Grantee Research Project Results
Final Report: Virulence Factors in Cryptosporidium and Infective Dose in Humans
EPA Grant Number: R824759Title: Virulence Factors in Cryptosporidium and Infective Dose in Humans
Investigators: Chappell, Cynthia L. , Okhuysen, Pablo C. , DuPont, Herbert L. , Sterling, Charles R.
Institution: The University of Texas at Houston , The University of Texas Health Science Center at San Antonio
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 30, 1998 (Extended to September 30, 2000)
Project Amount: $829,551
RFA: Human Health Risk Assessment (1995) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The objectives are to: (1) identify, collect, and propagate four C. parvum isolates; (2) perform laboratory analyses of the isolates (mouse infectivity, biochemical, immunological, and genetic); (3) establish dose response of three isolates in human subjects; and (4) characterize the serologic response to C. parvum in human subjects.Summary/Accomplishments (Outputs/Outcomes):
Infectivity Studies. In 1993, a study of Cryptosporidium infectivity and natural history of the infection in healthy adult volunteers was instituted at the University of Texas Health Science Center in Houston (EPA Cooperative Agreement). The studies accomplished under the initial agreement and subsequent STAR Program have generated information that has been used widely in risk assessment models for waterborne transmission of Cryptosporidium.Volunteers between the ages of 18 and 55 were enrolled after they had undergone a complete history and physical examination and a battery of tests to ensure that they were in excellent health. Importantly, all volunteers were proven HIV-negative and had normal T-cell subsets and no immunodeficiencies. After challenge with Cryptosporidium oocysts, volunteers were monitored daily for the first 14 days, and 3 times per week for an additional 4 weeks. Active surveillance of volunteers' households and/or other close contacts for diarrheal illness was maintained throughout the study period. When diarrhea occurred in the subjects or their contacts, an enteric microbiological work-up was performed. No secondary transmissions were documented.
Three geographically diverse, genotype 2 isolates have been studied for their infectivity in volunteers who had no serological evidence (by ELISA) of previous exposure. Challenge doses ranging from 10 to 1 million oocysts were given to volunteers, and infectivity was documented by the presence of fecal oocysts and/or the occurrence of a diarrheal illness. The dose necessary to cause infection in 50 percent of volunteers (ID50) varied with the isolate studied: the ID50 for the UCP isolate was 1,042 oocysts, the Iowa isolate was 87 oocysts, and the TAMU was 9 oocysts. Interestingly, the isolate with the lowest ID50 also was the most virulent when assessed for illness attack rate (TAMU, 86 percent versus Iowa, 52 percent and UCP, 59 percent). However, the onset, duration, and severity of illness did not differ significantly among infected individuals.
Taken together with earlier rechallenge experiments, these studies indicate that immunity to Cryptosporidium exists, but may be dependent on the time that has elapsed since the previous infection and the number of previous exposures. Persons with serum antibodies to the parasite demonstrated relative resistance to infection and illness when exposed to low oocyst concentrations. However, resistance could be overcome when high concentrations of oocysts were encountered. In such cases, infection and illness may occur, but oocyst excretion is suppressed to low levels and would presumably result in fewer secondary transmissions.
A fourth isolate, Peru, has been amplified in calves. This isolate showed a mixed genotype 1/genotype 2 pattern initially, but has reverted to a classical genotype 2 pattern after two calf passages. Three volunteers were challenged with this isolate before this polymorphism reverted to the genotype 2 pattern. The initial results yielded infectivity data similar to the Iowa isolate, but insufficient numbers of volunteers were studied to firmly establish a true ID50. However, this isolate along with the other three isolates discussed above were used to determine their relative infectivity in cultured human enterocytes and in CD1 neonatal mice.
Serum Antibodies to C. parvum Oocyst Extracts. Blood was collected before challenge and at days 5, 10, 30, and 45 post-challenge. Serum antibodies were detected by ELISA using disrupted oocysts. Post-challenge sera were examined from 19 volunteers receiving a primary challenge and compared to sera collected from the same volunteers after a second challenge 1 year later. All of these sera were tested separately for IgM, IgG, and IgA reactivity to antigens derived from the Iowa isolate.
After the initial challenge, 11 volunteers and 5 volunteers showed IgM and IgA responses, respectively. However, these responses were not correlated with oocyst shedding or diarrheal illness. Further, serum IgA reactivity showed no correlation with individuals who had a demonstrable specific fecal IgA response. Surprisingly, no IgG responses were detected in any of the volunteers following primary challenge, even those who were shedding oocysts and/or who developed diarrhea.
Volunteers were re-screened prior to receiving a second oocyst challenge 1 year after the initial challenge. None of the volunteers had demonstrable serum antibodies to C. parvum prior to the second challenge, even those who had IgM and/or IgA reactivities during the first study period. Also, those who had an IgM or IgA response after primary challenge were not protected from infection or illness after a second oocyst challenge. After rechallenge, six volunteers had an IgM response, a slight decrease from that seen after primary challenge. Likewise, six volunteers had an IgA response, a number similar to that seen after primary exposure. Interestingly, six subjects showed significant increases in IgG after the second challenge, when none had detectable antibody after the initial challenge. This finding suggests that a serum IgG response may require two or more challenges to yield a detectable response.
Persons who had detectable serum IgG to Cryptosporidium prior to exposure were studied for serological reactivity after oocyst challenge. Those with the highest IgG levels before challenge showed little to no increase in specific IgG after challenge. In contrast, subjects that had lower (but positive) specific IgG levels prior to challenge showed a significant increase after challenge. These findings suggest that there may be a maximal level of reactivity that is not increased with an additional challenge; whereas, those who have lower IgG levels will be "boosted" with a subsequent challenge.
Fecal IgA Response to C. parvum Oocyst Antigens. Fecal extracts were prepared from stool samples taken on multiple days after challenge with the Iowa isolate. Of those with confirmed infections, extracts from multiple days were positive for specific fecal IgA. Specific reactivity was detected as early as day 5 post-challenge, and persisted in some for the duration of the study. In contrast, two individuals who were presumably uninfected each had only a single positive sample at day 34 and day 35, respectively. Statistical analysis revealed that infection (oocyst shedding) and challenge dose were significantly associated with specific fecal IgA, while diarrhea alone was not. Further analysis of specific fecal IgA showed no significant association with the duration of oocyst shedding or the intensity of the infection. Thus, specific fecal IgA was an indicator of active infection in volunteers shedding oocysts regardless of their illness outcome. The antibody appeared to remain high throughout the study period of 6 weeks, but because later time points were not examined, it is unclear how long the antibody persists.
Conclusions:
1. Cryptosporidium parvum genotype 2 oocysts vary widely in their ability to cause infection and illness in healthy persons.
2. Overall, after exposure about 24 percent of infected persons shed oocysts without having a diarrheal illness. Potentially, these individuals would likely be responsible for secondary transmission, especially in non-household settings.
3. Persons who have serological evidence of prior infection (i.e., anti-C. parvum IgG) are relatively resistant to re-infection with low numbers of oocysts, as would be encountered in water sources. However, those who do become infected and develop diarrhea often experience a more severe illness than naive persons.
4. Specific fecal IgA was associated with oocyst shedding and challenge dose in seronegative volunteers exposed to Cryptosporidium ostensibly for the first time. In contrast, none of these individuals developed serum IgG.
5. Serum IgG was detected in 33 percent of individuals re-exposed to Cryptosporidium oocysts, indicating two or more exposures may be necessary to stimulate a detectable level of specific serum IgG.
6. Serum IgG levels in the "low detectable" range were increased after a subsequent exposure to Cryptosporidium oocysts.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 1 publications | 1 publications in selected types | All 1 journal articles |
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Chappell C, Darkoh C, Schimmin L, Farhana N, Kim D, Okhuysen P, Hixson J. Fecal Indole as a Biomarker of Sceptibility to Cryptosporidium Infection. INFECTION AND IMMUNITY 2016;84(8):2299-2306. |
R824759 (Final) R828035 (Final) |
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Supplemental Keywords:
risk assessment, health effects, human health, dose response, mammalian, organism, genetic polymorphisms, susceptibility, pathogens, biology, pathology., RFA, Health, Scientific Discipline, Water, Genetics, Health Risk Assessment, Risk Assessments, Biochemistry, Drinking Water, vaccine development, detection, infective dose, dose-response, genetic analysis, virulence factors, antigens, human exposure, microbial exposure, biomarker, cryptosporidium, genetic susceptibility, human health risk, biochemical researchProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.