2000 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
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, 1999 through February 19, 2000
Project Amount: $332,084
RFA: Drinking Water (1997) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water

Objective:

The project's goal is to increase knowledge and understanding of risk factors to Cryptosporidium parvum, a protozoan parasite that causes a disease called "cryptosporidiosis." We are using a gnotobiotic pig model to: (1) assess and compare the pathogenesis (infectivity and virulence) of C. parvum in neonatal versus older gnotobiotic pigs; (2) evaluate the susceptibility and clinical responses of immunosuppressed gnotobiotic pigs to C. parvum; and (3) characterize the humoral (B cell), cellular (T cell), and cytokine immune responses in gnotobiotic pigs with cryptosporidiosis.

We have utilized two genotype 2 (bovine) C. parvum isolates GCH1 (Grafton Compton Human I) originally obtained by Dr. Saul Tzipori, Tufts University, Grafton, MA, from an AIDS patient, and OH (Ohio) obtained by the PI (Dr. L. Ward) from an immune competent adult laboratory worker with clinical cryptosporidiosis. Both genotype 2 strains are now being maintained in neonatal gnotobiotic pigs after one (OH) to several (GCH1) years of passage in neonatal calves. As a result of collaborative studies with Dr. Lihua Xiao, CDC, Atlanta, GA, we recently begun studies using additional Cryptosporidium strains including isolates of genotype 1 (human) C. parvum, C. canis, and C. meleagridis. All these isolates originated from humans due to naturally-acquired childhood infections and/or infections associated with a US waterborne disease outbreak.

Progress Summary:

A. Genotyping Studies

In collaboration with Dr. Lihua Xiao at CDC in Atlanta, we have been enable to "subgenotype" our Cryptosporidium isolates and are showing relatively consistent genetic stability within our "cloned" type 2 isolates following sequential (low dose) passages in neonatal gnotobiotic pigs. We also have been able to propagate three different strains of genotype 1 (human) C. parvum, 1 strain of C. canis, and 1 strain of C. meleagridis in our neonatal gnotobiotic pig model, and are in the process of cloning these strains and testing for genetic stability. As found with our type 2 clones, molecular analyses of the first few gnotobiotic pig passages of two "cloned" human C. parvum strains (H2132 and H2265) suggests ?genetic' stability within the type 1 C. parvum strains as well. Further, we have been able to clearly demonstrate that there has been no "cross-contamination" of our clones during derivation and propagation by subgenotyping each passage pool.

B. Detection of C. parvum in Experimental Samples

Fecal Materials: We previously reported a procedure to enhance the Acid-Fast stain sensitivity to levels comparable with a commercially available IFA product (MERIFLOUR, Meridian Diagnostics, Inc., Cincinnati, OH) (Nielsen and Ward, 1999). We subsequently developed a rapid, inexpensive method for extracting PCR quality DNA from frozen fecal specimens which allows detection of a single C. parvum oocyst (Ward and Wang, 2001).

Intestinal Tissues: We developed 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). Using this standard curve QC-PCR assay, we have found that the number of C. parvum organisms per microgram of intestinal DNA varies significantly with the intestinal segment (p<0.016) and day PI (p<0.001). Following inoculation of gnotobiotic pigs with one or two cloned OH C. parvum oocysts, we found the greatest number of C. parvum organisms per microgram of intestinal DNA was 5.6 x 104 in the colon at 10 days PI whereas the lowest detectable number was 9 organisms/microgram DNA in the ileum at 5 days PI. We could not detect any parasitic DNA within the intestinal tissues at 0 and 3 days PI. The QC-PCR proved to be more sensitive and rapid than the conventional microscopic screening of AF stained tissue smears or H & E stained tissue sections. These results demonstrate the usefulness of the QC-PCR to monitor the developing intestinal parasitemia in the gnotobiotic piglet model of cryptosporidiosis.

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

Genotype 2 (bovine) C. parvum: We successfully derived single oocyst clones from our ?parental' OH and GCH1isolates (Wang 2000), and have maintained these clones through sequential low dose (5 to 10 oocysts) passages in gnotobiotic pigs. The infectivity of our clones has proven to be relatively stable up to four months at 4C. The MID for each clone was determined to be 1 oocyst and hence similar to that of the parental strains (<5 oocysts for both GCH1 and OH). However, each clone's DD50 and LD50 was found to be much lower than that of the parental isolates (DD50 and LD50 < 5 oocysts for both clones whereas DD50 and LD50 for parental GCH1 were 730 oocysts and 106 oocysts, and for parental OH, 6900 oocysts and 5 x 108 oocysts, respectively). These findings suggest the clones are more virulent in the neonatal gnotobiotic pig than the parental isolates. Further, variability in the clinical responses of gnotobiotic pigs to the different pools of our C.parvum clones are minimal to non-existent, suggesting that the cloning procedure effectively derived a genetically homogenous (clonal) population of parasites compared to the parental strains.

Genotype 1 (human) C. parvum: We have propagated three type 1 (human) C. parvum strains (H2132, H2265, and H2576) in neonatal gnotobiotic pigs. Similar to our type 2 parental strains, each type 1 parental strain has demonstrated a MID of 1 to 5 oocysts, and we have successfully cloned 2 of 3 strains to date (H2132 and H2265). Our preliminary data strongly suggest that our parental type 1 strains have significantly greater DD50's and LD50's in neonatal gnotobiotic pigs compared to our parental type 2 strains. We are currently estimating the DD50 for H2132 and H2265 to be greater than 106 oocysts and for H2576 to be >104 to 105 oocysts. We do not know if we will be able to successfully determine these strain's LD50's.

C. canis: We have successfully infected and propagated one C. canis strain (K9-2562) in neonatal gnotobiotic pigs using an initial infective dose of 104 oocysts. Cloning of this strain and determination of its' MID, DD50 and LD50 are underway.

C. meleagridis: We have successfully infected and propagated one C. meleagridis strain (Av2567) in neonatal gnotobiotic pigs using an initial infective dose of 105 oocysts. Cloning and determination of this strain's MID, DD50 and LD50 are underway.

D. Morphologic Studies

Genotype 2 (bovine) C. parvum:

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 days post-onset (DPO) of fecal oocyst shedding, more GCH1-infected pigs had AF-positive parasitic stages within their duodenal and jejunal mucosal smears than did OH infected pigs. By 10-14 days post-infection (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 not evaluated.

Hematoxylin & Eosin (H & E) stained tissue sections?We previously described the histologic changes in intestinal tissues of pigs inoculated with high doses (5 x 106 oocysts) of our parental type 2 isolates GCH1 and OH (Ward et al 1998; Pereira et al 2000). We have subsequently evaluated H & E stained tissue sections from pigs infected with 1 to 5 cloned GCH1 oocysts at 1 day of age and have found similar histologic changes BUT these changes/lesions do not develop until the onset of fecal oocyst shedding at 6 DPI. At 2-4 DPI (which corresponds to the time when we saw substantial lesion development in the high dose parental strain studies), mucosal parasitism is scarce to non-existent and little to no villous attenuation or lymphoid hyperplasia is observed. By 21-24 DPI (which corresponds to the time when mucosal parasitemia is virtually cleared in the high dose parental strain studies), moderate to high numbers of oocysts could still be observed along the mucosal surface of the ilea and colons. By 50 DPI, villi had returned to normal and there was no evidence of mucosal parasitemia (only lymphoid hyperplasia persisted). Preliminary studies of low dose cloned OH show similar onset and lesions as observed with low dose cloned GCH1 except that low dose cloned GCH1-infected pigs had greater infection rates in their upper small bowels than did low dose cloned OH-infected pigs during the patent period.

Genotype 1 (human) C. parvum:

Acid fast (AF)-stained mucosal smears?Mucosal smears were prepared from fresh tissue sections of duodenum, jejunum, ileum, and colon taken from type 1 C. parvum-infected pigs at necropsy. At 1 to 7 DPO, AF-positive parasitic stages were observed only in the ilea and colons of the H2132 and H2265-infected pigs but in all sections (duodenum, jejunum, ileum, colon) of H2576-infected pigs. At 15-22 DPO for strain H2265, AF-positive organisms remained detectable in all colonic mucosal smears but in only 1 of 3 ileal mucosal smears. Evaluation of mucosal smears obtained at other time points are underway.

Hematoxylin & Eosin (H & E)-stained tissue sections?Microscopic evaluation of H & E stained tissue sections from pigs infected with 1 to 105 oocysts of the type 2 C. parvum strains were evaluated. Evaluation of H & E stained tissue sections for strain H2132 at 1 to 7 DPO have revealed large numbers of oocysts in the lower ileum and colon with only mild villous attenuation and lymphoid hyperplasia. Only one section of duodenum from one pig had a single cluster of 7 oocysts. No other changes were noted in all other duodenal and jejunal sections. Evaluation of H & E stained tissue sections at other time points and for other isolates are underway.

E. Cytokine Responses

We previously developed a semi-quantitative RT-PCR assay to measure tissue cytokine mRNA levels during the course of cryptosporidiosis in the gnotobiotic pig model (Pereira, et al., 1999). We subsequently correlated cytokine mRNA expression with observed histological changes and clinical disease in gnotobiotic pigs infected with high dose type 2 parental C. parvum (Pereira, et al., 2000). The onset of oocyst shedding and diarrhea occurred between 2- 3 DPI. Resolution of diarrhea and oocyst shedding ranged from 16-20 DPI. At 3 DPI (or 0-1 DPO), small intestinal cytokine activity was elevated with mixed neutrophilic, lymphocytic infiltrates and heavy mucosal oocyst infection rates were observed histologically whereas colonic cytokine activity and histologic changes were minimal. However, by 10 DPI (or 7-8 DPO), histologic changes similar to small intestinal changes were noted in the colon and all colonic cytokine activity had increased (except TNF). In addition, cytokine mRNA activity continued to increase in duodenum (except TNF) but decreased in the ileum (except IL-12). Compared to PID 10, cytokine activity increased at PID 21 in the colon and ileum (except ileal IFN and IL-12) and decreased in the duodenum (except TNF). By PID 50, all cytokine activity had decreased (except duodenal IL-12). These results indicate that C. parvum-elicited cytokine responses strongly and positively correlate with C. parvum-induced tissue pathology in cryptosporidiosis.

F. Cellular Immunity

No additional lymphocyte proliferation studies were performed during this period.

G. Humoral Immunity

An isotype-specific ELISA was developed to evaluate specific humoral immune responses in serum and intestinal fluids from gnotobiotic pigs inoculated with C.parvum. These studies are being done in collaboration with Drs. Karol Sestak and Saul Tzipori at Tufts University School of Veterinary Medicine. At 6-10 days post-infection (DPI) with high dose type 2 parental GCH1 and OH, 23 percent of neonatal gnotobiotic pigs developed IgM serum antibodies while only 2 percent developed IgG2 and IgA serum antibodies at 6-10 DPI. By 21 DPI, 26 to 34 percent of pigs developed detectable low titer (GMT = 1.8-2.4) IgM, IgG1 and IgG2 serum antibodies. No IgA serum antibodies were detected at this time. By 28 DPI, 75 percent-100 percent of pigs had developed higher titer IgM, IgG1 and IgG2 serum antibodies (GMT = 8 to 26) but no serum IgA antibodies. By 49 DPI, 75 percent of pigs had detectable IgM (GMT = 29), 100 percent of pigs had IgG1 & IgG2 (GMT = 80-112), and 50 percent of pigs had IgA (GMT = 4.5). Antibodies were rarely detected in the intestinal contents from these same pigs with very low IgM, IgG and/or IgA antibody titers (GMT 1.2 ? 1.6) detectable in 8-9 percent of all pigs at 6-10 DPI and 21 DPI only. These findings indicate that a very limited humoral immune response is elicited by high-dose type 2 C. parvum infections.

H. Significance

Water sampling has demonstrated that Cryptosporidium is ubiquitous in the environment and will always probably be present as a waterborne pathogen. The risk of transmission of the protozoan parasite Cryptosporidium parvum to humans via drinking water is multi-factorial and includes not only the conditions under which C.parvum is introduced and survives in water, but knowledge of the minimum infective dose, strain virulence variability and individual host factors. We have used a neonatal gnotobiotic (Gn) pig model to derive single oocyst clones of genotype 1 and 2 C. parvum isolates and to assess risk factors to cryptosporidial disease. Infection studies using our genotype 2 GCH1 and OH clones in Gn pigs clearly shows both clones to be more virulent than the parental strains, and variability in the clinical responses of Gn pigs to different pools of each C.parvum clone is minimal to non-existent, suggesting that the cloning procedure effectively derived a genetically homogenous (clonal) population of parasites compared to the parental strains. The mere availability of genotype 1 as well as genotype 2 C.parvum clones for genetic and immunologic study is an enormous advancement for Cryptosporidium research. In addition, we have propagated non-C. parvum species (originating from human infections) in our Gn pig model including C. canis and C. meleagridis. Preliminary comparative infection studies with our type 1 & 2 C. parvum and non-C. parvum strains suggest that differences exist in the DD50 and LD50 but not MID of these species/strains (MID's are consistently low at 1 to 5 oocysts). More detailed studies of type 1 & 2 C. parvum strains show marked differences in prepatent periods, clinical disease expression, intestinal site of replication, and types and severity of lesions induced. These findings provide further support to published genomic studies that suggest genotype 1 and 2 C. parvum isolates may in fact belong to two distinct species. We also contemplate that Cryptosporidium "virulence" is determined by the host species infected as well as the parasite species (and not so much the strain within a species). Comparative morphology and cytokine studies in our Gn pig model infected with high dose parental GCH1 and OH show a strongly positive correlation between cytokine mRNA levels and C. parvum-induced pathology, suggesting that the host immune response may contribute to lesion development and disease expression in genotype 2 strain infections. Humoral immunity studies of our genotype 2 strains indicate that, although most pigs develop serum antibody responses to C. parvum, these responses are generally slow to develop and are relatively low (GMT are low) especially when compared to other enteric pathogens such as rotavirus. These findings indicate that a very limited humoral immune response is elicited by high-dose C. parvum infections and these ?poor responses' may explain in part why previously exposed hosts remain susceptible to repeated infections.

Future Activities:

We will continue our attempts to derive genetic "clones" of a variety of Cryptosporidium species originating from humans. Our humoral immunity studies will continue with emphasis on delineating the humoral responses to genotype 1 and 2 C. parvum given at low doses (to mimic natural dose exposures). We also will standardize our quantitative RT-PCR assay (for cytokine analysis) using a PCR lightcycler and anticipate completing and reporting these findings by mid-year. Studies in immunosuppressed animals to determine the role of an intact immune system in risk to genotype 1 and 2 C. parvum-associated disease and death are underway.


Journal Articles on this Report : 2 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 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 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
  • 2001 Progress Report
  • Final Report