to the presumed correlation of this biomarker with infectious Cryptosporidium. The 10-OH 18:0 was putatively localized at the sn-2 position of phosphatidylethanolamine. High-performance liquid chromatography/electrospray ionization mass spectrometry revealed that the 10-OH 18:0 existed principally in the free fatty acid form. Herein, we establish that the free fatty acid 10-OH 18:0 was, in actuality, an artifact of the procedures for sample preparation.

Butt, A. A., K. E. Aldridge, et al. (2004). "Infections related to the ingestion of seafood. Part II: parasitic infections and food safety." Lancet Infect Dis 4(5): 294-300.
	Parasites are responsible for a substantial number of seafood-associated infections. The factor most commonly associated with infection is consumption of raw or undercooked seafood. People with underlying disorders, particularly liver disease, are more susceptible to infection. In the first part of this review, published last month, we discussed the viral and bacterial agents associated with consumption of seafood. In part II, we discuss the parasites commonly associated with seafood consumption. Parasites readily identifiable from both consumable seafood and infected human beings include nematodes, trematodes, cestodes, and protozoa. The salient features associated with seafood-related parasite infestations are discussed. To provide a safe product for consumers, the seafood industry and the government in the USA have undertaken specific measures, which include good manufacturing practices and hazards analysis and critical control points implemented by the government and regulatory agencies. Consumers should take common precautions including obtaining seafood from reputable sources especially if the seafood is to be consumed uncooked. Adequate cooking of seafood is the safest way of preventing related infections.

Caccio, S., W. Homan, et al. (2000). "A microsatellite marker reveals population heterogeneity within human and animal genotypes of cryptosporidium parvum." Parasitology 120 ( Pt 3): 237-44.
	Isolates of the protozoan parasite Cryptosporidium parvum have been differentiated into 2 genotypes: genotype 'H', which is associated only with human infections, and genotype 'C', which is associated with both human and animal infections. To date, the analysis of polymorphisms of genes and of the small subunit ribosomal DNA have revealed no heterogeneity within the 2 genotypes. In the present study, a locus containing simple sequence repeats (microsatellites) was PCR amplified and sequenced from 94 C. parvum isolates, which were collected from humans (immunocompetent and immunocompromized individuals, outbreak and single cases) and from several animal hosts in 3 continents. The analysis revealed that genotype 'H' can be further differentiated into 2 subgenotypes, and genotype 'C' can be further differentiated into 4 subgenotypes. The 6 subgenotypes differ in terms of expansions/contractions of the microsatellite repeats and by point mutations. Some subgenotypes showed a wide geographical distribution, whereas others were restricted to specific regions. Therefore, microsatellites are informative markers for more defined studies on the epidemiology, the transmission routes, and the population structure of this parasite.

Caccio, S., E. Pinter, et al. (2002). "Human infection with Cryptosporidium felis: case report and literature review." Emerg Infect Dis 8(1): 85-6.
	An infection with Cryptosporidium felis in an HIV-positive man from Italy was successfully treated with paromomycin, despite the patient's having a CD4+ cell count of 31/mL. Fourteen cases of human infection with C. felis have been described, all in the past 3 years, emphasizing the public health importance of Cryptosporidium parasites other than C. parvum.

Caccio, S. and E. Pozio (2001). "Molecular identification of food-borne and water-borne protozoa." Southeast Asian J Trop Med Public Health 32 Suppl 2: 156-8.
	Cryptosporidium and Giardia can be transmitted to humans by contaminated food and water, resulting in large outbreaks of diarrheal disease. Sensitive methods for detecting these parasites are needed to control and prevent infection. However, this issue is complicated by the fact that there is still uncertainty about the role played by different species/genotypes with respect to human disease. We are in the process of collecting samples from clinical cases (both sporadic and outbreak-related human infections) and from the environment (tap and waste water samples from different geographic regions), to test the efficacy of methods for detection and genotyping. Concerning Cryptosporidium parvum, we have developed new genotyping methods based on highly polymorphic microsatellite markers. The use of microsatellite markers allows the route of transmission to be traced; these methods can also be used not only to distinguish between anthroponotic and zoonotic transmission but also to identify the source(s) of infection. Regarding Giardia, which was found very frequently in environmental water samples, we are testing the beta-giardin gene as a marker to discriminate among species/genotypes.

Caccio, S., F. Spano, et al. (2001). "Large sequence variation at two microsatellite loci among zoonotic (genotype C) isolates of Cryptosporidium parvum." Int J Parasitol 31(10): 1082-6.
	The genetic polymorphism among 57 Cryptosporidium parvum isolates belonging to genotype 'C' was studied by PCR amplification and the sequencing of two microsatellite loci (ML1 and ML2). A comparative analysis of DNA sequences showed the presence of three (ML1-238, ML1-226, and ML1-220) and seven (ML2-231, ML2-229, ML2-227, ML2-213, ML2-193, ML2-191, and ML2-187) different alleles at these two loci. Alleles differed by expansions/contractions of the microsatellite repeats that generated length polymorphisms. Some alleles were found to be associated with infections of all examined hosts (calf, kid, lamb, and human), whereas others were either associated with a single host, or were geographically restricted. When considering the information from both loci, some preferential associations between alleles are apparent. These data confirm the utility of microsatellite markers for the molecular identification of C. parvum, which is of particular relevance in the investigation of the source of infection of outbreaks and single cases, as well as for genetic studies.

Caccio, S. M. (2005). "Molecular epidemiology of human cryptosporidiosis." Parassitologia 47(2): 185-92.
	Species within the genus Cryptosporidium are protozoan parasites that infect a wide range of vertebrates, and represent a significant cause of morbidity and mortality in those animals. In humans, cryptosporidiosis is a common cause of diarrhoeal disease with a global distribution. Unravelling the epidemiology of human infection has proven to be difficult, due to the existence of multiple transmission routes (person-to-person, animal-to-person, waterborne, foodborne and airborne transmission), and to the difficulties in identifying the different species using conventional criteria, such as oocyst morphology. The advent of molecular techniques has had a remarkable impact on the way the epidemiology of cryptosporidiosis can be studied. Molecular investigations have shown that the vast majority of human cases are caused by C. hominis and C. parvum. Interestingly, differences in geographical and temporal distribution, disease presentations and risk factors for infection have been identified for both C. hominis and C. parvum. Further, molecular analyses have revealed that other species, including C. meleagridis, C. felis, C. canis, C. suis, C. muris and two Cryptosporidium genotypes, can infect humans and may be linked to clinical disease, not only in immunocompromised but also in immunocompetent individuals.

Caccio, S. M., M. De Giacomo, et al. (2003). "Giardia cysts in wastewater treatment plants in Italy." Appl Environ Microbiol 69(6): 3393-8.
	Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O(2) and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P = 0.05, analysis of variance). To characterize the cysts at the molecular level, the beta-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater.

Caccio, S. M., R. C. Thompson, et al. (2005). "Unravelling Cryptosporidium and Giardia epidemiology." Trends Parasitol 21(9): 430-7.
	Molecular biology has provided insights into the taxonomy and epidemiology of Cryptosporidium and Giardia, which are major causes of protozoal diarrhoea in humans worldwide. For both genera, previously unrecognized differences in disease, symptomatology, zoonotic potential, risk factors and environmental contamination have been identified using molecular tools that are appropriate for species, genotype and subtype analysis. In this article, to improve understanding of the epidemiology of cryptosporidiosis and giardiasis, we consider specific requirements for the development of more-effective molecular identification and genotyping systems that should be applicable to both clinical and environmental samples.

Call, J. L., M. Arrowood, et al. (2001). "Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples." J Parasitol 87(1): 203-10.
	Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.

Calvo, M., M. Carazo, et al. (2004). "[Prevalence of Cyclospora sp., Cryptosporidium sp, microsporidia and fecal coliform determination in fresh fruit and vegetables consumed in Costa Rica]." Arch Latinoam Nutr 54(4): 428-32.
	The presence of Cyclospora sp., Cryptosporidium sp. and microsporidia and the levels of fecal coliforms were determined in lettuce, parsley, cilantro, strawberries and blackberries acquired in local agricultural markets of the Central Valley of Costa Rica, in order to establish the possible transmission risk of these microorganisms and other pathogens from the consumption of these raw products. During the second semester of 2001 and the first of 2002, 50 different samples of each product, 25 taken in the dry season and 25 in the rainy season and coming from five different local agricultural markets were evaluated. The fecal coliforms count was done according to the technique recommended by Vanderzant & Splittstoesser. The parasite determination was done using Zielh Nielsen and Weber staining techniques from a sediment obtained through the rinse of the mentioned products, using sterile peptonated water 0.1% and centrifuging at 900 G for 15 min. One hundred per cent of vegetable samples had fecal coliforms and the greatest prevalence was obtained during the rainy season. Although all vegetables presented fecal coliforms in high concentrations, lettuce and cilantro presented statistical difference between rainy and dry season, being greater during the rainy season. Fecal coliforms were not detected in strawberries and blackberries probablydue to its low pH. All products evaluated presented, at least once, Cryptosporidium sp., Cyclospora sp. and microsporidia, showing the risk they represent to Public Health. Cryptosporidium was present in all products but strawberries. Microsporidia was present in all products except blackberries and Cyclospora was only isolated from lettuce during the dry season. These results show the importance of introducing in the country Good Agricultural Practices, especially due to the resistance of Cryptosporidium and Cyclospora to disinfecting agents.

Camero, L., M. Arrowood, et al. (1999). "Characterization of new monoclonal antibodies against Cryptosporidium parvum sporozoites." J Eukaryot Microbiol 46(5): 58S-59S.
	
Campbell, A. T., L. J. Robertson, et al. (1992). "Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes." Appl Environ Microbiol 58(11): 3488-93.
	A viability assay for oocysts of Cryptosporidium parvum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples.

Campbell, I., A. S. Tzipori, et al. (1982). "Effect of disinfectants on survival of cryptosporidium oocysts." Vet Rec 111(18): 414-5.
	
Campbell, P. N. and W. L. Current (1983). "Demonstration of serum antibodies to Cryptosporidium sp. in normal and immunodeficient humans with confirmed infections." J Clin Microbiol 18(1): 165-9.
	Antibodies to Cryptosporidium sp. were detected in sera from 12 immunocompetent individuals recovered from cryptosporidiosis and from 5 subjects with an acquired immunodeficiency syndrome and persistent cryptosporidiosis by an indirect immunofluorescent (IIF) test. Marked seroconversion accompanied recovery from infection in immunocompetent individuals, and their IIF titers remained high (1:40 to 1:640) for at least 1 year. No antibodies to Cryptosporidium sp. were detected in sera from two subjects with hypogammaglobulinemia, normal T-cell function, and persistent cryptosporidiosis or in sera from individuals not previously exposed to Cryptosporidium sp. Very little or no cross-reactivity with the other coccidia--Toxoplasma, Sarcocystis, and Isospora spp.--occurred in the IIF test procedure. The application of this IIF procedure, along with recently developed techniques to detect oocysts in the feces, should provide the basis for a more accurate assessment of the number of individuals within any subject group with previous and active Cryptosporidium infections.

Canals, A., P. Pasquali, et al. (1998). "Local ileal cytokine responses in cattle during a primary infection with Cryptosporidium parvum." J Parasitol 84(1): 125-30.
	In the present study, localized changes in cytokine transcription profiles were examined in neonatal calves following a primary infection with Cryptosporidium parvum, using competitive reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was prepared from ileocecal lymph nodes (LN), lamina propria lymphocytes (LPL), and intraepithelial lymphocytes (IEL) isolated from neonatal calves 7 days after C. parvum infection. Competitive RT-PCR performed on cDNA samples containing internal cytokine gene competitor molecules showed increases in the levels of interferon-gamma and interleukin-12 (IL-12) (P40) mRNA in both LPL and IEL populations but not in the draining LN. In addition, the levels of mRNA of the newly identified growth factor IL-15 decreased in the IEL of the infected animals. No consistent differences were seen in any of the cell populations when the samples were analyzed for IL-10 and levels of mRNA for IL-2 and IL-4 were low and highly variable in both infected and control groups in all 3 lymphocyte populations.

Cann, K. J., R. M. Chalmers, et al. (2000). "Cyclospora infections in England and Wales: 1993 to 1998." Commun Dis Public Health 3(1): 46-9.
	The coccidian protozoon Cyclospora cayetanensis is a treatable cause of prolonged, watery diarrhoea in humans. Microbiology laboratories in England and Wales often restrict testing to those who have recently travelled abroad. Only 44 to 66 laboratory reports of C. cayetanensis are made in England and Wales each year and a large proportion are found to have visited developing countries. Large foodborne outbreaks of infection have arisen in North America among people who have not travelled abroad but no such outbreaks have been identified in the United Kingdom. Public health laboratories in England and Wales were surveyed in 1998 to investigate their procedures for identifying C. cayetanensis. Sixty-eight per cent actively looked for the protozoon, but only half used a recommended method of direct microscopy of formol ether concentrates. National external quality assurance results for all participating UK laboratories were reviewed to assess laboratory proficiency in identification. C. cayetanensis was correctly identified in a wet preparation by 58% of laboratories, the lowest rate for specimens containing a single parasite species. Cyclosporiasis could be acquired in the UK from imported food, but current laboratory procedures might fail to identify it. Ascertainment must improve and awareness needs to be raised among food handlers, public and environmental health workers, laboratory staff, and general practitioners. We recommend that laboratories test all patients with watery diarrhoea for > 1 week for cyclospora, use formol ether concentration and microscopy with a calibrated eyepiece graticule, and confirm diagnoses with the help of a reference laboratory.

Carey, C. M., H. Lee, et al. (2004). "Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst." Water Res 38(4): 818-62.
	Cryptosporidium parvum and Cryptosporidium hominis are obligate enteric protozoan parasites which infect the gastrointestinal tract of animals and humans. The mechanism(s) by which these parasites cause gastrointestinal distress in their hosts is not well understood. The risk of waterborne transmission of Cryptosporidium is a serious global issue in drinking water safety. Oocysts from these organisms are extremely robust, prevalent in source water supplies and capable of surviving in the environment for extended periods of time. Resistance to conventional water treatment by chlorination, lack of correlation with biological indicator microorganisms and the absence of adequate methods to detect the presence of infectious oocysts necessitates the development of consistent and effective means of parasite removal from the water supply. Additional research into improving water treatment and sewage treatment practices is needed, particularly in testing the efficiency of ozone in oocyst inactivation. Timely and efficient detection of infectious C. parvum and C. hominis oocysts in environmental samples requires the development of rapid and sensitive techniques for the concentration, purification and detection of these parasites. A major factor confounding proper detection remains the inability to adequately and efficiently concentrate oocysts from environmental samples, while limiting the presence of extraneous materials. Molecular-based techniques are the most promising methods for the sensitive and accurate detection of C. parvum and C. hominis. With the availability of numerous target sequences, RT-PCR will likely emerge as an important method to assess oocyst viability. In addition, a multiplex PCR for the simultaneous detection of C. parvum, C. hominis and other waterborne pathogens such as Giardia lamblia would greatly benefit the water industry and protect human health.

Carpenter, C., R. Fayer, et al. (1999). "Chlorine disinfection of recreational water for Cryptosporidium parvum." Emerg Infect Dis 5(4): 579-84.
	We examined the effects of chlorine on oocyst viability, under the conditions of controlled pH and elevated calcium concentrations required for most community swimming pools. We found that fecal material may alter the Ct values (chlorine concentration in mg/L, multiplied by time in minutes) needed to disinfect swimming pools or other recreational water for Cryptosporidium parvum.

Carreno, R. A., N. J. Pokorny, et al. (2001). "Decrease in Cryptosporidium parvum oocyst infectivity in vitro by using the membrane filter dissolution method for recovering oocysts from water samples." Appl Environ Microbiol 67(7): 3309-13.
	Exposure of Cryptosporidium parvum oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.

Casemore, D. P. (1994). "Cyclospora: another "new" pathogen." J Med Microbiol 41(4): 217-9.
	
Casemore, D. P., C. A. Gardner, et al. (1994). "Cryptosporidial infection, with special reference to nosocomial transmission of Cryptosporidium parvum: a review." Folia Parasitol (Praha) 41(1): 17-21.
	
Castro Hermida, J. A., F. Freire Santos, et al. (2000). "In vitro and in vivo efficacy of lasalocid for treatment of experimental cryptosporidiosis." Vet Parasitol 90(4): 265-70.
	In vitro viability of purified Cryptosporidium parvum oocysts, exposed for 30, 60, 90 and 120min to 0.27mg/ml lasalocid suspension was evaluated by inclusion or exclusion of two fluorogenic vital dyes and an excystation technique. Continuously, preventive and curative efficacies at different doses (9, 6.75, 5.625 and 4.5mg/kg body weight) and regimens of lasalocid against cryptosporidial infection were evaluated on an experimental neonatal mice model. In vitro assays demonstrated a decrease in the oocyst viability related to an increase in exposure time for exposure to the lasalocid suspension. The infection was eradicated when the suspension was administered with a dose of > or = 6.75mg/kg body weight. No apparent toxic effects were observed.

Castro-Hermida, J. A., F. Freire-Santos, et al. (2000). "Unexpected activity of beta-cyclodextrin against experimental infection by Cryptosporidium parvum." J Parasitol 86(5): 1118-20.
	An unexpected activity of beta-cyclodextrin, an excipient used in pharmaceutical technology, was observed against Cryptosporidium parvum. The viability and infectivity of purified oocysts, exposed for 24 hr to beta-cyclodextrin (2.5% suspension), were evaluated by inclusion/exclusion of 2 fluorogenic vital dyes and a suckling murine model, respectively. Results of the viability assay showed a high proportion of nonviable oocysts (81.5%). The intensity of experimental infection, determined 7 days postinoculation by examination of intestinal homogenates, was significantly lower (P < 0.05) than in the control litters. The preventive and curative efficacies of beta-cyclodextrin suspension were also evaluated in experimentally infected neonatal mice. Infection was prevented when the suspension was administered 2 hr before inoculated oocysts and on days 1 and 2 postinoculation.

Castro-Hermida, J. A., Y. Gonzalez-Losada, et al. (2001). "Evaluation of beta-cyclodextrin against natural infections of cryptosporidiosis in calves." Vet Parasitol 101(2): 85-9.
	The effectiveness of beta-cyclodextrin, excipient used in pharmaceutical industry, in the treatment of natural infection by Cryptosporidium parvum in suckling calves, was evaluated. Administration of the drug at a dose of 500 mg/kg body weight for 3 consecutive days from birth (prophylactically) or following confirmation of the infection (therapeutically) decreased the severity of diarrhoea and shortened the duration of oocyst shedding.

Causape, A. C., J. Quilez, et al. (1996). "Prevalence of intestinal parasites, including Cryptosporidium parvum, in dogs in Zaragoza city, Spain." Vet Parasitol 67(3-4): 161-7.
	Faecal samples from 81 dogs aged between 2 months and 13 years were collected in the small animal clinic (37 domestic dogs) and the animal shelter (44 stray dogs) located in the Faculty of Veterinary Sciences in Zaragoza city (northeast Spain) and screened for the presence of Cryptosporidium oocysts. Faeces were concentrated by the formalin-ethyl acetate method and smears of the sediment were stained by using the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were detected in six dogs (7.4%) aged from 2 months to 6 years. Infection was detected in both domestic (three) and stray (three) dogs and all of them excreted few oocysts (0-1 oocyst per 20 x field). No statistically significant differences in prevalence occurred between dogs younger than 6 months (11.8%) and the older dogs (6.2%). Prevalences were not significantly different between domestic (8.1%) and stray dogs (6.8%). Diarrhoea was recorded in three of the positive dogs (50%), although additional enteric parasites such as oocysts of Isospora spp. were also detected in their faeces. Nevertheless, prevalence was significantly higher in diarrhoeic (30%) versus non-diarrhoeic (4.2%) dogs (P < 0.05). Cryptosporidium was one of the parasites most frequently detected in the dogs surveyed.

Causape, A. C., J. Quilez, et al. (2002). "Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain)." Vet Parasitol 104(4): 287-98.
	An epidemiologic study was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Zaragoza (northeastern Spain). Faecal samples from 583 lambs aged from 1 day to 3 months and 205 ewes older than 1 year were collected at 89 farms in the two regions of the province of Zaragoza with the highest sheep population (Zaragoza and Ejea de los Caballeros). In every sheep farm, data of the factors potentially associated with the likelihood of C. parvum infection were analysed: geographical location, season, size of herd, number of lambs in the herd at sampling time, lambing period, cleaning of lambing area and presence of diarrhoeic lambs in the farm. C. parvum oocysts were identified by using the Ziehl-Neelsen technique in 344 lambs (59%) from 75 farms (84.4%). Infected lambs ranged from less than 7 days to 90 days of age, although the percentage of animals shedding oocysts peaked at 8-14 days of age (76.2%). Statistical analysis showed that infection rates were significantly higher in lambs aged between 1 and 21 days (66.4%) than in those aged between 22 and 90 days (23%) (P<0.0001, chi(2)). Analysis of correlation between excretion of oocysts and diarrhoea revealed a relationship in all age groups and the probability of presenting diarrhoea was significantly higher for lambs shedding oocysts (86.3%) than for those which did not excrete the parasite (32.2%) (P<0.0001, chi(2)). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (79.4%) than in non-diarrhoeic lambs (22.4%). Furthermore, infection intensity was correlated with the presence of clinical symptoms. Presence of diarrhoeic lambs in the farm was the only factor significantly associated with an increased risk of infection since the percentage of herds testing positive was significantly higher in farms with diarrhoeic lambs (91.3%) than in those without cases of neonatal diarrhoea (12.5%) (P<0.0001, chi(2)). Factors associated with a decreased risk of C. parvum infection in lambs included low numbers of lambs in the farm and cleaning of the lambing area. Additionally, lambs 8-14 days of age were less likely to be infected at the first lambing period and in spring/autumn. Cryptosporidial infection was also detected in 16 ewes (7.8%) which excreted few oocysts and without diarrhoea.

Cegielski, J. P., Y. R. Ortega, et al. (1999). "Cryptosporidium, enterocytozoon, and cyclospora infections in pediatric and adult patients with diarrhea in Tanzania." Clin Infect Dis 28(2): 314-21.
	Cryptosporidiosis, microsporidiosis, and cyclosporiasis were studied in four groups of Tanzanian inpatients: adults with AIDS-associated diarrhea, children with chronic diarrhea (of whom 23 of 59 were positive [+] for human immunodeficiency virus [HIV]), children with acute diarrhea (of whom 15 of 55 were HIV+), and HIV control children without diarrhea. Cryptosporidium was identified in specimens from 6/86 adults, 5/59 children with chronic diarrhea (3/5, HIV+), 7/55 children with acute diarrhea (0/7, HIV+), and 0/20 control children. Among children with acute diarrhea, 7/7 with cryptosporidiosis were malnourished, compared with 10/48 without cryptosporidiosis (P < .01). Enterocytozoon was identified in specimens from 3/86 adults, 2/59 children with chronic diarrhea (1 HIV+), 0/55 children with acute diarrhea, and 4/20 control children. All four controls were underweight (P < .01). Cyclospora was identified in specimens from one adult and one child with acute diarrhea (HIV-). Thus, Cryptosporidium was the most frequent and Cyclospora the least frequent pathogen identified. Cryptosporidium and Enterocytozoon were associated with malnutrition. Asymptomatic fecal shedding of Enterocytozoon in otherwise healthy, HIV children has not been described previously.

Chalmers, R. and K. Elwin (2000). "Implications and importance of genotyping cryptosporidium." Commun Dis Public Health 3(3): 155-8.
	
Chalmers, R. M., K. Elwin, et al. (2002). "Cryptosporidium in farmed animals: the detection of a novel isolate in sheep." Int J Parasitol 32(1): 21-6.
	We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.

Chalmers, R. M., K. Elwin, et al. (2002). "Cryptosporidium in farmed animals: the detection of a novel isolate in sheep." Int J Parasitol 32(1): 21-6.
	We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.

Chalmers, R. M., K. Elwin, et al. (2002). "Infection with unusual types of Cryptosporidium is not restricted to immunocompromised patients." J Infect Dis 185(2): 270-1.
	
Chalmers, R. M., C. Ferguson, et al. (2005). "Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species." Int J Parasitol 35(4): 397-410.
	A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined 'subgenotypes' in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and 'gp15' provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.

Chalmers, R. M., S. Hughes, et al. (2002). "Laboratory ascertainment of Cryptosporidium and local authority policies for investigating sporadic cases of cryptosporidiosis in two regions of the United Kingdom." Commun Dis Public Health 5(2): 114-8.
	To discover laboratory ascertainment and reporting practice for cases of cryptosporidiosis in two health authority regions, we surveyed laboratories serving Wales and the North West of England for faecal screening policies and methods for detection of Cryptosporidium. Forty-eight of the 49 laboratories responded, of which 39 (81%) screen all stool specimens from symptomatic individuals for Cryptosporidium and 9 (19%) screen selected specimens. Although laboratory screening is more complete than has been reported in other regions, we identified discrepancies where patient age was used as a selection criterion, and we make suggestions to amend this. Forty-two (88%) responding laboratories report confirmed cases to the regional Communicable Disease Surveillance Centre (CDSC) and 45 (94%) report to the local authority environmental health department. We also surveyed local authorities in both regions for policy and practice concerning the investigation of reported cases of cryptosporidiosis in the same regions. All 59 local authorities responded, of which 57 (97%) investigate cases by completion of an exposure questionnaire as well as providing advice on the prevention of spread of infection. Variation in case ascertainment may influence perception of incidence, clusters and outbreaks of cases of cryptosporidiosis.

Chalmers, R. M., G. Nichols, et al. (2000). "Foodborne outbreaks of cyclosporiasis have arisen in North America. Is the United Kingdom at risk?" Commun Dis Public Health 3(1): 50-5.
	Cyclospora cayetanensis is a parasitic protozoon that causes prolonged watery diarrhoea. It is endemic in some developing countries, and recent foreign travel is often used as a selection criterion for screening in the United Kingdom (UK). Epidemiological investigations of outbreaks of cyclosporiasis among people in the United States and Canada who had not travelled abroad showed the infection to be foodborne and often associated with foods eaten raw. These included raspberries imported from Guatemala, and pesto (made with basil) and lettuce from other sources. Such foods are also being imported in increasing amounts to the UK, but no outbreaks have been documented, perhaps because none has occurred or because of poor ascertainment. This paper reviews the outbreaks reported from North America, evaluates the risks to the UK population, and suggests how surveillance could be improved.

Chalmers, R. M., A. P. Sturdee, et al. (1997). "The prevalence of Cryptosporidium parvum and C. muris in Mus domesticus, Apodemus sylvaticus and Clethrionomys glareolus in an agricultural system." Parasitol Res 83(5): 478-82.
	Wild mice and voles were tested for Cryptosporidium during a 2-year survey at an agricultural site in Warwickshire, United Kingdom. C. parvum and C. muris, the two cryptosporidial species known to infect mammals, were detected. Prevalence figures of 22%, 21% and 13% noted for C. parvum for Mus domesticus, Apodemus sylvaticus and Clethrionomys glareolus, respectively, were higher than those recorded for C. muris at 10%, 6% and 2%. C. parvum causes the sometimes severe diarrhoeal disease cryptosporidiosis in many hosts, but the wild rodents were asymptomatic. The discovery of C. muris in A. sylvaticus and C. glareolus confirms a wider distribution in wild rodents than has previously been reported. Rodents may represent a significant reservoir of Cryptosporidium with a high potential for infection of man and livestock due to cohabitation.

Chappell, C. L., P. C. Okhuysen, et al. (1996). "Cryptosporidium parvum: intensity of infection and oocyst excretion patterns in healthy volunteers." J Infect Dis 173(1): 232-6.
	Data about human Cryptosporidium parvum infection have originated from travelers, community and day care center outbreaks, and persons infected with the human immunodeficiency virus. In addition, experimental infection in 29 antibody-negative, healthy, adult volunteers generated information on the dose-infection response of C. parvum (Iowa strain). In that report, low inocula were sufficient to cause infection in 18 and illness in 7 persons. To further define the duration and intensity of infection in this population, oocyst shedding patterns were investigated in the 18 subjects infected with C. parvum. Oocyst quantitation revealed that volunteers with diarrheal illness (n = 7) excreted more oocysts over the course of the infection than did volunteers without diarrhea (n = 11; P < .05). Symptomatic subjects were more likely to shed oocysts on consecutive days. Further, a statistical nonsignificant inverse trend (r2 = .330, P = .136) was seen between challenge dose and total excreted oocysts. This paradox may relate to receptor saturation or a toxic effect on cells, parasites, or both afforded by a high inoculum.

Chauret, C., K. Nolan, et al. (1998). "Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine." Can J Microbiol 44(12): 1154-60.
	Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2 microm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3); pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log(10).day(-1)) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.

Chen, W., J. A. Harp, et al. (2003). "Cryptosporidium parvum infection in gene-targeted B cell-deficient mice." J Parasitol 89(2): 391-3.
	The importance of B cells in host resistance to, and recovery from, Cryptosporidium parvum infection was examined in gene-targeted B cell-deficient (muMT-/-) mice. Neonatal muMT-/- mice infected with C. parvum at 5 days of age completely cleared the infection by day 20 PI. The kinetics of infection and clearance were similar to those seen with age-matched C57BL/6 control mice. Furthermore, B cells were not required to clear existing C. parvum infection in adult mice. Reconstitution of persistently infected Rag-1-/- adult mice with spleen cells from muMT-/- donor mice resulted in significant reduction of infection, as in the results seen with spleen cells from C57BL6 donors. These findings indicate clearly that B cells are not essential for host resistance to, and recovery from, C. parvum infection in mice.

Chesnot, T. and J. Schwartzbrod (2004). "Quantitative and qualitative comparison of density-based purification methods for detection of Cryptosporidium oocysts in turbid environmental matrices." J Microbiol Methods 58(3): 375-86.
	Purification methods for Cryptosporidium oocysts are usually selected on the basis of recovery yield, but the amount of particulate debris in environmental matrices could limit efficiency of oocyst detection by microscopic examination or PCR detection. Previous studies have shown that the standard immunomagnetic separation (IMS) procedure would not be the most suitable method for oocyst purification from turbid matrices. We compared the capacity of Percoll-sucrose flotation and six other density-based purification methods to achieve selective separation of Cryptosporidium oocysts from particulate debris. Rate of oocyst recovery and particulate loading in the purified suspensions were chosen as comparison criteria for the different purification methods. In most earlier studies, the chemical treatments employed to obtain a purified oocyst suspension modify the surface properties of oocysts in spiked samples. Assuming this produces unrealistic conditions affecting the evaluation of purification methods, we performed the present study with native oocysts. Flotation and gradient procedures were tested with and without formaldehyde ethyl acetate (FEA) separation. FEA separation was found to be unsuitable. Filtration and Percoll gradient did not allow selective oocyst separation from debris. Among the purification methods suitable for routine microscopic examination, Percoll-sucrose flotation provided the best recovery rates. For automated enumeration systems or PCR detection, potassium bromide and especially Nycodenz gradients appeared to be the most suitable purification methods. Potassium bromide and Nycodenz gradients provided the best balance between oocyst recovery and particulate load.

Choi, W. Y., H. W. Nam, et al. (1997). "Foodborne outbreaks of human toxoplasmosis." J Infect Dis 175(5): 1280-2.
	Two outbreaks of acute toxoplasmosis involving 8 adult patients in Korea were linked to eating uncooked pork. In the first outbreak, 3 patients developed unilateral chorioretinitis within 3 months of eating a meal consisting of raw spleen and liver of a wild pig. In the second outbreak, 5 of 11 soldiers who ate a meal consisting of raw liver of a domestic pig developed lymphadenopathy. All 8 patients had high levels of IgG Toxoplasma gondii antibodies (> or = 1:1024) in the Sabin-Feldman dye test, modified agglutination test incorporating mercaptoethanol, and latex agglutination test. T. gondii IgM antibodies persisted in these patients for several months. Most patients had a favorable response to anti-T. gondii chemotherapy with pyrimethamine and sulfanomides.

Chrisp, C. E., M. A. Suckow, et al. (1992). "Comparison of the host ranges and antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from guinea pigs." J Protozool 39(3): 406-9.
	Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in 125I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.

Chung, P. A., J. Johnson, et al. (2000). "Cloning and molecular characterization of a gene encoding a Cryptosporidium parvum putative 20S proteasome beta1-type subunit." DNA Seq 11(3-4): 309-14.
	A DNA sequence composed of 1281 nucleotides (nt) consisting of a single open reading frame (ORF) encoding a putative 20S proteasome beta1-type subunit was isolated from clones derived from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Southern blot analysis suggested that the sequenced DNA exists in the C. parvum genome as a single copy; transcription was verified through reverse transcription-polymerase chain reaction (RT-PCR) performed on total RNA isolated from C. parvum sporozoites. The predicted protein consists of 210 amino acids (aa), contains characteristic amino acids common to all proteasomal subunits, and shares stronger similarity to the beta1-type subunit of yeast than to other types of beta-subunits.

Cimon, K. Y., R. D. Oberst, et al. (1996). "Biliary cryptosporidiosis in two corn snakes (Elaphe guttata)." J Vet Diagn Invest 8(3): 398-9.
	
Clancy, J. L., Z. Bukhari, et al. (2000). "Using UV to inactivate Cryptosporidium." Journal of the American Water Works Association 92(9): 97-104.
	Their small size and resistance to chlorine make Cryptosporidium parvum oocysts a challenge for the water profession. Ultraviolet (UV) technologies, however, are doing what conventional treatment processes and chemical disinfection cannot: inactivating oocysts efficiently and cost-effectively. Although previous studies have investigated UV inactivation of C. parvum oocysts, the findings have raised as many questions as they've answered. This work zeroes in on medium- versus low-pressure UV and finds them equally effective, even at low dosages. At 3 mJ/cm super(2), for example, medium-pressure UV showed a 3.4-log inactivation of oocysts, whereas low-pressure UV performed nearly as well, demonstrating a 3.0-log inactivation. And in the first trials testing UV effectiveness in water with turbidity &gt;1 ntu, medium-pressure UV continued to achieve high levels of oocyst inactivation. Water suppliers are always on the lookout for new technologies that will safeguard customers from waterborne disease without overstraining limited financial resources. With Stage 2 of the Disinfectants/Disinfection By-products Rule looming, UV promises to be a best available technology (BAT) to control Cryptosporidium in surface waters. Low capital and low operations and maintenance costs make UV a BAT for utilities as well.

Clark, D. P. (1999). "New insights into human cryptosporidiosis." Clin Microbiol Rev 12(4): 554-63.
	Cryptosporidium parvum is an important cause of diarrhea worldwide. Cryptosporidium causes a potentially life-threatening disease in people with AIDS and contributes significantly to morbidity among children in developing countries. In immunocompetent adults, Cryptosporidium is often associated with waterborne outbreaks of acute diarrheal illness. Recent studies with human volunteers have indicated that Cryptosporidium is highly infectious. Diagnosis of infection with this parasite has relied on identification of acid-fast oocysts in stool; however, new immunoassays or PCR-based assays may increase the sensitivity of detection. Although the mechanism by which Cryptosporidium causes diarrhea is still poorly understood, the parasite and the immune response to it probably combine to impair absorption and enhance secretion within the intestinal tract. Important genetic studies suggest that humans can be infected by at least two genetically distinct types of Cryptosporidium, which may vary in virulence. This may, in part, explain the clinical variability seen in patients with cryptosporidiosis.

Cole, R. A., D. S. Lindsay, et al. (2000). "Biological and molecular characterizations of Toxoplasma gondii strains obtained from southern sea otters (Enhydra lutris nereis)." J Parasitol 86(3): 526-30.
	Toxoplasma gondii was isolated from brain or heart tissue from 15 southern sea otters (Enhydra lutris nereis) in cell cultures. These strains were used to infect mice that developed antibodies to T. gondii as detected in the modified direct agglutination test and had T. gondii tissue cysts in their brains at necropsy. Mouse brains containing tissue cysts from 4 of the strains were fed to 4 cats. Two of the cats excreted T. gondii oocysts in their feces that were infectious for mice. Molecular analyses of 13 strains indicated that they were all type II strains, but that they were genetically distinct from one another.

Collins, M. V., G. J. Flick, et al. (2005). "The Effect of High-Pressure Processing on Infectivity of Cryptosporidium parvum Oocysts Recovered from Experimentally Exposed Eastern Oysters (Crassostrea virginica)." J Eukaryot Microbiol 52(6): 500-4.
	Shellfish have been identified as a potential source of Cryptosporidium infection for humans. The inactivation of Cryptosporidium parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The present study identified the efficacy of a non-thermal alternative food-processing treatment, high hydrostatic pressure processing (HPP), on the viability of C. parvum oocysts in the Eastern oysters Crassostrea virginica. Oysters were artificially exposed to 2 x 10(7) oocysts of the Beltsville strain of C. parvum in seawater and subjected to HPP treatments. The effects of the treatments were evaluated by inoculation of the processed oyster tissues into neonatal mice. High-pressure processing of shucked Eastern oysters at all pressures tested (305, 370, 400, 480, and 550 MPa) was significantly effective (P<0.05) in reducing the numbers of positive mouse pups fed treated oyster tissues exposed to C. parvum oocysts. A dose of 550 MPa at 180 s (s) of holding time produced the maximum decrease in numbers of C. parvum positive mouse pups (93.3%). Measurement of tristimulus color values of HPP-treated raw oysters at extended processing times from 120 s to 360 s at 550 MPa showed a small increase in whiteness of oyster meat. This non-thermal processing treatment shows promise for commercial applications to improve safety of seafood and reduce public health risks from cryptosporidiosis.

Collins, M. V., G. J. Flick, et al. (2005). "The Effects of E-beam Irradiation and Microwave Energy on Eastern Oysters (Crassostrea virginica) Experimentally Infected with Cryptosporidium parvum." J Eukaryot Microbiol 52(6): 484-8.
	Shellfish have been identified as a potential source of Cryptosporidium infection for humans. The inactivation of C. parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The present study examined the efficacy of two alternative food-processing treatments, e-beam irradiation and microwave energy, on the viability of C. parvum oocysts in Eastern Oysters (Crassostrea virginica), which were artificially infected with the Beltsville strain of C. parvum. The effects of the treatments were evaluated by oral feeding of the processed oyster tissues to neonatal mice. Significant reductions (P<0.05) in infectivity were observed for in-shell and shucked oysters treated with e-beam irradiation at doses of 1.0, 1.5, or 2 kGy vs. untreated controls. A dose of 2 kGy completely eliminated C. parvum infectivity and did not adversely affect the visual appearance of the oysters. Oyster tissue treated with microwave exposures of 1 s (43.2 degrees C), 2 s (54.0 degrees C), and 3 s (62.5 degrees C) showed a reduction in C. parvum mouse infectivity, but the effects were not significantly different (P>0.05) from controls. Microwave energy treatments at 2 and 3 s showed extensive changes in oyster meat texture and color. Thus, because of lack of efficacy and unacceptable tissue changes, microwave treatment of oysters is not considered a viable food-processing method.

Cook, A. J., R. E. Gilbert, et al. (2000). "Sources of toxoplasma infection in pregnant women: European multicentre case-control study. European Research Network on Congenital Toxoplasmosis." Bmj 321(7254): 142-7.
	OBJECTIVE: To determine the odds ratio and population attributable fraction associated with food and environmental risk factors for acute toxoplasmosis in pregnancy. DESIGN: Case-control study. SETTING: Six large European cities. PARTICIPANTS: Pregnant women with acute infection (cases) detected by seroconversion or positive for anti-Toxoplasma gondii IgM were compared with pregnant women seronegative for toxoplasma (controls). MAIN OUTCOME MEASURES: Odds ratios for acute infection adjusted for confounding variables; the population attributable fraction for risk factors. RESULTS: Risk factors most strongly predictive of acute infection in pregnant women were eating undercooked lamb, beef, or game, contact with soil, and travel outside Europe and the United States and Canada. Contact with cats was not a risk factor. Between 30% and 63% of infections in different centres were attributed to consumption of undercooked or cured meat products and 6% to 17% to soil contact. CONCLUSIONS: Inadequately cooked or cured meat is the main risk factor for infection with toxoplasma in all centres. Preventive strategies should aim to reduce prevalence of infection in meat, improve labelling of meat according to farming and processing methods, and improve the quality and consistency of health information given to pregnant women.

Cook, N., C. A. Paton, et al. (2006). "Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: development and optimization of methods." Int J Food Microbiol 109(3): 215-21.
	No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts.

Cook, N., C. A. Paton, et al. (2006). "Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 2: validation." Int J Food Microbiol 109(3): 222-8.
	We report the results of interlaboratory collaborative trials of methods to detect oocysts of the protozoan parasite Cryptosporidium parvum on lettuce and raspberries. The trials involved eight expert laboratories in the United Kingdom. Samples comprised 30 g lettuce, and 60 g raspberries. Lettuce samples were artificially contaminated at three levels: low (8.5-14.2 oocysts), medium (53.5-62.6 oocysts), and high (111.3-135.0 oocysts). Non-contaminated lettuce samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated lettuce samples) of 89.6%, and a specificity (correct identification of non-contaminated samples) of 85.4%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 30.4%. The method was just as reproducible between laboratories, as repeatable within a laboratory. Raspberry samples were artificially contaminated at three levels: low (8.5-26.8 oocysts), medium (29.7-65.7 oocysts), and high (53.9-131.3 oocysts). Non-contaminated raspberry samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated raspberry samples) of 95.8%, and a specificity (correct identification of non-contaminated samples) of 83.3%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 44.3%. The method was just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that these assays can be used effectively in analytical microbiological laboratories.

Cordell, R. L., P. M. Thor, et al. (1997). "Impact of a massive waterborne cryptosporidiosis outbreak on child care facilities in metropolitan Milwaukee, Wisconsin." Pediatr Infect Dis J 16(7): 639-44.
	OBJECTIVE: We describe the impact of the 1993 waterborne cryptosporidiosis outbreak on metropolitan Milwaukee child care homes and centers. METHODS: Information on outbreak-related illness and changes in policies and practices was collected from directors of 117 facilities. Stool specimens from 129 diapered children from 11 centers were screened for Cryptosporidium. RESULTS: Most (74%) facility directors reported children or staff with diarrhea during the outbreak; however, only 4 (3.4%) facilities closed because of illness among staff or children. During the outbreak child care homes were less likely to exclude children with diarrhea than were child care centers. Among diapered children attending centers the Cryptosporidium prevalence was 30%; 29% of infected children had no history of diarrhea associated with the Milwaukee outbreak. CONCLUSIONS: Facilities continued to operate during the outbreak despite considerable illness among children and staff. The news media were effective means for providing public health information to child care facilities. Although secondary transmission undoubtedly took place in child care facilities, the presence of children with asymptomatic Cryptosporidium infections did not result in an increased risk of diarrhea in infant and toddler rooms.

Corso, P. S., M. H. Kramer, et al. (2003). "Cost of illness in the 1993 waterborne Cryptosporidium outbreak, Milwaukee, Wisconsin." Emerg Infect Dis 9(4): 426-31.
	To assess the total medical costs and productivity losses associated with the 1993 waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, including the average cost per person with mild, moderate, and severe illness, we conducted a retrospective cost-of-illness analysis using data from 11 hospitals in the greater Milwaukee area and epidemiologic data collected during the outbreak. The total cost of outbreak-associated illness was 96.2 million US dollars: 31.7 million US dollars in medical costs and 64.6 million US dollars in productivity losses. The average total costs for persons with mild, moderate, and severe illness were 116 US dollars, 47 US dollars, and 7,808 US dollars, respectively. The potentially high cost of waterborne disease outbreaks should be considered in economic decisions regarding the safety of public drinking water supplies.

Craik, S. A., G. R. Finch, et al. (2000). "Inactivation of Giardia muris cysts using medium-pressure ultraviolet radiation in filtered drinking water." Water Research 34(18): 4325-4332.
	The effect of medium pressure ultraviolet radiation on Giardia muris was studied using a collimated beam apparatus with filtered surface water from the Grand River, Kitchener, Ontario, Canada. UV doses ranged from 5 to 83 mJ/cm super(2) and resulted in 2-3 log-units of reduction in infectivity measured by a C3H/HeN mouse infectivity model. In vitro excystation and nucleic acid staining with Live/Dead BacLight greatly underestimated the inactivation of Giardia when compared with animal infectivity. Medium pressure ultraviolet radiation is a potential alternative to conventional chemical disinfection methods.

Craun, G. F. (1986). "Waterborne giardiasis in the United States 1965-84." Lancet 2(8505): 513-4.
	
Current, W. L. and P. L. Long (1983). "Development of human and calf Cryptosporidium in chicken embryos." J Infect Dis 148(6): 1108-13.
	Cryptosporidium is a newly recognized, zoonotic protozoan that produces short-term, flu-like, gastrointestinal illness in immunocompetent humans and prolonged, severe, diarrhea in immunocompromised individuals. Successful completion of the life cycle, from sporozoite to infective oocyst, of isolates of Cryptosporidium from humans and calves was demonstrated in endoderm cells of the chorioallantoic membrane (CAM) of chicken embryos maintained at 37 C. The human and calf isolates of Cryptosporidium were morphologically and developmentally indistinguishable when grown in chicken embryos. The human isolate also completed its entire life cycle in the CAMs of chicken embryos maintained at 35 C and 41 C. Oocysts recovered from endoderm cells of infected CAMs produced heavy infections in suckling mice. The timing, presence, and morphology of developmental stages in CAM cells during the first eight days after inoculation of sporozoites were similar to those in enterocytes of mice inoculated with oocysts. The method described is safe and convenient for cultivating and studying Cryptosporidium in a bacteria-free environment; the system also lends itself to well-established procedures for evaluating antiprotozoan drugs.

Current, W. L., N. C. Reese, et al. (1983). "Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies of an outbreak and experimental transmission." N Engl J Med 308(21): 1252-7.
	Infection with cryptosporidium occurred in 12 immunocompetent persons who had direct contact with the feces of infected calves during three unrelated outbreaks of calf cryptosporidiosis. Nine of the twelve subjects had diarrhea and abdominal cramps that lasted 1 to 10 days. Infections were diagnosed and monitored by detection of oocysts in feces, with a modified Sheather's flotation technique and phase-contrast microscopy. Oocysts of cryptosporidium were isolated from calves but not from other animals with which these subjects had been in contact. Oocysts of cryptosporidium were also detected during repeated examinations of feces from two immunodeficient patients with persistent cryptosporidiosis. An apparently identical infection was transmitted to calves and mice, using oocysts from infected calves and human beings. Oocysts from an immunodeficient person also produced infections in kittens, puppies, and goats. This study shows that cryptosporidium may produce a moderate self-limited illness in immunocompetent persons, which contrasts sharply with the prolonged severe diarrhea in immunocompromised patients who contract cryptosporidiosis. Calves with diarrhea should be considered a potential source of human infection, and immunocompromised persons should avoid contact with such animals.

Curry, A. and H. V. Smith (1998). "Emerging pathogens: Isospora, Cyclospora and microsporidia." Parasitology 117 Suppl: S143-59.
	Isospora belli, Cyclospora cayetanensis as well as several species of microsporidia are recognized as emerging protozoan pathogens of humans. All are obligate intracellular parasites, with Isospora and the microsporidia being primarily associated with immunocompromised hosts. Cyclospora is a cause of traveller's diarrhoea, and is responsible for water-borne and food-borne outbreaks of disease. Drug treatment is available for these infections. Improved diagnostic methods including the autofluorescence of I. belli and C. cayetanensis oocysts have assisted in the routine detection of these pathogens. Since the recognition of immunosuppression due to HIV infection, microsporidia have become recognized as important human pathogens with a continuing expansion of the parasite-associated clinico-pathological spectrum. The small size, intracellular nature and poor staining properties with many histological stains result in under-reporting of microsporidial infections. Trichrome stain and optical brighteners are used to detect spores in faeces, urines, respiratory secretions and other aspirates. Electron microscopy remains an important diagnostic method but its sensitivity is relatively poor. Molecular techniques should overcome current diagnostic limitations. The ability to extract DNA and amplify by PCR directly from clinical samples has increased the usefulness of molecular methods. Restriction fragment length polymorphism analysis of amplicons can be used to determine genus, species and strain types of various microsporidia. Increased specificity is required in primer design because current primers used for amplifying non-microsporidian DNA also amplify microsporidian DNA. Diagnosis and pathogen characterisation rely increasingly on PCR-based approaches, and the sequence analysis approach becomes increasingly feasible and affordable. However, robust, reliable and sensitive methods are still required for dissecting pathogenesis, epidemiology, transmission routes and sources of infections.

da Silva, A. J., S. Caccio, et al. (2003). "Molecular and morphologic characterization of a Cryptosporidium genotype identified in lemurs." Vet Parasitol 111(4): 297-307.
	This study reports the molecular and morphologic characterization of a Cryptosporidium sp., identified in stools of captive lemurs Propithecus verreauxi coquereli. Stool samples were collected from seven animals (n=7) presenting episodes of diarrhea. Bright-field light microscopy of stool smears stained with modified acid-fast technique revealed the presence of Cryptosporidium sp. oocysts in four of the stool samples analyzed. All microscopically positive samples were confirmed by PCR using primers designed to amplify DNA fragments from two independent loci, i.e. the Cryptosporidium oocyst wall protein (COWP) gene and the small subunit ribosomal RNA (ssrRNA) gene. Phylogenetic analysis based on the full-length ssrRNA gene placed this isolate within a clade that contains all currently known C. parvum species/genotypes, closely related to the C. parvum pig genotype. Comparison with partial ssrRNA sequences available in the GenBank revealed 100% sequence identity with the genotype previously identified in Canadian patients. This finding was confirmed further by comparison of the COWP gene partial sequences.

Dalton, C., A. D. Goater, et al. (2001). "Viability of Giardia intestinalis cysts and viability and sporulation state of Cyclospora cayetanensis oocysts determined by electrorotation." Appl Environ Microbiol 67(2): 586-90.
	Electrorotation is a noninvasive technique that is capable of detecting changes in the morphology and physicochemical properties of microorganisms. Electrorotation studies are reported for two intestinal parasites, Giardia intestinalis and Cyclospora cayetanensis. It is concluded that viable and nonviable G. intestinalis cysts can be differentiated by this technique, and support for this conclusion was obtained using a fluorogenic vital dye assay and morphological indicators. The viability of C. cayetanensis oocysts (for which no vital dye assay is currently available) can also be determined by electrorotation, as can their sporulation state. Modeling of the electrorotational response of these organisms was used to determine their dielectric properties and to gain an insight into the changes occurring within them. Electrorotation offers a new, simple, and rapid method for determining the viability of parasites in potable water and food products and as such has important healthcare implications.

Daniels, M. J., M. R. Hutchings, et al. (2003). "The risk of disease transmission to livestock posed by contamination of farm stored feed by wildlife excreta." Epidemiol Infect 130(3): 561-8.
	Livestock feed is susceptible to contamination from wildlife excreta during on farm storage. Pathogens associated with diseases such as paratuberculosis, salmonella and cryptosporidiosis are present in wild rodent and bird excreta. Feed stores on four farms in the east of Scotland were monitored monthly over the winter of 1998/9 to quantify the levels of wildlife faecal contamination. A mean of 79.9 rodent (95% confidence interval: 37.5-165.9) and 24.9 (14.3-41.7) bird faeces were deposited per m2 of stored feed per month. It was estimated that individual cattle and sheep could encounter 1626 and 814 wildlife faeces over the winter. A model based on the numbers of infected faeces consumed per annum was used to estimate 'infectious probabilities' (Pinf) required to account for the reported prevalence of paratuberculosis, salmonella and cryptosporidiosis in sheep and cattle in the east of Scotland in 1998. Based on empirical data for input variables [the number of faeces encountered (Fe), the number ingested (Fi) and the prevalence of infection in wildlife species (Ip)], Pinf estimates ranged from 1.6 x 10(-8) for cryptosporidiosis in sheep to 8.2 x 10(-8) for paratuberculosis in cattle. The model suggested that ingestion of feed contaminated by wildlife faeces could account for the prevalence of all three diseases. Wildlife faecal contamination of stored feed should be given serious consideration as a potential source of infection to livestock.

D'Antonio, R. G., R. E. Winn, et al. (1985). "A waterborne outbreak of cryptosporidiosis in normal hosts." Ann Intern Med 103(6 ( Pt 1)): 886-8.
	In July 1984, an outbreak of gastroenteritis occurred in a suburban community in Texas. A random telephone survey of 100 of 1791 households in the community identified an attack rate of 34%. The outbreak was traced to contamination of the community water supply, an artesian well. Fecal coliforms were identified in untreated drinking water from the well during July. Stool examinations and serologic tests identified Cryptosporidium as the etiologic agent. Cryptosporidium should be added to the list of waterborne organisms capable of causing outbreaks of gastroenteritis.

Davis, L. J., H. L. Roberts, et al. (1998). "A survey of risk factors for cryptosporidiosis in New York City: drinking water and other exposures." Epidemiol Infect 121(2): 357-67.
	We conducted a survey to determine the prevalence of known and theoretical exposure risks for cryptosporidiosis among selected New York City residents. Subjects were recruited from outpatients attending either a practice for persons with HIV infection (n=160), or other medical practices (n=153), at The New York Hospital-Cornell Medical Center. Despite a greater concern for waterborne infection, 82% of HIV-infected subjects reported consuming municipal tap water compared to 69% of subjects from other medical clinics (OR 2.1, 95% Cl 1.2-3.6, P=0.006). Although 18% and 31% of subjects, respectively, denied any tap water consumption at home or work, all but one from each cohort responded positively to having at least one possible alternate source of tap water ingestion such as using tap water to brush teeth or drinking tap water offered in a restaurant. 78% and 76% of subjects, respectively, had at least one potential risk for exposure other than municipal water consumption, such as swimming in pools or contact with animals. Our findings indicate that it is possible to stratify the population into subsets by the amount of tap water consumed. This suggests that an observational epidemiologic study of the risk of contracting cryptosporidiosis from everyday tap water consumption is feasible.

Dawson, D. (2005). "Foodborne protozoan parasites." Int J Food Microbiol 103(2): 207-27.
	This report addresses Cryptosporidium, Giardia, Cyclospora, and more briefly, Toxoplasma as the main parasitic protozoa of concern to food production worldwide. Other parasitic protozoa may be spread in food or water but are not considered as great a risk to food manufacture. The protozoan parasites Cryptosporidium, Giardia, and Cyclospora have proven potential to cause waterborne and foodborne disease. Toxoplasma gondii has been considered a risk in specific cases, but humans are not its primary host. Cryptosporidium and Giardia are widespread in the environment, particularly the aquatic environment, and major outbreaks of cryptosporidiosis and giardiasis have occurred as a result of contaminated drinking water. Large outbreaks of waterborne cyclosporiasis have not been identified. Cryptosporidium, Giardia, and Cyclospora have potential significance in the preparation and consumption of fresh produce and in catering practice, in which ready-to-eat foods may be served that have not received heat treatment. None of the three organisms Cryptosporidium, Giardia, and Cyclospora has been shown to be a problem for heat processed food or tap water that has undergone appropriate treatment at a water treatment works. All three are sensitive to standard pasteurisation techniques. Although humans are not a primary host for T. gondii, the potential exists for both waterborne and foodborne toxoplasmosis. Parasitic protozoa do not multiply in foods, but they may survive in or on moist foods for months in cool, damp environments. Their ecology makes control of these parasites difficult. For general control of parasitic protozoa in the food chain, the following steps are necessary: - Follow good hygienic practice in food service and catering industries.- Minimise dissemination of cysts and oocysts in the farming environment and via human waste management.- Include these microorganisms in Hazard Analysis Critical Control Point (HACCP) plans of water suppliers, industries or sectors that use fresh produce, and operations in which contaminated process or ingredient water could end up in the product (e.g., where water supplies may become contaminated).

Dawson, D. J., C. M. Samuel, et al. (2004). "Survival of Cryptosporidium species in environments relevant to foods and beverages." J Appl Microbiol 96(6): 1222-9.
	AIMS: To provide data on the survival of Cryptosporidium oocysts in a range of conditions relevant to foods and beverages. METHODS AND RESULTS: Cryptosporidium parvum and C. hominis oocysts were stored in buffered media at different pH values and with various acids. In addition, neutral solutions with high salt (4.5% w/v), glycerol (20% v/v), sucrose (50% w/v) or ethanol (9 and 40% v/v) were used to determine their effects on survival. After storage periods of between 1 h and 14 days, viability was assessed using sporozoite ratio or infection of MRC-5 cell monolayers (not previously reported for culture of this organism). With all treatments, and with both assay techniques, viable oocysts were found at the end of the storage periods. However, treatments with one of the following additions: high salt, glycerol, sucrose or ethanol showed a negative and statistically significant effect on survival. Decline was noted after 1 day or even 1 h of treatment. CONCLUSIONS: MRC-5 cells are suitable for infection by C. parvum and C. hominis. Both tissue culture and sporozoite ratio gave broadly similar survival results and the greatest effects were seen with addition of components which reduced water activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful additional information to the food industry when considering the risk posed by this organism.

de Graaf, D. C., F. Spano, et al. (1999). "Speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy." Int J Parasitol 29(8): 1289-306.
	In this paper the authors question whether the development of a vaccine against cryptosporidiosis could be taken into consideration. The necessity and feasibility of such a vaccine for human and veterinary application is discussed. Developmental stages within the life cycle of the parasite that might act as possible targets for vaccine development are summarised, as well as the target antigens offered by molecular biology and immunology studies. Vaccination trials against cryptosporidiosis carried out so far, including the active and passive immunisation approach, are also overviewed. It seems that with respect to a Cryptosporidium vaccine two target groups can be considered: children of the developing world and neonatal ruminants. Antigens representing possible candidates for a subunit vaccine were identified based on their function, location and/or the immune response they evoke. While the active vaccination of newborn calves, lambs and goat kids has to face a number of important limitations, the passive immunisation approach, where dams were immunised to protect their progeny by colostral transfer, was proven to be a valuable alternative. Finally, a number of points of action for the near future are put forward.

de Graaf, D. C., E. Vanopdenbosch, et al. (1999). "A review of the importance of cryptosporidiosis in farm animals." Int J Parasitol 29(8): 1269-87.
	Cryptosporidium species are coccidian parasites with a large capacity to reproduce and to disseminate. Several species are known to infect farm animals, although the economic importance of cryptosporidiosis is highly host species dependent. This paper reviews the impact of cryptosporidial infections in livestock and poultry. For different farm animals, the Cryptosporidium spp. that occur, as well as their clinical and pathological features, and their interactions with other pathogens, are described. In addition, data concerning the prevalence, the transmission and the epidemiology of the disease are mentioned and a description of the economic losses associated with cryptosporidiosis in each of the hosts is given. Cryptosporidiosis seems to be mainly a problem in neonatal ruminants. Cryptosporidium parvum is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome of calves, lambs and goat kids, causing considerable direct and indirect economic losses. Avian cryptosporidiosis is an emerging health problem in poultry, associated with respiratory disease in chickens and other Galliformes, and with intestinal disease in turkeys and quails. Because of limited availability of effective drugs, the control of cryptosporidiosis relies mainly on hygienic measures and good management.

de Graaf, D. C., K. Walravens, et al. (1998). "A Cryptosporidium parvum oocyst low molecular mass fraction evokes a CD4+ T-cell-dependent IFN-gamma response in bovine peripheral blood mononuclear cell cultures." Int J Parasitol 28(12): 1875-80.
	T-Cell antigens that induce the in-vitro interferon-gamma response during Cryptosporidium parvum infection of neonatal calves were identified. A total oocyst extract was separated into a high and a low Mr fraction by a microfiltration technique. Both the high and low Mr fractions evoked an in-vitro interferon-gamma response in naturally infected animals, although strong individual differences between the hosts were observed. Using a complement-mediated technique CD4+ T-cells or WC1+gammadelta T-cells were depleted, whereupon the remaining lymphocyte cultures were stimulated with the different antigen preparations. It was shown that the in-vitro interferon-gamma response of Cryptosporidium-infected calves is CD4+ T-cell-dependent.

de la Fuente, C., J. M. San Miguel, et al. (2000). "Pharyngeal bot flies in Cervus elaphus in central Spain: prevalence and population dynamics." J Parasitol 86(1): 33-7.
	The prevalence and intensity of infestations by bot flies Pharyngomyia picta and Cephenemyia auribarbis in red deer (Cervus elaphus) from Quintos de Mora (Toledo, Spain) were determined over a 1-yr period. Bots were present all year. No clear correlations were found between age or sex of the host and parasitization levels (prevalence and intensity). Considerable variation was found in prevalence and intensity, with larger values from December to March. Cephenemyia auribarbis was restricted from November to March, with maximum numbers of L-3 in February. Pharyngomyia picta showed a more complex profile with 2 peaks (March and August), indicating 2 generations per year.

de la Fuente, R., M. Luzon, et al. (1999). "Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain." Vet Parasitol 80(3): 179-85.
	Faeces samples from 218, 1 to 30-day-old, diarrheic dairy calves in 65 dairy herds were screened for the presence of Cryptosporidium and concurrent infections with rotavirus, coronavirus, F5 Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1-7, 8-14, 15-21 and 22-30 days. Cryptosporidium infection was detected in 43.8%, 71.9%, 63.2% and 6.9% of the calves in the respective age groups. Significant differences in the detection rate of Cryptosporidium were found between the age group 22-30 days and all other age groups, and between the age group 1-7 days and the age groups 8-14 days and 15-21 days. Cryptosporidium was the only enteropathogen detected in 60 of the 114 (52.6%) diarrheic calves. Concurrent infections with other enteropathogen(s) were detected in 64.3%, 46.3%, 39.5% and 0% of the Cryptosporidium-infected calves in the age groups 1-7, 8-14, 15-21 and 22-30 days, respectively. A significant age-associated decrease in the detection rate of mixed infections (p < 0.05) was found. The detection rates of the other enteropathogens considered in calves with Cryptosporidium infection were 87% for rotavirus, 11.1% for coronavirus, 27.8% for F5+ E. coli and 1.8% for Salmonella.

de Wit, M. A., M. P. Koopmans, et al. (2001). "Gastroenteritis in sentinel general practices,The Netherlands." Emerg Infect Dis 7(1): 82-91.
	From 1996 to 1999, the incidence of gastroenteritis in general practices and the role of a broad range of pathogens in the Netherlands were studied. All patients with gastroenteritis who had visited a general practitioner were reported. All patients who had visited a general practitioner for gastroenteritis (cases) and an equal number of patients visiting for nongastrointestinal symptoms (controls) were invited to participate in a case-control study. The incidence of gastroenteritis was 79.7 per 10,000 person years. Campylobacter was detected most frequently (10% of cases), followed by Giardia lamblia (5%), rotavirus (5%), Norwalk-like viruses (5%) and Salmonella (4%). Our study found that in the Netherlands (population 15.6 million), an estimated 128,000 persons each year consult their general practitioner for gastroenteritis, slightly less than in a comparable study in 1992 to 1993. A pathogen could be detected in almost 40% of patients (bacteria 16%, viruses 15%, parasites 8%).

de Wit, M. A., M. P. Koopmans, et al. (2001). "Etiology of gastroenteritis in sentinel general practices in the netherlands." Clin Infect Dis 33(3): 280-8.
	Data from a general practice-based, case-control study on gastroenteritis and the pathogens related to this disease were used to study the association between specific pathogens and the infected patients' ages and symptoms. For comparison, the occurrence of these pathogens in control patients, stratified by age, also is presented. In children with gastroenteritis who were <5 years of age, rotavirus (in 21% of patients) and Norwalk-like virus (NLV; in 15%) were the most common pathogens. Among patients who were 5-14 years of age, Campylobacter species (in 16% of patients) and Giardia lamblia (in 10%) were the most common pathogens. In the older patients, Campylobacter species was also the most common pathogen (8% to 15% of patients). In addition, several symptoms in case patients were associated with specific pathogens. Blood in the stool was associated with infection with Campylobacter species. In patients with fever, Salmonella species, Campylobacter species, and rotavirus were detected relatively often. Vomiting was associated with NLV and rotavirus. This is the first study in The Netherlands and one of the first studies in the world that has investigated a broad range of pathogens recovered from an unselected population of patients who had consulted general practitioners because of gastroenteritis.

de Wit, M. A., M. P. Koopmans, et al. (2001). "Sensor, a population-based cohort study on gastroenteritis in the Netherlands: incidence and etiology." Am J Epidemiol 154(7): 666-74.
	A prospective population-based cohort study with a nested case-control study was conducted to estimate the incidence of gastroenteritis and the associated pathogens in the general Dutch population. Follow-up of two consecutive cohorts was performed by weekly reporting cards from December 1998 to December 1999. Cases and controls in the case-control study supplied a questionnaire and stool samples. The standardized gastroenteritis incidence was 283 per 1,000 person-years. The incidence rose with increasing level of education and was higher for persons with a history of diarrhea and for young children. Bacterial pathogens accounted for 5% of cases, bacterial toxins for 9%, parasites for 6%, and viral pathogens for 21%, with Norwalk-like virus (NLV) as the leading pathogen in 11% of cases. The gastroenteritis incidence was higher than that reported for England, but lower than for the United States. In community cases, viral pathogens are the leading cause of gastroenteritis, with NLV being the number one cause of illness in all age groups but one. In many countries, preventive measures are implemented to decrease bacterial infections. However, additional prevention of viral infections, especially NLV, might significantly decrease the number of gastroenteritis cases in the community.

de Wit, M. A., L. M. Kortbeek, et al. (2001). "A comparison of gastroenteritis in a general practice-based study and a community-based study." Epidemiol Infect 127(3): 389-97.
	We compared gastroenteritis cases that consulted a general practitioner (GP) with those who did not in a community-based study and also with those in a GP-based study. We aimed to identify factors associated with consultation, and with inclusion of cases by GPs, and secondly to study the effects on the frequency of detection of pathogens. Furthermore, we estimated the under-ascertainment by GPs. Both studies were performed in The Netherlands in the same population in an overlapping time-period. Overall, 5% of community cases consulted a GP. Cases who consulted suffered from more severe episodes than non-consulting cases. Inclusion of cases by GPs, instead of a study team, caused a selection of more severe cases with more chronic symptoms. When extrapolating data from GP-based studies, it should be taken into account that, in general practice, gastroenteritis due to bacteria and Giardia lamblia is a relatively large proportion of that in the community and gastroenteritis due to Norwalk-like viruses is a relatively small proportion. The incidence of gastroenteritis in general practices was estimated between 14 and 35 per 1000 person years.

Deng, M. and M. S. Abrahamsen (2001). "Microarray-based expression analysis of human epithelial cell response to Cryptosporidium parvum infection." J Eukaryot Microbiol Suppl: 42S-43S.
	
Deng, M., C. A. Lancto, et al. (2004). "Cryptosporidium parvum regulation of human epithelial cell gene expression." Int J Parasitol 34(1): 73-82.
	Cryptosporidium parvum is an obligate intracellular protozoan capable of causing life-threatening diarrhoeal disease in immunocompromised individuals. Efforts to develop novel therapeutic strategies have been hampered by the lack of understanding of the pathogenesis of infection. To better understand the host response to C. parvum infection, gene expression profiles of infected human ileocecal adenocarcinoma cells were analysed by using Affymetrix oligonucleotide microarrays containing probe sets for 12,600 human genes. Statistical analysis of expression data from three independent experiments identified 223 genes whose expression was reproducibly regulated by C. parvum infection at 24 h post-inoculation (125 up-regulated and 98 down-regulated), 13 of which were validated by quantitative reverse transcriptase polymerase chain reaction analysis. This analysis revealed the consistent up-regulation of host heat-shock genes and genes for pro-inflammatory chemokines IL-8, RANTES, and SCYB5. Multiple genes for host actin and tubulin genes were up-regulated whereas genes for actin binding proteins were down-regulated, confirming previous observations of host cytoskeleton rearrangement in response to C. parvum infection. In addition, host genes associated with cell proliferation and apoptosis were differentially regulated, reflecting the complexity of host-parasite interaction. Together, this study demonstrated that C. parvum infection results in significant changes in host biochemical pathways and provides new insights into specific biological processes of infectious disease caused by an intracellular protozoan parasite.

Deng, M., S. Nuanualsuwan, et al. (2001). "Inactivation of Cryptosporidium parvum oocysts by bacterial strains." J. Eukaryot. Microbiol. 2001(Suppl): 37S-39S.
	
Deng, M., M. S. Rutherford, et al. (2004). "Host intestinal epithelial response to Cryptosporidium parvum." Adv Drug Deliv Rev 56(6): 869-84.
	Cryptosporidium parvum is an obligate intracellular protozoan parasite that is a well-recognized cause of diarrhea in humans and animals throughout the world, and is associated with a substantial degree of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). C. parvum primarily infects epithelial cells of the gastrointestinal tract, resulting in acute watery diarrhea for which there is no effective therapy. During infection, all parasite development, sexual or asexual, occurs within epithelial cells of the host. This requires a unique and complex association between two distinct eukaryotic organisms. Conversely, due to the intracellular nature of C. parvum, epithelial cells appear to play a key role in activating and communicating with the host immune system. Delineation of the biochemical processes that are regulated within infected epithelial cells is crucial for understanding the pathology of C. parvum infection, the process by which the host clears and ultimately develops resistance to infection, and the development of chemotherapeutic strategies to intercede infections.

Deng, M. Q. and D. O. Cliver (1998). "Cryptosporidium parvum development in the BS-C-1 cell line." J Parasitol 84(1): 8-15.
	Cryptosporidium parvum is a worldwide parasitic protozoon capable of causing life-threatening disease in immunocompromised patients. In vitro cultivation of C. parvum has been under investigation for development of a well-defined in vitro model for C. parvum infectivity assay. This is the first report of C. parvum completing its life cycle in BS-C-1, an African green monkey kidney cell line. Both sodium hypochlorite-stimulated oocysts and purified sporozoites were able to initiate infection that led to completion of the whole life cycle, although inoculating purified sporozoites was less efficient. Beside Giemsa staining and normal light microscopy, an indirect fluorescent antibody staining method was developed to facilitate the detection and interpretation of C. parvum life stages developed in vitro. A C. parvum-specific polymerase chain reaction was also applied to detect and confirm the presence of C. parvum life stages in liquid from oocyst-infected cell cultures. In addition to the 452-base-pair (bp) product that can be specifically amplified from C. parvum oocysts and sporozoites, a fragment near 280 bp was also obtained. Various applications of this in vitro culture system are envisioned.

Deng, M. Q. and D. O. Cliver (1998). "Differentiation of Cryptosporidium parvum isolates by a simplified randomly amplified polymorphic DNA technique." Appl Environ Microbiol 64(5): 1954-7.
	Genomic DNA was isolated from Cryptosporidium parvum oocysts by a specific immunomagnetic separation-in vitro excystation procedure and subjected to randomly amplified polymorphic DNA analysis using sequence-independent primers. An estuary C. parvum isolate was easily differentiated from several bovine isolates, while five bovine isolates of the same origin were indistinguishable from each other.

Deng, M. Q., D. O. Cliver, et al. (1997). "Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples." Appl Environ Microbiol 63(8): 3134-8.
	A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.

Deng, M. Q., R. P. Peterson, et al. (2000). "First findings of Cryptosporidium and Giardia in California sea lions (Zalophus californianus)." J Parasitol 86(3): 490-4.
	We report the detection and identification of Cryptosporidium and Giardia from 1 of 3 species of pinnipeds. Fecal samples were collected from Pacific harbor seal (Phoca vitulina richardsi), northern elephant seal (Mirounga angustirostris), and California sea lion (Zalophus californianus) in the northern California coastal area. By means of fluorescently labeled monoclonal antibodies, Cryptosporidium oocysts were detected in 3 samples from California sea lions, 1 of which also contained Giardia cysts. Oocysts of Cryptosporidium and cysts of Giardia were morphologically indistinguishable from oocysts of C. parvum and cysts of G. duodenalis from other animal origins. Oocysts and cysts were then purified using immunomagnetic separation techniques and identified by polymerase chain reaction (PCR), from which species-specific products were obtained. Sequence analysis revealed that the 452-bp and 358-bp PCR products of Cryptosporidium isolated from California sea lion had identities of 98% with sequences of their template fragments of C. parvum obtained from infected calves. Based on morphological, immunological, and genetic characterization, the isolates were identified as C. parvum and G. duodenalis, respectively. The findings suggested that California sea lions could serve as reservoirs in the environmental transmission of Cryptosporidium and Giardia.

Deng, M. Y. and D. O. Cliver (1992). "Degradation of Giardia lamblia cysts in mixed human and swine wastes." Appl Environ Microbiol 58(8): 2368-74.
	This study was conducted to determine the persistence of Giardia lamblia cysts in mixed septic tank effluent and swine manure slurry and to correlate fluorescein diacetate-propidium iodide staining of G. lamblia cysts with their morphology under low-voltage scanning electron microscopy. Under field conditions, G. lamblia cysts were degraded more rapidly in the mixed waste than in the control Dulbecco's phosphate-buffered saline (PBS). For total and viable cysts, the mixed waste had D values (time for a 90% reduction in number of cysts) of 18.3 and 15.5 days, and the Dulbecco's PBS control had D values of 41.6 and 26.8 days. The rates of cyst degradation in septic tank effluent and in Dulbecco's PBS were similar. Increasing the proportion of swine manure slurry in the mixed waste favored degradation of the parasite. These results indicate that the mixed waste treatment was the predominant factor affecting the cyst persistence and that it was swine manure slurry that played the role of degrading the parasite. Visualization of viable and nonviable Giardia cysts with low-voltage scanning electron microscopy revealed an excellent correlation between the viability of the cysts determined by fluorescein diacetate-propidium iodide staining and their electron microscopic morphology.

Deng, M. Y. and D. O. Cliver (1994). "LVSEM morphology of Giardia lamblia cysts as related to viability determined by fluorogenic dye staining. ." Midwest Microscopy 23: 24-32.
	
Deselliers, L. P., D. T. M. Tan, et al. (1997). "Effects of Giardia Lamblia Infection on Gastrointestinal Transit and Contractility in Mongolian Gerbils." Digestive Diseases & Sciences 42(12): 2411-2419.
	To determine if Giardia lamblia infection is associated with altered gastrointestinal transit and smooth muscle contractile function, Mongolian gerbils were infected orogastrically with 2 x 10(5) trophozoites (infected) or vehicle (uninfected controls). At the time of peak colonization, control and infected animals were infused either orogastrically or intraduodenally with Cr-51. Gastric emptying of isotope and intestinal transit (measured by the geometric center of distribution of intestinal Cr-51 transit) were significantly (P < 0.05) greater in the infected compared to control animals in both the fasted and the fed states. Then, to determine whether Giardia lamblia has an effect on the contractility of longitudinal and circular smooth muscle, isometric tension of jejunal segments was recorded. The development of active tension with stretch and the dose-response curve to bethanechol were significantly increased in the longitudinal muscle of infected animals compared to controls. However, the circular smooth muscle did not show a similar increase in contractility. These findings suggest that an altered gastrointestinal transit and smooth muscle contractility may be involved in the pathophysiology of giardiasis. [References: 31] 31

Dias, R. A., I. T. Navarro, et al. (2005). "Toxoplasma gondii in fresh pork sausage and seroprevalence in butchers from factories in Londrina, Parana State, Brazil." Rev Inst Med Trop Sao Paulo 47(4): 185-9.
	The aims of this study were to verify the presence of Toxoplasma gondii cysts in fresh pork sausage and the presence of antibodies against T. gondii in serum of workers from factories with Municipal Inspection Service, in Londrina, PR, Brazil. 149 samples of sausage were collected from eight factories and blood samples from 47 workers. We also took information about the practices that were adopted in the factories and the workers' habits that could influence the prevalence of toxoplasmosis. After bioassay in mice, 13 (8.7%) sausage samples were positive, in one of them T. gondii was isolated and in the other 12 the mice seroconverted. Of 47 workers, 36 (76.6%) worked in sausage production and 11 (23.4%) were involved in other functions; 59.5% (28/47), 55.5% (20/36) and 72.7% (8/11), respectively, had T. gondii antibodies. There were no significant differences in the variables of industries' practices and workers' habits related to T. gondii infection. We concluded that fresh pork sausage could be important in the transmission of toxoplasmosis.

Diaz, V., M. Campos, et al. (1996). "Aspects of animal giardiosis in Granada province (southern Spain)." Vet Parasitol 64(3): 171-6.
	An epidemiological study of animal giardiosis was carried out in the province of Granada. In this study, 912 samples from dogs, 592 from cows, 1165 from sheep, and 574 from goats were analyzed, obtaining prevalences of 12.09%, 0.16%, 6.26% and 4.00%, respectively. Prevalence was studied in relation to sex of host, area within the province and seasonal change; in addition, the age factor was taken into account in the case of dogs.

DiGiorgio, C. L., D. A. Gonzalez, et al. (2002). "Cryptosporidium and giardia recoveries in natural waters by using environmental protection agency method 1623." Appl Environ Microbiol 68(12): 5952-5.
	Relatively few studies have examined recoveries from source waters by using Environmental Protection Agency method 1623 with organism spike doses that are environmentally realistic and at turbidity levels commonly found in surface waters. In this study, we evaluated the filtration capacities and recovery efficiencies of the Gelman Envirochek (standard filter) and the Gelman Envirochek high-volume (HV) sampling capsules under environmental conditions. We also examined the performance of method 1623 under ambient conditions with matrix spike experiments using 10 organisms/liter. Under turbid conditions, the HV capsule filtered approximately twice the volume filtered by the standard filter, but neither could filter 10 liters without clogging. In low-turbidity waters, oocyst, but not cyst, recoveries were significantly higher when the HV capsule was used. In turbid waters, organism recoveries were lower than those in nonturbid waters and were not significantly different for the different filters. When the HV capsule was used, Cryptosporidium recoveries ranged from 36 to 75%, and Giardia recoveries ranged from 0.5 to 53%. For both organisms, recoveries varied significantly by site. Turbidity could explain variation in Giardia recoveries (r(2) = 0.80) but not variation in Cryptosporidium recoveries (r(2) = 0.16). The inconsistent recoveries across sites suggested that the background matrix of the ambient water affected recovery by method 1623. A control sample collected at the height of the winter rainy season detected one organism, highlighting the difficulty of using this method to accurately measure pathogen abundance under natural conditions. Our findings support the use of the HV filter under field conditions but suggest that designing a cost-effective and statistically valid monitoring program to evaluate sources and loads of protozoan pathogens may be difficult.

Dillingham, R. A., A. A. Lima, et al. (2002). "Cryptosporidiosis: epidemiology and impact." Microbes Infect 4(10): 1059-66.
	Cryptosporidium was first recognized in humans in 1976 and came to prominence in the 1980s and 1990s as a cause of severe diarrheal illness in patients with AIDS. Its hardy, chlorine-resistant oocysts, tiny size, low infectious dose, fully infectious development when shed and zoonotic potential make it a threat in drinking and recreational water, contaminated food, day care centers, hospitals, and in persons with exposure to animals or unsanitary conditions, with potentially huge, long-term impact in malnourished children, as reviewed herein.

Dubey, J. P. (1995). "Duration of immunity to shedding of Toxoplasma gondii oocysts by cats." J Parasitol 81(3): 410-5.
	Cats that have shed Toxoplasma gondii oocysts are considered to be immune to reshedding of oocysts. To investigate if this immunity persists in cats for 6 yr, 12 4-6-mo-old cats without T. gondii antibodies were inoculated orally with tissue cysts of the ME-49 strain (6 cats) and the TS-2 strain (6 cats) of T. gondii. All of them shed > or = 20 million oocysts between 4 and 13 days after feeding tissue cysts. Two cats became ill between 11 and 13 days after primary infection; 1 died on the 13th day, and the other had to be killed on the 11th day because of generalized acute toxoplasmosis. Toxoplasma gondii oocysts were not found on the hair of 10 cats examined 7 days after cats had shed millions of oocysts. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. None of the challenged cats shed oocysts. One cat died due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Four of these 9 cats re-shed T. gondii oocysts; 3 of them had been challenged also at 39 days after primary infection. Two control cats housed together with chronically infected cats for 6 yr remained seronegative for T. gondii; both of these shed oocysts after challenge with the P89 strain.

Dubey, J. P. (1996). "Infectivity and pathogenicity of Toxoplasma gondii oocysts for cats." J Parasitol 82(6): 957-61.
	Toxoplasma gondii oocysts are highly infective to intermediate hosts including humans, pigs, and mice, but are considered less infective for cats, the definitive host. To determine infectivity of T. gondii oocysts for cats, 20 2- to 3-mo-old T. gondii-free cats in groups of 4 were fed graded doses of oocysts estimated to have 1, 10, 100, 1,000, or 10,000 mouse infective oocysts of the VEG strain of T. gondii. Feces of cats were examined for at least 35 days after feeding oocysts. All cats were killed, necropsied, their sera were tested for T. gondii antibodies, and tissues were bioassayed in mice. Three of the 4 cats fed 10,000 oocysts, 3 of the 4 cats fed 1,000 oocysts, and 2 of the 4 cats each fed 100 oocysts shed 7.3-162 million T. gondii oocysts in their feces, with a prepatent period of 18-44 days. Based on bioassay and antibody production, all 4 cats fed 10,000 oocysts, 3 of 4 cats fed 1,000 oocysts, 2 of 4 cats fed 100 oocysts, and 0 of 8 cats fed 1 or 10 oocysts acquired T. gondii infection. Antibodies to T. gondii were detected by the modified agglutination test in all 9 bioassay-proven T. gondii-infected cats and in none of the 11 cats without demonstrable T. gondii. In a series of other experiments, the age of the cat at the time of oocyst feeding and the administration of corticosteroids were found to have no influence on the prepatent periods after ingestion of oocysts. A review of published and unpublished data indicated that the minimum prepatent period to shedding of oocysts after the ingestion of oocysts by cats is 18 days.

Dubey, J. P. (1996). "Pathogenicity and infectivity of Toxoplasma gondii oocysts for rats." J Parasitol 82(6): 951-6.
	Rats are considered to be 1 of the most resistant hosts for Toxoplasma gondii infection, but relative infectivity of T. gondii for rats is not known. Therefore, infectivity and pathogenicity of oocysts of the VEG strain of T. gondii were studied in Sprague Dawley weaned rats (approximately 130 g). Groups of 5 rats were each inoculated orally with 1 to 1 million infective oocysts. Three of the 5 rats fed 1 million oocysts died of acute toxoplasmosis between 6 and 9 days after ingesting oocysts; all other rats survived. Tissue cysts were found in brains of all rats fed > or = 10 oocysts and in 3 of 6 rats fed 1 oocyst. The average number of tissue cysts in brains of rats was 300, 180, 528, 600, 396, 1,200, and 2,650 in rats fed 1, 10, 100, 1,000, 10,000, 100,000 or 1 million oocysts, respectively. Microscopic lesions were seen in brains of all T. gondii-infected rats and the frequency of lesions was usually proportional to the dose. Antibodies (> or = 1:512) to T. gondii were detected in sera of all infected rats 29 days after ingestion of oocysts by the modified agglutination test, the commercially available latex agglutination test, and the indirect hemagglutination test.

Dubey, J. P. (1997). "Bradyzoite-induced murine toxoplasmosis: stage conversion, pathogenesis, and tissue cyst formation in mice fed bradyzoites of different strains of Toxoplasma gondii." J Eukaryot Microbiol 44(6): 592-602.
	The development of Toxoplasma gondii was studied in mice fed bradyzoites. At one hour after oral inoculation (HAI), bradyzoites were found in cells of the surface epithelium and the lamina propria of the small intestine, primarily the ileum. Division into two tachyzoites was first observed at 18 HAI in the intestine. At 24 HAI, organisms were also seen in mesenteric lymph nodes. Organisms were first detected in the brain at six days after oral inoculation with bradyzoites (DAI) but not consistently until 10 DAI. Immunohistochemical staining with bradyzoite specific (BAG-5 antigen) anti-serum showed that bradyzoites retained their BAG-5 reactivity even after the first division into two tachyzoites in the intestine at 18 HAI. BAG-5 positive organisms were not seen 2-5 DAI. BAG-5 antigens reappeared in T. gondii at 6 DAI. Whole mice and individual tissues of mice fed bradyzoites were bioassayed in cats and mice for the presence of bradyzoites. Feces of cats fed murine tissues were examined for oocyst shedding for short prepatent periods. Bradyzoites were present in the intestines of mice up to 12 HAI but not at 18 HAI, and tachyzoites and not bradyzoites disseminated to other tissues from the intestine. Bradyzoites were again detected 6 DAI. Using the mouse bioassay, T. gondii was first detected in peripheral blood at 24 HAI and more consistently at 48 HAI. Using a pepsin-digestion procedure and mouse bioassay, organisms were demonstrated in many tissues of mice 15 and 49 DAI.

Dubey, J. P. (1997). "Distribution of tissue cysts in organs of rats fed Toxoplasma gondii oocysts." J Parasitol 83(4): 755-7.
	Toxoplasma gondii-infected rats are considered important in the epidemiology of toxoplasmosis because they can serve as a source of infection for pigs and possibly for cats. To study the distribution of tissue cysts, 10 Sprague-Dawley female adult rats were fed 1 oocyst (3 rats, group A) or 10(5) oocysts (3 rats, group B) of the VEG strain or 10(4) oocysts of the GT-1 strain (4 rats, group C) of T. gondii. All rats in a group were killed at 1 time: 76 (group A), 240 (group B), and 443 (group C) days after oocyst inoculation (DAI). Tissue cysts were seen in the brains of all 10 rats by direct microscopic examination. Portions or whole organs from heart, lung, liver, spleen, small intestines, kidneys, skeletal muscle, eyes, mesenteric lymph nodes, stomach uterus, and tongue from all rats in a group were pooled by organ, digested in acid-pepsin solution for 60 min, washed in saline, and then bioassayed in mice. Based on bioassay in mice, tissue cysts were present in 3 extraneural tissues of rats from group A, 6 extraneural organs of group B, and in 10 extraneural organs of rats of group C. Tissue cysts were present in skeletal muscles and kidneys of all 3 groups. Thus, tissue cysts are formed both in neural and extraneural tissues of rats. Therefore portions of infected rats, excluding the head, can be a source of infection for pigs and cats.

Dubey, J. P. (1997). "Tissue cyst tropism in Toxoplasma gondii: a comparison of tissue cyst formation in organs of cats, and rodents fed oocysts." Parasitology 115 ( Pt 1): 15-20.
	The persistence of Toxoplasma gondii tissue cysts in organs of cats (definitive host) and rodents (intermediate hosts) was studied. Nine cats, 12 rats, and 12 mice were fed T. gondii oocysts and their organs were digested in pepsin and then bioassayed for bradyzoites in mice. Of 9 cats killed 37 or 51 days after feeding 10(2) (2 cats), 10(3) (3 cats) or 10(4) (4 cats) oocysts of the VEG strain, tissue cysts were found in each cat; in the tongue of 9, in the heart of 5, in the brain of 4, and in the eyes of 1 cat. The dose had no effect on the distribution of tissue cysts in cats. Twelve rats were each fed 10(5) oocysts of the VEG strain of T. gondii and killed 21, 29, 64 or 237 days later. At each time-period, 11 tissues of 3 rats were pooled and bioassayed in mice. Tissue cysts were found in the brain, skeletal muscle, heart and kidneys of rats at each killing time; in the lungs, intestines, and mesenteric lymph nodes in 3 of 4 instances; in the tongue, liver, and eyes in 2 instances and in the spleen in 1 instance. Also, using the same procedures and sampling the same 11 tissues as used for rats, tissue cysts were seen in all organs except in the tongue and liver of 3 mice killed on day 82 after feeding the VEG strain. In 9 mice (3 with each strain) fed oocysts of the ME-49, GT-1, or P89 T. gondii strain and killed 62-130 days later, tissue cysts were found consistently only in the brain. Thus, in rats and mice, most tissue cysts were found in the brain and rarely in the tongue. This was in marked contrast to the distribution of tissue cysts in cats.

Dubey, J. P., B. C. Barr, et al. (2002). "Redescription of Neospora caninum and its differentiation from related coccidia." Int J Parasitol 32(8): 929-46.
	Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Dubey, J. P., D. S. Lindsay, et al. (1998). "Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts." Clin Microbiol Rev 11(2): 267-99.
	Infections by the protozoan parasite Toxoplasma gondii are widely prevalent world-wide in animals and humans. This paper reviews the life cycle; the structure of tachyzoites, bradyzoites, oocysts, sporocysts, sporozoites and enteroepithelial stages of T. gondii; and the mode of penetration of T. gondii. The review provides a detailed account of the biology of tissue cysts and bradyzoites including in vivo and in vitro development, methods of separation from host tissue, tissue cyst rupture, and relapse. The mechanism of in vivo and in vitro stage conversion from sporozoites to tachyzoites to bradyzoites and from bradyzoites to tachyzoites to bradyzoites is also discussed.

Dubey, J. P., J. K. Lunney, et al. (1996). "Infectivity of low numbers of Toxoplasma gondii oocysts to pigs." J Parasitol 82(3): 438-43.
	To define the infectiousness of the VEG strain of Toxoplasma gondii, 42 pigs were fed doses estimated at 10, 1, or < 1 mouse infective oocysts. They were killed 38-99 days after inoculation and 50 g of tissues from their tongue, heart, and brain were individually homogenized in acidic pepsin solution and bioassayed in mice. Pools of brain, heart, tongue, and skeletal muscle (total 500 g) were bioassayed in cats. Toxoplasma gondii was isolated by bioassays in mice and in cats from 13 of 14 pigs fed 10 oocysts, 13 of 14 pigs fed 1 oocyst, and 4 of 14 pigs fed "less than" 1 oocyst, indicating high infectivity of VEG strain of T. gondii to pigs. All infected pigs developed modified agglutination test antibodies (> 1:50). Control pigs (n = 6) remained seronegative (< 1:20) and T. gondii was not isolated from their tissues. Toxoplasma gondii was isolated from tongues of 27 (93%), brains of 21 (72%), and hearts of 13 (45%) of 29 experimentally infected pigs by bioassay in mice. The number of T. gondii-positive mice after inoculation of tongue, brain, and heart from infected pigs was 240 (80%), 84 (28%), and 36 (12%) of 300 mice inoculated with each organ, respectively. Thus, the VEG strain of T. gondii was localized more often and in higher numbers in the tongue than in the brain and the heart of pigs. The apparent muscle localization after infection with the low dose of the VEG strain of T. gondii agrees with other studies in livestock that suggest T. gondii is more neurotropic in mice than in livestock.

Dubey, J. P., S. K. Shen, et al. (1997). "Toxoplasmosis in rats (Rattus norvegicus): congenital transmission to first and second generation offspring and isolation of Toxoplasma gondii from seronegative rats." Parasitology 115 ( Pt 1): 9-14.
	To study congenital transmission of Toxoplasma gondii during acute and chronic infections, 4 pregnant Sprague-Dawley rats were each fed 10,000 oocysts of the VEG strain. Toxoplasma gondii was recovered from 33, 55, 83 and 57% of rats (F1) when dams were inoculated at 6, 9, 12 or 15 days of gestation, respectively. Progeny of 15 congenitally infected female rats were examined for T. gondii. Toxoplasma gondii was recovered from tissues of 1 of 155 rats (F2) born to congenitally infected dams. A total of 4 (F2) females were mated; 0 of 40 (F3) rats born to them were infected. None of the acutely infected 4 dams that had given birth to congenitally infected litters produced congenitally infected offspring during the second pregnancy. Thus, unlike mice, evidence for repeated congenital transmission of T. gondii in the rat was found in < 1% of cases. Of the 16 congenitally T. gondii infected pups with demonstrable tissue cysts, 5 were seronegative (< 1:4) in the Sabin-Feldman dye test and 5 were seronegative (< 1:20) in the modified agglutination test by the use of whole formalinized tachyzoites and mercaptoethanol.

Dubey, J. P., D. W. Thayer, et al. (1998). "Effect of gamma irradiation on unsporulated and sporulated Toxoplasma gondii oocysts." Int J Parasitol 28(3): 369-75.
	The effect of 137Cs irradiation on unsporulated and sporulated Toxoplasma gondii oocysts was investigated as a model system for sterilisation of fruit contaminated with other coccidia such as Cyclospora or Cryptosporidium. Unsporulated oocysts irradiated at > or = 0.4 to 0.8 kGy sporulated but were not infective to mice. Sporulated oocysts irradiated at > or = 0.4 kGy were able to excyst, and sporozoites were infective but not capable of inducing a viable infection in mice. Toxoplasma gondii was detected in histologic sections of mice up to 5 days but not at 7 days after feeding oocysts irradiated at 0.5 kGy. Transmission electron microscopy revealed that sporozoites from irradiated oocysts penetrated enterocytes and all cells in the lamina propria except for red blood cells. Sporozoites appeared normal ultrastructurally and formed a typical parasitophorous vacuole containing a well-developed tubulovesicular membrane network. Raspberries inoculated with sporulated T. gondii oocysts were rendered innocuous after irradiation at 0.4 kGy. Results indicate that irradiation at 0.5 kGy is effective in "killing" coccidian oocysts on fruits and vegetables.

Dubey, J. P., P. Thulliez, et al. (1995). "Toxoplasma gondii in Iowa sows: comparison of antibody titers to isolation of T. gondii by bioassays in mice and cats." J Parasitol 81(1): 48-53.
	Hearts of 1,000 pigs killed at an abattoir in Iowa were bioassayed for the prevalence of tissue cysts of Toxoplasma gondii. One hundred grams of cardiac muscle from each pig was homogenized, digested in pepsin solution, and bioassayed in 10 mice. Five hundred grams of heart tissue from each of a subset of 183 pigs was also bioassayed in cats. Serum collected from the heart from each pig was assayed for anti-T. gondii antibodies in the modified agglutination test using formalin-fixed whole tachyzoites. Anti-T. gondii antibodies were found in 22.2% of pigs. Viable T. gondii was isolated from a total of 170 pigs; from 50 hearts by bioassay in mice, from 58 hearts by bioassay in both mice and cats, and from 62 pigs by bioassay in cats only. The success of isolation in cats (65.6%) was approximately twice that in mice (31.7%). Percentage of isolations of T. gondii with respect to reciprocal antibody titers (in parentheses) in pigs was: 3.7% (< 20), 37.1% (20), 38.1% (40), 60% (80), 75% (200), 77% (400), 83% (800), and 75.8% (> or = 2,000 to 16,000).

DuPont, H. L. (1985). "Cryptosporidiosis and the healthy host." N Engl J Med 312(20): 1319-20.
	
DuPont, H. L., C. L. Chappell, et al. (1995). "The infectivity of Cryptosporidium parvum in healthy volunteers." N Engl J Med 332(13): 855-9.
	BACKGROUND. Small numbers of Cryptosporidium parvum oocysts can contaminate even treated drinking water, and ingestion of oocysts can cause diarrheal disease in normal as well as immunocompromised hosts. Since the number of organisms necessary to cause infection in humans is unknown, we performed a study to determine the infective dose of the parasite in healthy adults. METHODS. After providing informed consent, 29 healthy volunteers without evidence of previous C. parvum infection, as determined by the absence of anti-cryptosporidium-specific antibodies, were given a single dose of 30 to 1 million C. parvum oocysts obtained from a calf. They were then monitored for oocyst excretion and clinical illness for eight weeks. Household contacts were monitored for secondary spread. RESULTS. Of the 16 subjects who received an intended dose of 300 or more oocysts, 14 (88 percent) became infected. After a dose of 30 oocysts, one of five subjects (20 percent) became infected, whereas at a dose of 1000 or more oocysts, seven of seven became infected. The median infective dose, calculated by linear regression, was 132 oocysts. Of the 18 subjects who excreted oocysts after the challenge dose, 11 had enteric symptoms and 7 (39 percent) had clinical cryptosporidiosis, consisting of diarrhea plus at least one other enteric symptom. All recovered, and there were no secondary cases of diarrhea among household contacts. CONCLUSIONS. In healthy adults with no serologic evidence of past infection with C. parvum, a low dose of C. parvum oocysts is sufficient to cause infection.

Eberhard, M. L., A. J. da Silva, et al. (1999). "Morphologic and molecular characterization of new Cyclospora species from Ethiopian monkeys: C. cercopitheci sp.n., C. colobi sp.n., and C. papionis sp.n." Emerg Infect Dis 5(5): 651-8.
	In recent years, human cyclosporiasis has emerged as an important infection, with large outbreaks in the United States and Canada. Understanding the biology and epidemiology of Cyclospora has been difficult and slow and has been complicated by not knowing the pathogen s origins, animal reservoirs (if any), and relationship to other coccidian parasites. This report provides morphologic and molecular characterization of three parasites isolated from primates and names each isolate: Cyclospora cercopitheci sp.n. for a species recovered from green monkeys, C. colobi sp.n. for a parasite from colobus monkeys, and C. papionis sp.n. for a species infecting baboons. These species, plus C. cayetanensis, which infects humans, increase to four the recognized species of Cyclospora infecting primates. These four species group homogeneously as a single branch intermediate between avian and mammalian Eimeria. Results of our analysis contribute toward clarification of the taxonomic position of Cyclospora and its relationship to other coccidian parasites.

Eberhard, M. L., Y. R. Ortega, et al. (2000). "Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals." J Parasitol 86(3): 577-82.
	Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

Elliot, A., U. M. Morgan, et al. (1999). "Improved staining method for detecting Cryptosporidium oocysts in stools using malachite green." J Gen Appl Microbiol 45(3): 139-142.
	
Elsasser, T. H., J. L. Sartin, et al. (1998). "Changes in somatotropic axis response and body composition during growth hormone administration in progressive cachectic parasitism." Domest Anim Endocrinol 15(4): 239-55.
	A multistage protozoan parasitic disease was used as a cachexia model to study the effects of daily administration of bovine growth hormone (GH) on endocrine and body composition changes of young calves from the onset of the acute phase response (APR). Male calves averaging 127.5 +/- 2.0 kg body weight were assigned to control, ad libitum fed, noninfected (C); ad libitum fed, infected (250,000 oocysts Sarcocystis cruzi, per os, I); noninfected, pair-fed (PF) to matched I-treatment calves and these respective same treatments in calves injected daily with GH (USDA-bGH-B1), 12.5 mg/calf/day, im) designated as CGH, IGH and PFGH. GH injections were initiated on Day 20 postinfection (PI), 3 to 4 d before the onset of clinical signs of APR, and continued to Day 56 PI, at which time animals were euthanized for tissue collections. Abrupt increases in rectal temperature commensurate with up to 70% reduction in voluntary feed intake were observed in I and IGH beginning 23-25 d PI. For the trial period between Days 20 and 56 PI, average daily carcass protein gains were 123, 52, 109, 124, 48, and 67 g/d and average daily carcass fat gains were 85, 11, 43, 71, -23, and 29 g/d for C, I, PF, CGH, IGH, and PFGH, respectively. Effects of GH were significant for fat accretion and plasma urea depression. Rectus femoris was highly refractory to catabolic effects of infection while psoas major was significantly catabolized during infection. Plasma concentrations of insulin-like growth factor-I (IGF-I) increased significantly in all GH-treated calves between Day 20 and 23 PI. Plasma IGF-I declined well below Day 20 values in all infected calves from the onset of the APR through the end of the study. The decrease in plasma IGF-I concentrations in I and IG was highly correlated with the magnitude of the fever response. Hepatic mRNA for GH receptor and IGF-I was decreased in infected calves. Hepatic microsomal membrane binding of 125I-GH did not differ between groups. The data suggest that effects of GH and parasitism on tissue metabolism during disease may vary among different specific tissue pools. The data demonstrate that daily GH administration in young calves does not prevent lean tissue losses and may accelerate fat depletion associated with cachectic parasitism. Furthermore, the onset of APR overrode the capacity for GH to maintain elevated plasma concentrations of IGF-I, an effect not readily explained through changes of GH-receptor binding.

Enemark, H. L., P. Ahrens, et al. (2003). "Cryptosporidium parvum: infectivity and pathogenicity of the 'porcine' genotype." Parasitology 126(Pt 5): 407-16.
	Genetic studies have demonstrated profound differences between the 'porcine' genotype of Cryptosporidium parvum, versus 'human' and 'bovine' genotypes. The study analysed infectivity and pathogenicity of the 'porcine' genotype (CPP-13 isolate) of C. parvum, and compared the results with published data on the 'bovine' genotype (CPB-0 isolate). This was investigated in calves and piglets from commercial herds. Piglets were mildly affected by the CPP-13 isolate, contrary to piglets infected with the CPB-0 isolate, which caused diarrhoea of a mean duration of 3.5 days. CPP-13 produced no or very mild clinical signs in piglets despite the excretion of high numbers of oocysts. Concomitant infection with rotavirus, however, caused a dramatic aggravation of the clinical signs, and 5 of 6 experimentally infected piglets died. CPP-13 appeared to be adapted to porcine hosts as illustrated by the lack of infectivity to 1 experimentally inoculated calf, and the absence of clinical signs, the long pre-patent period (15 days), and the excretion of very low numbers of oocysts following experimental infection of another calf. Thus, in accordance with other molecular studies, our results support the genetic evidence for the existence of a new species of Cryptosporidium adapted to pigs.

Enemark, H. L., P. Ahrens, et al. (2002). "Molecular characterization of Danish Cryptosporidium parvum isolates." Parasitology 125(Pt 4): 331-41.
	The genetic polymorphism among 271 Danish Cryptosporidium isolates of human and animal origin was studied by partial amplification and sequencing of the Cryptosporidium oocyst wall protein (COWP) gene, the 1 8S rDNA, and a microsatellite locus. Furthermore, the microsatellite locus was studied directly using fragment analysis. A comparative analysis of DNA sequences showed the presence of 3 different subgenotypes (Cl, C2 and C3) in C. parvum isolates from Danish cattle, with prevalences of 16.7, 17.2 and 73.1% including 13 (7.0%) mixed infections. Subgenotype Cl was significantly more prevalent (P < 0.001) in the southern part of Denmark. In Cryptosporidium isolates of human origin the anthroponotic subgenotype H1 was identified, in addition to the zoonotic subgenotypes C1, C2, and C3. Of 44 human samples, 56.8% were anthroponotic, whereas 40.9% were zoonotic genotypes. One human isolate was characterized as C. meleagridis. The porcine Cryptosporidium isolates (N = 4) revealed a pattern which was genetically distinct from human and bovine isolates. Cryptosporidium in a hedgehog (Erinaceus europaeus L.) was identified for the first time. By microsatellite sequencing the hedgehog isolate showed a subgenotype distinct from the previously detected types. The assignment to subgenotype by microsatellite sequencing and fragment typing was 100% identical in samples where results were achieved by both methods. In addition, the fragment analysis proved more sensitive, easier, faster, and less expensive compared to sequencing.

Enemark, H. L., V. Bille-Hansen, et al. (2003). "Pathogenicity of Cryptosporidium parvum--evaluation of an animal infection model." Vet Parasitol 113(1): 35-57.
	With the intention of developing a standardised method for assessment of pathogenicity of Cryptosporidium parvum, the CPB-0 isolate was studied by propagation in 1-day-old calves followed by inoculation into specific pathogen free (SPF) piglets. The experiment was repeated. Diarrhoea and shedding of oocysts were seen in all animals infected with the CPB-0 isolate. Clinical signs included depression, inappetence, vomiting (exclusively in the piglets), and death. Histological examination at 17 and 19 days post-infection revealed parasitic stages and microscopic changes primarily restricted to colon and rectum.The unintended presence of rotavirus in some of the experimental animals revealed an additive or synergistic effect between rotavirus and C. parvum as indicated by prolonged diarrhoea, increased oocyst shedding, decreased weight gain and elevated levels of serum haptoglobin and serum amyloid A (SAA) in piglets infected simultaneously with both pathogens. The difference in daily weight gain between infected and control animals was significant only for piglets co-infected with rotavirus. The acute phase response of haptoglobin and SAA was characterised by a large individual variation. In piglets, co-infected with rotavirus, the levels of serum haptoglobin were 3.5 and 4.6 times higher in the infected versus the controls 6 and 9dpi, respectively (mean values: 2411microg/ml+/-S.D. 2023 and 1840 microg/ml+/-S.D. 1697). In the controls infected with rotavirus, peak haptoglobin concentration was seen 3dpi (mean: 1022 microg/ml+/-S.D. 425). Elevated levels of SAA were seen in 1 of 6 piglets infected with C. parvum, and in 5 of 6 piglets co-infected with rotavirus. Tumour necrosis factor alpha (TNFalpha) was undetectable in all serum samples from piglets.The obvious advantages of the SPF pig model are the naturally acquired intestinal microflora, the development of distinct clinical signs similar to cryptosporidiosis in humans and calves, the size of the animals, and the accessibility of individuals born within a short time span. This makes the model ideal for dose-response studies, evaluation of therapeutic agents as well as for assessment of differences in the clinical response to isolates of diverse genetic background. In conclusion, it was shown that the CPB-0 isolate was pathogenic to calves and piglets at a dose of 2.5 x 10(5) oocysts, and that the clinical signs could be replicated during separate experiments. Moreover, diarrhoea, oocyst shedding, body weight changes, histological alterations, and the acute phase response of haptoglobin and SAA were identified as useful parameters for discrimination of isolate-specific differences of pathogenicity.

Entrala, E. and C. Mascaro (1997). "Glycolytic enzyme activities in Cryptosporidium parvum oocysts." FEMS Microbiol Lett 151(1): 51-7.
	Oocysts of Cryptosporidium parvum were obtained from an experimentally infected newborn goat. After purification, the oocysts were homogenised and the activities of the glycolytic enzymes measured in the different subcellular fractions. All of the activities of the Embden-Meyerhoff pathway were located in the non-sedimentable, cytoplasmic fraction. Under the conditions used, hexokinase activity was below the limits of detection. The pathway is also characterised by the presence of a pyrophosphate-dependent phosphofructokinase and a carbon dioxide-fixing cycle comprising phosphoenolpyruvate carboxylase, malate dehydrogenase and malate dehydrogenase (decarboxylating) activities. The data presented in this paper suggest that the infective stage of this parasite probably relies on substrate-level phosphorylation for energy generation.

Entrala, E., C. Mascaro, et al. (1997). "Anti-oxidant enzymes in Cryptosporidium parvum oocysts." Parasitology 114 ( Pt 1): 13-7.
	Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4.8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H2O2 scavenging system was detected in the insoluble fraction.

Erickson, M. C. and Y. R. Ortega (2006). "Inactivation of protozoan parasites in food, water, and environmental systems." J Food Prot 69(11): 2786-808.
	Protozoan parasites can survive under ambient and refrigerated storage conditions when associated with a range of substrates. Consequently, various treatments have been used to inactivate protozoan parasites (Giardia, Cryptosporidium, and Cyclospora) in food, water, and environmental systems. Physical treatments that affect survival or removal of protozoan parasites include freezing, heating, filtration, sedimentation, UV light, irradiation, high pressure, and ultrasound. Ozone is a more effective chemical disinfectant than chlorine or chlorine dioxide for inactivation of protozoan parasites in water systems. However, sequential inactivation treatments can optimize existing treatments through synergistic effects. Careful selection of methods to evaluate inactivation treatments is needed because many studies that have employed vital dye stains and in vitro excystation have produced underestimations of the effectiveness of these treatments.

Evengard, B., K. Petersson, et al. (2001). "Low incidence of toxoplasma infection during pregnancy and in newborns in Sweden." Epidemiol Infect 127(1): 121-7.
	To estimate the burden of disease due to congenital toxoplasmosis in Sweden the incidence of primary infections during pregnancy and birth prevalence of congenital toxoplasmosis in 40,978 children born in two regions in Sweden was determined. Women possibly infected during pregnancy were identified based on: 1, detection of specific IgG based on neonatal screening of the phenylketonuria (PKU) card blood spot followed by retrospective testing of stored prenatal samples to detect women who acquired infection during pregnancy and follow up of their children to 12 months: 2, detection of specific IgM on the PKU blood spot. The birth prevalence of congenital toxoplasmosis was 0.73/10,000 (95 % CI 0.15-2.14) (3/40,978). The incidence of primary infection during pregnancy was 5.1/10,000 (95% CI 2.6-8.9) susceptible pregnant women. The seroprevalence in the southern part was 25.7% and in the Stockholm area 14.0%. The incidence of infection during pregnancy was low, as the birth prevalence of congenital toxoplasmosis. Neonatal screening warrants consideration in view of the low cost and feasibility.

Ey, P. L., T. Bruderer, et al. (1996). "Comparison of genetic groups determined by molecular and immunological analyses of Giardia isolated from animals and humans in Switzerland and Australia." Parasitol Res 82(1): 52-60.
	Nine axenic isolates of Giardia originating from four different host species in Switzerland were subjected to genetic analysis using the polymerase chain reaction (PCR) to amplify segments of genes encoding different trophozoite variant surface proteins (VSPs). Three genotypes were identified on the basis of product yield, size, and restriction-fragment-length polymorphisms (RFLPs). Five G. duodenalis isolates (O1, B1, B2, B3-1A1 and C1--from a sheep, three calves and a dog, respectively) were classified as belonging to genetic group I of Andrews et al. (1989). DNA amplified from the VSP genes tsp11, tsa417 and vsp1267 of these isolates was indistinguishable in size and restriction characteristics from that amplified from group-I Giardia isolated from humans in Australia. One human-derived Swiss isolate (H2-17A1), typed as belonging to genetic group II, yielded a vsp1267-specific PCR product that was indistinguishable by size or restriction sites from the equivalent 1.6-kb product amplified from human-derived Australian group-II organisms. This isolate also yielded 1.8-kb tsp11 and 0.52-kb tsa417/tsp11-like PCR products possessing RFLPs typical of group-II organisms. Three isolates (O2-4A1, O3 and H3-15K2--originating from two sheep and a human, respectively) represent a novel genotype that is closely related to genetic groups I and II. These three isolates exhibited identical RFLPs in their tsp11 PCR products and failed to yield a vsp1267 PCR product. An antiserum specific for the 90-kDa VSP of the sheep-derived clone O2-4A1 reacted strongly by immunofluorescence and on Western blots with surface proteins from the O2, O3 and H3 isolates only--consistent with the genetic classification determined above. The data provide no evidence for the occurrence of host-specific genotypes.

Ey, P. L., M. Mansouri, et al. (1997). "Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes." J Eukaryot Microbiol 44(6): 626-35.
	Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture or growth in suckling mice, were compared genetically by analysis of DNA amplified from loci encoding variant surface proteins or the enzyme glutamate dehydrogenase and by allozyme analysis. The isolates were heterogeneous, but all showed affinity with genetic Assemblage A--one of two major assemblages defined previously by analysis of Giardia from humans. Three distinct genotypes were evident. Ten isolates (eight axenic and two established in suckling mice) from an alpaca, pig, horse, cattle and sheep were indistinguishable from human-derived G. intestinalis belonging to a previously designated genetic group (Group I). This genotype seems to have broad host specificity, including a zoonotic potential for humans. Five isolates (two axenic and three established in suckling mice) from an alpaca, a horse and sheep had close affinity with human-derived Group I and Group II G. intestinalis genotypes. The other 16 isolates (comprising both axenic and suckling mouse-propagated cultures derived from cattle, sheep, alpaca, a goat and pigs in Australia and Europe) differed from all other Giardia with "duodenalis" morphology that have been examined by these methods and they segregated as a highly distinct sublineage (referred to herein as 'Novel livestock') within genetic Assemblage A. The predominance of 'Novel livestock' genotypes in the test panel and their apparent exclusive association with artiodactyl hosts indicates that they may be confined to this group of mammals. Assemblage B genotypes, which are prevalent in humans and some other animal species, were not detected.

Fayer, R. (1992). "Activity of sulfadimethoxine against cryptosporidiosis in dairy calves." J Parasitol 78(3): 534-7.
	Of 13 neonatal calves inoculated orally with 1.5 x 10(6) oocysts of Cryptosporidium parvum, 7 in group A were fed 5-g boluses of sulfadimethoxine for 21 consecutive days beginning 1 day before infection, and 6 calves in group B were untreated controls. Calves in group A had diarrhea for 6-18 days (mean = 11 days); those in group B had diarrhea for 4-14 days (mean = 8.7 days). The severity of diarrhea, based on a daily numerical scoring system, was similar for both groups. Calves in group A shed an average of 18 x 10(6) oocysts/ml of feces for 3.9 days; those in group B shed an average of 2.4 x 10(6) oocysts/ml of feces for 5.3 days. By 28 days of age, calves in group A vs. group B gained an average of 8.9 kg vs. 15.7 kg. These findings indicate that sulfadimethoxine did not significantly reduce the number of days or severity of diarrhea, or the number of oocysts or patent period, nor did it improve weight gains.

Fayer, R. (1994). "Development of a precocious strain of Cryptosporidium parvum in neonatal calves." J Eukaryot Microbiol 41(5): 40S.
	
Fayer, R. (1994). "Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water." Appl Environ Microbiol 60(8): 2732-5.
	Cryptosporidium parvum oocysts suspended in 0.5 ml of distilled water were pipetted into plastic vials which were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 60 to 100 degrees C. At each temperature setting four vials containing C. parvum oocysts were placed into wells and held for 15 s before time was recorded as zero, and then pairs of vials were removed 1 and 5 min later. Upon removal, all vials were immediately cooled on crushed ice. Also, at each temperature interval one vial containing 0.5 ml of distilled water was placed in a well and a digital thermometer was used to record the actual water temperature at 30-s intervals. Heated oocyst suspensions as well as unheated control suspensions were orally inoculated by gavage into 7- to 10-day-old BALB/c mouse pups to test for infectivity. At 96 h after inoculation the ileum, cecum, and colon from each mouse were removed and prepared for histology. Tissue sections were examined microscopically. Developmental-stage C. parvum was found in all three gut segments from all mice that received oocysts in unheated water and in water that reached temperatures of 54.4, 59.9, and 67.5 degrees C at 1 min when vials were removed from the heat source. C. parvum was also found in the ileum of one of six mice that received oocysts in water that reached a temperature of 59.7 degrees C at 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Fayer, R. (1995). "Effect of sodium hypochlorite exposure on infectivity of Cryptosporidium parvum oocysts for neonatal BALB/c mice." Appl Environ Microbiol 61(2): 844-6.
	Oocysts of Cryptosporidium parvum suspended in 5.25, 2.63, or 1.31% aqueous sodium hypochlorite (Clorox laundry bleach) for 10, 30, 60, or 120 min at 21 degrees C were administered by gastric intubation to neonatal BALB/c mice. Microscopic examination of intestinal tissue sections revealed developmental stages of C. parvum in all of the mice.

Fayer, R. (2004). "Cryptosporidium: a water-borne zoonotic parasite." Vet Parasitol 126(1-2): 37-56.
	Of 155 species of mammals reported to be infected with Cryptosporidium parvum or C. parvum-like organisms most animals are found in the Orders Artiodactyla, Primates, and Rodentia. Because Cryptosporidium from most of these animals have been identified by oocyst morphology alone with little or no host specificity and/or molecular data to support identification it is not known how many of the reported isolates are actually C. parvum or other species. Cryptosporidiosis is a cause of morbidity and mortality in animals and humans, resulting primarily in diarrhea, and resulting in the most severe infections in immune-compromised individuals. Of 15 named species of Cryptosporidium infectious for nonhuman vertebrate hosts C. baileyi, C. canis, C. felis, C. hominis, C. meleagridis, C. muris, and C. parvum have been reported to also infect humans. Humans are the primary hosts for C. hominis, and except for C. parvum, which is widespread amongst nonhuman hosts and is the most frequently reported zoonotic species, the remaining species have been reported primarily in immunocompromised humans. The oocyst stage can remain infective under cool, moist conditions for many months, especially where water temperatures in rivers, lakes, and ponds remain low but above freezing. Surveys of surface water, groundwater, estuaries, and seawater have dispelled the assumption that Cryptosporidium oocysts are present infrequently and in geographically isolated locations. Numerous reports of outbreaks of cryptosporidiosis related to drinking water in North America, the UK, and Japan, where detection methods are in place, indicate that water is a major vehicle for transmission of cryptosporidiosis.

Fayer, R. (2004). "Infectivity of microsporidia spores stored in seawater at environmental temperatures." J Parasitol 90(3): 654-7.
	To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.

Fayer, R. (2004). "Sarcocystis spp. in human infections." Clin Microbiol Rev 17(4): 894-902, table of contents.
	Sarcocystis species are intracellular protozoan parasites with an intermediate-definitive host life cycle based on a prey-predator relationship. Asexual stages develop in intermediate hosts after they ingest the oocyst stage from definitive-host feces and terminate with the formation of intramuscular cysts (sarcocysts). Sarcocysts in meat eaten by a definitive host initiate sexual stages in the intestine that terminate in oocysts excreted in the feces. Most Sarcocystis species infect specific hosts or closely related host species. For example, humans and some primates are definitive hosts for Sarcocystis hominis and S. suihominis after eating raw meat from cattle and pigs, respectively. The prevalence of intestinal sarcocystosis in humans is low and is only rarely associated with illness, except in volunteers who ingest large numbers of sarcocysts. Cases of infection of humans as intermediate hosts, with intramuscular cysts, number less than 100 and are of unknown origin. The asexual stages, including sarcocysts, can stimulate a strong inflammatory response. Livestock have suffered acute debilitating infections, resulting in abortion and death or chronic infections with failure to grow or thrive. This review provides a summary of Sarcocystis biology, including its morphology, life cycle, host specificity, prevalence, diagnosis, treatment, and prevention strategies, for human and food animal infections.

Fayer, R., J. R. Barta, et al. (1991). "Immunogold labeling of stages of Cryptosporidium parvum recognized by immunoglobulins in hyperimmune bovine colostrum." J Parasitol 77(3): 487-90.
	Ultrathin sections of mouse ileum infected with Cryptosporidium parvum were stained by immunogold techniques. Sections first were stained with polyvalent antibodies in whey from hyperimmune bovine colostrum (HBC), then stained by secondary antibodies in rabbit antibovine IgA, IgM, IgG1, and IgG2, and lastly labeled by goat anti-rabbit gold conjugate. Examination of the immunostained specimens by electron microscopy revealed that each bovine immunoglobulin isotype in the whey recognized antigens in meronts, merozoites, microgametocytes, microgametes, and macrogamonts. Based on these findings it is hypothesized that antigens in all stages of C. parvum provide targets of opportunity for the antiparasitic activity of HBC whey antibodies thereby accounting for its efficacy as an immunotherapeutic agent.

Fayer, R. and J. P. Dubey (1987). "Comparative epidemiology of coccidia: clues to the etiology of equine protozoal myeloencephalitis." Int J Parasitol 17(2): 615-20.
	
Fayer, R., J. P. Dubey, et al. (2004). "Zoonotic protozoa: from land to sea." Trends Parasitol 20(11): 531-6.
	Attention to worldwide pollution of the coastal marine environment has focused primarily on toxic algal blooms and pathogenic bacteria that multiply in nutrient-rich waters. However, massive but unseen amounts of feces from humans, their pets, and their domesticated animals are discharged, dumped, or carried in runoff, bringing encysted zoonotic protozoan parasites to estuaries and coastal waters. Here, they contaminate bathing beaches, are filtered and concentrated by shellfish eaten by humans and marine mammals, and infect a wide range of marine animal hosts, resulting in morbidity and mortality to some populations. This review addresses the extent of contamination and the animals affected by three genera of important zoonotic protozoa: Giardia, Cryptosporidium and Toxoplasma.

Fayer, R. and W. Ellis (1993). "Glycoside antibiotics alone and combined with tetracyclines for prophylaxis of experimental cryptosporidiosis in neonatal BALB/c mice." J Parasitol 79(4): 553-8.
	Glycoside antibiotics including the macrolide antibiotics azithromycin, clarithromycin, and erythromycin and the aminoglycoside paromomycin were administered alone or combined with doxycycline, minocycline, or tetracycline to neonatal BALB/c mice experimentally infected with Cryptosporidium parvum. Glycosides at 100 or 200 mg/kg of body weight and tetracyclines at 50 mg/kg of body weight were dissolved in dimethylsulfoxide (DMSO), which was then diluted with phosphate-buffered saline (PBS) and given orally by gavage. Drugs were administered at 0, 24, 48, and 72 hr postinfection (PI) for prophylaxis. Histologic sections of ileum, cecum, and colon from tissues fixed at 96 hr PI were examined microscopically to determine the number of developing parasites and assign a quantitative score based on infectivity. All groups that received glycosides had significantly (P < 0.01) lower scores than controls that received only DMSO/PBS. A range in efficacy was apparent. None or extremely few parasites were found in paromomycin- and azithromycin-treated groups, whereas few to moderate numbers of parasites were found in erythromycin- and clarithromycin-treated groups. The addition of tetracyclines did not consistently result in significantly lower scores.

Fayer, R. and W. Ellis (1993). "Paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves." J Parasitol 79(5): 771-4.
	Of 16 experimentally infected neonatal dairy calves, 12 were fed paromomycin twice daily in their milk for 11 consecutive days beginning 1 day before oral inoculation with 1.5-2.0 x 10(6) oocysts of Cryptosporidium parvum. Four calves each in groups A, B, C, and D received total daily doses of 100, 50, 25, and 0 mg of paromomycin per kilogram of body weight, respectively. From birth until 28 days of age feces from each calf were examined for diarrhea, and oocysts were enumerated, rectal temperature was recorded, and weight gain was determined. Total days of diarrhea, severity of diarrhea, the total number of days oocysts were shed, and the number of oocysts shed were significantly less in group A than in the unmedicated group D. The severity of diarrhea was also significantly less in groups B and C than in group D. Oocysts were not detected in feces from calves in group A. Except for 1 calf, oocysts were not detected from calves in groups B and C during the first week the drug was administered and those calves that shed oocysts began shedding at or near the end of paromomycin administration or more than 1 wk after treatment ended. Frequency of fever and weight gains did not vary significantly between the unmedicated and medicated groups except for group C, calves of which gained significantly less weight than those in all other groups.

Fayer, R. and W. Ellis (1994). "Qinghaosu (artemisinin) and derivatives fail to protect neonatal BALB/c mice against Cryptosporidium parvum (Cp) infection." J Eukaryot Microbiol 41(5): 41S.
	
Fayer, R. and T. H. Elsasser (1991). "Bovine sarcocystosis: How parasites negatively affect growth." Parasitol Today 7(9): 250-5.
	Growth, though genetically encoded, is markedly influenced in healthy animals by the interaction of hormonal and nutritional factors. The uptake and use of nutrients by specific tissues is regulated by a priority system that modulates physiological processes. Nutritional, hormonal and immunological consequences of parasitism often lead to partitioning of nutrients away from growth. In this article, Ron Foyer and Ted Elsosser use a bovine sarcocystosis model to show that changes in plasma concentrations of insulin-like growth factor-I (IGF-I), growth hormone (GH) and somotostotin (SSN), as well as the host's immunological response to the parasite via cytokine interactions with the endocrine system, are modulators of perturbed growth.

Fayer, R. and R. Fetterer (1995). "Activity of benzimidazoles against cryptosporidiosis in neonatal BALB/c mice." J Parasitol 81(5): 794-5.
	The need for an effective compound for the prevention and treatment of cryptosporidiosis in humans and animals has led to the testing of benzimidazoles based on reports that albendazole was clinically effective against related protozoan parasites causing microsporidiosis in humans. Albendazole and other benzimidazole derivatives were tested for prophylactic efficacy against cryptosporidiosis at dosage levels 1-3x the levels found effective for treatment of cattle or sheep for helminth infections. Daily dosage levels of thiabendazole, parbendazole, oxibendazole, mebendazole, and albendazole, as high as 200, 30, 10, 15, and 15 mg/kg of body weight, respectively, were not efficacious in neonatal mice. Although the number of parasites in histologic sections of intestine from mice mediated with 15 mg albendazole/kg of body weight was significantly lower than in unmedicated control mice, suggesting activity against the parasite, a high percentage of epithelial cells in the medicated mice were infected.

Fayer, R., J. R. Fischer, et al. (1996). "Spontaneous cryptosporidiosis in captive white-tailed deer (Odocoileus virginianus)." J Wildl Dis 32(4): 619-22.
	In August 1994, cryptosporidiosis was diagnosed in a diarrheic fawn from a captive white-tailed deer (Odocoileus virginianus) herd maintained for research purposes at The University of Georgia's Warnell School of Forest Resources in Athens, Georgia (USA). From June through August 1995, 11 captive female white-tailed deer were housed in individual barn stalls where they gave birth to 18 fawns. Feces collected at 2 or 3 day intervals from the 18 neonatal fawns for at least 21 days and from 11 adult females once from 1 to 30 days before fawns were born and on three to 12 occasions after their birth were examined for oocysts of Cryptosporidium spp. Feces from all animals appeared normal throughout the period of examination. Oocysts morphologically indistinguishable from those of Cryptosporidium parvum were detected intermittently in the feces of one adult female from 1 to 25 days after parturition and in the feces of her fawn from 11 to 22 days of age. Oocysts also were detected intermittently in feces from twin fawns from 9 to 20 days of age, but not from their mother. Oocysts from deer were infectious for neonatal mice as determined histologically, and for calves as determined by clinical signs and excretion of oocysts.

Fayer, R., L. Gasbarre, et al. (1998). "Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns." Int J Parasitol 28(1): 49-56.
	Twenty-six experimentally infected calves were monitored daily for oocyst excretion. All began excreting oocysts 3-6 days p.i. Most calves (n = 23) excreted oocysts for 6-9 days, with a daily range from 4 x 10(2) to 4.15 x 10(7) oocysts g(-1) of faeces. Over half the calves excreted peak numbers of oocysts 6-8 days p.i. Diarrhoea, observed intermittently beginning as early as day 3 p.i., lasted 4-16 days and varied greatly in severity from calf to calf. In a second study, nine of 18 calves were orally inoculated with 5 x 10(6) oocysts between birth and 2 days of age and nine remained uninfected. Monoclonal antibodies for cell surface markers indicated substantial increases in CD4+ and CD8+ T cells in the intraepithelial lymphocyte population of the ilea of infected calves at 7-9 days of age. RT-PCR demonstrated increases in mRNA for interleukin-12 and interferon-gamma that correlated with increases in both CD4+ and CD8 + intraepithelial lymphocyte cells. Increased mRNA for interleukin-12 and interferon-gamma from lamina propria lymphocytes correlated with increased numbers of CD8+ cells. No changes were found in interleukin-2, interleukin-4 or interleukin-10 mRNA levels. However, interleukin-15 mRNA, possibly from epithelial cells contaminating intraepithelial lymphocytes, was decreased in infected calves and had a negative correlation with increases in CD4+ and CD8+ cells. No differences were detected in mRNA levels for cytokines from lymph node lymphocytes.

Fayer, R., T. K. Graczyk, et al. (1995). "Multiple heterogenous isolates of Cryptosporidium serpentis from captive snakes are not transmissible to neonatal BALB/c mice (Mus musculus)." J Parasitol 81(3): 482-4.
	Oral inoculations of 9 litter-groups of 3 5-day-old suckling BALB/c mouse pups (Mus musculus) with 6.7 x 10(3) to 1.2 x 10(5) per pup of viable, Cryptosporidium serpentis oocysts from snakes resulted in no transmission. Mice showed normal development; the litter-group weight gain was not altered significantly (P > 0.05) relative to the total number of C. serpentis oocysts inoculated or to the initial group weight (P > 0.05). Histological sections of stomach, duodenum, jejunum, ileum, cecum, and colon 4 days postinoculation did not contain life-cycle stages of Cryptosporidium in any inoculated mice. Because these neonatal, C. parvum-susceptible BALB/c mice were resistant to infection it is unlikely that C. serpentis transmission to the snakes "via infected prey" results when captive snakes are maintained on a diet of BALB/c mice.

Fayer, R., T. K. Graczyk, et al. (1996). "Gaseous disinfection of Cryptosporidium parvum oocysts." Appl Environ Microbiol 62(10): 3908-9.
	Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.

Fayer, R., T. K. Graczyk, et al. (1998). "Survival of infectious Cryptosporidium parvum oocysts in seawater and eastern oysters (Crassostrea virginica) in the Chesapeake Bay." Appl Environ Microbiol 64(3): 1070-4.
	Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10 degrees C and at 10 ppt at 20 degrees C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20 degrees C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.

Fayer, R., A. Guidry, et al. (1990). "Immunotherapeutic efficacy of bovine colostral immunoglobulins from a hyperimmunized cow against cryptosporidiosis in neonatal mice." Infect Immun 58(9): 2962-5.
	Infection with Cryptosporidium parvum, a ubiquitous protozoan parasite of virtually all mammals, can cause mild to severe diarrhea in immunocompetent hosts and life-threatening diarrhea in immunocompromised hosts. Passive immunotherapy of experimentally infected animals and naturally infected humans with hyperimmune bovine colostrum has been reported to be efficacious, whereas chemotherapy has not. In this study, the efficacy of specific immunoglobulin isotypes purified from bovine colostrum from a cow hyperimmunized with Cryptosporidium parvum was assessed in neonatal BALB/c mice. Mice were orally infected with oocysts and treated with whole whey immunoglobulin G1 (IgG1), IgG2, IgA, or IgM at six intervals from 22 to 66 h postinfection. In histologic sections of intestine examined at 72 h postinfection, the reduction in number of intestinal stages in treated mice versus untreated controls was very highly significant (P less than 0.0001). The greatest reduction in parasite number was found in mice treated with IgG1, IgA, or whey.

Fayer, R. and M. C. Jenkins (1992). "Colostrum from cows immunized with Eimeria acervulina antigens reduces parasite development in vivo and in vitro." Poult Sci 71(10): 1637-45.
	Experiments were undertaken to determine whether passive immunization utilizing hyperimmune bovine colostrum (HBC) specific for Eimeria acervulina (EA) antigens conferred protection against coccidiosis in chickens. The HBC was produced by immunizing three pregnant, nonmilking Jersey cows with EA antigens administered via one intramuscular injection followed by three intramammary infusions at approximately 10, 8, 6, and 4 wk before parturition. One cow was immunized with sporozoites (SZ), the second with merozoites (MZ), and the third with recombinant merozoite antigen (rMZ). A fourth cow, unimmunized, provided normal colostrum (NC) for control purposes. Colostral whey from each cow was tested by ELISA for antibody against SZ, MZ, and rMZ antigens. In all immunized cows, antiparasite titers were elevated above those of the control. Antibodies from MZ- and rMZ-immunized cows recognized both MZ and rMZ antigen. Separate groups of 2-wk-old chickens received two oral doses of anti-SZ, -MZ, or -rMZ HBC or NC or PBS daily from 1 day before through 6 days after oral inoculation (DAI) with EA oocysts. Feces from each group were examined for oocysts. Intestines were examined for lesions 6 DAI. Histologic sections of duodenum were examined for asexual stages and gametocytes utilizing monoclonal antibody and fluorescence microscopy. In Experiments 1 and 2, oocyst production was reduced in all HBC-treated groups, except one treated with rMZ HBC, compared with PBS- or NC-treated groups. In Experiment 2, the severity of lesions was significantly reduced in all HBC-treated groups compared with those that received NC or PBS. Significantly fewer developmental stages were found in histological sections from all chickens treated with anti-SZ and anti-rMZ HBC than from controls. Anti-SZ HBC significantly reduced the number of intracellular SZ found 24 h after their inoculation into cultures of primary chicken kidney cells. These results suggest that HBC specific for certain EA antigens can inhibit parasite development and reduce severity of parasite-related gut lesions.

Fayer, R., E. J. Lewis, et al. (1999). "Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay." Emerg Infect Dis 5(5): 706-10.
	Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption.

Fayer, R., I. G. Mayhew, et al. (1990). "Epidemiology of equine protozoal myeloencephalitis in North America based on histologically confirmed cases. A report." J Vet Intern Med 4(2): 54-7.
	Following a workshop on equine protozoal myeloencephalitis (EPM) convened at the Veterinary Medical Forum of the American College of Veterinary Internal Medicine in 1988, this survey of EPM in North America was developed. It is based upon 364 histologically confirmed case records from California, Florida, Illinois, Kentucky, New York, Ohio, Oklahoma, Ontario, Pennsylvania, and Texas up to 1988. The highest rate of infection was found in young Thoroughbred, Standardbred, and quarter horses. Differences in geographic location, sex, and month (season) of infection were not discernible. This report, the first comprehensive survey of EPM in North America, is intended to serve as a basis for evaluating future changes in prevalence and spread of EPM.

Fayer, R., U. Morgan, et al. (2000). "Epidemiology of Cryptosporidium: transmission, detection and identification." Int J Parasitol 30(12-13): 1305-22.
	There are 10 valid species of Cryptosporidium and perhaps other cryptic species hidden under the umbrella of Cryptosporidium parvum. The oocyst stage is of primary importance for the dispersal, survival, and infectivity of the parasite and is of major importance for detection and identification. Because most oocysts measure 4-6 microm, appear nearly spherical, and have obscure internal structures, there are few or no morphometric features to differentiate species and in vitro cultivation does not provide differential data as for bacteria. Consequently, we rely on a combination of data from three tools: morphometrics, molecular techniques, and host specificity. Of 152 species of mammals reported to be infected with C. parvum or an indistinguishable organism, very few oocysts have ever been examined using more than one of these tools. This paper reviews the valid species of Cryptosporidium, their hosts and morphometrics; the reported hosts for the human pathogen, C. parvum; the mechanisms of transmission; the drinking water, recreational water, and food-borne outbreaks resulting from infection with C. parvum; and the microscopic, immunological, and molecular methods used to detect and identify species and genotypes.

Fayer, R. and T. Nerad (1996). "Effects of low temperatures on viability of Cryptosporidium parvum oocysts." Appl Environ Microbiol 62(4): 1431-3.
	Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.

Fayer, R., T. Nerad, et al. (1991). "Studies on cryopreservation of Cryptosporidium parvum." J Parasitol 77(3): 357-61.
	Neonatal BALB/c mice received oocysts or sporozoites of Cryptosporidium parvum pretreated by a variety of cryopreservation protocols. Histologic sections of infected and control mice were examined to determine if pretreated organisms established infection in the intestine. Sporozoites were inoculated rectally, oocysts orally. Freshly excysted sporozoites were frozen in Hanks' balanced salt solution (HBSS) containing dimethylsulfoxide (DMSO) or glycerol at concentrations of 5%, 10%, or 15% at cooling rates of -1 C and -10 C per min. Other sporozoites were frozen to -70 C in the absence of cryoprotectant without controlled reduction of temperature, others placed in HBSS with 10% DMSO but not subjected to freezing, whereas others were placed in vitrification media containing 5.5 M propylene glycol, 6.5 M glycerol, or 8 M ethylene glycol for 1 min before resuspension in fresh HBSS and inoculation into mice. Intact oocysts were frozen without controlled reduction of temperature directly to -70 C in HBSS containing no cryoprotectant or in HBSS that contained 10% DMSO. Others were cooled at -0.3 C per min from 4 C to -70 C in HBSS with 5% or 10% DMSO. Still others were cooled at a rate of -1 C per min until reaching -40 C and then cooled at -10 C per min until reaching -70 C in HBSS with 7.5% DMSO. Oocysts and sporozoites not exposed to cryoprotectants were inoculated into mice orally and rectally, respectively, for control purposes. Only unfrozen oocysts and sporozoites not exposed to cryoprotectant, and some of the unfrozen oocysts and sporozoites exposed to 10% DMSO, successfully established infections in mice.

Fayer, R., M. Santin, et al. (2003). "Comparison of microscopy and PCR for detection of three species of Encephalitozoon in feces." J Eukaryot Microbiol 50 Suppl: 572-3.
	
Fayer, R., M. Santin, et al. (2003). "Detection of Encephalitozoon hellem in feces of experimentally infected chickens." J Eukaryot Microbiol 50 Suppl: 574-5.
	
Fayer, R., M. Santin, et al. (2003). "First detection of microsporidia in dairy calves in North America." Parasitol Res 90(5): 383-6.
	Fecal specimens were obtained from a total of 413 dairy calves from farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, specimens were examined by fluorescence microscopy, polymerase chain reaction (PCR), and DNA sequencing analysis for the presence of microsporidia. Microscopic examination revealed no spores. PCR using generic primers for microsporidia revealed 70 positive calves. PCR was then conducted using specific primers for Enterocytozoon bieneusi, the most frequently found microsporidian in human infections. These primers revealed 13 positive calves from six farms in five states. DNA sequencing analysis of the 13 E. bieneusi-positive specimens confirmed the PCR results and indicated 96.8-99.8% similarity with E. bieneusi sequences in GenBank. This is the first report of E. bieneusi in cattle in North America.

Fayer, R., M. Santin, et al. (2006). "Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States." Vet Parasitol 135(2): 105-12.
	The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.

Fayer, R., M. Santin, et al. (2005). "Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)." J Parasitol 91(3): 624-9.
	A new species of Cryptosporidium, C. bovis, is described. Oocysts of C. bovis, previously identified as Cryptosporidium genotype Bovine B (GenBank AY120911), are morphologically indistinguishable from those of C. parvum. They are excreted fully sporulated and contain 4 sporozoites, but lack sporocysts. Oocysts measure 4.76-5.35 microm (mean = 4.89 microm) x 4.17-4.76 microm (mean = 4.63 microm), with a length-to-width ratio of 1.06 (n = 50). Oocysts were not infectious for neonatal BALB/ c mice, but were infectious for 2 calves that were previously infected with C. parvum. Oocysts were not infectious for 2 experimentally exposed lambs less than 1 wk of age and were not detected in 42 lambs 2-3 mo of age, but were detected in a 2-wk-old lamb. In an earlier study, 79 of 840 calves on 14 dairy farms in 7 states were found infected with the new species. Most calves were 2-7 mo of age and none exhibited signs of diarrhea. This new species has been found in 10 of 162 calves aged 9 to 11 mo on a beef farm in Maryland. Fragments of the 18S rDNA, HSP-70, and actin genes were amplified by PCR, and purified PCR products were sequenced. Multilocus analysis of the 3 unlinked loci demonstrated the new species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further evidence of species status. Based on these biological and molecular data, we consider this highly prevalent Cryptosporidium that infects primarily postweaned calves to be a new species and propose the name Cryptosporidium bovis n. sp. for this parasite.

Fayer, R., M. Tilley, et al. (1991). "Production and preparation of hyperimmune bovine colostrum for passive immunotherapy of cryptosporidiosis." J Protozool 38(6): 38S-39S.
	Pregnant cows were immunized to produce hyperimmune bovine colostrum (HBC) by intramuscular injection or intramammary infusion (TI) followed by 3 successive TI boosters with Cryptosporidium parvum (Cp) oocyst antigen mixed with Freund's (F) or Ribi (R) adjuvant. Control cows received no Cp. Colostrum from all cows was skimmed of butterfat and tested for specific anti-Cp immunoglobulin isotypes by ELISA. The HBC from Cp-F and Cp-R immunized cows had IgG1 titers exceeding 1:400,000 and 1:800,000, respectively. Some HBC from Cp-F immunized cows was freeze-dried to facilitate storage and some were irradiated at 42.5 kGy to kill potentially contaminating pathogens. Freeze-drying, but not irradiation, reduced IgG1 titers by only one dilution. Neither treatment affected Western blot banding patterns.

Fayer, R., J. Trout, et al. (1996). "Effects of a wide range of temperatures on infectivity of Cryptosporidium parvum oocysts." J Eukaryot Microbiol 43(5): 64S.
	
Fayer, R., J. M. Trout, et al. (2000). "Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms." Vet Parasitol 93(2): 103-12.
	The prevalence of Cryptosporidium, Giardia and Eimeria, in healthy, asymptomatic, post-weaned and mature cattle was investigated on three Maryland farms. One farm, a dairy research facility, had 150 multiparous Holstein milking cows; 24 were examined and Cryptosporidium andersoni was detected in three (12.5%) but neither Giardia nor Eimeria was detected. The second farm, a commercial dairy, had 57 multiparous Holstein milking cows and an equal number of heifers. Of 19 cows examined, C. parvum, Giardia duodenalis, and Eimeria bovis and/or E. ellipsoidalis were detected in two (10.5%), two (10.5%) and one (5.26%) cow, respectively. Of 23 heifers examined, C. parvum, Giardia, and E. bovis and E. ellipsoidalis, was detected in two (8.7%), four (17.4%), and five (21.7%), heifers, respectively. The third farm, a beef cattle breeding and genetics research facility, had 180 7- to 9-month old purebred black Angus. Of 118 examined for C. parvum and Giardia, 34 (28.8%) and 44 (37.3%) were positive, respectively, of 97 examined for E. bovis and/or E. ellipsoidalis 32 (33.0%) were positive. These findings, based on a method with a minimum detection level of 100 oocysts of C. parvum/g of feces, which underestimates the number of infected cattle, clearly demonstrate the presence of low level, asymptomatic infections in post-weaned and adult cattle in the United States and indicate the potential role of such cattle as reservoirs of infectious parasites.

Fayer, R., J. M. Trout, et al. (1998). "Infectivity of Cryptosporidium parvum oocysts stored in water at environmental temperatures." J Parasitol 84(6): 1165-9.
	Oocysts of Cryptosporidium parvum obtained from calves were cleaned of fecal debris by density gradient centrifugation and suspended in deionized water in microcentrifuge tubes. The tubes were placed in circulating water baths at temperatures of -10, -5, 0, 5, 10, 15, 20, 25, 30, or 35 C, and 2 tubes were removed from each water bath 1, 2, 4, 8, 12, 16, 20, and 24 wk later. Oocysts from 1 tube were administered at the rate of 1.5 x 10(5) oocysts per mouse to 2 litters of neonatal BALB/c mice and were considered infective when developmental stages were found in histologic sections of mouse gut and/or a positive polymerase chain reaction (PCR) was obtained for C. parvum DNA in mouse ileum. The second tube was held at -70 C until tubes from all time periods were available, then oocysts within the tubes were assayed for amylopectin concentration. Oocysts held at -10 C were infectious up to 1 wk of storage, and those held at -5 C were infectious up to 8 wk of storage, as determined by PCR but not histology. Oocysts held at 0, 5, 10, 15, and 20 C were still infectious after 24 wk of storage. By microscopic examination of mouse tissue, oocysts held at 20 C infected only 1 of 10 mice after 24 wk of storage, and the number of developmental stages began declining after 4 wk of storage; those held at 25 and 30 C each produced infections up to 12 wk after storage in 1 of 10 mice with reduced numbers of developmental stages beginning 4 wk after storage. Those held at 35 C produced light infections in 2 of 10 mice only up to 1 wk of storage. Amylopectin concentration decreased with increasing length of storage time or temperature. These findings provide a guide for estimating the potential duration of oocyst infectivity within a wide range of environmental temperatures and demonstrate the relationship between amylopectin concentration and infectivity.

Fayer, R., J. M. Trout, et al. (2003). "Contamination of Atlantic coast commercial shellfish with Cryptosporidium." Parasitol Res 89(2): 141-5.
	Shellfish (oysters and/or clams) were obtained from 37 commercial harvesting sites in 13 Atlantic coast states from Maine to Florida and one site in New Brunswick, Canada. Gill washings from each of 25 shellfish at each site were examined by immunofluorescence microscopy (IFA) for oocysts of Cryptosporidium. Gill washings from another 25 shellfish at each site were grouped into five pools of five shellfish each. DNA from each pool was utilized for PCR and genotyping. Oocysts were found in 3.7% of 925 oysters and clams examined by IFA in shellfish from New Brunswick and 11 of 13 states. Cryptosporidium DNA was detected by PCR in 35.2% of 185 pools. Cryptosporidium parvum genotypes 1 and 2, and Cryptosporidium meleagridis,all of which have been identified in infected humans, were identified at 37.8% of the sites. Gill washings from every site were tested for the presence of infectious oocysts by biological assay in neonatal BALB/c mice but no mice were found infected, suggesting that either the oocysts were no longer infectious or infections in mice were below the level of detection. Collectively, these findings indicate that Cryptosporidium species, indicative of pollution from human and animal feces and potentially infectious for humans, were found in commercial shellfish from 64.9% of sites examined along the Atlantic coast by either microscopy or molecular testing. Previous reports link periods of high rainfall with the elevated numbers of pathogen contaminated shellfish. Because shellfish in the present study were examined during a period of exceptionally low precipitation, the data are thought to underestimate the number of Cryptosporidium contaminated shellfish likely to be found during periods of normal or above normal precipitation.

Fayer, R., J. M. Trout, et al. (2002). "Temporal variability of Cryptosporidium in the Chesapeake Bay." Parasitol Res 88(11): 998-1003.
	Although Cryptosporidium has been found worldwide in molluscan shellfish from waters contaminated with human and animal feces, little or no related environmental data have been obtained. In the present study, oysters ( Crassostrea virginica) were collected eight times over 3 years from seven sites in the Chesapeake Bay or its tributaries, with accompanying data on water temperature, salinity, rainfall, and streamflow. Oyster gill washings were examined by immunofluorescence microscopy for Cryptosporidium oocysts. Of 1,590 oysters collected, 19.6% had detectable oocysts. Of 53 collections, oocysts were detected 81% of the time. The time when the greatest percentage of oysters at most sites had detectable oocysts coincided with the time of greatest weekly and monthly rainfall, greatest streamflow into the Bay, and lowest water temperatures. In 28% of 53 collections, C. parvum genotypes 1 and 2 and C. baileyi were identified by PCR and gene sequencing. Oocyst infectivity was confirmed from 37.5% of 40 collections by initiating C. parvum genotype 2 infections in mice.

Fayer, R., J. M. Trout, et al. (2000). "Rotifers ingest oocysts of Cryptosporidium parvum." J Eukaryot Microbiol 47(2): 161-3.
	Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.

Fayer, R., J. M. Trout, et al. (2001). "Cryptosporidium canis n. sp. from domestic dogs." J Parasitol 87(6): 1415-22.
	Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.

Feng, X., S. M. Rich, et al. (2002). "Experimental evidence for genetic recombination in the opportunistic pathogen Cryptosporidium parvum." Mol Biochem Parasitol 119(1): 55-62.
	Cryptosporidium parvum is an intracellular protozoan parasite causing intestinal malabsorption and diarrhea in humans. The infection is usually self-limiting, although persistent cryptosporidosis is observed in immunocompromised and malnourished individuals. As with other Apicomplexa, the life cycle of Cryptosporidium is thought to comprise a sexual phase, during which a motile microgamont fuses with a sessile macrogamont. The four sporozoites found within each oocyst (the infectious form excreted in the feces) are thought to be the product of a meiotic division taking place immediately following fertilization, but the existence of a meiotic cycle in this genus has not been tested experimentally. To substantiate the occurrence of meiotic recombination in this species, we performed a genetic cross between two distinct isolates of C. parvum co-infected in INF-gamma knockout mice. We found that mixed infections produced recombinant progeny characterized by multilocus genotypes comprising alleles inherited from each parental line. This observation represents the first demonstration of sexual recombination in this pathogen. Together with the occurrence of genetically heterogeneous infections, this finding suggests that outcrossing between genotypes may occur in nature. Experimental crosses among Cryptosporidium populations will facilitate mapping of clinically relevant genes, the delineation of Cryptosporidium species, and defining the taxonomical status of C. parvum subtypes and host-specific genotypes.

Feng, Y. Y., S. L. Ong, et al. (2003). "Effect of particles on the recovery of cryptosporidium oocysts from source water samples of various turbidities." Appl Environ Microbiol 69(4): 1898-903.
	Cryptosporidium parvum can be found in both source and drinking water and has been reported to cause serious waterborne outbreaks which threaten public health safety. The U.S. Environmental Protection Agency has developed method 1622 for detection of Cryptosporidium oocysts present in water. Method 1622 involves four key processing steps: filtration, immunomagnetic separation (IMS), fluorescent-antibody (FA) staining, and microscopic evaluation. The individual performance of each of these four steps was evaluated in this study. We found that the levels of recovery of C. parvum oocysts at the IMS-FA and FA staining stages were high, averaging more than 95%. In contrast, the level of recovery declined significantly, to 14.4%, when the filtration step was incorporated with tap water as a spiking medium. This observation suggested that a significant fraction of C. parvum oocysts was lost during the filtration step. When C. parvum oocysts were spiked into reclaimed water, tap water, microfiltration filtrate, and reservoir water, the highest mean level of recovery of (85.0% +/- 5.2% [mean +/- standard deviation]) was obtained for the relatively turbid reservoir water. Further studies indicated that it was the suspended particles present in the reservoir water that contributed to the enhanced C. parvum oocyst recovery. The levels of C. parvum oocyst recovery from spiked reservoir water with different turbidities indicated that particle size and concentration could affect oocyst recovery. Similar observations were also made when silica particles of different sizes and masses were added to seeded tap water. The optimal particle size was determined to be in the range from 5 to 40 micro m, and the corresponding optimal concentration of suspended particles was 1.42 g for 10 liters of tap water.

Finch, G. R., E. K. Black, et al. (1993). "Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity." Appl Environ Microbiol 59(12): 4203-10.
	Inactivation of Cryptosporidium parvum oocysts by ozone was performed in ozone demand-free 0.05 M phosphate buffer (pH 6.9) in bench-scale batch reactors at 7 and 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times ranging from 5 to 15 min. The viability of the control and treated oocysts was determined by using in vitro excystation and infection in neonatal CD-1 mice. It was found that excystation consistently underestimated inactivation when compared with animal infectivity (P < or = 0.05). As inactivations increased, the difference between excystation and infectivity also increased. The inactivation kinetics of C. parvum by ozone deviated from the simple first-order Chick-Watson model and was better described by a nonlinear Hom model. The use of the Hom model for predicting inactivation resulted in a family of unique concentration and time values for each inactivation level rather than the simple CT product of the Chick-Watson model.

Finch, G. R., E. K. Black, et al. (1993). "Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone." Appl Environ Microbiol 59(11): 3674-80.
	Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.

Finch, G. R., C. W. Daniels, et al. (1993). "Dose response of Cryptosporidium parvum in outbred neonatal CD-1 mice." Appl Environ Microbiol 59(11): 3661-5.
	Cryptosporidium parvum infectivity in a neonatal CD-1 mouse model was used to determine the dose needed to infect 50% of the population. The 50% infective dose was estimated to be 79 oocysts. It was observed that a mean oral inoculum of 23 oocysts produced infection in 2 of 25 neonatal mice 7 days postinoculation. All animals became infected when the mean oral dose exceeded 310 oocysts per animal. The dose response of C. parvum was modeled with a logit dose-response model suitable for use in water disinfection studies.

Flanagan, P. A. (1992). "Giardia--diagnosis, clinical course and epidemiology. A review." Epidemiol Infect 109(1): 1-22.
	Infection with giardia may be associated with significant ill-health and while the reported incidence of infection is increasing in the United Kingdom, the true prevalence of infection and extent of morbidity due to this organism is unknown. Diagnosis is made difficult by non-specificity of symptoms and low sensitivity of traditional diagnostic techniques. Immunological methods of diagnosis hold promise for the future, but in the meantime, more routine testing by laboratories and multiple faecal testing by clinicians may prevent unnecessary morbidity. The late summer/autumn peak in reported infection is difficult to explain while the age distribution is typical of an organism which is spread faeco-orally. The importance of potable water supplies as a source of infection in this country is not clear, nor is the role of zoonotic spread. The apparent susceptibility to infection of certain population groups requires further exploration as does the role of the asymptomatically infected in transmission.

Fleta, J., C. Sanchez-Acedo, et al. (1995). "Detection of Cryptosporidium oocysts in extra-intestinal tissues of sheep and pigs." Vet Parasitol 59(3-4): 201-5.
	Extra-intestinal infections by Cryptosporidium parvum have been detected in pigs and sheep. Detection was carried out by imprints of the mucosa of different organs and viscera in 55 sheep and 57 pigs slaughtered at three abattoirs in Zaragoza (northeast Spain). Imprints were stained by using a modified Ziehl-Neelsen technique. In addition to intestinal infections, cryptosporidial oocysts were found in the gall-bladders of two pigs which were 2 months old, and in some organs of sheep aged 5 days or more, including the gall-bladder (5), mesenteric lymph nodes (2), trachea (7), lung (3) and the uterus of one lamb.

Fontaine, M. and E. Guillot (2003). "An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples." J Microbiol Methods 54(1): 29-36.
	The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.

Francy, D. S., O. D. Simmons, 3rd, et al. (2004). "Effects of seeding procedures and water quality on recovery of Cryptosporidium oocysts from stream water by using U.S. Environmental Protection Agency Method 1623." Appl Environ Microbiol 70(7): 4118-28.
	U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

Freire-Santos, F., H. Gomez-Couso, et al. (2002). "Survival of Cryptosporidium parvum oocysts recovered from experimentally contaminated oysters (Ostrea edulis) and clams (Tapes decussatus)." Parasitol Res 88(2): 130-3.
	Samples of two species of shellfish that form part of the human food chain (the oyster Ostrea edulis and the marine clam Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. Changes in the viability of oocysts subsequently recovered from the shellfish were evaluated by means of an immunofluorescent antibody technique (IFAT) and inclusion/exclusion of the fluorogenic vital dye propidium iodide. There was a sharp decrease in oocyst viability during the first 4 days, with 15-25% viable oocysts remaining thereafter. In addition the infectivity of these oocysts at 10 and 31 days post-contamination was demonstrated using a suckling murine model.

Freire-Santos, F., A. M. Oteiza-Lopez, et al. (2001). "Viability and infectivity of oocysts recovered from clams, Ruditapes philippinarum, experimentally contaminated with Cryptosporidium parvum." Parasitol Res 87(6): 428-30.
	This study confirms the important role of marine bivalve molluscs, destined for human consumption, as transmitters of cryptosporidiosis, zoonotic diarrhoeal disease caused by Cryptosporidium parvum. C. parvum oocysts recovered from seawater clams (Ruditapes philippinarum) were viable and infective in five of eight infected neonatal CD-1 Swiss mice. Oocysts were observed in clam gill and gastrointestinal tract tissue homogenates as well as in gill histological sections, by an immunofluorescent antibody technique. In vitro viability of recovered oocysts was also determined using fluorogenic vital dyes (75% viability).

Freire-Santos, F., A. M. Oteiza-Lopez, et al. (2000). "Study of the combined influence of environmental factors on viability of cryptosporidium parvum oocysts in water evaluated by fluorogenic vital dyes and excystation techniques." Vet Parasitol 89(4): 253-9.
	Using a factorial experimental design, the combined effect of salinity, temperature and storage time on the viability of Cryptosporidium parvum oocysts in water was evaluated by fluorogenic vital dyes (4',6-diamidino-2-phenylindole and propidium iodide) and an excystation technique. Salinity, storage time and their interaction seemed to be the most influential factors, whereas temperature was not a significant factor. Under unfavourable conditions (salinity 35 per thousand, storage time 40 days), even more than 20% of oocysts remain viable, indicating a high risk of infection for immunocompromised individuals.

Freire-Santos, F., A. M. Oteiza-Lopez, et al. (2000). "Detection of Cryptosporidium oocysts in bivalve molluscs destined for human consumption." J Parasitol 86(4): 853-4.
	Clams (Dosinia exoleta, Ruditapes philippinarum, Venerupis pullastra, Venerupis rhomboideus, Venus verrucosa), mussels (Mytilus galloprovincialis), and oysters (Ostrea edulis) were tested for the presence of Cryptosporidium sp. oocysts using various stain techniques and a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. All molluscs were harvested in northwest Spain (Galicia) except for R. philippinarum, which was from Italy, and 1 of the 6 oyster samples, which was from England. The results showed the presence of Cryptosporidium sp. oocysts in all of the molluscan species destined for human consumption.

Freire-Santos, F., A. M. Oteiza-Lopez, et al. (1999). "Effect of salinity, temperature and storage time on mouse experimental infection by Cryptosporidium parvum." Vet Parasitol 87(1): 1-7.
	Cryptosporidium parvum oocysts collected from a naturally infected calf were exposed to different salinity and temperature for 2, 21 and 40 days, and then inoculated intragastrically into coccidium-free neonatal mice. The intensity of infection as determined seven days later by examination of intestinal homogenates were statistically analysed. Salinity, time and salinity-time interaction were the only factors with significant effect on the infection intensity.

Gajadhar, A. A., J. J. Aramini, et al. (1998). "Prevalence of Toxoplasma gondii in Canadian market-age pigs." J Parasitol 84(4): 759-63.
	During 1991 and 1992, 2,800 market-age pigs were sampled at federally inspected abattoirs from across Canada. Anti-Toxoplasma gondii IgG at titers of > or =1:32 were found in 240 pigs examined by a commercial, latex agglutination test. Seroprevalences ranged from 3.5 to 13.2% in the different regions of the country. Tissue hybridization studies using a previously developed probe demonstrated T. gondii ribosomal RNA in 9 of 36 animals, whereas mouse bioassay testing of heart muscle and diaphragm from all 2,800 pigs failed to demonstrate the presence of infective stages of T. gondii in tissues. Although serology results from this study indicated that Canadian market-age pigs are infected with T. gondii at rates similar to those reported from other parts of North America, mouse bioassay results suggested that Canadian pork products contain low levels of infective organisms. This apparent discrepancy suggests that serological evidence of T. gondii infection in pigs alone does not accurately assess the public health risks associated with consuming improperly cooked pork products.

Gale, P. (2005). "Land application of treated sewage sludge: quantifying pathogen risks from consumption of crops." J Appl Microbiol 98(2): 380-96.
	AIMS: To predict the number of humans in the UK infected through consumption of root crops grown on agricultural land to which treated sewage sludge has been applied in accordance with the current regulations and guidance (Safe Sludge Matrix). METHODS AND RESULTS: Quantitative risk assessments based on the source, pathway, receptor approach are developed for seven pathogens, namely salmonellas, Listeria monocytogenes, campylobacters, Escherichia coli O157, Cryptosporidium parvum, Giardia, and enteroviruses. Using laboratory data for pathogen destruction by mesophilic anaerobic digestion, and not extrapolating experimental data for pathogen decay in soil to the full 30-month harvest interval specified by the Matrix, predicts 50 Giardia infections per year, but less than one infection per year for the other six pathogens. Assuming linear decay in the soil, a 12-month harvest interval eliminates the risks from all seven pathogens; the highest predicted being one infection of C. parvum in the UK every 45 years. Computer simulations show that a protective effect from binding of pathogens to particulate matter could potentially exaggerate the observed rate of decay in experimental systems. CONCLUSIONS: The results confirm, assuming pathogens behave according to our current understanding, that the risks to humans from consumption of vegetable crops are remote. Furthermore the harvest intervals stipulated by the Safe Sludge Matrix compensate for potential lapses in the operational efficiency of sludge treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The models demonstrate the huge potential impact of decay in the soil over the 12/30-month intervals specified by the Matrix, although lack of knowledge on the exact nature of soil decay processes is a source of uncertainty. The models enable the sensitivity of the predicted risks to changes in the operational efficiency of sewage sludge treatment to be assessed.

Gale, P. and G. Stanfield (2000). "Cryptosporidium during a simulated outbreak." Journal of the American Water Works Association 92(9): 105-116.
	Protozoa and viruses have in recent years replaced bacterial pathogens as the agents of primary concern in waterborne disease. Unfortunately, so-called "spot" sampling for Cryptosporidium may underestimate the risk of infection. Continuous large-volume sampling of treated water may help overcome the variation in oocyst counts in treated water. Gale and Stanfield provide a model to help utility managers interpret data during outbreaks and to design strategies to monitor for Cryptosporidium. The authors' model also helps explain why some, and often all, samples collected during an outbreak contain zero oocysts. They show that there is no clear association between measured oocyst densities in the drinking water supply and the outbreak of illness. Instead they emphasize the need to accommodate the spatial and temporal variation in oocyst counts in treated water in order to fully assess the net oocyst removal by treatment processes. The model they present promises to enhance water professionals' understanding of the association between measured oocyst densities and the risk of illness in the population.

Gale, P., P. A. H. Van Dijk, et al. (1997). "Drinking water treatment increases micro-organism clustering; the implications for microbiological risk assessment." AQUA: J. WATER SUPPLY RES. TECHNOL., vol. 46(3): 117-126.
	Current models to assess the risks to public health from waterborne pathogens assume that micro-organisms are randomly dispersed within drinking water samples. One manifestation of such an assumption is that, under non-outbreak conditions, models predict that consumers are only exposed to daily doses of either zero pathogens or just one pathogen. This paper presents evidence against this assumption and demonstrates that in drinking water bacteria spores are spatially associated or clustered to some degree. The variability between spore counts in most 10-mL or 100-mL sub-samples within 100 L treated water volumes was accommodated by a negative binomial distribution. However, a few sub-samples contained extremely high counts which could not be so accommodated. A comparison of spore counts within 100 L volumes before and after alum coagulation and rapid gravity filtration demonstrated that drinking water treatment not only removed 95-99% of spores but also appeared to promote their clustering. More extreme clustering could occur from tight association, where micro-organisms are bound to particles such as metal hydroxides from coagulation. This was not investigated. Through clustering, a few drinking water consumers could be exposed to higher doses of pathogen than just one a day. This is an important consideration in risk models for waterborne pathogens such as Cryptosporidium parvum with an ID sub(50) for healthy human volunteers of about 200 oocysts. The data presented here contribute to modelling the effect of treatment on raw water pathogen density data (measured in 100 L volumes) and simulating pathogen counts in the finished waters at the resolution of unboiled volumes consumed daily by consumers (0.1-1 L).

Garber, L. P., M. D. Salman, et al. (1994). "Potential risk factors for Cryptosporidium infection in dairy calves." J Am Vet Med Assoc 205(1): 86-91.
	Fecal samples from 7,369 calves on 1,103 farms were examined for cryptosporidia in a nationwide survey, using monoclonal antibody technique. Cryptosporidium oocysts were found in calves from 652 (59.1%) of the farms and in 1,648 (22.4%) of the tested calves. Almost half the calves between 7 and 21 days of age had cryptosporidia in their fecal samples. Prevalence was highest during the summer. Farms with multiple-cow maternity facilities and farms with > 100 milking cows were the most likely to have calves with cryptosporidia.

Garcia, A., W. Yanko, et al. (2002). "Giardia cysts in tertiary-treated wastewater effluents: are they infective?" Water Environ Res 74(6): 541-4.
	The infectivity of Giardia lamblia cysts recovered in primary- and tertiary-treated wastewater reclamation plant effluents was assessed in Mongolian gerbils (Meriones unguiculatus). Infections in gerbils inoculated with cysts from primary effluent concentrates demonstrated the presence of infectious G. lamblia cysts. No infectious cysts were detected by this method in concentrates of tertiary-treated effluents. This study found that determination of cyst concentrations without viability or infectivity assessment may significantly overestimate the potential health risks associated with protozoan cysts in tertiary-treated wastewater effluents.

Gasser, R. B., X. Zhu, et al. (2001). "Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions." Electrophoresis 22(3): 433-7.
	Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

Gatei, W., R. W. Ashford, et al. (2002). "Cryptosporidium muris infection in an HIV-infected adult, Kenya." Emerg Infect Dis 8(2): 204-6.
	We describe a case of Cryptosporidium muris infection in an HIV-infected adult with diarrhea in Kenya. Sequence analysis of an 840-bp region of the 18S rRNA gene locus demonstrated the isolate had 100% nucleotide identity with C. muris recovered from a rock hyrax, 98.8% with a C. muris "calf" isolate, 95.5% with C. serpentis, but only 87.8% with C. parvum "human" type.

Gatei, W., J. Greensill, et al. (2003). "Molecular analysis of the 18S rRNA gene of Cryptosporidium parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam." J Clin Microbiol 41(4): 1458-62.
	An 840-bp fragment of the 18S rRNA gene was used to identify Cryptosporidium spp. recovered from human immunodeficiency virus (HIV)-infected and -uninfected patients from Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. Initial identification was by Ziehl-Neelsen acid-fast staining. Confirmation was by nested PCR, targeting the most polymorphic region of the 18S rRNA gene. Genotyping was by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing. Among 63 isolates analyzed, four genotypes of Cryptosporidium were identified; 75% of the isolates were of the C. parvum human genotype, while the potentially zoonotic species were of the C. parvum bovine genotype (21.7%), the C. meleagridis genotype (1.6% [one isolate]), and the C. muris genotype (1.6% [one case]). HIV-infected individuals were more likely to have zoonotic genotypes than the HIV-uninfected individuals. Among the C. parvum group, strains clustered distinctly into either human or bovine genotypes regardless of the geographical origin, age, or HIV status of the patients. The intragenotypic variation observed in the C. parvum human genotype was extensive compared to that within the C. parvum bovine genotype group. The variation within genotypes was conserved in all geographical regions regardless of the patients' HIV status. The extensive diversity within genotypes at the 18S rRNA gene locus may limit its application to phylogenetic analyses.

Gatei, W., Y. Suputtamongkol, et al. (2002). "Zoonotic species of Cryptosporidium are as prevalent as the anthroponotic in HIV-infected patients in Thailand." Ann Trop Med Parasitol 96(8): 797-802.
	The epidemiology of chronic diarrhoea in adults with late-stage HIV infection was investigated in a prospective study in Bangkok, Thailand. During this investigation, 34 Cryptosporidium isolates were obtained from the faeces of 36 patients, with mean CD4(+) counts of only 14 x 10(6) CD4(+) cells/litre (range = 2 x 10(6) - 53 x 10(6)/litre), who had symptomatic cryptosporidiosis. Genotyping of these isolates, by RFLP analysis and DNA sequencing of the hypervariable region of the 18S rRNA gene, indicated that only 17 (50%) were of the C. parvum human genotype. The rest were of C. meleagridis (seven), the C. parvum 'bovine' genotype (five), C. felis (three) and C. canis (two). Extensive genotypic heterogeneity was observed among the C. parvum isolates, and two other isolates, one of C. meleagridis and the other of C. felis, produced atypical restriction patterns and were only identified by sequencing. This appears to represent the first report of C. canis and the 'bovine' genotype of C. parvum in HIV-infected Thai patients.

Gelletlie, R., J. Stuart, et al. (1997). "Cryptosporidiosis associated with school milk." Lancet 350(9083): 1005-6.
	
Gerba, C. P., D. C. Johnson, et al. (1997). "Efficacy of iodine water purification tablets against Cryptosporidium oocysts and Giardia cysts." Wilderness Environ Med 8(2): 96-100.
	The ability to control water-borne diseases is critical for soldiers, hikers, and others who may need to drink directly from an outdoor source. Water-borne protozoan parasites that are specifically of concern are Giardia and Cryptosporidium because of their resistance to halogen disinfection. The purpose of this study was to determine the effectiveness of iodine tablets against Giardia and Cryptosporidium under general- and worst-case water conditions that might be found in the field. Giardia cysts and Cryptosporidium oocysts were exposed to iodine according to manufacturer's instructions (two tablets/L = 13-18 mg/L for 20 minutes). This dose inactivated 3-log10 of Giardia in general-case water and pH 9. In worst-case water, however, only about 35% of cysts were inactivated at pH 5. Fifty minutes were required to achieve a 3-log10 reduction at pH 5. Cryptosporidium oocysts were more difficult to inactivate. Only 10% were inactivated after a 20-minute exposure to iodine according to manufacturer's instructions; even after 240 minutes of exposure to iodine only 66-81% oocysts were inactivated. These data strongly suggest that iodine disinfection is not effective in inactivating Cryptosporidium oocysts in water. Because this organism is common in all surface waters, it is recommended that another method of treatment be used before ingestion.

Gilbert, R., D. Dunn, et al. (2001). "Ecological comparison of the risks of mother-to-child transmission and clinical manifestations of congenital toxoplasmosis according to prenatal treatment protocol." Epidemiol Infect 127(1): 113-20.
	We compared the relative risks of mother-to-child transmission of Toxoplasma gondii and clinical manifestations due to congenital toxoplasmosis associated with intensive prenatal treatment in Lyon and Austria, short term treatment in 51% of Dutch women, and no treatment in Danish women. For each cohort, relative risks were standardized for gestation at seroconversion. In total, 856 mother-child pairs were studied: 549 in Lyon, 133 in Austria, 123 in Denmark and 51 in The Netherlands. The relative risk for mother-to-child transmission compared to Lyon was 1.24 (95% CI: 0.88, 1.59) in Austria; 0.59 (0.41, 0.81) in Denmark; and 0.65 (0.37, 1.01) in The Netherlands. Relative risks for clinical manifestations compared with Lyon (adjusted for follow-up to age 3 years) were: Austria 0.19 (0.04, 0.51); Denmark 0.60 (0.13, 1.08); and The Netherlands 1.46 (0.51, 2.72). There was no clear evidence that the risk of transmission or of clinical manifestations was lowest in centres with the most intensive prenatal treatment.

Gilbert, R. and L. Gras (2003). "Effect of timing and type of treatment on the risk of mother to child transmission of Toxoplasma gondii." Bjog 110(2): 112-20.
	OBJECTIVE: To determine the effects on mother to child transmission of the timing and type of prenatal treatment, taking into account gestational age at maternal seroconversion. DESIGN: Prospective cohort study. SETTING: European centres offering prenatal screening for toxoplasmosis. POPULATION:Children born to a cohort of pregnant women with toxoplasma infection. METHODS: We determined the effects on mother to child transmission of the interval between seroconversion and start of treatment (treatment delay), and the type of treatment, taking into account gestational age at maternal seroconversion. MAIN OUTCOME MEASURE: Congenital infection status confirmed by toxoplasma IgG results at one year postnatal age. RESULTS: Of 1208 women analysed, 72% were first prescribed spiramycin, 19% pyrimethamine-sulphonamide and 9% (mostly infected during the last trimester) were untreated. The odds ratios for mother to child transmission for all women treated after a delay of four to seven weeks was 0.77 (95% CI 0.34-1.69), and after eight weeks or more was 1.33 (0.56-2.89) compared with less than four weeks. The odds ratio per week of treatment delay was 1.01 (0.93-1.08). There was no evidence that transmission risk differed in women first treated with pyrimethamine-sulphonamide versus spiramycin: odds ratio 1.10 (0.63-1.91) or in untreated versus treated women: odds ratio 0.57 (0.27-1.17). CONCLUSION: We were unable to demonstrate a beneficial effect of the timing or type of prenatal treatment on the risk of mother to child transmission but we could not exclude a clinically important effect. Randomised controlled trials are required to determine the effect of prenatal treatment on mother to child transmission.

Gilbert, R. E., D. T. Dunn, et al. (1999). "Incidence of symptomatic toxoplasma eye disease: aetiology and public health implications." Epidemiol Infect 123(2): 283-9.
	Ocular disease is the commonest disabling consequence of toxoplasma infection. Incidence and lifetime risk of ocular symptoms were determined by ascertaining affected patients in a population-based, active reporting study involving ophthalmologists serving a population of 7.4 million. Eighty-seven symptomatic episodes were attributed to toxoplasma infection. Bilateral visual acuity of 6/12 or less was found in seven episodes (8%) and was likely to have been transient in most cases. Black people born in West Africa had a 100-fold higher incidence of symptoms than white people born in Britain. Only two patients reported symptoms before 10 years of age. The estimated lifetime risk of symptoms in British born individuals (52% of all episodes) was 18/100000 (95% confidence interval: 10.8-25.2). The low risk and mild symptoms in an unscreened British population indicate limited potential benefits of prenatal or postnatal screening. The late age at presentation suggests a mixed aetiology of postnatally acquired and congenital infection for which primary prevention may be appropriate, particularly among West Africans.

Gile, M., D. C. Warhurst, et al. (2002). "A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2." Parasitology 125(Pt 1): 35-44.
	A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Glaberman, S., J. E. Moore, et al. (2002). "Three drinking-water-associated cryptosporidiosis outbreaks, Northern Ireland." Emerg Infect Dis 8(6): 631-3.
	Three recent drinking-water-associated cryptosporidiosis outbreaks in Northern Ireland were investigated by using genotyping and subgenotyping tools. One Cryptosporidium parvum outbreak was caused by the bovine genotype, and two were caused by the human genotype. Subgenotyping analyses indicate that two predominant subgenotypes were associated with these outbreaks and had been circulating in the community.

Glaberman, S., I. M. Sulaiman, et al. (2001). "A multilocus genotypic analysis of Cryptosporidium meleagridis." J Eukaryot Microbiol Suppl: 19S-22S.
	Cryptosporidium meleagridis is a common cause of cryptosporidiosis in birds. In addition, recent reports have described the parasite as an etiologic agent of cryptosporidiosis in both immunocompetent and immunocompromised humans. Therefore, it is important to genetically characterize isolates of C. meleagridis from different hosts and geographic areas, and to develop molecular tools to differentiate isolates from various hosts or areas. In this study, a total of 11 isolates of Cryptosporidium meleagridis from both human and avian hosts were examined at three genetic loci: the small-subunit rRNA, 60-kDa glycoprotein precursor, and 70-kDa heat shock protein genes. Two genotypes of C. meleagridis were seen at the small-subunit rRNA locus. These differed from each other by the presence or lack of a heterogeneous copy of the gene and an ATT repeat. The 60-kDa glycoprotein precursor gene divided these eleven isolates of C. meleagridis into six genotypes with high sequence diversity between groups. The highest genetic heterogeneity, however, was seen at the 70-kDa heat shock protein locus, and was primarily present at the 3' end of the gene. This heterogeneity separated eight isolates of C. meleagridis into six genotypes. These data could be useful in the development of molecular tools to promote understanding of the transmission of C. meleagridis in humans.

Gomez-Bautista, M., L. M. Ortega-Mora, et al. (2000). "Detection of infectious Cryptosporidium parvum oocysts in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule)." Appl Environ Microbiol 66(5): 1866-70.
	Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10(3) oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.

Gomez-Couso, H., F. Freire-Santos, et al. (2004). "Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR." Int J Food Microbiol 91(3): 279-88.
	A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.

Gomez-Couso, H., F. Freire-Santos, et al. (2003). "Contamination of bivalve molluscs by Cryptosporidium oocysts: the need for new quality control standards." Int J Food Microbiol 87(1-2): 97-105.
	A yearlong study was carried out to investigate the presence and viability of Cryptosporidium oocysts in 203 samples of cultured shellfish from Galicia (NW Spain) and 38 samples imported from other European Union (EU) countries. Shellfish samples included mussels, oysters, clams and cockles. Cryptosporidium oocysts were detected, using a direct immunofluorescence antibody test (IFAT), in 34.4% of the samples analyzed; use of the fluorogenic dye propidium iodide (PI) revealed viable potentially infective oocysts in 53.0% of these samples. There was no relation between the presence of Cryptosporidium oocysts and the microbiological contamination detected in the samples expressed as Most-Probable-Number (MPN) of fecal coliforms, the different species of mollusc, or the month of sampling. One important finding was that the depuration process was ineffective in totally removing oocyst contamination. Furthermore, the existence of viable oocysts in samples with microbiological contamination levels lower than 300 fecal coliforms/100 g, which in accordance with current legislation are considered suitable for human consumption, suggests the need to include parasitological analyses in the quality control for these molluscs.

Gomez-Couso, H., F. Freire-Santos, et al. (2003). "Environmental dispersal of Cryptosporidium parvum oocysts and cross transmission in cultured bivalve molluscs." Parasitol Res 90(2): 140-2.
	Two commercially valuable mollusc species ( Ostrea edulisand Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. A direct immunofluorescent antibody technique and inclusion/exclusion of the fluorogenic vital dye propidium iodide were used to test for the presence and viability of the oocysts, showing that transmission of contamination occurred between coexisting species. There was a decrease in the viability of oocysts in the initially uncontaminated molluscs as well as a large decrease in the number of oocysts retained when dead molluscs were used as the source of contamination. The results show the potentially important role that these molluscs play in spreading contamination in depuration plants and areas where aquatic organisms are cultivated.

Gomez-Couso, H., F. Mendez-Hermida, et al. (2006). "Cooking mussels (Mytilus galloprovincialis) by steam does not destroy the infectivity of Cryptosporidium parvum." J Food Prot 69(4): 948-50.
	The consumption of shellfish has increased considerably worldwide, with an associated increase in foodborne illnesses. Among the bivalves, the mussels are usually cooked by steam, which constitutes a typical dish in several regions. In this article, we demonstrate that this preparation is not sufficient to destroy completely the infectivity of Cryptosporidium parvum. Oocysts recovered from experimentally contaminated mussels (Mytilus galloprovincialis) were infectious to neonatal mice after cooking. Although, to date, no official cases of cryptosporidiosis linked to shellfish consumption have been reported, we recommend that people with reduced immunity avoid this type of food because they are at high risk of being infected with Cryptosporidium spp. after eating raw or undercooked contaminated bivalves.

Gomez-Couso, H., F. Mendez-Hermida, et al. (2006). "Cryptosporidium contamination in harvesting areas of bivalve molluscs." J Food Prot 69(1): 185-90.
	Cryptosporidium contamination was evaluated in areas in Galicia (northwestern Spain) where bivalve molluscs are harvested. Galicia is the main mussel-producing region in Europe. Data were collected on water contamination of effluents that are discharged into these areas. Cryptosporidium spp. were detected by immunofluorescence microscopy and molecular methods in 71% of the river water samples (n = 7), 64% of raw sewage samples (n = 11), 50% of effluents from wastewater treatment plants (n = 16), and 29.3% of the mussel samples (Mytilus galloprovincialis, n = 184). Cryptosporidium parvum was identified in all samples of contaminated mussels, Cryptosporidium muris was found in three samples of effluent from wastewater treatment plants, and Cryptosporidium baileyi was found in a sample of raw sewage. Further studies are needed to determine the parasitological quality of water in these shellfish harvesting and recreational areas. Cryptosporidium could be a public health risk from consumption of raw or undercooked contaminated molluscs and use of contaminated waters for recreational purposes.

Gostin, L. O., Z. Lazzarini, et al. (2000). "Water quality laws and waterborne diseases: Cryptosporidium and other emerging pathogens." Am J Public Health 90(6): 847-53.
	Waterborne diseases, such as cryptosporidiosis, cause many cases of serious illness in the United States annually. Water quality is regulated by a complex system of federal and state legal provisions and agencies, which has been poorly studied. The authors surveyed state and territorial agencies responsible for water quality about their laws, regulations, policies, and practices related to water quality and surveillance of cryptosporidiosis related to drinking water. In this commentary they review the development and current status of federal drinking water regulations, identify conflicts or gaps in legal authority between federal agencies and state and territorial agencies, and describe court-imposed limitations on federal authority with regard to regulation of water quality. Recommendations are made for government actions that would increase the efficiency of efforts to ensure water quality; protect watersheds; strengthen waterborne disease surveillance; and protect the health of vulnerable populations.

Graczyk, T. K., D. B. Conn, et al. (2003). "Accumulation of human waterborne parasites by zebra mussels (Dreissena polymorpha) and Asian freshwater clams (Corbicula fluminea)." Parasitol Res 89(2): 107-12.
	Zebra mussels (Dreissena polymorpha) and Asian freshwater clams (Corbicula fluminea) are nonindigenous invasive bivalves present in North American fresh waters that are frequently contaminated with human enteric parasites, Cryptosporidium parvum and Giardia lamblia. Six-week laboratory exposure of D. polymorpha and Corbicula fluminea to both parasites seeded daily at concentrations reported from surface waters demonstrated efficient removal of Cryptosporidium parvum oocysts and G. lamblia cysts by both bivalve species. The number of parasites in mollusk tissue progressively increased in relation to the concentration of waterborne contamination, and decreased after cessation of the contamination. Oocysts outnumbered cysts in the tissue of both bivalves, and more parasites were identified in D. polymorpha than in Corbicula fluminea; overall 35.0% and 16.3% of the parasites seeded, respectively. Because C. fluminea and D. polymorpha can accumulate human waterborne parasites in proportion to ambient concentrations, these species of bivalves can be effective bioindicators of contamination of freshwater habitats with Cryptosporidium and Giardia.

Graczyk, T. K., M. R. Cranfield, et al. (1996). "Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (FA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum." Am J Trop Med Hyg 54(3): 274-9.
	A commercial enzyme immunoassay (EIA) (ProSpect Rapid Assay), a direct immunofluorescence antibody (IFA) test for stool testing (MERIFLUOR Cryptosporidium/Giardia), and an indirect IFA test for environmental testing (Hydrofluor-Combo Cryptosporidium/Giardia) were evaluated for detection of low public health risk Cryptosporidium oocyst isolates, and for C. parvum oocyst isolates from human and bovine feces that represent a high public health risk. There was no cross-reactivity of EIA with ova of eight medically important helminths, three Eimeria species oocysts, Sarcocystis cruzi sporocysts, and two Candida sp. isolates. All nine snake oocyst isolates (C. serpentis), two of seven lizard oocyst isolates, one turtle oocyst isolate, two avian oocyst isolates (turkey, C. meleagridis), one C. wrairi oocyst isolate from guinea pigs, one C. muris oocyst isolate from hyrax, one heifer C. muris isolate, and two C. muris-like oocyst isolates from a camel were positive by both IFA tests; six of these 19 oocyst isolates were EIA-positive. There was no difference in the sensitivity and specificity between direct and indirect IFA tests. The sensitivity of the EIA and both IFA tests to the C. parvum oocysts was 100%. The EIA showed less cross-reactivity with the non-C. parvum oocysts (24%) than direct or indirect IFA (76%), and was less sensitive to those isolates (20%) than both IFA tests (63%). A simulated sampling model for high and low public health risk Cryptosporidium oocysts showed that the low risk oocyst isolates may constitute up to 35% of all positive environmental samples by direct or indirect IFA determination, and up to 12% of all EIA positive samples. This study indicates a superiority of direct and indirect IFA and EIA for screening of human-or-bovine-origin fecal specimens, whereas testing of environmental samples may lead to misidentification of medically important isolates. The results demonstrated that the EIA kit can more accurately identify environmental samples containing oocytes pathogenic for humans than both IFA tests. The specificity of commercially available diagnostic kits to C. parvum should be critically examined for cross-species identification before they are recommended or adopted for use in testing environmental samples.

Graczyk, T. K., M. R. Cranfield, et al. (1997). "Recovery of waterborne oocysts of Cryptosporidium from water samples by the membrane-filter dissolution method." Parasitol Res 83(2): 121-5.
	The cellulose-acetate membrane (CAM)-filter dissolution method implemented into a Millipore Glass Microanalysis system was used for recovery of Cryptosporidium parvum oocysts seeded into 25 l of drinking water in polyethylene carboy aspirator bottles. CAM-entrapped oocysts were detected by immunofluorescence microscopy. From 65 to 94 oocysts/l (mean 75 oocysts/l), 34.7% overall of the inoculated oocysts, were unrecovered as determined after the water had been drained from the bottle, rinsed with 1 l of eluting fluid (EF), and CAM-filtered. Efficiency rates of oocyst recovery ranged from 24.0% to 64.0% (mean 44.1%), without the use of EF and from 72.1% to 82.3% (mean 78.8%) when EF was used. To ensure a high recovery efficiency of Cryptosporidium oocysts from sampled water by the CAM-filter dissolution method, it is recommended that 1 l of EF per 25 l of water be used.

Graczyk, T. K., M. R. Cranfield, et al. (1998). "Oocysts of Cryptosporidium from snakes are not infectious to ducklings but retain viability after intestinal passage through a refractory host." Vet Parasitol 77(1): 33-40.
	Six 2-week-old Cryptosporidium-free Peking ducklings (Anas platyrhynchos) each received 2.0 x 10(6) viable Cryptosporidium serpentis oocysts from 6 naturally infected captive snakes. Histological sections of digestive (stomach, jejunum, ileum, cloaca, and cecum) and respiratory tract tissues (larynx, trachea, and lungs) did not contain life-cycle stages of Cryptosporidium in any of the inoculated ducklings. Because ducklings were refractory to infection, C. serpentis transmission via a diet of Peking ducklings is improbable. Viable (per in vitro excystation assay) inoculum-derived oocysts were detected in duckling feces up to 7 days post-inoculation (PI); the number of intact oocysts excreted during the first 2 days PI was significantly higher than for the remaining 5 days PI (P < 0.01). The dynamics of oocyst shedding showed that overall the birds released a significantly higher number of intact oocysts than oocyst shells (P < 0.01). Retention of the viability of C. serpentis oocysts following intestinal passage through a refractory avian species may have epizootiological implications. Under certain circumstances such as after the ingestion of C. serpentis-infected prey, herpetivorous birds may disseminate C. serpentis oocysts in the environment.

Graczyk, T. K., M. R. Cranfield, et al. (1996). "Viability and infectivity of Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host." Appl Environ Microbiol 62(9): 3234-7.
	Six Cryptosporidium-free Peking ducks (Anas platyrhynchos) were each orally inoculated with 2.0 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. Histological examination of the stomachs jejunums, ilea, ceca, cloacae, larynges, tracheae, and lungs of the ducks euthanized on day 7 postinoculation (p.i.) revealed no life-cycle stages of C. parvum. However, inoculum-derived oocysts extracted from duck feces established severe infection in eight neonatal BALB/c mice (inoculum dose, 2.5 x 10(5) per mouse). On the basis of acid-fast stained direct wet smears, 73% of the oocysts in duck feces were intact (27% were oocyst shells), and their morphological features conformed to those of viable and infectious oocysts of the original inoculum. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the feces-recovered oocysts (the majority were 3+ to 4+). The dynamics of oocyst shedding showed that the birds released a significantly higher number of intact oocysts than the oocyst shells (P < 0.01). The number of intact oocysts shed (87%) during the first 2 days p.i. was significantly higher than the number shed during the remaining 5 days p.i. (P < 0.01) and significantly decreased from day 1 to day 2 p.i. (P < 0.01). The number of oocyst shells shed during 7 days p.i. did not vary significantly (P > 0.05). The retention of infectivity of C. parvum oocysts after intestinal passage through an aquatic bird has serious epidemiological and epizootiological implications. Waterfowl may serve as mechanical vectors for the waterborne oocysts and may enhance contamination of surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to watershed management practices, the watershed protection program should consider waterfowl as a potential factor enhancing contamination of the source water with C. parvum.

Graczyk, T. K., M. R. Cranfield, et al. (1999). "House flies (Musca domestica) as transport hosts of Cryptosporidium parvum." Am J Trop Med Hyg 61(3): 500-4.
	Refuse and promiscuous-landing synanthropic filth flies, such as house flies (Musca domestica), are recognized as transport hosts for a variety of protozoan and metazoan parasites in addition to viral and bacterial pathogens of public health importance. Exposure of adult M. domestica to 20 ml of bovine diarrheal feces containing Cryptosporidium parvum oocysts (2.0 x 10(5) oocysts/ml) resulted in intense deposition of the oocysts through fly feces on the surfaces visited by the flies (mean = 108 oocysts/cm2). Cryptosporidium parvum oocysts were detected by immunofluorescent antibodies on the exoskeleton of adult flies and in their digestive tracts. An average of 267, 131, 32, 19, and 14 oocysts per adult fly were eluted from its exoskeleton on days 3, 5, 7, 9, and 11 after they emerged, respectively. Approximately 320 C. parvum oocysts per pupa were eluted from the external surface of the pupae derived from maggots that breed in a substrate contaminated with the bovine feces; the oocysts were numerous on maggots (approximately 150 oocysts/maggot). Adult and larval stages of house flies breeding or having access to C. parvum-contaminated substrate will mechanically carry the oocysts in their digestive tracts and on their external surfaces.

Graczyk, T. K., M. R. Cranfield, et al. (1997). "Infectivity of Cryptosporidium parvum oocysts is retained upon intestinal passage through a migratory water-fowl species (Canada goose, Branta canadensis)." Trop Med Int Health 2(4): 341-7.
	Five Cryptosporidium-free Canada geese (Branta canadensis) were individually orally dosed with 3.5 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. After intestinal passage, inoculum-derived oocysts extracted from goose faeces established severe infection in 14 neonatal BALB/c mice (inoculum dose 2.5 x 10(5)/mouse). The inoculum-derived oocysts were detected in goose faeces up to 9 days post-inoculation (PI); the number of intact oocysts and oocyst shells shed during the first 3 days PI was significantly higher than for the remaining 6 days PI (P < 0.01). Based on acid-fast stained air-dried direct wet smears, 62% of the oocysts in goose faeces were intact (oocyst shells) constituted 38%) and conformed to morphological features of viable and infectious inoculum oocysts. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the faeces-recovered oocysts (majority 3+ to 4+). The dynamics of oocyst shedding showed that overall, the birds released a significantly higher number of intact oocysts than oocyst (P < 0.01). Retention of the viability and infectivity of C. parvum oocysts following intestinal passage through a migratory water-fowl species has serious epidemiological implications. Water-fowl can serve as mechanical vectors for the water-borne oocysts and can contaminate surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to water-shed management practices, water-shed protection programme officials should consider water-fowl as a potential factor enhancing contamination of the source water with Cryptosporidium.

Graczyk, T. K., M. R. Cranfield, et al. (1998). "Therapeutic efficacy of hyperimmune bovine colostrum treatment against clinical and subclinical Cryptosporidium serpentis infections in captive snakes." Vet Parasitol 74(2-4): 123-32.
	Therapy based on the protective passive immunity of Hyperimmune Bovine Colostrum (HBC) (raised against Cryptosporidium parvum in dairy cows immunized during gestation) was tested for heterologous efficacy in subclinical and clinical infections of 12 captive snakes with C. serpentis. Six gastric HBC treatments of 1% snake weight at 1-week intervals each, have histologically cleared C. serpentis in three subclinically infected snakes, and regressed gastric histopathological changes in one of these snakes. In all snakes, each subsequent HBC treatment significantly decreased the number of oocysts recovered in gastric lavage eluants (P < 0.03). The treatments induced oocyst-negative gastric eluants and stools in all snakes, and improved clinical signs of infection. Clinically infected snakes displayed severe histopathological changes in the gastric region; however, the numbers of developmental stages of C. serpentis were moderate. Considering the severity of pathology, much lower than expected pathogen numbers were observed, and it is believed that clinically infected snakes did not have enough time to repair tissue damage that had occurred over the years of infection. As the HBC treatment was safe and highly efficacious, it is recommended to gastrically administer the HBC therapeutically to snakes that are clinically or subclinically infected with C. serpentis. Hyperimmune bovine colostrum can also be used in snake supportive therapy or prophylaxis.

Graczyk, T. K., C. A. Farley, et al. (1998). "Detection of Cryptosporidium oocysts and Giardia cysts in the tissues of eastern oysters (Crassostrea virginica) carrying principal oyster infectious diseases." J Parasitol 84(5): 1039-42.
	The potential cross-reactivity of the combined Cryptosporidium/Giardia direct immunofluorescence antibodies (IFA) of MERIFLUOR and HYDROFLUOR-COMBO tests was examined against tissues containing known developmental stages of 12 pathogens causing the principal infectious diseases in oysters. Spores of Haplosporidium nelsoni and Haplosporidium costale produced positive acid-fast stain (AFS) reactions similar in intensity to Cryptosporidium parvum oocysts. Hexamia nelsoni trophozoites produced positive IFA reactions in both IFA tests; however, the intensity of fluorescence was considerably lower and the fluorescein-staining pattern different than those of Giardia cysts. The applicability of AFS for screening oysters for Cryptosporidium oocysts is low, and positive identification of Cryptosporidium oocysts cannot be accomplished based on the AFS. Presumptive IFA identification of the Cryptosporidium oocysts or Giardia cysts in the oyster tissue should fulfill 3 criteria, i.e., bright-green fluorescence of the same intensity as C. parvum oocysts and Giardia cysts in the positive control, correct size and shape of the fluorescein-stained objects, and oocyst or cyst shell clearly visible.

Graczyk, T. K., R. Fayer, et al. (1999). "Evaluation of the recovery of waterborne Giardia cysts by freshwater clams and cyst detection in clam tissue." Parasitol Res 85(1): 30-4.
	The Asian freshwater clam, Corbicula fluminea, inhabits environments recognized to be contaminated with waterborne Giardia cysts. Sixty-four tissue samples of Giardia-free clams were spiked with various numbers of Giardia duodenalis cysts within the range of 50-700 cysts. Regression analysis showed that paired numbers of spiked (x) versus recovered (y) cysts regressed significantly (P < 0.01) according to the equation y = 42.57 +/- 1.81x (+/- 64.3). The cyst detection threshold was 43 cysts/clam, the coefficient of determination was 77%, and the overall sensitivity of cyst detection was 42.9%. All 20 values of cyst numbers in clam tissue samples that were processed blind were located within the 95% prediction limits of the linear regression equation. The cyst retention rate of 160 clams kept in an aquarium with 38 l of water spiked with 1.00 x 10(5) G. duodenalis cysts was approximately 1.3 x 10(3) cysts/clam. No waterborne cysts were detected by the membrane filtration method 90 min after spiking the aquarium water. G. duodenalis cysts were detected in clam tissue up to 3 weeks post-exposure. Filtration of water by clams substantially depleted the aquarium water of its particulate matter. The sampling program demonstrated that the population of 160 clams examined during the study could be accurately assessed for exposure to waterborne Giardia cysts by random sampling of 86 (54%) clams. The results indicate that C. fluminea clams can be used for biological monitoring of contamination with Giardia.

Graczyk, T. K., R. Fayer, et al. (1996). "Cryptosporidium parvum is not transmissible to fish, amphibians, or reptiles." J Parasitol 82(5): 748-51.
	A recent report suggested that an isolate of Cryptosporidium parvum had established infections in fish, amphibians, and reptiles and raises concern that animals other than mammals might be a potential source of waterborne Cryptosporidium oocysts. To test this possibility, viable C. parvum oocysts, infectious for neonatal BALB/c mice, were delivered by gastric intubation to bluegill sunfish, poison-dart frogs, African clawed frogs, bearded dragon lizards, and corn snakes. Histological sections of the stomach, jejunum, ileum, and cloaca prepared from tissues collected on days 7 and 14 postinoculation (PI) were negative for Cryptosporidium developmental stages. However, inoculum-derived oocysts were detectable by fluorescein-labeled monoclonal antibody in feces of inoculated animals from day 1 to day 12 PI in fish and frogs, and up to day 14 PI in lizards. Snakes did not defecate for 14 days PI. Impression smears taken at necropsy on days 7 and 14 PI revealed C. parvum oocysts in the lumen of the cloaca of 2 fish and 1 lizard on day 7 PI only. Because tissue stages of the pathogen were not found, it appears that C. parvum was not heterologously transmitted to lower vertebrates. Under certain circumstances, however, such as after the ingestion of C. parvum-infected prey, lower vertebrates may disseminate C. parvum oocysts in the environment.

Graczyk, T. K., R. Fayer, et al. (1997). "Zoonotic transmission of Cryptosporidium parvum: Implications for water-borne cryptosporidiosis." Parasitol Today 13(9): 348-51.
	The emergence of Cryptosporidium parvum-associated cryptosporidiosis as a worldwide zoonosis has stimulated interest in the modes of pathogen transmission. Here, Thaddeus Graczyk, Ronald Fayer and Michael Cranfield discuss the complex epidemiology of C. parvum, emphasizing the crosstransmission potential of the pathogen, mechanical vectors involved in water-borne transmission of the oocysts, and factors contributing to contamination of pristine waters with Cryptosporidium. They also outline the public health importance of proper interpretation of positive detection of Cryptosporidium oocysts at water-treatment facilities and identify means by which watersheds can be protected from Cryptosporidium contamination.

Graczyk, T. K., R. Fayer, et al. (1998). "Recovery of waterborne Cryptosporidium parvum oocysts by freshwater benthic clams (Corbicula fluminea)." Appl Environ Microbiol 64(2): 427-30.
	Asian freshwater clams, Corbicula fluminea, exposed for 24 h to 38 liters of water contaminated with infectious Cryptosporidium parvum oocysts (1.00 x 10(6) oocysts/liter; approximately 1.9 x 10(5) oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used--acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MERIFLUOR test kit).

Graczyk, T. K., R. Fayer, et al. (1999). "Filth flies are transport hosts of Cryptosporidium parvum." Emerg Infect Dis 5(5): 726-7.
	
Graczyk, T. K., R. Fayer, et al. (1997). "Cryptosporidium parvum oocysts recovered from water by the membrane filter dissolution method retain their infectivity." J Parasitol 83(1): 111-4.
	Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice were processed by the cellulose-acetate membrane (CAM) filter dissolution method to determine if the procedure that utilizes acetone incubation and alcohol centrifugations alters their viability (determined by in vitro excystation) or infectivity (determined by infectivity bioassay). In addition, most oocysts with altered viability by desiccation, heat inactivation, and snap freezing that were processed by the CAM filter dissolution method were nonrefractile, unstained oocyst ghosts. The remaining organisms, oocyst shells, were lightly stained with the acid-fast stain. Infectious oocysts retained their infectivity and nonviable oocysts (oocyst shells) retained their morphology when processed by the CAM dissolution method. Infectious oocysts, oocyst shells, and oocyst ghosts produced positive reactions of similar intensity in direct immunofluorescence antibody staining, utilizing the MERIFLUOR Cryptosporidium/Giardia test kit. Cryptosporidium oocysts recovered from finished drinking water by the CAM dissolution method can be subjected to testing for their viability and infectivity.

Graczyk, T. K., R. Fayer, et al. (2000). "Mechanical transport and transmission of Cryptosporidium parvum oocysts by wild filth flies." Am J Trop Med Hyg 63(3-4): 178-83.
	Over the course of six months wild filth flies were collected from traps left for 7-10 days in a barn with or without a calf shedding Cryptosporidium parvum Genotype 2 oocysts in diarrheic feces. The oocysts of C. parvum transported on the flies' exoskeletons and eluted from their droplets left on visited surfaces were infectious for mice. The mean number of oocysts carried by a fly varied from 4 to 131, and the total oocyst number per collection varied from 56 to approximately 4.56 x 10(3). Fly abundance and intensity of mechanical transmission of infectious C. parvum oocysts were positively correlated, and both increased significantly when an infected calf was in the barn. Molecular data showed that the oocysts shed by infected calves were carried by flies for at least 3 weeks. Filth flies can acquire infectious C. parvum oocysts from unsanitary sites, deposit them on visited surfaces, and therefore may be involved in human or animal cryptosporidiosis.

Graczyk, T. K., R. Fayer, et al. (1997). "In vitro interactions between hemocytes of the eastern oyster, Crassostrea virginica Gmelin, 1791 and Cryptosporidium parvum oocysts." J Parasitol 83(5): 949-52.
	It was demonstrated by an in vitro slide phagocytosis assay that hemocytes of the Eastern oyster, Crassostrea virginica Gmelin, 1791 are capable of rapid recognition and internalization of infectious Cryptosporidium parvum (AUCP-1 strain) oocysts. The incubation of hemocyte monolayers (8.5 x 10(4) cells) that had received 6.8 x 10(5) or 3.4 x 10(5) oocysts was arrested at 5, 15, 30, 60, 90, and 120 min and the oocytes detected by acid-fast stain and immunofluorescent antibody (IFAT). An average of 20.5, 38.3, 50.2, 58.9, 69.0, and 75.0% oocysts were phagocytosed after 5, 15, 30, 60, 90, and 120 min, respectively. The intensity of fluorescence of phagocytosed oocysts significantly decreased over time (P < 0.01), and their round shape was altered. The number of cells containing oocysts and the mean number of ingested oocysts (range: 1.2-4.5 per cell) increased significantly over time (P < 0.01), whereas the numbers of nonphagocytosed oocysts that were adherent to the glass slides significantly decreased (P < 0.05). By extrapolation, the results indicate that Cr. virginica is capable of internalizing up to 6.4 x 10(6) Cryptosporidium oocysts per ml of its hemolymph.

Graczyk, T. K., R. Fayer, et al. (1999). "Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay." Parasitol Res 85(7): 518-21.
	Filter-feeding molluscan shellfish can concentrate environmentally derived waterborne pathogens of humans, which can be utilized in the sanitary assessment of water quality. In the present study, oocysts of Cryptosporidium were detected in Bent mussels (Ischadium recurvum) at two Chesapeake Bay sites from which C. parvum-contaminated oysters had previously been collected. Spiking of Cryptosporidium-free blue mussel (Mytilus edulis) tissue with C. parvum oocysts showed a 51.1% recovery rate of oocysts, giving an oocyst detection limit of 19 oocysts/0.7 ml of mussel tissue homogenate. The results indicate that Bent mussels, which are common throughout the Chesapeake Bay region, may prove to be useful as biological indicators of water contamination with Cryptosporidium oocysts.

Graczyk, T. K., R. Fayer, et al. (2000). "Susceptibility of the Chesapeake Bay to environmental contamination with Cryptosporidium parvum." Environ Res 82(2): 106-12.
	
Graczyk, T. K., R. Fayer, et al. (1998). "Giardia sp. cysts and infectious Cryptosporidium parvum oocysts in the feces of migratory Canada geese (Branta canadensis)." Appl Environ Microbiol 64(7): 2736-8.
	Fecal droppings of migratory Canada geese, Branta canadensis, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of Cryptosporidium parvum and Giardia spp. Cryptosporidium sp. oocysts were found in feces at seven of nine sites, and Giardia cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of Cryptosporidium parvum. Waterfowl can disseminate infectious C. parvum oocysts in the environment.

Graczyk, T. K., A. S. Girouard, et al. (2006). "Recovery, bioaccumulation, and inactivation of human waterborne pathogens by the Chesapeake Bay nonnative oyster, Crassostrea ariakensis." Appl Environ Microbiol 72(5): 3390-5.
	The introduction of nonnative oysters (i.e., Crassostrea ariakensis) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8-ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 x 10(5) transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters.

Graczyk, T. K., B. H. Grimes, et al. (2003). "Detection of Cryptosporidium parvum and Giardia lamblia carried by synanthropic flies by combined fluorescent in situ hybridization and a monoclonal antibody." Am J Trop Med Hyg 68(2): 228-32.
	Wild-caught synanthropic flies were tested for the presence of Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their digestive tracks by fluorescent in situ hybridization and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MAb) against Cryptosporidium and Giardia cell wall epitopes. The levels of C. parvum were positively correlated with the levels of G. lamblia, indicating a common source of contamination. The majority of oocysts and cysts were potentially viable (C. parvum = 80% and G. lamblia = 69%). More G. lamblia cysts occurred on the exoskeleton of the flies than within the digestive tracts; the opposite relationship was observed for C. parvum. No genotype other than C. parvum G2 was found to be associated with flies. Because filth flies carry viable C. parvum oocysts and G. lamblia cysts acquired naturally from unhygienic sources, they can be involved in the epidemiology of cryptosporidiosis and giardiasis. Fluorescent oligonucleotide probes used together with FITC-conjugated MAb represent a convenient and cost-effective technique for rapid and specific identification of human-infectious species of Cryptosporidium and Giardia mechanically transported by flies, and for the assessment of the viability of these pathogens.

Graczyk, T. K., Y. R. Ortega, et al. (1998). "Recovery of waterborne oocysts of Cyclospora cayetanensis by Asian freshwater clams (Corbicula fluminea)." Am J Trop Med Hyg 59(6): 928-32.
	Asian freshwater clams (Corbicula fluminea) were exposed for 24 hr in 38 liters of water contaminated with 1.0 x 10(5) Cyclospora cayetanensis oocysts (2.6 x 10(3) oocysts/L). The hemolyph and gill smears of 30 clams were examined by acid-fast stain on days 1, 3, 5, 7, 10, 13, and 18 postexposure (PE). Since no oocysts were detected in the water 24 hr after contamination by the membrane filter-dissolution method, the oocyst retention rate was 4.6 X 10(2) oocysts/clam. The prevalence of oocyst-positive clams significantly decreased (P < 0.01) from 93% to 47% during 13 days PE. None of the clams contained oocysts on day 18 PE; no oocysts were detected in the clam feces. The numbers of oocysts recovered from six clam size classes varied and significantly decreased with smaller clam size (P < 0.01). The lowest prevalence values of oocyst-positive clams, 45% and 34%, were observed in the two lowest size classes: 12.1-14.0 mm and 14.1-16.0 mm, respectively. The prevalence values in the remaining four classes ranged from 84% to 100%. The sampling program demonstrated that the population of 180 clams examined during the study up to 13 day PE could be assessed for C. cayetanensis positivity by random testing of a minimum of 75 clams (42%). When the two lowest clam size classes are eliminated, the population of 114 clams could be assessed by sampling a minimum of 32 clams (28%). The results demonstrate that Corbicula fluminea can recover waterborne oocysts of C. cayetanensis, and could be used as biological indicators of contamination of water with C. cayetanensis oocysts.

Graczyk, T. K., R. C. Thompson, et al. (1999). "Giardia duodenalis cysts of genotype A recovered from clams in the Chesapeake Bay subestuary, Rhode River." Am J Trop Med Hyg 61(4): 526-9.
	Filter-feeding molluscan shellfish can concentrate zoonotic and anthroponotic waterborne pathogens. Cysts of Giardia sp. were detected by immunofluorescent antibodies in tissues of the clams Macoma balthica and M. mitchelli from Rhode River, a Chesapeake Bay (Maryland) subestuary. Molecular tests identified the cysts as Giardia duodenalis Genotype A, the most common genotype recovered from humans. Macoma clams are burrowers in mud or sandy-mud substrata and preferentially feed on the surface sediment layer. Waterborne Giardia cysts settle rapidly to the bottom in slow-moving waters and contaminate the sediment. Macoma clams do not have economic value, but can serve as biologic indicators of sediment contamination with Giardia sp. cysts of public health importance. These clams can be used for sanitary assessment of water quality.

Granstrom, D. E., J. P. Dubey, et al. (1993). "Equine protozoal myeloencephalitis: antigen analysis of cultured Sarcocystis neurona merozoites." J Vet Diagn Invest 5(1): 88-90.
	Antigens of cultured Sarcocystis neurona merozoites were examined using immunoblot analysis. Blotted proteins were probed with S. cruzi, S. muris, and S. neurona antisera produced in rabbits, S. fayeri (pre- and post-infection) and S. neurona (pre- and post-inoculation) sera produced in horses, immune sera from 7 histologically confirmed cases of equine protozoal myeloencephalitis (EPM), and pre-suckle serum from a newborn foal. Eight proteins, 70, 24, 23.5, 22.5, 13, 11, 10.5, and 10 Kd, were detected only by S. neurona antiserum and/or immune serum from EPM-affected horses. Equine sera were titered by the indirect immunofluorescent antibody (IFA) method using air-dried, cultured S. neurona merozoites. Anti-Sarcocystis IFA titers were found in horses with or without EPM. Serum titers did not correspond to the number of specific bands recognized on immunoblots.

Gras, L., R. E. Gilbert, et al. (2004). "Duration of the IgM response in women acquiring Toxoplasma gondii during pregnancy: implications for clinical practice and cross-sectional incidence studies." Epidemiol Infect 132(3): 541-8.
	We followed up a cohort of 446 toxoplasma-infected pregnant women to determine the median and variability of the duration of positive toxoplasma-IgM (immunoglobulin M) results measured by an immunofluorescence test (IFT) and an immunosorbent agglutination assay (ISAGA). IgM antibodies were detected for longer using the ISAGA test [median 12.8 months, interquartile range (IQR) 6.9-24.9] than the IFT (median 10.4, IQR 7.1-14.4), but the variability between individuals in the duration of IgM positivity was greatest for the ISAGA test. IgM-positive results persisted beyond 2 years in a substantial minority of women (27.1% ISAGA, 9.1% IFT). Variation in the duration of the IgM response measured by ISAGA and IFT limit their usefulness for predicting the timing of infection in pregnant women. However, measurement of IgM and IgG antibodies in a cross-sectional serosurvey offers an efficient method for estimating the incidence of toxoplasma infection.

Green, S. L., D. M. Bouley, et al. (2003). "Cryptosporidiosis associated with emaciation and proliferative gastritis in a laboratory-reared South African clawed frog (Xenopus laevis)." Comp Med 53(1): 81-4.
	A 2-year-old emaciated female South African clawed frog (Xenopus laevis) was euthanized because of chronic weight loss. At necropsy, there was no evidence of bacterial, fungal or viral disease; however, the histopathologic findings indicated a proliferative gastritis and the presence of numerous cryptosporidial stages throughout the intestinal tract. Crytosporidial oocysts were present in the water taken from the aquarium housing the infected frog and were likely shed by the sick frog; however, the exact source of the oocysts could not be identified. Water samples from other frog aquaria in the facility did not contain cryptosporidial oocysts. Some Cryptosporidium species are important zoonotic pathogens and, to our knowledge, this is the first report of disease associated with Cryptosporidium infection in a laboratory Xenopus laevis.

Grigg, M. E. and J. C. Boothroyd (2001). "Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene." J Clin Microbiol 39(1): 398-400.
	Sequence analysis at the 35-fold-repetitive B1 locus identified three restriction sites capable of discriminating type I (mouse-virulent) from type II or III (mouse-avirulent) strains of Toxoplasma gondii. B1 PCR-restriction fragment length polymorphism analysis of 8 type I, 17 type II, and 8 type III strains confirms the specificity of the assay. It should now be possible to ask whether strain genotype affects the severity and type of clinical disease in humans.

Grimason, A. M., H. V. Smith, et al. (1993). "Occurrence of Giardia sp. cysts and Cryptosporidium sp. oocysts in faeces from public parks in the west of Scotland." Epidemiol Infect 110(3): 641-5.
	One hundred faecal specimens, randomly collected from various locations within seven public parks in the west of Scotland, were examined for the presence of Giardia sp. cysts and Cryptosporidium sp. oocysts. Eleven percent of samples contained Giardia sp. cysts and 1% contained Cryptosporidium sp. oocysts. Occurrence data from individual parks varied from 0 to 40% for Giardia and 0 to 2.4% for Cryptosporidium. The occurrence of parasitic organisms in public parks, especially in the vicinity of children's playing areas is a matter of concern for public health officials and regulators of leisure and recreation amenities.

Guerrant, R. L. (1997). "Cryptosporidiosis: an emerging, highly infectious threat." Emerg Infect Dis 3(1): 51-7.
	Cryptosporidium parvum, a leading cause of persistent diarrhea in developing countries, is a major threat to the U.S. water supply. Able to infect with as few as 30 microscopic oocysts, Cryptosporidium is found in untreated surface water, as well as in swimming and wade pools, day-care centers, and hospitals. The organism can cause illnesses lasting longer than 1 to 2 weeks in previously healthy persons or indefinitely in immunocompromised patients; furthermore, in young children in developing countries, cryptosporidiosis predisposes to substantially increased diarrheal illnesses. Recent increased awareness of the threat of cryptosporidiosis should improve detection in patients with diarrhea. New methods such as those using polymerase chain reaction may help with detection of Cryptosporidium in water supplies or in asymptomatic carriers. Although treatment is very limited, new approaches that may reduce secretion or enhance repair of the damaged intestinal mucosa are under study.

Gumbo, T., S. Sarbah, et al. (1999). "Intestinal parasites in patients with diarrhea and human immunodeficiency virus infection in Zimbabwe." Aids 13(7): 819-21.
	OBJECTIVES: To determine the prevalence of intestinal parasites and risk factors for infection associated with diarrhea in HIV-infected patients in Harare, Zimbabwe. DESIGN: Prospective observational study. METHODS: Single stool samples were collected from 88 HIV-infected individuals presenting with diarrhea of greater than 1 week duration. Stools were examined for intestinal parasites using modified acid fast stain, fluorescence- labeled monoclonal antibody for Cryptosporidium parvum, as well as a modified trichrome stain and a PCR-based protocol for Enterocytozoon bieneusi. RESULTS: C. parvum was detected in 9% (seven out of 82) of samples evaluated, but no Cyclospora was detected. E. bieneusi was detected in 18% (10 out of 55) of stool by trichrome staining and in 51% (28 out of 55) of stool examined by PCR. Risk factors for E. bieneusi infection were: living in rural areas, consumption of nonpiped water, contact with cow dung and household contact with an individual with diarrhea. CONCLUSION: E. bieneusi infection was common in HIV-infected patients with diarrhea in Zimbabwe and may be acquired through person-to-person and fecal-oral transmission.

Guselle, N. J., A. J. Appelbee, et al. (2003). "Biology of Cryptosporidium parvum in pigs: from weaning to market." Veterinary Parasitology 113(1): 7-18.
	Cryptosporidium parvum is commonly identified as infecting domestic livestock and humans. Prevalence of C. parvum in pigs has been reported, however, the duration and infection pattern of naturally acquired Cryptosporidium infections in pigs has not been reported. This study was undertaken to investigate the age of oocyst shedding and duration of natural Cryptosporidium parvum infections in pigs from weaning to market weight. Fecal samples were collected from weaned Yorkshire-Landrace piglets (n = 33) twice per week until Cryptosporidium oocysts were detected. Upon oocyst detection, fecal samples were collected three times per week and pigs were monitored throughout the study for diarrhea and examined after concentration and immunofluroescent staining. Cryptosporidium isolates were genotyped by polymerase chain reaction to amplify the HSP70 gene which was subsequently sequence analyzed. All 33 pigs shed oocysts some time during the study. The mean age of initial oocyst detection was 45.2 days post-weaning with the mean duration of infection 28.7 days. Mean number of Cryptosporidium oocysts was low and declined to zero prior to study completion. Episodes of diarrhea were not associated with oocyst excretion. Genetic sequences were obtained for 10 of the pigs. All of the 10 isolates aligned as the Cryptosporidium parvum 'pig' genotype. This study demonstrates that the age and duration of oocyst shedding in pigs infected with C. parvum porcine genotype is different from other livestock species. (C) 2003 Elsevier Science B.V. All rights reserved. [References: 53] 53

Guy, R. A., P. Payment, et al. (2003). "Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage." Appl Environ Microbiol 69(9): 5178-85.
	The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

Guyot, K., A. Follet-Dumoulin, et al. (2001). "Molecular characterization of Cryptosporidium isolates obtained from humans in France." J Clin Microbiol 39(10): 3472-80.
	Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom Cryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of Cryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.

Guyot, K., A. Follet-Dumoulin, et al. (2002). "PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin." Appl Environ Microbiol 68(4): 2071-6.
	Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.

Hallier-Soulier, S. and E. Guillot (2000). "Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction." J Appl Microbiol 89(1): 5-10.
	Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inefficient. We recently described an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS-PCR assay to detect all cryptosporidial oocysts was developed, and both IMS-PCR assays were optimized on river water samples. A comparative study of the two IMS-PCR assays and the classical detection method based on an immunofluorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS-PCR took the form of IFA-negative/IMS-PCR-positive results, and was caused mainly by the greater sensitivity of IMS-PCR as compared with IFA. Of the 50 water samples, only five tested positive for C. parvum using IMS-PCR, and could constitute a threat to human health. These results show that both IMS-PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts.

Hamedi, Y., O. Safa, et al. (2005). "Cryptosporidium infection in diarrheic children in southeastern Iran." Pediatr Infect Dis J 24(1): 86-8.
	In a cross-sectional study conducted in children referred to Bandar Abbas Pediatric Hospital in southeastern Iran, the prevalence of Cryptosporidium infection was 7%. Diarrhea lasted significantly longer in children infected with Cryptosporidium. There were also a significant association between Cryptosporidium infection and underweight children and no association with parent's occupation, breast-feeding, source of drinking water, vicinity or presence of sewage or animal exposure.

Harp, J. A. (2003). "Cryptosporidium and host resistance: historical perspective and some novel approaches." Anim Health Res Rev 4(1): 53-62.
	Cryptosporidium parvum is recognized as a major cause of diarrheal disease in neonatal bovine calves. In addition, this protozoan parasite has emerged as an important cause of disease in both immunocompromised and immunocompetent humans. Despite years of research, no consistently effective means of prevention or treatment are readily available for cryptosporidiosis in any species. Infection through ingestion of contaminated water has been widely documented; C. parvum was reported to be responsible for the largest waterborne outbreak of infectious disease in US history. In addition to its role as a primary disease agent, C. parvum has potential to initiate or exacerbate other gastrointestinal disorders, such as inflammatory bowel disease. Thus, control of C. parvum infection in both animals and humans remains an important objective. Research in our laboratory has focused on understanding mechanisms of resistance to C. parvum. We have demonstrated that acquisition of intestinal flora increases resistance to C. parvum. Substances present in the intestinal mucosa of adult animals can transfer resistance when fed to susceptible infants. Both expression of intestinal enzymes and rate of proliferation of epithelial cells may be altered following C. parvum infection. These and other changes may have profound effects on host resistance to C. parvum.

Harp, J. A. (2003). "Parasitic infections of the gastrointestinal tract." Curr Opin Gastroenterol 19(1): 31-6.
	Intestinal parasites continue to be a significant health problem in both developed and developing countries. In developed countries, protozoans are more commonly the cause of gastrointestinal infections than are helminths. Some protozoan parasites have stages in which, in addition to being resistant to chemicals used for water treatment, they are small enough to pass through commonly used filtration processes. The relatively large size of helminth eggs increases the likelihood of their removal during water filtration. The direct impact of protozoan parasites on both human and animal health is considerable, and there is some evidence that infection may contribute to the development of various forms of intestinal dysregulation as well as disseminated infection, especially in AIDS patients. Protozoans of special interest, due to either their frequency of isolation or their role as emerging pathogens, include Giardia duodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, and the microsporidians, Enterocytozoon bieneusi and Encephalitozoon intestinalis.

Harp, J. A., R. Fayer, et al. (1996). "Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk." Appl Environ Microbiol 62(8): 2866-8.
	Cryptosporidium parvum is a major cause of diarrheal disease in humans and has been identified in 78 other species of mammals. The oocyst stage, excreted in feces of infected humans and animals, has been responsible for recent waterborne outbreaks of human cryptosporidiosis. High temperature and long exposure time have been shown to render oocysts (suspended in water) noninfectious, but for practical purposes, it is important to know if high-temperature--short-time conditions (71.7 degrees C for 15 s) used in commercial pasteurization are sufficient to destroy infectivity of oocysts. In this study, oocysts were suspended in either water or whole milk and heated to 71.7 degrees C for 15, 10, or 5 s in a laboratory-scale pasteurizer. Pasteurized and nonpasteurized (control) oocysts were then tested for the ability to infect infant mice. No mice (0 of 177) given 10(5) oocysts pasteurized for 15, 10, or 5 s in either water or milk were found to be infected with C. parvum on the basis of histologic examination of the terminal ileum. In contrast, all (80 of 80) control mice given nonpasteurized oocysts were heavily infected. These data indicate that high-temperature--short-time pasteurization is sufficient to destroy the infectivity of C. parvum oocysts in water and milk.

Harp, J. A., P. Jardon, et al. (1996). "Field testing of prophylactic measures against Cryptosporidium parvum infection in calves in a California dairy herd." Am J Vet Res 57(11): 1586-8.
	OBJECTIVE: To test the ability of oral vaccination or probiotic treatment with lactic acid-producing bacteria to protect calves from Cryptosporidium parvum infection under field conditions. ANIMALS: 134 Holstein calves born on a dairy farm where cryptosporidiosis was endemic. PROCEDURE: Calves were randomly assigned to 1 of 3 treatment groups at birth. Calves in the vaccine group received an oral dose of C parvum vaccine within several hours of birth. Calves in the bacteria group received an oral dose of lactic acid-producing bacteria daily for the first 10 days after birth. Control calves were not treated. All calves were monitored for diarrhea and fecal shedding of C parvum oocysts for 3 weeks. RESULTS: There were no significant differences in the incidence of diarrhea and oocyst shedding among the 3 groups. CONCLUSIONS: Neither vaccination nor probiotic treatment was effective in preventing C parvum infection in calves under field conditions. High numbers of C parvum in the environment may have overwhelmed any potential benefits of these regimens. Further work is necessary to develop effective prophylaxis against C parvum under field conditions.

Harp, J. A., D. B. Woodmansee, et al. (1990). "Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure." Infect Immun 58(7): 2237-40.
	Cryptosporidium parvum is a coccidian parasite that causes diarrheal disease in many vertebrate species, including young (less than or equal to 1 month old) calves. Older calves and adult cattle are resistant to infection. In this study, newborn calves were raised in isolation from C. parvum for 1 week to 3 months before experimental challenge with the parasite. Calves orally challenged with C. parvum at 1 week of age shed oocysts in their feces and had diarrhea after challenge exposure. When these calves were rechallenged at 1 and 3 months of age, they neither shed oocysts nor had diarrhea. There was no significant increase in the mean anticryptosporidium enzyme-linked immunosorbent assay serum antibody titer in these calves following any of the challenge exposures. Calves orally inoculated with C. parvum for the first time at 1 month of age shed oocysts, had diarrhea after challenge exposure, and were resistant to rechallenge at 3 months of age. These calves had a twofold increase in serum antibody titer after the first challenge and no increase after the second challenge. Calves orally inoculated with C. parvum for the first time at 3 months of age shed oocysts, and two of seven animals had diarrhea. These calves had a 10-fold increase in serum antibody to C. parvum after exposure. This study demonstrates that calves raised in isolation from C. parvum remain susceptible to challenge until at least 3 months of age. Furthermore, within this time period, initial exposure and recovery renders calves resistant to further challenge with the parasite. The data also suggest that exposure of young calves to C. parvum may inhibit the development of a serum antibody response to the parasite.

Harris, J. R., M. Adrian, et al. (2004). "Amylopectin: a major component of the residual body in Cryptosporidium parvum oocysts." Parasitology 128(Pt 3): 269-82.
	Amylopectin is used for carbohydrate storage in different life-stages of a number of apicomplexan parasites. We have performed an ultrastructural analysis of amylopectin granules from the oocyst residual body and sporozoites of Cryptosporidium parvum. Amylopectin granules were studied in situ and after isolation from 'French' press disrupted parasites, by conventional transmission electron microscopy (TEM) of sectioned oocysts and various negative staining and cryoelectron microscopy techniques. Within the membrane-enclosed oocyst residuum large amylopectin granules (0.1-0.3 microm) can be found besides a characteristic large lipid body and a crystalline protein inclusion. Smaller granules were detected in sectioned sporozoites. Negative staining of isolated amylopectin granules revealed some ultrastructural features not readily visible in sectioned material. The large amylopectin granules had a smooth surface with a 'ball of string'-like inner structure. Granules isolated from sporozoites were more irregularly shaped and showed a rod-like particulate composition. With the exception of alpha-amylase, which led to some degree of damage of the surface of the particles, treatment of amylopectin granules with other glycohydrolases had little effect on the overall structure. However, granules adhered to one another. Only when the granules were boiled did the 'ball of string' structure gradually dissolve.

Harris, J. R. and F. Petry (1999). "Cryptosporidium parvum: structural components of the oocyst wall." J Parasitol 85(5): 839-49.
	Cryptosporidium parvum, an enteropathogenic parasite, infects a wide range of mammals including man and constitutes a substantial veterinary and medical threat due to its ubiquitous distribution and the stability of the oocyst stage. The oocyst wall of C. parvum is known to be extremely resistant to chemical and mechanical disruption. Isolated oocyst walls are shown by both thin sectioning and negative staining transmission electron microscopy to possess a filamentous array on the inner surface. This filamentous array can be greatly depleted by digestion with proteinase K and trypsin, but pepsin has less effect. Ultrasonication of the untreated oocyst walls produced almost no fragmentation, but extension of the suture resulted in inward spiraling of the wall to generate ellipsoid and cigar-shaped multilayer bodies, with the filamentous array still present. When ultrasonicated, proteinase K-digested oocyst walls progressively fragmented into small sheets. These wall fragments, depleted of filaments, are shown by negative staining to possess a pronounced linearity, indicative of an integral highly complex lattice structure.

Hashimoto, A., T. Hirata, et al. (2001). Occurrence of Cryptosporidium oocysts and Giardia cysts in a conventional water purification plant. 10. International Symposium on Health-related Water Microbiology, Paris (France), 3-6 Jul 2000.
	A one-year monitoring of Cryptosporidium oocysts and Giardia cysts was conducted at a water purification plant. A total of thirteen 50 L samples of river source water and twenty-six 2,000 L samples of filtered water (treated by coagulation-flocculation, sedimentation and rapid filtration) were concentrated using a hollow fibre ultrafiltration membrane module at a purification plant. Cryptosporidium occysts were detected in all raw water samples with a geometric mean concentration of 400 oocysts/m super(3) (range 160-1,500 oocysts m super(3)). Giardia cysts were detected in 12/13 raw water (92%) with a geometric mean concentration of 170 cysts/m super(3) (range 40-580 oocysts/m super(3)). Probability distributions of both Cryptosporidium oocyst and Giardia cyst concentration in raw water were nearly log-normal. In filtered water samples, Cryptosporidium oocysts were detected in 9/26 samples (35%) with a geometric mean concentration of 1.2 oocysts/m super(3) (range 0.5-8 oocysts/m super(3)) and Giardia cysts in three samples (12%) with 0.8 cysts/m super(3) (range 0.5-2 ooctsts/m super(3)). The estimated removal of Cryptosporidium oocysts and Giardia cysts was, respectively, 2.54 log 10 and 2.53 log sub(10) on the basis of geometric means, 3.20 and 3.57 log sub(10) on the basis of 50% observation level and 2.70 and 2.90 log sub(10) on the basis of 90% observation level.

Hayes, E. B., T. D. Matte, et al. (1989). "Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply." N Engl J Med 320(21): 1372-6.
	Between January 12 and February 7, 1987, an outbreak of gastroenteritis affected an estimated 13,000 people in a county of 64,900 residents in western Georgia. Cryptosporidium oocysts were identified in the stools of 58 of 147 patients with gastroenteritis (39 percent) tested during the outbreak. Studies for bacterial, viral, and other parasitic pathogens failed to implicate any other agent. In a random telephone survey, 299 of 489 household members exposed to the public water supply (61 percent) reported gastrointestinal illness, as compared with 64 of 322 (20 percent) who were not exposed (relative risk, 3.1; 95 percent confidence interval, 2.4 to 3.9). The prevalence of IgG to cryptosporidium was significantly higher among exposed respondents to the survey who had become ill than among nonresident controls. Cryptosporidium oocysts were identified in samples of treated public water with use of a monoclonal-antibody test. Although the sand-filtered and chlorinated water system met all regulatory-agency quality standards, sub-optimal flocculation and filtration probably allowed the parasite to pass into the drinking-water supply. Low-level cryptosporidium infection in cattle in the watershed and a sewage overflow were considered as possible contributors to the contamination of the surface-water supply. We conclude that current standards for the treatment of public water supplies may not prevent the contamination of drinking water by cryptosporidium, with consequent outbreaks of cryptosporidiosis.

Hayes, S. L., E. W. Rice, et al. (2003). "Low pressure ultraviolet studies for inactivation of Giardia muris cysts." J Appl Microbiol 94(1): 54-9.
	AIMS: The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low-pressure u.v. irradiation. METHODS AND RESULTS: Cysts of Giardia muris were inactivated using a low-pressure u.v. light source. Giardia muris was employed as a surrogate for the human pathogen Giardia lamblia. Cyst viability was determined by both in vitro excystation and animal infectivity. Cyst doses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ cm(-2) did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, u.v. fluences of 1.4, 1.9 and 2.3 mJ cm(-2) yielded log10 reductions ranging from 0.3 to >or= 4.4. CONCLUSIONS: Results indicate that in vitro excystation is not a reliable indicator of G. muris cyst viability after u.v. disinfection. Very low doses of u.v. light rendered G. muris cysts non-infective in the mouse model employed. SIGNIFICANCE AND IMPACT OF THE STUDY: Data presented represent the only complete u.v. inactivation curve for G. muris. This research provides evidence that u.v. can be an effective barrier against Giardia spp. in the treatment of drinking water supplies.

Heitman, T. L., L. M. Frederick, et al. (2002). "Prevalence of Giardia and Cryptosporidium and characterization of Cryptosporidium spp. isolated from wildlife, human, and agricultural sources in the North Saskatchewan River Basin in Alberta, Canada." Can J Microbiol 48(6): 530-41.
	The environmental distribution of Giardia spp. and Cryptosporidium spp. is dependent upon human, agricultural, and wildlife sources. The significance of each source with regard to the presence of parasites in the environment is unknown. This 2-year study examined parasite prevalence in human sewage influent, wildlife, and agricultural sources associated with the North Saskatchewan River Basin in Alberta, Canada. Fecal samples were collected from cow-calf, dairy, and hog operations in the watershed area. Sewage-treatment facilities were sampled bimonthly during the 2-year study, and wildlife scat was collected at locations along tributaries of the North Saskatchewan River. All samples were analyzed for the presence of Giardia and Cryptosporidium, using sucrose-gradient separation followed by immunofluorescent microscopy. Giardia and Cryptosporidium were detected in all three sources. The lowest prevalence of both Giardia (3.28%) and Cryptosporidium (0.94%) was found in wildlife, with 6 of 19 species testing positive. Sewage influent had the highest prevalence of Giardia (48.80%) and Cryptosporidium parvum-like oocysts (5.42%); however, the concentration of both parasites was minimal compared with the concentration detected in cattle feces. Cow-calf sources contained the highest concentration of Giardia (mean 5800/g feces, P < 0.01), and dairy sources contained the highest concentration of C. parvum-like oocysts (mean 295/g feces, P < 0.01). Although prevalence and concentration are higher in cattle feces than in sewage, the Giardia and Cryptosporidium in animal manure do not have direct access to water draining into the North Saskatchewan River. PCR-based characterization of rDNA from isolates of Cryptosporidium collected from Alberta human, pig, calf, mature steer, dog, cat, and beaver hosts revealed distinct genetic differences that may reflect host specificity.

Heitman, T. L., L. M. Frederick, et al. (2002). "Prevalence of Giardia and Cryptosporidium and characterization of Cryptosporidium spp. isolated from wildlife, human, and agricultural sources in the North Saskatchewan River Basin in Alberta, Canada." Canadian Journal of Microbiology 48(6): 530-541.
	The environmental distribution of Giardia spp. and Cryptosporidium spp. is dependent upon human, agricultural, and wildlife sources. The significance of each source with regard to the presence of parasites in the environment is unknown. This 2-year study examined parasite prevalence in human sewage influent, wildlife, and agricultural sources associated with the North Saskatchewan River Basin in Alberta, Canada. Fecal samples were collected from cow-calf, dairy, and hog operations in the watershed area. Sewage-treatment facilities were sampled bimonthly during the 2-year study, and wildlife scat was collected at locations along tributaries of the North Saskatchewan River. All samples were analyzed for the presence of Giardia and Crytosporidium, using sucrose-gradient separation followed by immunofluorescent microscopy. Giardia and Cryptosporidium were detected in all three sources. The lowest prevalence of both Giardia (3.28%) and Cryptosporidium (0.94%) was found in wildlife, with 6 of 19 species testing positive. Sewage influent had the highest prevalence of Giardia (48.80%) and Cryptosporidium parvum-like oocysts (5.42%); however, the concentration of both parasites was minimal compared with the concentration detected in cattle feces. Cow-calf sources contained the highest concentration of Giardia (mean 5800/g feces, P < 0.01), and dairy sources contained the highest concentration of C. parvum-like oocysts (mean 295/g feces, P < 0.01). Although prevalence and concentration are higher in cattle feces than in sewage, the Giardia and Cryptosporidium in animal manure do not have direct access to water draining into the North Saskatchewan River. PCR-based characterization of rDNA from isolates of Cryptosporidium collected from Alberta human, pig, calf, mature steer, dog, cat, and beaver hosts revealed distinct genetic differences that may reflect host specificity. [References: 39] 39

Henriksen, S. A. and J. F. Pohlenz (1981). "Staining of cryptosporidia by a modified Ziehl-Neelsen technique." Acta Vet Scand 22(3-4): 594-6.
	
Heriveau, C., I. Dimier-Poisson, et al. (2000). "Inhibition of Eimeria tenella replication after recombinant IFN-gamma activation in chicken macrophages, fibroblasts and epithelial cells." Vet Parasitol 92(1): 37-49.
	We have previously shown that activation of primary cultures of chicken bone-marrow macrophages and embryo fibroblasts with supernatants of concanavaline A-stimulated or reticuloendotheliosis virus (REV)-transformed chicken spleen cells as source of IFN-gamma significantly decreases Eimeria tenella growth in vitro. In the present study, we used various chicken cell lines, HD11 macrophages and DU24 fibroblasts, both virally transformed, CHCC-OU2 fibroblasts and LMH hepatic epithelial cells, both chemically transformed, to replicate E. tenella in vitro. We confirmed the previous results by showing that HD11 macrophages pre-treated for 24h with recombinant chicken IFN-gamma (either produced in E. coli or by transfected COS cells), at doses ranging from 1000 to 10U/ml, drastically inhibited E. tenella replication as measured by [3H] uracil uptake after a further 70h of culture, as when treated with REV supernatant. Likewise the fibroblast and epithelial cell lines exhibited significant inhibitory activity on E. tenella replication after pre-treatment with recombinant chicken IFN-gamma, but were less sensitive (1000-100U/ml) than when treated with REV supernatant. Recombinant chicken IFN-alpha pre-treatment of all cell lines had no inhibitory effect on parasite development.

Herwaldt, B. L. (2000). "Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s." Clin Infect Dis 31(4): 1040-57.
	Cyclospora cayetanensis, a coccidian parasite that causes protracted, relapsing gastroenteritis, has a short recorded history. In retrospect, the first 3 documented human cases of Cyclospora infection were diagnosed in 1977 and 1978. However, not much was published about the organism until the 1990s. One of the surprises has been the fact that a parasite that likely requires days to weeks outside the host to become infectious has repeatedly caused foodborne outbreaks, including large multistate outbreaks in the United States and Canada. In this review, I discuss what has been learned about this enigmatic parasite since its discovery and what some of the remaining questions are. My focus is the foodborne and waterborne outbreaks of cyclosporiasis that were documented from 1990 through 1999. The occurrence of the outbreaks highlights the need for health care personnel to consider that seemingly isolated cases of infection could be part of widespread outbreaks and should be reported to public health officials. Health care personnel should also be aware that stool specimens examined for ova and parasites usually are not examined for Cyclospora unless such testing is specifically requested and that Cyclospora infection is treatable with trimethoprim-sulfamethoxazole.

Higgins, J. A., R. Fayer, et al. (2001). "Real-time PCR for the detection of Cryptosporidium parvum." J Microbiol Methods 47(3): 323-37.
	Real time, TaqMan PCR assays were developed for the Cp11 and 18S rRNA genes of the protozoan parasite Cryptosporidium parvum. The TaqMan probes were specific for the genus Cryptosporidium, but could not hybridize exclusively with human-infectious C. parvum species and genotypes. In conjunction with development of the TaqMan assays, two commercial kits, the Mo Bio UltraClean Soil DNA kit, and the Qiagen QIAamp DNA Stool kit, were evaluated for DNA extraction from calf diarrhea and manure, and potassium dichromate and formalin preserved human feces. Real-time quantitation was achieved with the diarrhea samples, but nested PCR was necessary to detect C. parvum DNA in manure and human feces. Ileal tissues were obtained from calves at 3, 7, and 14 days post-infection, and DNA extracted and assayed. Nested PCR detected C. parvum DNA in the 7-day post-infection sample, but neither of the other time point samples were positive. These results indicate that real-time quantitation of C. parvum DNA, extracted using the commercial kits, is feasible on diarrheic feces, with large numbers of oocysts and small concentrations of PCR inhibitor(s). For samples with few oocysts and high concentrations of PCR inhibitor(s), such as manure, nested PCR is necessary for detection.

Higgins, J. A., M. C. Jenkins, et al. (2001). "Rapid extraction of DNA From Escherichia coli and Cryptosporidium parvum for use in PCR." Appl Environ Microbiol 67(11): 5321-4.
	The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum. All methods provided satisfactory DNA from E. coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C. parvum.

Higgins, J. A., J. M. Trout, et al. (2003). "Recovery and detection of Cryptosporidium parvum oocysts from water samples using continuous flow centrifugation." Water Res 37(15): 3551-60.
	Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) and immunofluorescence microscopy (IFA) and nested PCR to recover and detect oocysts of Cryptosporidium parvum and cysts of Giardia intestinalis from 10L volumes of source water samples. Using a spiking dose of 100 oocysts, nine of 10 runs were positive by IFA, with a mean recovery of 4.4+/-2.27 oocysts; when another 10 runs were analyzed using nested PCR to the TRAP C-1 and Cp41 genes, nine of 10 were positive with both PCR assays. When the spiking dose was reduced to 10 oocysts in 10L, 10 of 12 runs were positive by IFA, with a mean oocyst recovery of 3.25+/-3.25 oocysts. When 10 cysts of Giardia intestinalis were co-spiked with oocysts into 10L of source water, five of seven runs were positive, with a mean cyst recovery of x=0.85+/-0.7. When 10 oocysts (enumerated using a fluorescence activated cell sorter) were spiked into 10L volumes of tap water, one of 10 runs was positive, with one oocyst detected. For the majority of the source water samples, turbidities of the source water samples ranged from 1.1 to 22 NTU, but exceeded 100 NTU for some samples collected when sediment was disturbed. The turbidities of pellets recovered using CFC and resuspended in 10 mL of water were very high (exceeding 500 NTU for the source water-derived pellets and 100 NTU for the tap water-derived pellets). While not as efficient as existing capsule-filtration based methods (i.e., US EPA methods 1622/1623), CFC and IMS may provide a more rapid and economical alternative for isolation of C. parvum oocysts from highly turbid water samples containing small quantities of oocysts.

Hijjawi, N. S., B. P. Meloni, et al. (2001). "Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture." Int J Parasitol 31(10): 1048-55.
	This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.

Hijjawi, N. S., B. P. Meloni, et al. (2004). "Complete development of Cryptosporidium parvum in host cell-free culture." Int J Parasitol 34(7): 769-77.
	The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6-7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.

Hijjawi, N. S., B. P. Meloni, et al. (2002). "Successful in vitro cultivation of Cryptosporidium andersoni: evidence for the existence of novel extracellular stages in the life cycle and implications for the classification of Cryptosporidium." International Journal for Parasitology 32(14): 1719-1726.
	The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72 h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72 h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. [References: 30] 30

Hirata, T., A. Shimura, et al. (2001). The effect of temperature on the efficacy of ozonation for inactivating Cryptosporidium parvum oocysts. 10. International Symposium on Health-related Water Microbiology, Paris (France), 3-6 Jul 2000.
	Examination of the effects of water temperature on the inactivation of Cryptosporidium parvum oocysts with ozone, ozonation experiments were conducted in a semi-batch mode with a wide temperature range of 3-30 degree C. Inactivation was assessed in terms of mice infectivity and in vitro excystation. The temperature dependency of the CT products by a reduction in infectivity of 2 log sub(10) could be described successfully by the Arrhenius equation, 1/CT=1.086x10 super(18)e super(-12520/K) where CT is the integrated ozone concentration over the contact time (mg/min/L) and K is the Kelvin temperature of water. As for the reduction in viability assessed by the excystation assay, protocol B, the obtained regression equation, 1/CT=1.802x10 super(18)e super(-12640/K), was almost identical to that observed for the infectivity. Thus, the CT products required for a 2 log sub(10) reduction in both infectivity and viability increased by an average factor of 4.2 for every 10 degree C decrease in water temperature. Additionally, our findings suggested that the viability, as determined by protocol B, could substitute for animal infectivity in evaluating the effects of environmental factors on the efficacy of ozonation.

Hoar, B. R., E. R. Atwill, et al. (1999). "Comparison of fecal samples collected per rectum and off the ground for estimation of environmental contamination attributable to beef cattle." Am J Vet Res 60(11): 1352-6.
	OBJECTIVES: To determine whether sampling feces off the ground replicates prevalence estimates for specific pathogens obtained from fecal samples collected per rectum of adult cows, and to determine characteristics of feces on the ground (fecal pats) that are associated with subsequent identification of Campylobacter spp, Cryptosporidium parvum, and Giardia duodenalis. ANIMALS: A random sample of adult beef cattle from 25 herds located throughout California. PROCEDURE: 1,115 rectal and ground fecal samples were obtained. Samples were submitted for culture of Campylobacter spp and examined, using a direct fluorescent antibody assay, to detect C parvum oocysts and G duodenalis cysts. Characteristics of fecal pats, such as volume and consistency, were recorded. RESULTS: Prevalence of Campylobacter spp was 5.0% (20/401) for rectal fecal samples, which was significantly greater than prevalence determined for ground fecal samples (2/402; 0.5%). Most isolates were C jejuni subsp jejuni. Prevalence of C parvum was higher in rectal fecal samples (6/557; 1.1%) than in ground fecal samples (1/558; 0.2%), but this difference was not significant. Prevalence of G duodenalis did not differ for rectal (36/557; 6.5%) versus ground (26/558; 4.7%) fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Evaluation of ground fecal samples may not accurately indicate the prevalence of Campylobacter spp or C parvum in cattle but may reflect prevalence of G duodenalis. Differences in prevalence estimates between the 2 methods suggest inactivation of pathogens in feces after cattle have defecated. Prevalence estimates generated by evaluation of ground fecal samples, however, may more accurately estimate environmental pathogen burden.

Hobbs, R. P., L. E. Twigg, et al. (1999). "Factors influencing the fecal egg and oocyst counts of parasites of wild European rabbits Oryctolagus cuniculus (L.) in Southern Western Australia." J Parasitol 85(5): 796-802.
	Abundance of intestinal parasites was monitored by fecal egg and oocyst counts for samples of wild rabbits Oryctolagus cuniculus with different levels of imposed female sterility from 12 populations in southwestern Australia. Differences in egg counts of Trichostrongylus retortaeformis between seasons and age groups were dependent on the sex of the host. Pregnancy may have been responsible for these differences because egg counts were consistently higher in intact females than in females surgically sterilized by tubal ligation. Egg counts for Passalurus ambiguus were influenced by season and host age but there were no differences between sexes or between intact and sterilized female rabbits. No differences were detected in the oocyst counts of the 8 species of Eimeria between male and female rabbits or between intact and sterilized females. Seasonal differences were detected in oocyst counts of Eimeria flavescens and Eimeria stiedai. The overwhelming determinant of coccidian oocyst counts was host age, with 6 species being much more abundant in rabbits up to 4 mo of age. There was a suggestion that egg counts of T. retortaeformis and oocyst counts of several species of Eimeria were reduced in populations where rabbit numbers had been depressed for at least 2 yr, but there was no evidence that short-term variations in rabbit numbers had a measurable effect on parasite abundance.

Hoge, C. W., D. R. Shlim, et al. (1995). "Placebo-controlled trial of co-trimoxazole for Cyclospora infections among travellers and foreign residents in Nepal." Lancet 345(8951): 691-3.
	Cyclospora is a coccidian (previously referred to as cyanobacterium-like bodies) that has been implicated in cases of prolonged diarrhoea. The average duration of symptoms is more than three weeks, and no specific treatment has been shown to shorten the illness. A case report suggested that co-trimoxazole may be effective. Expatriate persons with gastrointestinal complaints and cyclospora detected on examination of faeces were recruited from two clinics in Kathmandu, Nepal, between May and August, 1994. Participants were assigned in a randomised, double-blinded manner to receive either cotrimoxazole (160 mg trimethoprim, 800 mg sulphamethoxazole) or placebo tablets twice daily for 7 days. Of 40 patients included in the study, 21 received cotrimoxazole and 19 placebo. There were no significant differences between these two groups in age, sex, time in Nepal, duration or severity of illness, or presence of other enteric pathogens. After 3 days, 71% of patients receiving co-trimoxazole still had cyclospora detected, compared with 100% of patients receiving placebo (p = 0.016). After 7 days, cyclospora was detected in 1 (6%) of 16 patients treated with co-trimoxazole who submitted stool specimens compared with 15 (88%) of 17 patients receiving placebo (p < 0.0001). Eradication of the organism was correlated with clinical improvement. There was no evidence of relapse of infection among treated patients followed for an additional 7 days. Treatment with co-trimoxazole for 7 days was effective in curing cyclospora infection among an expatriate population in Nepal.

Hoge, C. W., D. R. Shlim, et al. (1993). "Epidemiology of diarrhoeal illness associated with coccidian-like organism among travellers and foreign residents in Nepal." Lancet 341(8854): 1175-9.
	A newly described organism called CLB (coccidian-like or cyanobacterium-like body) has been identified in cases of prolonged diarrhoea. To confirm an association of CLB with disease and identify risk factors for transmission, we conducted a case-control study of travellers and foreign residents at two outpatient clinics in Kathmandu, Nepal. Patients without diarrhoea were matched to CLB cases by clinic and date of visit. For comparison, patients with other causes of diarrhoea were also studied. Stools were examined for enteric pathogens with standard microbiological and molecular genetic techniques. CLB was identified in 108 (11%) of 964 individuals with gastrointestinal symptoms compared with only 1 (1%) of 96 symptom-free controls (p = 0.003). 7% of residents in the US Embassy community acquired the infection. The diarrhoeal illness associated with CLB lasted a median of 7 weeks (interquartile range 4-9) compared with 9 days (4-19) for individuals with other causes of diarrhoea (p < 0.0001). The prevalence of other enteric pathogens was no higher among CLB cases than among symptom-free controls. Patients with CLB infection were more likely than controls to report consumption of untreated water (odds ratio 3.98; 95% CI 1.29-13.14); organisms of the same appearance were identified in an epidemiologically implicated water sample. The significant association of CLB with prolonged diarrhoea, and the low rate of other enteropathogens in CLB cases, strongly supports the hypothesis that CLB is a new pathogen. Epidemiological and environmental data suggest that the organism is waterborne.

Homan, W., T. van Gorkom, et al. (1999). "Characterization of Cryptosporidium parvum in human and animal feces by single-tube nested polymerase chain reaction and restriction analysis." Parasitol Res 85(8-9): 707-12.
	A DNA isolation and purification method is described that produced DNA free of inhibitory substances in 148 of the 159 analyzed fecal samples. The polymerase chain reaction (PCR) product from a sensitive single-tube nested PCR that amplifies a part of an oocyst protein was used to characterize Cryptosporidium parvum genotypes by a simple restriction analysis. Genotype 1 was solely detected in human-derived oocysts, genotype 2 was present in both animal and human-derived oocysts. The ratio between both genotypes in humans in The Netherlands varied markedly between samples obtained during a period of augmented cases of cryptosporidiosis in the western part of the country and randomly selected samples from gastroenteritis patients. Sequence analysis of a 581-bp fragment from the nested PCR product revealed 12 nucleotide substitutions between the two genotypes. Sequences from isolates in each genotype group were identical.

Homan, W. L., M. Gilsing, et al. (1998). "Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting." Parasitol Res 84(9): 707-14.
	The development of a polymerase chain reaction (PCR) based fingerprinting method for the characterization of Giardia duodenalis isolates is described. The method uses three different PCRs; one is specific for the A ("Polish") major group of G. duodenalis isolates, another is specific for the B ("Belgian") group of isolates; and one amplifies a fragment of the glutamate dehydrogenase gene present in all G. duodenalis isolates. The PCRs perform highly sensitively on DNA from cultured trophozoites. Isolates were further characterized by restriction-fragment-length polymorphism (RFLP) analysis of the PCR products. In this way, representative isolates from the A and B groups could be grouped together into a number of subgroups. The stability of the genotypes with time and the reproducibility of the two methods were tested on cloned and subcloned lines from a number of isolates and proved to be highly satisfactory. The PCR/RFLP method was evaluated on cysts derived from a number of human patients. It is concluded that the PCR fingerprinting method described in this paper provides a reliable characterization method for Giardia isolates and has the potential to be used as a direct method of typing G. duodenalis cysts from feces.

Homan, W. L., L. Limper, et al. (1997). "Comparison of the internal transcribed spacer, ITS 1, from Toxoplasma gondii isolates and Neospora caninum." Parasitol Res 83(3): 285-9.
	The internal transcribed spacer (ITS1) region and the 5' part of the 5.8S ribosomal RNA gene of the ribosomal DNA repeat from 20 Toxoplasma gondil isolates was sequenced and found to be identical in all isolates, independent of host origin or virulence to mice. The ITS1 region from the closely related coccidian parasite Neospora caninum differed in 22% of its nucleotides. Hence, the ITS1 region provides a good marker for the distinction of T. gondii and N. caninum but is not useful for epidemiology studies of T. gondii.

Homan, W. L., F. H. van Enckevort, et al. (1992). "Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes." Parasitol Res 78(4): 316-23.
	A total of 13 new Giardia isolates were established in axenic culture. All of the new isolates were obtained by excystation of Giardia cysts from the feces of patients in Dutch hospitals. These isolates were subjected to isoenzyme and DNA analysis together with isolates from Poland, Belgium, and various other parts of the world. Isoenzyme analysis revealed that nearly all of the newly established isolates exhibited unique zymodemes. Isolates obtained from individuals from Belgium and Poland, on the other hand, displayed single zymodemes. Genomic DNA libraries were constructed from isolates belonging to the latter two zymodemes; specific and common recombinant DNA clones were selected from these libraries. Differential screening revealed that the two isolates had only 80% of the clones in common. Restriction-fragment-length polymorphism analysis using three different probes together with two synthetic probes that are complementary to Giardia structural protein genes led to the separation of all isolates into two major groups; within these groups, a further division could be made by application of other techniques or probes. The results of DNA analysis and zymodeme classification were in general agreement; in the present report they are compared with the data in the literature and discussed.

Hopkins, R. M., B. P. Meloni, et al. (1997). "Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality." J Parasitol 83(1): 44-51.
	A polymerase chain reaction-based method for genotyping Giardia duodenalis isolates using a polymorphic region near the 5' end of the small subunit ribosomal (SSU) RNA gene is described. Analysis was performed using Giardia cysts purified directly from feces. Isolates were collected from humans and dogs living in isolated Aboriginal communities where Giardia infections are highly endemic. This is the first report of the genetic characterization of Giardia from dogs and humans living in the same locality. Comparison of the SSU-rRNA sequences from 13 human and 9 dog isolates revealed 4 different genetic groups. Groups 1 and 2 contained all of the human isolates, whereas groups 3 and 4 consisted entirely of Giardia samples recovered from dogs. One dog sample contained templates from both groups 2 and 3. These results suggest that zoonotic transmission of Giardia infections between humans and dogs does not occur frequently in these communities. The dog-associated SSU-rRNA sequences have not been reported before, suggesting a new G. duodenalis subgroup. A genetic basis for the differences observed between the groups was supported by sequence analysis of 9 in vitro cultured isolates that were placed into the same genetic groups established by enzyme electrophoresis.

Horman, A., H. Korpela, et al. (2004). "Meta-analysis in assessment of the prevalence and annual incidence of Giardia spp. and Cryptosporidium spp. infections in humans in the Nordic countries." Int J Parasitol 34(12): 1337-46.
	We aimed to apply the meta-analysis in the studies of protozoan pathogens in order to obtain a general overview of the prevalence and annual incidence of Giardia spp. and Cryptosporidium spp. infections in asymptomatic and symptomatic human populations in the Nordic countries of Denmark, Finland, Norway and Sweden. In combining the data of 13 clinically and methodologically non-heterogeneous studies published before 2004 using the random effects model with DerSimonian-Laird estimator, we estimated the prevalence (% prevalence: 95% confidence limits) of Giardia cases in the asymptomatic (i.e. no gastroenteric symptoms) general population to be 2.97% (2.64; 3.31) and in the symptomatic population 5.81% (5.34; 6.30). For Cryptosporidium the prevalences were 0.99% (0.81; 1.19) and 2.91% (2.71; 3.12), respectively. In analyzing the data, we estimated that there will be 4670 (4300; 5060) symptomatic cases of Giardia and 3340 (3110; 3580) symptomatic cases of Cryptosporidium annually per 100,000 general population in the Nordic countries. The vast majority of cases will remain unregistered in the national registers of infectious diseases, since for single registered cases there will be 254-867 cases of Giardia undetected/unregistered and 4072 to 15,181 cases of Cryptosporidium, respectively.

Horman, A., R. Rimhanen-Finne, et al. (2004). "Campylobacter spp., Giardia spp., Cryptosporidium spp., noroviruses, and indicator organisms in surface water in southwestern Finland, 2000-2001." Appl Environ Microbiol 70(1): 87-95.
	A total of 139 surface water samples from seven lakes and 15 rivers in southwestern Finland were analyzed during five consecutive seasons from autumn 2000 to autumn 2001 for the presence of various enteropathogens (Campylobacter spp., Giardia spp., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, Escherichia coli, Clostridium perfringens, and F-RNA bacteriophages) and for physicochemical parameters (turbidity and temperature); this was the first such systematic study. Altogether, 41.0% (57 of 139) of the samples were positive for at least one of the pathogens; 17.3% were positive for Campylobacter spp. (45.8% of the positive samples contained Campylobacter jejuni, 25.0% contained Campylobacter lari, 4.2% contained Campylobacter coli, and 25.0% contained Campylobacter isolates that were not identified), 13.7% were positive for Giardia spp., 10.1% were positive for Cryptosporidium spp., and 9.4% were positive for noroviruses (23.0% of the positive samples contained genogroup I and 77.0% contained genogroup II). The samples were positive for enteropathogens significantly (P < 0.05) less frequently during the winter season than during the other sampling seasons. No significant differences in the prevalence of enteropathogens were found when rivers and lakes were compared. The presence of thermotolerant coliforms, E. coli, and C. perfringens had significant bivariate nonparametric Spearman's rank order correlation coefficients (P < 0.001) with samples that were positive for one or more of the pathogens analyzed. The absence of these indicators in a logistic regression model was found to have significant predictive value (odds ratios, 1.15 x 10(8), 7.57, and 2.74, respectively; P < 0.05) for a sample that was negative for the pathogens analyzed. There were no significant correlations between counts or count levels for thermotolerant coliforms or E. coli or the presence of F-RNA phages and pathogens in the samples analyzed.

Hoskins, D., C. E. Chrisp, et al. (1991). "Effect of hyperimmune bovine colostrum raised against Cryptosporidium parvum on infection of guinea pigs by Cryptosporidium wrairi." J Protozool 38(6): 185S-186S.
	Oocysts shedding was markedly reduced in guinea pigs inoculated intraintestinally with Cryptosporidium wrairi sporozoites that had been incubated with hyperimmune bovine colostrum raised to C. parvum when compared with shedding in guinea pigs inoculated with sporozoites incubated in either non-immune bovine colostrum or buffered saline. However oocyst shedding was apparently not reduced in guinea pigs inoculated by gavage with oocysts of C. wrairi and subsequently treated twice daily per os with hyperimmune bovine colostrum. Similarly, oocyst shedding was apparently not reduced by oral treatment with hyperimmune bovine colostrum when treatment was begun simultaneously with inoculation of C. wrairi oocysts.

Hou, L., X. Li, et al. (2004). "Neonatal-mouse infectivity of intact Cryptosporidium parvum oocysts isolated after optimized in vitro excystation." Appl Environ Microbiol 70(1): 642-6.
	We reexamined the finding of Neumann et al. that intact Cryptosporidium parvum oocysts obtained after in vitro excystation were infectious for neonatal CD-1 mice. We used both established excystation protocols and our own protocol that maximized excystation. Although intact oocysts isolated after any of three protocols were infectious for neonatal CD-1 mice, the infectivity of intact oocysts isolated with our optimized excystation protocol was significantly lower than the infectivity of intact oocysts isolated after established protocols or from fresh oocysts. Excystation should not be considered a valid measure of C. parvum viability, given that it is biologically implausible for oocysts to be nonviable and yet infectious.

Howe, A. D., S. Forster, et al. (2002). "Cryptosporidium oocysts in a water supply associated with a cryptosporidiosis outbreak." Emerg Infect Dis 8(6): 619-24.
	An outbreak of cryptosporidiosis occurred in and around Clitheroe, Lancashire, in northwest England, during March 2000. Fifty-eight cases of diarrhea with Cryptosporidium identified in stool specimens were reported. Cryptosporidium oocysts were identified in samples from the water treatment works as well as domestic taps. Descriptive epidemiology suggested that drinking unboiled tap water in a single water zone was the common factor linking cases. Environmental investigation suggested that contamination with animal feces was the likely source of the outbreak. This outbreak was unusual in that hydrodynamic modeling was used to give a good estimate of the peak oocyst count at the time of the contamination incident. The oocysts' persistence in the water distribution system after switching to another water source was also unusual. This persistence may have been due to oocysts being entrapped within biofilm. Despite the continued presence of oocysts, epidemiologic evidence suggested that no one became ill after the water source was changed.

Hsu, B. M. (2003). "Evaluation of analyzing methods for Giardia and Cryptosporidium in a Taiwan water treatment plant." J Parasitol 89(2): 369-71.
	Giardia sp. and Cryptosporidium sp. have emerged as waterborne pathogens of concern in Taiwan. This study examined both parasites in the actual water samples in southern Taiwan. Method 1623 was characterized by a higher recovery rate and lower detection limit compared with the information collection requirement protozoan method. A significant correlation between water turbidity and Cryptosporidium sp. in raw water samples was found in this study.

Hu, J., Y. Feng, et al. (2004). "Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623." J Microbiol Methods 58(3): 321-5.
	The U.S. Environmental Protection Agency has developed method 1623 for simultaneous detection of Cryptosporidium oocysts and Giardia cysts in water. Method 1623 includes four major steps: filtration, immunomagnetic separation (IMS), fluorescent antibody (FA) staining and microscopic examination. It was noted that the recovery levels following IMS-FA and FA staining were high, averaging more than 92.0% and 89.0% for C. parvum oocysts and G. lamblia cysts, respectively. In contrast, when the filtration step was incorporated, the recovery level of C. parvum oocysts declined significantly to 18.1% in seeded tap water, while a relatively high recovery level of 77.2% for G. lamblia cysts could still be achieved. Further study indicated that the recovery level of C. parvum oocysts could be enhanced significantly when an appropriate amount of silica particles was added to a water sample. The recovery level of C. parvum oocysts was affected by particle size and concentration. The optimal silica particle size was determined to be within the range of 5-40 microm, and the corresponding optimal silica concentration was 1.42 g for 10-l tap water. When both G. lamblia cysts and C. parvum oocysts were spiked into the tap water sample containing the optimum amount of silica particles, the average recovery levels of oocysts and cysts were 82.7% and 75.4%, respectively. The results obtained clearly suggested that addition of an appropriate amount of silica particles could improve the recovery level of C. parvum oocysts significantly and yet there was no noticeable deleterious effect on the recovery level of G. lamblia cysts. Further study indicated that the rotation time in the IMS procedure using the Dynal GC-Combo IMS kit (which was recommended in method 1623) was important for G. lamblia cyst detection. In contrast, the recovery level of C. parvum oocysts was not affected by the rotation time. Furthermore, it was found that the recovery levels of C. parvum oocysts using methods 1622 and 1623 were quite close although different IMS kits were used in the two methods.

Huamanchay, O., L. Genzlinger, et al. (2004). "Ingestion of Cryptosporidium oocysts by Caenorhabditis elegans." J Parasitol 90(5): 1176-8.
	Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.

Huang, P., J. T. Weber, et al. (1995). "The first reported outbreak of diarrheal illness associated with Cyclospora in the United States." Ann Intern Med 123(6): 409-14.
	OBJECTIVE: To investigate and characterize the epidemiology of a diarrheal outbreak associated with a potentially new pathogen, Cyclospora species (previously referred to as Cyanobacteria [blue-green algae]-like bodies). DESIGN: Three retrospective cohort studies supported by laboratory studies, environmental investigation, and community surveillance. SETTING: A hospital in Chicago. PARTICIPANTS: Housestaff physicians and hospital administrative staff. MEASUREMENTS: Identification of clinical features associated with illness and potential risks for acquisition of infection. RESULTS: Illness was characterized by watery diarrhea, abdominal cramping, decreased appetite, and low-grade fever. Symptoms typically occurred in a distinctive cycle of remissions and exacerbations lasting up to several weeks. Stool cultures and examinations for known ova and parasites were negative. Microscopic examination of stool specimens from 11 ill persons showed many spherical bodies, 8 to 10 microns in diameter, that were identified as Cyclospora organisms. The organisms disappeared by 9 weeks after onset of illness in the 7 patients from whom follow-up specimens were obtained. Epidemiologic studies implicated tap water from a physicians' dormitory as the most likely source of the outbreak. Environmental investigation suggested that stagnant water in a storage tank may have contaminated the water supply after a pump failure. CONCLUSIONS: This is the first reported outbreak of diarrhea associated with Cyclospora in the United States. Cyclospora may be a human enteric pathogen able to produce bouts of acute and relapsing diarrhea, and it should be considered in assessments of patients with unexplained, prolonged diarrheal illness.

Huffman, D. E., A. Gennaccaro, et al. (2002). "Low- and medium-pressure UV inactivation of microsporidia Encephalitozoon intestinalis." Water Res 36(12): 3161-4.
	Newly recognized waterborne pathogens such as microsporidia are being detected in the world's water supplies with increasing frequency. Many of these organisms have been shown to cause negative health impacts for both immunocompetent as well as immunocompromised individuals. It is imperative that these emerging pathogens be investigated for their ability to resist both traditional and novel disinfection technologies that are currently in use or under consideration for drinking water treatment. Low- and medium pressure UV light is at the cutting edge of disinfection technologies for the drinking water industry. While previous UV disinfection studies have focused on the inactivation of Cryptosporidium and Giardia as well as viruses and common bacteria, this research reports the ability of low- and medium pressure UV light to inactivate > 3.6 log10 of microsporidia Encephalitozoon intestinalis spores at a dose of 6 mJ/cm2 or higher as determined using a cell culture approach.

Hunt, D. A., S. Sebugwawo, et al. (1994). "Cryptosporidiosis associated with a swimming pool complex." Commun Dis Rep CDR Rev 4(2): R20-2.
	Twelve children and one adult in Gloucestershire fell ill with cryptosporidiosis in March 1992. Ten cases lived in or near a particular Cotswold town. Eight of these had visited a swimming pool within 24 hours of a suspected faecal accident, and two may have been secondary cases. Of the three cases who lived elsewhere, one was probably unrelated to the outbreak, one had visited the pool, and one was a contact of a boy who may have been the source of the outbreak.

Hunter, P., Q. Syed, et al. (2001). "Possible undetected outbreaks of cryptosporidiosis in areas of the north west of England supplied by an unfiltered surface water source." Commun Dis Public Health 4(2): 136-8.
	We report a ten-year retrospective analysis of laboratory reports of cryptosporidium infection in the North West of England. Weekly report data from six health authorities known to have been affected by outbreaks associated with a single supply were compared with data from other health authorities in the North West. Following graphical representation of report rates, it would appear that outbreaks in the six health authorities were considerably more common than the average recorded in the national outbreak surveillance system.

Hunter, P. R., R. M. Chalmers, et al. (2003). "Foot and mouth disease and cryptosporidiosis: possible interaction between two emerging infectious diseases." Emerg Infect Dis 9(1): 109-12.
	During 2001, a large outbreak of foot and mouth disease occurred in the United Kingdom, during which approximately 2,030 confirmed cases of the disease were reported, >6 million animals were slaughtered, and strict restrictions on access to the countryside were imposed. We report a dramatic decline in the reported incidence of human cryptosporidiosis in northwest England during weeks 13-38 in 2001, compared with the previous 11 years. This decline coincided with the period of foot and mouth restrictions. No similar reduction occurred in the other 26 weeks of the year. We also noted a substantial decline in the proportion of human infections caused by the bovine strain (genotype 2) of Cryptosporidium parvum during weeks 13-38 in that year but not during the other weeks.

Hunter, P. R., J. M. Colford, et al. (2001). "Waterborne diseases." Emerg Infect Dis 7(3 Suppl): 544.
	
Hunter, P. R., S. Hughes, et al. (2004). "Sporadic cryptosporidiosis case-control study with genotyping." Emerg Infect Dis 10(7): 1241-9.
	We report a case-control study of sporadic cryptosporidiosis with genotyping of isolates from case-patients. A postal questionnaire was completed by 427 patients and 427 controls. We obtained genotyping data on isolates from 191 patients; 115 were Cryptosporidium hominis, and 76 were C. parvum. When all cryptosporidiosis cases were analyzed, three variables were strongly associated with illness: travel outside the United Kingdom, contact with another person with diarrhea, and touching cattle. Eating ice cream and eating raw vegetables were both strongly negatively associated with illness. Helping a child <5 years of age to use the toilet and the number of glasses of tap water drunk at home each day were also independently positively associated with risk. Eating tomatoes was negatively associated. For C. hominis infections, the strongly significant risk factors were travel abroad and changing diapers of children <5 years of age. For C. parvum, eating raw vegetables and eating tomatoes were strongly negatively associated with illness; touching farm animals was associated with illness.

Hunter, P. R. and G. Nichols (2002). "Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients." Clin Microbiol Rev 15(1): 145-54.
	Cryptosporidium spp. are a major cause of diarrheal disease in both immunocompetent and immunodeficient individuals. They also cause waterborne disease in both the United States and United Kingdom. Studies on the mechanisms of immunity to cryptosporidiosis indicate the importance of the T-cell response. The spectrum and severity of disease in immunocompromised individuals with cryptosporidiosis reflect this importance since the most severe disease is seen in individuals with defects in the T-cell response. The most commonly studied group is that of patients with AIDS. These patients suffer from more severe and prolonged gastrointestinal disease that can be fatal; in addition, body systems other than the gastrointestinal tract may be affected. The widespread use of antiretroviral therapy does appear to be having a beneficial effect on recovery from cryptosporidiosis and on the frequency of infection in human immunodeficiency virus-positive patients. Other diseases that are associated with increased risk of severe cryptosporidiosis, such as primary immunodeficiencies, most notably severe combined immunodeficiency syndrome, are also predominantly associated with T-cell defects. Of the remaining groups, children with acute leukemia seem to be most at risk from cryptosporidiosis. There is less evidence of severe complications in patients with other malignant diseases or in those receiving immunosuppressive chemotherapy.

Hunter, P. R. and R. C. Thompson (2005). "The zoonotic transmission of Giardia and Cryptosporidium." Int J Parasitol 35(11-12): 1181-90.
	The molecular characterisation of Giardia and Cryptosporidium has given rise to a more epidemiological meaningful and robust taxonomy. Importantly, molecular tools are now available for 'typing' isolates of the parasites directly from clinical and environmental samples. As a consequence, information on zoonotic potential has been obtained although the frequency of zoonotic transmission is still poorly understood. Analysis of outbreaks and case-control studies, especially when coupled with genotyping data, is slowly providing information on the public health significance of zoonotic transmission. Such studies support the hypothesis that Cryptosporidium hominis is spread only between humans but that the major reservoir for Cryptosporidium parvum is domestic livestock, predominantly cattle, and that direct contact with infected cattle is a major transmission pathway along with indirect transmission through drinking water. The situation is less clearcut for Giardia duodenalis but the evidence does not, in general, support zoonotic transmission as a major risk for human infections. However, for both parasites there is a need for molecular epidemiological studies to be undertaken in well-defined foci of transmission in order to fully determine the frequency and importance of zoonotic transmission.

Hutchison, M. L., L. D. Walters, et al. (2005). "Fate of pathogens present in livestock wastes spread onto fescue plots." Appl Environ Microbiol 71(2): 691-6.
	Fecal wastes from a variety of farmed livestock were inoculated with livestock isolates of Escherichia coli O157, Listeria monocytogenes, Salmonella, Campylobacter jejuni, and Cryptosporidium parvum oocysts at levels representative of the levels found in naturally contaminated wastes. The wastes were subsequently spread onto a grass pasture, and the decline of each of the zoonotic agents was monitored over time. There were no significant differences among the decimal reduction times for the bacterial pathogens. The mean bacterial decimal reduction time was 1.94 days. A range of times between 8 and 31 days for a 1-log reduction in C. parvum levels was obtained, demonstrating that the protozoans were significantly more hardy than the bacteria. Oocyst recovery was more efficient from wastes with lower dry matter contents. The levels of most of the zoonotic agents had declined to below detectable levels by 64 days. However, for some waste types, 128 days was required for the complete decline of L. monocytogenes levels. We were unable to find significant differences between the rates of pathogen decline in liquid (slurry) and solid (farmyard manure) wastes, although concerns have been raised that increased slurry generation as a consequence of more intensive farming practices could lead to increased survival of zoonotic agents in the environment.

Ignatius, R., M. Lehmann, et al. (1997). "A new acid-fast trichrome stain for simultaneous detection of Cryptosporidium parvum and microsporidial species in stool specimens." J Clin Microbiol 35(2): 446-9.
	The detection in stool specimens of Cryptosporidium parvum and microsporidia, the most frequent parasitic pathogens causing diarrhea in AIDS patients, until now has depended on two different staining methods. However, since double infections occur and minimization of laboratory costs is mandatory, development of a method for simultaneous detection of these parasites appeared desirable. We report on a new, inexpensive, and easy-to-perform staining procedure to demonstrate both acid-fast oocysts of C. parvum and other coccidia, as well as microsporidial spores. This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination.

Isaac-Renton, J., J. Blatherwick, et al. (1999). "Epidemic and endemic seroprevalence of antibodies to Cryptosporidium and Giardia in residents of three communities with different drinking water supplies." Am J Trop Med Hyg 60(4): 578-83.
	This study was carried out to compare cryptosporidiosis and giardiasis seroprevalence rates in residents of three communities. Community (Com 1) uses drinking water from deep wells, community 2 (Com 2) uses surface water from a protected watershed, and community 3 (Com 3) uses surface water frequently containing Cryptosporidium oocysts and Giardia cysts. Unfiltered drinking water from each community was collected at the tap and tested for Cryptosporidium oocysts and Giardia cysts during the 12 months in which sera were collected for testing. No oocysts or cysts were detected in the water from the Com 1 deep wells; oocysts and cysts were detected intermittently in the drinking water from the other two communities. A waterborne outbreak of cryptosporidiosis occurred in a municipality adjacent to Com 3 six months into this 12-month study. Sera from residents of each of the communities were collected proportionately by month and by population size. Coded sera were tested for IgG to Cryptosporidium using a previously developed Western blotting method. The presence or absence of bands at 15-17 kD and/or 27 kD was recorded for the 1,944 sera tested. Definite bands at 15-17 kD and/or 27 kD were detected in 981 (50.5%) of the sera. A total of 33.2% of sera from Com 1 (community using deep wells) were positive using the same criteria compared with 53.5% (Com 2) and 52.5% (Com 3) of sera from the two communities using surface drinking water. Both bands (15-17 kD plus 27 kD) were detected in 582 sera (29.9%) from the three communities: 14.1% of sera from Com 1 compared with 32.7% from Com 2 and 31.5% from Com 3. These findings are consistent with a lower risk of exposure to Cryptosporidium from drinking water obtained from deep well sources. However, analysis of results by calendar quarter showed a significant (P < 0.001) increase in the number of Com 3 positive sera (compared with Com 1) following the waterborne outbreak. Without this outbreak-related observation, a significant overall difference in seropositivity would not have been seen. We also observed that in sera from the community affected by the outbreak, the presence on immunoblots of both Cryptosporidium bands appeared to be the best indicator of recent infection. Seroprevalence rates using an ELISA to detect IgG to Giardia were estimated using the same sera. Overall 30.3% (590 of 1,944) of sera were positive by the ELISA. A total of 19.1% of sera from Com 1, 34.7% from Com 2 and 16.0% from Com 3 were seropositive. Rates for both Com 3 and Com 1 did not change significantly over time. In Com 2, rates decreased significantly (P < 0.001) during the last half of the study period (third and fourth calendar quarters). The reasons for the decrease in seroprevalence in Com 2 sera are presently not known. These studies show intriguing associations between seroprevalence, outbreak-related laboratory serologic data, and patterns of parasite contamination of drinking water. Further studies are required to validate the serologic approach to risk assessment of waterborne parasitic infections at a community level.

Isaac-Renton, J., W. R. Bowie, et al. (1998). "Detection of Toxoplasma gondii oocysts in drinking water." Appl Environ Microbiol 64(6): 2278-80.
	The world's largest outbreak of waterborne toxoplasmosis occurred in a municipality in the western Canadian province of British Columbia. When drinking water emerged as a possible source of infection during the outbreak investigation, a laboratory method was needed to attempt detection of the parasite, Toxoplasma gondii. The method developed was based on the current U.S. Environmental Protection Agency method for detection of Cryptosporidium oocysts. Collection of large-volume drinking water samples and cartridge filter processing were unchanged, although identification of Toxoplasma oocysts in the filter retentate was carried out by using a previously described rodent model. Validation of the method developed was tested by using oocysts from a well-characterized Toxoplasma strain.

Isaac-Renton, J., W. Moorehead, et al. (1996). "Longitudinal studies of Giardia contamination in two community drinking water supplies: cyst levels, parasite viability, and health impact." Appl Environ Microbiol 62(1): 47-54.
	Giardia cyst concentrations were determined in an inventory of 153 raw and 91 chlorinated drinking water samples collected at 86 sites from throughout the western Canadian province of British Columbia. Sixty-four percent of raw water samples were cyst positive (69% of sites). Cyst concentrations were lower in chlorinated than in raw water. The viability of cysts in drinking water samples assessed by infectivity in Mongolian gerbils (Meriones unguiculatus) was decreased in chlorinated water. Two rural communities using Giardia-contaminated surface drinking water sources were selected for longitudinal studies including drinking water testing and serological studies of residents. Three hundred thirty-six raw and treated samples from these communities were collected over 24 months. Cyst concentrations and viability were assessed in a 12-month study of each community. Parasite concentrations were lower in chlorinated water than in raw water in both communities. Cyst concentrations were lower in reservoir-settled water than in raw water. Viability, assessed by animal infectivity and corrected for inoculum, decreased following reservoir settling as well as after chlorination. A bolus or spiking phenomenon of cysts was observed in both community drinking water systems and deserves further study. A striking seasonal pattern was seen in one community but not in the second. The seroprevalence data and number of laboratory-confirmed cases identified in each year-long community study are consistent with the possibility that low-level endemic transmission is occurring.

Isaac-Renton, J. L., C. Cordeiro, et al. (1993). "Characterization of Giardia duodenalis isolates from a waterborne outbreak." J Infect Dis 167(2): 431-40.
	Isolates were retrieved from drinking water and from animal and human sources associated with a waterborne outbreak of giardiasis. This is the first report of water-source and epidemic-associated Giardia isolates being adapted to in vitro propagation. Outbreak-associated, non-out-break-associated, and reference isolates were characterized using isoenzyme electrophoresis and pulsed-field gel electrophoresis (PFGE). All outbreak-associated and 2 other isolates were in one of eight zymodemes. The chromosomal complement of the outbreak-associated isolates was relatively homogeneous; this PFGE karyotype was distinguishable from other karyotypes. Overall results of both characterization methods were similar, although PFGE appears to be a more discriminating biotyping technique. Banding patterns of the outbreak-associated Giardia isolates remained the same even though the parasite passed through different hosts during the outbreak. Heterogeneity of isolates was also demonstrated for the first time within a single community not associated with the outbreak.

Isaac-Renton, J. L., C. P. Fung, et al. (1986). "Evaluation of a tangential-flow multiple-filter technique for detection of Giardia lamblia cysts in water." Appl Environ Microbiol 52(2): 400-2.
	A system of tangential-flow filtration was evaluated for use in the detection of Giardia cysts in drinking water. This method was more sensitive in recovering cysts than a frequently used wound-orlon system of through-filtration.

Isaac-Renton, J. L., H. Shahriari, et al. (1992). "Comparison of an in vitro method and an in vivo method of Giardia excystation." Appl Environ Microbiol 58(5): 1530-3.
	An in vitro method and an in vivo method of excystation were compared to determine the most useful method for the retrieval of Giardia duodenalis isolates. Cysts from 11 Giardia strains were used. In vitro excystation produced motile trophozoites in 16 sets, while in vivo excystation produced trophozoites in all of the 21 comparative sets of excystations. Few cultures were lost because of contamination by either method (17% of in vitro-derived trophozoites versus 23% of in vivo-derived trophozoites; P greater than 0.05). Both methods demonstrated comparable isolate retrieval rates (15% of in vitro-derived trophozoites adapting to culture compared with 29% of in vivo-derived trophozoites; P greater than 0.05), although analysis of the strains retrieved showed that two isolates were retrieved from in vitro excystation alone, compared with four from in vivo excystation. Analysis that included results of extra in vivo cultures showed that a total of nine isolates were retrieved by using this type of excystation. Despite the disadvantages of cost and labor, in vivo excystation appears to be more useful than in vitro excystation for isolate retrieval at the present time.

Istre, G. R., T. S. Dunlop, et al. (1984). "Waterborne giardiasis at a mountain resort: evidence for acquired immunity." Am J Public Health 74(6): 602-4.
	In November 1981, an outbreak of waterborne giardiasis occurred at a popular ski resort in Colorado. Stratification of illness by consumption of municipal tap water showed a striking dose-response, with an attack rate of 42 per cent among persons who drank six or more glasses of water per day. Filtered water samples revealed Giardia cysts in specimens both before and after treatment, and several deficiencies were found in the water treatment facility. Residents who had lived in the area greater than 2 years had a lower attack rate for illness than short-term residents.

Jakubowski, W., S. Boutros, et al. (1996). "Environmental methods for cryptosporidium." Journal American Water Works Association 88(9): 107-121.
	This report was prepared by the Working Group on Waterborne Cryptosporidiosis (Technical Task Force E, Developmental Status of Environmental Sampling, Water Testing, and Surrogate Indicators). Methods for detecting Cryptosporidium oocysts in water have centered around microscopic examination of fluorescent antibody-stained concentrates from large-volume water samples. The limitations of these antibody-based methods include the need for experienced analysts, lengthy analytical time, expense, lack of specificity, erratic efficiency, low precision, and difficulty in determining viability. A number of methods, assays, and procedures that have the potential for ameliorating some of these limitations are currently being evaluated. How successful such processes will. be remains to be demonstrated by the scientific community.

Jellison, K. L., D. L. Distel, et al. (2004). "Phylogenetic analysis of the hypervariable region of the 18S rRNA gene of Cryptosporidium oocysts in feces of Canada geese (Branta canadensis): evidence for five novel genotypes." Appl Environ Microbiol 70(1): 452-8.
	To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.

Jellison, K. L., H. F. Hemond, et al. (2002). "Sources and species of cryptosporidium oocysts in the Wachusett Reservoir watershed." Appl Environ Microbiol 68(2): 569-75.
	Understanding the behavior of Cryptosporidium oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCR-restriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen Cryptosporidium parvum from other Cryptosporidium species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.

Jenkins, M., J. Higgins, et al. (2004). "Protection of calves against cryptosporiosis by oral inoculation with gamma-irradiated Cryptosporidium parvum oocysts." J Parasitol 90(5): 1178-80.
	The purpose of this study was to determine whether gamma-irradiated Cryptosporidium parvum oocysts could elicit protective immunity against cryptosporidiosis in dairy calves. Cryptosporidium parvum Iowa strain oocysts (1 x 10(6) per inoculation) were exposed to various levels of gamma irradiation (350-500 Gy) and inoculated into 1-day-old dairy calves. The calves were examined daily for clinical signs of cryptosporidiosis, and fecal samples were processed for the presence of C. parvum oocysts. At 21 days of age, the calves were challenged by oral inoculation with 1 x 10(5) C. parvum oocysts and examined daily for oocyst shedding and clinical cryptosporidiosis. Calves that were inoculated with C. parvum oocysts exposed to 350-375 Gy shed C. parvum oocysts in feces. Higher irradiation doses (450 or 500 Gy) prevented oocyst development, but the calves remained susceptible to C. parvum challenge infection. Cryptosporidium parvum oocysts exposed to 400 Gy were incapable of any measurable development but retained the capacity to elicit a protective response against C. parvum challenge. These findings indicate that it may be possible to protect calves against cryptosporidiosis by inoculation with C. parvum oocysts exposed to 400-Gy gamma irradiation.

Jenkins, M., D. Kerr, et al. (1995). "Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen." Vaccine 13(17): 1658-64.
	In an effort to generate high titer colostrum for immunotherapy of cryptosporidiosis, a study was conducted to test the efficacy of immunizing sheep with recombinant plasmid DNA (pCMV-CP15/60) encoding epitopes of 15 and 60 kDa surface antigens of Cryptosporidium parvum sporozoites. The plasmid DNA was used to immunize preparturient ewes at three dose levels by jet-injection into either hind limb muscle (IM) or mammary tissue (IMAM). Regardless of route of injection, a dose-dependent anti-CP15/60 immunoglobulin response was observed in sera and colostrum from sheep immunized with pCMV-CP15/60 plasmid DNA. High titer antibody responses were observed in one of three animals per group receiving an IM injection of 100 or 1000 micrograms pCMV-CP15/60. IMAM immunization with 100 or 1000 micrograms pCMV-CP15/60 plasmid DNA elicited higher titer colostrum responses and more consistent serum responses compared to IM injections. A negligible serum and colostrum anti-CP15/60 response was observed in ewes injected IM with 10 micrograms pCMV-CP15/60 or 1000 micrograms control plasmid DNA. Immunoblotting of native C. parvum sporozoite/oocyst protein with hyperimmune serum and colostrum corroborated the increased titers against CP15/60 antigen. Serum and colostrum antibodies from pCMV-CP15/60-immunized sheep were eluted from native CP15 protein and bound a surface antigen of C. parvum sporozoites as indicated by indirect immunofluorescence staining.

Jenkins, M., J. M. Trout, et al. (2003). "Comparison of tests for viable and infectious Cryptosporidium parvum oocysts." Parasitol Res 89(1): 1-5.
	The purpose of this study was to compare different assays for viable Cryptosporidium parvum incubated in water at a temperature commonly found in the environment. C. parvum oocysts were stored in sterile water for 9 months at 15 degrees C. A sample was removed monthly and analyzed by five different assays to determine oocyst viability. Mouse infection and cell culture showed that C. parvum oocysts remained viable and infectious when stored for 7 months at this temperature. Fluorescence in situ hybridization (FISH) using probes directed to ribosomal RNA was also applied to these oocysts. The proportion of FISH-positive oocysts was 70-80% for the first 2 months of storage, decreased and remained nearly constant at 40-50% for 3-7 months, then decreased to 20% by 8 months, and to 0% by 9 months. Amylopectin content and mRNA for amyloglucosidase (CPAG), as measured by RT-PCR, decreased much more rapidly. By 3 months and for the remainder of the incubation period, amylopectin content was 20% of the original amount present in the oocysts. The CPAG RT-PCR signal at 3 months was 50% of that observed after 1 month storage, 20% at 4 months, and was not detected thereafter. Thus, results from cell culture and mouse infection assay exhibited the best agreement, the FISH assay showed modest agreement with these assays, and CPAG RT-PCR and the amylopectin assay displayed marginal agreement with the other three assays.

Jenkins, M. B., L. J. Anguish, et al. (1997). "Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts." Appl Environ Microbiol 63(10): 3844-50.
	The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeable to fully permeable was accelerated at temperatures above 4 degrees C. This sequence of oocyst permeability changes was taken as an indicator of the oocyst inactivation pathway. Using the dye permeability results, inactivation rates of oocysts in two fecal pools stored in the dark at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day-1, respectively. The excystation assay gave similar inactivation rates of 0.0046 and 0.0079 oocyst day-1. These results demonstrate the utility of the dye permeability assay as an indicator of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments.

Jenkins, M. B., M. J. Walker, et al. (1999). "Use of a sentinel system for field measurements of Cryptosporidium parvum oocyst inactivation in soil and animal waste." Appl Environ Microbiol 65(5): 1998-2005.
	A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purified Cryptosporidium parvum oocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50 degrees C and decreases in soil water potential (-0.003 to -3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect of soil freeze-thaw cycles was evident in the large proportion of empty sentinel oocysts. The potentially infective sentinel oocysts were reduced to <1% while the proportions in controls did not decrease below 50% potentially infective during the first field experiment. Microscopic observations of empty oocyst fragments indicated that abrasive effects of soil particles were a factor in oocyst inactivation. A similar pattern was observed in a second field experiment at the same site.

Jenkins, M. C. and R. Fayer (1995). "Cloning and expression of cDNA encoding an antigenic Cryptosporidium parvum protein." Mol Biochem Parasitol 71(1): 149-52.
	
Jenkins, M. C., R. Fayer, et al. (1993). "Cloning and expression of a cDNA encoding epitopes shared by 15- and 60-kilodalton proteins of Cryptosporidium parvum sporozoites." Infect Immun 61(6): 2377-82.
	A cDNA (CP15/60) encoding epitopes of Cryptosporidium parvum 15- and 60-kDa sporozoite proteins was isolated and expressed in Escherichia coli toward the goal of developing an immunogen for producing high-titer anticryptosporidial colostrum. Antisera prepared in rats to native C. parvum 15-kDa protein and used to identify the CP15/60 bacteriophage clone recognized both 15- and 60-kDa in vitro translation products derived from sporozoite RNA. Antisera specific for recombinant CP15/60 antigen recognized native 15- and 60-kDa C. parvum sporozoite proteins by immunoblotting and identified both surface and internal antigens on C. parvum sporozoites by immunofluorescence staining. Northern (RNA) and Southern blot hybridization experiments using sporozoite RNA and DNA indicated that CP15/60 DNA is transcribed as a single 1.4-kb RNA species from a single-copy gene. Recombinant CP15/60 antigen was recognized by hyperimmune colostrum from cows immunized with C. parvum oocyst-sporozoite protein and by convalescent-phase sera from C. parvum-infected calves.

Jenkins, M. C., C. O'Brien, et al. (1999). "Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice." Vaccine 17(19): 2453-60.
	Preparturient cows were immunized three times over a six-week period with recombinant plasmid DNA encoding the Cryptosporidium parvum CP15/60 antigen by injecting the DNA in the mammary gland. Serum was collected at each immunization and first colostrum was collected after parturition; all were assayed for Cryptosporidium-specific antibodies (Ab). A serological response to C. parvum sporozoite and oocyst antigen was detected in cows immunized with pCP15/60 plasmid DNA. Colostrum from these cows, unlike colostrum from normal controls, contained Ab specific for C. parvum sporozoites and oocysts as indicated by immunofluorescence Ab (IFA) staining. Colostrum was also tested for conferring passive immunity against C. parvum infection by oral administration to immunosuppressed adult inbred mice. Immune colostrum and control colostrum were administered to separate groups of dexamethasone (DEX)-treated adult C57BL/6NCr mice beginning 12 h before and at 12 h intervals for 3 days after oral C. parvum oocyst infection. Cryptosporidium development was assayed in ilea of immune- and control-colostrum-treated mice 96 h postinfection by semiquantitative PCR. Mice receiving immune colostrum showed partial protection (about 50% reduction) against intestinal C. parvum development compared to mice receiving control colostrum. This protection was evident at a challenge dose of 10(3) C. parvum oocysts per mouse; no differences were noted in parasite development between groups receiving immune or control colostrum and infected with 10(4) oocysts. This study showed that serum and colostrum Ab response to C. parvum can be elicited in preparturient cows by direct injection of recombinant pCP15/60 plasmid DNA and that passive protection against cryptosporidiosis can be obtained by treating immunosuppressed mice with immune colostrum before and after C. parvum infection.

Jenkins, M. C., J. Trout, et al. (2000). "Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase." J Microbiol Methods 43(2): 97-106.
	The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected RNA from as few as 10(3) C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts stored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG RT-PCR may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro.

Jenkins, M. C., J. Trout, et al. (1998). "Development and application of an improved semiquantitative technique for detecting low-level Cryptosporidium parvum infections in mouse tissue using polymerase chain reaction." J Parasitol 84(1): 182-6.
	An improved semiquantitative technique was developed for measuring low infectious doses of Cryptosporidium parvum in neonatal mice using polymerase chain reaction (PCR). Separate litters of neonatal mice were inoculated with 0, 10(2), 10(3), or 10(4) C. parvum oocysts and killed 96 hr postinfection. A segment of the ileum or the entire whole intestine was then removed from subgroups of mice in each litter and total DNA was extracted using standard procedures. By employing a CP15/60-based semiquantitative PCR technique, C. parvum DNA was detected in mice infected with as few as 10(2) oocysts. DNA isolated from the ileum of infected mice produced a more intense PCR signal than DNA isolated from the whole intestine. This technique was used to study the intracellular development of C. parvum sporozoites that had been exposed in the oocyst stage to either 0, 15, 20, 25, or 30 kRad gamma-irradiation. A CP15/60 PCR signal was observed in ileum tissue from mice infected with 0-kRad- or 15-kRad-irradiated C. parvum oocysts. A very slight PCR signal was generated by PCR on ileum tissue DNA from mice infected with 20-kRad-irradiated oocysts, whereas no signal was observed in PCR on intestinal DNA from mice infected with oocysts exposed to higher radiation doses.

Jenkins, M. C., J. Trout, et al. (1999). "Cloning and expression of a DNA sequence encoding a 41-kilodalton Cryptosporidium parvum oocyst wall protein." Clin Diagn Lab Immunol 6(6): 912-20.
	This study was conducted to produce a recombinant species-specific oocyst wall protein of Cryptosporidium parvum. Antigens unique to C. parvum were identified by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of oocyst proteins from several different Cryptosporidium species. Antiserum was then prepared against a 41-kDa antigen unique to C. parvum and used to identify a recombinant DNA clone, designated rCP41. Expression of CP41 mRNA in C. parvum oocysts was confirmed by reverse transcriptase PCR (RT-PCR). Although the CP41 sequence was shown by PCR to be present in the genome of C. baileyi, CP41 mRNA was not detected in this species by RT-PCR. Immunofluorescence staining with antiserum against recombinant CP41 detected native CP41 antigen on the surface of C. parvum oocysts but failed to detect CP41 on C. baileyi oocysts. Immunoelectron microscopy demonstrated that native CP41 was distributed unevenly on the C. parvum oocyst surface and was associated with amorphous oocyst wall material. In an enzyme-linked immunosorbent assay, purified rCP41 performed as well as native C. parvum oocyst protein in measuring the serological responses of young calves and adult cows to experimental and natural C. parvum infections. These results indicate that recombinant CP41 antigen may have potential in the immunodiagnosis of cryptosporidiosis.

Jiang, J., K. A. Alderisio, et al. (2005). "Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors." Appl Environ Microbiol 71(3): 1135-41.
	Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/mul or 25 ng of T4 gene 32 protein/mul to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.

Jiang, J., K. A. Alderisio, et al. (2005). "Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York." Appl Environ Microbiol 71(8): 4446-54.
	To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.

Jiang, J. and L. Xiao (2003). "An evaluation of molecular diagnostic tools for the detection and differentiation of human-pathogenic Cryptosporidium spp." J Eukaryot Microbiol 50 Suppl: 542-7.
	The performance of 10 commonly used genotyping tools in the detection and differentiation of 7 human-pathogenic Cryptosporidium spp. (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris and Cryptosporidium pig genotype 1) was evaluated. All 3 SU rRNA gene-based tools could amplify the DNA of 7 Cryptosporidium spp. efficiently. However, the tools based on the antigens TRAP-C1, TRAP-C2 and COWP genes, the housekeeping genes HSP70 and DHFR, or a genomic sequence, failed to detect the DNA of C. felis, C. canis, Cryptosporidium pig genotype I, and C. muris. With the exception of 1 tool based on the TRAP-C2 gene, the PCR-RFLP or the PCR sequencing tools evaluated in this study could differentiate C. hominis, C. parvum and C. meleagridis from each other, and 2 SSU rRNA gene-based tools could differentiate all 7 Cryptosporidium spp. Thus, a thorough understanding of the strength and weakness of each technique is needed when using molecular diagnostic tool in epidemiological investigations of human cryptosporidiosis.

John, D. E., C. N. Haas, et al. (2005). "Chlorine and ozone disinfection of Encephalitozoon intestinalis spores." Water Res 39(11): 2369-75.
	Microsporidia are intracellular eukaryotic parasites which have the potential for zoonotic and environmental, including waterborne, transmission. Encephalitozoon intestinalis is a microsporidian pathogen of humans and animals and has been detected in surface water. It is also on the Contaminant Candidate List of potential emerging waterborne pathogens for the US EPA. We performed disinfection studies using chlorine and ozone on E. intestinalis spores with a cell-culture most-probable-number assay to determine infectivity. Chlorine experiments were performed at 5 degrees C at pH of 6, 7, and 8 with 1mg/L initial chlorine concentrations, while ozone experiments were performed at 5 degrees C and pH 7 with initial ozone doses of 1 and 0.5mg/L, both in buffered water. A derivation of Hom's model for disinfection kinetics under dynamic disinfectant concentrations was used to fit observed data and calculate concentration-time product (C*t) values. Chlorine C*t values varied with pH such that 99% (2-log(10)) C*t ranged from 12.8 at pH 6 to 68.8 at pH 8 (mg min/L). Ozone C*t values were approximately an order of magnitude less at 0.59--0.84 mg min/L, depending on initial concentration.

John, D. E., N. Nwachuku, et al. (2003). "Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation." J Microbiol Methods 52(2): 183-96.
	Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).

Johnson, A. M., K. Linden, et al. (2005). "UV inactivation of Cryptosporidium hominis as measured in cell culture." Appl Environ Microbiol 71(5): 2800-2.
	The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.

Johnson, C. H., M. M. Marshall, et al. (2003). "Chlorine inactivation of spores of Encephalitozoon spp." Appl Environ Microbiol 69(2): 1325-6.
	This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Johnson, D. C., C. E. Enriquez, et al. (1997). "Survival of Giardia, Cryptosporidium, poliovirus and Salmonella in marine waters." HEALTH-RELATED WATER MICROBIOLOGY 1996: 261-268.
	Discharge of sewage into the ocean is still a common method of disposal worldwide. Both treated and untreated sewage may contain significant concentration of waterborne pathogens, such as Giardia, Cryptosporidium, poliovirus and Salmonella. Limited studies exist on the survival of poliovirus and Salmonella in marine waters; however, almost no information exists on the survival of protozoan parasites in marine waters. This study examined the survival of Giardia muris cysts, Cryptosporidium parvum oocysts, poliovirus-1 and Salmonella typhimurium in marine waters. The survival of the microorganisms varied according to the presence of light, salinity and water quality (as determined by quantity of enterococci). All microorganisms survived longer in the dark than in sunlight, the order of survival in sunlight being: Cryptosporidium > poliovirus > Giardia > Salmonella.

Johnson, D. C., K. A. Reynolds, et al. (1995). Detection of Giardia and Cryptosporidium in marine waters. International Symposium, IAWQ Specialist Group on Health Related Microbiology. 17. Biennial Conference of the International Association on Water Quality, Budapest (Hungary), 24-30 Jul 1994.
	Raw sewage disposal in marine waters is a common practice in many countries. This practice raises health risk concerns of possible transmission of Giardia and Cryptosporidium. Both of these protozoa have been shown to be transmitted by recreational swimming. To date no studies have determined the efficiency of their detection and concentration in marine waters. This study evaluated the efficiency of their detection in tap water and from marine waters in Hawaii with two different filter types. This study compared a polypropylene fiber cartridge filter, DPPPY (1.0 mu m nominal porosity) (Cuno, Meriden CT) which is typically used for parasite detection and the Filterite negatively charged filter (0.45 mu m) (Filtemp Sales, Inc., Phoenix, AZ). The latter would allow for both viruses and parasites to be concentrated simultaneously. The organisms were removed from the filter by passing the eluent through the filters in the opposite direction of collection and detected by indirect immunofluorescence antibody staining specific for Giardia and Cryptosporidium. Processing was simpler and faster with the Filterite filter and the overall efficiency for both Giardia and Cryptosporidium detection was greater. These methods are currently being used for the detection of the oocysts and cysts at bathing beaches in Hawaii impacted by marine sewage discharge.

Johnson, D. W., N. J. Pieniazek, et al. (1995). "Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples." Appl Environ Microbiol 61(11): 3849-55.
	The development of a reliable method of using PCR for detection of Cryptosporidium oocysts in environmental samples with oligonucleotide primers which amplify a portion of the sequence encoding the small (18S) subunit of rRNA producing a 435-bp product was demonstrated. The PCR assay was found to provide highly genus-specific detection of Cryptosporidium spp. after release of nucleic acids from oocysts by a simple freeze-thaw procedure. The assay routinely detected 1 to 10 oocysts in purified oocyst preparations, as shown by direct microscopic counts and by an immunofluorescence assay. The sensitivity of the PCR assay in some seeded environmental water samples was up to 1,000-fold lower. However, this interference was eliminated by either flow cytometry or magnetic-antibody capture. Sensitivity was also improved 10- to 1,000-fold by probing of the PCR product on dot blots with an oligonucleotide probe detected by chemiluminescence. Confirmation of the presence of Cryptosporidium oocysts in water samples from the outbreak in Milwaukee, Wis., was obtained with this technique, and PCR was found to be as sensitive as immunofluorescence for detection of oocysts in wastewater concentrates.

Jongwutiwes, S., R. Tiangtip, et al. (2002). "Simple method for long-term copro-preservation of Cryptosporidium oocysts for morphometric and molecular analysis." Trop Med Int Health 7(3): 257-64.
	Preservation of Cryptosporidium oocysts in faecal specimens containing 75% ethanol is suitable for subsequent morphometric and molecular analysis. No significant morphologic alteration occurred after storage at ambient temperatures, ranging from 22 to 38 degrees C, for more than 2 years. After washing, sugar floatation and DNA extraction, a nested polymerase chain reaction targeting the small subunit ribosomal RNA gene successfully amplified Cryptosporidium DNA in all 15 isolates examined. The sensitivity of detection by polymerase chain reaction (PCR) was found to be as high as 1.25 oocysts per reaction (mean=3.01, SD=1.14). Importantly, a 2.2-kb of the complete DNA sequence of a gene encoding Cryptosporidium thrombospondin-related adhesive protein (TRAP-C1) was also consistently amplified by PCR in all isolates. The PCR-amplified product can be used as a good template for sequencing. Therefore, this simple procedure should be useful for epidemiological analysis of clinical samples from outbreaks, endemic or sporadic cases of cryptosporidiosis when long-term storage of oocysts is required.

Jonnalagadda, P. R. and R. V. Bhat (1995). "Parasitic contamination of stored water used for drinking/cooking in Hyderabad." Southeast Asian J Trop Med Public Health 26(4): 789-94.
	A study was undertaken to investigate the parasitic contamination of water in Hyderabad city, India. A total of 232 samples of water were collected from different places; social welfare hostels, small restaurants, different households, public places like railway stations, bus depots, street food vendors, hand washings from the food handlers, and vegetable washings from vegetable vendors. Of these 232 samples 61 samples indicated the presence of pathogenic parasites which include protozoans (cysts of Giardia lamblia, Entamoeba histolytica, adult stages of G. lamblia, Balantidium coli) and nematode eggs, (Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura), rhabditiform and filariform larvae and adult stages of Strongyloides stercoralis and Enterobius vermicularis. The source of the samples in all places was the water stored in overhead tanks and various other containers. Hand washings from food handlers also showed the presence of pathogenic parasites although the original water used for such washings were free from contamination.

Jordan, C. N., J. A. Dicristina, et al. (2006). "Activity of bleach, ethanol and two commercial disinfectants against spores of Encephalitozoon cuniculi." Vet Parasitol 136(3-4): 343-6.
	Encephalitozoon cuniculi is a small protist parasite in the phylum Microspora. Hosts are infected by ingestion or inhalation of spores passed in the urine or feces. Infection with E. cuniculi is usually asymptomatic, except in young or immunocompromised hosts. This study examined the effects of various disinfectants on in vitro infectivity of E. cuniculi spores. Spores of E. cuniculi were exposed to several dilutions of commercial bleach, 70% ethanol and dilutions of commercial disinfectants HiTor and Roccal for 10 min and then loaded onto human fibroblast cells (Hs68 cells). Ten minutes of exposure to these disinfectants was lethal to E. cuniculi spores. Additional exposure time studies were done using dilutions of bleach at 0.1, 1 and 10%, and 70% ethanol. Exposure of E. cuniculi spores to 1 or 10% bleach for 30s rendered them non-infectious for Hs68 cells. Growth of E. cuniculi was observed in Hs68 cells inoculated with spores treated with 0.1% bleach for 30s or 1, 3 and 5 min, but not with spores treated for 7 min or longer. Exposure of E. cuniculi spores to 70% ethanol for 30s rendered them non-infectious for Hs68 cells. Spores of E. cuniculi are more sensitive to disinfectants than are coccidial oocysts and other parasite cysts. The relatively short contact time needed to kill spores indicates that disinfection of animal housing may be a viable means to reduce exposure of animals to E. cuniculi spores.

Jordan, C. N., A. M. Zajac, et al. (2006). "Direct agglutination test for Encephalitozoon cuniculi." Vet Parasitol 135(3-4): 235-40.
	Encephalitozoon cuniculi is a small protozoan parasite in the phylum Microspora. It has been shown to naturally infect several host species, including humans. Infection with microsporidia is usually asymptomatic, except in young or immunocompromised hosts. Currently, serological diagnosis of infection is made using the indirect immunofluorescent antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA). Although these methods are sensitive and reliable, there are several drawbacks to the IFA and ELISA tests. Cross-reactivity between other Encephalitozoon species is common, and specialized equipment is required to conduct these tests. This paper reports the development of a direct agglutination test for detecting IgG antibodies to E. cuniculi. The utility of the agglutination test was examined in CD-1 and C3H/He mice infected with E. cuniculi or one of 2 other Encephalitozoon species. Test sera were incubated overnight with eosin-stained microsporidia spores in round-bottom microtiter plates. In positive samples, agglutination of spores with antibodies in test sera resulted in an opaque mat spread across the well. The results indicate that the agglutination test is 86% sensitive and 98% specific for E. cuniculi, with limited cross-reactivity to Encephalitozoon intestinalis. No cross-reactivity to Encephalitozoon hellem was observed. The test is fast and easy to conduct, and species-specific antibodies are not required.

Joshi, N. V., H. Medina, et al. (2001). "Effect of an electric field on the motility of Entamoeba histolytica examined by multiple-beam interference microscopy." Exp Parasitol 97(4): 179-85.
	A recently developed multiple-beam interference microscopic technique has been used to visualize submicroscopic structures of Entamoeba histolytica and their movements in applied external electric fields. The movements were videorecorded and it was found that at low current (120 microA) pseudopods are filled with hyaline ectoplasm. At slightly higher current (about 150 microA), the amoeba stops extending the pseudopods and loosens its attachment to the surface. At higher currents (200 microA), it forms a cyst and remains immobile for a time. Before this stage is reached a narrow ring is formed around the nucleus due to alterations in the proteins to protect it.

Juranek, D. D. (1997). "Cryptosporidium and water: a public health handbook--1997." Clin Lab Sci 10(5): 272.
	
Katsumata, T., D. Hosea, et al. (2000). "Short report: possible Cryptosporidium muris infection in humans." Am J Trop Med Hyg 62(1): 70-2.
	Oocysts of cryptosporidia whose morphology resembled that of Cryptosporidium muris were found in the stool of 2 healthy girls in Surabaya, Indonesia. The oocysts were predominantly oval and measured 7.75+/-0.17 x 5.55+/-0.13 microm (mean+/-SD). The number of oocysts excreted were more than 10(5) per gram of stool. The oocysts were well stained with fluorescein-conjugated monoclonal antibody to Cryptosporidium. The specimens from both girls containing the oocysts showed a positive result by the polymerase chain reaction (PCR) using primers specific for the genus Cryptosporidium, but a negative result by the PCR using primers specific for C. parvum. The 2 girls passed oocysts for 5 and 6 days, respectively. They did not complain of any symptoms during the passage of oocysts.

Katsumata, T., D. Hosea, et al. (1998). "Cryptosporidiosis in Indonesia: a hospital-based study and a community-based survey." Am J Trop Med Hyg 59(4): 628-32.
	Hospital-based and community-based studies were conducted to understand the prevalence and mode of transmission of Cryptosporidium parvum infection in Surabaya, Indonesia. In both studies people with and without diarrhea were examined for oocysts. A community-based survey included questionnaires to a community and stool examination of cats. Questionnaires covered demographic information, health status, and hygienic indicators. In the hospital, C. parvum oocysts were found in 26 (2.8%) of 917 patients with diarrhea and 15 (1.4%) of 1,043 control patients. The most susceptible age was less than two years old. The prevalence was higher during the rainy season. A community-based study again showed that C. parvum oocysts were frequently detected in diarrhea samples (8.2%), exclusively during rainy season. Thirteen (2.4%) of 532 cats passed C. parvum oocysts. A multiple logistic regression model indicated that contact with cats, rain, flood, and crowded living conditions are significant risk factors for Cryptosporidium infection.

Katz, D. E. and D. N. Taylor (2001). "Parasitic infections of the gastrointestinal tract." Gastroenterol Clin North Am 30(3): 797-815, x.
	This article updates recent advances in the body of knowledge of diagnosis and treatment of intestinal parasites. The articles focus on the manifestations of disease in the immunocompetent adult host from developed countries. Specific pathogens discussed are Giardia lamblia and Dientamoeba fragilis, Entamoeba histolytica, Entamoeba dipar, Blastocystis hominis, Cyclospora cayetanensis, and Cryptosporidium parvum.

Kaucner, C. and T. Stinear (1998). "Sensitive and rapid detection of viable Giardia cysts and Cryptosporidium parvum oocysts in large-volume water samples with wound fiberglass cartridge filters and reverse transcription-PCR." Appl Environ Microbiol 64(5): 1743-9.
	We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-microliter packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardia cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting that Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.

Kayser, O., W. R. Waters, et al. (2001). "Evaluation of in vitro activity of aurones and related compounds against Cryptosporidium parvum." Planta Med 67(8): 722-5.
	The efficacy of a series of aurones, auronols and 4-methoxy-alpha-pyrones has been screened for the ability to inhibit the intracellular growth of the parasitic protist Cryptosporidium parvum using an in vitro enzyme linked immunosorbent assay (ELISA). All aurones of this series were active at 25 to 100 microM. 10 of 19 aurones inhibited the intracellular growth of C. parvum by > 90 % with moderate to no toxicity. The most active of these was 3',4',6-trihydroxy-2-[phenylmethylene]-3(2H)-benzofuranone.

Kayser, O., W. R. Waters, et al. (2002). "Evaluation of in vitro and in vivo activity of benzindazole-4,9-quinones against Cryptosporidium parvum." J Antimicrob Chemother 50(6): 975-80.
	A series of benzindazole-4,9-quinones was tested for growth-inhibitory effects on Cryptosporidium parvum in vitro and in vivo. Most compounds showed considerable activity at concentrations from 25 to 100 micro M. For instance, at 25 micro M the derivatives 5-hydroxy-8-chloro-N1-methylbenz[f]-indazole-4,9-quinone and 5-chloro-N2-methylbenz[f]indazole-4,9-quinone inhibited growth of C. parvum 78-100%, and at 50 micro M seven of the 23 derivatives inhibited growth > or = 90%. The activity of the former two compounds was confirmed in a T-cell receptor alpha (TCR-alpha)-deficient mouse model of chronic cryptosporidiosis. In these mice, the mean infectivity scores (IS) in the caecum were 0.63-0.20, whereas in sham-treated mice the score was 1.44 (P < 0.05). There were similar differences in IS in the ileum, where the score for treated mice was 1.12-0.20 and that for mice receiving no drug was 1.32. There was no acute or chronic toxicity for any compound tested in vivo.

Keegan, A. R., S. Fanok, et al. (2003). "Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection." Appl Environ Microbiol 69(5): 2505-11.
	Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.

Keithly, J. S., G. Zhu, et al. (1997). "Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy." Mol Biochem Parasitol 88(1-2): 35-42.
	This study demonstrates that polyamine biosynthesis in Cryptosporidium parvum occurs via a pathway chiefly found in plants and some bacteria. The lead enzyme of this pathway, arginine decarboxylase (ADC) was sensitive to the specific, irreversible inhibitor DL-alpha-difluoromethyl-arginine (IC50 30 microM), and intracellular growth of C. parvum was significantly reduced by inhibitors of ADC. No activity was detected using ornithine as substrate, and the irreversible inhibitor of ornithine decarboxylase, DL-alpha-difluoromethyl-ornithine, had no effect upon ADC activity or upon growth of the parasite. Back-conversion of spermine to spermidine and putrescine via spermidine:spermine-N1-acetyltransferase (SSAT) was also detected. Compounds such as his(ethyl)norspermine, which have been demonstrated to down-regulate SSAT activity in tumor cells, were synergistic in the inhibition of growth when used in combination with inhibitors of the forward pathway. Thus, C. parvum differs fundamentally in its polyamine metabolism from the majority of eukaryotes, including humans. Such differences indicate that polyamine metabolism may serve as a chemotherapeutic target in this organism.

Khramtsov, N. V., D. S. Blunt, et al. (1996). "The putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-CoA synthetases." J Parasitol 82(3): 423-7.
	We determined the nucleotide (nt) sequence of the putative gene encoding acetyl-coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum. The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns. The gene is characterized by low codon usage bias and encodes a 694-amino acid (aa) protein with a predicted molecular size of 78 kDa, similar to other ACSs from different prokaryotic and eukaryotic species. Comparison of multiple protein alignments of ACSs revealed a new conserved sequence motif PKT(R/V/L)SGK(I/V/T)(T/M/V/K)R(R/N) near the C-terminus, which may be a signature for ACSs. This motif shares significant homology with sequences from other members of the AMP-binding family, has secondary structure similar to the purine-binding motif of ATP- and GTP-ases, and may play a role in the enzymatic activity of proteins from the AMP-binding family.

Khramtsov, N. V., P. A. Chung, et al. (2000). "Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum." J Parasitol 86(2): 275-82.
	We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Khramtsov, N. V., M. Tilley, et al. (1995). "Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70." J Eukaryot Microbiol 42(4): 416-22.
	An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

Khramtsov, N. V. and S. J. Upton (2000). "Association of RNA polymerase complexes of the parasitic protozoan Cryptosporidium parvum with virus-like particles: heterogeneous system." J Virol 74(13): 5788-95.
	RNA polymerase complexes were purified from Cryptosporidium parvum, a parasitic protozoan known to infect many species of mammals including humans. Western blot analysis revealed the association of the complexes with two different proteins, encoded by large and small segments of viral double-stranded RNAs. Each complex was found to contain only double-stranded RNA, both double- and single-stranded RNA, or only single-stranded RNA. Maximum RNA-dependent RNA polymerase activity was observed within the complexes containing both double- and single-stranded RNAs. These complexes possessed both transcriptase and replicase polymerase activities. Virus-like particles with a diameter of 31 nm were copurified with RNA polymerase complexes, and buoyant density and polymerase studies suggest that C. parvum harbors a putative double-stranded RNA virus which separately encapsidates the large and small RNA segments. The mechanism of replication and other characteristics of this virus are similar to those of the viruses of the family Partitiviridae, previously identified only in fungi and plants.

Khramtsov, N. V., K. M. Woods, et al. (1997). "Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum." Mol Microbiol 26(2): 289-300.
	We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.

Kijlstra, A., O. A. Eissen, et al. (2004). "Toxoplasma gondii infection in animal-friendly pig production systems." Invest Ophthalmol Vis Sci 45(9): 3165-9.
	PURPOSE: Consumption of undercooked pork meat products has been considered a major risk factor for contracting toxoplasmosis in humans. Indoor farming and improved hygiene have drastically reduced Toxoplasma infections in pigs over the past decades. Whether introduction of animal-friendly production systems will lead to a reemergence of Toxoplasma infections in pigs is not yet known. Investigating this possibility was the purpose of this study. METHODS: Blood was obtained from pigs raised for slaughter and tested for Toxoplasma antibodies by using latex agglutination and indirect immunofluorescence testing, with confirmation by immunoblotting. RESULTS: None of the slaughter pigs (n = 621) from conventional farms (n = 30) were positive, whereas 38 (2.9%) of 1295 animals from animal-friendly systems tested positive (n = 33 farms; 13 [39%] farms positive). CONCLUSIONS: The following conclusions may be derived from this study: Conventionally (indoors) raised pigs are free from Toxoplasma infection, and (2) animal-friendly production systems may lead to a reemergence of Toxoplasma infections, although many of these farms remain Toxoplasma free. Slaughterhouse monitoring of pigs from animal-friendly production systems combined with on-farm prevention strategies should be applied to ensure safety for consumers of the meat products obtained from these animals.

King, B. J., A. R. Keegan, et al. (2005). "Environmental temperature controls cryptosporidium oocyst metabolic rate and associated retention of infectivity." Appl Environ Microbiol 71(7): 3848-57.
	Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4 degrees C and 15 degrees C remained infective over the 12-week holding period, we observed a 4 log(10) reduction in infectivity for both 20 and 25 degrees C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37 degrees C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.

Kistemann, T., T. Classen, et al. (2002). "Microbial load of drinking water reservoir tributaries during extreme rainfall and runoff." Appl Environ Microbiol 68(5): 2188-97.
	Hygienic and microbiological examinations of watercourses are usually not carried out during heavy rainfall and runoff events. After rainfall or snowmelt, there are often massive increases in turbidity in flooding creeks in mountain ranges, which are frequently interpreted as an indication of microbial contamination. The aim of this study was to quantify the microbial loads of watercourses during such runoff events and to compare these loads with loads occurring during regular conditions. In a 14-month monitoring period we investigated the microbial loads of three tributaries of different drinking water reservoirs. A total of 99 water samples were taken under different runoff conditions and analyzed to determine physical, chemical, bacterial, and parasitic parameters. Thirty-two water samples were considered event samples during nine measuring series. The criteria for events, based on duration and intensity of precipitation, water depth gauge measurements, and dynamics, had been fixed before the investigation for each creek individually. Of the physical and chemical parameters examined, only the turbidity, pH, and nitrate values differed clearly from the values obtained for regular samples. Most of the bacteriological parameters investigated (colony, Escherichia coli, coliform, fecal streptococcal, and Clostridium perfringens counts) increased considerably during extreme runoff events. If relevant sources of parasitic contamination occurred in catchment areas, the concentrations of Giardia and Cryptosporidium rose significantly during events. The results show that substantial shares of the total microbial loads in watercourses and in drinking water reservoirs result from rainfall and extreme runoff events. Consequently, regular samples are considered inadequate for representing the microbial contamination of watercourse systems. The procedures for raw water surveillance in the context of multiple-barrier protection and risk assessment ought to include sampling during extreme runoff situations.

Kniel, K. E., J. A. Higgins, et al. (2004). "Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts." J Microbiol Methods 58(2): 189-95.
	The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.

Kniel, K. E. and M. C. Jenkins (2005). "Detection of Cryptosporidium parvum oocysts on fresh vegetables and 1 herbs using antibodies specific for a Cryptosporidium parvum viral antigen." J Food Prot 68(5): 1093-6.
	The purpose of this study was to determine if the viral symbiont of Cryptosporidium parvum (CPV) sporozoites could be used as a target for sensitive detection of the parasite in food samples. Polyclonal sera specific to the recombinant viral capsid protein (rCPV40) was used in a dot blot hybridization assay to detect oocysts recovered from green onions and cilantro. Small batches of chopped green onions and cilantro leaves were artificially contaminated with three different concentrations of oocysts: 10(6), 10(2), and 10(1). rCPV40 was superior in detecting oocysts compared with other antibodies directed toward total oocyst protein and oocyst surface antigens. This study provides evidence that CPV is an excellent target for sensitive detection of C. parvum oocysts in foods.

Kniel, K. E., S. S. Sumner, et al. (2003). "Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices." J Food Prot 66(9): 1650-7.
	Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.

Kniel, K. E., S. S. Sumner, et al. (2004). "Effect of hydrogen peroxide and other protease inhibitors on Cryptosporidium parvum excystation and in vitro development." J Parasitol 90(4): 885-8.
	This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by >90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.

Koch, K. L., T. V. Shankey, et al. (1983). "Cryptosporidiosis in a patient with hemophilia, common variable hypogammaglobulinemia, and the acquired immunodeficiency syndrome." Ann Intern Med 99(3): 337-40.
	A 36-year-old man had chronic, debilitating diarrhea due to cryptosporidiosis. This patient had longstanding common variable hypogammaglobulinemia and recurrent bacterial infections. Immunologic evaluation after discovery of Cryptosporidium showed lymphopenia with persistently reduced numbers of helper/inducer cells (OKT-4), variable numbers of suppressor/cytotoxic cells (OKT-8), OKT-4/OKT-8 ratio of 0.09, and increased levels of serum alpha-interferon, all of which describe the acquired immunodeficiency syndrome. Oocysts of Cryptosporidium were found in feces from the patient's cat, thus identifying a possible source of his infection. The patient had disseminated candidiasis, cytomegalovirus pneumonia, and cryptosporidiosis when he died.

Korich, D. G., J. R. Mead, et al. (1990). "Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability." Appl Environ Microbiol 56(5): 1423-8.
	Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.

Kosek, M., C. Alcantara, et al. (2001). "Cryptosporidiosis: an update." Lancet Infect Dis 1(4): 262-9.
	Cryptosporidiosis was recognised in human beings in 1976, and was prominent in the 1980s and 1990s as a cause of severe diarrhoeal illness in patients with AIDS. It is now additionally recognised as a major cause of waterborne diarrhoeal illness in developed regions, and as a pathogen with long-term effect on childhood growth and development in impoverished areas. This update focuses on recent changes in our understanding of the taxonomy of cryptosporidium, its epidemiology, effects, pathogenesis, diagnosis, and treatment.

Kostrzynska, M., M. Sankey, et al. (1999). "Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples." J Microbiol Methods 35(1): 65-71.
	Cryptosporidium parvum is a protozoan parasite responsible for an increasing number of outbreaks of gastrointestinal illness worldwide. In this report, we describe development of sample preparation protocols for polymerase chain reaction (PCR)-based detection of C. parvum in fecal material and environmental water samples. Two of these methods were found adequate for isolation of Cryptosporidium DNA from filtered water pellet suspensions. The first involved several filtration steps, immunomagnetic separation and freeze-thaw cycles. The second method involved filtration, addition of EnviroAmp lysis reagent, freeze-thaw cycles and precipitation of the DNA with isopropanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pellet suspension, with either of the above sample preparation procedures. Nested PCR increased sensitivity of the assay by two to three orders of magnitude as compared to the primary PCR. The detection limit for seeded fecal samples was 10-fold higher than for filtered environmental water pellet suspension. Nested PCR results showed 62.4 and 91.1% correlation with immunofluorescence assay (IFA) for fecal samples and filtered environmental water pellet suspensions, respectively. This correlation decreased to 47.2% and 44.4%, respectively, when only IFA positive samples were analyzed. However, in fecal samples contaminated with a high number (> 10(5)/g) of C. parvum oocysts, this correlation was 100%.

Kramer, M. H., F. E. Sorhage, et al. (1998). "First reported outbreak in the United States of cryptosporidiosis associated with a recreational lake." Clin Infect Dis 26(1): 27-33.
	In the summer of 1994, an outbreak of cryptosporidiosis occurred among visitors to a state park in New Jersey. We enrolled 185 persons in a cohort study, 38 (20.5%) of whom had laboratory-confirmed cryptosporidiosis or gastrointestinal illness that met our clinical case definition. Having any exposure to lake water (e.g., swimming) was strongly associated with illness (P < .001). The outbreak lasted 4 weeks and affected an estimated 2,070 persons. The most likely sources of the outbreak were contaminated runoff of rainwater and infected bathers. This outbreak of cryptosporidiosis is the first reported to be associated with recreational exposure to lake water. Our investigation shows that even a large and ongoing outbreak may not be detected for several weeks. Health professionals and persons at high risk for severe cryptosporidiosis should be aware that recreational water can be a source of cryptosporidium infection.

Kreader, C. A. (1996). "Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein." Appl Environ Microbiol 62(3): 1102-6.
	The benefits of adding bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to PCR were evaluated with reaction mixtures containing substances that inhibit amplification. Whereas 10- to 1,000-fold more FeCl3, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces, freshwater, or marine water were accommodated in PCR when either 400 ng of BSA per microl or 150 ng of gp32 per microl was included in the reactions, neither BSA nor gp32 relieved interference significantly when minimum inhibitory levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate, or Triton X-100 were present. Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors.

Kuczynska, E. and D. R. Shelton (1999). "Method for detection and enumeration of Cryptosporidium parvum oocysts in feces, manures, and soils." Appl Environ Microbiol 65(7): 2820-6.
	Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 10(2), 10(3), 10(4), and 10(5) oocysts g of bovine feces-1. The percentages of recovery ranged from 10.8% (25 oocysts g-1) to 17.0% (10(4) oocysts g-1) (r2 = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces-1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (10(4) oocysts g-1). The percentages of recovery were highest for bovine feces (17. 0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris-0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil-1 depending on the soil type. The percentages of recovery without dispersant (distilled H2O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.

Laberge, I. and M. W. Griffiths (1996). "Prevalence, detection and control of Cryptosporidium parvum in food." Int J Food Microbiol 32(1-2): 1-26.
	The role of Cryptosporidium parvum as a foodborne pathogen has not been well documented. Epidemiological features of this parasitic protozoon lead to the assumption that the incidence of cryptosporidiosis due to contaminated food is under-estimated. The high prevalence of C. parvum among dairy herds has increased the spread of oocysts in the farm environment, and their potential presence in raw milk and other raw foods. In October 1993, the first well-documented foodborne outbreak was reported in Maine, USA, and was caused by contaminated hand-pressed apple cider. Although various cases of cryptosporidiosis among humans have pointed to raw milk and other raw foods as possible sources of infection, a conclusive demonstration of foodborne cryptosporidiosis has rarely been established. The limited numbers of oocysts in the suspected samples and the lack of sensitive detection methods adapted for oocyst detection in food contribute to this under-reporting. This review paper discusses various aspects of Cryptosporidium spp. and cryptosporidiosis, including the routes of transmission, the control of oocysts in food, and the available detection methods. The polymerase chain reaction (PCR) combined with DNA probe hybridization is a promising detection method. Recent knowledge on the molecular biology of the parasite for the development of new PCR assays and their potential use in the detection of C. parvum in food are described.

Laberge, I., A. Ibrahim, et al. (1996). "Detection of Cryptosporidium parvum in raw milk by PCR and oligonucleotide probe hybridization." Appl Environ Microbiol 62(9): 3259-64.
	Cryptosporidium spp. are potential contaminants of food. Suspected cases of food-borne cryptosporidiosis are rarely confirmed because of the limited numbers of oocysts in the samples and the lack of sensitive detection methods adaptable to food. PCR was investigated as a means of overcoming this problem. A PCR assay was designed for the specific amplification of a previously sequenced portion of an oocyst protein gene fragment of Cryptosporidium parvum (N. C. Lally, G. D. Baird, S. J. McQuay, F. Wright, and J. J. Oliver, Mol. Biochem. Parasitol. 56:69-78, 1992) and compared with the primer set of Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991). The PCR products were hybridized with digoxigenin-labeled internal probes and detected by chemiluminescence to enhance sensitivity. The two sets of primers were compared with regard to their sensitivity and specificity by using a variety of human and animal isolates of C. parvum and related parasites. Both assays enabled the detection of 1 to 10 oocysts in 20 ml of artificially contaminated raw milk. The assay based on the PCR set and probe of Laxer et al. detected DNAs from Eimeria acervulina and Giardia intestinalis. The new assay has good specificity for C. parvum bovine isolates and hence has a better potential for monitoring the prevalence of C. parvum in raw milk and other environmental samples.

Lalle, M., E. Jimenez-Cardosa, et al. (2005). "Genotyping of Giardia duodenalis from humans and dogs from Mexico using a beta-giardin nested polymerase chain reaction assay." J Parasitol 91(1): 203-5.
	Cysts of Giardia duodenalis were collected in Mexico from symptomatic children (n = 9) and from pet dogs (n = 5), and they were directly characterized by nested polymerase chain reaction (PCR) amplification of the beta-giardin gene. Eight isolates of human origin established as in vitro cultures and 2 reference strains, representing assemblages A and B of G. duodenalis, were also analyzed. PCR-restriction fragment length polymorphism showed that all isolates belonged to assemblage A. Sequence analyses indicated that the large majority of isolates were of the A1 genotype; interestingly, 2 human isolates displayed the A3 genotype, which has been previously identified in human isolates from Italy. The presence of cysts of the A1 and A3 genotypes in isolates from pet dogs is consistent with their role as reservoirs for human infection, although further studies are needed to confirm the occurrence of zoonotic transmission. Remarkably, cysts of assemblage B have not been found in any of the Mexican isolates studied to date.

Lalle, M., E. Pozio, et al. (2005). "Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardiaduodenalis and identification of potentially zoonotic subgenotypes." Int J Parasitol 35(2): 207-13.
	Human giardiasis, caused by the intestinal flagellate Giardia duodenalis, is considered a zoonotic infection, although the role of animals in the transmission to humans is still unclear. Molecular characterisation of cysts of human and animal origin represents an objective means to validate or reject this hypothesis. In the present work, cysts were collected in Italy from humans (n=37) and animals (dogs, one cat and calves, n=46), and were characterised by PCR amplification and sequencing of the beta-giardin gene. As expected, only Assemblages A and B were identified among human isolates. The host-specific Assemblages C and D were found in the majority of dog isolates; however, 6 dog isolates were typed as Assemblage A. The cat-specific Assemblage F has been identified in the single feline isolate available. Among calf isolates, most were typed as Assemblages A (n=12) and B (n=5), whereas the host-specific Assemblage E was rarely found (n=3). Sequence heterogeneity in the beta-giardin gene allowed a number of subgenotypes to be identified within Assemblage A (8 subgenotypes), B (6 subgenotypes), D (2 subgenotypes), and E (3 subgenotypes). Five of these subgenotypes, namely A1, A2, A3, A4 and B3, were found to be associated with infections of humans, of dogs and of calves; these data, therefore, supported the role of these animals as a source of infection for humans.

Lapham, S. C., R. S. Hopkins, et al. (1987). "A prospective study of giardiasis and water supplies in Colorado." Am J Public Health 77(3): 354-5.
	A prospective study of 484 visitors to Vail and Aspen/Snowmass, Colorado, was conducted to determine the risk of acquiring giardiasis. Of the 259 visitors to Vail, no cases of giardiasis were confirmed and only one of 12 water filtrates were positive for Giardia cysts. Of 225 visitors to Aspen/Snowmass two cases of giardiasis were confirmed and 12 of 20 water filtrates were positive for Giardia cysts. The regular occurrence of Giardia cysts in Aspen and Snowmass water was associated with lower rates of giardiasis acquisition than reported during outbreaks of waterborne giardiasis.

Laxer, M. A., A. K. Alcantara, et al. (1990). "Immune response to cryptosporidiosis in Philippine children." Am J Trop Med Hyg 42(2): 131-9.
	An ELISA was used to measure the Cryptosporidium-specific IgA, IgG, and IgM antibody levels in serum, stool, and duodenal fluid of 15 Filipino children. Antibody levels were measured on admission to the hospital, 1 week later, and at a 6 week follow-up examination. Delayed type hypersensitivity skin tests were used to assay cell mediated immunity (CMI), iron status was measured by serum iron tests and total iron binding capacity, and the degree of malnutrition was determined by clinical examination. Antibody response to Cryptosporidium was qualitatively and quantitatively strong and maintained over time. All subjects showed impaired CMI early with some reconstitution after 6 weeks. All subjects showed some degree of malnutrition and/or depleted iron status.

Laxer, M. A., M. E. D'Nicuola, et al. (1992). "Detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue by the polymerase chain reaction." Am J Trop Med Hyg 47(4): 450-5.
	The objective of this project was to demonstrate detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue using the polymerase chain reaction (PCR). DNA was purified from six samples of fixed, paraffin-embedded tissue that were histologically positive for C. parvum and used in the PCR. Previously developed oligonucleotide primers specific for C. parvum were used to amplify a 452-base target sequence, and a 20-base synthetic probe labeled with digoxigenin-11-dUTP was used to detect the amplification product by chemiluminescence. All six samples were positive by PCR; negative controls showed no amplification or hybridization. This approach could provide a sensitive and specific method for detection of parasite material in fixed, paraffin-embedded tissue samples, and prove to be of significant value in retrospective studies of archival material.

Laxer, M. A., B. K. Timblin, et al. (1991). "DNA sequences for the specific detection of Cryptosporidium parvum by the polymerase chain reaction." Am J Trop Med Hyg 45(6): 688-94.
	The objective of this project was to construct specific and sensitive molecular probes and amplification primers for Cryptosporidium parvum that could be used in diagnosis, retrospective tissue studies, and in epidemiologic surveys. Whole genomic DNA was extracted from oocysts of C. parvum purified from human and bovine feces. A genomic library was constructed in plasmid pUC18 and propagated in Escherichia coli DH5 alpha. Transformants were screened by colony hybridization and autoradiography. The 2.3-kilobase segment in plasmid pHC1, a clone specific for C. parvum, was sequenced by the Sanger method. Computer analysis gave a G+C content of 35%. A 400-base region (bases 470-870) was selected as an amplification target because it contained a unique restriction endonuclease site that could serve as a useful marker. Primers of 26 nucleotides each were synthesized. Sensitive and specific amplification of the target sequence was demonstrated both by ethidium bromide staining of agarose and acrylamide gels, and by hybridization with chemiluminescence-labeled synthetic oligonucleotide probes.

Le Blancq, S. M., N. V. Khramtsov, et al. (1997). "Ribosomal RNA gene organization in Cryptosporidium parvum." Mol Biochem Parasitol 90(2): 463-78.
	The cytoplasmic ribosomal RNA (rRNA) genes of the Apicomplexan protozoan parasite Cryptosporidium parvum have been analyzed with respect to size, copy number, organization and structure. The small and large subunit rRNAs are 1.7 and 3.6 kb, respectively. A 151 bp putative 5.8S rRNA gene was identified. The rDNA unit is 5' small subunit rRNA internal transcribed spacer 1-5.8S rRNA-internal transcribed spacer 2-large subunit rRNA 3'. There are five copies of the rDNA unit per haploid genome and they are not organized in a conventional head to tail tandem array with a conserved external transcribed spacer. The rDNA units are dispersed through the genome to at least three chromosomes. At least two of the rDNA units are single unlinked copies on different chromosomes. There are two structurally distinct types of rDNA unit, Type A and B, with marked differences in the internal transcribed spacer regions. There are four copies of the Type A rDNA unit and one copy of the Type B rDNA unit.

Learmonth, J. J., G. Ionas, et al. (2004). "Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand." Appl Environ Microbiol 70(7): 3973-8.
	Little is known about the genetic characteristics, distribution, and transmission cycles of Cryptosporidium species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing Cryptosporidium oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall, Cryptosporidium hominis accounted for 47% of the isolates, with the remaining 53% being the C. parvum bovine genotype. A difference, however, was observed between the Cryptosporidium species from rural and urban isolates, with C. hominis dominant in the urban region, whereas the C. parvum bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel Cryptosporidium sp., which on DNA sequence analysis showed a close relationship with C. canis, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.

Leav, B. A., M. R. Mackay, et al. (2002). "Analysis of sequence diversity at the highly polymorphic Cpgp40/15 locus among Cryptosporidium isolates from human immunodeficiency virus-infected children in South Africa." Infect Immun 70(7): 3881-90.
	Cryptosporidium sp. is a significant cause of diarrheal disease, particularly in human immunodeficiency virus (HIV)-infected patients in developing countries. We recently cloned and sequenced several alleles of the highly polymorphic single-copy Cryptosporidium parvum gene Cpgp40/15. This gene encodes a precursor protein that is proteolytically cleaved to yield mature cell surface glycoproteins gp40 and gp15, which are implicated in zoite attachment to and invasion of enterocytes. The most-striking feature of the Cpgp40/15 alleles and proteins is their unprecedented degree of sequence polymorphism, which is far greater than that observed for any other gene or protein studied in C. parvum to date. In this study we analyzed nucleic acid and amino acid sequence polymorphism at the Cpgp40/15 locus of 20 C. parvum isolates from HIV-infected South African children. Fifteen isolates exhibited one of four previously identified genotype I alleles at the Cpgp40/15 locus (Ia, Ib, Ic, and Id), while five displayed a novel set of polymorphisms that defined a new Cpgp40/15 genotype I allele, designated genotype Ie. Surprisingly, only 15 of these isolates exhibited concordant type I alleles at the thrombospondin-related adhesive protein of Cryptosporidium and Cryptosporidium oocyst wall protein loci, while five isolates (all of which displayed Cpgp40/15 genotype Ic alleles) displayed genotype II alleles at these loci. Furthermore, the last five isolates also manifested chimeric genotype Ic/Ib or Ic/II alleles at the Cpgp40/15 locus, raising the possibility of sexual recombination within and between prototypical parasite genotypes. Lastly, children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other genotype I alleles.

Leav, B. A., M. Yoshida, et al. (2005). "An early intestinal mucosal source of gamma interferon is associated with resistance to and control of Cryptosporidium parvum infection in mice." Infect Immun 73(12): 8425-8.
	Resistance to and control of Cryptosporidium parvum infection in mice in the absence of adaptive immunity appears to be gamma interferon (IFN-gamma) dependent. Using an IFN-gamma-neutralizing antibody in a murine model, we demonstrated increased susceptibility to infection within 24 h. We correlated this early resistance and control with increased mucosal expression of IFN-gamma and demonstrate that CD8+ T-cell receptor alphabeta intestinal intraepithelial lymphocytes express and secrete this cytokine shortly after infection. The rapid kinetics of IFN-gamma expression and secretion by naive CD8+ T cells in response to a protozoan pathogen have not previously been demonstrated.

LeChevallier, M. W., G. D. Di Giovanni, et al. (2003). "Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters." Appl Environ Microbiol 69(2): 971-9.
	Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods. The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06). Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623. Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique. There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites. The other two sites had 16.3 and 24% correspondence between the methods. Infectious oocysts were detected in all of the watersheds. Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious. DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes. More than 90% of the C. parvum isolates were identified as having the bovine or bovine-like genotype. The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods. The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.

LeChevallier, M. W., W. D. Norton, et al. (1991). "Giardia and Cryptosporidium spp. in filtered drinking water supplies." Appl Environ Microbiol 57(9): 2617-21.
	Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.

LeChevallier, M. W., W. D. Norton, et al. (1991). "Occurrence of Giardia and Cryptosporidium spp. in surface water supplies." Appl Environ Microbiol 57(9): 2610-6.
	Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.

Lee, M. B. (1992). "The effectiveness of commercially available disinfectants upon Giardia lamblia cysts." Can J Public Health 83(2): 171-2.
	
Lefay, D., M. Naciri, et al. (2000). "Prevalence of Cryptosporidium infection in calves in France." Vet Parasitol 89(1-2): 1-9.
	Two multicentre surveys were conducted in France to estimate the prevalence of Cryptosporidium infection in calves using qualitative ELISA for detection of Cryptosporidium coproantigens and oocysts. The first survey involved 4-12-day-old calves in six dairy-calf distribution centres, collecting calves from seven Administrative Regions (Aquitaine, Bretagne, Franche-Comte, Lorraine, Normandie, Nord, Pays de Loire). For each region, 20 calves were selected every month for 12 consecutive months (October 1995-September 1996). Prevalence of Cryptosporidium infection was 17.9% (Confidence Intervals (C.I.) 95%=[16.1%; 19.8%]) among the 1628 selected calves, of which only 5.3% had diarrhoea. The second survey conducted between November 1995 and May 1996 involved 4-21-day-old calves examined by veterinary practitioners who selected 189 livestock farms of dairy- or suckler-type in ten Administrative Departments (Allier, Cantal, Creuse, Doubs, Ille-et-Vilaine, Maine-et-Loire, Manche, Pas-de-Calais, Saone-et-Loire, Vendee). Cryptosporidia were detected in 105 (55.6%) of the farms. Among the 440 calves examined, of which 398 (90.5%) presented diarrhoea, cryptosporidia were found in 191 animals, i.e. a prevalence of 43.4% (C.I. 95%=[38. 8%; 48.0%]). Breed of calves and type of housing had very little impact on prevalence in this survey. Some regional variations could be noticed, even if cryptosporidia infection is widespread. Monthly variations could be related to seasonal peaks in calving with a lower infection rate during summer.

Leland, D., J. Mcanulty, et al. (1993). "A Cryptosporidiosis Outbreak in a Filtered-Water Supply." Journal American Water Works Association 85(6): 34-42.
	From January to June 1992, a large outbreak of cryptosporidiosis occurred in Jackson County, Oregon. Epidemiologic investigations associated 43 cases of cryptosporidiosis with the drinking water system in the small community of Talent, Ore. Mechanical and operational deficiencies at one of the city's water filtration plants along with unusually poor raw-water supply conditions because of low stream flows were possible cocauses of the outbreak. Correction of the deficiencies in the filter plant resulted in substantial improvement in treated-water quality, and this improvement coincided with the end of the Talent-related outbreak.

Lemarchand, K. and P. Lebaron (2003). "Occurrence of Salmonella spp and Cryptosporidium spp in a French coastal watershed: relationship with fecal indicators." FEMS Microbiol Lett 218(1): 203-9.
	Cryptosporidium and Salmonella are pathogenic microorganisms that can cause severe gastrointestinal illness in humans. Because these organisms are potentially transmitted through natural waters, this study was carried out to estimate the concentrations of both pathogens in a French coastal watershed and to determine the relationships with fecal indicators. Water samples from nine wastewater treatment plants and eight rivers were analyzed. Although both pathogens and indicators are released from sewage effluents, no clear correlation was found between the two enteric pathogens nor between a given pathogen and fecal indicators. These results suggest that fecal indicators do not adequately indicate the presence of Cryptosporidium and Salmonella in natural waters and that pathogens and indicators may have different behaviors in the aquatic environment.

Lemmon, J. M., J. M. McAnulty, et al. (1996). "Outbreak of cryptosporidiosis linked to an indoor swimming pool." Med J Aust 165(11-12): 613-6.
	OBJECTIVE: To determine the extent and source of a community outbreak of cryptosporidiosis. DESIGN: Questionnaire-based survey and matched case-control study. SETTING: Sutherland area in southern Sydney, September 1994 to January 1995. PARTICIPANTS: 70 patients reported by pathology laboratories to have stool specimens positive for cryptosporidia, of whom 43 were surveyed; 35 were compared with age- and neighbourhood-matched controls. MAIN OUTCOME MEASURES: Demographic characteristics and potential risk factors in the two weeks before onset of illness. RESULTS: Laboratories reported 70 cases of cryptosporidiosis between September 1994 and January 1995. We found no association between illness and foods consumed or contact with people with diarrhoea or sick animals in the two weeks before onset. Seventeen of the case group (49%) reported swimming in a particular indoor swimming pool, compared with only seven controls (20%) (odds ratio, 3.7; P = 0.015). Cryptosporidial oocysts were detected in water from the swimming pool in January 1995. CONCLUSIONS: The outbreak of cryptosporidiosis was probably associated with ingestion of water from the indoor swimming pool, presumably contaminated by infected bathers. RECOMMENDATIONS: As it is difficult to eradicate cryptosporidia from swimming pools by either disinfection or filtration, we recommend that: People with recent diarrhoea should avoid public swimming pools; and Non-toilet-trained and faecally incontinent swimmers should be provided with alternative swimming facilities with separate water and filtration systems. To enable appropriate public health responses: Doctors and pathology laboratories should consider cryptosporidiosis in patients with diarrhoea lasting longer than three days; and Laboratory reporting of cryptosporidia to local health departments should be mandatory in all States and Territories.

Lemos, V., T. K. Graczyk, et al. (2005). "Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies." Parasitol Res 98(1): 48-53.
	In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

Li, X., R. Fayer, et al. (2001). "Effects of gamma irradiation on the survival of Encephalitozoon intestinalis spores." J Eukaryot Microbiol Suppl: 91S.
	
Li, X., K. Guyot, et al. (2006). "Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France)." Int J Food Microbiol 108(3): 321-5.
	Cultured mussels (Mytilus edulis) were collected seasonally during one year from three sites on the Northwestern coastal area of Normandy (France). Flesh, gills and innerwater were examined for Cryptosporidium oocyst detection using immunomagnetic separation and immunofluorescence assay. Oocysts were present in all samples for all sites and seasons and flesh was the most contaminated part. Oocyst rates were apparently related with seasonal rain precipitation variations. Molecular analysis revealed that oocysts belonged to the species Cryptosporidium parvum (formerly genotype 2 or <<bovine genotype>>). Oocyst infectivity was assessed by oral administration to suckling NMRI-mice, and developmental stages were observed in only one mouse infected with oocysts from one location. The detection of potentially infectious C. parvum oocysts of likely cattle-breeding origin in cultured edible mussels confirms their resistance to sea environments, and underlines the potential risk of food-borne infection. This work reports for the first time the presence of infectious Cryptosporidium oocysts in shellfish from France.

Li, X., R. Palmer, et al. (2003). "Infectivity of microsporidia spores stored in water at environmental temperatures." J Parasitol 89(1): 185-8.
	To determine how long waterborne spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis could survive at environmental temperatures, culture-derived spores were stored in water at 10, 15, 20, 25, and 30 C and tested for infectivity in monolayer cultures of Madin Darby bovine kidney (MDBK) cells. At 10 C, spores of E. intestinalis were still infective after 12 mo, whereas those of E. hellem and E. cuniculi were infective for 9 and 3 mo, respectively. At 15 C, spores of the same species remained infective for 10, 6, and 2 mo, and at 20 C, for 7, 5, and 1 mo, respectively. At 25 C, spores of E. intestinalis and E. hellem were infective for 3 mo, but those of E. cuniculi were infective for only 3 wk. At 30 C, the former 2 species were infective for 3 wk and 1 mo, respectively, and the latter species for only 1 wk. These findings indicate that spores of different species of Encephalitozoon differ in their longevity and temperature tolerance, but at temperatures from 10 to 30 C, all 3 have the potential to remain infective in the environment long enough to become widely dispersed.

Lind, P., J. Haugegaard, et al. (1997). "The time course of the specific antibody response by various ELISAs in pigs experimentally infected with Toxoplasma gondii." Vet Parasitol 71(1): 1-15.
	With the aim of developing routine serological tests for monitoring the Toxoplasma infection status of Danish swine herds, four ELISAs based on tachyzoite antigen were set up: (1) an indirect ELISA for IgG-antibody; (2) a blocking ELISA for antibody to the membrane antigen, P30; (3) an indirect ELISA for IgM; (4) a reverse, antibody-catching IgM-ELISA. Groups of pigs (number between 6 and 10) were inoculated with tachyzoites of the RH-strain, tissue cysts of two complete strains, or oocysts in two doses (10(3) and 10(4). All inoculations were tolerated well. Irrespective of strain and stage used for inoculation, specific IgG and anti-P30 blocking activity appeared after 1-2 weeks, with OD-values stabilizing after 3-6 weeks and persisting throughout the study period (3-4 months). Specific IgM appeared quickly, but was short-lived (approximately 2 weeks). A cut-off OD-value of 0.36 for positive seroreaction in the indirect IgG-ELISA was determined on the basis of 69 sera from four herds, investigated in the dye-test (serum dilution 1:10) and ELISA. The chosen cut-off gave optimal combined sensitivity and specificity of 0.94 and 0.92, respectively, using the dye-test as a standard. Corresponding figures for the blocking ELISA were 37% inhibition as cut-off, with sensitivity and specificity of 0.94 and 0.94, respectively. Sera from a total of 87 pigs, experimentally infected with bacteria of the genera Salmonella, Yersinia or Actinobacillus and with the parasites Isospora suis, Trichinella spiralis or Ascaris suum, in no case produced cross-reactions in the IgG-ELISA. However, 3/9 pigs inoculated with 50 000 sporocysts of Sarcocystis miescheriana gave maximal OD-readings of 0.40-0.45 during the 13-15 weeks observation period. None of the sera from heterologously infected animals produced inhibitions in the anti-P30 blocking ELISA exceeding 36%.

Linden, K. G., G. A. Shin, et al. (2002). "UV disinfection of Giardia lamblia cysts in water." Environ Sci Technol 36(11): 2519-22.
	The human and animal pathogen Giardia lamblia is a waterborne risk to public health because the cysts are ubiquitous and persistent in water and wastewater, not completely removed by physical-chemical treatment processes, and relatively resistant to chemical disinfection. Given the recently recognized efficacy of UV irradiation against Cryptosporidium parvum oocysts, the inactivation of G. lamblia cysts in buffered saline water at pH 7.3 and room temperature by near monochromatic (254 nm) UV irradiation from low-pressure mercury vapor lamps was determined using a "collimated beam" exposure system. Reduction of G. lamblia infectivity for gerbils was very rapid and extensive, reaching a detection limit of >4 log within a dose of 10 JM-2. The ability of UV-irradiated G. lamblia cysts to repair UV-induced damage following typical drinking water and wastewater doses of 160 and 400 JM(-2) was also investigated using experimental protocols typical for bacterial and eucaryotic DNA repair under both light and dark conditions. The infectivity reduction of G. lamblia cysts at these UV doses remained unchanged after exposure to repair conditions. Therefore, no phenotypic evidence of either light or dark repair of DNA damage caused by LP UV irradiation of cysts was observed at the UV doses tested. We conclude that UV disinfection at practical doses achieves appreciable (much greater than 4 log) inactivation of G. lamblia cysts in water with no evidence of DNA repair leading to infectivity reactivation.

Lindergard, G., S. E. Wade, et al. (2001). "Detection of Cryptosporidium oocysts in soil samples by enzyme-linked immunoassay." Vet Parasitol 94(3): 163-76.
	An ELISA protocol was adapted for detection of Cryptosporidium parvum oocysts in soil samples and the limit of detection of the test was determined. A modified indirect antigen capture ELISA protocol was developed using monoclonal antibodies against the oocyst outer wall. The accuracy of the ELISA was compared to spiked soil samples and measured in terms of sensitivity and specificity of the test. The performance of the ELISA was evaluated in field soil samples and measured using the kappa-statistics. Similarly, the performance of the ELISA was compared to the concentration flotation method, to a modified concentration flotation method and to a commercial ELISA (ProSpecT) in field fecal and soil samples.The limit of detection of the test was selected to be 10,000 oocysts/g. At this limit of detection, the ELISA had a sensitivity of 95% and specificity of 100%. The agreement between the ELISA and the modified flotation-concentration method in detecting Cryptosporidium oocysts in soil samples was 32% (kappa=0.32). The ELISA had the same relative sensitivity (82%) in comparison to both the flotation and ProSpecT in determining Cryptosporidium-infection status of an animal. The kappa-statistics was 0.26 for both tests. The developed ELISA proved to be a valuable diagnostic test for detecting oocysts in soil samples and has a potential application in determining the infection status of animals.

Lindsay, D. S., W. L. Current, et al. (1983). "Excystation of Isospora suis Biester, 1934 of swine." Z Parasitenkd 69(1): 27-34.
	The in vitro excystation of sporozoites of Isospora suis Biester 1934 is described. Sporocysts of I. suis lack a Stieda body. Upon incubation in 0.75% sodium taurocholate or in 0.25% trypsin + 0.75% sodium taurocholate excystation solutions, sporozoites were released by separation of the sporocyst wall into four plates. Occasionally, the sporocyst wall did not separate completely but opened partially and released the sporozoite. At the time of excystation, sporozoites were short and broad but became elongated after 5 to 10 min in the excystation fluids. Elongate sporozoites measuring 11.7 x 3.8 micrometers, had a pointed anterior end and a nucleus located in the posterior half of the cell. Living sporozoites exhibited gliding movements, side-to-side flexion, and probed with their anterior ends. Incubation in 5.25% sodium hypochlorite removed the oocyst walls from most oocysts. Sporozoites did not excyst from sporocysts that were released during treatment with sodium hypochlorite.

Lindsay, D. S., W. L. Current, et al. (1983). "Motility of Isospora suis meronts." J Parasitol 69(4): 783-4.
	
Lindsay, D. S., J. P. Dubey, et al. (1997). "Mechanical transmission of Toxoplasma gondii oocysts by dogs." Vet Parasitol 73(1-2): 27-33.
	Two experiments were conducted to determine if dogs could mechanically transmit Toxoplasma gondii after ingesting cat feces or by rolling in cat feces containing oocysts. In the first experiment, two dogs were fed sporulated T. gondii oocysts; viable sporulated oocysts were present in dog feces for up to 2 days postinoculation (PI). Both dogs seroconverted to T. gondii but did not develop clinical signs of toxoplasmosis. In the second experiment, nonsporulated oocysts were placed on dog skin and fur, and fur clippings were bioassayed for T. gondii in mice. Oocysts did not sporulate on dog fur. The results of this study support the hypothesis that dogs may be involved in the mechanical transmission of T. gondii to humans.

Lindsay, D. S., J. P. Dubey, et al. (1990). "Extraintestinal stages of Eimeria bovis in calves and attempts to induce relapse of clinical disease." Vet Parasitol 36(1-2): 1-9.
	Monoclonal antibodies to all life cycle stages of Eimeria bovis were used in an avidin-biotin immunoperoxidase test to examine extraintestinal tissues from experimentally infected calves for developmental stages of the parasite. First-generation meronts of Eimeria bovis were found in the mesenteric lymph nodes (MLNs) of three of four calves examined during the first 18 days of primary experimental infection. No extraintestinal stages were found in the MLNs of two calves examined 69 days after a primary infection or in five calves examined 6 months after a challenge infection. Tissue homogenates of liver, spleen, and MLNs from immune calves were not infectious for nonimmune recipient calves following oral or intraperitoneal inoculation indicating that infectious stages were not present in these extraintestinal tissues at the time of inoculations. Relapse of clinical coccidiosis and reoccurrence of oocyst shedding were not seen in E. bovis immune calves that were stressed by being put on a 16-week corticosteroid treatment program.

Lindsay, D. S., J. P. Dubey, et al. (1991). "Immunohistologic examination of monoclonal antibodies generated against Eimeria bovis sporozoites for reactivity to meronts and sexual stages of E bovis and other eimerian parasites." Am J Vet Res 52(2): 239-42.
	Seven monoclonal antibodies (MAB) generated against sporozoites of Eimeria bovis were tested for reactivity against immature and mature first-generation meronts, sexual stages, and oocysts in tissues from experimentally infected calves by use of an avidin-biotin peroxidase complex (ABPC) immunohistologic test. Three of the 7 MAB reacted in the ABPC test. One of these, MAB-4FB4, reacted only with mature E bovis meronts. The other 2 MAB, MAB-2AE7 and MAB-4AD7, reacted with all the developmental stages of E bovis tested. Asexual stages and sexual stages of E tenella from chickens and E papillata from mice also were examined in the ABPC test. Monoclonal antibodies MAB-2AE7 and MAB-4AD7 reacted with all stages of these eimerian protozoa. None of the other 5 MAB reacted with these parasites. Results of this study suggested that antigens are shared among the asexual and sexual stages of several diverse Eimeria species.

Lindsay, D. S., R. McKown, et al. (1996). "Prevalence and identity of Sarcocystis infections in armadillos (Dasypus novemcinctus)." J Parasitol 82(3): 518-20.
	Little is known about the prevalence or identity of Sarcocystis species infecting armadillos in North America. Sarcocysts were observed in the tongues of 23 (96%) of 24 armadillos collected between 1989 and 1994 from Texas, Oklahoma, Kansas, and Arkansas. The identity of the species present was determined in histological sections of tongue from armadillos. Sarcocystis dasypi was present in 21 (88%) and Sarcocystis diminuta was present in 5 (21%). Mixed infections with S. dasypi and S. diminuta were present in 3 (13%) armadillos. A single sarcocyst with ultrastructural features distinct from S. dasypi and S. diminuta was observed with transmission electron microscopy.

Lindsay, D. S., R. R. Mitschler, et al. (1993). "Association of host cell mitochondria with developing Toxoplasma gondii tissue cysts." Am J Vet Res 54(10): 1663-7.
	Ultrastructure of the interactions of host cell mitochondria with developing Toxoplasma gondii tissue cysts was examined in cultured cells, using transmission electron microscopy of infected cells and rhodamine 123 (a mitochondria-specific vital fluorescent dye) staining of isolated tissue cysts. Structurally mature T gondii tissue cysts were observed as early as 2 days after inoculation of cultured cells. During development of T gondii, host cell mitochondria were observed surrounding the parasitophorous vacuole membrane. Mitochondria became flat and elongated in the vicinity of the parasitophorous vacuole membrane. These mitochondria were also closely associated with T gondii tissue cysts. Incubation of tissue cysts from cultured cells and tissue cysts from mouse brains with rhodamine 123 revealed fluorescence of the tissue cyst wall in living specimens. Incubation of tissue cysts with 10 microM rotenone caused diminished fluorescence of the tissue cyst walls, and 100 microM rotenone caused complete inhibition. Mouse RBC, and tissue cysts fixed in 100% methanol did not fluoresce after exposure to rhodamine. Tissue cysts in 9 isolates of T gondii from mouse brains were examined, using rhodamine 123, and the tissue cysts walls of all isolates fluoresced, indicating no isolate effects. Our results indicate that host cell mitochondria may be closely associated with the tissue cysts of T gondii in cell cultures and in mice.

Lindsay, D. S., S. J. Upton, et al. (1999). "A structural study of the Neospora caninum oocyst." Int J Parasitol 29(10): 1521-3.
	Oocysts of Neospora caninum were collected from the faeces of a dog fed mouse brains containing tissue cysts of the NC-beef strain of N. caninum. Sporulated oocysts were spherical to subspherical, and were 11.7x11.3 microm. The length/width ratio was 1.04. No micropyle or oocyst residuum was present. Polar granules were not present, although occasionally tiny refractile granules were seen among sporocysts. Sporocysts were ellipsoidal, did not contain a Stieda body, and were 8.4x6.1 microm. The length/width ratio for sporocysts was 1.37. A spherical or subspherical sporocyst residuum was present, and was composed of a cluster of small, compact granules of 4.3x3.9 microm, or was represented by many dispersed granules of similar size. Sporozoites were elongate and 7.0-8.0x2.0-3.0 microm in situ. No refractile bodies were present and the nucleus was centrally or slightly posteriorly located. The features of the oocyst of N. caninum are similar to those of Hammondia heydorni oocysts from dog faeces and Toxoplasma gondii and Hammondia hammondi oocysts from cat faeces.

Lindsay, D. S., S. J. Upton, et al. (1999). "Descriptions of two new species of coccidia (Protozoa: Eimeriidae) and redescriptions of Eimeria ivensae and Eimeria odocoilei from captive white-tailed deer, Odocoileus virginianus." J Parasitol 85(6): 1120-5.
	Two new species of Eimeria were observed in the feces of captive white-tailed deer fawns, Odocoileus virginianus, from Alabama. The first new species was easily recognized because of its small size. Sporulated oocysts are spherical, average 10.2 by 10.0 microm, and lack a micropyle and oocyst residuum. Oocysts contain a polar granule and elongate-ellipsoidal sporocysts that measure 6.7 by 3.1 microm. A Stieda body is present on the sporocysts. Oocysts were observed in the feces, and gamonts and oocysts were observed in the jejunum of a month-old fawn from Minnesota that died from enteritis due to this species. Oocysts of this small species were present in 5 of the 6 white-tailed deer fawns examined. Oocysts of a second new species are ellipsoidal and average 29.5 by 24.6 microm. The oocyst encloses an oocyst residuum, polar granule, and elongate-ellipsoidal sporocysts that average 16.0 by 9.0 microm. A Stieda body and substieda body are present on the sporocysts. Oocysts of the second new species were present in 4 of the 6 white-tailed deer fawns examined. Oocysts of E. ivensae are ovoid or flask-like and average 32.0 by 20.8 microm. The oocyst wall is rough, contains a micropyle, and encloses elongate-ellipsoidal sporocysts that average 16.5 by 7.8 microm. A Stieda body is present on the sporocysts. Oocysts of E. ivensae were present in 4 of the 6 white-tailed deer fawns. Oocysts of E. odocoilei are spherical or slightly subspherical and measure 24.7 by 21.5 microm. They enclose ovoid sporocysts that average 12.7 by 8.8 microm. A Stieda and substieda body are present on the sporocyst. Oocysts of E. odocoilei were present in 4 of the 6 white-tailed deer fawns.

Lindsay, D. S., S. J. Upton, et al. (2000). "Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus." J Eukaryot Microbiol 47(1): 91-5.
	A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.

Lindsay, D. S., K. M. Woods, et al. (2000). "Activity of decoquinate against Cryptosporidium parvum in cell cultures and neonatal mice." Vet Parasitol 89(4): 307-11.
	Cryptosporidium parvum is an apicomplexan parasite that is an important cause of diarrhea in neonatal calves and humans. No treatment is currently available for neonatal calves. We have recently learned from colleagues in the pharmaceutical industry that dairy practitioners are sometimes using decoquinate for the treatment of neonatal bovine cryptosporidiosis. Therefore, the present study was undertaken to determine whether the clinical observations in calves can be substantiated by laboratory investigation. Oocysts of the KSU-1 isolate of C. parvum were used to infect human ileocecal epithelial cells in vitro to measure the efficacy of treatment using an ELISA based assay. No activity was observed at 10 or 50microM decoquinate, but at 100microM an 8% inhibition of development was seen. Oocysts of the AUCp-1 isolate of C. parvum were then used to infect suckling mice. The numbers of oocysts observed in suckling mice treated with 2.5 or 5.0mg/kg decoquinate were not significantly different from untreated control suckling mice (p0.05). The results of our study suggest that decoquinate should have little efficacy for treatment of neonatal bovine cryptosporidiosis if administered once per day and that any clinical improvement observed in treated calves may be due to factors unrelated to decoquinate's effect on C. parvum.

Lisle, J. T. and J. B. Rose (1995). "Cryptosporidium contamination of water in the USA and UK: A mini-review." AQUA, vol. 44(3): 103-117.
	During the past 10 years the protozoan parasite Cryptosporidium has been recognised as a public health threat in drinking waters. Recently, the largest outbreak to date occurred in Milwaukee, Wisconsin, USA. Over 1.5 million consumers were exposed to this intestinal pathogen, of which 403 000 became ill. Many of those who were immunocompromised died. The probability of an outbreak of cryptosporidiosis occurring in drinking water systems, relative to that of bacterial and viral pathogens, is increased due to the resistant nature of oocysts to concentrations of disinfectants routinely used in drinking-water treatment. Surveys of surface and drinking waters in the USA and UK have shown Cryptosporidium oocysts to be present in polluted, pristine and drinking waters at concentrations that may put the consumer at risk of infection, based upon current risk assessment models. This mini-review is an attempt to present the most recent literature concerning Cryptosporidium in regard to outbreaks, occurrence, monitoring and detection, and regulatory implications.

Liu, C. and M. S. Abrahamsen (1999). "Identification of a putative telomeric repeat DNA binding factor of Cryptosporidium parvum." J Eukaryot Microbiol 46(5): 50S-51S.
	
Lloyd, A. and D. Drury (2002). "Continuous monitoring for Cryptosporidium--a novel approach to public health protection." Water Sci Technol 46(11-12): 297-301.
	Regulations requiring water utilities in England and Wales to carry out continuous monitoring for Cryptosporidium in treated water were introduced in June 1999. The rationale of the Regulations and the technical basis for the sampling and analysis are reviewed. The results of the first nine months of monitoring are summarised and compared with other equivalent data sets. The impact of the Regulations on water utilities operational practices and the implications for public health protection are discussed.

Lonigro, A., A. Pollice, et al. (2006). "Giardia cysts and Cryptosporidium oocysts in membrane-filtered municipal wastewater used for irrigation." Appl Environ Microbiol 72(12): 7916-8.
	A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.

Lopez, A. B., M. T. Hossain, et al. (2002). "Giardia intestinalis glucosamine 6-phosphate isomerase: the key enzyme to encystment appears to be controlled by ubiquitin attachment." J Eukaryot Microbiol 49(2): 134-6.
	The cyst wall of the parasitic protozoan, Giardia intestinalis, is composed of a polymer of N-acetylgalactosamine, the precursor of which is synthesized by an inducible enzyme pathway. The first enzyme in this pathway, glucosamine 6-phosphate isomerase, is transcriptionally regulated. During encystment and in mature cysts this isomerase appears to be modified by ubiquitin attachment. Thus, it might be targeted for destruction by an ubiquitin-mediated pathway, suggesting that glucosamine 6-phosphate isomerase expression is tightly regulated.

Lopez, A. S., D. R. Dodson, et al. (2001). "Outbreak of cyclosporiasis associated with basil in Missouri in 1999." Clin Infect Dis 32(7): 1010-7.
	During the summer of 1999, an outbreak of cyclosporiasis occurred among attendees of 2 events held on 24 July in different counties in Missouri. We conducted retrospective cohort studies of the 2 clusters of cases, which comprised 62 case patients. The chicken pasta salad served at one event (relative risk [RR], 4.25; 95% confidence interval [CI], 1.80-10.01) and the tomato basil salad served at the other event (RR, 2.95; 95% CI, 1.72-5.07) were most strongly associated with illness. The most likely vehicle of infection was fresh basil, which was included in both salads and could have been grown either in Mexico or the United States. Leftover chicken pasta salad was found to be positive for Cyclospora DNA by means of polymerase chain reaction analysis, and 1 sporulated Cyclospora oocyst was found by use of microscopy. This is the second documented outbreak of cyclosporiasis in the United States linked to fresh basil and the first US outbreak for which Cyclospora has been detected in an epidemiologically implicated food item.

Lowery, C. J., J. E. Moore, et al. (2000). "Detection and speciation of Cryptosporidium spp. in environmental water samples by immunomagnetic separation, PCR and endonuclease restriction." J Med Microbiol 49(9): 779-85.
	Current methods for the detection of Cryptosporidium oocysts in water samples are both time-consuming and subject to variation in sensitivity. A genus-specific PCR assay was designed for the specific amplification of a 552-bp region of the 18S rRNA gene. Postamplification endonuclease restriction generated unique digest patterns that enabled differentiation between the three species, C. muris, C. baileyi and C. parvum, the major human pathogen. Theoretical restriction profiles for other Cryptosporidium species were also predicted. The assay routinely detected 10 oocysts in 10-ml purified oocyst preparations, but sensitivity was found to be 10(3)-10(4) -fold lower in environmental water samples. The use of Chelex resin and an immunomagnetic separation procedure overcame this inhibition. This provided detection levels of 10(1)-10(3) oocysts, depending on water turbidity. Rapid and sensitive pathogen detection methods are essential for the water industry. The results of this study demonstrate that PCR has the potential to improve current detection capabilities greatly by differentiating the major human pathogens from non-pathogenic species. This will greatly facilitate a closer examination of the epidemiology of this important pathogen.

Lowery, C. J., J. E. Moore, et al. (2001). "Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern Ireland." J Appl Microbiol 91(5): 774-9.
	AIMS: To investigate the incidence and genotype of Cryptosporidium parvum oocysts in drinking water sources in Northern Ireland for the period 1996-1999, and to compare conventional and molecular methods of detection. METHODS AND RESULTS: Four hundred and seventy-four waters were investigated by conventional methods, namely immuno-fluorescent antibody detection (IFA; 380) and immuno-magnetic separation-IFA (IMS-IFA; 94), of which 14/474 (3%) were positive. Two hundred and fourteen samples (214/474) were also investigated by PCR techniques, targeting both the 18S rRNA and TRAP-C2 genes, of which 11/214 (5.1%) were positive. These 11 samples were classified as genotype II following sequence analysis of the TRAP-C2 amplicon. CONCLUSIONS: This study demonstrated the low incidence of oocysts of C. parvum in water sources in Northern Ireland. SIGNIFICANCE AND IMPACT OF THE STUDY: Such molecular-based techniques offer a number of advantages over conventional detection methodologies, namely greater sensitivity and specificity as well as the ability to provide accurate genotyping data rapidly, which may be valuable in directing operational management in potential outbreak situations.

Lowery, C. J., P. Nugent, et al. (2001). "PCR-IMS detection and molecular typing of Cryptosporidium parvum recovered from a recreational river source and an associated mussel (Mytilus edulis) bed in Northern Ireland." Epidemiol Infect 127(3): 545-53.
	PCR-IMS was used to detect Cryptosporidium spp. in environmental water samples in Northern Ireland which had previously tested negative by a conventional IFA staining method. Oocysts of C. parvum detected in river water and final treated sewage effluent collected from various sites along the river Lagan were identified as genotype 2 (animal origin) based on polymorphisms observed at the thrombospondin related adhesion protein gene locus. Similarly, genotype 1 (human origin) oocysts of C. parvum were detected in the marine filter feeder mussel, Mytilus edulis, collected from the shores of Belfast Lough. Detection of the human genotype of Cryptosporidium in mussels destined for human consumption identifies the organism's serious potential as a foodborne pathogen. This work highlights the possible value of monitoring filter feeder systems, in conjunction with specific molecular epidemiological tools, as an alternative monitoring system for the parasite within the aquatic environment.

Mac Kenzie, W. R., N. J. Hoxie, et al. (1994). "A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply." N Engl J Med 331(3): 161-7.
	BACKGROUND. Early in the spring of 1993 there was a widespread outbreak of acute watery diarrhea among the residents of Milwaukee. METHODS. We investigated the two Milwaukee water-treatment plants, gathered data from clinical laboratories on the results of tests for enteric pathogens, and examined ice made during the time of the outbreak for cryptosporidium oocysts. We surveyed residents with confirmed cryptosporidium infection and a sample of those with acute watery diarrhea consistent with cryptosporidium infection. To estimate the magnitude of the outbreak, we also conducted a survey using randomly selected telephone numbers in Milwaukee and four surrounding counties. RESULTS. There were marked increases in the turbidity of treated water at the city's southern water-treatment plant from March 23 until April 9, when the plant was shut down. Cryptosporidium oocysts were identified in water from ice made in southern Milwaukee during these weeks. The rates of isolation of other enteric pathogens remained stable, but there was more than a 100-fold increase in the rate of isolation of cryptosporidium. The median duration of illness was 9 days (range, 1 to 55). The median maximal number of stools per day was 12 (range, 1 to 90). Among 285 people surveyed who had laboratory-confirmed cryptosporidiosis, the clinical manifestations included watery diarrhea (in 93 percent), abdominal cramps (in 84 percent), fever (in 57 percent), and vomiting (in 48 percent). We estimate that 403,000 people had watery diarrhea attributable to this outbreak. CONCLUSIONS. This massive outbreak of watery diarrhea was caused by cryptosporidium oocysts that passed through the filtration system of one of the city's water-treatment plants. Water-quality standards and the testing of patients for cryptosporidium were not adequate to detect this outbreak.

MacRae, M., C. Hamilton, et al. (2005). "The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish." J Microbiol Methods 60(3): 395-401.
	Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12-34% in mussels, 48-69.5% in oysters and 30-65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 degrees C. Increasing enrichment temperatures from 37 to 42 degrees C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.

Madore, M. S., J. B. Rose, et al. (1987). "Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters." J Parasitol 73(4): 702-5.
	An existing method for the detection of Cryptosporidium oocysts in water was modified to investigate oocyst prevalence in large volumes of water. Surface waters and sewage effluents were filtered, eluted from the filter, and concentrated using centrifugation. The resultant pellet was then homogenized, sonicated, and placed on a sucrose gradient to separate oocysts from the sediment. The uppermost gradient layer was then examined by immunofluorescence using a labeled monoclonal antibody. Using this technique, average numbers of oocysts detected in raw and treated sewage were 5.18 X 10(3) and 1.30 X 10(3)/L, respectively. Filtered sewage effluents had significantly lower numbers of oocysts (10.0/L). These data show that sand filtration may reduce the concentrations of this parasite in waste waters. Highly variable oocyst numbers were encountered in surface waters. Since Cryptosporidium oocysts are frequently present in environmental waters, they could be responsible for waterborne outbreaks of disease.

Mahbubani, M. H., A. K. Bej, et al. (1991). "Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts." Appl Environ Microbiol 57(12): 3456-61.
	A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.

Mahbubani, M. H., F. W. Schaefer, 3rd, et al. (1998). "Detection of Giardia in environmental waters by immuno-PCR amplification methods." Curr Microbiol 36(2): 107-13.
	Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR-amplified following "direct extraction" of Giardia DNA from seeded Cahaba river water concentrate with moderate turbidity (780 JTU's), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 x 10(5) JTUs) failed to amplify. However, if the cysts were first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex(R)100 treatment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 x 10(0) or 3 x 10(1) cysts/ml, respectively. If, however, the G. muris cysts used to seed even moderately turbid river waters (780 JTUs) were formalin treated (which is conventionally used for microscopic examination), neither direct extraction nor IMB purification methods yielded amplifiable DNA. Use of immunomagnetic beads to separate Giardia cysts from complex matrices of environmental surface waters followed by DNA release and PCR amplification of the target giardin gene improved the reliability of detection of this pathogen with the required sensitivity.

Mahdi, N. K. and N. H. Ali (2002). "Cryptosporidiosis among animal handlers and their livestock in Basrah, Iraq." East Afr Med J 79(10): 550-3.
	OBJECTIVE: To investigate the prevalence of cryptosporidiosis among groups at risk (animal handlers) and among domestic animals. DESIGN: Comparative study with zoonotic aspect. METHOD: Stool samples were collected from 60 animal handlers, 175 non-animal handlers and 198 domestic animals (60 cows, 45 sheep, 45 goats, 25 horses and 23 camels). Direct smear method and then formalin-ether sedimentation method were carried out for stool samples to detect intestinal parasites. Faecal smears were prepared from the sediment and stained by the modified Ziehl-Neelsen method for the recovery of red-pink oocysts of Cryptosporidium. RESULTS: Out of the 60 animal handlers, 30 (50%) were found to be positive for intestinal parasites compared to 26 (14.8%) of non-animal handlers (P < 0.01). Cryptosporidium oocysts were found to be excreted by three (5%) animal handlers and two (1.14%) of the non-animal handlers (P > 0.05). Cryptosporidiosis was also diagnosed in 20%, 13.3%, 17.7% and 12% of cattle, sheep, goats and horses respectively. No single positive case was detected among the examined camels. CONCLUSION: Veterinarians, butchers and breeders should be aware of the disease among farm animals in order to avoid great losses and to prevent its transmission to humans.

Mahdi, N. K. and N. H. Ali (2002). "Intestinal parasites, including Cryptosporidium species, in Iraqi patients with sickle-cell anaemia." East Mediterr Health J 8(2-3): 345-9.
	Stool samples were obtained from individuals admitted to three hospitals in Basra during November 1997-May 1998. Of 40 patients with sickle-cell anaemia, 25 (62.5%) had parasitic infections. In the apparently healthy comparison group, 26 of 175 individuals (14.8%) had intestinal parasitic infections, a statistically significant difference. The most common intestinal parasites isolated in the sickle-cell patients were Blastocystis hominis (36%) and Giardia lamblia (28%). The isolation rate of Cryptosporidium species in sickle-cell patients (5%) was not significantly different from that in apparently healthy individuals (1.14%). We report for the first time the isolation of Isospora belli from a sickle-cell patient in Iraq and the Mediterranean region.

Majewska, A. C., A. Werner, et al. (2000). "Prevalence of Cryptosporidium in sheep and goats bred on five farms in west-central region of Poland." Vet Parasitol 89(4): 269-75.
	Faecal specimens were taken from 205 sheep and goats housed in five different localities in the west-central part of Poland. All faecal specimens were examined for Cryptosporidium by using microscopy screening of smears stained by modified Ziehl-Neelsen technique and commercial enzyme immunoassay. PCR technique using genus specific primers was additionally applied in the surveys of 10 faecal specimens collected from lambs. C. parvum infection was identified in 16 of 159 sheep (10.1%). Lambs were more often infected than adult sheep, and the intensity of infection was higher in lambs than in sheep, as a rule. Both lambs and sheep examined in the study were asymptomatically infected with Cryptosporidium. Both microscopy and enzyme immunoassay methods gave one false negative result. The examination of 10 faecal samples revealed 100% agreement among the results obtained by microscopic, immunologic and molecular methods. None of the goats raised on three farms were infected with Cryptosporidium.

Mancassola, R., A. Richard, et al. (1997). "Evaluation of decoquinate to treat experimental cryptosporidiosis in kids." Vet Parasitol 69(1-2): 31-7.
	The purpose of this trial was to evaluate the effects of decoquinate at 2.5 mg/kg/day for 21 days to prevent an experimental cryptosporidiosis in kids. Twenty 1-day-old male kids (French Alpin), fed initially goat colostrum heated 1 h at 56 degrees C and fed twice daily with nonmedicated milk replacer, were assigned into 2 groups. Kids of both groups were orally inoculated with 10(6) Cryptosporidium parvum (D0 = inoculation day). Group A kids were kept as nonmedicated controls and group B kids were orally medicated with 2.5 mg/kg/day of decoquinate (Deccox L. Rhone Poulenc Animal Nutrition) for 21 days from D-3 to D17. The studied criteria were body weight gain, oocyst shedding and specific anti-C. parvum immune response. In group A, the inoculation was not followed by mortality; but only by diarrhea and high oocyst shedding. Decoquinate reduced the severity of cryptosporidiosis in group B kids. The treatment prevented episodes of diarrhea and weight gain decrease for the D0-D7 and D0-D14 disease periods but did not allow a better final weight gain. The oocyst shedding was decreased in number and in duration. This parasitic development has induced a specific anti-C. parvum immune response. This drug is well-tolerated by animals and may be recommended in the prevention of ruminant cryptosporidiosis, a disease which has very limited treatment options.

Mansfield, L. S. and A. A. Gajadhar (2004). "Cyclospora cayetanensis, a food- and waterborne coccidian parasite." Vet Parasitol 126(1-2): 73-90.
	Food- and waterborne coccidia including Cryptosporidium parvum, Cyclospora cayetanensis, Sarcocystis hominis and Sarcocystis suihominis, and Isospora belli are cyst-forming apicomplexan protozoa that cause intracellular infections, predominantly in the epithelial cells of the intestine. They are transmitted by oocysts from person-to-person by the fecal-oral route or via contaminated water or food. The most common symptom of infection is diarrhea, however, asymptomatic infections occur. Infections are associated with intestinal inflammation, with pathological lesions such as villus blunting, and abnormal function such as malabsorption. Mild-to-moderate, self-limiting diarrhea is common in healthy individuals ingesting infective stages of these organisms. However, patients with immune dysfunction can have severe intestinal injury and prolonged diarrhea. Diagnosis in many cases is made by a microscopic examination of the stool, and the use of appropriate staining techniques, but more recently molecular methods for detection are used increasingly. Effective antimicrobial treatment for prolonged infection in immunocompromised patients is available for most of these infections. These gastrointestinal coccidial pathogens have important similarities in epidemiology, disease pathogenesis, clinical manifestations, diagnosis, and treatment. Although there are many other cyst-forming coccidia of public health, veterinary and/or economic importance, discussion in this chapter will be limited to C. cayetanensis, as an important example of the group. Aspects of the biology, epidemiology, diagnosis, disease, treatment and control are considered. This parasite is considered to be an emerging pathogen. From 1990 to 2000, there were 11 foodborne outbreaks of cyclosporosis in North America that affected at least 3600 people. There are many outstanding questions regarding this parasite and under-reporting is common because general diagnostic methods for intestinal parasites are inadequate for detection of Cyclospora.

Marshall, M. M., S. Hayes, et al. (2003). "Comparison of UV inactivation of spores of three encephalitozoon species with that of spores of two DNA repair-deficient Bacillus subtilis biodosimetry strains." Appl Environ Microbiol 69(1): 683-5.
	When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m(2), respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.

Marshall, M. M., D. Naumovitz, et al. (1997). "Waterborne protozoan pathogens." Clin Microbiol Rev 10(1): 67-85.
	Protozoan parasites were the most frequently identified etiologic agents in waterborne disease outbreak from 1991 to 1994. The waterborne parasites Giardia lamblia, Naegleria fowleri, Acanthamoeba spp., Entamoeba histolytica, Cryptosporidium parvum, Cyclospora cayetanesis, Isospora belli, and the microsporidia are reviewed. For each parasite, the review includes history, life cycle, incidence, symptoms, and therapy. Clinical detection methods are compared, and emerging technologies are discussed. Information on the association of these parasites with waterborne outbreaks is reviewed. Current information on protozoan parasites identified as etiological agents in waterborne outbreaks is discussed. Water industry issues related to recent disease outbreaks are examined in the context of water quality testing regulations for G. lamblia and those proposed for C. parvum. The review identifies the limitations of the American Society of Testing and Materials water-testing method for these parasites. An overview of federal regulations affecting the water industry and laboratories that test for water quality is also provided. The article highlights the importance of the clinical laboratory as a frontline defense for the detection of infectious organisms. The review points to the need for clinical laboratories, physicians, and public health personnel to cooperatively plan and assess the challenge of meeting this potential public health threat.

Mathieu, E., D. A. Levy, et al. (2004). "Epidemiologic and environmental investigation of a recreational water outbreak caused by two genotypes of Cryptosporidium parvum in Ohio in 2000." Am J Trop Med Hyg 71(5): 582-9.
	In August 2000, the Ohio Department of Health requested assistance to investigate a cryptosporidiosis outbreak with more than 700 clinical case-patients. An epidemiologic and environmental investigation was conducted. Stool specimens, pool water, and sand filter samples were analyzed. A community-based case-control study showed that the main risk factor was swimming in pool A (odds ratio [OR] = 42, 95% confidence interval [CI] = 12.3-144.9). This was supported by results of polymerase chain reaction (PCR) analysis, which showed the presence of both the human and bovine genotypes of Cryptosporidium parvum in case-patients and samples from the filter of pool A. A pool-based case-control study indicated that the highest risk was related to exposure to pool water via the mouth (OR = 5.1, 95% CI = 2.1-12.5) or to pool sprinklers (OR = 2.5, 95% CI = 1.3-4.7). Fecal accidents at the pool were documented. Records indicated that the pool met local health regulations. The outbreak, caused by co-infection with two C. parvum genotypes (human and bovine), underscores the need for concerted action to improve public health policies for recreational water facilities and enhanced education regarding the potential for disease transmission through pools.

Mathison, B. A. and O. Ditrich (1999). "The fate of Cryptosporidium parvum oocysts ingested by dung beetles and their possible role in the dissemination of cryptosporidiosis." J Parasitol 85(4): 678-81.
	The fate of oocysts of Cryptosporidium parvum ingested by dung beetles and the possible role these beetles serve in the dissemination of cryptosporidiosis were tested on the following species: Anoplotrupes stercorosus, Aphodius rufus, and Onthophagus fracticornis. Ten specimens of each species were offered cattle dung supplemented with 5.9 x 10(6) oocysts of C. purvum. After 24 hr of feeding, the beetles were examined for the presence of oocysts on their external surfaces, in their gastrointestinal tracts, and in feces passed during the experiment. Results indicate that although many oocysts pass safely through the mouthparts and gastrointestinal tracts of the beetles, the majority of them are destroyed. Coprophagous insects can, therefore, be considered an important aspect in the ecology of gastrointestinal diseases of man and livestock, as both agents of control and dissemination.

McAllister, C. T., S. J. Upton, et al. (1995). "A description of Isospora amphiboluri (Apicomplexa: Eimeriidae) from the inland bearded dragon, Pogona vitticeps (Sauria: Agamidae)." J Parasitol 81(2): 281-4.
	Fecal samples from 50 captive inland bearded dragons, Pogona vitticeps (Ahl, 1926), bred in California, were examined for coccidian parasites. Sixteen (32%) of the lizards were found to be passing oocysts of Isospora amphiboluri Cannon, 1967, previously described from bearded dragons Pogona barbata (Cuvier, 1829) from Australia. Sporulated oocytes were spherical to subspherical, 25.3 x 25.1 (23-26 x 23-26) microns, with a shape index (length/width) of 1.0 (1.0-1.1). A micropyle, oocyst residuum, and polar granule were absent. Sporocyts were ovoidal, 17.0 x 11.4 (16-18 x 11-12) microns, with a shape index of 1.5 (1.4-1.7). A sporocyst residuum, Stieda, and substieda bodies were present, but parastieda bodies were absent. Sporozoites were elongated, 13.9 x 3.5 (12-15 x 3-4) microns in situ, containing spherical anterior and posterior refractile bodies. The occurrence of I. amphiboluri in P. vitticeps is a new host and geographic record for the parasite. Photomicrographs of the oocysts and endogenous life cycle stages of I. amphiboluri are presented for the first time.

McAllister, C. T., S. J. Upton, et al. (1995). "Coccidian parasites (Apicomplexa) from snakes in the southcentral and southwestern United States: new host and geographic records." J Parasitol 81(1): 63-8.
	Four hundred thirty-five leptotyphlopid, colubrid, elapid, and viperid snakes were collected from various localities in Arkansas, New Mexico, Oklahoma, and Texas, and their feces were examined for coccidian parasites. Of these, 131 (30%) were passing oocysts or sporocysts of at least 1 coccidian; 88 (67%) of the infected snakes had only 1 species of coccidian when they were examined. Aquatic and semiaquatic snakes accounted for 48% of the infections, whereas strictly terrestrial snakes comprised the other 52%. There was more than a 2-fold difference in prevalence among these 2 groups as 63 of 129 (49%) of the aquatic and semiaquatic snakes versus 68 of 306 (22%) of the terrestrial snakes harbored coccidia. Most terrestrial snakes were infected by species of Caryospora and Sarcocystis that are either facultatively or obligatorily heteroxenous. The aquatic and semiaquatic species most often harbored eimerians. Attempts to transmit some of the Sarcocystis spp. experimentally from Crotalus atrox to Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, or Microtus ochrogaster were unsuccessful. This report documents 27 new host and several distributional records for coccidians from snakes in the southcentral and southwestern United States.

McAllister, T. A., C. B. Annett, et al. (2001). "Studies on the use of Yucca schidigera to control giardiosis." Veterinary Parasitology 97(2): 85-99.
	The potential anti-giardial activity of a powdered preparation of Yucca schidigera (yucca powder) was investigated in vitro, in a modified adherence inhibition assay, and in vivo, by enumeration of trophozoites (intestinal) or cysts (fecal) in experimentally infected gerbils and lambs receiving oral doses of whole or butanol-extracted yucca powder. Yucca powder, butanol-, acetone- and chloroform-extracted powder, and the butanol-insoluble fraction of the powder were required in concentrations of 22, 15, 62, 135 and 250 mug/ml, respectively, to reduce in vitro trophozoite adherence by 50%. Ethyl ether extract exhibited no anti-giardial activity. Virtually no trophozoites were tolerant of butanol extract at greater than or equal to 90 mug/ml. Butanol extract at 1500 mug/ml exerted effects on trophozoites similar to the nitroimidazole, metronidazole, at 40 mug/ml during a 27-h incubation. Reducing trophozoite adherence to 50% of the controls required 5-10 h of exposure to butanol extract or metronidazole. Oral administration of butanol extract (6.1 mg) or metronidazole (1 mg) once daily for 3 days reduced the number of trophozoites in the small intestine of infected gerbils, significantly (P < 0.05) in the jejunum and ileum, and numerically (P > 0.05) in the duodenum (n = 8). Oral dosing of 50 mg of butanol extract in eight doses over 3 days reduced (P < 0.05) trophozoites in the duodenum and jejunum, and eliminated them from the ileum. Including 4.5% (w/w) yucca powder in the diet did not alter Giardia trophozoite recovery from the duodenum or jejunum of infected gerbils, but trophozoite reduction (P = 0.051) was observed in the ileum (n = 9), Jejunum gut loop data were inconclusive, possibly due to insufficient duration of exposure of trophozoites to butanol extract. Compared to controls (0 g yucca powder per day) lambs receiving 10 g of yucca powder per day in their diet shed fewer (P < 0.05) Giardia cysts in their feces after 5, 9, 12 and 19 days of treatment, but a corresponding decline in the prevalence of infection was not observed (n = 10). After 26 days, Giardia infections persisted in 90% of the lambs in both treatment groups. At the dosing levels studied in vivo, yucca powder did not affect the extent of cyst shedding by experimentally infected lambs, but Further purification and concentration of the saponin fraction from Y. schidigera may provide the anti-giardial effects observed in vitro and in dosing trials. Published by Elsevier Science B.V. [References: 51] 51

McAllister, T. A., C. B. Annett, et al. (1996). "Effect of Salinomycin on Giardiasis and Coccidiosis in Growing Lambs." Journal of Animal Science 74(12): 2896-2903.
	An experiment was undertaken to determine the effect of salinomycin on Giardia in vitro and on Giardia and coccidia in growing lambs. Concentrations of salinomycin above (3.9 mu g/mL) reduced the adherence (index of viability) of Giardia S2 trophozoites by more than 50%. This strain did not develop resistance after repeated exposure to sublethal concentrations of salinomycin. Five of 40 lambs escaped natural infection by Giardia, and these were inoculated with greater than or equal to 500,000 cysts. Giardiasis (presence of cysts in feces) was confirmed in all lambs before commencement of the experiment. Coccidiosis (presence of oocysts) developed by natural exposure. Lambs were assigned randomly to diets containing 0, 4, 10, or 16 ppm of salinomycin. Giardia cyst and coccidia oocyst excretions were determined on 6 d during the first week and weekly thereafter. Giardia cysts were detected at each sampling date in all treatments (highest release, 2.3 x 10(6) cysts/g feces). The number of Giardia cysts shed in feces was not affected(P >.05) by salinomycin but did decline (P <.05) with time. Average infection rates remained above 50% until d 41 of the experiment and declined linearly(P <.05) with salinomycin concentration and time. The number of coccidia oocysts in feces was low !highest release, 6.8 x 10(4) oocysts/g feces), but shedding occurred in 38 of the 40 lambs. Treatment with salinomycin had a cubic effect (P <.05) on coccidia oocyst excretion, and no oocysts were detected beyond d 28. Treatment effect on average daily gain (P <.002), dry matter intake (P <.02), and final live weight(P <.07) was cubic, whereas carcass weight (P <.003) and dressing percentage (P <.08) were linearly affected by salinomycin concentration. Although a beneficial effect of 10 ppm salinomycin on lamb performance was apparent, the development of natural resistance makes it difficult to attribute this response to the control of coccidiosis or giardiasis. [References: 39] 39

McCuin, R. M., Z. Bukhari, et al. (2001). "Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation." J Microbiol Methods 45(2): 69-76.
	Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.

McDonnell, P. A., K. G. E. Scott, et al. (2003). "Giardia duodenalis trophozoites isolated from a parrot (Cacatua galerita) colonize the small intestinal tracts of domestic kittens and lambs." Veterinary Parasitology 111(1): 31-46.
	This study examines the ability of Giardia duodenalis trophozoites, isolated from a wild bird, to colonize the intestinal tracts of companion animals (kittens) and domestic ruminants (lambs). Trophozoites colonized the intestinal tracts of intraduodenally inoculated animals as demonstrated by increasing parasite burdens within the duodenum and jejunum and by fecal passage of cysts within 4 days post-inoculation. The pathogenesis of the trophozoites was further investigated in kittens. In these animals, infection significantly reduced jejunal brush border microvillous length and density, which resulted in a loss of overall epithelial brush border surface area. This injury was associated with the production of diarrhea in four of five infected kittens. These findings indicate that some bird species may carry G. duodenalis that represent a possible health threat to companion animals and livestock. Our results describe the first successful colonization of avian-derived G. duodenalis trophozoites in the small intestines of domestic kittens and lambs. (C) 2002 Elsevier Science B.V. All rights reserved. [References: 62] 62

McLauchlin, J., C. Amar, et al. (2000). "Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals." J Clin Microbiol 38(11): 3984-90.
	Cryptosporidium present in 1,705 fecal samples from humans and 105 from livestock animals were analyzed by PCR-restriction fragment length polymorphism of the Cryptosporidium oocyst wall protein. Overall, genotype 1 (human exclusive type) was detected in 37.8% of the samples from humans, genotype 2 (broad host range) was detected in 61.5%, a third genotype designated genotype 3 (Cryptosporidium meleagridis) was detected in 0.3%, and both genotypes 1 and 2 were recovered from 0.4%. All samples from livestock yielded genotype 2. Among 469 patients infected during eight drinking water-related outbreaks, five outbreaks were predominantly due to genotype 1, and three were due to genotype 2. Fifty-four samples were collected from patients involved with five swimming pool-associated outbreaks: two outbreaks were due to genotype 1, one was due to genotype 2, and the remaining two involved both genotypes 1 and 2. Among 26 family outbreaks and 1 children's nursery outbreak (2 to 3 members per group), the same genotype was recovered from the different members of each outbreak: 13 were due to genotype 1, and 14 were due to genotype 2. In eighteen patients reporting contact with animals and/or farms, genotype 1 was recovered from one patient and genotype 2 was recovered from the remaining 17. Among the sporadic cases, there were distinct geographical and temporal variations in the distribution of the genotypes. The spring peak in cases was due to genotype 2. Genotype 1 was significantly more common in patients infected during the late-summer-autumn peak and in those with a history of foreign travel.

McRoberts, K. M., B. P. Meloni, et al. (1996). "Morphological and molecular characterization of Giardia isolated from the straw-necked ibis (Threskiornis spinicollis) in Western Australia." J Parasitol 82(5): 711-8.
	Following the first report of avian Giardia infection in Australia, isolates of the parasite recovered from naturally infected straw-necked ibis (Theskiornis spinicollis) were characterized using median body morphology, scanning electron microscopy, multilocus enzyme electrophoresis, random amplified polymorphic DNA (RAPD), and small subunit ribosomal RNA (SSU-rRNA) analyses. Results were compared with Giardia from other birds and mammals, and the extent of genetic diversity between a range of ibis isolates collected in Western Australia was determined. The ibis isolates of Giardia were genetically relatively homogeneous, which is in contrast to the extensive genetic heterogeneity often displayed by mammalian Giardia isolates. Morphologically, Giardia from ibis were similar to Giardia ardeae although they differed genetically and by the fact that the ibis isolates could not be established in in vitro culture. Sequence data of the DNA coding for the SSU-rRNA found a 96% homology between the ibis isolates from Western Australia and G. ardeae, suggesting that they represent distinct strains of the same species. In contrast, the ibis isolates were genetically and morphologically very different than Giardia duodenalis and Giardia muris from mammals.

Mead, J. R., M. J. Arrowood, et al. (1988). "Field inversion gel electrophoretic separation of Cryptosporidium spp. chromosome-sized DNA." J Parasitol 74(3): 366-9.
	Chromosomal DNA from 5 isolates of Cryptosporidium parvum and 1 of C. baileyi were compared by field-inversion gel electrophoresis (FIGE). FIGE analyses of parasite DNA prepared from purified sporozoites versus intact oocysts showed no observable differences. Chromosomal DNA migration patterns of the 5 C. parvum isolates were indistinguishable, whereas similar but distinct differences were evident between C. baileyi and the isolates of C. parvum. Oocyst-reactive monoclonal antibodies differentiated oocysts of C. parvum from those of C. baileyi but were unable to distinguish oocysts of 1 isolate of C. parvum from another.

Mead, J. R., M. J. Arrowood, et al. (1988). "Antigens of Cryptosporidium sporozoites recognized by immune sera of infected animals and humans." J Parasitol 74(1): 135-43.
	The humoral response of humans, calves, and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans, and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sodium dodecyl sulfate polyacrylamide gel-electrophoresed (SDS-PAGE) sporozoite antigens. The number of antigens recognized by immune sera from humans and animals increased with time postinfection. A 20-kDa antigen appears to be a major sporozoite surface determinant labeled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20-kDa band occurred in 3-wk postinfection (PI) sera from all species tested. Reactivity to the 20-kDa band diminished significantly in sera 5 mo PI or longer from infected humans with no known recurrence of cryptosporidial diarrhea. In contrast, 12-mo PI sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3-wk convalescent sera. Serum reactivity to the 20-kDa antigen appears to be a good indicator of exposure to Cryptosporidium.

Mead, J. R., M. T. Bonafonte, et al. (1996). "In vitro expression of mRNA coding for a Cryptosporidium parvum oocyst wall protein." J Eukaryot Microbiol 43(5): 84S-85S.
	
Mead, J. R., R. M. Lloyd, Jr., et al. (1994). "Isolation and partial characterization of Cryptosporidium sporozoite and oocyst wall recombinant proteins." J Eukaryot Microbiol 41(5): 51S.
	
Mead, P. S., L. Slutsker, et al. (1999). "Food-related illness and death in the United States." Emerg Infect Dis 5(5): 607-25.
	To better quantify the impact of foodborne diseases on health in the United States, we compiled and analyzed information from multiple surveillance systems and other sources. We estimate that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year. Known pathogens account for an estimated 14 million illnesses, 60, 000 hospitalizations, and 1,800 deaths. Three pathogens, Salmonella, Listeria, and Toxoplasma, are responsible for 1,500 deaths each year, more than 75% of those caused by known pathogens, while unknown agents account for the remaining 62 million illnesses, 265,000 hospitalizations, and 3,200 deaths. Overall, foodborne diseases appear to cause more illnesses but fewer deaths than previously estimated.

Meamar, A. R., M. Rezaian, et al. (2006). "Cryptosporidium parvum bovine genotype oocysts in the respiratory samples of an AIDS patient: efficacy of treatment with a combination of azithromycin and paromomycin." Parasitol Res 98(6): 593-5.
	Cryptosporidium has been recognized as an emerging zoonotic agent of intestinal cryptosporidiosis leading to diarrhea, malabsorption syndrome, and weight loss in AIDS patients. In the present case, oocysts of zoonotic Cryptosporidium parvum were detected in the sputum and stool samples of an AIDS patient with a 3-month history of intestinal cryptosporidiosis. The oocysts were detected by modified Ziehl-Neelsen staining; confirmation was achieved by nested polymerase chain reaction (PCR), targeting the most polymorphic region of the 18S rRNA gene. Genotyping was done by restriction endonuclease digestion of the PCR product. The zoonotic C. parvum bovine genotype was identified in both intestinal and respiratory samples. Treatment with both azithromycin and paromomycin resulted in improvement of both intestinal and respiratory symptoms, as well as the elimination of the parasite. This is the first report of the identification of Cryptosporidium sp. oocysts in the respiratory samples obtained from an AIDS patient in Iran. Pulmonary cryptosporidiosis should be considered whenever an AIDS patient with intestinal cryptosporidiosis develops respiratory symptoms.

Medema, G. J., M. Bahar, et al. (1997). "Survival of Cryptosporidium parvum, Escherichia coli, faecal enterococci and Clostridium perfringens in river water: Influence of temperature and autochthonous microorganisms." HEALTH-RELATED WATER MICROBIOLOGY 1996: 249-252.
	Oocysts of Cryptosporidium parvum can survive for several months in surface water, one of the main factors determining their success in environmental transmission and thus their health hazard via water. Several factors in the environment, e.g. temperature, presence of predators and exo-enzymes will probably influence oocyst survival. The high persistence of oocysts may also limit the value of traditional faecal indicator bacteria. The aim of this study was to determine the rate at which C parvum oocysts, E coli, faecal enterococci and C. perfringens spores die in surface water and the influence of temperature and the presence of autochthonous (micro)organisms on the die-off rate. Microcosms with autoclaved river water were inoculated with the organisms. Microcosms with untreated river water were inoculated with concentrated primary effluent containing the bacteria and with C. parvum oocysts. Microcosms were incubated at 5 degree C or 15 degree C at 100rpm. Viability of oocysts was monitored by in vitro excystation and dye-exclusion; viability of the bacteria was determined on appropriate selective media. When pseudo first-order die-off kinetics were assumed, the die-off rate of oocysts at 5 degree C was 0.010 log sub(10)/d and at 15 degree C, 0.006-0.024 log sub(10)/d. These rates underestimate die-off since oocyst disintegration was not accounted for. Incubation in autoclaved or untreated water did influence the die-off rate of oocysts at 15 degree C but not at 5 degree C. The die-off rate of E coli and enterococci was faster in the non-sterile river water than in autoclaved water at both temperatures. At 15 degree C, E coli (and possibly E faecium) even multiplied in autoclaved water. In untreated river water, the die-off of E coli and enterococci was approximately 10x faster than die-off of oocysts but die-off rates of C perfringens were lower than those of oocysts. As for oocysts, die-off of the bacteria and spores was faster at 15 degree C than at 5 degree C. Oocysts are very persistent in river water: the time required for a 10x reduction in viability being 40-160d at 15 degree C and 100d at 5 degree C. Biological/biochemical activity influenced oocyst survival at 15 degree C and survival of both vegetative bacteria at 5 and 15 degree C. The rapid die-off of E coli and enterococci makes them less suitable as indicators of oocyst presence in water. As C perfringens survived longer in untreated river water than oocysts, it may prove useful as an indicator of the presence of C parvum.

Medema, G. J., F. M. Schets, et al. (1998). "Sedimentation of free and attached Cryptosporidium oocysts and Giardia cysts in water." Appl Environ Microbiol 64(11): 4460-6.
	Experimental analysis of the sedimentation velocity of Cryptosporidium parvum oocysts and Giardia lamblia cysts was compared with mathematical description of their sedimentation velocities by using measurements of (oo)cyst size and density and the density and viscosity of the sedimentation medium to determine if the sedimentation kinetics of freely suspended oocysts of C. parvum and cysts of G. lamblia can be described by Stokes' law. The theoretically calculated sedimentation kinetics showed a good agreement with the experimentally observed kinetics. Both showed a decline in sedimentation velocity over time, caused primarily by variation in (oo)cyst density. The initial apparent sedimentation velocities in Hanks balanced salt solution at 23 degrees C was 0.35 micron . s-1 for oocysts and 1.4 micron . s-1 for cysts. (Oo)cysts that enter the surface water environment by discharges of biologically treated sewage may be attached to sewage particles, and this will affect their sedimentation kinetics. Therefore, (oo)cysts were mixed with settled secondary effluent. (Oo)cysts readily attached to the (biological) particles in effluent; 30% of both cysts and oocysts attached during the first minutes of mixing, and this fraction increased to approximately 75% after 24 h. The sedimentation velocity of (oo)cysts attached to secondary effluent particles increased with particle size and was (already in the smallest size fraction [1 to 40 micron]) determined by the sedimentation kinetics of the effluent particles. The observed sedimentation velocities of freely suspended (oo)cysts are probably too low to cause significant sedimentation in surface water or reservoirs. However, since a significant proportion of both cysts and oocysts attached readily to organic biological particles in secondary effluent, sedimentation of attached (oo)cysts after discharge into surface water will probably be a significant factor in the environmental ecology of C. parvum and G. lamblia. Attachment to particles influences not only sedimentation of (oo)cysts in surface water but also their behavior in drinking water treatment processes.

Medina, H., J. M. Barboza, et al. (2001). "Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope." Mem Inst Oswaldo Cruz 96(7): 983-6.
	A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 microm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Meinhardt, P. L., D. P. Casemore, et al. (1996). "Epidemiologic aspects of human cryptosporidiosis and the role of waterborne transmission." Epidemiol Rev 18(2): 118-36.
	
Meloni, B. P. and R. C. Thompson (1996). "Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro." J Parasitol 82(5): 757-62.
	Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 micrograms, 4-aminobenzoic acid 100 micrograms, calcium pantothenate 50 micrograms, ascorbic acid 875 micrograms, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously.

Meyer, E. A. and E. L. Jarroll (1980). "Giardiasis." Am J Epidemiol 111(1): 1-12.
	
Millard, P. S., K. F. Gensheimer, et al. (1994). "An outbreak of cryptosporidiosis from fresh-pressed apple cider." Jama 272(20): 1592-6.
	BACKGROUND--Recent waterborne outbreaks have established Cryptosporidium as an emerging enteric pathogen, but foodborne transmission has rarely been reported. In October 1993, an outbreak of cryptosporidiosis occurred among students and staff attending a 1-day school agricultural fair in central Maine. DESIGN--Environmental/laboratory investigation and cohort study. PARTICIPANTS--Attendees of the fair and their household members. MAIN OUTCOME MEASURES--Clinical or laboratory-confirmed cryptosporidiosis. Clinical cryptosporidiosis was defined as 3 days of either diarrhea (three loose stools in a 24-hour period) or vomiting. RESULTS--Surveys were completed for 611 (81%) of the estimated 759 fair attendees. Among attendees who completed the survey, there were 160 (26%) primary cases. Cryptosporidium oocysts were detected in the stools of 50 (89%) of 56 primary and secondary case patients tested. The median incubation period was 6 days (range, 10 hours to 13 days); the median duration of illness was 6 days (range, 1 to 16 days). Eighty-four percent of primary case patients had diarrhea and 82% had vomiting. Persons drinking apple cider that was hand pressed in the afternoon were at increased risk for cryptosporidiosis (154 [54%] of 284 exposed vs six [2%] of 292 unexposed; relative risk, 26; 95% confidence interval, 12 to 59). Cryptosporidium oocysts were detected in the apple cider, on the cider press, and in the stool specimen of a calf on the farm that supplied the apples. The secondary household transmission rate was 15% (53/353). CONCLUSIONS--This is the first large cryptosporidiosis outbreak in which foodborne transmission has been documented. It underscores the need for agricultural producers to take measures to avoid contamination of foodstuffs with infectious agents common to the farm environment.

Miller, W. A., E. R. Atwill, et al. (2005). "Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California." Int J Parasitol 35(6): 673-84.
	This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.

Miller, W. A., M. A. Miller, et al. (2005). "New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast." Int J Parasitol 35(10): 1103-13.
	A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.

Mintz, E. D., M. Hudson-Wragg, et al. (1993). "Foodborne giardiasis in a corporate office setting." J Infect Dis 167(1): 250-3.
	Giardiasis is the most commonly reported intestinal protozoal infection worldwide, but its relatively long incubation period and often insidious onset make detection of common-source outbreaks difficult. Few well-documented foodborne outbreaks of giardiasis have been reported. In November 1990, such an outbreak among insurance company employees resulted in 18 laboratory-confirmed and 9 suspected cases of giardiasis. A case-control study of 26 ill and 162 well employees implicated raw sliced vegetables served in the employee cafeteria and prepared by a food handler infected with Giardia lamblia as the probable vehicle (odds ratio, 5.1; 95% confidence interval, 1.4-22.7). This outbreak illustrates the potential for transmission of Giardia organisms to occur in commercial establishments through a frequently served food item.

Mitschler, R. R., R. Welti, et al. (1994). "A comparative study of lipid compositions of Cryptosporidium parvum (Apicomplexa) and Madin-Darby bovine kidney cells." J Eukaryot Microbiol 41(1): 8-12.
	Membrane lipid compositions of Cryptosporidium parvum and Madin-Darby bovine kidney cells, an epithelial-like cell line commonly used to study coccidia in vitro, were analyzed using both thin-layer chromatography and gas-liquid chromatography. Phosphatidylcholine was the predominant lipid in both C. parvum and Madin-Darby bovine kidney cells, comprising 65% and 41% of the total phospholipids, respectively. Phospholipids of C. parvum contained twice the level of 16:0 and twenty-fold more 18:2 than the Madin-Darby bovine kidney cell line. We suggest that the parasite may be capable of sequestering specific complex membrane lipids at concentrations greater than those in the host cells. This study constitutes the first report of the lipid composition of C. parvum.

Miyahira, Y. and T. Takeuchi (1991). "Application of ATP measurement to evaluation of the growth of parasitic protozoa in vitro with a special reference to Pneumocystis carinii." Comp Biochem Physiol A 100(4): 1031-4.
	1. There was a significant correlation between the increase in the number of Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia and Leishmania donovani in culture, and their ATP contents determined by luciferase reaction. 2. The similar correlation was also demonstrated between the decreased number of E. histolytica in the presence of an anti-amebic quassinoid and the nucleotide content in vitro. 3. In the case of Pneumocystis carinii, the numbers of the organism remained relatively constant in culture for at least 7 days without growth; however, the ATP content dropped rapidly in 1 to 3 days except in RPMI 1640. 4. The possibility that ATP determination of P. carinii is complicated by the host cell nucleotide seemed to be excluded, since the concentration of this nucleotide in normal lung was almost negligible. These observations suggest that the present procedure is useful for evaluating the growth and viability of these organisms in vitro.

Mohammed, H. O., S. E. Wade, et al. (1999). "Risk factors associated with Cryptosporidium parvum infection in dairy cattle in southeastern New York State." Vet Parasitol 83(1): 1-13.
	An observational analytical epidemiologic study was carried out to identify factors associated with the risk of infection with Cryptosporidium parvum in dairy herds in southeastern New York state. A random sample of 2943 cattle on 109 farms was selected from the target population. Fecal samples were collected from animals in three different age groups and examined for the presence of C. parvum using a quantitative centrifugation concentration flotation method. Data on intrinsic, preweaning, postweaning, maternity, and general management factors were collected and evaluated for their association with the risk of infection with C. parvum. Indices for each of these categories of management were developed from factors significantly associated with the risk of infection with C. parvum. Significant factors were identified using the logistic regression statistical technique. A final analysis, including the indices, age, and season, was performed to identify factors significantly associated with the risk of infection with C. parvum while simultaneously controlling for the effect of other factors. The farm effect was evaluated using a mixed effect model. Preweaning factors found to be significantly associated with a decreased risk of infection were: use of ventilation in calf rearing areas, daily addition of bedding, feeding of milk replacer, daily disposal and cleaning of bedding, and use of antibiotics. Postweaning factors such as moving of the animals after weaning, cleaning of soiled bedding, and use of antibiotics and ionophores as preventive measures were significantly associated with the decreased risk of an infection with C. parvum. Consideration of maternity management factors showed that winter housing of cows individually within 2 months of calving, use of fresh colostrum to feed calves, and having a concrete floor in the calving area were significantly associated with decreased risk of C. parvum infection. The total number of dairy cattle, total number of other species of agricultural animals on the farm, and the distance of the barn water source from the septic system were found to be significantly associated with increased risk of C. parvum infection.In the final analysis, the risk of infection with C. parvum was significantly decreased with an increased value of the maternity management index score. The general management significantly affected the risk of infection with C. parvum where the risk increased with the increase of the value of the index. The risk of infection significantly decreased with increase in the age of the animal.

Monge, R. and M. L. Arias (1996). "[Presence of various pathogenic microorganisms in fresh vegetables in Costa Rica]." Arch Latinoam Nutr 46(4): 292-4.
	This study reports the occurrence of some pathogenic microorganisms in vegetable consumed on a daily basis by Costa Ricans. Cryptosporidium sp. oocysts were found in 5.2% (4/80) of cilantro leaves, in 8.7% (7/80) of cilantro roots and 2.5% of lettuce samples. A 1.2% (1/80) incidence was found in other vegetables samples (carrot, cucumber, radish and tomatoe). Oocysts of this parasite were absent in cabbage. Giardia intestinalis was only detected in 5.2% (4/80) of cilantro leaves and in 2.5% (2/ 80) of cilantro roots. Entamoeba histolytica cysts were found in 6.2% (5/80) of cilantro leaves, in 2.5% (2/80) cilantro roots, in 3.8% (3/80) lettuce and in 2.5% (2/80) radish samples. At least a 2% incidence of this amoeba was found in other vegetable samples (carrot, cucumber, cabbage and tomatoe). Listeria monocytogenes was isolated in 20% (10/50) of the samples of cabbage salad. Hepatitis A virus and Rotavirus were evidenciated in three of the lettuce pooles, suggesting that at least three of the samples were contaminated with these viruses.

Monge, R. and M. Chinchilla (1996). "Presence of Cryptosporidium oocysts in fresh vegetables." Journal of Food Protection 59(2): 202-203.
	In Costa Rica, a total of 640 samples from eight different vegetables used for raw consumption were analyzed for the presence of Cryptosporidium spp. oocysts, fecal coliforms, and Escherichia coli. Cryptosporidium spp. oocysts were found in 5.0% (4 samples) of cilantro leaves, 8.7% (7 samples) of cilantro roots and 2.5% (2 samples) of lettuce samples. A 1.2% contamination rate was detected in samples of other vegetables (radish, tomato, cucumbers and carrot). Oocysts of this parasite were absent from cabbage. A greater percentage of positive samples was found during the rainy season, and only in cilantro roots and lettuce was a positive linear correlation (P < 0.05) established between the presence of Cryptosporidium spp. oocysts and fecal coliforms and E. coli.

Monge, R., M. Chinchilla, et al. (1996). "[Seasonality of parasites and intestinal bacteria in vegetables that are consumed raw in Costa Rica]." Rev Biol Trop 44(2A): 369-75.
	In Costa Rica, a total of 640 samples from eight different vegetables used for raw consumption, were analyzed for the presence of intestinal parasites and fecal coliforms. Eighty samples of each vegetable were analyzed, forty during the dry season and forty in the rainy. A greater, but unsignificant (p > 0.05) level of fecal coliforms was found during the dry season. Levels of Escherichia coli, were higher (p < 0.05) during the dry season in lettuce (Latuca sativa) and cilantro (Coleandrum sativum) leaves. Cysts of Endolimax nana, Entamoeba coli, Entamoeba histolytica, Giardia intestinalis and Cryptosporidium sp. were found in all vegetables. The greater percentage of positive samples was found during the dry season, although these relation was only corroborated (p < 0.05) in radish (Raphanus sativus) and cilantro leaves. Only lettuce and cilantro levels showed a positive linear correlation (p < 0.05) between occurrences of intestinal parasites and fecal coliforms.

Monis, P. T., G. Mayrhofer, et al. (1996). "Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus." Parasitology 112 ( Pt 1): 1-12.
	Samples of DNA from a panel of Giardia isolated from humans and animals in Europe and shown previously to consist of 2 major genotypes--'Polish' and 'Belgian'--have been compared with human-derived Australian isolates chosen to represent distinct genotypes (genetic groups I-IV) defined previously by allozymic analysis. Homologous 0.52 kilobase (kb) segments of 2 trophozoite surface protein genes (tsa417 and tsp11, both present in isolates belonging to genetic groups I and II) and a 1.2 kb segment of the glutamate dehydrogenase (gdh) gene were amplified by the polymerase chain reaction (PCR) and examined for restriction fragment length polymorphisms (RFLPs). Of 21 'Polish' isolates that were tested, all yielded tsa417-like and tsp11-like PCR products that are characteristic of genetic groups I or II (15 and 6 isolates respectively) in a distinct assemblage of G. intestinalis from Australia (Assemblage A). Conversely, most of the 19 'Belgian' isolates resembled a second assemblage of genotypes defined in Australia (Assemblage B) which contains genetic groups III and IV. RFLP analysis of gdh amplification products showed also that 'Polish' isolates were equivalent to Australian Assemblage A isolates (this analysis does not distinguish between genetic groups I and II) and that 'Belgian' isolates were equivalent to Australian Assemblage B isolates. Comparison of nucleotide sequences determined for a 690 base-pair portion of the gdh PCR products revealed > or = 99.0% identity between group I and group II (Assemblage A/'Polish') genotypes, 88.3-89.7% identity between Assemblage A and Assemblage B genotypes, and > or = 98.4% identity between various Assemblage B/'Belgian' genotypes. The results confirm that the G. duodenalis isolates examined in this study (inclusive of G. intestinalis from humans) can be divided into 2 major genetic clusters: Assemblage A (= 'Polish' genotype) containing allozymically defined groups I and II, and Assemblage B (= 'Belgian' genotype) containing allozymically defined groups III and IV and other related genotypes.

Montemayor, M., F. Valero, et al. (2005). "Occurrence of Cryptosporidium spp. oocysts in raw and treated sewage and river water in north-eastern Spain." J Appl Microbiol 99(6): 1455-62.
	AIMS: To determine the occurrence and levels of Cryptosporidium parvum oocysts in wastewater and surface waters in north-eastern Spain. METHODS AND RESULTS: Samples from five sewage treatment plants were taken monthly and quarterly during 2003. In addition, water was collected monthly from the River Llobregat (NE Spain) during the period from 2001 to 2003. All samples were analysed by filtration on cellulose acetate filters or through Envirocheck using EPA method 1623, followed by immunomagnetic separation and examination by laser scanning cytometry. All raw sewage, secondary effluent and river water samples tested were positive for Cryptosporidium oocysts. Of the tertiary sewage effluents tested, 71% were positive for Cryptosporidium oocysts. The proportion of viable oocysts varied according to the sample. CONCLUSIONS: Two clear maxima were observed during spring and autumn in raw sewage, showing a seasonal distribution and a correlation with the number of cryptosporidiosis cases and rainfall events. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of Cryptosporidium oocysts in natural waters in north-eastern Spain.

Montoya, J. G. and O. Liesenfeld (2004). "Toxoplasmosis." Lancet 363(9425): 1965-76.
	Toxoplasma gondii is a protozoan parasite that infects up to a third of the world's population. Infection is mainly acquired by ingestion of food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts. Primary infection is usually subclinical but in some patients cervical lymphadenopathy or ocular disease can be present. Infection acquired during pregnancy may cause severe damage to the fetus. In immunocompromised patients, reactivation of latent disease can cause life-threatening encephalitis. Diagnosis of toxoplasmosis can be established by direct detection of the parasite or by serological techniques. The most commonly used therapeutic regimen, and probably the most effective, is the combination of pyrimethamine with sulfadiazine and folinic acid. This Seminar provides an overview and update on management of patients with acute infection, pregnant women who acquire infection during gestation, fetuses or infants who are congenitally infected, those with ocular disease, and immunocompromised individuals. Controversy about the effectiveness of primary and secondary prevention in pregnant women is discussed. Important topics of current and future research are presented.

Morgan, U., R. Weber, et al. (2000). "Molecular characterization of Cryptosporidium isolates obtained from human immunodeficiency virus-infected individuals living in Switzerland, Kenya, and the United States." J Clin Microbiol 38(3): 1180-3.
	A total of 22 Cryptosporidium isolates from human immunodeficiency virus-infected patients from Kenya, Switzerland, and the United States were examined at three genetic loci: the 18S ribosomal DNA, HSP-70, and acetyl coenzyme A synthetase genes. Four distinct Cryptosporidium genotypes were identified: (i) the Cryptosporidium parvum "human" genotype, (ii) the C. parvum "cattle" genotype, (iii) Cryptosporidium felis, and (iv) Cryptosporidium meleagridis. This is the first report of C. meleagridis in a human host. These results and those of others indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes. Future studies are required to understand the full public health significance of Cryptosporidium genotypes and species in immunocompromised hosts.

Morgan, U. M., J. R. Buddle, et al. (1999). "Molecular and biological characterisation of Cryptosporidium in pigs." Aust Vet J 77(1): 44-7.
	OBJECTIVE: Genetic and biological characterisation of 12 isolates of Cryptosporidium from pigs and comparing them with Cryptosporidium isolates from humans and cattle. DESIGN: Cryptosporidium isolates from pigs were compared with those obtained from human and cattle using rDNA sequence analysis. The infectivity of two of the porcine isolates was determined in neonatal mice and the clinical history of the infected pigs recorded. RESULTS: Pig-derived isolates of Cryptosporidium exhibited two distinct genotypes; a porcine genotype and a bovine genotype, which is common to cattle and other livestock. The porcine genotype did not produce any infection in neonatal mice whereas the bovine genotype did. CONCLUSION: Two distinct genetically and biologically differing strains of Cryptosporidium appeared to be associated with acute diarrhoea in pigs. Whether Cryptosporidium was a primary or secondary pathogen is unclear but warrants further investigation. As the bovine genotype is known to infect humans, the results suggest that pigs can act as reservoirs of cryptosporidial infections for humans and other live-stock. The zoonotic potential of the pig-adapted genotype is uncertain and requires further study.

Morgan, U. M., C. C. Constantine, et al. (1997). "Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis." J Parasitol 83(5): 825-30.
	Sequence analysis of a polymerase chain reaction (PCR)-amplified 298-bp region of the Cryptosporidium parvum 18S rRNA gene was carried out on 10 human and 9 animal isolates. Eight of the 9 animal isolates and 3 human isolates displayed the recognition sequence TATATTT, whereas 7/10 human isolates exhibited the recognition sequence TTTTTTTTTTT. Sequence analysis of the ninth animal isolate, which was recovered from a Koala, revealed this isolate to be different from both human and animal isolates. The AT richness of the rDNA recognition sequences rendered them unsuitable for primer design and therefore a diagnostic randomly amplified polymorphic DNA fragment previously developed in our laboratory was also sequenced. Analysis of 2 human and 2 animal isolates again revealed distinct differences between animal and human isolates. On the basis of this sequence information, diagnostic primers were designed that could directly differentiate between animal and human isolates on the basis of the size of the PCR product. The ability to differentiate directly between human and animal isolates has important implications for studies of the transmission and zoonotic potential of this organism. These results also raise further doubts about the uniformity of the species C. parvum.

Morgan, U. M., C. C. Constantine, et al. (1995). "Molecular characterization of Cryptosporidium isolates from humans and other animals using random amplified polymorphic DNA analysis." Am J Trop Med Hyg 52(6): 559-64.
	Genetic variation in 25 Cryptosporidium isolates was analyzed using the random amplified polymorphic DNA (RAPD) technique. Simple reproducible polymorphisms were generated (using five primers) from Cryptosporidium DNA that was free of contaminating bacterial DNA. The results generated by four of the five primers were statistically correlated (P < 0.001). The combined data from three primers were used to construct a phenogram using Jaccard's distance. Four groupings could be distinguished. Two C. serpentis isolates from snakes formed a distinct group of their own, whereas C. parvum isolates were divided into two main groups: one containing most human isolates and the other containing mostly domestic animals plus two remaining human isolates. Due to the sensitivity of the RAPD technique, isolates can now be analyzed genetically, directly from fecal samples without further biological amplification. This represents a significant advance on current techniques.

Morgan, U. M., P. T. Monis, et al. (1999). "Phylogenetic relationships among isolates of Cryptosporidium: evidence for several new species." J Parasitol 85(6): 1126-33.
	Isolates of Cryptosporidium were characterized using nucleotide sequence analysis of the 18S rRNA and dihydrofolate reductase genes and also random-amplified polymorphic DNA analysis. Phylogenetic analysis confirmed the validity of the species of Cryptosporidium examined in this study such as Cryptospordium muris and Cryptosporidium baileyi, and also reinforced evidence from numerous researchers worldwide suggesting that Cryptosporidium parvum is not a single uniform species. The data obtained provided strong support for the validity of Cryptosporidium felis. Evidence suggests that the newly identified marsupial and pig genotypes may also be distinct and valid species, but biological studies are required for confirmation.

Morgan, U. M., P. T. Monis, et al. (2001). "Molecular and phylogenetic characterisation of Cryptosporidium from birds." Int J Parasitol 31(3): 289-96.
	Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

Morgan, U. M., L. Pallant, et al. (1998). "Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: clinical trial." J Clin Microbiol 36(4): 995-8.
	PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.

Morgan, U. M., A. P. Sturdee, et al. (1999). "The Cryptosporidium "mouse" genotype is conserved across geographic areas." J Clin Microbiol 37(5): 1302-5.
	A 298-bp region of the Cryptosporidium parvum 18S rRNA gene and a 390-bp region of the acetyl coenzyme A synthetase gene were sequenced for a range of Cryptosporidium isolates from wild house mice (Mus domesticus), a bat (Myotus adversus), and cattle from different geographical areas. Previous research has identified a distinct genotype, referred to as the "mouse"-derived Cryptosporidium genotype, common to isolates from Australian mice. Comparison of a wider range of Australian mouse isolates with United Kingdom and Spanish isolates from mice and cattle and also an Australian bat-derived Cryptosporidium isolate revealed that the "mouse" genotype is conserved across geographic areas. Mice are also susceptible to infection with the "cattle" Cryptosporidium genotype, which has important implications for their role as reservoirs of infection for humans and domestic animals.

Morgan, U. M., L. Xiao, et al. (1999). "Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis." J Parasitol 85(3): 525-30.
	Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

Morgan, U. M., L. Xiao, et al. (1999). "Variation in Cryptosporidium: towards a taxonomic revision of the genus." Int J Parasitol 29(11): 1733-51.
	Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the difficulty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from different species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed.

Morgan, U. M., L. Xiao, et al. (2000). "Epidemiology and strain variation of Cryptosporidium parvum." Contrib Microbiol 6: 116-39.
	
Morgan, U. M., L. Xiao, et al. (2000). "Detection of the Cryptosporidium parvum "human" genotype in a dugong (Dugong dugon)." J Parasitol 86(6): 1352-4.
	The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.

Morgan, U. M., L. Xiao, et al. (2000). "Cryptosporidium meleagridis in an Indian ring-necked parrot (Psittacula krameri)." Aust Vet J 78(3): 182-3.
	OBJECTIVE: To perform a morphological and genetic characterisation of a Cryptosporidium infection in an Indian ring-necked parrot (Psittacula krameri) and to compare this with C meleagridis from a turkey. DESIGN: Tissue and intestinal sections from an Indian ring-necked parrot were examined microscopically for Cryptosporidium. The organism was also purified from the crop and intestine, the DNA extracted and a portion of the 18S rDNA gene amplified, sequenced and compared with sequence and biological information obtained for C meleagridis from a turkey as well as sequence information for other species of Cryptosporidium. RESULTS: Morphological examination of tissue sections from an Indian ring-necked parrot revealed large numbers of Cryptosporidium oocysts attached to the apical border of enterocytes lining the intestinal tract. Purified Cryptosporidium oocysts measured about 5.1 x 4.5 microns, which conformed morphologically to C meleagridis. The sequence obtained from this isolate was identical to sequence information obtained from a C meleagridis isolate from a turkey. CONCLUSION: Cryptosporidium meleagridis was detected in an Indian ring-necked parrot using morphological and molecular methods. This is the first time that this species of Cryptosporidium has been reported in a non-galliform host and extends the known host range of C meleagridis.

Morgan, U. M., L. Xiao, et al. (2000). "Cryptosporidium spp. in domestic dogs: the "dog" genotype." Appl Environ Microbiol 66(5): 2220-3.
	Genetic and phylogenetic characterization of Cryptosporidium isolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derived Cryptosporidium isolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the "dog" genotype is, in fact, a valid species.

Morgan, U. M., L. Xiao, et al. (2000). "Molecular and phylogenetic analysis of Cryptosporidium muris from various hosts." Parasitology 120 ( Pt 5): 457-64.
	Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris; (1) C. muris genotype A; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.

Morgan-Ryan, U. M., A. Fall, et al. (2002). "Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens." J Eukaryot Microbiol 49(6): 433-40.
	The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.

Morita, S., A. Namikoshi, et al. (2002). "Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts." Appl Environ Microbiol 68(11): 5387-93.
	To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log(10) reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm(2) at 20 degrees C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm(2) for a 2-log(10) reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm(2). Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10 degrees C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log(10) reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.

Moss, D. M. and M. J. Arrowood (2001). "Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry." J Parasitol 87(2): 406-12.
	Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.

Moss, D. M., S. N. Bennett, et al. (1994). "Kinetic and isotypic analysis of specific immunoglobulins from crew members with cryptosporidiosis on a U.S. Coast Guard cutter." J Eukaryot Microbiol 41(5): 52S-55S.
	
Moss, D. M., C. L. Chappell, et al. (1998). "The antibody response to 27-, 17-, and 15-kDa Cryptosporidium antigens following experimental infection in humans." J Infect Dis 178(3): 827-33.
	Previous studies have suggested that persons infected with Cryptosporidium parvum develop antibody responses to 27-, 17-, and 15-kDa C. parvum antigens. Studies of volunteers infected with Cryptosporidium species provided an opportunity to evaluate the relationship between antibody reactivity to these antigens and infection outcome. As monitored by immunoblot, increases in specific antibody reactivity were more prevalent among volunteers who developed signs and symptoms of cryptosporidiosis (n = 11) than among asymptomatic infected (n = 7; P = .05) or oocyst-negative volunteers (n = 11; P = .02). Volunteers with preexisting IgG antibody to the 27-kDa antigen excreted fewer oocysts than volunteers without this antibody (P = .003). IgG reactivity to the 17-kDa antigens and IgM reactivity to the 27-kDa antigens were higher at day 0 for asymptomatic infected persons than for those who developed symptoms (P = .03 and P = .04, respectively). These results suggest that characteristic antibody responses develop following C. parvum infection and that persons with preexisting antibodies may be less likely to develop illness.

Mtambo, M. M., E. Wright, et al. (1996). "Infectivity of a Cryptosporidium species isolated from a domestic cat (Felis domestica) in lambs and mice." Res Vet Sci 60(1): 61-4.
	Two neonatal lambs were inoculated orally with purified Cryptosporidium species oocysts isolated from a farm cat. Oocysts first appeared in the faeces of the two lambs three and 10 days after infection. Two distinct sizes of oocysts were observed in the faeces of both the cat and the lambs, the smaller measuring approximately 5.0 x 4.5 microns and the larger measuring approximately 6.0 x 5.0 microns in diameter. The smaller type predominated. Histological examination of the alimentary tract of the lambs revealed endogenous stages of Cryptosporidium in the epithelial borders of the ileum. In addition, Cryptosporidium oocysts were detected in impression smears from the jejunum, ileum, caecum and colon. Suspensions of 10(3) oocysts from the faeces of the farm cat were inoculated into each of 10 newborn mice and 10(4) oocysts from the two experimentally infected lambs were inoculated into each of 20 newborn mice. Cryptosporidium oocysts were detected in gut homogenates from 19 of the 20 mice inoculated with oocysts from the lambs but in none of the mice inoculated with oocysts from the cat.

Muthusamy, D., S. S. Rao, et al. (2006). "Multilocus genotyping of Cryptosporidium sp. isolates from human immunodeficiency virus-infected individuals in South India." J Clin Microbiol 44(2): 632-4.
	This study characterized cryptosporidial infections in 48 human immunodeficiency virus-infected individuals in India by multilocus genotyping. Cryptosporidium hominis, C. parvum, C. felis, C. muris, and C. meleagridis were identified. Cpgp40/15 PCR-restriction fragment length polymorphism identified six subgenotypes. Cryptosporidial diarrhea was associated with decreased CD4 counts, below 200 (P = 0.009), but not high viral loads.

Naciri, M., M. P. Lefay, et al. (1999). "Role of Cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in France." Vet Parasitol 85(4): 245-57.
	This study was carried out to find the importance of Cryptosporidium parvum in diarrhoea of neonatal calves in two types of breeding - suckling and dairy calves - in France. Different agents causing neonatal diarrhoea, E. coli, rotavirus, coronavirus, Salmonella and Cryptosporidium were systematically researched in faeces. 1. Suckling calves: In 40 livestock farms selected for diarrhoea, 311 calves 4 to 10 days old which had diarrhoea for less than 24h or no diarrhoea, were included in the study. A prophylaxis of neonatal diarrhoea had been carried out in 21 of the 40 livestock farms. On D0 (inclusion day), the mean age was 6 days, 82% presented a good initial general condition and 76.2% had a good appetite; 48.6% were diarrhoeic but 91.3% presented no sign of dehydration. Only 6.1% were infected by E. coli K99, 14.3% by rotavirus, 6.8% by coronavirus, 0.3% by Salmonella but 50% excreted C. parvum oocysts. This later percentage increases up to 84% and 86% by D3 and D7, respectively . We note that 16% of the 4-day-old calves on D0 are excreting oocysts and this percentage increases as a function of the age of the calf on D0 to reach 90% to 95% by the age of 8 days. 10 out of 12 dead calves excreted C. parvum oocysts. From D0 to D14 the other pathogen agents show a relative or a decreasing stability. 2. Dairy calves: 382 calves which had diarrhoea for less than 24 h or no diarrhoea, aged 8 to 15 days coming from six industrial livestock farms were included in the study. On D0, 99% of the calves presented a good initial general condition, 99.7% had a good appetite and no calf was dehydrated. At this date (D0), 16.8% of the calves excreted cryptosporidia. This percentage increases up to 23% and 51.8% on D3 and D8, respectively, then decreases to 31.9% on D14. The pressure of the other pathogenicagents remains relatively stable, excepted for rotavirus on D7 (from 9.9% on D0 to 27.2% on D7, then 12.6% on D14) which does not explain the concomitantpeak in diarrhoea because the infection by rotavirus on D7 is more frequent in non-diarrhoeic calves than in diarrhoeic calves. Our results show that Cryptosporidium prevalence is higher in suckling than in dairy calves and C. parvum constitutes actually in both cases the major aetiological agent of neonatal diarrhoea.

Naciri, M., R. Mancassola, et al. (1994). "Treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum." Vet Parasitol 53(3-4): 173-90.
	Ovine or bovine colostrums with different antibody titers were tested for their ability to prevent cryptosporidiosis in five groups of neonatal lambs experimentally infected with 10(6) Cryptosporidium parvum oocysts 2 days after birth (Day 0). In a control group (Group 1), six lambs were deprived of ewe colostrum and exclusively fed with milk replacer. Two groups of six lambs were allowed to suckle their non-hyperimmunized (Group 2) or hyperimmunized (Group 3) dams throughout the experiment. Two groups of seven lambs were separated from their dams at birth before suckling and fed with non-hyperimmune (Group 4) or hyperimmune (Group 5) bovine colostrum; for 7 days they received 50 ml of colostrum completed by milk replacer twice a day, then they were fed with milk replacer exclusively. Control lambs became infected and developed clinical cryptosporidiosis with diarrhea on Days 4-9 post inoculation, oocyst shedding and mortality (2/6). In all the treated groups, the colostrum prevented mortality and clinical cryptosporidiosis. The mortality (5/7) observed in Group 5 was not due to cryptosporidiosis but anemia. In treated groups, specific antibodies were detected on Day 0 after 2 days of colostrum intake and varied little in time for IgM and IgG in spite of the parasite development, whereas they appeared later in the control group, on Day 4 for IgM, Day 11 for IgA and Day 14 for IgG. In all groups, the response which was the most consistent was the IgA response which evolved from Days 11 to 18 in association with the decline of oocyst shedding. Our results show that whatever the serum antibody titers were, the specific C. parvum circulating antibodies have no influence on the control of cryptosporidiosis. The prophylaxis or the treatment of cryptosporidiosis require high titers of specific C. parvum antibodies in the gut lumen during a sufficiently long period.

Naciri, M., R. Mancassola, et al. (1993). "The effect of halofuginone lactate on experimental Cryptosporidium parvum infections in calves." Vet Parasitol 45(3-4): 199-207.
	The chemoprophylactic effects of halofuginone lactate were tested against calf experimental cryptosporidiosis. Twenty 2-day-old calves, divided into four groups, were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium parvum. The infected control group was unmedicated whereas the three other groups were medicated with the drug at 30, 60 and 120 micrograms kg-1 day-1, respectively, for 7 days, from Day (D) 2 to D8 post-inoculation (D 0 was inoculation day). The calves were weighed twice weekly and disease development and drug efficacy were assessed daily from D0 to D30 from consistency of feces, shedding of oocysts and mortality. Experimental C. parvum infection caused a severe clinical disease with profuse watery diarrhea, high oocyst shedding and mortality (3 out of 5) in the unmedicated group. The results clearly demonstrated the efficacy of halofuginone lactate in reducing the severity of clinical cryptosporidiosis. This efficacy was dose-dependent. The lowest dose (30 micrograms kg-1 day-1) was not able to prevent clinical disease and mortality (3 out of 5). No clinical signs were observed with the 60 and 120 micrograms kg-1 day-1 doses, but the animals shed oocysts after drug withdrawal. This shedding was more delayed the higher the dose of drug administered, but the delayed shedding had no effect on the growth of the animals.

Navin, T. R. and D. D. Juranek (1984). "Cryptosporidiosis: clinical, epidemiologic, and parasitologic review." Rev Infect Dis 6(3): 313-27.
	Cryptosporidium, an intestinal protozoan parasite, is a well-known cause of diarrhea in animals but has been recognized only recently as a cause of human disease. Since 1976, 58 cases of cryptosporidiosis in humans have been reported; 18 of the patients had normal immune function, and 40 had various immunologic abnormalities, the most common of which, acquired immune deficiency syndrome (AIDS), occurred in 33 patients. Patients with normal immune function had self-limited diarrhea, but patients with immunologic abnormalities often developed severe, irreversible diarrhea; 22 patients have died. The diagnosis of cryptosporidiosis can now be made noninvasively, but increased diagnostic proficiency has led to little improvement in control or treatment of the disease. Although 23 compounds have been evaluated in experimentally infected animals and 20 drugs have been used in human clinical trials, no effective chemotherapeutic agent for cryptosporidiosis has been identified to date.

Nesterenko, M. V., M. Tilley, et al. (1995). "A metallo-dependent cysteine proteinase of Cryptosporidium parvum associated with the surface of sporozoites." Microbios 83(335): 77-88.
	A proteinase of 24 kD was found associated with sporozoites of Cryptosporidium parvum. Optimal hydrolysis of azocasein, casein, bovine serum albumin, and gelatin occurred at a pH of 6.5-7.0. Activity against azocasein was inhibited by ethylenediaminotetraacetic acid (EDTA), iodoacetic acid (IAA), trans-epoxysuccinyl-L-leucylamido(4-guanido) butane (E-64), and phosphoramidon, suggesting that the enzyme was a metallo-dependent cysteine proteinase. Both serine and aspartate protease inhibitors failed to inhibit enzyme activity. The enzyme was partially purified by preparative isoelectric focusing of parasite membrane proteins. Polyclonal antiserum to parasite membrane proteins was generated in rats. The enzyme-containing fraction was subjected to SDS-PAGE and probed with antiserum, and the antibodies against the protease were eluted directly from nitrocellulose blots. An indirect immunofluorescence assay using these monospecific antibodies revealed that the protease occurred on the surface of sporozoites, but was not associated with oocyst walls, rhoptries, or micronemes.

Nesterenko, M. V., K. Woods, et al. (1999). "Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell." Biochim Biophys Acta 1454(2): 165-73.
	The ability of membrane antigens on sporozoites of the intestinal pathogen, Cryptosporidium parvum, to bind host cell surface antigens was investigated. A novel membrane-associated protein of approximately 47 kDa, designated CP47, was found to possess significant binding affinity for the surface of both human and animal ileal cells. This protein was purified by a combination of anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified CP47 demonstrated competitive binding with parasite-associated membrane antigens to membranes of HCT-8 and ileal cells in a dose-dependent manner. Furthermore, the binding activity of CP47 was found to be Mn2+-sensitive, and was completely inhibited in the presence of 10 mM MnCl2. These results were consistent with earlier findings demonstrating the inhibitory effect of Mn2+ ions on Cryptosporidium infection both in vitro and in vivo (Nesterenko et al., Biol. Trace Elem. Res. 56 (1997) 243-253). Immunoelectron microscopy using gold-conjugated antibodies revealed CP47 to be localized at the apical end of the sporozoites. A single protein with an electrophoretic mobility of 57 kDa was purified from host cell membranes using CP47-Affigel. Similarly, affinity purification of this protein was abrogated in the presence of Mn2+. These data suggest that a novel parasite protein, CP47, may play an important role in sporozoite/host cell attachment.

Nesterenko, M. V., K. M. Woods, et al. (1997). "Effective non-radioactive method of surface labeling Cryptosporidium parvum sporozoites." Acta Trop 65(1): 53-7.
	
Nesterenko, M. V., K. M. Woods, et al. (1997). "Effects of manganese salts on the AIDS-related pathogen, Cryptosporidium parvum in vitro and in vivo." Biol Trace Elem Res 56(3): 243-53.
	The authors examined the effects of manganese salts on the interaction of the AIDS-related pathogen, Cryptosporidium parvum, with human ileoadenocarcinoma (HCT-8) cells in vitro. Manganese (Mn) inhibited binding of C. parvum sporozoite membrane antigens to intact, fixed HCT-8 cells in a dose-dependent fashion, whereas Ca++, Mg++, and Zn++ salts had no effect. Manganese was also found to affect sporozoite penetration of live HCT-8 cells, which resulted in a dose-dependent inhibition of parasite development. However, the levels of Mn++ needed in the live cell assays was approx 10-fold greater than in the fixed-cell assays. This inhibition of parasite development was not reversible when Ca++ or Mg++ were used as competitors. Oral supplementation of suckling mice infected with C. parvum with MnSO4 resulted in significant reductions and, in some cases, elimination of intestinally derived oocysts.

Neumann, N. F., L. L. Gyurek, et al. (2000). "Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice." FEMS Microbiol Lett 183(2): 331-6.
	In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.

Neumann, N. F., L. L. Gyurek, et al. (2000). "Comparison of animal infectivity and nucleic acid staining for assessment of Cryptosporidium parvum viability in water." Appl Environ Microbiol 66(1): 406-12.
	Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.

Ng, J., I. Pavlasek, et al. (2006). "Identification of novel Cryptosporidium genotypes from avian hosts." Appl Environ Microbiol 72(12): 7548-53.
	A total of 430 avian-derived fecal specimens were randomly collected from selected Western Australian commercial aviaries, poultry farms, hatcheries, wildlife parks, and the Perth Zoo and screened for the presence of Cryptosporidium by PCR. Of these, 27 Cryptosporidium-positive isolates were detected, characterized, and compared with 11 avian-derived isolates from the Czech Republic at the 18S rRNA and actin gene loci. Sequence and phylogenetic analysis identified four genetically distinct genotypes, avian genotypes I to IV, from various avian hosts. In addition, the host range for Cryptosporidium galli was extended. Cryptosporidium muris and Cryptosporidium andersoni were also identified in a tawny frogmouth and a quail-crested wood partridge, respectively.

Nichols, R. A., B. M. Campbell, et al. (2003). "Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay." Appl Environ Microbiol 69(7): 4183-9.
	We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65 degrees C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

Nichols, R. A., B. M. Campbell, et al. (2006). "Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring." Appl Environ Microbiol 72(8): 5428-35.
	We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.

Nichols, R. A., C. A. Paton, et al. (2004). "Survival of Cryptosporidium parvum oocysts after prolonged exposure to still natural mineral waters." J Food Prot 67(3): 517-23.
	The survival kinetics of purified Cryptosporidium parvum oocysts of both human and ovine origin, immersed in four still natural mineral waters (total dissolved salts ranging from 91 mg/liter to 430 mg/liter) and reverse osmosis water was assessed by inclusion or exclusion of the fluorogenic vital dyes 4',6-diamidino-2-phenylindole and propidium iodide over a 12-week period. Semipermeable chambers were used to contain the oocysts while immersed in each mineral water type, permitting both intimate interactions between oocysts and matrices and straightforward sampling for viability assessments. The viability of both oocyst types, assessed at weekly intervals, remained unaltered after 12 weeks at 4 degrees C, whereas a progressive decline in the viability of both oocyst isolates was observed when immersed in mineral waters at 20 degrees C. At 20 degrees C, approximately 30% of oocysts remained viable after 12 weeks incubation. Here, temperature was the major factor that adversely affected oocyst survival, although higher mineral content was also proportionally and significantly associated with this increased oocyst inactivation. The prolonged survival of oocysts at 4 degrees C in our studies indicates that they could survive for prolonged periods of time in U.K. groundwaters (average temperature approximately 10 degrees C) and thus represent a potential public health hazard if contamination of mineral water sources by viable oocysts were to occur.

Nichols, R. A. and H. V. Smith (2004). "Optimization of DNA extraction and molecular detection of Cryptosporidium oocysts in natural mineral water sources." J Food Prot 67(3): 524-32.
	The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65 degrees C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65 degrees C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.

Nouri, M. and M. Karami (1991). "Asymptomatic cryptosporidiosis in nomadic shepherds and their sheep." J Infect 23(3): 331-3.
	The formed stool samples of 276 asymptomatic members of 36 households of a group of nomadic shepherds and 215 non-diarrhoeal faecal samples from their sheep were examined for cryptosporidial infection by a modified Ziehl-Nielsen technique. Thirty-six (13%) human samples and 37 (17.2%) samples from sheep were positive. A strong possibility of asymptomatic zoonotic transmission of cryptosporidiosis due to prolonged and constant association of shepherds and their sheep was an important finding.

Nouri, M. and R. Toroghi (1991). "Asymptomatic cryptosporidiosis in cattle and humans in Iran." Vet Rec 128(15): 358-9.
	
Ochiai, Y., C. Takada, et al. (2005). "Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR." Appl Environ Microbiol 71(2): 898-903.
	Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.

O'Connor, R. M., C. M. Thorpe, et al. (2002). "Expression of the highly polymorphic Cryptosporidium parvum Cpgp40/15 gene in genotype I and II isolates." Mol Biochem Parasitol 119(2): 203-15.
	The enteric protozoan Cryptosporidium parvum infects intestinal epithelial cells in a wide range of hosts, causing severe gastrointestinal disease. The invasive sporozoite stage most likely attaches to and invades host cells through multiple host receptor/parasite ligand interactions. Preliminary evidence suggests that the glycoprotein products of the Cpgp40/15 gene, gp40 and gp15, are involved in these interactions. In addition, the Cpgp40/15 gene that encodes these glycopeptides is highly polymorphic in genotype I isolates, suggesting that the gene products may be subject to immune selection. In this study, we characterized the Cpgp40/15 gene in a genotype I isolate and compared expression of the Cpgp40/15 gene in isolates of both genotype. Cpgp40/15 is a single copy gene in both TU502 (genotype I) and GCH1 (genotype II) isolates. However, Northern blot analysis revealed the presence of two transcripts, 2.3 and 1.5 kb in size, in mRNA from GCH1 as well as TU502-infected Caco-2A cells. Accumulation of the two Cpgp40/15 mRNAs peaked 12-24 h post-infection. Using 3'RACE analysis, three polyadenylation sites were identified 371, 978 and 1002 bp downstream of the GCH1 Cpgp40/15 stop codon. Two of these polyadenylation sites were also used in TU502. The sequences of the GCH1 Cpgp40/15 3'untranslated regions (3'UTRs) were identical to genomic sequence and shared 96.7% homology with TU502 3'UTRs. Actinomycin D treatment of GCH1-infected Caco-2A cells followed by Northern blot analysis, revealed that the stability of the 1.5 kb message was considerably greater than that of the 2.3 kb transcript.

O'Donoghue, P. J. (1995). "Cryptosporidium and cryptosporidiosis in man and animals." Int J Parasitol 25(2): 139-95.
	
O'Handley, R. M., A. G. Buret, et al. (2001). "Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function." International Journal for Parasitology 31(1): 73-79.
	Twelve Giardia duodenalis-infected Holstein dairy calves were allocated into a treatment (n = 6) and placebo group (n = 6) according to pre-study faecal cyst counts. Calves in the treatment group received an oral dose of 5 mg/kg fenbendazole once daily for 3 days, while placebo calves received a sterile saline solution. Calves were euthanised 7 days following the initiation of treatment and intestinal were collected and prepared for trophozoite quantitation, histology, electron microscopy, and disaccharidase assays. In all calves treated with fenbendazole, intestinal trophozoites were below detection limits, while in saline-treated calves, trophozoites were observed in all intestinal segments. Histologically, no significant difference was observed between treatment groups with respect to intestinal villus height or crypt depth. However, a significant decline in the number of intraepithelial lymphocytes (IEL) was observed in fenbendazole-treated calves when compared with placebo-treated calves in the duodenum (13.9 +/- 1.2 vs. 17.0 +/- 1.1 IEL/100 enterocytes) and jejunum (21.6 +/- 0.8 vs. 30.7 +/- 1.0 IEL/100 enterocytes). In addition, measurements from TEM micrographs demonstrated a significant increase in microvillus surface area in the jejunum of fenbendazole-treated calves compared with saline-treated calves (31.2 +/- 10.2 vs. 22.8 +/- 7.6 mum(2)). This increase in microvillus surface area was also associated with an increase in jejunal maltase activity in fenbendazole-treated calves compared with calves treated with saline. These results demonstrate that fenbendazole is an effective treatment for giardiasis in calves. fenbendazole treatment eliminated Giardia trophozoites from the small intestine of calves resulting in increased microvillus surface area and greater intestinal enzyme activity. This study also demonstrates that the pathogenesis of giardiasis in calves is similar to that observed in humans and laboratory animals, and provides further evidence that Giardia is a pathogen of cattle with potential economic importance. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. [References: 32] 32

O'Handley, R. M., H. Ceri, et al. (2003). "Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections." Veterinary Parasitology 113(2): 89-98.
	In a previous study, Giardia infection patterns were studied in newborn dairy calves over a 4-month period. Chronic Giardia infections were observed in all calves with initial cyst excretion occurring at approximately 1 month of age. In the work presented here, the passive immunity and serological immune response associated with these Giardia infections were examined. Colostrum and milk samples were collected from the dams of these calves, and monthly serum samples were collected from each calf. The colostrum, milk and sera samples were analyzed by ELISA and Western blot for the presence of anti-Giardia IgG antibodies. In addition, the in vitro anti-Giardia activity of milk and colostrum was examined using a miniculture adherence assay. When examined by ELISA, mean anti-Giardia antibody titres were found to be significantly higher in colostrum compared to milk. The monthly mean serum antibody titres in the calves were not found to differ significantly at any time point during the study. Western blot analysis revealed that colostrum from the dams reacted strongly with many different Giardia antigens between 205 and 7.5 kDa, while milk reacted with few antigens in the same size range. Sera collected from the calves when 30 and 60 days of age reacted with few Giardia antigens, but as the calves aged, IgG antibodies in their sera began to react with antigens of 21, 50, 65, 73 and 79 kDa. The miniculture adherence assay demonstrated that colostrum had significantly more anti-Giardia activity in vitro compared to milk. These results suggest that the calves in this dairy did not mount a significant humoral immune response against Giardia following infection. However, colostrum contained a high level of anti-Giardia antibodies and exhibited anti-Giardia activity in vitro. Therefore, colostrum may have the potential to provide initial protection against Giardia infections in calves, but the lack of a strong, specific humoral immune response by these calves could account for the high prevalence and chronic duration of the infections. (C) 2003 Elsevier Science B.V. All rights reserved. [References: 31] 31

O'Handley, R. M., M. E. Olson, et al. (2000). "Prevalence and genotypic characterisation of Giardia in dairy calves from Western Australia and Western Canada." Veterinary Parasitology 90(3): 193-200.
	In this study, the prevalence of Giardia duodenalis infections was determined in Western Canadian and Western Australian dairy calves. Faecal samples were collected from Holstein calves located on a commercial dairy near Lethbridge, Alta., Canada (N=28) and from calves located on two commercial dairies located near Perth, WA, Australia (N=36). Faecal samples were examined for the presence of Giardia cysts using sucrose gradient centrifugation, followed by immunofluoresence microscopy. DNA was then extracted from Giardia isolates obtained from positive samples. A PCR based method was employed to amplify and sequence a 292 bp region of the 16S-rRNA gene. Genetic sequences obtained from Giardia isolates were compared to each other and to previously sequenced isolates. Following a single faecal sample, 58% of Western Australian calves and 57% of Western Canadian calves were positive for Giardia. Geometric mean cyst counts/g of faeces were 839 for Western Australian calves and 3475 for Western Canadian calves, but these values did not differ significantly. Genetic sequences were obtained from 10 calves from Western Canada, while six sequences were obtained from Western Australian calves. Of the Western Canadian isolates, eight aligned with the proposed 'Hoofed livestock' genotype. Of the five isolates obtained from Western Australian calves, four sequences were identical to the 'Hoofed-livestock' genotype. Two isolates from the Western Canadian calves and one isolate from the Western Australian calves had the identical genetic sequence to the Genotype (Assemblage) A sequence, a common human genotype. The results of this study demonstrate, for the first time, that Giardia infections occur in Western Australian calves. Also, calves from different geographical locations appear to be primarily infected with a Giardia genotype unique to hoofed livestock. However, calves can shed Giardia cysts potentially infective for humans. Thus, Giardia infections should be considered important to Australian dairy producers, and infections in calves may pose a risk to public health regardless of geographical location. (C) 2000 Elsevier Science B.V. All rights reserved. [References: 29] 29

Ohandley, R. M., M. E. Olson, et al. (1997). "Efficacy of Fenbendazole for Treatment of Giardiasis in Calves." American Journal of Veterinary Research 58(4): 384-388.
	Objective-To determine efficacy of fenbendazole for treatment of giardiasis in calves. Animals-Twenty male and 15 female Holstein calves (100 to 180 kg), naturally infected with Giardia sp. Procedure-In vitro fenbendazole susceptibility and resistance development was determined for a ruminant Giardia isolate by use of an adherence assay. Carves were treated as follows. group 1, a single administration of 5 mg of fenbendazole/kg of body weight; group 2, a single administration of 10 mg of fenbendazole/kg; group 3, 5 mg of fenbendazole/kg, every 24 hours for 3 days; group 4, 10 mg of fenbendazole/kg, every 24 hours for 3 days; group 5, 20 mg of fenbendazole/kg, every 24 hours for 3 days; group 6, 0.833 mg of fenbendazole/kg, even/ 24 hours for 6 days; and group 7, saline solution. Fecal Giardia cysts were counted on days -3 through -1 and 1 through 7, 9, 11, 13, 21, and 28 by use of sucrose gradient concentration and staining with a fluorescent monoclonal antibody. Results-The 50% adherence inhibition concentration was 0.024 +/- 0.002 mu g/ml, and resistance could not be detected after 5 weeks of continuous culture at sublethal concentration of fenbendazole (0.01 mu g/ kg). Fenbendazole was 100% effective in eliminating cysts from the feces within 6 days for calves in treatment groups 2-6. Reinfection was observed in some calves within the 28-day study period. Conclusions- Fenbendazole is effective in the elimination of Giardia infections in calves, but repeat treatments may be required in reinfected animals. Clinical Relevance-Fenbendazole is an effective and economical treatment for Giardia-associated diarrhea and growth rate reduction in calves. [References: 38] 38

Okhuysen, P. C., C. L. Chappell, et al. (1999). "Virulence of three distinct Cryptosporidium parvum isolates for healthy adults." J Infect Dis 180(4): 1275-81.
	The infectivity of three Cryptosporidium parvum isolates (Iowa [calf], UCP [calf], and TAMU [horse]) of the C genotype was investigated in healthy adults. After exposure, volunteers recorded the number and form of stools passed and symptoms experienced. Oocyst excretion was assessed by immunofluorescence. The ID50 differed among isolates: Iowa, 87 (SE, 19; 95% confidence interval [CI], 48.67-126); UCP, 1042 (SE, 1000; 95% CI, 0-3004); and TAMU, 9 oocysts (SE, 2.34; 95% CI, 4.46-13.65); TAMU versus Iowa, P=.002 or UCP, P=.019. Isolates also differed significantly (P=.045) in attack rate between TAMU (86%) and Iowa (52%) or UCP (59%). A trend toward a longer duration of diarrhea was seen for the TAMU (94.5 h) versus UCP (81.6 h) and Iowa (64.2 h) isolates. C. parvum isolates of the C genotype differ in their infectivity for humans.

Okhuysen, P. C., S. M. Rich, et al. (2002). "Infectivity of a Cryptosporidium parvum isolate of cervine origin for healthy adults and interferon-gamma knockout mice." J Infect Dis 185(9): 1320-5.
	The infectivity of a Cryptosporidium parvum isolate of cervine origin (type 2, Moredun) propagated in calves was investigated simultaneously in healthy adult human volunteers and in interferon-gamma knockout (GKO) mice. After exposure to 100-3000 oocysts, 16 volunteers recorded, for a duration of 6 weeks, the number and form of stools that they passed and any symptoms that they experienced. Oocyst excretion was assessed by enzyme-linked immunosorbent assay and direct immunofluorescence assay. Eleven subjects (69%) became ill, and 8 subjects (50%) shed oocysts in stool. The median duration of illness was 169 h, and the median number of unformed stools passed was 24. The duration and intensity of symptoms were more severe than were those associated with previously studied isolates. The median infectious dose was estimated to be 300 oocysts for humans and 1 oocyst for the GKO mouse model. The Moredun isolate was more pathogenic than the reference GCH-1 isolate. The GKO mouse model of cryptosporidiosis is useful for discerning isolate-specific differences in pathogenicity.

Olson, E. J., W. B. Epperson, et al. (1998). "Effects of an allicin-based product on cryptosporidiosis in neonatal calves." J Am Vet Med Assoc 212(7): 987-90.
	OBJECTIVE: To evaluate effectiveness of an allicin-based product in neonatal calves inoculated with Cryptosporidium parvum. DESIGN: Randomized controlled study. ANIMALS: 43 neonatal calves. PROCEDURE: Calves were inoculated with 1.5 x 10(8) or 7.5 x 10(5) C parvum oocysts within 2 days after birth. Calves were given an allicin-based product once after inoculation or daily for 7 days after inoculation or were not treated. Calves that developed diarrhea were treated by administration of the product. Fecal consistency scores and weight gains were statistically evaluated. RESULTS: Mean daily weight gain and severity of diarrhea in calves 4 to 21 days old were unaffected by prophylactic use of the product. However, intensive prophylactic administration may have delayed onset of C parvum-induced diarrhea in calves inoculated with the lower dose of oocysts. CLINICAL IMPLICATIONS: Administration of an allicin-based product did not alter duration of C parvum-induced diarrhea or enhance weight gain in neonatal calves. However, intensive prophylactic administration of an allicin-based product may delay onset of diarrhea in calves exposed to C parvum oocysts.

Olson, M. E., H. Ceri, et al. (2000). "Giardia vaccination." Parasitology Today 16(5): 213-217.
	Recently, a Giardia vaccine has become commercially available in the USA for prevention of clinical signs of giardiasis and reduction of cyst shedding in dogs and cats. The vaccine is based upon the current state of knowledge of Giardia antigenicity and immunology. Here, Merle Olson, Howard Ceri and Douglas Morck describe studies that led to the development of this vaccine and subsequent efficacy studies. Immunoprophylaxis and immunotherapeutic application of the vaccine are discussed. [References: 27] 27

Olson, M. E., J. Goh, et al. (1999). "Giardia cyst and Cryptosporidium oocyst survival in water, soil, and cattle feces." Journal of Environmental Quality 28(6): 1991-1996.
	Giardiasis and cryptosporidiosis are gastrointestinal diseases caused by protozoan parasites that mag infect domestic animals, wildlife and human beings. The ability of cysts and oocysts of these parasites to persist in the environment was determined because agricultural fecal waste has the potential to contaminate municipal water supplies. The degradation rate and viability of Giardia cysts and Cryptosporidium oocysts in water, cattle (Bos taurus) feces, and soil was evaluated at temperatures of -4, 4, and 25 degrees C for up to 12 wk. Cysts and oocysts were enumerated after staining samples with a specific fluorescent monoclonal antibody and the viability was determined using propidium iodide dye exclusion and mouse infectivity assays. Giardia cysts were noninfective in water, feces, and soil following 1 wk of freezing to -4 degrees C and within 2 wk at 25 degrees C. At 4 degrees C Giardia cysts were infective for 11 wk in water, 7 wk in soil, and 1 wk in cattle feces. Cryptosporidium oocysts were more environmentally resistant. At -4 and 4 degrees C, the oocysts could survive in water and soil for >12 wk but degradation was accelerated at 25 degrees C. Cryptosporidium oocysts also were degraded more rapidly in feces and in soil containing natural microorganisms. Contaminated cattle feces should be distributed on fields during warmer weather and after 12 wk of storage to reduce potential waterborne transmission following heavy runoffs. [References: 16] 16

Olson, M. E., N. J. Guselle, et al. (1997). "Giardia and Cryptosporidium in Dairy Calves in British Columbia." Canadian Veterinary Journal - Revue Veterinaire Canadienne 38(11): 703-706.
	A study was undertaken to determine the prevalence of Giardia infections in dairy calves and to compare Giardia and Cryptosporidium infections in calves of different ages. Fresh fecal samples were collected from 386 male and female Holstein calves (newborn to 24 wk) in 20 dairies located in the lower Fraser river valley area of British Columbia. Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium muris were enumerated in each sample after concentration by sucrose gradient centrifugation and immunofluorescent staining. Giardia was identified at all farm locations. The overall prevalence of Giardia in calves was 73% with a geometric mean cyst count of 1180 cysts per gram of feces (CI, 41 to 5014). Cryptosporidium parvum and C. muris were identified in 80% and 40% of the farms, respectively. The prevalence of C. parvum was 59%, and the geometric mean for oocysts was 457 oocysts per gram of feces (CI, 18 to 160). The prevalence of C. muris was only 2% and the mean oocyst counts were 54 oocysts per gram of feces. Giardiasis was not age dependent, and approximately 80% of the calves from 2 to 24 wk were infected. In contrast, C. parvum infections were predominant in calves 2 to 4 wk, while C. muris was demonstrated in calves older than 4 wk. Fourty-seven percent of calves with diarrhea had high numbers of Giardia cysts in their feces. Giardia infections are highly prevalent in dairy calves and should be considered in animals with diarrhea or failure to thrive. [References: 22] 22

Olson, M. E., C. J. Hannigan, et al. (2001). "The use of a Giardia vaccine as an immunotherapeutic agent in dogs." Canadian Veterinary Journal - Revue Veterinaire Canadienne 42(11): 865-868.
	Dogs (n = 13), which had failed to be cured of giardiosis following chemotherapeutic measures, were treated with a Giardia vaccine (2-3 injections). Clinical signs resolved between 16 and 42 days postvaccination and cessation of fecal cyst shedding was between 21 and 70 days. Vaccination is a potential method of treating giardiosis in dogs. [References: 13] 13

Olson, M. E., T. A. McAllister, et al. (1995). "Effects of Giardiasis on Production in a Domestic Ruminant (Lamb) Model." American Journal of Veterinary Research 56(11): 1470-1474.
	Objective-To examine the effects of giardiasis on production and carcass quality, using growing lambs as a domestic ruminant model. Design-Randomized block. Animals-Giardia-free lambs: 23 in infected group, 24 in control group. Procedure-Six-week-old, specific-pathogen-free lambs were infected with Giardia trophozoites; control lambs received saline solution. Clinical signs of infection, body weight, and feed intake were determined for 10 weeks. Carcass weight and quality were determined at slaughter weight of 45 kg. Results-Giardia infection persisted from weeks 7 to 16. For 5 weeks after challenge exposure, abnormal feces were more frequently observed in infected lambs. Giardia infection was associated with a decrease in rate of weight gain and impairment in feed efficiency. Time to reach slaughter weight was extended in infected lambs, and the carcass weight of Giardia-infected lambs was lower than that of control lambs. Conclusion-Giardiasis has a negative effect on domestic ruminant production. Clinical Relevance-Giardiasis in domestic ruminants is an economically important disease, thus necessitating control or elimination of the infection. [References: 31] 31

Olson, M. E., A. W. Morck, et al. (1997). "Preliminary Data on the Efficacy of a Giardia Vaccine in Puppies." Canadian Veterinary Journal - Revue Veterinaire Canadienne 38(12): 777-779.
	Twenty puppies were vaccinated with a trophozoite-derived Giardia vaccine on day 0 and boosted on day 21 (Group 1); 10 control puppies received only saline (Group 2). Both groups were experimentally infected on day 35 with 1 X 10(6) Giardia duodenalis trophozoites by intraduodenal injection. Immunization provided protection to experimental Giardia infection. [References: 11] 11

Olson, M. E., D. W. Morck, et al. (1996). "The Efficacy of a Giardia Lamblia Vaccine in Kittens." Canadian Journal of Veterinary Research - Revue Canadienne de Recherche Veterinaire 60(4): 249-256.
	Twenty kittens were vaccinated with a Giardia lamblia vaccine prepared on a commercial scale on day 0 and boosted on day 21 (group 1); while 10 kittens received only saline (group 2). These kittens were challenged on day 35 with 10(6) Giardia lamblia trophozoites by a surgical intraduodenal injection. Three control kittens were not vaccinated and not challenged (group 3). Following challenge, Giardia vaccinated kittens had significantly fewer days in which abnormal stools were observed and reduced food intake occurred compared to saline injected animals. The rate of weight gain between group 1 and group 2 animals was not different in the prechallenge period (day 0 to day 35), but vaccinated animals had a significantly higher weight gain in the postchallenge period (P < 0.05). On day 56, all vaccinated animals were not passing cysts in their feces, while 40% of saline injected kittens had Giardia cysts in their feces. In vaccinated kittens, cysts were never demonstrated in 45% of the animals, while cysts were detected in 90% of the saline injected kittens. Viability of the cysts in vaccinated kittens was 38% while the cysts viability in saline injected kittens was 99%. On postmortem examination, trophozoites could be detected in 5% of vaccinated kittens and 60% of saline injected kittens. Vaccination produced an elevated Giardia specific serum IgG and IgA response prior to challenge and throughout the postinfection period. The Giardia infection in the saline injected group did not induce an elevated specific serum response. Giardia vaccination of kittens provides protection in kittens from an experimental challenge by reducing or eliminating intestinal trophozoites and fecal cyst excretion. [References: 37] 37

Olson, M. E., R. M. O'Handley, et al. (2004). "Update on Cryptosporidium and Giardia infections in cattle [Review]." Trends in Parasitology 20(4): 185-191.
	Cattle are frequently parasitized with Giardia duodenalis, Cryptosporidium parvum and Cryptosporidium andersoni. These parasites cause diarrhoea and impair gain of body weight. Giardia and Cryptosporidium from cattle are potential zoonotic pathogens, and contact with animals, manure or contaminated water is believed to lead to infections in humans. Molecular epidemiology has suggested that cattle are not as significant a reservoir for human infections as was once believed. Most G. duodenalis from cattle (Assemblage E) are different from those in humans (Assemblages A and B), and C. andersoni does not infect humans. However, molecular tools have shown that humans can be infected with zoonotic C. parvum, as well as anthroponotic Cryptosporidium hominis. [References: 73] 73

Olson, M. E., P. D. Roach, et al. (1997). "Giardiasis in Ringed Seals from the Western Arctic." Journal of Wildlife Diseases 33(3): 646-648.
	Sixteen beluga whales (Delphinapterus leucas) and fifteen ringed seals (Phoca hispida) from the western arctic region of Canada were examined for giardiasis and cryptosporidiosis. Intestinal contents from the rectum and colon were collected from animals slaughtered by Inuit hunters. A fluorescent monoclonal antibody identified Giardia sp. cysts in three of 15 (20%) seals. Thus, ringed seals are implicated as a potential reservoir for this zoonosis in the arctic. [References: 11] 11

Olson, M. E., C. L. Thorlakson, et al. (1997). "Giardia and Cryptosporidium in Canadian Farm Animals." Veterinary Parasitology 68(4): 375-381.
	Giardia intestinalis and Cryptosporidium spp, are commonly identified intestinal pathogens in humans and animals. In light of the clinical disease, production losses and zoonotic potential of both Giardia and Cryptosporidium infections, a study was undertaken to investigate the prevalence of these parasites in cattle, sheep, pigs and horses in Canadian farms at different geographical locations. A total of 104 cattle, 89 sheep, 236 pigs and 35 horses were sampled from 15 different Canadian geographical locations. Fecal samples were examined after concentration and immunofluorescent staining. Giardia and Cryptosporidium were present in cattle and sheep in six out of six sites sampled. In cattle the overall prevalence was 29% for Giardia and 20% for Cryptosporidium. Giardia was identified in 38% of sheep while 23% of sheep were positive for Cryptosporidium. Giardia and Cryptosporidium were identified in four out of six hog operations with an overall prevalence of 9% for Giardia and 11% for Cryptosporidium. All horse sampling locations (4/4) were positive for Giardia with 20% of animals infected. Cryptosporidium was identified in three out of four sampling sites with a prevalence of 17%. The prevalence of Giardia and Cryptosporidium was greater in calves and lambs compared to adults. This study demonstrates that both Giardia and Cryptosporidium appear to be prevalent in farm livestock. [References: 16] 16

Omata, Y., C. Dilorenzo, et al. (1994). "Correlation between antibody levels in Toxoplasma gondii infected pigs and pathogenicity of the isolated parasite." Vet Parasitol 51(3-4): 205-10.
	Sera and diaphragm muscle tissues were obtained from 109 commercial pigs between September 1991 and May 1992 from the slaughterhouse at La Plata, Provincia Buenos Aires, Argentina. Anti-Toxoplasma gondii IgG antibody reactivity to T. gondii antigens were assayed using sera by indirect immunofluorescence assay and immunoblotting technique. Anti-T. gondii IgG titers at serum dilutions of 1:1024 and higher were noted in 11.0% of the tested sera, and at dilutions of 1:16 and lower in 36.7% of the serum samples. Using mouse inoculation test, T. gondii was isolated from 14 pig diaphragm samples. Of five samples derived from pigs with antibodies at dilutions of 1:1024 and higher, four contained trophozoites which, when inoculated into mice intraperitoneally, killed all recipient hosts within 15 days post inoculation. Parasites detected in seven out of eight samples from pigs with antibodies at serum dilutions of 1:64 and lower formed cysts in the brain, and mice survived longer than 13 days post inoculation. Immunoblotting demonstrated antibody reactivity in pig sera samples with relatively high titers for parasite antigens. Results of the present study suggest that antibody production in infected pigs is apparently dependent on the pathogenicity of the parasite strain.

Ong, C., W. Moorehead, et al. (1996). "Studies of Giardia spp. and Cryptosporidium spp. in two adjacent watersheds." Appl Environ Microbiol 62(8): 2798-805.
	Two adjacent British Columbia, Canada, watersheds with similar topographical features were studied. Both the Black Mountain Irrigation District (BMID) and the Vernon Irrigation District (VID) serve rural agricultural communities which are active in cattle ranching. The present study was carried out in five phases, during which a total of 249 surface water samples were tested in the study watersheds. The aims of these phases were to determine levels of parasite contamination in raw water samples collected from the intakes as well as from other sites in each watershed and to investigate cattle in the watersheds as potential sources of parasite contamination of surface drinking water supplies. Giardia cysts were not detected in the raw water samples collected from lake sources at the headwaters of both watersheds but were found in 100% (70 or 70) of water samples collected at the BMID intake and 97% (68 of 70) of water samples collected at the VID intake. Significantly higher levels (P < 0.05) of Giardia cysts were found at the BMID intake (phase 1, 7 to 2,215 cysts per 100 liters; phase 3, 4.6 to 1,880 cysts per 100 liters) when compared with that of the VID intake (2 to 114 cysts per 100 liters). The BMID watershed has a more complex system of surface water sources than the VID watershed. Cattle have access to creeks in the BMID watershed, whereas access is restricted in the VID watershed. Collection of raw water samples from a creek upstream and downstream of a cattle ranch in the BMID watershed showed that the downstream location had significantly higher (P < 0.05) levels (0.6 to 42.9 cysts per 100 liters and 1.4 to 300.0 oocysts per 100 liters) of both Giardia cysts and Cryptosporidium oocysts than those of the upstream location (0.5 to 34.4 cysts per 100 liters and 0.5 to 34.4 oocysts per 100 liters). Peak concentrations of both parasites coincided with calving activity. Fecal samples, collected from cattle in both watersheds, showed 10% (3 of 30) in the BMID and 50% (5 of 10) in the VID watersheds to be Giardia positive. No Cryptosporidium-positive fecal samples were found. Giardia cysts isolated from the BMID watershed were repeatedly infective to gerbils in contrast to those from the VID watershed. The 10 BMID drinking water Giardia isolates retrieved into culture and biotyped showed zymodeme and karyotype heterogeneity. The differences in patterns of parasite contamination and cattle management practices contribute to the unique watershed characteristics observed between two areas which are topographically similar and geographically adjacent.

Ong, C. S., D. L. Eisler, et al. (2002). "Novel cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype." Emerg Infect Dis 8(3): 263-8.
	In this study, we genotyped parasites from the fecal specimens of sporadic cryptosporidiosis cases in British Columbia from 1995 to 1999. Genotyping was conducted by polymerase chain amplification of the internal transcribed spacer region, a hypervariable region in the 18S rRNA gene and the Cryptosporidium oocyst wall protein gene. Subsequent analysis was by restriction fragment length polymorphism and DNA sequencing. We identified two new Cryptosporidium genotypes in humans. One of these genotypes has been found recently in deer in New York state. The other genotype has not been identified in humans or animals. These results have important implications for drinking water quality strategies, especially for communities that obtain drinking water supplies from surface sources located in forested regions with deer populations.

Ong, C. S., D. L. Eisler, et al. (1999). "Molecular epidemiology of cryptosporidiosis outbreaks and transmission in British Columbia, Canada." Am J Trop Med Hyg 61(1): 63-9.
	Isolates from 25 (13 sporadic and 12 outbreak) cryptosporidiosis cases, 24 of which were from British Columbia, Canada, were characterized using nested polymerase chain reaction amplification of the polymorphic internal transcribed spacer 1 locus. Two predominant Cryptosporidium parvum genotypes were found. Twelve (8 sporadic and 4 outbreak) isolates amplified with the cry7/cry21 primer pair and 12 (5 sporadic and 7 outbreak) isolates amplified with the cry7/cryITS1 primer pair. Multi-locus gene analysis using sequence polymorphisms on 3 other loci, i.e., the thrombospondin-related adhesion protein gene, the dihydrofolate reductase gene, and the 18S rRNA gene on 8 (4 outbreak and 4 sporadic) isolates showed non-random association among the human and animal alleles of the 4 different C. parvum gene loci. Associations between these 2 parasite genotypes and different routes of cryptosporidiosis transmission such as zoonotic, anthroponotic, and waterborne transmission were studied using municipal population and agricultural information, as well as detection of C. parvum oocysts in municipal drinking water specimens of the residential communities of sporadic and outbreak cases.

Ong, C. S., A. S. Li, et al. (2005). "Enzyme immunoassay of Cryptosporidium-specific immunoglobulin G antibodies to assess longitudinal infection trends in six communities in British Columbia, Canada." Am J Trop Med Hyg 73(2): 288-95.
	A newly developed enzyme-linked immunosorbent assay (ELISA) that detects immunoglobulin G antibodies to the 27-kDa Cryptosporidium parvum sporozoite surface antigen was used to test 4,097 sera collected from pregnant women in 6 communities in British Columbia, Canada, between January 1996, and December 1997. Waterborne outbreaks of cryptosporidiosis occurred in two of the study communities during the period of follow-up, and ELISA seropositivity was high in all six communities during the study period (77% positive to 92% positive). In the community with the largest outbreak, levels of antibody to the 27-kDa antigen increased rapidly and then decayed to background levels within 3-4 months of the peak of the epidemic curve. Trends in serologic reactivity were complex in all communities, and increased antibody levels not related temporally to known waterborne outbreaks were also observed. Serological assays may provide more accurate information regarding community levels of Cryptosporidium infection.

Ono, K., H. Tsuji, et al. (2001). "Contamination of river water by Cryptosporidium parvum oocysts in western Japan." Appl Environ Microbiol 67(9): 3832-6.
	In Japan, only a few rivers have been inspected for Cryptosporidium parvum contamination, and the methods used had low sensitivity. In 1998 and 1999, we used a method with higher sensitivity to examine all large rivers used as sources of water supply in one prefecture (which we divided into four areas) in western Japan for Cryptosporidium oocysts. One sample was collected at each of 156 sites along 18 rivers, and samples were tested for Cryptosporidium oocysts by immunomagnetic separation. Samples were classified as being obtained on an island with livestock and fishing industries, a densely populated urban area, a western region including farming villages, or a still more rural northern area with agriculture and fishing. Restriction fragment length polymorphism analysis was used for identification of the C. parvum found as the bovine or human type. C. parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the samples from each area contained C. parvum oocysts. The number of C. parvum oocysts per 20 liters of river water varied in the same pattern as the number of cattle kept in the four kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected in rivers probably came from cattle kept in that valley. As we had expected, when tested with a more sensitive method, river water in western Japan was found to be greatly contaminated with C. parvum oocysts, as reported in other countries.

Ortega, Y. R. and R. D. Adam (1997). "Giardia: overview and update." Clin Infect Dis 25(3): 545-9; quiz 550.
	
Ortega, Y. R. and D. Bonavia (2003). "Cryptosporidium, Giardia, and Cyclospora in ancient Peruvians." J Parasitol 89(3): 635-6.
	Twenty-two coprolites of human origin, collected from excavations along the north-central coast of Peru, were examined using fluorescent microscopy for the presence of fecal parasites, with emphasis on Cryptosporidium sp., Giardia sp., and Cyclospora sp. Three samples were positive. One coprolite dated between ca. 2,375 and 1,525 BC contained Giardia sp. cysts. This coprolite corresponded to the Peruvian preceramic period. Another positive coprolite ca. AD 770-830 corresponded to Epoch 3 of the Middle Horizon and contained Cryptosporidium sp. oocysts. The third positive coprolite (corresponding to the Middle Horizon. ca. AD 500-900) contained Giardia sp. cysts. This report demonstrates that Giardia sp. and Cryptosporidium sp. were present in Peruvian coastal populations for at least 4,300 and 1,100 BP.

Ortega, Y. R., R. H. Gilman, et al. (1994). "A new coccidian parasite (Apicomplexa: Eimeriidae) from humans." J Parasitol 80(4): 625-9.
	A new coccidian parasite has been found in stool specimens of humans with and without diarrhea. The oocyst of this parasite measures 8.6 microns in diameter (7.7-9.9 microns), with ovoid sporocysts 4.0 x 6.3 (3.3-4.4 x 5.5-7.1) microns. Each oocyst has 2 sporocysts and each sporocyst contains 2 sporozoites. Based on these characteristics and the structures observed by electron microscopy, this parasite has been classified in the genus Cyclospora. We propose the name Cyclospora cayetanensis n. sp. for this new human parasite.

Ortega, Y. R. and J. Liao (2006). "Microwave inactivation of Cyclospora cayetanensis sporulation and viability of Cryptosporidium parvum oocysts." J Food Prot 69(8): 1957-60.
	The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23 degrees C for 2 weeks, and sporulation rates were then determined. The 4',6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80 degrees C or higher were reached in the microwave ovens.

Ortega, Y. R., R. Nagle, et al. (1997). "Pathologic and clinical findings in patients with cyclosporiasis and a description of intracellular parasite life-cycle stages." J Infect Dis 176(6): 1584-9.
	Cyclospora cayetanensis has been observed in the feces of persons with prolonged diarrhea. A description of the symptoms and histopathologic findings for patients with cyclosporiasis is presented. The intracellular life-cycle stages of these parasites in the enterocytes of patients will also be described. Seventeen Peruvian patients positive for Cyclospora organisms were surveyed and underwent endoscopy, and their symptoms were recorded. Patients presented with gastrointestinal symptoms, including diarrhea, flatulence, weight loss, abdominal discomfort, and nausea. Jejunal biopsies showed an altered mucosal architecture with shortening and widening of the intestinal villi due to diffuse edema and infiltration by a mixed inflammatory cell infiltrate. There was reactive hyperemia with vascular dilatation and congestion of villous capillaries. Parasitophorous vacuoles contained sexual and asexual forms. Type I and II meronts, with 8-12 and 4 fully differentiated merozoites, respectively, were found at the luminal end of epithelial cells. These findings demonstrate the complete developmental cycle associated with host changes due to Cyclospora organisms.

Ortega, Y. R., C. R. Roxas, et al. (1997). "Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetables collected in markets of an endemic region in Peru." Am J Trop Med Hyg 57(6): 683-6.
	Cryptosporidium parvum and Cyclospora cayetanensis are protozoan pathogens that cause prolonged diarrhea in both immunocompetent and immunocompromised hosts. Cryptosporidium parvum can be transmitted via the fecal-oral route, while the exact mechanisms of transmission of Cyclospora cayetanensis have not been fully determined. Humans appear to be the sole host for the latter and a distinct seasonality has been observed in endemic areas around the world. Samples of vegetables were collected at several small markets in a periurban slum in Peru during the seasons of high and low incidence. The vegetables were washed, the supernatants were collected and centrifuged, and the pellets were resuspended in a solution of 2.5% potassium dichromate. Pellets were examined using direct microscopic observation, acid-fast staining, and immunofluorescent assays for C. parvum and Cyclospora cayetanensis oocysts. Samples were collected during three time periods: the season of low incidence, the beginning of the season of high incidence, and end of the season of high incidence. Of the total vegetables examined, 14.5% contained C. parvum oocysts and 1.8% had Cyclospora oocysts. Thus, market vegetables may provide a route by which Cryptosporidium and Cyclospora can be transmitted. Our study also suggests that washing vegetables does not completely remove Cryptosporidium and Cyclospora oocysts.

Ortega, Y. R., C. R. Sterling, et al. (1998). "Cyclospora cayetanensis." Adv Parasitol 40: 399-418.
	Cyclospora cayetanensis is a coccidian pathogen in humans. Cyclosporiasis is characterized by mild to severe nausea, anorexia, abdominal cramping, and watery diarrhea. Cyclospora has now been described from patients with protracted diarrheal illness in North, Central and South America, the Caribbean, Africa, Bangladesh, south-east Asia, Australia, England, and eastern Europe, and is characterized by marked seasonality. Routes of transmission are still unknown, although the fecal-oral route, either directly or via water, is probably the major one. A recent outbreak in the USA suggested transmission of Cyclospora by ingestion of contaminated berries. Cyclospora oocysts can be detected by phase contrast microscopy, modified acid-fast staining, autofluorescence, and amplification by the polymerase chain reaction. Oocysts are not sporulated when excreted in the feces, and sporulated oocysts are needed for infection. Each sporulated oocyst contains two sporocysts and each sporocyst contains two sporozoites. Humans seem to be the only host for this parasite. Histopathological examination of jejunal biopsies from infected individuals showed mild to moderate acute inflammation of the lamina propria and surface epithelial disarray. Parasitophorous vacuoles containing sexual and asexual forms of Cycl. cayetanensis were located in the cytoplasm of epithelial cells. Cyclospora infections can be treated successfully with trimethoprim-sulfamethoxazole.

Ortega-Mora, L. M., J. M. Troncoso, et al. (1992). "Cross-reactivity of polyclonal serum antibodies generated against Cryptosporidium parvum oocysts." Infect Immun 60(8): 3442-5.
	Polyclonal antibodies raised against Cryptosporidium parvum oocysts were found to cross-react with Eimeria spp. oocyst antigens in an indirect immunofluorescence assay, and sera from Eimeria spp.-infected lambs reacted with some antigens from sonicated C. parvum oocysts (between 29 to 30 and 66 to 69 kDa) by Western blot (immunoblot). No cross-reaction was observed with cystozoites of Toxoplasma and Sarcocystis spp. These results show the existence of epitopes common to C. parvum and various Eimeria spp.

Ortega-Mora, L. M. and S. E. Wright (1994). "Age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response." Infect Immun 62(11): 5003-9.
	The phenomenon of age-related resistance to infection with Cryptosporidium parvum has been well characterized in rodent models, and its existence has been demonstrated in calves. To determine whether this is a genuine age effect in a fully susceptible animal model or the result of infection with related pathogens inducing a nonspecific immunity, and to examine several parameters associated with severity of clinical diseases, lambs maintained in a parasite-free environment were infected with C. parvum oocysts at increasing ages. A marked decrease in the severity of clinical symptoms was observed as the age at infection increased, though the kinetics of both fecal and serum antibody responses were similar in all age groups, suggesting that mechanisms other than humoral response may play an important role in the development of age-related resistance. This study demonstrates the first experimental evidence for age-related resistance to ovine cryptosporidiosis and examines parameters which may influence the acquisition of resistance to infection.

Ottoson, J., A. Hansen, et al. (2006). "Removal of viruses, parasitic protozoa and microbial indicators in conventional and membrane processes in a wastewater pilot plant." Water Res 40(7): 1449-57.
	The aim of this study was to investigate variations in the occurrence and removal of enterovirus and norovirus genomes, Giardia cysts, Cryptosporidium oocysts and the most commonly used faecal indicators in a Swedish wastewater pilot plant. Paired samples were taken from the inlet and outlet of each treatment line: tertiary filtration, membrane bioreactor (MBR) and upflow anaerobic sludge blankets (UASB). (Oo)cysts and indicators were enumerated using standard methods and viruses using RT-PCR. Giardia cysts and enteroviruses were constantly detected, mean numbers 10(3.11) cysts and 10(4.0) PCR units L(-1), respectively. Oocysts were found in 5/19 samples, mean number 5 L(-1). Noroviruses were found in 6/7 influent samples, with an average titre of 10(3.28)L(-1), during winter, but only in 2/15 in the rest of the year (mean 200 L(-1)). MBR treatment removed indicators more efficiently than did the other two lines, with 5log removal of E. coli. Human virus genome removal did not differ between the MBR and tertiary treatment line. Microorganism removal in UASB was significantly lower for all the organisms studied. E. coli, enterococci and Cl. perfringens removal was correlated (p<0.05) with enterovirus genome removal, with R-values around 0.4. However, values for removal of indicators were more strongly correlated to each other. Removal of viruses based on enumeration using RT-PCR probably underestimates infectious virion removal.

Pal, S., S. K. Bhattacharya, et al. (1989). "Occurrence and significance of Cryptosporidium infection in Calcutta." Trans R Soc Trop Med Hyg 83(4): 520-1.
	During a 2-year study, Cryptosporidium oocysts were detected in 32 (5.6%) of 566 hospitalized paediatric diarrhoea cases and 2 (1.2%) of 167 non-diarrhoeic individuals. Cryptosporidium was the sole pathogen detected in 17 (3.0%) of the 32 positive cases; in the other 15 it occurred in combination with one or more other established enteropathogen(s). The frequency of detection of the parasite was highest in the 0-6 months age group; no sex-specific difference was discernible. The detection rate of the parasite was highest during the monsoon and post-monsoon months. Most of the patients had watery stools with a mild to moderate degree of dehydration, with the diarrhoea lasting for less than 7 d.

Palmer, C. J., L. Xiao, et al. (2003). "Cryptosporidium muris, a rodent pathogen, recovered from a human in Peru." Emerg Infect Dis 9(9): 1174-6.
	Cryptosporidium muris, predominantly a rodent species of Cryptosporidium, is not normally considered a human pathogen. Recently, isolated human infections have been reported from Indonesia, Thailand, France, and Kenya. We report the first case of C. muris in a human in the Western Hemisphere. This species may be an emerging zoonotic pathogen capable of infecting humans.

Pasquali, P., R. Fayer, et al. (1997). "Lymphocyte dynamic patterns in cattle during a primary infection with Cryptosporidium parvum." J Parasitol 83(2): 247-50.
	Changes in intraepithelial (IEL), lamina propria (LPL), and draining lymph node (LNL) lymphocytes were assessed in 9-day-old calves during primary infection with Cryptosporidium parvum and in similarly aged noninfected calves. A very low percentage of both CD4+ and CD8+ T cells were found in IEL and LPL of noninfected calves. In infected compared to controls, percentages of CD2+, CD3+, CD4+, and CD8+ T cells in IEL exhibited a significant increase (P < 0.05), whereas the percentage of IL2R+ increased and the percentage of IgG+ cells decreased, but neither of these changes were statistically significant. In LPL, percentages of CD2+, CD3+, CD8+, and IL2R+ T cells were increased in infected compared to noninfected calves, whereas the percentage of IgG-bearing cells decreased; but only the increase in CD3+, CD8+, and IL2R+ cells was significant (P < or = 0.05). In LNL only minimal changes were seen. In fact, the percentage of CD2+ T cells increased whereas the percentage of CD8+ T cells decreased, but neither of these differences was statistically significant. These findings indicate that T cells subsets in the ileal mucosa of naive neonatal calves are different than those of adult cattle, and that the immune response to C. parvum infection differs in ileal mucosa when compared to the regional lymph nodes.

Pasquali, P., R. Fayer, et al. (2005). "Recombinant bovine interleukin-12 stimulates a gut immune response but does not provide resistance to Cryptosporidium parvum infection in neonatal calves." Vet Parasitol.
	This study was undertaken to determine if administration of recombinant bovine interleukin-12 (rBoIL-12) could stimulate a cellular immune response that protected calves from an oral challenge inoculation with Cryptosporidium parvum oocysts. In a first experiment, rBoIL-12 intraperitoneally administered as a single dose 1 day before challenge inoculation, did not alter the course of infection. The percentage of immune competent cells and levels of cytokine gene expression in the ileo-cecal mucosa and in the draining lymph nodes of treated calves were similar to those of untreated control calves. However, when rBoIL-12 was subcutaneously administered daily from 2 days before infection to 2 days after infection, a consistent increase of T lymphocytes and an higher expression of interferon-gamma (IFN-gamma) was detected. Again, treatment did not alter the course of infection. Similar results were obtained when rBoIL-12 was administered daily for 4 days beginning 2 days after oral inoculation. These data indicate that although rBoIL-12 stimulated a strong immune response in the gut of neonatal calves, the response was not able to provide protection from challenge inoculation with C. parvum oocysts.

Pedraza-Diaz, S., C. Amar, et al. (2001). "Unusual cryptosporidium species recovered from human faeces: first description of Cryptosporidium felis and Cryptosporidium 'dog type' from patients in England." J Med Microbiol 50(3): 293-6.
	DNA was extracted from faecal samples collected from 1680 patients in which Cryptosporidium oocysts were recognised by light microscopy. DNA from faeces from five of these patients failed to amplify by PCR three gene fragments--the Cryptosporidium oocyst wall protein (COWP) gene, the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) gene and the thrombospondin-related adhesive protein of Cryptosporidium-2 (TRAP-C2) gene--with primers designed from C. parvum sequences. However, DNA from these five patients did amplify cryptosporidial 18S rDNA gene fragments and a heat-shock protein (HSP70) gene fragment was also amplified from four of them. The purpose of this study was to characterise further the Cryptosporidium associated with infection in these patients. DNA sequence analysis of 18S rDNA genes showed that four of these patients were infected by C. felis, and the remaining one by an as yet un-named Cryptosporidium species designated the 'dog type' (C. dt). Infection by C. felis was further confirmed in all four patients by DNA sequence analysis of the HSP70 gene. Oocysts present in all five samples reacted strongly with two anti-cryptosporidial oocyst monoclonal antibodies, except for the C. dt, which was tested with only one of the antibodies. Two of the patients infected by C. felis had underlying illness; one 8-year-old male had an undefined severe inherited underlying condition, and the second patient, a 32-year-old male, was HIV positive. Two of the remaining three patients (two females aged 1 and 2 years, respectively) were apparently immunocompetent (one infected with C. felis and one with the C. dt). No information was obtained for the fifth patient. The patient infected by C. dt had a recent history of travel to Africa. This is the first report of infection with these two Cryptosporidium species in immunocompetent patients, and in any patient in the UK.

Pedraza-Diaz, S., C. Amar, et al. (2000). "The identification and characterisation of an unusual genotype of Cryptosporidium from human faeces as Cryptosporidium meleagridis." FEMS Microbiol Lett 189(2): 189-94.
	An unusual genotype of Cryptosporidium was identified in the faeces of six human patients by PCR/RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene. Conventional microscopy showed oocysts indistinguishable in size from those of Cryptosporidium parvum, which reacted with two different commercially available anti-oocyst monoclonal antibodies. The isolates were further characterised by PCR/RFLP analysis of the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) genes as well as by DNA sequencing of the COWP and the TRAP-C1 gene fragments and of two regions of the 18S rRNA gene. Sequence analysis of the COWP, TRAP-C1, and 18S rRNA gene fragments confirmed that this genotype is genetically distinct from C. parvum. 18S rRNA gene sequences were found to be identical to those published for Cryptosporidium meleagridis.

Pedraza-Diaz, S., C. F. Amar, et al. (2001). "Cryptosporidium meleagridis from humans: molecular analysis and description of affected patients." J Infect 42(4): 243-50.
	OBJECTIVES: To genetically characterize an unusual genotype of Cryptosporidium from the stools of humans with diarrhoea and to identify risk factors in the affected patients. METHODS: DNA was extracted from human faeces where Cryptosporidium oocysts were detected by light microscopy. Cryptosporidial gene fragments from six different loci were analysed by PCR alone, PCR/RFLP and by DNA sequencing. Oocysts were characterized by light and immunofluorescence microscopy and epidemiological data was collected from the affected patients. RESULTS: Analysis of the Cryptosporidium oocyst wall protein (COWP) gene amplified from > 2000 human faecal samples identified 19 patients all of which produced an unusual RFLP profile. Subsequent DNA sequence analysis of this and an additional four genetic loci (including 18S rRNA sequences) confirmed these as a homogeneous group which was genetically distinct from Cryptosporidium parvum. The isolates were identified as Cryptosporidium meleagridis since the gene sequences were identical to those from this species recovered from birds. Conventional microscopy showed oocysts indistinguishable from C. parvum and reacted strongly with two different commercially available anti-oocyst monoclonal antibodies. None of the patients showed risk factors unusual for cryptosporidiosis; however, ten of the cases occurred during the summer/autumn, six had a history of foreign travel, four were co-infected with Giardia, two were HIV positive, and six were without identifiable immunocompromising factors. CONCLUSIONS: This study further confirms that C. meleagridis, in addition to C. parvum, is involved in human disease. The study also highlights the lack of basic information on the host range of this genus of parasites, the complexity of the transmission routes involved in human cryptosporidiosis, and the value of molecular techniques in identify hitherto unrecognised differences in Cryptosporidium from human faeces.

Peeters, J. E., E. A. Mazas, et al. (1989). "Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts." Appl Environ Microbiol 55(6): 1519-22.
	Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.

Peng, M. M., O. Matos, et al. (2001). "A comparison of Cryptosporidium subgenotypes from several geographic regions." J Eukaryot Microbiol Suppl: 28S-31S.
	
Peng, M. M., S. R. Meshnick, et al. (2003). "Molecular epidemiology of cryptosporidiosis in children in Malawi." J Eukaryot Microbiol 50 Suppl: 557-9.
	Few studies have examined the molecular epidemiology of cryptosporidiosis in developing countries. In this study, DNA of 69 microscopy-positive human fecal samples collected from Malawi were examined by multilocus genetic analyses. From 43, 27 and 28 of the samples, the SSU rRNA, 70 kDa heat shock protein (HSP70) and 60 kDa glycoprotein (GP60) genes, respectively, were successfully PCR-amplified. Restriction analysis of the SSU PCR products showed that 41 of the 43 PCR-positive samples had C. hominis and 2 had C. parvum. Sequence analysis of the HSP70 and GP60 gene confirmed the species identification by SSU rRNA PCR-RFLP analysis, but also revealed high intraspecific variations. Altogether, six HSP70 subtypes and six GP60 subtypes (belonging to four subtype alleles) of C. hominis were found. Linkage disequilibrum analysis of the two genetic loci showed possible intraspecific recombination. Thus, cryptosporidiosis in the study area was largely caused by anthroponotic transmission. The high intraspecific variation and existence of genetic recombination were probably results of high transmission of cryptosporidiosis in this area.

Peng, M. M., M. L. Wilson, et al. (2003). "Genetic diversity of Cryptosporidium spp. in cattle in Michigan: implications for understanding the transmission dynamics." Parasitol Res 90(3): 175-80.
	Epidemiological and molecular data on 248 bovine, 17 human, and 16 water samples of Cryptosporidium spp. collected from the lower peninsula of Michigan between 1997 and 2000 were analysed. Cryptosporidium parvum bovine genotype and Cryptosporidium andersoni were found in 56 and four cattle samples, respectively. A total of six C. parvum subgenotypes were found in 34 bovine samples, and five of the eight farms had two or three subgenotypes in cattle. Six water samples from these farms had C. andersoni, five had the C. parvum bovine genotype, and one had Cryptosporidium muris. In contrast, four PCR-positive human samples produced the C. parvum bovine genotype and two had the C. parvum human genotype. Among the C. parvum bovine genotype samples, two human samples and one water sample had subgenotypes identical to those found on cattle farms. The results of this study demonstrate the potential use of molecular methods in tracking the transmission of Cryptosporidium.

Peng, M. M., L. Xiao, et al. (1997). "Genetic polymorphism among Cryptosporidium parvum isolates: evidence of two distinct human transmission cycles." Emerg Infect Dis 3(4): 567-73.
	We report the results of molecular analysis of 39 isolates of Cryptosporidium parvum from human and bovine sources in nine human outbreaks and from bovine sources from a wide geographic distribution. All 39 isolates could be divided into either of two genotypes, on the basis of genetic polymorphism observed at the thrombospondin-related adhesion protein (TRAP-C2) locus. Genotype 1 was observed only in isolates from humans. Genotype 2, however, was seen in calf isolates and in isolates from a subset of human patients who reported direct exposure to infected cattle or consumed items thought to be contaminated with cattle faces. Furthermore, experimental infection studies showed that genotype 2 isolates were infective to mice or calves under routine laboratory conditions, whereas genotype 1 isolates were not. These results support the occurrence of two distinct transmission cycles of C. parvum in humans.

Pereira, M. D., E. R. Atwill, et al. (1999). "Comparison of sensitivity of immunofluorescent microscopy to that of a combination of immunofluorescent microscopy and immunomagnetic separation for detection of Cryptosporidium parvum oocysts in adult bovine feces." Appl Environ Microbiol 65(7): 3236-9.
	A direct immunofluorescence assay (DFA) (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared to an immunomagnetic separation (IMS) assay (Dynabeads; Dynal, Inc., Lake Success, N.Y.) coupled with immunofluorescent microscopy (Waterborne, Inc., New Orleans, La.) for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine fecal material. IMS-DFA resulted in a 2-log-unit increase in sensitivity (10 oocysts/g) compared to DFA alone (1,000 oocysts/g). The higher sensitivity obtained with IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA; the increased sensitivity was not attributable to a higher percent recovery.

Pereira, M. d. G., E. R. Atwill, et al. (1998). "DNA sequence similarity between California isolates of Cryptosporidium parvum." Appl Environ Microbiol 64(4): 1584-6.
	We evaluated whether nucleic acid amplification with primers specific for Cryptosporidium parvum followed by automated DNA sequence analysis of the PCR amplicons could differentiate between California isolates of C. parvum obtained from livestock, humans, and feral pigs. Almost complete sequence identity existed among the livestock isolates and between the livestock and human isolates. DNA sequences from feral pig isolates differed from those from livestock and humans by 1.0 to 1.2%. The reference sequence obtained by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991.) differed from California isolates of C. parvum by 1.8 to 3.2%. These data suggest that DNA sequence analysis of the amplicon of Laxer et al. does not allow for differentiation between various strains of C. parvum or that our collection of isolates obtained from various hosts from across California was limited to one strain of C. parvum.

Pereira, S. J., N. E. Ramirez, et al. (2002). "Pathogenesis of human and bovine Cryptosporidium parvum in gnotobiotic pigs." J Infect Dis 186(5): 715-8.
	To compare the pathogenesis of human genotype 1 (HuG1) and bovine genotype 2 (BoG2) Cryptosporidium parvum, neonatal gnotobiotic pigs were given 1-10 HuG1 or BoG2 oocysts. The prepatent and patent periods were significantly longer for HuG1 than for BoG2 C. parvum (prepatent, 8.6 vs. 5.6 days; patent, 16.6 vs. 10.3 days). BoG2-infected pigs developed significantly more severe disease than did HuG1-infected pigs. BoG2 parasites were seen microscopically throughout the intestines during the prepatent and patent periods. HuG1 parasites were only detected during the patent period in the ileum and colon but colonized the mucosal surface in significantly larger numbers than did BoG2. Moderate-to-severe villus/mucosal attenuation with lymphoid hyperplasia was seen throughout the intestines of BoG2-infected pigs, whereas lesions in HuG1-infected pigs were mild to moderate and restricted to the ileum and colon. These findings provide additional support for the hypothesis that human and bovine C. parvum genotypes may be separate species.

Perryman, L. E., M. W. Riggs, et al. (1990). "Kinetics of Cryptosporidium parvum sporozoite neutralization by monoclonal antibodies, immune bovine serum, and immune bovine colostrum." Infect Immun 58(1): 257-9.
	Monoclonal antibodies, immune bovine serum, and immune bovine colostral whey neutralized infectivity of Cryptosporidium parvum sporozoites for mice in a time-dependent manner. Immune colostral whey neutralized sporozoites more rapidly and completely than immune serum, monoclonal antibody (MAb) 18.44, or a combination of MAb 18.44 and MAb 17.41. Mice were partially protected against oral challenge with C. parvum oocytes when treated with immune colostral whey, MAb 17.41, or a combination of MAb 17.41 and MAb 18.44.

Petry, F. and J. R. Harris (1999). "Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites." Int J Parasitol 29(8): 1249-60.
	Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120-180 kDa range.

Phelps, K. K., D. S. Lindsay, et al. (2001). "Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability." J Eukaryot Microbiol Suppl: 40S-41S.
	Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.

Pieniazek, N. J., F. J. Bornay-Llinares, et al. (1999). "New cryptosporidium genotypes in HIV-infected persons." Emerg Infect Dis 5(3): 444-9.
	Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes.

Pieniazek, N. J. and B. L. Herwaldt (1997). "Reevaluating the molecular taxonomy: is human-associated Cyclospora a mammalian Eimeria species?" Emerg Infect Dis 3(3): 381-3.
	Human-associated Cyclospora is a coccidian parasite that causes diarrheal disease. A reevaluation of the parasite's molecular taxonomy that takes into account newly published data for seven Eimeria species shows that Cyclospora belongs to the Eimeria clade (Eimeriidae family). The Cyclospora branch on the phylogenetic tree is between the branches of the eight avian and two mammalian Eimeria species that have been evaluated to date. Furthermore, preliminary results indicate that Cyclospora and Isospora belli, another coccidian parasite that causes diarrheal disease in humans, belong to different families. To improve our understanding of the taxonomy of human-associated Cyclospora, molecular evaluation of isolates of additional Cyclospora and Eimeria species is needed.

Pieniazek, N. J., S. B. Slemenda, et al. (1996). "PCR confirmation of infection with Cyclospora cayetanensis." Emerg Infect Dis 2(4): 357-9.
	
Porter, J. D., C. Gaffney, et al. (1990). "Food-borne outbreak of Giardia lamblia." Am J Public Health 80(10): 1259-60.
	An outbreak of giardiasis occurred following a family party for 25 persons. Nine who had eaten fruit salad became ill, compared with one who had not eaten the salad (Relative Risk = 7.4, 95% CI = 1.4, 169.3). The fruit salad preparer had a diapered child and a pet rabbit at home who were both positive for Giardia lamblia. This outbreak emphasizes the importance of good hygienic practices in food preparation and the possibility of domestic-animal-to-person transmission in Giardia outbreaks.

Porter, J. D., H. P. Ragazzoni, et al. (1988). "Giardia transmission in a swimming pool." Am J Public Health 78(6): 659-62.
	In the fall of 1985, an outbreak of giardiasis occurred among several swimming groups at an indoor pool in northeast New Jersey. Nine clinical cases were identified, eight of whom had Giardia positive stool specimens. All were female; seven were adults (greater than 18 years) and two were children. The attack rate was highest (39 per cent, 5/13) for the ladies lap group who had exposure on one day. These cases had no direct contact with children or other risk factors for acquiring Giardia. Infection most likely occurred following the ingestion of swimming pool water contaminated with Giardia cysts. The source of Giardia contamination was a handicapped child who had a fecal accident in the pool. He was a member of a group that swam at the same time as the ladies lap group. A stool survey of the handicapped group showed that of the 20 persons tested, nine were positive for Giardia, including the specimen from this child. Examination of the pool records showed that no chlorine levels had been taken on the day of the fecal accident and that on the following day the chlorine level was zero. This is the second report of Giardia transmission among swimming pool attendees. It emphasizes the need to maintain appropriate chlorine levels in swimming pools and to institute measures to clear pools after a fecal accident.

Potasman, I., A. Paz, et al. (2002). "Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective." Clin Infect Dis 35(8): 921-8.
	Outbreaks of shellfish-associated infection have been reported for more than a century. Since the early 1970s, the global consumption of shellfish has increased considerably--and with it, the reports of outbreaks of infection. Most of these reports have originated from the United States, but Europe and, to a lesser extent, Asia and Australia have also been represented. The majority of outbreaks have been linked to oysters, followed by clams and mussels. Hepatitis A virus caused the largest ever shellfish-associated outbreak, but caliciviruses have caused the highest number of outbreaks; Vibrio species lead the list of bacterial pathogens. The prognosis of shellfish-associated infections is generally good, except for outbreaks of Vibrio vulnificus infection, which have a mortality rate of up to 50% in vulnerable people. Conventional and molecular techniques should be applied to better identify the causative agents, thereby enabling more-targeted control measures in growing, harvesting, and shipping bivalves.

Power, M. L., M. B. Slade, et al. (2004). "Genetic characterisation of Cryptosporidium from a wild population of eastern grey kangaroos Macropus giganteus inhabiting a water catchment." Infect Genet Evol 4(1): 59-67.
	Molecular characterisation of Cryptosporidium oocysts isolated from faeces collected from eastern grey kangaroos Macropus giganteus inhabiting an Australian water catchment revealed that this host was susceptible to three types of Cryptosporidium. Nucleotide sequence analysis of the 18S rDNA, Cryptosporidium oocyst wall protein (COWP) and a 70kDa heat shock protein (HSP70) identified an isolate identical to the described Cryptosporidium 'marsupial' genotype. A second isolate had less than 0.5% variation, compared to the described Cryptosporidium 'marsupial' genotype, within the sequences of the 18S rDNA, COWP and HSP70 and 10% variation in the internal transcribed spacer 1 (ITS1). Multilocus analysis of the third Cryptosporidium revealed a novel genotype that had a degree of genetic variation, at the four loci characterised, which was greater than or equivalent to that used to discriminate between currently recognised Cryptosporidium species. These findings have increased our current understanding on the molecular epidemiology of Cryptosporidium in Australian wildlife and have provided information on the types of Cryptosporidium marsupials may shed into the environment.

Putignani, L., A. Tait, et al. (2004). "Characterization of a mitochondrion-like organelle in Cryptosporidium parvum." Parasitology 129(Pt 1): 1-18.
	Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.

Quilez, J., C. Sanchez-Acedo, et al. (2005). "Efficacy of two peroxygen-based disinfectants for inactivation of Cryptosporidium parvum oocysts." Appl Environ Microbiol 71(5): 2479-83.
	Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.

Quilez, J., C. Sanchez-Acedo, et al. (1996). "Prevalence of Cryptosporidium infections in pigs in Aragon (northeastern Spain)." Vet Parasitol 67(1-2): 83-8.
	Faecal samples from 620 pigs randomly selected from 27 farms throughout Aragon were examined to determine the prevalence of Cryptosporidium infections. Detection of oocysts was performed using the ethyl-acetate stool concentration method and the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were identified in 136 (21.9%) pigs from 21 (77.8%) farms. Infected animals ranged from 1 to 6 months old and oocysts were not detected in suckling piglets or adults. Infection rates were significantly higher in weaned, 1-2 month old piglets (59.2%) than in fattening, 2-6 month old pigs (34.3%) (P < 0.001). Cryptosporidial infections were asymptomatic in most of the pigs (90.4%) and usually of low intensity, since 92.6% of the infected pigs excreted few oocysts (0-1 oocysts per field at x 200 magnifications). Although 24.1% of weaned and 5.6% of fattening pigs infected by C. parvum had diarrhoea, it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic pigs, in both weaned (64.7% and 46.7%, respectively) and fattening pigs (34.3% and 33.3%).

Quilez, J., C. Sanchez-Acedo, et al. (1996). "Prevalence of Cryptosporidium and Giardia infections in cattle in Aragon (northeastern Spain)." Vet Parasitol 66(3-4): 139-46.
	Faecal samples from 554 bovines randomly selected at 30 farms in Aragon were examined to investigate the prevalence of Cryptosporidium and Giardia infections. C. parvum oocysts were identified by using the Ziehl-Neelsen modified technique in 109 (19.7%) bovines ranging from 3 days old to adults. Positive animals were found in 19 (63.3%) farms. As much as 44.4% of calves aged 3-4 days were infected, but infection rates peaked at 6-15 days of age (76.7%). Nevertheless, prevalence was also high in weanling calves aged 1.5-4 months (14%), fattening calves and heifers 4-24 months old (7.7%) and adults (17.8%). Diarrhoea was recorded in 78.6% of suckling and 29.4% of weanling calves infected by C. parvum, but it was only found to be statistically associated with infection in suckling calves (P < 0.01). All calves shedding moderate or many oocysts had diarrhoea, whereas asymptomatic infection was always correlated with few oocysts in faeces. Cryptosporidial infections were always asymptomatic in bovines older than 4 months. Giardia cysts were identified in 65 bovines (11.7%) from 16 (53.3%) of the farms surveyed. Infection rates were significantly higher in suckling (14.1%) and weanling calves (38%) than in bovines older than 4 months (2.2%) (P < 0.001). Diarrhoea was recorded in 45.5% of suckling and 10.9% of weanling calves infected by Giardia, but it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic calves.

Quilez, J., C. A. Vergara-Castiblanco, et al. (2002). "Serum antibody response and Cryptosporidium parvum oocyst antigens recognized by sera from naturally infected sheep." Vet Parasitol 104(3): 187-97.
	The response of specific serum immunoglobulins (IgG, IgM and IgA) and the major antigens of Cryptosporidium parvum recognized by these isotypes were investigated by using enzyme-linked immunosorbent assay and immunoblot techniques in lambs and ewes naturally infected throughout an outbreak of cryptosporidiosis. Serum samples were collected from 20 lambs the first day they showed diarrhoea (D1), and Days 11 and 22, in addition to single serum samples from 17 of their dams. Serum anti-C. parvum IgG, IgM and/or IgA antibodies were detected in lambs as early as Day 1. Levels of IgM antibodies remained steady from D1 to D11 and increased at D22, whereas the IgG response decreased from D1 to D11 and subsequently increased. In contrast, IgA antibodies rapidly fell from D1 and all lambs were seronegative at D11 and D22. The highest levels of specific antibodies were detected in sera from ewes. In fact, all ewes were seropositives for IgM and IgA isotypes and most (16/17) showed positive levels of IgG. Four protein fractions (37-39, 42-48, 51-57 and 60-69 kDa) were the most frequently recognized by IgG and IgM from lamb sera. A low molecular weight fraction (12-14 kDa) reacting with IgG and IgA in most lamb sera was scarcely recognized by IgM and three broad bands were frequently recognized by IgA antibodies (23-25, 51-57 and 90-95 kDa). The recognition pattern of 23-25 kDa peptides by IgA from lamb sera clearly increased with the age. Peptides of 42-48, 51-57, 60-69 and 71-78 kDa were most frequently recognized by IgG and IgM from ewe sera. In relation to IgA antibodies from ewe sera, a frequent immunoreactivity was found with proteins in the intervals between 12 and 22 kDa as well as between 32 and 34 kDa and practically all sera reacted with fractions from 42 to 95 kDa.

Quintero-Betancourt, W., A. L. Gennaccaro, et al. (2003). "Assessment of methods for detection of infectious Cryptosporidium oocysts and Giardia cysts in reclaimed effluents." Appl Environ Microbiol 69(9): 5380-8.
	This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.

Quintero-Betancourt, W., E. R. Peele, et al. (2002). "Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites." J Microbiol Methods 49(3): 209-24.
	Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps.To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment. Meanwhile, molecular methods (e.g. molecular markers such as intervening transcribed spacer regions), which can identify different genotypes of C. cayetanensis, show good promise for detection of this emerging coccidian parasite in water.

Quiroz, E. S., C. Bern, et al. (2000). "An outbreak of cryptosporidiosis linked to a foodhandler." J Infect Dis 181(2): 695-700.
	In September and October 1998, a cryptosporidiosis outbreak occurred on a Washington, DC, university campus. In a case-control study of 88 case patients and 67 control subjects, eating in 1 of 2 cafeterias was associated with diarrheal illness (P<.001). Morbidity was associated with eating dinner on 22 September (odds ratio, 8.1; 95% confidence interval, 3.4-19.5); weaker associations were found for 6 other meals. Cryptosporidium parvum was detected in stool specimens of 16 (70%) of 23 ill students and 2 of 4 ill employees. One ill foodhandler with laboratory-confirmed C. parvum prepared raw produce on 20-22 September. All 25 Cryptosporidium isolates submitted for DNA analysis, including 3 from the ill foodhandler, were genotype 1. This outbreak illustrates the potential for cryptosporidiosis to cause foodborne illness. Epidemiologic and molecular evidence indicate that an ill foodhandler was the likely outbreak source.

Rabold, J. G., C. W. Hoge, et al. (1994). "Cyclospora outbreak associated with chlorinated drinking water." Lancet 344(8933): 1360-1.
	
Ralston, B. J., C. L. Cockwill, et al. (2003). "Prevalence of Giardia and Cryptosporidium andersoni and their effects on performance in feedlot beef cattle." Canadian Journal of Animal Science 83(1): 153-159.
	Sixty individually housed Charolais crossbred steers originating from one ranch source had a 12-d (days 0-12) adaptation period in their pens to adjust to their ration and surroundings, followed by two consecutive backgrounding periods (85.5% roughage, 12% concentrate rations) with durations of 84 d (days 13-97) and 63 d (days 98-153), respectively. Steers had a 21-d adaptation period (days 154-174), followed by a 77-d (days 175-257) finishing period (20% roughage, 75% concentrate ration). Fecal samples and animal weights were collected from each steer every 28 d initially, then every 21 d during a test duration of 257 d. Feed weigh-backs were performed weekly for each steer. Fecal samples were processed, and Giardia duodenalis cysts and Cryptosporidium andersoni oocysts were counted. ADG, DMI and FE were calculated for each of the periods (Backgrounding Period 1, Backgrounding Period 2, Finishing Period 3 and Overall). Overall prevalence of C. andersoni and G. duodenalis was 85 and 82%, respectively. There was a decrease (P < 0.05) in the percentage of G. duodenalis infected steers from day 132 to the completion of the trial. The percentage of C. andersoni infected steers decreased (P < 0.05) from day 97 to the completion of the trial (day 257). Shedding of G. duodenalis cysts and C. andersoni oocysts in the feces was intermittent throughout the trial period. A comparison between the ADG, DMI and FE of G. duodenalis infected and non-infected steers demonstrated no overall differences (P > 0.05). A similar comparison between C andersoni infected and non-infected steers showed no overall difference (P > 0.05) with the exception of a lower (P < 0.05) DMI for infected steers. The degree of Giardia or C. andersoni infection observed in the present study did not effect DMI, ADG or FE of feedlot steers. [References: 28] 28

Read, C., J. Walters, et al. (2002). "Correlation between genotype of Giardia duodenalis and diarrhoea." Int J Parasitol 32(2): 229-31.
	
Read, C. M., P. T. Monis, et al. (2004). "Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP." Infect Genet Evol 4(2): 125-30.
	A PCR-RFLP genotyping tool was developed and used to characterise morphologically identical isolates of Giardia duodenalis from a variety of host species. Primers were designed to amplify a 432bp region of the glutamate dehydrogenase gene (gdh) from genetic Assemblages AI, AII, BIII, BIV, C, D and E of G. duodenalis. DNA extracted from cultured Giardia trophozoites, Giardia cysts purified from faeces and directly from whole faeces was amplified and sequenced at the gdh and 18SrDNA loci. The gdh sequences were identical with published gdh sequences for each assemblage with a few exceptions. However, in some cases genotyping results obtained using gdh differed from 18SrDNA genotyping results. From gdh sequence information a PCR-RFLP profile was identified for each of the genetic assemblages. PCR-RFLP is a reproducible, reliable and sensitive method for genotyping Giardia. Eight human, 12 cat, 9 dog and 16 cattle faecal isolates were genotyped using PCR-RFLP. This method allows G. duodenalis isolates from human-beings, their companion animals and livestock to be genotyped directly from faeces, leading to valuable information about Giardia genotypes in population without the need for in vitro/in vivo amplification.

Reduker, D. W. and C. A. Speer (1985). "Factors influencing excystation in Cryptosporidium oocysts from cattle." J Parasitol 71(1): 112-5.
	
Reed, C., G. D. Sturbaum, et al. (2002). "Cryptosporidium parvum mixed genotypes detected by PCR-restriction fragment length polymorphism analysis." Appl Environ Microbiol 68(1): 427-9.
	Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.

Reif, J. S., L. Wimmer, et al. (1989). "Human cryptosporidiosis associated with an epizootic in calves." Am J Public Health 79(11): 1528-30.
	An outbreak of human cryptosporidiosis occurred among previously healthy persons in a veterinary medical teaching hospital. Human illness began after admission of calves from a farm which had been experiencing an epizootic of neonatal diarrhea due to Cryptosporidium. The clinical syndrome in humans was characterized by watery diarrhea, abdominal cramping, flatulence, and headache. Cryptosporidiosis was confirmed by zinc sulfate flotation of fecal specimens in four persons, three of whom had been responsible for the care and treatment of infected calves. A fourth patient had washed her husband's soiled clothing and appeared to have been infected indirectly through fomite contamination. Among 112 persons surveyed, 26 (23.2 percent) had a diarrheal illness during the outbreak and nine met the case definition of a diarrheal illness lasting at least three days. These persons were more likely to have had contact with a calf with diarrhea than were 52 referents who did not become ill during the outbreak.

Reperant, J. M., M. Naciri, et al. (1994). "Major antigens of Cryptosporidium parvum recognised by serum antibodies from different infected animal species and man." Vet Parasitol 55(1-2): 1-13.
	Serum humoral immune response to Cryptosporidium parvum was evaluated in six species: mouse, rabbit, lamb, calf, pig and man. Electrophoretic and immunoblot analysis showed that specific animal antibody response appeared between Day 4 and Day 15 post inoculation. The two main target antigens had apparent molecular weights of 15-17 and 23 kDa. They were recognised by each species studied. Serum IgA intensively recognised the 15-17 kDa antigen, except in rabbit. This study demonstrates that these two antigens are consistent targets of humoral immune response and can therefore be of great interest in studies of therapy/prophylaxis.

Reynolds, D. T., R. B. Slade, et al. (1999). "Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data." J Appl Microbiol 87(6): 804-13.
	When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.

Ripabelli, G., A. Leone, et al. (2004). "Detection of Cryptosporidium parvum oocysts in experimentally contaminated lettuce using filtration, immunomagnetic separation, light microscopy, and PCR." Foodborne Pathog Dis 1(4): 216-22.
	Recovery of Cryptosporidium parvum oocysts in a fecal suspension that experimentally contaminated onto lettuce leaves was investigated. Material recovered from the lettuce samples by washing in detergent solutions were concentrated by filtration using the Envirochek Sampling Capsule. Oocysts were concentrated by immunomagnetic separation (IMS) and detected by microscopy following modified Ziehl-Neelsen (MZN) staining. Cryptosporidal DNA was detected using a nested-PCR assay for amplification of a fragment of the Cryptosporidium oocyst wall protein (COWP) gene, which was applied to DNA extracted from both filtrates, and material recovered from MZN stained smears on glass slides after microscopy. No Cryptosporidium were detected by microscopy or by PCR of un-inoculated lettuce leaves. After IMS, means of 0-6.5% of the total numbers of oocysts inoculated were recovered and detected by microscopy. Detection by PCR was less sensitive than microscopy. There was a strong association between successful PCR amplification, the numbers of oocysts detected by microscopy and the numbers of oocysts in the inoculum. This study confirms that C. parvum oocysts can be recovered from contaminated lettuce using filtration and IMS, and detected by microscopy and PCR. However, further developments are required to improve recovery of this parasite.

Roach, P. D., M. E. Olson, et al. (1993). "Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada." Appl Environ Microbiol 59(1): 67-73.
	Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)

Robertson, L. J., A. T. Campbell, et al. (1992). "Survival of Cryptosporidium parvum oocysts under various environmental pressures." Appl Environ Microbiol 58(11): 3494-500.
	The survival of various isolates of Cryptosporidium parvum oocysts under a range of environmental pressures including freezing, desiccation, and water treatment processes and in physical environments commonly associated with oocysts such as feces and various water types was monitored. Oocyst viability was assessed by in vitro excystation and by a viability assay based on the exclusion or inclusion of two fluorogenic vital dyes. Although desiccation was found to be lethal, a small proportion of oocysts were able to withstand exposure to temperatures as low as -22 degrees C. The water treatment processes investigated did not affect the survival of oocysts when pH was corrected. However, contact with lime, ferric sulfate, or alum had a significant impact on oocyst survival if the pH was not corrected. Oocysts demonstrated longevity in all water types investigated, including seawater, and when in contact with feces were considered to develop an enhanced impermeability to small molecules which might increase the robustness of the oocysts when exposed to environmental pressures.

Robertson, L. J., A. T. Campbell, et al. (1993). "In vitro excystation of Cryptosporidium parvum." Parasitology 106 ( Pt 1): 13-9.
	Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

Robertson, L. J., A. T. Campbell, et al. (1993). "Induction of folds or sutures on the walls of Cryptosporidium parvum oocysts and their importance as a diagnostic feature." Appl Environ Microbiol 59(8): 2638-41.
	The proportion of oocysts of Cryptosporidium parvum showing a fold on oocyst walls when incubated with either fluorescent monoclonal antibody or a surface-reactive fluorescent dye was increased by incubating suspensions of oocysts with dimethyl sulfoxide, sucrose, or Hanks' balanced salt solution. Further incubation of sucrose-incubated oocysts with water showed this to be a reversible phenomenon. Oocysts demonstrating this fold after incubation in dimethyl sulfoxide were of the same viability as control oocysts and followed the same excystation dynamics. Despite this fold having been previously described as a suture, we were unable to find any evidence that this pattern of fluorescence highlighted the same suture that has been described in ultrastructural studies. Furthermore, oocysts were observed in which this fold was not always continuous with the gape in the oocyst wall through which the sporozoites had emerged. We propose that this fluorescently highlighted region or fold should no longer be described as a suture and question its validity as a diagnostic feature. When environmental and other samples are being examined for the presence of C. parvum oocysts, objects of appropriate size, shape, and fluorescence which do not demonstrate a surface fold should not necessarily be excluded.

Robertson, L. J., A. T. Campbell, et al. (1998). "Viability of Cryptosporidium parvum oocysts: assessment by the dye permeability assay." Appl Environ Microbiol 64(9): 3544-5.
	
Robertson, L. J., T. Forberg, et al. (2006). "Cryptosporidium parvum infections in Bergen, Norway, during an extensive outbreak of waterborne giardiasis in autumn and winter 2004." Appl Environ Microbiol 72(3): 2218-20.
	During a large waterborne giardiasis outbreak in Norway, many diarrheic patients were found to have Cryptosporidium infections. Gene sequencing identified these infections as Cryptosporidium parvum infections, although they were not identical. Whether these infections were due to a simultaneous outbreak of waterborne cryptosporidiosis or reflected background levels not normally detected is discussed.

Robertson, L. J. and B. Gjerde (2000). "Effect of sample holding time on recovery of Cryptosporidium oocysts and Giardia cysts from water samples." Appl Environ Microbiol 66(4): 1724-5.
	U.S. Environmental Protection Agency methods for analysis of water for Cryptosporidium and Giardia stipulate maximum sample holding times which are not always practical to comply with. A spiking experiment indicated that holding times of up to 2 weeks had no significant effect on recovery of these parasites from 10-liter samples of raw water in plastic carboys.

Robertson, L. J. and B. Gjerde (2000). "Isolation and enumeration of Giardia cysts, cryptosporidium oocysts, and Ascaris eggs from fruits and vegetables." J Food Prot 63(6): 775-8.
	Published techniques for recovering parasites from fruit and vegetables are generally inadequate, with low and variable recovery efficiencies. Here we describe an improved methodology for analyzing fruit and vegetables for Giardia cysts, Cryptosporidium oocysts, and Ascaris eggs. The method includes washing procedures, sonication, and, for Giardia and Cryptosporidium, immunomagnetic separation. Identification is by immunofluorescence (Giardia and Cryptosporidium) or brightfield microscopy (Ascaris). Recovery efficiencies from lettuce, Chinese leaves, and strawberries were found to be approximately 67% for Giardia, 42% for Cryptosporidium, and 72% for Ascaris. Recovery efficiencies from bean sprouts tended to be more variable and lower. This could be due to material removed with the parasites during the washing procedures, which, in turn, appeared related to the age of the bean sprouts. It is therefore recommended that fruit and vegetables should be as fresh as possible when analyzed for parasites.

Robertson, L. J. and B. Gjerde (2001). "Factors affecting recovery efficiency in isolation of Cryptosporidium oocysts and Giardia cysts from vegetables for standard method development." J Food Prot 64(11): 1799-805.
	While recently published techniques for recovering parasites from fruits and vegetables demonstrate a marked increase in efficiency and utility, there is still scope for further improvement in developing a standard method, particularly with difficult, but important, sample matrices such as bean sprouts. Herein, a number of parameters used in published techniques are investigated more closely. While sample size reduction may improve recovery efficiency because of a range of factors, it is important to keep the sample large enough for detection of low-level contamination. Age of sample is also important, and samples should be as fresh as possible. Elution procedures may contribute to losses of Giardia and should be more thoroughly investigated. Improved immunomagnetic separation techniques currently coming onto the market also have the potential to increase recovery efficiency substantially, even with difficult samples such as aged bean sprouts. However, merely increasing magnetic strength of the capturing magnet does not affect recovery efficiency, which must be reliant on a superior bead system, buffering system, or both.

Robertson, L. J. and B. Gjerde (2001). "Occurrence of Cryptosporidium oocysts and Giardia cysts in raw waters in Norway." Scand J Public Health 29(3): 200-7.
	AIMS: This paper reports the first investigation into the occurrence of Cryptosporidium and Giardia in Norwegian raw water sources. METHODS: Between June 1998 and October 1999, 408 raw water samples, collected from 147 different sites across Norway, were analysed for these parasites. Analysis was based upon US EPA Method 1623. RESULTS: In 305 samples (75%), parasites were not detected. In 55 samples (13.5%), Cryptosporidium only was detected. In 38 samples (9%), Giardia only was detected. In 10 samples (2.5%) both Cryptosporidium and Giardia were detected. Of the sites sampled, parasites were not detected at 100 (68%) of them, Cryptosporidium only was detected at 20 (13.5%). Giardia only was detected at 11 (7.5%), and both Cryptosporidium and Giardia were detected at 16 (11%). Concentrations of parasites were low; usually one cyst/oocyst detected per 10 litres of water. CONCLUSIONS: Significant associations were demonstrated for these samples between the detection of these parasites and (a) turbidity > or =2.0 NTU, and (b) high numbers of domestic animals within the catchment area. No association between seasonality and the occurrence of these parasites could be detected. The results are discussed in relation to other studies and the potential public health implications for Norway.

Robertson, L. J. and B. Gjerde (2001). "Occurrence of parasites on fruits and vegetables in Norway." J Food Prot 64(11): 1793-8.
	Between August 1999 and January 2001, samples of various fruits and vegetables obtained within Norway were analyzed by published methods for parasite contamination. Neither Cyclospora oocysts nor Ascaris (or other helminth) eggs were detected on any of the samples examined for these parasites. However, of the 475 samples examined for Cryptosporidium oocysts and Giardia cysts, 29 (6%) were found to be positive. No samples were positive for both parasites. Of the 19 Cryptosporidium-positive samples. 5 (26%) were in lettuce, and 14 (74%) in mung bean sprouts. Of the 10 Giardia-positive samples, 2 (20%) were in dill, 2 (20%) in lettuce, 3 (30%) in mung bean sprouts, 1 (10%) in radish sprouts, and 2 (20%) in strawberries. Mung bean sprouts were significantly more likely to be contaminated with Cryptosporidium oocysts or Giardia cysts than the other fruits and vegetables. Concentrations of Cryptosporidium and Giardia detected were generally low (mean of approximately 3 [oo]cysts per 100 g produce). Although some of the contaminated produce was imported (the majority, if sprouted seeds are excluded), there was no association between imported produce and detection of parasites. Crvptosporidium oocysts and Giardia cysts were also detected in water samples concerned with field irrigation and production of bean sprouts within Norway. This is the first time that parasites have been detected on vegetables and fruit obtained in a highly developed. wealthy country, without there being an outbreak situation. These findings may have important implications for global food safety.

Robertson, L. J., B. Gjerde, et al. (2000). "Isolation of Cyclospora oocysts from fruits and vegetables using lectin-coated paramagnetic beads." J Food Prot 63(10): 1410-4.
	Published techniques for recovering parasites from fruit and vegetables are generally inadequate, with low and variable recovery efficiencies. Herein, we describe an improved method for analyzing fruit and vegetables for Cyclospora oocysts. The technique includes washing procedures, sonication, and separation using lectin-coated paramagnetic beads. Identification is by microscopy (differential interference contrast and fluorescence). Oocyst recovery efficiencies from mushrooms, lettuce, and raspberries were approximately 12%. Recovery efficiencies from bean sprouts were approximately 4%. Although no significant difference in recovery efficiency could be detected between samples processed using the lectin-coated beads and samples processed without this procedure, distinct advantages were apparent when the lectin-coated beads were used. A considerably smaller, cleaner final volume remained for microscopy, which increases the sensitivity of the technique and reduces operator time.

Robertson, L. J., J. D. Greig, et al. (2005). "The potential for acquiring cryptosporidiosis or giardiosis from consumption of mung bean sprouts in Norway: a preliminary step-wise risk assessment." Int J Food Microbiol 98(3): 291-300.
	The current work evolved from a microbial survey of fruits and vegetables conducted in Norway between 1999 and 2001. This survey found that mung bean sprouts were more likely to be contaminated with Cryptosporidium and Giardia than the other produce included in the survey. To support this observation and to demonstrate to public health officials that this might be a risk warranting further attention, a simple risk assessment was initiated. Assuming that 60,000 people in Norway consume a single serving of bean sprouts per week, and contamination levels are similar to those found in the survey, it was calculated that there could be in the order of 20 cases of Giardia or Cryptosporidium infection per 100,000 population attributable to consumption of mung bean sprouts. A number of assumptions were made for the calculations, including parasite factors (e.g. viability, genotype), product factors (e.g. extent of product contamination) and host factors (e.g. composition and extent of consumer group). These assumptions and areas of uncertainty, where further data would improve the risk assessment, are highlighted throughout. Not only does the risk assessment identify new areas of research, but it also demonstrates how risk assessment can be used as a tool to try to influence public health surveillance.

Robertson, L. J., L. Hermansen, et al. (2006). "Occurrence of cryptosporidium oocysts and giardia cysts in sewage in norway." Appl Environ Microbiol 72(8): 5297-303.
	Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of >20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and >80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.

Robertson, L. J., L. Hermansen, et al. (2006). "Application of genotyping during an extensive outbreak of waterborne giardiasis in Bergen, Norway, during autumn and winter 2004." Appl Environ Microbiol 72(3): 2212-7.
	During the autumn and winter of 2004 and 2005, an extensive outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1,500 patients were diagnosed with giardiasis. Analysis of water from the implicated source revealed low numbers of Giardia cysts, but the initial contamination event probably occurred up to 10 weeks previously. While sewage leakage from a residential area is now considered to be the probable source of contamination, during the episode waste from one particular septic tank was thought to be a possible source. Genotyping of cysts from the septic tank demonstrated that they were assemblage A cysts, although the sequences were not identical to any previously published sequences. For the beta-giardin gene, the closest published subgenotype was subgenotype A3; for the gdh gene, the closest published subgenotype was subgenotype A2. Genotyping of cysts from 21 patient samples revealed that they were assemblage B cysts; thus, the septic tank was unlikely to be the contamination source. Sequencing of the beta-giardin and gdh genes from patient samples and a comparison of the sequences gave complex results. For the beta-giardin gene, three isolates had sequences identical to subgenotype B3 sequences. However, other isolates had between one and four single-nucleotide polymorphisms (SNPs). For the gdh gene, none of the sequences were identical to the sequence published for subgenotype B3, and the sequences had between one and three SNPs. One isolate, which was identical to subgenotype B3 at the beta-giardin gene, was more similar to subgenotype B2 at the gdh gene. Grouping the isolates on the basis of SNPs resulted in different groups for the two genes. The results are discussed in relation to giardiasis in Norway and to other Giardia genotyping studies.

Robertson, L. J., G. S. Johannessen, et al. (2002). "Microbiological analysis of seed sprouts in Norway." Int J Food Microbiol 75(1-2): 119-26.
	As part of larger survey of microbial contamination of fruits and vegetables in Norway, four different sprouted seed products were analysed for bacterial and parasitic contaminants (n = 300 for bacterial analyses and n = from 17 to 171 for parasite analyses, depending on parasite). Escherichia coli O157, Salmonella, Listeria monocytogenes, Cyclospora oocysts, Ascaris eggs and other helminth parasites were not detected in any of the sprout samples. Thermotolerant coliform bacteria (TCB) were isolated from approximately 25% of the sprout samples, with the highest percentage of TCB positive samples in alfalfa sprouts. Most TCB were Enterobacter spp. and Klebsiella. E. coli was isolated from 8 of 62 TCB positive mung bean sprout samples. Cryptosporidium oocysts were detected in 8% of the sprout samples and Giardia cysts were detected in 2% of the samples. All the Cryptosporidium positive samples, and most of the Giardia positive samples, were mung bean sprouts. Parasite concentrations in positive samples were low (between 1 and 3 oocysts/cysts per 50 g sprouts). Sprout irrigation water was also analysed for microbial contaminants. E. coli O157 and L. monocytogenes were not detected. TCB were isolated from approximately 40% of the water samples. Salmonella reading was isolated from three samples of spent irrigation water on 3 consecutive days. Cryptosporidium and Giardia were also isolated from spent irrigation water. Additionally, eight samples of unsprouted mung bean seed were analysed for Cryptosporidium oocysts and Giardia cysts. One or both of these parasites were detected in six of the unsprouted seed samples at concentrations of between 1 and 5 oocysts/cysts per 100 g unsprouted seed. Whilst our results support spent irrigation water as the most suitable matrix for testing for bacteria, unsprouted seed is considered the more useful matrix for analysing for parasite contaminants.

Robinson, P., P. C. Okhuysen, et al. (2003). "Substance P expression correlates with severity of diarrhea in cryptosporidiosis." J Infect Dis 188(2): 290-6.
	Cryptosporidiosis, caused by Cryptosporidium parvum, is self-limited in immunocompetent hosts but may cause chronic diarrhea in patients with acquired immunodeficiency syndrome (AIDS). Substance P (SP), a neuropeptide belonging to the tachykinin family, is expressed in gastrointestinal tract and can cause electrogenic chloride anion secretion. Therefore, we studied SP mRNA and protein expression in jejunal tissue samples of patients with AIDS with naturally occurring chronic cryptosporidiosis and healthy volunteers with mild cryptosporidiosis or asymptomatic infection after experimental C. parvum challenge. SP mRNA was associated with symptoms in cryptosporidiosis. SP protein levels were greater in symptomatic than asymptomatic volunteers. Similarly, greater expression of SP mRNA and protein were noted in patients with AIDS with chronic cryptosporidiosis versus immunocompetent volunteers with self-limited infection. This study demonstrates a direct correlation between SP levels and disease severity and may imply that SP plays a role in diarrhea mediation.

Roche, J. K., C. A. Martins, et al. (2000). "Transforming growth factor beta1 ameliorates intestinal epithelial barrier disruption by Cryptosporidium parvum in vitro in the absence of mucosal T lymphocytes." Infect Immun 68(10): 5635-44.
	Exposure to oocysts of the protozoan Cryptosporidium parvum causes intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor beta1 (TGF-beta1)-a cytokine synthesized and released by cells in the intestine-to preserve the barrier function of human colonic epithelia when challenged with C. parvum oocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-beta1 were localized by confocal microscopy, and TGF-beta1-induced protein kinase C activation was detected intracellularly by translocation of its alpha isozyme. TGF-beta1 alone enhanced intestinal epithelial barrier function, while exposure to C. parvum oocysts (> or =10(5)/monolayer) markedly reduced barrier function to < or =40% of that of the control. When epithelial monolayers were pretreated with TGF-beta1 at 5.0 ng/ml, the barrier-disrupting effect of C. parvum oocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-beta1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-beta1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-beta1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-beta1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa alpha isozyme within 30 s of epithelial exposure to TGF-beta1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-beta1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-beta1 when human colonic epithelium is exposed to an important human pathogen, C. parvum.

Rochelle, P. A., R. De Leon, et al. (1999). "Evaluation of immunomagnetic separation for recovery of infectious Cryptosporidium parvum oocysts from environmental samples." Appl Environ Microbiol 65(2): 841-5.
	Two commercial immunomagnetic separation (IMS) kits for Cryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity.

Rochelle, P. A., R. De Leon, et al. (1997). "Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water." Appl Environ Microbiol 63(1): 106-14.
	Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.

Rochelle, P. A., D. Fallar, et al. (2004). "Irreversible UV inactivation of Cryptosporidium spp. despite the presence of UV repair genes." J Eukaryot Microbiol 51(5): 553-62.
	Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92-98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

Rochelle, P. A., D. M. Ferguson, et al. (1997). "An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum." Appl Environ Microbiol 63(5): 2029-37.
	The presence of Cryptosporidium in drinking water supplies is a significant problem faced by the water industry. Although a variety of methods exist for the detection of waterborne oocysts, water utilities currently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to public health by waterborne Cryptosporidium. In this paper, the development of an infectivity assay for waterborne Cryptosporidium parvum is described. Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the heat shock protein 70 (hsp70) gene. A single infectious oocyst was detected by this experimental procedure. The use of concentrated samples obtained from 250 liters of finished water had no observable effect on the integrity of cell monolayers or on the infectivity of oocysts seeded into the concentrate. Intracellular developmental stages of the parasite were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parvum, while a second pair recognized all species of Cryptosporidium tested. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samples and were compatible with multiplex PCR for the simultaneous detection of C. parvum and Giardia lambia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly, demonstrates the potential of an in vitro infectivity assay.

Rochelle, P. A., M. M. Marshall, et al. (2002). "Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum." Appl Environ Microbiol 68(8): 3809-17.
	In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

Rogers, K. A., A. B. Rogers, et al. (2006). "MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice." Infect Immun 74(1): 549-56.
	Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.

Rose, J. B. (1997). "Environmental ecology of Cryptosporidium and public health implications." Annual Review of Public Health 18: 135-161.
	Cryptosporidium has become the most important contaminant found in drinking water and is associated with a high risk of waterborne disease particularly for the immunocompromised. There have been 12 documented waterborne outbreaks in North America since 1985; in two of these (Milwaukee and Las Vegas) mortality rates in the immunocompromised ranged from 52% to 68%. The immunofluorescence antibody assay (IFA) using epifluorescence microscopy has been used to examine the occurrence of Cryptosporidium in sewage (1 to 120 oocysts/liter), filtered secondary treated wastewater (0.01 to 0.13 oocysts/liter), surface waters (0.001 to 107 oocysts/liter), groundwater (0.004 to 0.922 oocysts/liter) and treated drinking water (0.001 to 0.72 oocysts/liter). New rules are being developed (Information Collection Rule and Enhanced Surface Water Treatment Rule) to obtain more occurrence data for drinking water systems for use with new risk assessment models. Public health officials should consider a communication program to physicians treating the immunocompromised, nursing homes, develop a plan to evaluate cases of cryptosporidiosis in the community, and contribute to the development of public policies that limit contamination of source waters, improve water treatment, and protect public health.

Rose, J. B. and T. R. Slifko (1999). "Giardia, Cryptosporidium, and Cyclospora and their impact on foods: a review." J Food Prot 62(9): 1059-70.
	While the risk from pathogenic microorganisms in foods has been recognized for hundreds of years, bacterial agents are generally implicated as the contaminants. Although many outbreaks of gastroenteritis caused by protozoan pathogens have occurred, it is only in the last 3 years that attention has focused on protozoan association with foodborne transmission. Recognized as waterborne parasites, Giardia, Cryptosporidium, and Cyclospora have now been associated with several foodborne outbreaks. The oocysts and cysts of these organisms can persist and survive for long periods of time both in water and on foods. While Cyclospora oocysts require a maturation period, Cryptosporidium oocysts and Giardia cysts are immediately infectious upon excretion from the previous host. As a result, these parasites have emerged as public health risks and have become a concern to the food industry. More than 200 cases of foodborne giardiasis (seven outbreaks) were reported from 1979 to 1990. Four foodborne Cryptosporidium outbreaks (with a total of 252 cases) have been documented since 1993. Cyclospora caused a series of sporadic outbreaks of cyclosporasis throughout North America that have affected over 3,038 people since 1995. Control and prevention of protozoan foodborne disease depends upon our ability to prevent, remove, or kill protozoan contaminants. This review will address the biology, foodborne and waterborne transmission, survival, and methods for detection and control of Giardia, Cryptosporidium, and Cyclospora.

Roy, S. L., S. M. DeLong, et al. (2004). "Risk factors for sporadic cryptosporidiosis among immunocompetent persons in the United States from 1999 to 2001." J Clin Microbiol 42(7): 2944-51.
	Many studies have evaluated the role of Cryptosporidium spp. in outbreaks of enteric illness, but few studies have evaluated sporadic cryptosporidiosis in the United States. To assess the risk factors for sporadic cryptosporidiosis among immunocompetent persons, a matched case-control study was conducted in seven sites of the Foodborne Diseases Active Surveillance Network (FoodNet) involving 282 persons with laboratory-identified cryptosporidiosis and 490 age-matched and geographically matched controls. Risk factors included international travel (odds ratio [OR] = 7.7; 95% confidence interval [95% CI] = 2.7 to 22.0), contact with cattle (OR = 3.5; 95% CI = 1.8 to 6.8), contact with persons >2 to 11 years of age with diarrhea (OR = 3.0; 95% CI = 1.5 to 6.2), and freshwater swimming (OR = 1.9; 95% CI = 1.049 to 3.5). Eating raw vegetables was protective (OR = 0.5; 95% CI = 0.3 to 0.7). This study underscores the need for ongoing public health education to prevent cryptosporidiosis, particularly among travelers, animal handlers, child caregivers, and swimmers, and the need for further assessment of the role of raw vegetables in cryptosporidiosis.

Ruecker, N. J., N. Bounsombath, et al. (2005). "Molecular forensic profiling of Cryptosporidium species and genotypes in raw water." Appl Environ Microbiol 71(12): 8991-4.
	The emerging concept of host specificity of Cryptosporidium spp. was exploited to characterize sources of fecal contamination in a watershed. A method of molecular forensic profiling of Cryptosporidium oocysts on microscope slides prepared from raw water samples processed by U.S. Environmental Protection Agency Method 1623 was developed. The method was based on a repetitive nested PCR-restriction fragment length polymorphism-DNA sequencing approach that permitted the resolution of multiple species/genotypes of Cryptosporidium in a single water sample.

Rulofson, F. C., E. R. Atwill, et al. (2001). "Fecal shedding of Giardia duodenalis, Cryptosporidium parvum, Salmonella organisms, and Escherichia coli O157:H7 from llamas in California." Am J Vet Res 62(4): 637-42.
	OBJECTIVE: To evaluate fecal shedding of Giardia duodenalis, Cryptosporidium parvum, Salmonella organisms, and Escherichia coli O157:H7 from llamas in California with respect to host factors and management practices. ANIMALS: 354 llamas from 33 facilities. PROCEDURE: Fecal specimens were collected and examined for G. duodenalis and C. parvum by means of immunofluorescent microscopy. Salmonella organisms were cultured by placing feces into selenite enrichment broth followed by selective media. Escherichia coli O157:H7 was cultured by use of modified tryptocase soy broth followed by sorbitol MacConkey agar, with suspect colonies confirmed by means of immunofluorescent microscopy. RESULTS: 12 of 354 fecal specimens (3.4%) had G. duodenalis cysts. Younger llamas (crias) were more likely to be shedding cysts, compared with older llamas. Farm-level factors that increased the risk of shedding were large numbers of yearlings on the property (> 10), smaller pen sizes, large numbers of crias born during the previous year (> 10), and large pen or pasture populations (> 20). None of the 354 fecal specimens had C. parvum oocysts. Seventy-six (from 7 facilities) and 192 (from 22 facilities) llamas were tested for Salmonella organisms and E. coli O157:H7, respectively. All fecal specimens had negative results for these bacteria. CONCLUSIONS AND CLINICAL RELEVANCE: Shedding of G. duodenalis was primarily limited to crias 1 to 4 months old. Llamas from properties with large numbers of crias born in the previous year, resulting in large numbers of yearlings in the current year, were at greater risk of infection. In addition, housing llamas in smaller pens or pastures and managing llamas and crias in large groups also increased the risk of G. duodenalis shedding.

Ryan, U., A. O'Hara, et al. (2004). "Molecular and Biological Characterization of a Cryptosporidium molnari-Like Isolate from a Guppy (Poecilia reticulata)." Appl Environ Microbiol 70(6): 3761-5.
	Histological, morphological, genetic, and phylogenetic analyses of a Cryptosporidium molnari-like isolate from a guppy (Poecilia reticulata) identified stages consistent with those of C. molnari and revealed that C. molnari is genetically very distinct from all other species of Cryptosporidium. This study represents the first genetic characterization of C. molnari.

Ryan, U., C. Read, et al. (2005). "Genotypes of Cryptosporidium from Sydney water catchment areas." J Appl Microbiol 98(5): 1221-9.
	AIMS: Currently cryptosporidiosis represents the major public health concern of water utilities in developed nations and increasingly, new species and genotypes of Cryptosporidium are being identified in which the infectivity for humans is not clear. The complicated epidemiology of Cryptosporidium and the fact that the majority of species and genotypes of Cryptosporidium cannot be distinguished morphologically makes the assessment of public health risk difficult if oocysts are detected in the raw water supplies. The aim of this study was to use molecular tools to identify sources of Cryptosporidium from the Warragamba catchment area of Sydney, Australia. METHODS AND RESULTS: Both faecal and water samples from the catchment area were collected and screened using immunomagnetic separation (IMS) and immunofluorescence microscopy. Samples that contained Cryptosporidium oocysts were genotyped using sequence and phylogenetic analysis of the 18S rDNA, and the heat-shock (HSP-70) gene. Analysis identified five Cryptosporidium species/genotypes including C. parvum (cattle genotype), C. suis, pig genotype II, the cervid genotype and a novel goat genotype. CONCLUSIONS: Monitoring and characterization of the sources of oocyst contamination in watersheds will aid in the development and implementation of the most appropriate watershed management policies to protect the public from the risks of waterborne Cryptosporidium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that quantification by IMS analysis can be combined with the specificity of genotyping to provide an extremely valuable tool for assessing the human health risks from land use activities in drinking water catchments.

Ryan, U., L. Xiao, et al. (2003). "Identification of novel Cryptosporidium genotypes from the Czech Republic." Appl Environ Microbiol 69(7): 4302-7.
	Isolates of Cryptosporidium from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.

Ryan, U. M., C. Bath, et al. (2005). "Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites." Appl Environ Microbiol 71(9): 4992-7.
	Little is known of the prevalence of Cryptosporidium and Giardia parasites in sheep and the genotypes that they harbor, although potentially sheep may contribute significantly to contamination of watersheds. In the present study, conducted in Western Australia, a total of 1,647 sheep fecal samples were screened for the presence of Cryptosporidium and Giardia spp. using microscopy, and a subset (n = 500) were screened by PCR and genotyped. Analysis revealed that although both parasites were detected in a high proportion of samples by PCR (44% and 26% for Giardia and Cryptosporidium spp., respectively), with the exception of one Cryptosporidium hominis isolate, the majority of isolates genotyped are not commonly found in humans. These results suggest that the public health risk of sheep-derived Cryptosporidium and Giardia spp. in catchment areas and effluent may be overestimated and warrant further investigation.

Ryan, U. M., P. Monis, et al. (2004). "Cryptosporidium suis n. sp. (Apicomplexa: Cryptosporidiidae) in pigs (Sus scrofa)." J Parasitol 90(4): 769-73.
	Molecular and biological characteristics of a new species of Cryptosporidium from the feces of pigs (Sus scrofa) is described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum; they are passed fully sporulated, lack sporocysts, and measure 4.9-4.4 microm (mean = 4.6 microm) x 4.0-4.3 microm (mean = 4.2 microm); length to width ratio 1.1 (n = 50). Cryptosporidium suis is not transmissible to nude mice and is poorly infectious for cattle. Molecular and phylogenetic analyses at the 18S ribosomal RNA, heat shock protein 70, and actin gene loci demonstrate C. suis to be genetically distinct from all known species and genotypes of Cryptosporidium, and thus is named as Cryptosporidium suis.

Ryan, U. M., B. Samarasinghe, et al. (2003). "Identification of a novel Cryptosporidium genotype in pigs." Appl Environ Microbiol 69(7): 3970-4.
	Over a 3-year period, a total of 646 fecal samples from pigs in 22 indoor and outdoor herds from Western Australia were screened for Cryptosporidium spp. by microscopy. Results revealed that 39 of 646 samples (6.03%) were positive for Cryptosporidium. Cryptosporidium was much more common in outdoor herds (17.2%) than in indoor herds (0.5%) and was more common in animals between the ages of 5 and 8 weeks (69.2%) than in younger animals (P < 0.0001). Molecular characterization of the positive samples at the 18S ribosomal DNA locus identified two distinct genotypes of Cryptosporidium: the previously identified pig genotype I and a novel pig genotype (pig genotype II), both of which warrant species status.

Ryan, U. M., L. Xiao, et al. (2003). "A redescription of Cryptosporidium galli Pavlasek, 1999 (Apicomplexa: Cryptosporidiidae) from birds." J Parasitol 89(4): 809-13.
	Cryptosporidium galli Pavlasek, 1999, described from the feces of birds, is redescribed with additional molecular and biological data. Oocysts are ellipsoidal, are passed fully sporulated, lack sporocysts, and measure 8.25 x 6.3 microm (range 8.0-8.5 x 6.2-6.4 microm) with a length-width ratio of 1.30 (n = 50). Oocysts are structurally similar to those of Cryptosporidium baileyi described from chickens, but in addition to being considerably larger than oocysts of C. baileyi, these oocysts infect the proventriculus in a variety of birds and not the respiratory tract. Oocysts were successfully transmitted from chickens to chickens, and morphologically similar oocysts also were observed in a variety of exotic and wild birds (Order Passeriformes, Phasianidae, Fringillidae, and Icteridae). Molecular and phylogenetic analyses at the 18S rRNA, HSP70, and actin gene loci demonstrate that this species is genetically distinct from all known species and genotypes of Cryptosporidium and, thus, was named C. galli.

Saavedra, G. M. and Y. R. Ortega (2004). "Seroprevalence of Toxoplasma gondii in swine from slaughterhouses in Lima, Peru, and Georgia, U.S.A." J Parasitol 90(4): 902-4.
	Toxoplasma gondii is an important pathogen transmitted by food, with raw or undercooked meat as the main foodborne source of toxoplasmosis. In Peru, 2-4 million people have antibodies to T. gondii. It is believed that more than 60 million people in the United States are infected with T. gondii. In this study, the prevalence of T. gondii in pigs from Peru and the United States was determined by Western blot. The presence of IgG antibodies to T. gondii from serum samples was determined. Blood samples were collected from 137 pigs at a slaughterhouse in Lima, Peru, and 152 pigs at a slaughterhouse in Georgia. Of the serum samples collected from swine, 27.7% (n = 38) from Peru and 16.4% (n = 25) from the United States were positive for T. gondii. Swine represent a significant source of human infection with T. gondii in Peru and the United States.

Santin, M., B. R. Dixon, et al. (2005). "Genetic characterization of Cryptosporidium isolates from ringed seals (Phoca hispida) in Northern Quebec, Canada." J Parasitol 91(3): 712-6.
	This study reports the molecular characterization of Cryptosporidium spp. isolates identified from intestinal contents of ringed seals (Phoca hispida) from Nunavik (Quebec, Canada). Cryptosporidium spp. fragments of 18S rRNA, HSP-70, and actin loci were amplified by PCR from seal intestinal contents. PCR-positive specimens were sequenced and compared with other Cryptosporidium species and genotypes reported previously. Sequence analysis showed the presence of C. muris and 2 novel genotypes in ringed seals.

Santin, M., K. Ludwig, et al. (2003). "First report of Giardia in coyotes (Canis latrans)." J Eukaryot Microbiol 50 Suppl: 709.
	
Santin, M., J. M. Trout, et al. (2004). "Prevalence of Enterocytozoon bieneusi in post-weaned dairy calves in the eastern United States." Parasitol Res 93(4): 287-9.
	Fecal specimens were obtained from 3- to 8-month-old post-weaned dairy calves on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, 59 of 452 calves (13%) from 11 farms in six states were found positive for Enterocytozoon bieneusi by PCR and DNA sequence analysis. Based on gene sequence data this genotype of E. bieneusi found in post-weaned calves was 100% identical to that found in pre-weaned calves in North America and differed by only two positions in 1,069 base pairs from specimens analyzed from humans. However, compared with previous reports, the prevalence of E. bieneusi was significantly higher in post-weaned than in pre-weaned calves from many of the same farms.

Santin, M., J. M. Trout, et al. (2004). "Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves." Vet Parasitol 122(2): 103-17.
	Fifteen dairy farms in seven states on the east coast of the US were each visited on two consecutive years to determinate the prevalence of Cryptosporidium species in pre-weaned (5 days to 2 months) and post-weaned calves (3-11 months), respectively. After each of 971 fecal specimens collected directly from each calf was sieved and subjected to density gradient centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy, and polymerase chain reaction (PCR). For all PCR-positive specimens the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified from all farms. Types of housing appeared to have no influence with regard to prevalence of infection. Of 971 calves, 345 were infected with Cryptosporidium (35.5%), but more pre-weaned calves (253 of 503; 50.3%) than post-weaned calves (92 of 468; 19.7%) were found to be infected. A total of 278 PCR-positive specimens characterized by gene sequencing revealed Cryptosporidium parvum, Cryptosporidium andersoni, and two unnamed Cryptosporidium genotypes Bovine B (AY120911) and deer-like genotype (AY120910). The prevalence of these Cryptosporidium species and genotypes appeared to be age related between pre- and post-weaned calves. C. parvum, the only zoonotic species/genotype, constituted 85% of the Cryptosporidium infections in pre-weaned calves but only 1% of the Cryptosporidium infections in post-weaned calves. These findings clearly demonstrate that earlier reports on the presence and prevalence of C. parvum in post-weaned cattle that were based solely on oocyst morphology must be reassessed using molecular methods to validate species and genotype. This finding also indicates that persons handling or otherwise exposed to calves under 2 months of age are at greater risk of zoonotic infection from Cryptosporidium than the risk of infection from exposure to older calves.

Sathyanarayanan, L. and Y. Ortega (2004). "Effects of pesticides on sporulation of Cyclospora cayetanensis and viability of Cryptosporidium parvum." J Food Prot 67(5): 1044-9.
	Disease outbreaks caused by the coccidian parasite Cyclospora cayetanensis in food have been linked to consumption of raspberries that may have been contaminated through exposure to water mixed with insecticides and fungicides that may have been sprayed onto the berries. Three different fungicides (captan 50% W.P., benomyl 50% W.P., and zineb 75% W.P.) and two different insecticides (malathion 25% W.P. and diazinon 4E 47.5%) were evaluated at five different concentrations and for exposure times of 30 min to 1 week. Sporulation of C. cayetanensis did not decrease with use of any of the pesticides from time periods of 30 min to 24 h at all concentrations. Sporulation percentage was reduced with the fungicide benomyl at 1 week of exposure. The growth of the parasite Cryptosporidium parvum was also evaluated using captan 50% W.P., benomyl 50% W.P., and diazinon 4E 47.5%. Oocyst infectivity was reduced only after 7 days of exposure. These results indicate that these pesticides used at recommended concentration levels do not affect the sporulation of Cyclospora.

Sathyanarayanan, L. and Y. Ortega (2006). "Effects of temperature and different food matrices on Cyclospora cayetanensis oocyst sporulation." J Parasitol 92(2): 218-22.
	Effects of temperature on the sporulation of the parasite Cyclospora cayetanensis were studied in 2 food substrates, dairy and basil. Unsporulated Cyclospora oocysts were subjected to freezing and heating conditions for time periods ranging from 15 min to 1 wk. Oocysts were then removed from the food substrates and placed in 2.5% potassium dichromate for 2 wk to allow viable unsporulated oocysts to differentiate and fully sporulate, and to determine the percentage sporulation as an indicator of viability. Sporulation occurred when oocysts resuspended in dairy substrates were stored within 24 hr at -15 C. When oocysts were placed in water or basil, sporulation occurred after incubation for up to 2 days at -20 C, and up to 4 days at 37 C. Few oocysts sporulated when incubated for 1 hr at 50 C. Sporulation was not observed in basil leaves or water at -70 C, 70 C, and 100 C. Sporulation was not affected when incubated at 4 C and 23 C for up to 1 wk, which was the duration of the experiment in both of the tested substrates.

Schets, F. M., G. B. Engels, et al. (2005). "Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples." Appl Environ Microbiol 71(11): 6793-8.
	In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.

Schrum, D. P., S. Alugupalli, et al. (1997). "Structural characterization of a "signature" phosphatidylethanolamine as the major 10-hydroxy stearic acid-containing lipid of Cryptosporidium parvum oocysts." Lipids 32(7): 789-93.
	A 10-hydroxy stearic acid-containing lipid from Cryptosporidium parvum was purified by thin-layer chromatography and analyzed by infrared spectroscopy, fast-atom bombardment mass spectrometry, 1H and 31P nuclear magnetic resonance spectroscopy, and was identified as phosphatidyl-ethanolamine.

Schulzig, H. S. and K. Fehlhaber (2005). "[Longitudinal study on the seroprevalence of Toxoplasma gondii infection in four German pig breeding and raising farms]." Berl Munch Tierarztl Wochenschr 118(9-10): 399-403.
	There are very few current data on the prevalence of Toxoplasma (T.) gondii in German pig farms. Consequently a reliable risk assessment of human Toxoplasmosis caused by ingesting raw or improperly cooked pork and pork products is not available. The aim of this study was to show current data on T. gondii prevalence in German pig farms. In four pig farms with different management systems (three conventional, one organic) 100 animals each were selected and tested for T. gondii antibodies. The test was done four times during the period from birth to slaughtering. In one farm 20 mother sows were tested additionally. The slaughtered pigs from conventional farms showed seroprevalences between 0 and 15.2% (mean value 5.6%). At the organic system T. gondii antibodies were not detected. All slaughtered seropositive pigs (6 months old) were tested negatively at the age of 9 weeks, but shortly after birth high titres of T. gondii antibodies had been detected in the same animals. Comparing the results gained in different seasons significantly more pigs were found to be infected during the autumn/winter than in the spring/summer period. In order to assess the current risk of Toxoplasmosis more pig farms should be tested. From the point of view of consumer protection the detection of highly infected pig herds is necessary.

Searcy, K. E., A. I. Packman, et al. (2005). "Association of Cryptosporidium parvum with suspended particles: impact on oocyst sedimentation." Appl Environ Microbiol 71(2): 1072-8.
	The association of Cryptosporidium parvum oocysts with suspended particles can alter the oocysts' effective physical properties and influence their transport in aquatic systems. To assess this behavior, C. parvum oocysts were mixed with various suspended sediments under a variety of water chemical conditions, and the resulting settling of the oocysts was observed. Direct microscopic observations showed that oocysts attached to suspended sediments. Settling column and batch experiments demonstrated that oocysts are removed from suspension at a much higher rate when associated with sediments. The rate of oocyst sedimentation depended primarily on the type of sediment with which the oocysts were mixed. Changes in background water conditions had a relatively small impact on the extent of oocyst-particle association and the resulting oocyst deposition. We believe that the ubiquitous association of C. parvum oocysts with suspended particles enhances the sedimentation of oocysts in natural waters and that this interaction should generally be considered when predicting the migration of pathogens in the environment.

Shepherd, K. M. and A. P. Wyn-Jones (1996). "An evaluation of methods for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water." Appl Environ Microbiol 62(4): 1317-22.
	Methods for the simultaneous detection of Cryptosporidium parvum oocysts and Giardia cysts from water are described and their relative recovery efficiencies are assessed for seeded samples of both tap and river water. Cartridge filtration, membrane filtration, and calcium carbonate flocculation were evaluated, and steps to optimize the concentration procedures were undertaken. Increasing centrifugation to 5,000 x g, coupled with staining in suspension, was found to increase the overall efficiency of recovery of both cysts and oocysts. Cartridge filtration for both cysts and oocysts was examined by use of 100-liter volumes of both tap and river water. Improvements in recovery were observed for Cryptosporidium oocysts after extra washes of the filters. Calcium carbonate flocculation gave the maximum recovery for both Cryptosporidium oocysts and Giardia cysts and for both water types. A variety of 142-mm membranes was examined by use of 10-liter seeded samples of tap and river water. Cellulose acetate with a 1.2-micron pore size provided the best results for Cryptosporidium oocysts, and cellulose nitrate with a 3.0-micron pore size did so for Giardia cysts.

Shield, J., J. H. Baumer, et al. (1990). "Cryptosporidiosis--an educational experience." J Infect 21(3): 297-301.
	Eleven children aged 7 to 8 years from one school class developed diarrhoea and vomiting after an educational visit to a dairy farm. Three required hospital admission and intravenous fluid replacement for dehydration. Cryptosporidium oocytes were found in the faeces of these three children and from one classmate when the remainder of the class was tested between 16 and 21 days after the visit. At the farm some children tasted pelleted cow feed, silage and dried milk powder. A case-control study showed a significant correlation between diarrhoea and the tasting of silage and pelleted feed. Guidance on the safe conduct of educational visits to farms is given.

Shin, G. A., K. G. Linden, et al. (2001). "Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts." Appl Environ Microbiol 67(7): 3029-32.
	Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25 degrees C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm(2) (=30 J/m(2)), the reduction reached the cell culture assay detection limit of approximately 3 log(10). At UV doses of 1.2 and 3 mJ/cm(2), the log(10) reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.

Simmons, O. D., 3rd, M. D. Sobsey, et al. (2001). "Concentration and detection of cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration." Appl Environ Microbiol 67(3): 1123-7.
	The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4',6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.

Sinclair, J. L. (2000). "Enumeration of Cryptosporidium spp. in water with US EPA method 1622, USA." J AOAC Int 83(5): 1108-14.
	The occurrence of Cryptosporidium parvum or other pathogenic Cryptosporidium species in water must be known in order to assess risk and determine the treatment needed to reduce Cryptosporidium oocysts to acceptable levels in finished drinking water. Because Cryptosporidium oocyst occurrence may be sparse, methods must concentrate a large volume of water and correctly identify oocysts in the concentrate. The U.S. Environmental Protection Agency Information Collection Rule (ICR) protozoan method gives low and variable recoveries of Cryptosporidium oocysts, making risk assessment difficult. Therefore, a method giving better oocyst recovery and more consistent results was needed. Method 1622 was developed with existing materials and procedures, and improvements were made in filtration, cleanup, and detection. Absolute porosity filters were used, with cleanup by immunomagnetic separation and detection by direct fluorescent antibody stain with 4',6-diamidino-2-phenylindole (DAPI) staining for additional cell structures. Both the level and consistency of oocyst recovery were improved compared to recovery with the ICR method.

Singh, A., I. Bairy, et al. (2003). "Spectrum of opportunistic infections in AIDS cases." Indian J Med Sci 57(1): 16-21.
	Human Immunodeficiency viruses are the initial causative agents in AIDS, but most of the morbidity and mortality in AIDS cases result from opportunistic infections, Identification of such pathogen is very important for clinicians and health planners to tackle the AIDS epidemic in more effective manner. The present study describes the clinical and laboratory profile of 100 AIDS causes who presented to a referral hospital. Oral candidiasis (59.00%) was found to be the most common opportunistic infection, followed by tuberculosis (56.00%), Cryptosporidium infection (47.00%) and Pneumocystis carinii (7.00%). Presence of oral candidiasis and weight loss is highly predictive of low DC4 count and can be considered as a marker of HIV disease progression. The patients coinfected with HIV and tuberculosis are also on rise. Recognition of dual infection and taking adequate steps to deal with this epidemic is needed. As Cryptosporidium infection was detected in large number, provision of safe drinking water and maintaining good hygiene is important for prevention. Early diagnosis of opportunistic infection and prompt treatment, delays the progression towards AIDS. 91.00% of patients were infected with HIV1 and 4.00% had HIV2 infection and 5.00% were dully infected. 87.00% of patients were males and 13.00% were belonging to 21-40 years of age. Majority of them were belonging to lower socioeconomic status and heterosexual route of transmission was the commonest mode of spread.

Sinski, E., M. Bednarska, et al. (1992). "Biological characterization of Cryptosporidium parvum isolates in suckling and immunosuppressed mice." Acta Parasitologica 37(3): 139-143.
	Suckling mice and immunosuppressed adult mice were used as model hosts to compare the endogenous development of two isolates of C. parvum obtained from naturally infected calves in different seasons of the year. There were no differences in these two isolates as to their location, time of appearance, intensity of infection and antigenicity. Present study indicates that Cryptosporidium infection may be easily established in 5-7 day old outbred Swiss mice. The peak of infection is shown on day 8 after inoculation and shortly after that a self-limiting process of infection takes place. However, C. parvum does not develop endogenously in adult immunosuppressed mice. The lack of establishment of infection in these animals, even in latent state, indicates that age dependent mechanisms of immunity, developed in mice, are not impaired by prednisolone or cyclophosphamide. There was no difference in biological characteristics of two isolates C. parvum. Moreover, the present study illustrates that oocysts, spread by naturally infected calves in autumn and winter, are infective for suckling mice, which might be associated in transmissibility of C. parvum.

Sinski, E. and M. Czarnogrecka (1989). "Cryptosporidium-sp infection in snakes." Acta Parasitologica Polonica 34(4): 307-310.
	This is the first record of Cryptosporidium sp. infection in snakes of genus Elaphe (E. obsoleta, E. quatuorlineata, E. shrencki, E. guttata) in Poland. Fecal smears from snakes contained spherical organisms, confirmed by specific method of Ziehl-Neelson to be oocyst of Cryptosporidium. The examined snakes had severe chronic gastritis.

Sinski, E., E. Hlebowicz, et al. (1993). "Occurrence of Cryptosporidium parvum infection in wild small mammals in District of Mazury Lake Poland." Acta Parasitologica 38(2): 59-61.
	This study was conducted in population of wild small mammals in northern Poland, District of Mazury Lake, Urwitalt, near Mikolajki. Samples were collected four times, from autumn 1989 until spring 1991. Twenty percent (66 of 330) of the examined mammals were positive for Cryptosporidium, i.e., 55 of 275 Clethrionomys glareolus, 6 of 39 Apodemus flavicollis, 5 of 16 Sorex araneus were naturally infected with Cryptosporidium. There was no infection of Cryptosporidium assessed in A. agrarius and S. minutus. Our histological data clearly indicate that the population of C. glareolus was infected with C. parvum. This population of rodents appears to have maintained an infection throughout two years. These findings indicate that C. glareolus and possibly other rodents have potential to act as a reservoir for C. parvum, a pathogen of man and ruminants.

Sivapalasingam, S., C. R. Friedman, et al. (2004). "Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997." J Food Prot 67(10): 2342-53.
	Fresh produce is an important part of a healthy diet. During the last three decades, the number of outbreaks caused by foodborne pathogens associated with fresh produce consumption reported to the Centers for Disease Control and Prevention has increased. To identify trends, we analyzed data for 1973 through 1997 from the Foodborne Outbreak Surveillance System. We defined a produce-associated outbreak as the occurrence of two or more cases of the same illness in which epidemiologic investigation implicated the same uncooked fruit, vegetable, salad, or juice. A total of 190 produce-associated outbreaks were reported, associated with 16,058 illnesses, 598 hospitalizations, and eight deaths. Produce-associated outbreaks accounted for an increasing proportion of all reported foodborne outbreaks with a known food item, rising from 0.7% in the 1970s to 6% in the 1990s. Among produce-associated outbreaks, the food items most frequently implicated included salad, lettuce, juice, melon, sprouts, and berries. Among 103 (54%) produce-associated outbreaks with a known pathogen, 62 (60%) were caused by bacterial pathogens, of which 30 (48%) were caused by Salmonella. During the study period, Cyclospora and Escherichia coli O157:H7 were newly recognized as causes of foodborne illness. Foodborne outbreaks associated with fresh produce in the United States have increased in absolute numbers and as a proportion of all reported foodborne outbreaks. Fruit and vegetables are major components of a healthy diet, but eating fresh uncooked produce is not risk free. Further efforts are needed to better understand the complex interactions between microbes and produce and the mechanisms by which contamination occurs from farm to table.

Slifko, T. R., D. Friedman, et al. (1997). "An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture." Appl Environ Microbiol 63(9): 3669-75.
	Current assay methods to detect Cryptosporidium oocysts in water are generally not able to evaluate viability or infectivity. A method was developed for low-level detection of infective oocysts by using HCT-8 cells in culture as hosts to C. parvum reproductive stages. The infective foci were detected by labeling intracellular developmental stages of the parasite in an indirect-antibody assay with a primary antibody specific for reproductive stages and a secondary fluorescein isothiocyanate-conjugated antibody. The complete assay was named the focus detection method (FDM). The infectious foci (indicating that at least one of the four sporozoites released from a viable oocyst had infected a cell) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series experiments demonstrated that the autoreinfective life cycle in host HCT-8 cells began after 12 h of incubation. Through dilution studies, levels as low as one infectious oocyst were detected. The cell culture FDM compared well to other viability assays. Vital stains and excystation demonstrated that oocyst populations less than 1% viable (by vital dyes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an oocyst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cell culture method described demonstrate two important applications: (i) an infectivity assay that may be used in conjunction with current U.S. Environmental Protection Agency-mandated detection methodologies, and (ii) a method to evaluate oocyst infectivity in survival and disinfection studies.

Slifko, T. R., D. E. Friedman, et al. (1997). "Unique cultural methods used to detect viable Cryptosporidium parvum oocysts in environmental samples." HEALTH-RELATED WATER MICROBIOLOGY 1996: 363-368.
	Cryptosporidium parvum is an infectious enteric protozoan parasite that causes waterborne disease, severe gastroenteritis and is associated with high mortality in immunocompromised individuals. Detection of oocysts in water is very difficult and current methodologies do not determine viability. This project has focused on low level detection of Cryptosporidium parvum in environmental samples using a unique cultural method. Previously, cell culture methods have been used the developmental stages of Cryptosporidium; however, no cultural methods have been employed with environmental samples. The percentage of viable oocysts can be estimated by detecting intracellular developmental stages of the parasite using fluorescently labelled antibodies. Other methods are not capable of low level detection or high sensitivity. We are evaluating detection of single foci of infection, indicating that one of the four sporozoites released from the viable oocysts has infected a single cell.

Slifko, T. R., E. Raghubeer, et al. (2000). "Effect of high hydrostatic pressure on Cryptosporidium parvum infectivity." J Food Prot 63(9): 1262-7.
	The incidence of foodborne disease outbreaks caused by contaminated low-pH fruit juices is increasing. With recent mandatory pasteurization of apple juice and the industry's concerns of food safety, fruit juice processors are showing more interest in alternative nonthermal technologies that can kill >99.99% of microbial pathogens present in foods. The association of the coccidian protozoan, Cryptosporidium, with diarrheal disease outbreaks from contaminated tap water and fruit juice raises a safety concern in the food and beverage industries. The objective of this study was to evaluate the effects of high hydrostatic pressure (HHP) on C. parvum oocysts. Oocysts were suspended in apple and orange juice and HHP treated at 5.5 x 10(8) Pa (80,000 psi) for 0, 30, 45, 60, 90, and 120 s. Oocyst viability was assessed by excystation using bile salts and trypsin while the cell culture foci detection method was used to assess infectivity. Results indicated that HHP inactivated C. parvum oocysts by at least 3.4 log10 after 30 s of treatment. No infectivity was detected in samples exposed to > or =60 s of HHP and >99.995% inactivation was observed. This study demonstrated that HHP efficiently rendered the oocysts nonviable and noninfectious after treatment at 5.5 x 10(8) Pa.

Slifko, T. R., H. V. Smith, et al. (2000). "Emerging parasite zoonoses associated with water and food." Int J Parasitol 30(12-13): 1379-93.
	The environmental route of transmission is important for many protozoan and helminth parasites, with water, soil and food being particularly significant. Both the potential for producing large numbers of transmissive stages and their environmental robustness, being able to survive in moist microclimates for prolonged periods of time, pose a persistent threat to public and veterinary health. The increased demands on natural resources increase the likelihood of encountering environments and produce contaminated with parasites. For waterborne diseases, the protozoa, Cryptosporidium, Giardia and Toxoplasma, are the most significant causes, yet, with the exception of Toxoplasma, the contribution of zoonotic transmission remains unclear due to the absence of 'standardised' methods. The microsporidia have been documented in one waterborne outbreak, but the role of animals as the cause of contamination was not elucidated. In foods, surface contamination is associated with the faecal-oral pathogens, and some data are available to indicate that animal wastes remain an important source of contamination (e.g. cattle faeces and apple cider outbreaks), however, further work should focus on examining the source of contamination on fruit and vegetables. Increasing recognition of the burden of human fascioliasis has occurred; it is now recognised as an emerging zoonosis by the WHO. Toxoplasma, Trichinella and Taenia spp. remain important meatborne parasites, however, others, including Pleistophora-like microsporidians may be acquired from raw or lightly cooked fish or crustaceans. With increased international travel, the public health importance of the foodborne trematodiases must also be realised. Global sourcing of food, coupled with changing consumer vogues, including the consumption of raw vegetables and undercooking to retain the natural taste and preserve heat-labile nutrients, can increase the risk of foodborne transmission. A greater awareness of parasite contamination of our environment and its impact on health has precipitated the development of better detection methods. Robust, efficient detection, viability and typing methods are required to assess risks and to further epidemiological understanding.

Smerdon, W. J., T. Nichols, et al. (2003). "Foot and mouth disease in livestock and reduced cryptosporidiosis in humans, England and Wales." Emerg Infect Dis 9(1): 22-8.
	During the 2001 epidemic of foot and mouth disease (FMD) in livestock in England and Wales, we discovered a corresponding decrease in laboratory reports of cryptosporidiosis in humans. Using a regression model of laboratory reports of cryptosporidiosis, we found an estimated 35% (95% confidence interval [CI] 20% to 47%) reduction in reports during the weeks spanning the period from the first and last cases of FMD. The largest reduction occurred in northwest England, where the estimated decrease was 63% (95% CI 31% to 80%). Genotyping a subgroup of human isolates suggested that the proportion of Cryptosporidium genotype 2 strain (animal and human) was lower during the weeks of the FMD epidemic in 2001 compared with the same weeks in 2000. Our observations are consistent with livestock making a substantial contribution to Cryptosporidium infection in humans in England and Wales; our findings have implications for agriculture, visitors to rural areas, water companies, and regulators.

Smith, A. L. and H. V. Smith (1989). "A comparison of fluorescein diacetate and propidium iodide staining and in vitro excystation for determining Giardia intestinalis cyst viability." Parasitology 99 Pt 3: 329-31.
	The viability of 4 human isolates of Giardia intestinalis cysts using either the fluorogenic vital dyes fluorescein diacetate (FDA) and propidium iodide (PI) or in vitro excystation was assessed. Whereas viable cysts, as defined by in vitro excystation were present in each of the 4 isolates, cysts from only 3 of the 4 isolates took up the vital dyes. FDA consistently over-estimated cyst viability whilst PI under-estimated non-viable cysts when compared with in vitro excystation. Following in vitro excystation, both FDA and PI stained a proportion of unexcysted cysts indicating that FDA stained cysts which were incapable of excystation, whereas PI did not stain all cysts which were incapable of excystation. One human cyst isolate, which underwent in vitro excystation, could not be stained with either FDA or PI. In the absence of currently more specific fluorescent indicators of viability, PI alone could be used to determine the lower limit of nonviability in positive water-related samples, where small numbers of cysts are to be expected.

Smith, H. V. (1998). "Detection of parasites in the environment." Parasitology 117 Suppl: S113-41.
	The environmental route of transmission is important for many protozoan and helminth parasites, with water, soil and food being particularly significant. Both the potential for producing large numbers of transmissive stages and their environmental robustness (with the ability to survive in moist microclimates for prolonged periods of time) pose persistent threats to public and veterinary health. Increased demands made on natural resources increase the likelihood of encountering environments and produce contaminated with parasites. In the last 30 years, endemic and epidemic waterborne and foodborne outbreaks in developed countries have led to a reappraisal of conventional isolation and detection methods. While these methods have proved invaluable in our understanding of environmental transmission routes for helminths, they have been less effective for the parasitic protozoa. Robust, efficient detection, viability and typing methods are required to assess risk and to further epidemiological understanding. Greater awareness of parasite contamination of our environment and its impact on health has precipitated the development of better detection methods. Currently, nowhere is this more apparent than with Cryptosporidium, with a broad range of immunological, microscopical and molecular methods available. The upsurge in molecular techniques, particularly the polymerase chain reaction, for determining occurrence and viability have brought with them the added benefits of increased sensitivity and specificity, yet many methods still have to be shown to address these issues consistently in the field. Rapid commercialization of reagents and standardization of methods provide consistency. The advances identified in non-destructive and destructive methods for the protozoa have application for helminths and emerging pathogens and should determine the importance of the matrices involved in the environmental transmission of parasites, further safeguarding public and veterinary health.

Smith, H. V., J. Brown, et al. (1993). "Occurrence of oocysts of Cryptosporidium sp. in Larus spp. gulls." Epidemiol Infect 110(1): 135-43.
	Between November 1990 and February 1991 101 gull faecal samples, collected in central Scotland, and 50 cloacal lavages, from gulls captured at two refuse tips near Durham, England were examined for the presence of Cryptosporidium sp. oocysts. Five of 101 (c 5%) of faecal samples and 11 of 50 (22%) of cloacal lavages contained oocysts, of which 64% and 83%, respectively were considered viable when examined with propidium iodide and 4'-6-diamidino-2-phenylindole. Since there is insufficient evidence to ascribe these oocysts to a recognized species they are therefore referred to as Cryptosporidium sp. oocysts. There were significant differences in the occurrence of oocysts between gulls captured at the different refuse tips (P < or = 0.01), but no significant difference between the distribution of oocysts in two species of gull, Larus argentatus (Herring Gull) and L. ridibundus (Black-head Gull). The differences may be explained by different food sources and feeding habits. The contribution of gulls to environmental contamination with Cryptosporidium sp. oocysts is probably generally small, but may be more significant when large numbers roost on surface waters.

Smith, H. V., S. M. Caccio, et al. (2006). "Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans." Trends Parasitol 22(4): 160-7.
	Cryptosporidiosis and giardiasis are major public health concerns. The role of water and food in the epidemiology of these diseases is now well recognized. Molecular techniques are available to determine the species and genotypes of Cryptosporidium and Giardia and to distinguish human from non-human pathogens. Validated methods to determine the species, genotype and subgenotype that are present in heterologous mixtures should be applied to environmental samples to enable the monitoring and characterization of infection sources, disease tracking and the establishment of causative links to both waterborne and foodborne outbreaks. Meaningful interpretation of population structures and occurrence-prevalence baselines can be performed only by analysing a well-planned set of samples from all possible sources taken regularly over time, rather than focusing on outbreak investigations. For food, this includes such analyses in the country of origin.

Smith, H. V., B. M. Campbell, et al. (2002). "Significance of enhanced morphological detection of Cryptosporidium sp. oocysts in water concentrates determined by using 4',6'-diamidino-2-phenylindole and immunofluorescence microscopy." Appl Environ Microbiol 68(10): 5198-201.
	Of 2,361 water concentrates analyzed for the presence of Cryptosporidium spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-Cryptosporidium sp. monoclonal antibody and the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.

Smith, H. V., A. McDiarmid, et al. (1989). "An analysis of staining methods for the detection of Cryptosporidium spp. oocysts in water-related samples." Parasitology 99 Pt 3: 323-7.
	The ability of five staining techniques, originally developed for the rapid identification of Cryptosporidium spp. oocysts in faecal samples, to detect oocysts in water and water-related samples was assessed. All the stains used (modified Ziehl Neelsen, auramine-phenol (Lempert), Wright-Giemsa, safranin-methylene blue and FITC-labelled monoclonal antibody) stained oocysts after storage in water for 2 months at 4 degrees C (71-89% of control values). Storage of oocysts below 0 degrees C greatly reduced the staining ability of auramine-phenol. With the exception of oocysts stored in raw and final waters, the histochemical stains proved less useful in detecting oocysts than the monoclonal antibody. Organisms of similar size and shape took up these stains, causing confusion in interpretation. Cold Ziehl Neelsen and the FITC-labelled monoclonal antibody were best at identifying oocysts from a waterborne outbreak. Screening with a fluorescent antibody, followed by confirmation with cold Ziehl Neelsen, where possible, are the currently recommended procedures for the detection of oocysts in water-related samples.

Smith, H. V., C. A. Paton, et al. (1997). "Sporulation of Cyclospora sp. oocysts." Appl Environ Microbiol 63(4): 1631-2.
	Cyclospora sp. oocysts sporulated maximally at 22 and 30 degrees C for 14 days retarded sporulation. Up to 12% of human- and baboon-derived oocysts previously stored at 4 degrees C for 1 to 2 months sporulated when stored for 6 to 7 days at 30 degrees C.

Smith, H. V., W. J. Patterson, et al. (1989). "An outbreak of waterborne cryptosporidiosis caused by post-treatment contamination." Epidemiol Infect 103(3): 703-15.
	An outbreak of waterborne cryptosporidiosis affecting 27 persons, diagnosed stool positive, occurred in Ayrshire in April 1988. Twenty-one in 27 confirmed cases required some form of fluid replacement therapy. Local general practitioners indicated a two- to fivefold increase in diarrhoeal disease during the outbreak, and following enquiries made by Environmental Health Officers it became apparent that many hundreds of people had suffered a diarrhoeal illness at that time. Cryptosporidium spp. oocysts were detected in the treated chlorinated water supply system, in the absence of faecal bacterial indicators. Oocyst contamination of a break-pressure tank containing final water for distribution was the cause of this waterborne outbreak. An irregular seepage of oocyst-containing water, which increased during heavy rains, was the cause of the break-pressure tank contamination, rather than a failure of the water-treatment processes. The waterborne route should be considered when clusters of cryptosporidiosis-associated with potable water occur. Waterborne cryptosporidiosis can occur in the absence of other faecal indicators of contamination.

Smith, H. V. and J. B. Rose (1990). "Waterborne cryptosporidiosis." Parasitol Today 6(1): 8-12.
	Awareness of the importance of Cryptosporidium as a gastrointestinal parasite of developed countries not only stems from its prevalence in AIDS patients but also from its recent recognition as a possible contaminant of drinking water supplies. The importance of Cryptosporidium to public health has recently been revealed by a series of major epidemics of diarrhoeal disease in the USA and UK. In this review, Huw Smith and Joan Rose document what is known of the causes of some of these outbreaks and explain why this parasite can escape the battery of treatment processes normally used for drinking water supplies in these countries.

Smith, L. M., M. T. Bonafonte, et al. (2001). "Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice." Exp Parasitol 98(3): 123-33.
	Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines. To determine whether the addition of either a Th1 or Th2 cytokine could alter the outcome of infection, we treated GKO mice with either recombinant (r)IL-4 or rIL-12 1 day before infection (DBI) or daily. No effect on the oocyst shedding patterns in either strain nor an increase in survival of the C57BL/6-GKO mice was observed in the rIL-4-treated mice. Whereas one dose of 0.5 microg rIL-12 given 1 DBI had no effect on oocyst shedding, we found that daily doses of rIL-12 administered intraperitoneally exacerbated C. parvum infection in both animal models. Administration of rIL-12 shortened the survival time in the C57BL/6-GKO mice and prevented BALB/c-GKO mice from recovering from infection. Specific proliferation of T cells to cryptosporidial antigen and Th1 and Th2 mRNA cytokine expression was markedly decreased in rIL-12-treated mice. Nitric oxide (NO) may have played a minor role in the decreased proliferation observed since levels of NO present in the splenocyte cultures from rIL-12-treated mice in response to parasite antigen stimulation were higher than those observed in controls. Thus, we propose that resistance to and recovery from C. parvum infections involves a fine balance in the amount and timing of Th1 and Th2 cytokines.

Soave, R., B. L. Herwaldt, et al. (1998). "Cyclospora." Infect Dis Clin North Am 12(1): 1-12.
	Although Cyclospora infection has been documented in humans worldwide since at least 1977, it is only in the past 2 years that this organism has come into prominence as a result of major foodborne outbreaks in the United States and Canada. Cyclospora causes significant gastrointestinal disease in immunocompetent and immunocompromised hosts and can be successfully treated with trimethoprim-sulfamethoxazole. The infection is under-recognized because our methods for diagnosis are rudimentary and insensitive. The mechanisms by which the parasite causes disease, the range of animal hosts, and the natural reservoir are unknown. Cyclospora is a unique coccidian parasite that has just begun to emerge; as yet, we have no clue as to where it comes from or where it hides.

Somiya, I., S. Fujii, et al. (2000). "Development of a mathematical model of Cryptosporidium inactivation by ozonation." Water Science & Technology 41(7): 173-180.
	A new mathematical model was developed to express the processes of Cryptosporidium inactivation by ozonation. In this model, five different stages were considered as state variables of Cryptosporidium oocysts for accurate expression of the inactivation. ATP, in vitro excystation and DAPI/PI permeability assays were used to describe the oocyst amounts of different stages. Some reaction constants were estimated by the structure components of oocysts or by the stoichiometry of reactions, while the others were by the oocysts population changes in ozonation. The calculated values of this model were well consistent with experimental inactivation data. Three-log inactivation of sporozoites required about 0.04 mgO sub(3) per unit oocyst (mgC) from the simulation results. Before ozone reacts with sporozoites, more ozone was consumed to oxidize other parts of oocysts and DOC produced. The main path of inactivation of oocysts by ozonation was estimated to be P sub(1) (intact oocysts) arrow right P sub(2) (oocysts with damaged outer oocyst wall) arrow right P sub(4) (oocysts without excystation function) arrow right P sub(5) (oocysts with inactivated sporozoites and no excystation function) from experimental and simulated results.

Somiya, I., S. Fujii, et al. (2000). "Development of ATP assay as a surrogate indicator of viability of Cryptosporidium parvum oocysts." Water Science & Technology 41(7): 181-188.
	Adenosine Triphosphate (ATP) determination was applied to evaluate viability of Cryptosporidium oocysts. Three pretreatment methods, such as incubation in acidified Hanks balanced salt solution (HBSS), excystation and sonication were investigated for ATP extraction from oocysts. Incubation in acidified HBSS was insufficient to extract ATP from oocysts, but a linear relationship between the number of oocysts and the concentration of ATP extracted was observed in the test of excystation and sonication treatments. Sonication treatment was able to extract ATP from oocysts more rapidly and precisely than excystation treatment. ATP amount per oocyst by sonication treatment (ATPs) was evaluated to be 2.9x10 super(-8) mu g on average, and its detection limit was 500 oocysts/100 mu l. Ozone treatment experiments were conducted in batch condition to evaluate differences among ATP concentrations extracted, in vitro excystation and DAPI/PI permeability assays. ATPs assay was observed to have a linear relationship with DAPI/PI permeability assay (R super(2) identical with 0.98). As a result, ATP assay is applicable as a surrogate indicator of the viability of C. parvum, and is superior to in vitro excystation and DAPI/PI permeability assay, because of its rapid, accurate and simple procedure.

Sorel, N., E. Guillot, et al. (2003). "Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples." J Appl Microbiol 94(2): 273-9.
	AIMS: Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. METHODS AND RESULTS: The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. CONCLUSIONS: Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. SIGNIFICANCE AND IMPACT OF THE STUDY: The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.

Spano, F., L. Putignani, et al. (1998). "Multilocus genotypic analysis of Cryptosporidium parvum isolates from different hosts and geographical origins." J Clin Microbiol 36(11): 3255-9.
	The genetic analysis of oocysts recovered from the stools of humans and animals infected with Cryptosporidium parvum has consistently shown the existence of two distinct genotypes. One of the genotypes is found exclusively in some human infections, whereas the other genotype is found in human as well as in animal infections. On the basis of these observations and the results of published epidemiological studies with single polymorphic markers, the existence of two separate transmission cycles has been postulated, one exclusively anthroponotic and the other involving both animals and humans. To test this hypothesis, C. parvum isolates of different geographic and host origins were analyzed by using unlinked genetic polymorphisms. A total of 28 isolates originating from Europe, North and South America, and Australia were examined. Isolates clustered into two groups, one comprising both human and animal isolates and the other comprising isolates only of human origin. The absence of recombinant genotypes is consistent with two reproductively isolated populations within the species C. parvum.

Speer, C. A., S. Clark, et al. (1998). "Ultrastructure of the oocysts, sporocysts, and sporozoites of Toxoplasma gondii." J Parasitol 84(3): 505-12.
	Transmission and scanning electron microscopy were used to study the ultrastructure of the oocysts, sporocysts, and sporozoites of the VEG strain of Toxoplasma gondii and to compare the ultrastructure of sporozoites with tachyzoites (from the peritoneum of mice) and bradyzoites (from brain tissue cysts in mice). Oocysts were surrounded by a thin veil of finely reticulate material. The oocyst wall consisted of 3 layers and contained a previously unknown disc-shaped micropyle that appeared as a depression in the oocyst wall. The sporocyst contained 4 sporozoites and a residuum of lipid and amylopectin granules. The sporocyst wall was 3-layered with the innermost layer consisting of 4 curved plates held together at sutures by an interposed strip. Exposure to excysting fluid caused the interposed strip to separate from the curved plates, which curled inward releasing the sporozoites. Sporozoites had a posteriorly located nucleus and all the organelles typical for coccidian zoites. Sporozoites, tachyzoites, and bradyzoites had similar numbers of rhoptries but differed in the numbers and sizes of micronemes, dense granules, amylopectin granules, and lipid bodies.

Speer, C. A., J. P. Dubey, et al. (1999). "Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii." Int J Parasitol 29(10): 1509-19.
	The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural differences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5-4 microm, than those of T. gondii which were smooth and 0.5 microm thick.

Srivastava, S., S. Bhattacharya, et al. (2005). "Species- and strain-specific probes derived from repetitive DNA for distinguishing Entamoeba histolytica and Entamoeba dispar." Exp Parasitol 110(3): 303-8.
	Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable species that are found in the human gut. Of the two, E. histolytica is considered to be pathogenic while E. dispar is nonpathogenic. To generate molecular probes to detect and distinguish between the two species, we utilized repeat sequences present in Entamoeba genome. We have developed probes and primers from rDNA episomes, and unidentified Entamoeba EST1 repeat for this purpose, and used them for dot blot hybridization and PCR amplification. To investigate the possible existence of invasive and noninvasive strains of E. histolytica, the ability to differentiate individual isolates is necessary. For this purpose, we have utilized a modification of the AFLP procedure called 'Transposon display,' which generates and displays large number of genomic bands associated with a transposon. We have used the abundant retrotransposon, EhSINE1, for this purpose,and demonstrated its potential as a marker to study strain variation in E. histolytica. This technique could suitably be employed in carrying out significant molecular epidemiological studies and large-scale typing of this parasite.

Stanfield, G., E. Carrington, et al. (2000). An optimised and standardised test to determine the presence of the protozoa Cryptosporidium and Giardia in water. International Conference on Minimising Risk from Cryptosporidium and Other Waterborne Particles, Paris (France), 19-23 Apr 1999.
	With funding from the European Commission, a consortium of members of the European Water Research Institutes is carrying out a programme of work with the objective of optimising and standardising a method for determining the presence in water of (oo)cysts of Cryptosporidium and Giardia. Each of the stages of the conventional analysis procedure (initial concentration, recovery and identification and enumeration) are being investigated and the relative merits of existing and new methods are being assessed. Newly developed filters (Envirochek and Filta-Max) have been shown to be more efficient for initial recovery of (oo)cysts from water than the previously used Cuno cartridge filters. In addition, for the analysis of raw waters, flocculation with ferric sulphate has been shown to give recoveries similar to the Envirochek and Filta Max. Modern purification systems such as immunomagnetic separation have also been assessed and found to offer some advantages over flotation although optimisation of the latter has brought improved efficiency. Preliminary assessment of solid phase cytometry has indicated that this technique could offer significant time savings compared to conventional microscopic counting. The results of the study will be used to propose a revised standard method to CEN.

Stanfield, G., E. Carrington, et al. (2000). "Optimized and standardized test to determine the presence of the protozoa Cryptosporidium and Giardia in water." Water Science & Technology 41(7): 103-110.
	With funding from the European Commission, a consortium of members of the European Water Research Institutes is carrying out a programme of work with the objective of optimizing and standardizing a method for determining the presence in water of (oo)cysts of Cryptosporidium and Giardia. Each of the stages of the conventional analysis procedure (initial concentration, recovery and identification and enumeration) are being investigated and the relative merits of existing and new methods are being assessed. Newly developed filters (Envirochek and Filta-Max) have been shown to be more efficient for initial recovery of (oo)cysts from water than the previously used Cuno cartridge filters. In addition, for the analysis of raw waters, flocculation with ferric sulphate has been shown to give recoveries similar to the Envirochek and Filta Max. Modern purification systems such as immunomagnetic separation have also been assessed and found to offer some advantages over flotation although optimization of the latter has brought improved efficiency. Preliminary assessment of solid phase cytometry has indicated that this technique could offer significant time savings compared to conventional microscopic counting. The results of the study will be used to propose a revised standard method to CEN.

Starkey, S. R., S. E. Wade, et al. (2005). "Incidence of Cryptosporidium parvum in the dairy cattle population in a New York City Watershed." Vet Parasitol 131(3-4): 197-205.
	A longitudinal study of 2-year duration was conducted to determine the risk, as measured by incidence rate, of Cryptosporidium parvum infection among dairy cattle in the Catskill/Delaware Watershed of New York City (NYC), and the factors that predispose animals to the likelihood of infection. A proportional sampling scheme with follow up at quarterly farm visits was employed for heifers and cows. Additionally, all calves born on the 39 study farms were sampled once during the first four weeks of life and at least once more before weaning. Samples were analyzed for the presence of C. parvum using a quantitative centrifugation concentration flotation technique and a C. parvum-specific enzyme-linked immunosorbent assay (ELISA). Of the 9914 fecal samples collected, 747 were found to contain C. parvum. The average number of oocysts detected was 1.3x10(5)/g (range: 1.0/g--8.2x10(6)/g). The average age at time of first detection of the organism was 15.0 days with a standard deviation of 6.59 days. The age range of animals infected with C. parvum in the study population was 3--60 days (inclusive). The unadjusted (crude) incidence rate of C. parvum among the entire study population was 2.05 per 1000 animal-days. The unadjusted incidence rate among pre-weaned calves was 15.55 per 1000 animal-days. After controlling for age and prior protozoal risk level, no seasonal impact on the incidence of C. parvum was detected among animals less than 61 days by negative binomial regression. A seasonal impact was identified among the oocyst counts of infected animals after controlling for age and prior protozoal risk level.

Sterling, C. R. and Y. R. Ortega (1999). "Cyclospora: an enigma worth unraveling." Emerg Infect Dis 5(1): 48-53.
	In part, Cyclospora cayetanensis owes its recognition as an emerging pathogen to the increased use of staining methods for detecting enteric parasites such as Cryptosporidium. First reported in patients in New Guinea in 1977 but thought to be a coccidian parasite of the genus Isospora, C. cayetanensis received little attention until it was again described in 1985 in New York and Peru. In the early 1990s, human infection associated with waterborne transmission of C. cayetanensis was suspected; foodborne transmission was likewise suggested in early studies. The parasite was associated with several disease outbreaks in the United States during 1996 and 1997. This article reviews current knowledge about C. cayetanensis (including its association with waterborne and foodborne transmission), unresolved issues, and research needs.

Stinear, T., A. Matusan, et al. (1996). "Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR." Appl Environ Microbiol 62(9): 3385-90.
	Current methods for detection of Cryptosporidium parvum oocysts in water are time-consuming and difficult. We have developed a reverse transcription (RT)-PCR which can detect the presence of a single viable oocyst spiked into concentrated environmental water samples. The test is based on the detection of mRNA from a C. parvum heat shock protein (hsp). The synthesis of hsp was induced by a short 45 degrees C incubation followed by oocyst lysis by a freeze-thaw process. Hsp70 mRNA, produced only from viable oocysts, was then isolated by hybridization to oligo(dT)25-coated magnetic beads. Detection was achieved by RT-PCR amplification of a 590-bp region of hsp70 mRNA specific for C. parvum. To test the method, samples of reticulated, reservoir, bore, and river water were concentrated by chemical flocculation and Percoll-sucrose gradient centrifugation and then spiked with dilutions of oocysts. In all four of the water types examined, the detection of single oocysts was possible by RT-PCR combined with Southern hybridization. RT-PCR products were not obtained from formalin-inactivated oocysts. An RNA internal positive control fragment was synthesized that was included with each reaction to guard against RT-PCR false-negative results that may be caused by the presence of inhibitory substances. However, when the magnetic beads were used to extract and concentrate mRNA, no inhibition was observed. The technique is versatile, straightforward, and rapid (1 day) and provides a sensitive and economic means of screening concentrated water samples for the presence of C. parvum.

Stotish, R. L. and C. C. Wang (1975). "Preparation and purification of merozoites of Eimeria tenella." J Parasitol 61(4): 700-3.
	Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.

Stott, R., E. May, et al. (2001). Protozoan predation as a mechanism for the removal of Cryptosporidium oocysts from wastewaters in constructed wetlands. 7. International Conference on Wetland Systems for Water Pollution Control 2000, Lake Buena Vista, FL [USA], 11-16 Nov 2000.
	The removal of the protozoan parasite, Cryptosporidium parvum, from wastewaters is becoming of increasing importance in the UK, especially since contamination of raw waters by sewage effluents has been implicated in major waterborne outbreaks of cryptosporidiosis in recent years. Compared to conventional wastewater-treatment processes, constructed wetlands have demonstrated favourable removal rates for Cryptosporidium oocysts. The removal mechanisms however, remain unknown. Predation by free-living ciliated protozoa, which are commonly found in constructed wetlands, was investigated as a possible mechanism for oocyst removal. In laboratory feeding experiments, ciliates (Euplotes patella, Stylonychia mytilus, Paramecium caudatum and an unidentified wetland ciliate species), were exposed to doses ranging from 10 to 10 super(6) oocysts/ml for between 5 and 60 minutes. Ciliate predatory activities were assessed by enumerating fluorescently labelled ingested oocysts using epifluorescence microscopy. Oocysts were found to be ingested by all species investigated. Paramecium demonstrated the highest mean ingestion rates (up to 170 oocysts/hr) followed by Stylonychia (up to 60 oocysts/hour). Euplotes and the wetland ciliate had lower mean grazing rates (4 and 10 oocysts/hr respectively). These results indicate that protozoan predation may be an important factor in the removal of Cryptosporidium oocysts from wastewaters in constructed wetlands.

Stott, R., E. May, et al. (2003). Predation of Cryptosporidium oocysts by protozoa and rotifers: implications for water quality and public health. 11. International Symposium on Health-related Water Microbiology, Melbourne (Australia), 7-11 Apr 2002.
	Predation by free-living protozoa and rotifers was investigated as a possible mechanism for the removal of Cryptosporidium parvum oocysts in aquatic ecosystems including wastewater treatment plants. Free-living ciliated protozoa (Stylonychia mytilus, Paramecium caudatum and an unidentified wastewater wetland ciliate), an amoeba (Acanthamoeba culbertsoni) and rotifers, all commonly found in aquatic ecosystems, were exposed to varying doses of C. parvum oocysts. All organisms investigated ingested oocysts. Predation activity and rates of ingestion varied with predator species and prey density. Ciliated protozoa demonstrated greater predation activity than A. culbertsoni or rotifers when exposed to 2 x 10 super(5) oocyst/mL for up to 3 h. Greatest predation after 1 h exposure was observed in P. caudatum, the largest ciliate, with on average 1.9 oocysts/cell (range 0-9 oocysts/cell). Stylonychia mytilus and the wetland ciliate had a similar mean ingestion of around 0.3 oocysts/cell, with numbers internalised ranging from 0-3 oocysts/cell. Rotifers ingested on average 1.6 oocysts/individual (range 0-7 oocysts/individual) whilst amoebae ingested on average 1.8 oocysts/cell after 2 h exposure (up to 3 oocysts/cell). Grazing activity by P. caudatum was demonstrated at a variety of prey levels ranging from 9 to 9,000 oocysts. Numbers of oocysts internalised by Paramecium frequently exceeded the reported human infective dose of 30 oocysts. In general, numbers of internalised oocysts increased with incubation time of up to 20-30 min although the rate of accumulation was slower at lower dose levels. The significance of predation on the fate of Cryptosporidium oocysts in the environment is discussed.

Straub, T. M., D. S. Daly, et al. (2002). "Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray." Appl Environ Microbiol 68(4): 1817-26.
	We investigated the application of an oligonucleotide microarray to (i) specifically detect Cryptosporidium spp., (ii) differentiate between closely related C. parvum isolates and Cryptosporidium species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the hsp70 gene of Cryptosporidium parvum was constructed. Labeled hsp70 targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I C. parvum isolates (TU502 and UG502), two C. parvum genotype II isolates (Iowa and GCH1), and DNAs from 22 non-Cryptosporidium sp. organisms were used to test method specificity. Only DNAs from C. parvum isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.

Strohmeyer, R. A., P. S. Morley, et al. (2006). "Evaluation of bacterial and protozoal contamination of commercially available raw meat diets for dogs." J Am Vet Med Assoc 228(4): 537-42.
	OBJECTIVE: To evaluate bacterial and protozoal contamination of commercially available raw meat diets for dogs. DESIGN: Prospective longitudinal study. SAMPLE POPULATION: 240 samples from 20 raw meat diets for dogs (containing beef, lamb, chicken, or turkey), 24 samples from 2 dry dog foods, and 24 samples from 2 canned dog foods. PROCEDURE: Each product was purchased commercially on 4 dates approximately 2 months apart. Three samples from each product at each sampling period were evaluated via bacterial culture for non-type-specific Escherichia coli (NTSEC), Salmonella enterica, and Campylobacter spp. Antimicrobial susceptibility testing was performed on selected isolates. Polymerase chain reaction assays were used to detect DNA from Cryptosporidium spp, Neospora spp, and Toxoplasma spp in samples obtained in the third and fourth sampling periods. RESULTS: One hundred fifty-three of 288 (53%) samples were contaminated with NTSEC. Both raw and prepared foods contained NTSEC during at least 1 culture period. Salmonella enterica was recovered from 17 (5.9%) samples, all of which were raw meat products. Campylobacter spp was not isolated from any samples. In 91 of 288 (31.6%) samples, there was no gram-negative bacterial growth before enrichment and in 48 of 288 (16.7%) samples, there was no aerobic bacterial growth before enrichment. Susceptibility phenotypes were variable. Cryptosporidium spp DNA was detected in 3 samples. CONCLUSIONS AND CLINICAL RELEVANCE: Bacterial contamination is common in commercially available raw meat diets, suggesting that there is a risk of foodborne illness in dogs fed these diets as well possible risk for humans associated with the dogs or their environments.

Strong, W. B., J. Gut, et al. (2000). "Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products." Infect Immun 68(7): 4117-34.
	The apicomplexan parasite Cryptosporidium parvum is a major cause of serious diarrheal disease in both humans and animals. No efficacious chemo- or immunotherapies have been identified for cryptosporidiosis, but certain antibodies directed against zoite surface antigens and/or proteins shed by gliding zoites have been shown to neutralize infectivity in vitro and/or to passively protect against, or ameliorate, disease in vivo. We previously used monoclonal antibody 11A5 to identify a 15-kDa surface glycoprotein that was shed behind motile sporozoites and was recognized by several lectins that neutralized parasite infectivity for cultured epithelial cells. Here we report the cloning and sequence analysis of the gene encoding this 11A5 antigen. Surprisingly, the gene encoded a 330-amino-acid, mucin-like glycoprotein that was predicted to contain an N-terminal signal peptide, a homopolymeric tract of serine residues, 36 sites of O-linked glycosylation, and a hydrophobic C-terminal peptide specifying attachment of a glycosylphosphatidylinositol anchor. The single-copy gene lacked introns and was expressed during merogony to produce a 60-kDa precursor which was proteolytically cleaved to 15- and 45-kDa glycoprotein products that both localized to the surface of sporozoites and merozoites. The gp15/45/60 gene displayed a very high degree of sequence diversity among C. parvum isolates, and the numerous single-nucleotide and single-amino-acid polymorphisms defined five to six allelic classes, each characterized by additional intra-allelic sequence variation. The gp15/45/60 single-nucleotide polymorphisms will prove useful for haplotyping and fingerprinting isolates and for establishing meaningful relationships between C. parvum genotype and phenotype.

Strong, W. B. and R. G. Nelson (2000). "Gene discovery in Cryptosporidium parvum: expressed sequence tags and genome survey sequences." Contrib Microbiol 6: 92-115.
	
Strong, W. B. and R. G. Nelson (2000). "Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis." Mol Biochem Parasitol 107(1): 1-32.
	Cryptosporidium parvum is a protozoan enteropathogen that infects humans and animals and causes a pronounced diarrheal disease that can be life-threatening in immunocompromised hosts. No specific chemo- or immunotherapies exist to treat cryptosporidiosis and little molecular information is available to guide development of such therapies. To accelerate gene discovery and identify genes encoding potential drug and vaccine targets we constructed sporozoite cDNA and genomic DNA sequencing libraries from the Iowa isolate of C. parvum and determined approximately 2000 sequence tags by single-pass sequencing of random clones. Together, the 567 expressed sequence tags (ESTs) and 1507 genome survey sequences (GSSs) totaled one megabase (1 mb) of unique genomic sequence indicating that approximately 10% of the 10.4 mb C. parvum genome has been sequence tagged in this gene discovery expedition. The tags were used to search the public nucleic acid and protein databases via BLAST analyses, and 180 ESTs (32%) and 277 GSSs (18%) exhibited similarity with database sequences at smallest sum probabilities P(N)< or =10(-8). Some tags encoded proteins with clear therapeutic potential including S-adenosylhomocysteine hydrolase, histone deacetylase, polyketide/fatty-acid synthases, various cyclophilins, thrombospondin-related cysteine-rich protein and ATP-binding-cassette transporters. Several anonymous ESTs encoded proteins predicted to contain signal peptides or multiple transmembrane spanning segments suggesting they were destined for membrane-bound compartments, the cell surface or extracellular secretion. One-hundred four simple sequence repeats were identified within the nonredundant sequence tag collection with (TAA)(> or =6)/(TTA)(> or =6) and (TA)(> or = 10)/(AT)(> or =10 ) being the most prevalent, occurring 40 and 15 times, respectively. Various cellular RNAs and their genes were also identified including the small and large ribosomal RNAs, five tRNAs, the U2 small nuclear RNA, and the small and large virus-like, double-stranded RNAs. This investigation has demonstrated that survey sequencing is an efficient procedure for gene discovery and genome characterization and has identified and sequence tagged many C. parvum genes encoding potential therapeutic targets.

Sturbaum, G. D., P. T. Klonicki, et al. (2002). "Immunomagnetic separation (IMS)-fluorescent antibody detection and IMS-PCR detection of seeded Cryptosporidium parvum oocysts in natural waters and their limitations." Appl Environ Microbiol 68(6): 2991-6.
	Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.

Sturbaum, G. D., Y. R. Ortega, et al. (1998). "Detection of Cyclospora cayetanensis in wastewater." Appl Environ Microbiol 64(6): 2284-6.
	Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.

Sturbaum, G. D., C. Reed, et al. (2001). "Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts." Appl Environ Microbiol 67(6): 2665-8.
	Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

Sturdee, A. P., A. T. Bodley-Tickell, et al. (2003). "Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom." Vet Parasitol 116(2): 97-113.
	A longitudinal sample survey testing for Cryptosporidium in livestock and small wild mammals conducted over 6 years (1992-1997) on a lowland farm in Warwickshire, England, has shown the parasite to be endemic and persistently present in all mammalian categories. Faecal samples were taken throughout the year and oocysts concentrated by a formal ether sedimentation method for detection by immunofluorescence staining using a commercially available genus specific monoclonal antibody. Cryptosporidium parvum was identified by morphology and measurement of modified Ziehl-Neelsen stained oocysts. C. muris was rarely found in wild mammals and C. andersoni oocysts were never detected in livestock. Faecal samples from 3721 individuals gave cumulative 6-year prevalences for C. parvum as follows: bull beef, 3.6%; dairy cows, 3.5%; ewes, 6.4%; horses, 8.9%; calves (home bred), 52%; calves (bought-in) 23.2%; lambs, 12.9%; small wild mammals (rodents) living in and around farm buildings, 32.8%; small wild mammals (mainly rodents) living in areas of pasture, 29.9%. Animal categories with the highest prevalences also shed the highest average oocyst numbers per gram of faeces (ranging from 1.4 x 10(3) for bull beef to 1.1 x 10(5) for calves). Analysis of annual and seasonal data for each animal category revealed that patterns of infection were variable and sporadic; this means that short-term sampling was never likely to provide a true or representative picture. Seasonally combined data for adult livestock, young livestock and small wild mammals showed all three categories tended to have the highest Cryptosporidium prevalences in the autumn. Calves were separated from their dams within 24h of birth and yet showed high prevalence of infection in most years despite the low prevalence for the dairy herd. It is possible the coincidence of high autumn prevalence in mice with the main period for the rearing of calves contributed to the infection of the latter. The farming estate was used to teach students of agriculture and took pride in good land management and husbandry practices that produced well fed and healthy livestock. The data from this estate may represent, therefore, the baseline, the lowest possible levels to be expected, for Cryptosporidium infection and oocyst production on a lowland farm in the United Kingdom.

Sturdee, A. P., R. M. Chalmers, et al. (1999). "Detection of Cryptosporidium oocysts in wild mammals of mainland Britain." Vet Parasitol 80(4): 273-80.
	This paper combines the results from a preliminary survey of occurrence of Cryptosporidium species in faecal samples from a range of wild mammal species inhabiting mainland Britain with a tabulated literature review of world-wide reports of the parasite in those British mammals. In the literature, C. parvum was reported from 11 wild mammals found in Britain and elsewhere, mainly in rodents but also in insectivores, lagomorphs and ungulates. C. muris has been reported only in wild rodents. The sample survey detected C. parvum in seven additional British species, including carnivores. Overall, 12% of 184 faecal samples tested with a genus-specific monoclonal antibody contained oocysts of C. parvum. The results further emphasise the widespread distribution of Cryptosporidium amongst wild mammals in Britain, highlight the potential for transmission between host species and warn of the possibility of direct exposure for anybody using the countryside for professional or recreational purposes (e.g. farmers and ramblers) to previously unregarded sources of infection. It seems increasingly likely that most, if not all, mammalian species can be infected with C. parvum.

Sulaiman, I. M., R. Fayer, et al. (2003). "Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis." Emerg Infect Dis 9(11): 1444-52.
	To address the source of infection in humans and public health importance of Giardia duodenalis parasites from animals, nucleotide sequences of the triosephosphate isomerase (TPI) gene were generated for 37 human isolates, 15 dog isolates, 8 muskrat isolates, 7 isolates each from cattle and beavers, and 1 isolate each from a rat and a rabbit. Distinct genotypes were found in humans, cattle, beavers, dogs, muskrats, and rats. TPI and small subunit ribosomal RNA (SSU rRNA) gene sequences of G. microti from muskrats were also generated and analyzed. Phylogenetic analysis on the TPI sequences confirmed the formation of distinct groups. Nevertheless, a major group (assemblage B) contained most of the human and muskrat isolates, all beaver isolates, and the rabbit isolate. These data confirm that G. duodenalis from certain animals can potentially infect humans and should be useful in the detection, differentiation, and taxonomy of Giardia spp.

Sulaiman, I. M., R. Fayer, et al. (2003). "Molecular characterization of microsporidia indicates that wild mammals Harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi." Appl Environ Microbiol 69(8): 4495-501.
	Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.

Sulaiman, I. M., R. Fayer, et al. (2004). "Molecular characterization of Enterocytozoon bieneusi in cattle indicates that only some isolates have zoonotic potential." Parasitol Res.
	
Sulaiman, I. M., P. R. Hira, et al. (2005). "Unique endemicity of cryptosporidiosis in children in Kuwait." J Clin Microbiol 43(6): 2805-9.
	To understand the transmission of Cryptosporidium infection in children, fecal specimens from 62 Kuwaiti children with gastrointestinal symptoms found to be positive by microscopy were genotyped and subtyped with a small subunit rRNA-based PCR-restriction fragment length polymorphism analysis and a 60-kDa glycoprotein-based DNA sequencing tool. The median age of infected children was 4.5 years, and 77% of infections occurred during the cool season of November to April. Fifty-eight of the children (94%) had Cryptosporidium parvum, three (5%) had Cryptosporidium hominis, and one (1%) had both C. parvum and C. hominis. Altogether, 13 subtypes of C. parvum (belonging to four subtype allele families) and C. hominis (belonging to three subtype allele families) were observed, with 92% of specimens belonging to the common allele family IIa and the unusual allele family IId. Thus, the transmission of cryptosporidiosis in Kuwaiti children differed significantly from other tropical countries.

Sulaiman, I. M., J. Jiang, et al. (2004). "Distribution of Giardia duodenalis genotypes and subgenotypes in raw urban wastewater in Milwaukee, Wisconsin." Appl Environ Microbiol 70(6): 3776-80.
	Giardia cysts in 131 raw wastewater samples from Milwaukee, Wis., were genotyped by sequence analysis of the triosephosphate isomerase gene which showed the presence of two distinct genotypes (assemblages A and B) of Giardia duodenalis. Of the 131 samples, 111 belonged to assemblage A, and the remaining samples belonged to assemblage B. A high degree of genetic polymorphism was evident within the assemblage B cluster, with 10 distinct subgenotypes identified, eight of which have not been reported before.

Sulaiman, I. M., A. A. Lal, et al. (1999). "Biallelic polymorphism in the intron region of beta-tubulin gene of Cryptosporidium parasites." J Parasitol 85(1): 154-7.
	Nucleotide sequencing of polymerase chain reaction amplified intron region of the Cryptosporidium parvum beta-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is in agreement with our previous genotyping data based on the thrombospondin-related adhesion protein (TRAP-C2) gene, indicating these genotype characteristics are linked at 2 genetic loci. Characterization of Cryptosporidium muris and Cryptosporidium serpentis has further shown that non-parvum Cryptosporidium parasites have beta-tubulin intron sequences identical to bovine genotype of C. parvum. Thus, results of this study confirm the lineage of 2 genotypes of C. parvum at 2 genetic loci and suggest a need for extensive characterization of various Cryptosporidium spp.

Sulaiman, I. M., A. A. Lal, et al. (2002). "Molecular phylogeny and evolutionary relationships of Cryptosporidium parasites at the actin locus." J Parasitol 88(2): 388-94.
	To further validate previous observations in the taxonomy of Cryptosporidium parasites, the phylogenetic relationship was analyzed among various Cryptosporidium parasites at the actin locus. Nucleotide sequences of the actin gene were obtained from 9 putative Cryptosporidium species (C. parvum, C. andersoni, C. baileyi, C. felis, C. meleagridis, C. muris, C. saurophilum, C. serpentis, and C. wrairi) and various C. parvum genotypes. After multiple alignment of the obtained actin sequences, genetic distances were measured, and phylogenetic trees were constructed. Results of the analysis confirmed the presence of genetically distinct species within Cryptosporidium and various distinct genotypes within C. parvum. The phylogenetic tree constructed on the basis of the actin sequences was largely in agreement with previous results based on small subunit rRNA, 70-kDa heat shock protein, and Cryptosporidium oocyst wall protein genes. The Cryptosporidium species formed 2 major clades; isolates of C. andersoni, C. muris, and C. serpentis formed the first major group, whereas isolates of all other species, as well as various C. parvum genotypes, formed the second major group. Intragenotype variations were low or absent at this locus.

Sulaiman, I. M., U. M. Morgan, et al. (2000). "Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene." Appl Environ Microbiol 66(6): 2385-91.
	We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. The Cryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes of C. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus Cryptosporidium.

Sulaiman, I. M., L. Xiao, et al. (1999). "Evaluation of Cryptosporidium parvum genotyping techniques." Appl Environ Microbiol 65(10): 4431-5.
	We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.

Sulaiman, I. M., L. Xiao, et al. (1998). "Differentiating human from animal isolates of Cryptosporidium parvum." Emerg Infect Dis 4(4): 681-5.
	We analyzed 92 Cryptosporidium parvum isolates from humans and animals by a polymerase chain reaction/restriction fragment length polymorphism method based on the thrombospondin-related anonymous protein 2 gene sequence. Used as a molecular marker, this method can differentiate between the two genotypes of C. parvum and elucidate the transmission of infection to humans.

Sunnotel, O., C. J. Lowery, et al. (2006). "Cryptosporidium." Lett Appl Microbiol 43(1): 7-16.
	This review discusses characteristics of the genus Cryptosporidium and addresses the pathogenesis, reservoirs, public health significance and current applications for the detection and typing of this important pathogen. By increasing knowledge in key areas of Cryptosporidium research such as aetiology, epidemiology, transmission and host interactions, the numbers of cases of human cryptosporidiosis should be reduced.

Sunnotel, O., W. J. Snelling, et al. (2006). "Rapid and sensitive detection of single cryptosporidium oocysts from archived glass slides." J Clin Microbiol 44(9): 3285-91.
	In this study we report on the development and application of a novel method for efficiently extracting and detecting single Cryptosporidium oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of Cryptosporidium oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of Cryptosporidium and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type Cryptosporidium and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control.

Szostakowska, B., W. Kruminis-Lozowska, et al. (2004). "Cryptosporidium parvum and Giardia lamblia recovered from flies on a cattle farm and in a landfill." Appl Environ Microbiol 70(6): 3742-4.
	Filth flies associated with a cattle barn and a municipal landfill were tested positive by combined immunofluorescent antibody and fluorescent in situ hybridization for Cryptosporidium parvum and Giardia lamblia on their exoskeletons and in their guts. More pathogens were carried by flies from the cattle barn than from the landfill; 81% of C. parvum and 84% of G. lamblia pathogens were presumptively viable.

Taghi-Kilani, R., L. L. Gyurek, et al. (1996). "Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation." Int J Parasitol 26(6): 637-46.
	A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

Tamburrini, A. and E. Pozio (1999). "Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis)." Int J Parasitol 29(5): 711-5.
	Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters flow into the sea, potentially causing runoff of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in artificial seawater, to determine if the oocysts are retained in mussel tissues (Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in artificial seawater at 6-8 degrees C under moderate oxygenation and the infectivity of oocysts was tested five times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 10(5) oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing artificial seawater filtered out more than 4 x 10(8) oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be filtered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans.

Tanriverdi, S., A. Tanyeli, et al. (2002). "Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis." J Clin Microbiol 40(9): 3237-44.
	Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.

Tate, K. W., E. R. Atwill, et al. (2000). "Cryptosporidium parvum transport from cattle fecal deposits on California rangelands." Journal of Range Management 53(3): 295-299.
	Cryptosporidium parvum is a fecal borne protozoan parasite that can be carried by and cause gastrointestinal illness in humans, cattle, and wildlife. The illness, cryptosporidiosis, can be fatal to persons with compromised immune systems. At question is the potential for C. parvum in cattle fecal deposits on rangeland watersheds to contaminate surface water. First, C. parvum oocysts must be released from fecal deposits during rainfall, becoming available for transport. In 1996, we examined the transport of C. parvum oocysts in overland flow from fecal deposits under natural rainfall and rangeland conditions at the San Joaquin Experimental Range in Madera County, Calif. Our null hypothesis was that C. parvum oocysts are not released from fecal pats and transported 1 m downslope as overland flow with rainfall. Paired plots were located on 10, 20, and 30% slope sites. Each plot was loaded with four, 200 g fecal pats dosed with 10(5) oocysts g(-1). Fats were placed 1.0 m above the base of each plot. Composite runoff samples from each plot were analyzed for oocyst concentration following each of 4 storm events. Oocysts were transported during each storm. Slope was a significant factor in oocyst transport, with oocyst transport increasing with slope. Although not significant, there was an apparent flushing effect of oocysts across storms, with the majority transported in the first 2 storms. A pilot rainfall simulation experiment also revealed a flushing phenomenon from pats during individual rainfall events. C. parvum oocysts in fecal pats on rangeland can be transported from fecal deposits during rainfall events, becoming available for transport to water-bodies. Future studies need to examine surface and subsurface transport of oocysts on rangeland hillslopes for distances greater than 1 m. [References: 25] 25

Tate, K. W., E. R. Atwill, et al. (2003). "Spatial and temporal patterns of cattle feces deposition on rangeland." Journal of Range Management 56(5): 432-438.
	The objective of this study was to identify and model environmental and management factors associated with cattle feces deposition patterns across annual rangeland watersheds in the Sierra Nevada foothills. Daily cattle fecal load accumulation rates were calculated from seasonal fecal loads measured biannually on 40 m(2) permanent transects distributed across a 150.5 ha pasture in Madera County, Calif. during the 4 year period from 1995 through 1998. Associations between daily fecal load per season, livestock management, and environmental factors measured for each transect were determined using a linear mixed effects model. Cattle feces distribution patterns were significantly associated with location of livestock attractants, slope percentage, slope aspect, hydrologic position, and season. Transects located in livestock concentration areas experienced a significantly higher daily fecal load compared to transects outside of these concentration areas (P < 0.001). Percent slope was negatively associated with daily fecal load, but this association had a significant interaction with slope aspect (P = 0.02). Daily fecal load was significantly lower during the wet season compared to the dry season (P = 0.002). Daily fecal loading rates across hydrologic positions were dependent upon season. Our results illustrate the opportunities to reduce the risk of water quality contamination by strategic placement of cattle attractants, and provide a means to predict cattle feces deposition based upon inherent watershed characteristics and management factors. [References: 27] 27

Taylor, M. A., J. Catchpole, et al. (1993). "Giardiasis in lambs at pasture." Vet Rec 133(6): 131-3.
	Faecal samples from a group of lambs at pasture were screened at weekly intervals for nine weeks for the presence of Giardia species using a modified zinc sulphate flotation method. Fifty-nine of 86 lambs (68.6 per cent) excreted giardia cysts on one or more occasions. They were first detected at approximately three weeks of age and the highest incidence of excretion of cysts occurred when the lambs reached a mean age of 37 days. The lambs had diarrhoea but it was attributed to the presence of Eimeria ovinoidalis.

Tenter, A. M., A. R. Heckeroth, et al. (2000). "Toxoplasma gondii: from animals to humans." Int J Parasitol 30(12-13): 1217-58.
	Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. If first contracted during pregnancy, T. gondii may be transmitted vertically by tachyzoites that are passed to the foetus via the placenta. Horizontal transmission of T. gondii may involve three life-cycle stages, i.e. ingesting infectious oocysts from the environment or ingesting tissue cysts or tachyzoites which are contained in meat or primary offal (viscera) of many different animals. Transmission may also occur via tachyzoites contained in blood products, tissue transplants, or unpasteurised milk. However, it is not known which of these routes is more important epidemiologically. In the past, the consumption of raw or undercooked meat, in particular of pigs and sheep, has been regarded as a major route of transmission to humans. However, recent studies showed that the prevalence of T. gondii in meat-producing animals decreased considerably over the past 20 years in areas with intensive farm management. For example, in several countries of the European Union prevalences of T. gondii in fattening pigs are now <1%. Considering these data it is unlikely that pork is still a major source of infection for humans in these countries. However, it is likely that the major routes of transmission are different in human populations with differences in culture and eating habits. In the Americas, recent outbreaks of acute toxoplasmosis in humans have been associated with oocyst contamination of the environment. Therefore, future epidemiological studies on T. gondii infections should consider the role of oocysts as potential sources of infection for humans, and methods to monitor these are currently being developed. This review presents recent epidemiological data on T. gondii, hypotheses on the major routes of transmission to humans in different populations, and preventive measures that may reduce the risk of contracting a primary infection during pregnancy.

Teunis, P. F. M., G. J. Medema, et al. (1997). "Assessment of the risk of infection by Cryptosporidium or Giardia in drinking water from a surface water source." Water Research 31(6): 1333-1346.
	The significance of the presence in drinking water of the protozoan microparasites Cryptosporidium parvum and Giardia lamblia for public health may be analyzed by means of risk assessment. This requires quantitative knowledge of all the contributing factors, from the concentration of these organisms in the source water to the dose-response relation for the probability of infection or disease in a human host. The major contributing factors are: the concentration of cysts or oocysts in raw water, the recovery of the detection method, the viability of recovered cysts or oocysts, the removal of organisms in the treatment process, and the daily consumption of unboiled tap water. To enable analysis of the uncertainty in the calculated risk of infection, each of these factors is treated as a stochastic variable, for which a suitable distribution is proposed. A frequency distribution for the probability of infection is then constructed with standard sampling techniques. This first evaluation of the calculation of the risk of infection due to exposure to Cryptosporidium oocysts and Giardia cysts via drinking water, shows that the uncertainty in the estimated removal efficiency of the treatment process dominates over uncertainties in other contributing factors.

Thompson, R. C. (2000). "Giardiasis as a re-emerging infectious disease and its zoonotic potential." Int J Parasitol 30(12-13): 1259-67.
	The reasons for considering giardiasis as a re-emerging infectious disease are presented, with emphasis on Giardia infections in child care centres, livestock and pets, and the role of zoonotic transmission. However, the aetiology and control of giardiasis is complicated by the genetic and phenotypic variability of Giardia species infective to mammals. Of particular significance has been the uncertainty about host specificity and the question of zoonotic transmission. The recent application of molecular characterisation procedures based on PCR has made an enormous contribution to an understanding of the genetic structure of Giardia populations, and this is reviewed in the context of the zoonotic transmission and molecular epidemiology of Giardia infections.

Thompson, R. C. (2004). "The zoonotic significance and molecular epidemiology of Giardia and giardiasis." Vet Parasitol 126(1-2): 15-35.
	The taxonomy and molecular epidemiology of Giardia and Giardia infections are reviewed in the context of zoonotic and waterborne transmission. Evidence to support the zoonotic transmission of Giardia is very strong, but how frequent such transmission occurs and under what circumstances, have yet to be determined. Zoonotic origin for waterborne outbreaks of Giardia infection appears to be uncommon. Similarly, livestock are unlikely to be an important source of infection in humans. The greatest risk of zoonotic transmission appears to be from companion animals such as dogs and cats, although further studies are required in different endemic foci in order to determine the frequency of such transmission.

Thompson, R. C. and R. M. Chalmers (2002). "Cryptosporidium: from molecules to disease." Trends Parasitol 18(3): 98-100.
	The international conference, Cryptosporidium: from molecules to disease, was held 7-12 October 2001, in Fremantle, Western Australia, to discuss all aspects of Cryptosporidium and cryptosporidiosis.

Thompson, R. C., R. M. Hopkins, et al. (2000). "Nomenclature and genetic groupings of Giardia infecting mammals." Parasitol Today 16(5): 210-3.
	Giardia is a ubiquitous and well-known enteric parasite affecting humans and a range of domestic and wild mammals. It is one of the most common parasites of domestic dogs and dairy cattle and a frequently recognized waterborne pathogen. Giardiasis is considered to be a re-emerging infection because of its association with outbreaks of diarrhoea in child-care centres. Although only a single species has been recognized as causing disease in humans and most other mammals, molecular characterization of morphologically identical isolates from humans and numerous other species of mammals has confirmed the heterogeneity of this parasite and provided a basis for a clearer understanding of the taxonomy and zoonotic potential of Giardia.

Thompson, R. C., A. J. Lymbery, et al. (1996). "Giardia duodenalis: exposure to metronidazole inhibits competitive interactions between isolates of the parasite in vitro." J Parasitol 82(4): 679-83.
	The competitive interactions of genetically distinct isolates of Giardia duodenalis with different growth rates were studied in vitro. Electrophoretic analysis of mixed cultures showed that competition between 2 cloned isolates occurs under normal in vitro culture conditions, with faster-growing isolates outcompeting those with slower growth rates. The addition of sublethal concentrations of metronidazole to clonal mixtures in vitro prevented the competitive exclusion, which was seen in normal culture. This apparently occurred because the drug reduced the growth rate of the faster-growing but not the slower-growing clone.

Thompson, R. C., M. E. Olson, et al. (2005). "Cryptosporidium and cryptosporidiosis." Adv Parasitol 59: 77-158.
	Cryptosporidium is one of the most common enteric protozoan parasites of vertebrates with a wide host range that includes humans and domestic animals. It is a significant cause of diarrhoeal disease and an ubiquitous contaminant of water which serves as an excellent vehicle for transmission. A better understanding of the development and life cycle of Cryptosporidium, and new insights into its phylogenetic relationships, have illustrated the need to re-evaluate many aspects of the biology of Cryptosporidium. This has been reinforced by information obtained from the recent successful Cryptosporidium genome sequencing project, which has emphasised the uniqueness of this organism in terms of its parasite life style and evolutionary biology. This chapter provides an up to date review of the biology, biochemistry and host parasite relationships of Cryptosporidium.

Thurston-Enriquez, J. A., P. Watt, et al. (2002). "Detection of protozoan parasites and microsporidia in irrigation waters used for crop production." J Food Prot 65(2): 378-82.
	The occurrence of human pathogenic parasites in irrigation waters used for food crops traditionally eaten raw was investigated. The polymerase chain reaction was used to detect human pathogenic microsporidia in irrigation waters from the United States and several Central American countries. In addition, the occurrence of both Cryptosporidium oocysts and Giardia cysts was determined by immunofluorescent techniques. Twenty-eight percent of the irrigation water samples tested positive for microsporidia, 60% tested positive for Giardia cysts, and 36% tested positive for Cryptosporidium oocysts. The average concentrations in samples from Central America containing Giardia cysts and Cryptosporidium oocysts were 559 cysts and 227 oocysts per 100 liters. In samples from the United States, averages of 25 Giardia cysts per 100 liters and <19 (average detection limit) Cryptosporidium oocysts per 100 liters were detected. Two of the samples that were positive for microsporidia were sequenced, and subsequent database homology comparisons allowed the presumptive identification of two human pathogenic species, Encephalitozoon intestinalis (94% homology) and Pleistophora spp. (89% homology). The presence of human pathogenic parasites in irrigation waters used in the production of crops traditionally consumed raw suggests that there may be a risk of infection to consumers who come in contact with or eat these products.

Tiangtip, R. and S. Jongwutiwes (2002). "Molecular analysis of Cryptosporidium species isolated from HIV-infected patients in Thailand." Trop Med Int Health 7(4): 357-64.
	Cryptosporidium isolates from diarrhoeal stools of human immunodeficiency virus (HIV)-infected patients in Thailand were genetically analysed by sequencing the variable region in the 18S rRNA gene. Twenty-nine isolates from four children and 25 adults attending King Chulalongkorn Memorial Hospital in Bangkok during 1996 and 2000 were analysed. All patients suffered from chronic watery diarrhoea and had low CD4+ lymphocytes (mean +/- SD=105.5 +/- 133.2 cells/microl). Four Cryptosporidium species were identified, i.e. C. parvum (genotype 1), C. meleagridis, C. muris and C. felis occurring in 24, 3, 1 and 1 isolates, respectively. Oocysts of C. muris were significantly larger than oocysts of other species; C. felis was the smallest in these populations (P < 0.01). Sequences of the ITS1, 5.8S rRNA and ITS2 regions of C. muris and C. meleagridis identified in this study displayed unique sequences from those of other known species. Based on a limited number of isolates analysed, only C. meleagridis and C. muris were found in HIV-infected children, whereas the genotype 1 of C. parvum predominated in HIV-infected adults.

Tillett, H. E., J. de Louvois, et al. (1998). "Surveillance of outbreaks of waterborne infectious disease: categorizing levels of evidence." Epidemiol Infect 120(1): 37-42.
	Public health surveillance requires the monitoring of waterborne disease, but sensitive and specific detection of relevant incidents is difficult. The Communicable Disease Surveillance Centre receives information from various sources about clusters of cases of illness in England and Wales. The reporter may suspect that water consumption or recreational water exposure is the route of infection, or subsequent investigation may raise the hypothesis that water is associated with illness. It is difficult to prove beyond reasonable doubt that such a hypothesis is correct. Water samples from the time of exposure are seldom available, some organisms are difficult to detect and almost everyone has some exposure to water. Therefore, we have developed a method of categorizing the degree of evidence used to implicate water. The categories take into account the epidemiology, microbiology and water quality information. Thus outbreaks are classified as being associated with water either 'strongly', 'probably' or 'possibly'. This system allows a broad database for monitoring possible effects of water and is not confined to the few outbreaks which have been intensively investigated or have positive environmental microbiology. Thus, for reported incidents, the sensitivity of classifying it as water associated should be high but this may be at the expense of specificity, especially with the 'possible' association.

Tilley, M., M. T. Eggleston, et al. (1993). "Multiple oral inoculations with Cryptosporidium parvum as a means of immunization for production of monoclonal antibodies." FEMS Microbiol Lett 113(2): 235-40.
	Oral immunization of suckling mice with Cryptosporidium parvum results in a humoral response to a limited set of antigens. Six-day-old BALB/c mice were each inoculated orally with 1 x 10(6) viable oocysts and subsequently administered oral inoculations of 2 x 10(6) viable oocysts at 30 and 60 days following the primary infection. After 45 days, mice were boosted with 1 x 10(6) oocysts orally, plus soluble extracts equivalent to 2 x 10(6) and 1 x 10(6) oocysts given intravenously and intraperitoneally, respectively. Four days later, splenic lymphocytes were fused to Ag8 myeloma cells. Using this method, we have been able to select for monoclonal antibodies that predominantly recognize sporozoite surface and apical complex antigens.

Tilley, M., R. Fayer, et al. (1990). "Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocyst and sporozoite antigens recognized by bovine colostral antibodies." Infect Immun 58(9): 2966-71.
	Colostral whey from seven hyperimmunized and two control cows (hyperimmune bovine colostrum) was examined by Western immunoblotting for the presence of antibody against oocysts and sporozoites of Cryptosporidium parvum, using rabbit anti-bovine immunoglobulin A (IgA), IgG1, IgG2, and IgM antibodies, followed by a horseradish peroxidase goat anti-rabbit polyvalent antibody. Although considerable variation was found in binding activity between cows on different immunization protocols, IgA and IgG1 in whey recognized a greater variety of C. parvum antigens than did IgG2 and IgM. A band at 9 to 10 kilodaltons appeared unique in that it was recognized only by IgA.

Tilley, M. and S. J. Upton (1991). "Sporozoites and merozoites of Cryptosporidium parvum share a common epitope recognized by a monoclonal antibody and two-dimensional electrophoresis." J Protozool 38(6): 48S-49S.
	Sporozoites and merozoites of Cryptosporidium parvum were analyzed for the presence of a 15 kDa surface antigen using a monoclonal antibody probe. Both were found to possess the antigen by immunofluorescence, and further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed these observations. When separated by two-dimensional electrophoresis the isoelectric point was found to be similar, with major spots at 4.25 and minor spots at 4.15.

Tilley, M. and S. J. Upton (1994). "Both CP15 and CP25 are left as trails behind gliding sporozoites of Cryptosporidium parvum (Apicomplexa)." FEMS Microbiol Lett 120(3): 275-8.
	Sporozoites of Cryptosporidium parvum were examined after gliding upon glass microscope slides using monoclonal antibodies to the 15 and 25 kDa surface molecules and immunogold-silver enhancement. Both antibodies bound to surface antigen deposited as trials behind parasites, suggesting that both surface molecules are involved in substrate attachment.

Tilley, M., S. J. Upton, et al. (1990). "Identification of outer oocyst wall proteins of three Cryptosporidium (Apicomplexa: Cryptosporidiidae) species by 125I surface labeling." Infect Immun 58(1): 252-3.
	Autoradiography of oocyst wall surface proteins of three Cryptosporidium spp. revealed common bands at 285 to 290, 145 to 148, 120, 57, and 32 kilodaltons (kDa). Cryptosporidium baileyi and C. muris share proteins at 180, 100, 80 to 81, 29, and 18 to 19 kDa; C. baileyi and C. parvum share one protein at 46 to 47 kDa; and C. muris and C. parvum share a protein at 67 to 69 kDa. Additional protein bands, each unique to one species, were also observed.

Tilley, M., S. J. Upton, et al. (1991). "A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa)." Can J Microbiol 37(12): 949-952.
	Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.

Tilley, M., S. J. Upton, et al. (1991). "Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum." Infect Immun 59(3): 1002-7.
	An immunoglobulin A monoclonal antibody (MAb5C3) was developed against a 15-kDa surface glycoprotein (GP15) of Cryptosporidium parvum sporozoites. Indirect immunofluorescence and colloidal gold immunoelectron microscopy revealed that the antibody reacted with both the sporozoite and merozoite surface plasma membranes. On Western immunoblots, MAb5C3 binding was found to be strongly inhibited when 200 mM N-acetylglucosamine was used as a competing sugar. N-Acetylgalactosamine inhibited binding of the antibody only slightly, whereas glucose, mannose, and galactose failed to inhibit binding. MAb5C3 was found to recognize a similar 15-kDa epitope associated with a Cryptosporidium sp. isolated from guinea pigs. However, MAb5C3 failed to react with any proteins or glycoproteins associated with C. baileyi from chickens, Cryptosporidium sp. (= bovine C. muris) from cattle, C. serpentis from a rat snake, bradyzoites of Besnoitia darlingi from an opossum, sporozoite/oocyst extracts of Caryospora bigenetica from an eastern diamondback rattlesnake, sporozoites of Eimeria nieschulzi and E. papillata from rats and mice, or tachyzoites of Toxoplasma gondii (RH strain). When hybridoma supernatants containing MAb5C3 were administered orally to suckling mice experimentally infected with C. parvum, a 75% reduction in developmental stages was seen histologically at 72 h postinfection and a 67.5% reduction in mean oocyst output was found at 6 days postinfection.

Traub, R., S. Wade, et al. (2005). "Molecular characterization of potentially zoonotic isolates of Giardia duodenalis in horses." Vet Parasitol 130(3-4): 317-21.
	Giardia isolates from eight horses from New York State (NY), USA and two horses from Western Australia (WA) were genetically characterized at the SSU-rDNA and triose-phosphate isomerase (TPI) genes. Phylogenetic analysis of the TPI gene provided strong support for the placement of both isolates of Giardia from horses in WA and a single isolate from a horse in NY within the assemblage AI genotype of G. duodenalis. Another two isolates from horses in NY placed within the assemblage AII genotype of G. duodenalis. Phylogenetic analysis of the TPI gene also provided strong bootstrap support for the placement of four G. duodenalis isolates from horses in NY into a potentially host-specific sub-assemblage of assemblage BIV. The results of this study are consistent with previous studies showing that assemblages AI and AII of G. duodenalis provide the greatest potential zoonotic risk to humans. Horses may therefore constitute a potential source for human infection of Giardia either directly or via watersheds.

Traversa, D., A. Giangaspero, et al. (2004). "Genotyping of Cryptosporidium isolates from Chamelea gallina clams in Italy." Appl Environ Microbiol 70(7): 4367-70.
	Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.

Trout, J. M., M. Santin, et al. (2003). "Identification of assemblage A Giardia in white-tailed deer." J Parasitol 89(6): 1254-5.
	Fecal samples were collected from hunter-killed white-tailed deer (Odocoileus virginianus) during a managed hunt in a central Maryland county. Fecal samples were cleaned of debris and concentrated by CsCl density gradient centrifugation and stained with MerIFluor reagents. Stained samples were examined by fluorescent microscopy for the presence of Giardia sp. cysts. One of 26 samples was found to be positive for Giardia sp. Polymerase chain reaction amplification using primers directed to the beta-giardin and TPI genes identified the same sample as the only positive one. Sequencing of the beta-giardin and TPI genes revealed that the Giardia sp. belonged to assemblage A, a genotype infectious for humans and also reported in a small percentage of cattle. This is the first report of assemblage A Giardia sp. in deer and suggests that deer could be a potential source of infectious cysts for humans and cattle.

Trout, J. M., M. Santin, et al. (2004). "Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves." Vet Parasitol 124(3-4): 179-86.
	To determine the prevalence of Giardia genotypes in pre-weaned dairy calves, fecal samples were collected from a minimum of 18, 1-7-week-old dairy calves per farm on two farms each in the states of Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Samples cleaned of fecal debris and concentrated using CsCl density gradient centrifugation were stained and examined by immunofluorescence microscopy and also subjected to PCR and gene sequence analysis. Prevalence by PCR ranged from 9% on a farm in Pennsylvania to 93% on a farm in Vermont, with an average prevalence for 407 calves on 14 farms of 40%. Gene sequence analysis of the TPI, beta-giardin and 16S rRNA genes revealed 85% of the positive samples to be Assemblage E, while 15% were Assemblage A, although the percentages of these genotypes varied greatly from farm to farm. Some farms had no Assemblage A Giardia. Thus, while a majority of the calves were infected with a genotype that is not known to be infectious for humans, calves on 7 of 14 farms did harbor Assemblage A Giardia. Calves should be considered as a potential source of human infectious cysts in the environment.

Trout, J. M., M. Santin, et al. (2005). "Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves." Vet Parasitol 130(3-4): 177-83.
	To determine the prevalence of Giardia genotypes in post-weaned dairy calves, fecal specimens were collected from 3 to 11-month-old dairy calves per farm on two farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens cleaned of fecal debris and concentrated using CsCl density gradient centrifugation were stained and examined by immunofluorescence microscopy and also subjected to PCR and DNA sequence analysis. Overall, PCR provided more sensitive detection than IFA. Prevalence of Giardia infection, as detected by PCR ranged from 20% on NC-2 to 81% on VT-2, with an overall prevalence of 52% (237 positive samples out of 456 total). DNA sequence analysis of the 16S rRNA gene revealed 87% of the 237 Giardia isolates were Assemblage E, and 13% were Assemblage A although the prevalence of these genotypes varied greatly from farm to farm, with five farms having no Assemblage A Giardia. Therefore, Assemblage E was present in 45% of all animals tested and Assemblage A was present in 7% of the animals. Thus, while many of the calves were infected with a genotype that is not known to be infectious for humans, post-weaned calves on nine of 14 farms did harbor Assemblage A Giardia. Therefore calves should be considered as a potential source of human infectious cysts in the environment, with some farms representing a much higher risk than others.

Trout, J. M., E. J. Walsh, et al. (2002). "Rotifers ingest Giardia cysts." J Parasitol 88(5): 1038-40.
	Seven species of rotifers representing 6 genera, Epiphanes, Plationus, Asplanchna, Philodina species A, Philodina species B. Platyias, and Brachionus, were exposed to Giardia cysts isolated from the feces of experimentally infected holstein calves. Giardia cysts were prestained with a fluorescein isothiocyanate-conjugated monoclonal antibody and mixed with viable rotifers on 3-well Teflon-coated microscope slides. Organisms were observed with phase-contrast, differential interference contrast, and fluorescence microscopy. Five rotifer species, Epiphanes brachionus, Plationus patulus, Philodina (both A and B), and Platyias quadricornis, ingested varying numbers of cysts, which were retained within the rotifers' bodies throughout the observation period. Rotifer ingestion of Giardia cysts may represent a means of reducing water contamination.

Tumwine, J. K., A. Kekitiinwa, et al. (2003). "Cryptosporidium parvum in children with diarrhea in Mulago Hospital, Kampala, Uganda." Am J Trop Med Hyg 68(6): 710-5.
	A cross-sectional case-control study (ratio = 3:1) was conducted over a 15-month period to determine the prevalence and consequences of cryptosporidiosis in hospitalized diarrheic children (0-5 years old) at Mulago Hospital in Kampala, Uganda. Cryptosporidium parvum was detected and genotyped among 2,446 children of whom 1,779 (72.7%) had diarrhea, and 667 (27.3%) were age- and sex-matched controls. Of the 1,779 children with diarrhea, 532 (29.9%) had persistent (> 14 days) diarrhea and 1,247 (70.1%) had acute diarrhea. Overall, 444 (25.0%) of the 1,779 children with diarrhea had C. parvum, compared with only 57 (8.5%) of the 667 children without diarrhea (chi2 = 80.2, P < or = 0.0001). Within this group of infected children, 72.8% were infected with genotype 1, 18.4% with genotype 2, and 4.1% with a mixture of both genotypes, and 4.1% isolates were either unclassified or C. meleagridis. The prevalence was highest during the rainy months of April to June. Of the 532 children with persistent diarrhea, 166 (31.2%) had C. parvum compared with 278 (22.3%) of the 1,247 children with acute diarrhea (chi2 = 15.8, P < or = 0.0001). There was a significant association between C. parvum and malnutrition including stunting, being underweight, and wasting. Unfavorable outcome (death or failure to resolve within 14 days) occurred in 139 (72.8%) of the 191 children with C. parvum, and in only 65.1% of the 545 without (odds ratio = 1.117, 95% confidence interval = 1.005-1.243, P = 0.05), Of the 191 children with C. parvum, 24 (12.6%) died, compared with 34 (6.2%) of the 545 without C. parvum (P = 0.005). Mortality rates were higher among children with severe dehydration and persistent diarrhea, and in stunted or underweight children infected with C. parvum. Among Ugandan children, cryptosporidiosis, which remains untreatable, is frequently associated with diarrhea and other serious and unfavorable consequences.

Ungar, B. L., D. J. Ward, et al. (1990). "Cessation of Cryptosporidium-associated diarrhea in an acquired immunodeficiency syndrome patient after treatment with hyperimmune bovine colostrum." Gastroenterology 98(2): 486-9.
	Cryptosporidium is a parasite of the human gastrointestinal tract that can cause life-threatening diarrhea in immunodeficient patients. Although more than 80 agents have been tried with occasional anecdotal success, treatment remains primarily limited to hydration. A 38-yr-old homosexual man with antibody to human immunodeficiency virus and Cryptosporidium-related diarrhea is described. The patient excreted 6-12 L of stool per day for at least 3 mo, 2 of them spent in the hospital. Trials with more than 6 antidiarrheal medications were ineffective. The patient received bovine colostrum hyperimmune to Cryptosporidium by direct duodenal infusion. During infusion, the patient's fecal output decreased to less than 2 L per day, and 48 h after treatment, stools were formed and oocysts to Cryptosporidium were absent. The patient remained asymptomatic for 3 mo. Hyperimmune bovine colostrum offers an exciting new therapy for cryptosporidiosis; controlled trials to establish efficacy should be undertaken and the active factor(s) characterized.

Upton, S. J. and H. H. Gillock (1996). "Infection dynamics of Cryptosporidium parvum in ICR outbred suckling mice." Folia Parasitol (Praha) 43(2): 101-6.
	An ICR outbred suckling mouse model of cryptosporidiosis was used to explain some of the variability associated with experimental Cryptosporidium parvum infections in neonate mice. Fourty-four groups of 12 mice each, ranging in age from 4-12 days, each received 1.0 x 10(4) CsCl purified oocysts per os in 5 microns PBS. At 6 days post-inoculation (PI), mice were killed by CO2 overdose and individually weighed. Intestines were then homogenized and oocysts were quantified by hemacytometer. Results revealed that both age and weight have pronounced effects on numbers of oocysts produced in vivo, with larger and older mice producing higher numbers of parasites. Mice 8-9 days of age at the time of inoculation displayed the least amount of weight dependent variability, produced the highest numbers of oocysts, and were judged to be superior over other ages for pharmaceutical screening. Significant reductions in numbers of oocysts occurred in mice inoculated at 10 days of age, and only a few oocysts were found in mice inoculated at 11-12 days of age. These studies suggest that at least some data on Cryptosporidium generated from suckling mouse studies to date are probably unreliable and should be viewed skeptically.

Upton, S. J., M. A. Stamper, et al. (2000). "A new species of Eimeria (Apicomplexa, Eimeriidae) from the weedy sea dragon Phyllopteryx taeniolatus (Osteichthyes: Syngnathidae)." Dis Aquat Organ 43(1): 55-9.
	A new species of intestinal coccidian is described from the weedy or common sea dragon Phyllopteryx taeniolatus housed at the New England Aquarium in Boston and at the Shedd Aquarium in Chicago, USA. Live oocysts of Eimeria phyllopterycis sp. n. are spherical, 30.9 (28.0-34.4) microm, with a thin, single-layered wall. Both a micropyle and oocyst residuum are absent and a large polar granule is sometimes present. Sporocysts are ellipsoidal and elongate, 24.3 x 10.4 (23.4-25.6 x 9.2-11.2) microm, with Stieda and substieda bodies; shape index (length/width) 2.33 (2.14-2.70). A sporocyst residuum is present, consisting of numerous granules of various sizes. Sporozoites each possess 3 refractile bodies. Preliminary evidence suggests that the coccidian may affect the health of sea dragons; however, it could not be determined whether this parasite caused significant morbidity or mortality.

Upton, S. J. and M. Tilley (1992). "Effect of select media supplements on motility and development of Eimeria nieschulzi in vitro." J Parasitol 78(2): 329-33.
	A technique was developed to examine the effects of exogenous substances on apicomplexan sporozoite motility in vitro. Sporozoites of Eimeria nieschulzi were placed in the top compartments of blind well chemotactic chambers and separated from potential chemoattractant/chemokinetic agents by 8.0-microns pore size Millipore filters. After 3-hr incubations, the number of sporozoites migrating through the filters was assessed using a hemacytometer. Results revealed that several substances, especially albumin and fetuin, enhanced motility of coccidial sporozoites. Cell culture assays supported these data, with higher numbers of parasites found in cultures supplemented with albumin and/or fetuin.

Upton, S. J., M. Tilley, et al. (1994). "Comparative development of Cryptosporidium parvum (Apicomplexa) in 11 continuous host cell lines." FEMS Microbiol Lett 118(3): 233-6.
	Using standardized media, incubation, and parasite inoculating procedures, we compared development of Cryptosporidium parvum between Madin-Darby bovine kidney (MDBK) cells and 10 additional host cell lines available through the American Type Culture Collection. Parasite development was assessed by counting parasite numbers atop monolayers in 25 random oil fields 68 h post-infection using Nomarski interference-contrast optics. Results revealed that the human ileocecal adenocarcinoma (HCT-8) cell line supported nearly twice the number of parasite developmental stages than MDBK cells or any of the other host cell types.

Upton, S. J., M. Tilley, et al. (1995). "Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells." J Clin Microbiol 33(2): 371-5.
	Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and antibiotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed free of debris, fresh cell culture media containing select supplements were added and cultures were reincubated. Parasite growth was assessed 66 h later by counting the number of parasite developmental stages in 25 random x 100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, folic acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro. The addition of insulin and the sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with any of the vitamins. Using the above information, we developed a supplemental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 micrograms of ascorbic acid, 1.0 micrograms of folic acid, 4.0 micrograms of 4-aminobenzoic acid, 2.0 micrograms of calcium pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 micrograms of streptomycin, and 0.25 microgram of amphotericin B (Fungizone) per ml (pH 7.4). The growth of c. parvum in this medium was found to be enhanced approximately 10-fold compared with that in control medium without additional glucose, insulin, or vitamins.

Upton, S. J., M. Tilley, et al. (1991). "Incorporation of exogenous uracil by Cryptosporidium parvum in vitro." J Clin Microbiol 29(5): 1062-5.
	Oocysts of Cryptosporidium parvum were used to infect Madin-Darby bovine kidney cells. Cultures were incubated in a reduced-oxygen atmosphere in candle jars or in a 5% CO2-95% air atmosphere. At 72 h, parasites were quantitated microscopically and found to be enhanced 5.5-fold in the reduced-oxygen atmosphere. Using candle jars, we then determined that C. parvum was amenable to [3H]uracil incorporation assays and easily quantitated with this method.

Upton, S. J., M. Tilley, et al. (1994). "A simple and reliable method of producing in vitro infections of Cryptosporidium parvum (Apicomplexa)." FEMS Microbiol Lett 118(1-2): 45-9.
	A variety of techniques have been used to infect cell monolayers in culture with the protozoan, Cryptosporidium parvum. However, most of these methods rely on the use of trypsin and/or bile salts to excyst sporozoites in vitro, followed by washing sporozoites free of excystation solution prior to their addition to subconfluent monolayers. This method not only increases the amount of time required to establish infections in vitro, but also results in prolonged exposure of free sporozoites to environmental conditions. Here we report a simple, fast, and efficient method of obtaining consistent infections of C. parvum in cell monolayers. This technique relies on the ability of the parasite to excyst at 37 degrees C but not at room temperature following pretreatment with sodium hypochlorite. By adding surface-sterilized oocysts directly to monolayers, sporozoites have access to host cells immediately upon excystation.

Upton, S. J., S. C. Wilson, et al. (2001). "A new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the Bali (Rothschild's) mynah Leucopsar rothschildi (Passeriformes: Sturnidae), and comments concerning the genera Atoxoplasma Garnham, 1950 and Isospora." Syst Parasitol 48(1): 47-53.
	A new species of isosporan (Apicomplexa: Eimeriidae) is reported from the Bali mynah Leucopsar rothschildi (Passeriformes: Sturnidae). Oocysts of Isospora rothschildi n. sp. are spherical to subspherical, 22.3 x 21.6 (20-26 x 19-23) microm, with a shape index (length/width) of 1.03 (1.00-1.15). A micropyle and oocyst residuum are absent, but one to many polar granules are present. Sporocysts are ovoidal, 15.9 x 10.6 (15-18 x 9.5-11) microm, with Stieda and substieda bodies and a shape index of 1.50 (1.39-1.65). Each sporocyst contains an ellipsoidal sporocyst residuum, 8.0 x 5.8 (6-11 x 5-8), and each sporozoite contains two refractile bodies. No correlation was found between the presence of coccidian oocysts in the faeces of some of the birds and Atoxoplasma in blood smears.

Upton, S. J. and C. A. Zien (1997). "Description of a Giardia varani-like flagellate from a water monitor, Varanus salvator, from Malaysia." J Parasitol 83(5): 970-1.
	A Giardia varani Lavier, 1923-like flagellate was found in the feces of a captive water monitor, Varanus salvator, originally caught wild from an unknown location in Malaysia. The parasite is similar in size and shape to Giardia lamblia, except that median bodies are rare and cysts are binucleate. A description of both the trophozoite and cyst stage of this flagellate is provided.

Urban, J. F., Jr., R. Fayer, et al. (1996). "IL-12 protects immunocompetent and immunodeficient neonatal mice against infection with Cryptosporidium parvum." J Immunol 156(1): 263-8.
	The protozoan parasite Cryptosporidium parvum (Cp) causes diarrhea that can be acutely severe in immunocompetent persons and can become chronic and life-threatening in immunocompromised individuals. Because studies in mice have implicated IFN-gamma in protection against this parasite, and IL-12 can induce IFN-gamma production, we examined the ability of IL-12 to prevent or cure Cp infection in neonatal BALB/c and SCID mice. Treatment of both immunocompetent and immunodeficient mice with IL-12 before experimental inoculation with Cp oocysts prevented or greatly reduced the severity of infection. Intestinal epithelial cell invasion and/or early intracellular development of Cp was inhibited by exogenous IL-12. The protective effect of IL-12 was completely blocked by anti-IFN-gamma mAb. Established infections were associated with elevated IFN-gamma gene expression and were not ameliorated by IL-12 treatment, even though such treatment further enhanced IFN-gamma gene expression. The severity of Cp infection was, however, exacerbated by treatment with anti-IL-12 Ab. These observations provide the first evidence that treatment with exogenous IL-12 can prevent Cp infection through an IFN-gamma-dependent, specific immune system-independent mechanism, and that endogenous IL-12 production has a role in limiting Cp infection.

Urban, J. F., Jr., R. Fayer, et al. (1996). "Local TH1 and TH2 responses to parasitic infection in the intestine: regulation by IFN-gamma and IL-4." Vet Immunol Immunopathol 54(1-4): 337-44.
	Control of parasitic infections is dependent on the production of cytokines that activate mechanisms which limit invasion, reproduction or survival of the parasite. In contrast, conditions that induce inappropriate cytokine responses facilitate the spread of infection and ultimately exacerbate the level of disease. Measurement of local cytokine responses to different gastrointestinal parasites, such as the intracellular protozoan, Cryptosporidium parvum, and luminal dwelling nematodes like Nippostrongylus brasiliensis and Heligmosomoides polygyrus, reveal stereotype response patterns. In general, intracellular parasites stimulate type 1 responses where IFN-gamma is the predominant immune activator, while extracellular parasites stimulate type 2 responses where IL-4 plays a prominent role in elevating humoral immune mechanisms. Cytokines alter cellular function and the milieu of the intestinal lumen to affect the outcome of an infection. The importance of a particular response during the course of an infection can be studied by selective enhancement with an excess of exogenous recombinant cytokine or cytokine antagonists. For example, exogenous IL-12 enhances resistance to C.parvum, but suppresses the normally rapid cure of an infection with N. brasiliensis. Both mechanisms are dependent on expression of IFN-gamma. At the molecular level, exogenous IL-12 stimulates IFN-gamma production which elevates a protective type 1 response to C. parvum but converts the normally anti-worm type 2 response to a type 1 response that inappropriately regulates the infection. Alternatively, excess IL-4 plays a prominent role in modulating effector elements that change intestinal physiology to create a hostile environment for worm parasites. Exogenous IL-4 can cure chronic worm infection, while IL-4 antagonists interfere with protective responses to infection. These observations provide a paradigm for analysis of stereotype responses to different gastrointestinal parasites, and demonstrate how cytokine-induced immune system-dependent and independent effector mechanisms can limit parasitic infection, while inappropriate cytokine responses can exacerbate the state of disease.

van Keulen, H., P. T. Macechko, et al. (2002). "Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis." Vet Parasitol 108(2): 97-107.
	Giardia lamblia which parasitize humans belong to either of two genotypes, A or B, based on specific signature sequences in the 5' end of the small subunit (16S) ribosomal RNA (rRNA) gene. These two genotypes also were found in cysts from fecal samples of animal origin such as dogs, cats, some farm animals and wild animals. In addition, trophozoites recovered from cysts obtained from environmental samples belonged to these two genotypes as well, suggesting that the G. lamblia genotypes A and B are widespread and possibly zoonotic. Trophozoites were recovered from rats and these isolates might belong to another genotype of G. lamblia. Deer mice and one dog appeared to be parasitized by genotypes of Giardia with close affinity to G. microti. This species, therefore, also consists of a genotype complex.

van Knapen, F. (1989). "Toxoplasmosis, old stories and new facts." Int Ophthalmol 13(6): 371-5.
	In this introductory paper some general opinions are presented regarding the parasite Toxoplasma gondii, toxoplasma infection and toxoplasmosis. The transmission of the parasite through oocysts and/or infected meat is discussed with regard to possible prevention. Old stories have been replaced by new facts in the epidemiology of toxoplasma infection. Veterinary measurements in the field of prevention are not likely to be accepted in the near future. It is stressed that laboratory diagnosticians should clearly differentiate between diagnostic support in suspected cases of clinical toxoplasmosis or screening for certain characteristics such as IgG antibodies to toxoplasma in a healthy population.

Varma, M., J. D. Hester, et al. (2003). "Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay." J Microbiol Methods 53(1): 27-36.
	Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.

Vergara Castiblanco, C., S. Santos Nunez, et al. (2000). "[Cryptosporidiosis in the Andean region of Colombia: seroprevalence and recognition of antigens]." Rev Panam Salud Publica 8(6): 373-9.
	The objective of this study was to investigate, for the first time, the seroprevalence of cryptosporidiosis among urban and rural inhabitants in several departments of the Andean region of Colombia. The antigen recognition of Cryptosporidium parvum was also studied with sera. Between June 1996 and October 1998 1,778 serum samples were collected from people selected through convenience sampling. The detection of anti-C. parvum antibodies (IgM, IgA, and IgG) was carried out with enzyme-linked immunosorbent assay, and antigen recognition was done with immunoblotting. A prevalence of 83.3% was found, and the antibody percentages were 72.2% for IgM, 27.7% for IgA, and 27.6% for IgG. Higher seropositivity percentages were found among women, persons less than 30 years old, and residents of rural areas. IgM seroprevalence decreased with age, while IgG and IgA seroprevalences increased with age. These three immunoglobulin isotypes most frequently recognized the antigens from 51 to 69 kDa, which can be considered immunodominant. Of note was the immunoreactivity of IgM and IgA to protein fractions from 12 to 14 kDa and from 42 to 48 kDa, respectively, which could indicate exposure to the parasite. These results indicate that cryptosporidiosis is endemic in the Andean region of Colombia, and that it is possible to attribute many cases of diarrheal syndrome to C. parvum.

Vergara-Castiblanco, C. A., F. Freire-Santos, et al. (2000). "Viability and infectivity of two Cryptosporidium parvum bovine isolates from different geographical location." Vet Parasitol 89(4): 261-7.
	The viability of two Cryptosporidium parvum bovine isolates from Spain and Colombia was evaluated by in vitro excystation, inclusion/exclusion of two fluorogenic vital dyes (DAPI and PI) and infectivity assay in a suckling murine model. Excystation percentages were similar for both Spain and Colombia isolates (83% and 87%, respectively). The total viability of the Spain isolate, measured by inclusion/exclusion of two fluorogenic vital dyes, was 71% in comparison with that detected for oocysts of the Colombia isolate, 32.3%. The bovine C. parvum oocysts of both isolates were viable and infectious for suckling Swiss CD-1 mice. However, infectivity percentage and the mean intensity of infection were consistently higher in the Spain isolate than those from Colombia isolate. It was not possible to obtain a good correlation between in vitro excystation, inclusion/exclusion of vital dyes and in vivo infectivity for the Colombia isolate, while data obtained with the Spain isolate indicated that there was an apparent strong correlation between excystation efficiency, total viability and the infectivity. Although a comparative analysis of genetic variation among these isolates from different geographical location is necessary, variations observed between the both isolates seemed to be a result of parasite adaptation to environmental stresses such as temperature which appears to have a direct effect on the permeability of the oocysts.

Vergara-Castiblanco, C. A., J. Quilez-Cinca, et al. (2001). "Serological response to Cryptosporidium parvum in adult cattle from the Andean region of Colombia." Parasitol Res 87(6): 500-4.
	Single faecal and serum samples were individually collected from 135 asymptomatic adult cows on seven farms in Cundinamarca (Colombian Andean region). Tests for the presence of oocysts of Cryptosporidium parvum (carbol fuchsin stain) and Eimeria spp (flotation in saturated sal