1999 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, 1998 through February 19, 1999
Project Amount: $332,084
RFA: Drinking Water (1997) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water

Objective:

The goal of this research is to increase our 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. Two C. parvum isolates are being studied: GCH1 (Grafton Compton Human I) obtained by Saul Tzipori, Tufts University School of Veterinary Medicine, Grafton, MA, from an AIDS patient; and OH (Ohio) obtained by the Principal Investigator (Dr. L. Ward) from an adult laboratory worker with clinical cryptosporidiosis.

Progress Summary:

Genotyping Studies. Genotyping studies are being conducted in collaboration with Drs. Saul Tzipori and Giovanni Widmer at the Tufts University School of Veterinary Medicine. Genetic analyses demonstrate that both isolates are genotype C (bovine). Indepth analysis of the tubulin locus of OH also demonstrated a homogenous C genotype, which was unusual in that most C isolates from humans are heterogenous at this locus (including GCH1). The homogenous beta tubulin locus was lost after six passages in pigs and calves. This was not surprising in that large numbers of oocysts (106 to 107) were used for propagation. However, our previous studies showed the minimum infective dose for either GCH1 or OH to be low (<5 oocysts) in gnotobiotic pigs; thus, we currently are passing OH (from original stock) and GCH1 using 1 to 5 oocysts to obtain the first (and only) C. parvum "clones."

Detection of C. parvum in Experimental Samples. We previously reported a procedure to enhance the Acid-Fast stain sensitivity to levels comparable with a commercially available immunofluorescent assay (IFA) product (MERIFLOUR, Meridian Diagnostics, Inc., Cincinnati, OH). We also have developed a less expensive method for extracting polymerase chain reaction (PCR) quality DNA from frozen fecal specimens, which can be combined with a nested PCR assay for detection of a single C. parvum oocyst in fecal material.

Minimum Infective Dose (MID), Median (50%) Diarrheal Dose (DD50), and 50% Lethal Dose (LD50). The MID for GCH1 in neonatal (1 to 4 days old) gnotobiotic pigs is <5 oocysts which results in 25 percent diarrhea and 100 percent oocyst shedding for 10+ days. The DD50 is 730 oocysts that results in mild to moderate diarrhea for 5 to 10 days. Doses of >108 GCH1 oocysts were 100 percent lethal with onset of oocyst shedding and diarrhea observed by 24-36 hours post-inoculation (PI). By comparison, the OH isolate's MID in neonatal pigs is similarly low (<5 oocysts), but its DD50 was higher (6,900 oocysts), and relatively high doses (>108 OH oocysts) were lethal to only 20 percent of inoculated animals. Studies in older (3-4 weeks old) gnotobiotic pigs show a similarly low MID (<5 oocysts), but significantly higher DD50, and LD50 for both isolates with no death at doses as high as 109 oocysts.

Morphologic Studies. Microscopic evaluation of tissues from pigs infected with 5 x 106 GCH1 oocysts at 1-4 days of age show attenuation of small intestinal and colonic mucosa. At 3 days PI, large numbers of oocysts are present in the duodenum, jejunum, and ileum, with duodenum having the greatest infection rate. Ooocysts also can be found in the bile duct. By 10 days PI, most oocysts are observed in the ileum and colon. By 21 days PI, a few oocysts were still present in ileum, colon, and bile duct. By 50 days PI, no oocysts were observed. Initial comparative studies using 5 x 106 OH oocysts indicated differences in oocyst distribution and infection rates with greater predilection for the colon at 3 days PI. However, when OH doses were increased 10-fold, no difference in tissue pathology was observed.

Cytokine Responses. A reverse transcriptase-PCR (RT-PCR) assay with primers specific for porcine IL-12 (p40), IFN-g, TNF-a, and IL10 has been used for evaluating cytokine mRNA expression patterns in systemic and intestinal tissues of gnotobiotic pigs inoculated with C. parvum at 1-2 days of age. Total RNA was extracted from the spleen, small intestine (duodenum, ileum), and colon of C. parvum-infected pigs at selected intervals PI and compared to uninfected age-matched controls to assess cytokine activity over the course of disease. Splenic cytokine mRNA expression increased at the onset of diarrhea and oocyst shedding, decreased during the course of the disease, and increased after disease resolution. Small intestinal cytokine mRNA expression was similar to splenic cytokine expression at the onset of shedding and disease (increased compared to negative controls) but unlike spleen, increased even more (except TNF-a and ileal IL-10) during disease, and finally decreased upon disease resolution. Colonic cytokine expression (although higher than negative controls) was markedly lower than spleen and small intestine at the onset of disease (3 days PI) and did not obtain comparable levels to the small intestine until 10-21 days PI. Thus, small intestinal cytokine mRNA expression differs from the systemic and colonic cytokine expression in cryptosporidiosis.

Cellular Immunity. A lymphocyte proliferation (LP) assay has been developed to study T helper cell responses to C. parvum. Previous studies in gnotobiotic pigs using enteric human rotaviruses demonstrated that lymphocytes in the peripheral blood "mirrored" lymphocyte activity in the gut during the course of rotavirus infection and disease. Consequently, we are monitoring peripheral blood lymphocytes (PBLs) during the course of cryptosporidiosis to assess the T cell immune response to C. parvum. Six pigs were inoculated with 106 oocysts and three pigs were inoculated with low doses (5-100 oocysts). The PBLs were collected from pigs between 10-50 days PI. The PBLs from six age-matched control pigs (not exposed to C. parvum) also were collected. The PBLs (n = 15) were cultured in triplicate with a whole-cell C. parvum antigen for 4 days and labeled with tritiated thymidine for 18 hours. All PBLs from C.parvum-inoculated pigs showed significant LP responses, whereas control pigs did not.

Humoral Immunity. An isotype-specific ELISA has been developed to evaluate specific humoral immune responses in serum and intestinal fluids from gnotobiotic pigs inoculated with C. parvum. These studies are being conducted in collaboration with Drs. Saul Tzipori and Karol Sestak at the Tufts University School of Veterinary Medicine. Preliminary findings suggest that the development of serum C. parvum-specific IgG is related to the inoculating dose and/or severity of subsequent disease. Two pigs inoculated with 5 and 50 oocysts shed oocysts PI but did not develop significant diarrhea, and neither had developed serum IgG antibodies to C. parvum by 3 weeks PI. By comparison, 10 of 10 pigs that received 104 to 109 oocysts shed large numbers of oocysts and developed moderate to severe diarrhea PI, and 10 of 10 pigs had high titers of C. parvum-specific serum IgG antibodies by 3 weeks PI.

The significance of these findings is discussed below. 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 C. parvum to humans via drinking water is multifactorial and includes not only the conditions under which C. parvum is introduced and survives in water, but knowledge of the minimum infective dose, C. parvum strain virulence variability, and individual host factors (age, immune status, etc.). We are utilizing a gnotobiotic pig model to delineate risk factors to cryptosporidiosis. Our findings suggest that age is a significant risk factor for disease and death following infection with C. parvum; however, age alone does not appear to be a significant determinant of risk to infection in the susceptible host (which includes no previous C. parvum exposure). Our humoral and cellular immunity studies provide preliminary evidence to suggest that our gnotobiotic pig model closely parallels adult human volunteers as reported by Chappel and colleagues in terms of T cell and B cell responses to C. parvum following inoculation. It will be of great interest to see if this trend continues. Comparative analyses of our morphologic and cytokine studies suggest a strong relationship exists between cytokine mRNA expression and C. parvum-induced pathology. Delineation of the specific cytokines involved may allow for new and innovative forms of therapy and/or treatment of cryptosporidial disease.

The use of "clonal" C. parvum isolates would eliminate inherent variation due to the potentially huge genetic diversity of our current C. parvum pools propagated by traditional methodology (i.e., passage with high doses). Consequently, the mere availability of a C. parvum clone for genetic, pathologic, and immunologic study would be an enormous advancement for Cryptosporidium research.

Future Activities:

We will continue our attempts to derive a genetic "clone" of C. parvum. Our immunological studies will continue and will include standardization of our current quantitative RT-PCR assay (for cytokine analysis) from purified lymphocytes to whole tissues. We will begin the MID, DD50, and LD50 studies in immunosuppressed animals to determine the role of an intact immune system in risk to disease and death.


Journal Articles on this Report : 1 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)
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  • 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
  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report