2001 Progress Report: Understanding Risk Factors to Cryptosporidium parvum: Studies in Gnotobiotic Pigs

EPA Grant Number: R826138
Title: Understanding Risk Factors to Cryptosporidium parvum: Studies in Gnotobiotic Pigs
Investigators: Ward, Lucy A.
Institution: The Ohio State University - Main Campus
Current Institution: The Ohio State University
EPA Project Officer: Hiscock, Michael
Project Period: February 20, 1998 through February 19, 2001 (Extended to December 19, 2002)
Project Period Covered by this Report: February 20, 2000 through February 19, 2001
Project Amount: $332,084
RFA: Drinking Water (1997) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water

Objective:

The goal of this project is to increase our knowledge and understanding of risk factors to infection and disease caused by the waterborne protozoan parasite Cryptosporidium parvum. We are using the gnotobiotic pig model to: (1) assess and compare the pathogenesis (infectivity and virulence) of C. parvum in the young versus older host; (2) evaluate the susceptibility and clinical responses of the immunosuppressed host to C. parvum; and (3) characterize the infected host's immune response (B cell, T cell) to disease caused by C. parvum infection.

We are studying two bovine C. parvum strains in our gnotobiotic pig model: GCH1 (Grafton Compton Human I) strain was originally obtained by Dr. Saul Tzipori, Tufts University, Grafton, MA, from an AIDS patient, and the OH (Ohio) strain was obtained by the Principal Investigator (Dr. Lucy Ward) from an immune competent adult laboratory worker with clinical cryptosporidiosis. Both bovine strains have been "cloned" from a single oocyst and are now being maintained in neonatal gnotobiotic pigs following passage in neonatal calves for one (OH) to several (GCH1) years. We also are performing infection and pathogenesis studies in gnotobiotic pigs with human and pig C. parvum, C. meleagridis, and C. canis strains provided by Dr. Lihua Xiao, Centers for Disease Control and Prevention, Atlanta, GA. All these strains originated from infected human feces due to naturally acquired childhood infections and/or infections associated with U.S. waterborne disease outbreaks.

Progress Summary:

In collaboration with Dr. Lihua Xiao, we are genotyping/subgenotyping all Cryptosporidium isolates used in these gnotobiotic pig studies. It is important to note that all strains are maintained in our laboratory using sequential low-dose infection (inoculation doses of 1 to 10 oocysts) of gnotobiotic pigs. To date, we have successfully passaged two bovine (genotype 2) C. parvum strains [GCH1 and OH], four human (genotype 1) C. parvum strains [H2132, H2265, H2576, H3438], and two C. meleagridis strains [Av2567 and Av4500], and have derived single oocyst clones from most these isolates. Molecular analyses of sequential gnotobiotic pig passages have revealed genetically stable parasite populations. Subgenotyping of H2132 and H2265 currently suggests these two strains, though derived from U.S. waterborne disease outbreaks at geographically separated sites (one from Nebraska and one from North Carolina), are the same. Genotyping/subgenotyping studies also have shown that cross-contamination of strains has occurred (see C. canis studies below) although only once during initial pig passage; we have otherwise been able to propagate our single oocyst clones free of cross-contamination.

Detection of C. parvum in Experimental Samples

Fecal Materials: We previously reported and published an Acid Fast-Ultraviolet light (AF-UV) procedure that enhances the AF stain sensitivity to levels comparable with a commercially available IFA product (MERIFLOUR, Meridian Diagnostics, Inc., Cincinnati, OH) (Nielsen and Ward, 1999) More recently, we developed and published a relatively rapid, inexpensive method for extracting PCR quality DNA from frozen fecal specimens that allows detection of a single C. parvum oocyst (Ward and Wang, 2001). This procedure involves the use of glassmilk (silica suspension) to directly extract C. parvum DNA from feces and is thus useful for specimens in which the oocyst integrity is disrupted, such as frozen samples. We further showed that, when combined with nested PCR, our method repeatedly allowed for detection of a single C. parvum organism in frozen fecal materials (Ward and Wang, 2001).

Intestinal Tissues: We previously reported developing a standard curve quantitative competitive (QC)-PCR assay to determine the number of C. parvum organisms in the intestinal tissues (duodenum, jejunum, ileum, and colon) of gnotobiotic pigs following low-dose inoculations (Wang, 2000). We showed that the number of C. parvum organisms per microgram of intestinal DNA varied significantly by intestinal segment (p < 0.016) and day post-infection (PI) (p < 0.001). Following single bovine C. parvum oocyst inoculation, the greatest number of C. parvum organisms per microgram of intestinal DNA was found in the colon at 10 days PI (5.6 x 104 organisms) and the earliest but lowest detectable number of organisms/microgram DNA was in the ileum at 5 days PI (nine organisms). Parasitic DNA was not detectable in intestinal tissues at 0 and 3 days PI. The QC-PCR proved useful for monitoring the developing intestinal parasitemia in the gnotobiotic piglet model of cryptosporidiosis and was more sensitive and rapid than conventional microscopic screening of AF-stained smears or Haematoxylon & Eosin (H&E)-stained tissues.

Minimum Infective Dose (MID), Median (50 percent) Diarrhea Dose (DD50), Median Lethal Dose (LD50)

Bovine C. parvum: We have successfully maintained our OH and GCH1 single oocyst clones, which we derived from our parental stock (Wang, 2000) through sequential low dose (5 to 10 oocysts) passages in gnotobiotic pigs (Pereira, 2001; Pereira, et al., 2001). The infectivity of these clones has proven to be relatively stable up to 6 months at 4 C and their MID to be the same as that of the parental strains (a single oocyst). The DD50 and LD50 of our GCH1 and OH clones are low (< 5 oocysts and < 100 oocysts) and suggest to us that our clones are more virulent than their respective parental strains (parental GCH1 DD50 and LD50 = 730 oocysts and 106 oocysts, respectively; and for parental OH, 6900 oocysts and 5 x 108 oocysts, respectively). We also have found less variability in the clinical responses of gnotobiotic pigs to different passages of our clones, suggesting that our clone low-dose passage program is effective in maintaining relatively stable genetic parasite populations.

Human C. parvum: We currently have six human C. parvum strains available for study (H2132, H2265, H2546, H2576, H3438, and H3439) and have successfully derived single oocyst clones and/or low dose passages with four of these isolates (H2132, H2265, H2576, and H3438). We thus propose the MID of human C. parvum for neonatal gnotobiotic pigs also is a single oocyst. The DD50 for our cloned human C. parvum strains is similarly low (< 20 oocysts), but the diarrheal disease associated with the human C. parvum infections is not as severe as that observed with the bovine C. parvum (see Pathogenesis studies below). Consequently, the LD50 of human C. parvum for neonatal gnotobiotic pigs is high and currently known to be >105 oocysts for any given strain.

C. meleagridis: We have five C. meleagridis strains available for study (Av2567, Av4500, Av4501, Av4508, and Av4509) and have successfully propagated two of these strains (Av2567 and Av4500) in neonatal gnotobiotic pigs using an initial infective doses of 104 to 105 oocysts. Verification of these strains as C. meleagridis via genotyping/subgenotyping is pending. We have infected pigs with as few as 10 oocysts and thus predict the MID for C. meleagridis also to be a single oocyst and thus will be able to derive single oocyst clones from these strains as well. From our preliminary passages we further predict the DD50 and LD50 of C. meleagridis to be like that of human C. parvum with the DD50 being low and the LD50 relatively high.

C. canis: We previously reported successful propagation of C. canis (K9-2562 strain) in gnotobiotic pigs. This report was based on the detection of oocysts in pig feces by AF-UV (Nielsen & Ward, 1999) at 12 to 18 days PI (average 14.6 + 2.2 days) following inoculation with 104 C. canis oocysts at 1 day of age. However, molecular analysis (genotyping) of the Cryptosporidium-infected pig feces revealed only bovine C. parvum even though the C. canis genotype could be re-isolated from the human feces used to prepare the pig innocuously We thus concluded that at least one of the pig inocula had been contaminated with bovine C. parvum during laboratory preparation as bovine C. parvum also was being prepared for inoculation that same day and its MID is known to be a single oocyst. Consequently, two additional human fecal samples containing C. canis (K9-4495 and K9-4496) were obtained and 5 x 104 oocysts per strain per pig used for inoculation. This time, neither strain elicited productive infection as determined by AF-UV by 12 days PI; consequently, pigs were given a second inoculum of 105 C. canis oocysts each and monitored 2 additional weeks at which time the experiment was terminated. Pigs continued to fail to produce a productive infection as determined by AF-UV although PCR detection of C. canis in collected feces/intestinal contents is pending. From our preliminary findings thus far, we conclude that neonatal gnotobiotic pigs may not be readily susceptible to infection with C. canis and/or neonatal gnotobiotic pigs develop subclinical C. canis infections (and thus should be detectable by PCR) and/or the C. canis inocula have not been viable as the human fecal samples had been stored for 3 to 6 months prior to oocyst extraction (this also is difficult to prove/disprove without availability of an alternative infectivity assay).

Porcine C. parvum: The pig C. parvum inocula used for this study (strain P4193) had been stored at 4°C for 9+ months prior to oocyst extraction, and the viability of oocysts was unknown; hence the actual infective dose is probably much lower than what we report here. Approximately 104 pig genotype C. parvum (strain P4193) were inoculated into 2-day-old gnotobiotic pigs. The P4193 strain did not elicit productive infection as determined by AF-UV by 11 days PI at which time a second P4193 inoculum of 103 oocysts was given. A productive infection as determined by AF-UV failed to be detected following the second inoculation, and the experiment was terminated 2 weeks later. Molecular analysis (PCR detection and genotyping) of the collected feces/intestinal contents is pending. However, we have concluded from these preliminary studies that the gnotobiotic pigs were either not infected with the pig C. parvum most likely due to the innocuously being non-viable and/or pigs developed a subclinical infection with the pig C. parvum (and thus should be detectable by PCR). Development of a subclinical infection is further substantiated by pathogenesis studies (see below).

Pathogenesis Studies

Bovine and Human C. parvum: We are continuing to perform comparative C. parvum pathogenesis studies using bovine strains GCH1 and OH, and human strains H2132, H2265, H3438, and H34396 (Pereira, 2001; Pereira, et al., 2001 submitted; Morgan-Ryan, et al., 2002 in preparation). As noted earlier, our observations of different LD50s and disease severity between the bovine and human strains suggested to us that additional differences in the pathogenesis of these C. parvum were likely to exist. Our current findings are summarized in brief below. Our results clearly are providing support to the hypothesis that human and bovine C. parvum genotypes may indeed represent separate species.

(1) Clinical disease and oocyst shedding. Oocyst shedding was evaluated by AF-UV (Nielsen & Ward, 1999). The onset to oocyst shedding (or prepotent period) was consistently longer for human C. parvum (7 to 14 days) compared to bovine C. parvum (4 to 7 days). The length of oocyst shedding (or patent period) also was consistently longer for human C. parvum (13 to 26 days) compared to bovine C. parvum (5 to 16 days). Feces were scored daily from 0-3 where 0 = normal, 1 = pastey, 2 = semi-liquid, and 3 = liquid. Diarrhea was deemed present at scores > 2. The diarrheal disease in pigs during the first week post-onset of oocyst shedding was consistently less severe with human C. parvum strains (daily diarrhea scores ranged from 0.63 to 1.89) compared to bovine C. parvum (daily diarrhea scores ranged from 1.63 to 2.78). Uninfected control pigs had an average daily fecal score of 0.48 + 0.8 SE. Bovine C. parvum also induced moderate to severe dehydration, weight loss, protruding vertebrae, and sunken eye appearance in pigs during the patent (shedding) period whereas human C. parvum elicited only mild to no dehydration with minimal to no weight loss.

(2) Morphologic changes in the intestinal tissues. Microscopic examination of H & E stained tissue sections showed bovine C. parvum parasites throughout the duodenum, jejunum, ileum, and colon. Bovine C. parvum parasites were detectable in the intestinal tissues of the pigs killed during the prepatent (3 days post-infection [DPI]) and patent (0-21 days post-onset [DPO]) periods. By comparison, human C. parvum parasites of strain H2132 and H2265 were seen only during the patent period and in the ileum and colon, and no human C. parvum parasites have been found in the duodenum and upper jejunum. However, at the onset of shedding, consistently more human C. parvum parasites per villus can be observed in the ileum compared to bovine C. parvum (develop greater infection intensities). Bovine C. parvum elicits moderate lymphoid hyperplasia with scattered inflammatory cell (neutrophils) infiltrates at the onset of shedding in the duodenum, at 7 DPO in the jejunum and ileum, and 7 and 21 DPO in the colon. With human C. parvum, lymphoid hyperplasia generally is observed only during the patent period and only mild lymphocytic infiltrates with rare inflammatory cells are seen at this time in the ileum and colon. Mild to moderate villus attenuation with epithelial sloughing was seen at the onset of shedding and 7 DPO in the duodenum, and 21 DPO in the colon with bovine C. parvum, whereas human C. parvum elicited only mild villus/mucosa attenuation, which was generally restricted to the ileum and colon during the patent period. These preliminary studies suggest significant differences may exist between the virulence of human strains as H3438 and H2576 infected pigs had detectable parasites throughout their small and large intestines and developed more severe diarrhea than H2132 and H2265 infected pigs. However, because genotypic analysis of these pigs' feces/intestinal contents is pending, we cannot definitively conclude that lesions observed are due to human C. parvum infection.

(3) Acid fast (AF)-stained mucosal smears. Mucosal smears were prepared from fresh tissue sections of duodenum, jejunum, ileum, and colon taken from C. parvum-infected pigs at necropsy. At 1 to 7 DPO, more bovine C. parvum GCH1-infected pigs had AF-positive parasitic stages within their duodenal and jejunal mucosal smears than did OH infected pigs. By 10-14 DPI and 21-24 DPI, similar numbers of OH and GCH1 infected pigs showed AF-positive staining in ileal and colonic smears. Mucosal smears at 50 DPI were negative. At 1 to 7 DPO, AF-positive parasitic stages were observed only in the idea and colons of the human C. parvum H2132 and H2265-infected pigs but in all sections (duodenum, jejunum, ileum, colon) of H2576 and H3438-infected pigs (please note that genotyping studies on these pigs is pending and hence confirmation that they are human C. parvum infected remains to be determined). The AF-positive human C. parvum parasites consistently remained detectable in colonic mucosal smears at 15-22 DPO, but were variable in ileal mucosal smears at this time. Human C. parvum parasites were not observed in mucosal smears obtained at 50 DPO.

C. meleagridis:

(1) Clinical disease and shedding. Oocyst shedding was detected by AF-UV from 6 to 9 days PI and oocyst shedding was observed for 16 to 28 days. Diarrhea was moderate with daily fecal scores ranging from 0.5 to 2.75 (on a scale of 0 to 3 where 0 = normal, 1 = pastey, 2 = semi-liquid, 3 = liquid) and averaging 2.0 + 0.26.

(2) Morphologic changes in the intestinal tissues. Microscopic evaluation of H & E stained tissues collected at necropsy are pending.

(3) Acid fast (AF)-stained mucosal smears. Numerous C. parvum organisms were observed in the dital jejunum, ileum, and colonic mucosal smears taken at necropsy. Microscopic evaluation of H & E stained tissues and molecular analysis (PCR detection and genotyping) of collected feces/intestinal contents are pending.

Porcine C. parvum:

(1) Clinical disease and shedding. Oocyst shedding was not detected by AF-UV on feces; consequently, onset and duration of shedding could not be determined. However, feces were scored daily from 0-3 (0 = normal, 1 = pastey, 2 = semi-liquid, 3 = liquid) and softening of the stools (scores of 2-3) were noted for 2 to 3 days approximately 5 to 8 days after each inoculation.

(2) Morphologic changes in the intestinal tissues. Microscopic evaluation of H & E stained tissues collected at necropsy are pending.

(3) Acid fast (AF)-stained mucosal smears. Two AF-UV positive C. parvum organisms were observed in ileal mucosal smears taken at necropsy from one pig. Microscopic evaluation of H & E stained tissues and PCR detection of pig C. parvum in collected feces/intestinal contents are pending. We thus conclude from these preliminary studies that pigs may have been infected subclinical with pig C. parvum P4193 (as genotypic analysis of feces/intestinal contents is pending).

C. canis:

(1) Clinical disease and shedding. Oocyst shedding was not detected by AF-UV on feces; consequently, onset and duration of shedding could not be determined. Further, stools were scored a maximum of two for a day or two immediately after each inoculation.

(2) Morphologic changes in the intestinal tissues. Microscopic evaluation of H & E stained tissues collected at necropsy are pending.

(3) Acid fast (AF)-stained mucosal smears. No C. parvum organisms were observed in any of the mucosal smears taken at necropsy. Microscopic evaluation of H & E stained tissues and molecular analysis (PCR detection and genotyping) of collected feces/intestinal contents are pending.

Dual Infection Studies with Bovine and Human C. parvum

Distinct genotypic and phenotypic (see Pathogenesis Studies above) differences between human and bovine C. parvum have been identified that strongly suggest these genotypes are separate and distinct species (see Morgan-Ryan, et al., 2002). If the bovine and human C. parvum genotypes represent separate species, then we postulate that free exchange of genetic materials will not occur in a dually infected host as has been suggested by some. Thus, dual infection of gnotobiotic pigs with bovine and human C. parvum are underway to definitively assess the ability of these two genotypes to exchange genetic material. Pigs were dually infected with 10 GCH1 oocysts and 103 H2265 oocysts and killed at selected days post-onset of shedding to evaluate tissues and fecal contents. Oocyst shedding commenced between 5 to 7 days post-infection (consistent with bovine C. parvum prepotent period) and lasted 21 days (consistent with human C. parvum patent period). Pigs developed relatively severe diarrhea (average daily fecal score 2.68 + 0.17) the first week post-onset of shedding accompanied by dehydration and weight loss. Molecular analysis of the feces/intestinal contents collected at necropsy and microscopic evaluation of AF-stained mucosal smears and H & E stained tissues are pending.

Immunosuppression Studies

It was readily apparent from our infectivity studies that the status of a pig's immune system (i.e., immune competent vs. immunosuppressed) could not be a factor in risk of infection as the minimum infective dose for all our strains is one oocyst. Consequently, we focused our immunosuppression studies on the role of an intact immune system in the risk of developing disease. We are using an immunosuppression protocol of oral dexamethazone/prednisolone (dex/pred) in neonatal gnotobiotic pigs inoculated with low dose (10 oocysts) human (H2265) or bovine (GCH1) C. parvum. Preliminary findings reveal the prepotent period is shorter and the patent period longer in dex/pred treated GCH1-inoculated pigs compared to untreated GCH1-inoculated pigs (prepotent period: 5.5 vs. 8 days; patent period: 14 days vs. 8 days). The clinical disease observed, however, was similar between the dex/pred treated and untreated GCH1-infected pigs (average daily fecal scores 1 week DPO were 2.2 + 0.25 vs. 2.14 + 0.19 on a scale of 0 to 3 where 0 = normal, 1 = pastey, 2 = semi-liquid, and 3 = liquid). By comparison, H2265-inoculation of dex/pred treated pigs did not elicit a productive infection as determined by AF-UV of daily fecal smears although softening of the stools was observed for a few days post-inoculation. Further pigs are needed to determine the validity of this observation. Histologic evaluation of tissues and genotypic analysis of the feces/intestinal contents collected at necropsy are pending.

Intestinal Microflora Studies

Studies in mice show that intestinal microflora or specific probiotic bacteria (Lactobacillus reuteri) may shorten the duration of bovine C. parvum oocyst shedding. The effect of such bacteria on cryptosporidiosis (the disease caused by C. parvum) cannot be adequately assessed in mice because mice are only susceptible to disease for a short period of time (first several days of life), they require high doses of bovine C. parvum oocysts to get infected (> 1,000-10,000 oocysts), and they are not "colonized" by human intestinal microflora bacteria. Further, recent studies have shown that the majority (75 percent) of C. parvum infections in humans are due to genotype 1 (human) C. parvum, but mice are resistant to infection by human strains. Consequently, we initiated studies on the role of the intestinal microflora in risk to infection and disease by C. parvum. These studies have been done in collaboration with Dr. Polly Courtney, Food Science & Technology, The Ohio Sate University, Columbus, OH. Preliminary results from these studies using bovine C. parvum have been described (Foster, et al., 2001; Foster, et al., 2002) and are summarized below.

In Vitro Studies: We developed an in vitro flow cytometric assay based on the inclusion or exclusion of vital dyes to assess the effect of L. acidophilus, L. reuteri, B. breve, and B. longum cell supernatants on the viability of C. parvum oocysts (the infective stage of the parasite). We found that undiluted, 1:2, and 1:5 dilutions of both Lactobacillus spp. supernatants significantly reduced the percentage of viable C. parvum oocysts compared to broth controls. There was no significant difference between the percent of viable oocysts after incubation with L. acidophilus compared to L. reuteri supernatants except at a dilution of 1:2. Only the undiluted supernatant of B. longum significantly reduced the percent of viable oocysts compared to broth controls.

Microbial Colonization of Neonatal Pigs: Preliminary studies were performed in conventional and gnotobiotic neonatal pigs to determine whether animals could be "colonized" with human strains of L. acidophilus, L. reuteri, L. casei, and B. longum. Although several (70 percent) conventional neonatal colostrum-deprived pigs could be colonized with Lactobacilli, as many pigs developed secondary complications of sepsis and concurrent E. coli infections, which were fatal in 30 percent of animals. By comparison, neonatal gnotobiotic pigs remained healthy following a 3-day feeding regimen of the bacteria beginning at 1 day of age, and bacteria were re-isolated from segments of the lower small bowels at 1-2 weeks and 3-4 weeks later.

Probiotic Efficacy Studies: A small preliminary study has been performed in neonatal gnotobiotic pigs to determine the effect of feeding L. acidophilus, L. reuteri, B. breve, and B. longum on the pathogenesis of bovine (genotype 2) C. parvum. Day-old gnotobiotic pigs were fed 105 CFU bacteria or broth (controls) daily for 3 days and then orally challenged with 102 to 104 bovine (genotype 2) C. parvum oocysts. At 15 and 25 days post-inoculation, pigs from each group were killed and samples collected for analysis. Significant differences between groups were not obtained as the sample numbers were too small (n = 2-4 pigs per group). However, pigs fed the Lactobacilli developed less severe diarrhea and shed fewer oocysts for a shorter duration compared to controls, whereas pigs fed the Bifidiobacteria shed the same amount of oocysts for the same duration as controls but developed more severe diarrhea than controls.

Studies on Host Immunity to C. parvum

Cytokine Responses: We have investigated and are reporting on the tissue cytokine mRNA levels during the course of bovine C. parvum-induced disease in gnotobiotic pigs as assessed by a semi-quantitative RT-PCR of IL-12 (p40), IFNg, TNFa, IL-10, and IL-6 mRNA levels (Pereira, 2001; Pereira, et al., 2002 submitted). Briefly, day old gnotobiotic pigs were orally inoculated with approximately 5x106 (parental) bovine C. parvum oocysts and portions of their small and large intestines collected for morphologic evaluation and cytokine analysis at post-inoculation days (PID) 3, 10, 21, and 50. Clinical disease was observed in all pigs by PID 4 and was resolved by PID 16. At PID 3, all cytokine mRNA levels were higher than negative controls in the small intestinal tissues and heavy mucosal oocyst infection rates with villus atrophy and mixed neutrophilic, lymphocytic infiltrates in the lamina propria were observed histologically. However, colonic cytokine activity and histologic changes were no different than negative controls at this time. At PID 10, small intestinal cytokine mRNA levels also remained higher than negative controls at this time with duodenal activity being the greatest among tissues. Elevated cytokine mRNA activity was first observed in the colon, which also had the greatest mucosal oocyst infection rates and mucosal morphology changes. At PID 21, cytokine mRNA levels increased in colon and ileum, but decreased in the duodenum (compared to negative controls). Histologically, small intestinal tissues showed only mild attenuation and no oocysts observed, whereas colonic tissues showed prominent lymphoid hyperplasia and moderate oocysts infection rates. By PID 50, no oocysts were observed and cytokine mRNA activity in all tissues had reduced to levels similar to the negative controls. These results demonstrate a strong time-dependant correlation between bovine C. parvum-elicited cytokine responses and bovine C. parvum-associated pathology and disease.

Antibody Responses: We previously reported on the use of an isotype-specific ELISA to evaluate IgM, IgG, and IgA antibody responses in gnotobiotic pigs infected with bovine C.parvum. These studies were done in collaboration with Drs. Karol Sestak and Saul Tzipori at Tufts University School of Veterinary Medicine. They showed a very limited and slow to develop humoral immune response that was elicited by high-dose bovine (genotype 2) C. parvum inoculation. Analysis of sera from low-dose bovine and human C. parvum-inoculated pigs is underway. More recently, the sera from our high-dose bovine C. parvum-inoculated pigs were used to show the antigenic relatedness between human and bovine C. parvum surface glycoproteins (Sestak, et al., 2002).

Significance

The risk of transmission of the protozoan parasite Cryptosporidium to humans via drinking water is multi-factorial and includes not only the conditions under which Cryptosporidium is introduced and survives in water, but knowledge of the minimum infective dose, strain involved, strain virulence, and a variety of individual host factors. From these studies, we propose that the minimum infective dose of the genus Cryptosporidium for susceptible hosts is a single viable oocyst, regardless of the host's age or immune status, as ingestion of one human C. parvum, bovine C. parvum, or C. meleagridis oocyst by gnotobiotic pigs consistently elicits diarrheal disease and oocyst shedding. Our findings also support the hypothesis that human and bovine C. parvum represent separate and distinct species. Consequently, we are strong proponents for designation of a new Cryptosporidium species, C. hominis. Our findings also demonstrate that the risk of developing disease following Cryptosporidium infection is determined not only by parasite-specific factors (such as strain, genotype, or species) but also by host-specific factors (such as the presence/absence of an intact immune system and composition of the intestinal microflora). Our humoral immunity studies to date demonstrate that even the immune competent host suffering from moderate to severe cryptosporidial disease slowly develops an immune response of relatively low magnitude, especially when compared to other enteric pathogens such as rotavirus, and begin to provide insight into why most hosts are susceptible to multiple infections with this parasite.

Future Activities:

Studies in immunosuppressed animals are in progress and expected to be completed by fall 2002. We will derive and maintain the Cryptosporidium clones from those isolates that we currently have in hand. Pending pathogenesis studies should be completed by year's end. Completion of the dual infection studies is expected by fall and will help to conclusively establish the genetic relatedness between human and bovine C. parvum. Humoral immunity studies will focus on bovine and human C. parvum and should be completed by mid-year. We currently are determining cytokine mRNA responses to low dose bovine and human C. parvum using the real-time PCR assay and anticipate completion of these studies by mid-summer. If time permits, we also will assess cytokine mRNA responses to other cryptosporidial strains.


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 41 publications 8 publications in selected types All 6 journal articles
Type Citation Project Document Sources
Journal Article Morgan-Ryan, UM, Fall A, Ward LA, Hijjawi N, Sulaiman I, Fayer R, Thompson RCA, Olson M, Lal A , Xiao L. Cryptosporidium hominis n. sp (Apicomplexa: Cryptosporidiidae) from Homo sapiens. Journal of Eukaryotic Microbiology 2002;49(6):433-440. R826138 (2001)
R826138 (Final)
  • Abstract from PubMed
  • Journal Article Nielsen CK, Ward LA. Enhanced detection of Cryptosporidium parvum in the acid-fast stain. Journal of Veterinary Diagnostic Investigation 1999;11(6):567-569. R826138 (1999)
    R826138 (2000)
    R826138 (2001)
    R826138 (Final)
  • Abstract from PubMed
  • Other: JVDI PDF
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  • Journal Article Sestak K, Ward LA, Sheoran A, Feng X, Akiyoshi DE, Ward HD, Tzipori S. Variability among Cryptosporidium parvum genotype 1 and 2 immunodominant surface glycoproteins. Parasite Immunology 2002;24(4):213-219. R826138 (2001)
    R826138 (Final)
  • Abstract from PubMed
  • Full-text: InterScience Full Text
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  • Other: InterScience PDF
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  • Journal Article Ward LA, Wang Y. Rapid methods to isolate Cryptosporidium DNA from frozen feces for PCR. Diagnostic Microbiology and Infectious Disease 2001;41(1-2):37-42. R826138 (2000)
    R826138 (2001)
    R826138 (Final)
  • Abstract from PubMed
  • Supplemental Keywords:

    dose-response, drinking water, pathogens, immunology., RFA, Health, Scientific Discipline, Water, Environmental Chemistry, Health Risk Assessment, Chemistry, Risk Assessments, Biochemistry, Drinking Water, cryptosporidium parvum oocysts, pathogens, public water systems, risk factors, microbial risk assessment, waterborne disease, exposure and effects, disinfection byproducts (DPBs), exposure, dose response, community water system, gnotobiotic pigs, human exposure, susceptibility, treatment, dietary ingestion exposures, drinking water contaminants, infectivity, water treatment

    Progress and Final Reports:

    Original Abstract
  • 1998
  • 1999 Progress Report
  • 2000 Progress Report
  • Final Report