(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