Grantee Research Project Results
Final Report: Infectivity and Virulence of Cryptosporidium Non-parvum Species in Healthy Adult Volunteers
EPA Grant Number: R829180Title: Infectivity and Virulence of Cryptosporidium Non-parvum Species in Healthy Adult Volunteers
Investigators: Chappell, Cynthia L. , Okhuysen, Pablo C. , Widmer, Giovanni , Tzipori, Saul
Institution: The University of Texas at Houston
EPA Project Officer: Page, Angela
Project Period: September 1, 2001 through August 31, 2004 (Extended to August 31, 2005)
Project Amount: $524,540
RFA: Drinking Water (2000) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall goal of this project was to investigate the infectivity, illness, and immune response to non-parvum Cryptosporidium species in healthy adult volunteers. In addition, the project examined the infectivity and development of these isolates in an in vitro system (HCT-8 cells) that is a potential surrogate for human infection. These overall goals changed to some extent from the original proposal. Specifically, two species (instead of three) were studied because the third species was not available. The specific objectives of this research project were to: (1) establish infection in a laboratory host (gnotobiotic pig) with each of two non-parvum Cryptosporidium species; (2) compare infectivity and growth (cycling times) of Cryptosporidium species in HCT-8 (human enterocyte) cell cultures with infectivity in volunteers and further evaluate HCT-8’s usefulness as an in vitro surrogate for human infections; and (3) determine the potential for infectivity of two non-parvum Cryptosporidium species in healthy adults.
Summary/Accomplishments (Outputs/Outcomes):
Recent molecular studies have revealed that Cryptosporidium species once thought to cause infection only in animals are capable of infecting the human host, particularly immunocompromised individuals. C. meleagridis has been reported in at least 48 individuals, including both immunocompetent and immunocompromised individuals. In contrast, C. muris has been described in three HIV+ persons with diarrhea and in two HIV-, asymptomatic children. In these cases of C. meleagridis and C. muris in immunocompetent humans, the immune status was not typically studied beyond HIV testing, and possible immunodeficiencies (such as IgA deficiency) in these individuals were not examined. Thus, questions remain regarding the infectivity of these parasite species in healthy humans with intact immune systems as well as the potential of the parasites as agents of diarrhea in the general population.
The experimental design was to expose healthy adults to a high parasite inoculum that was expected to result in infection if the parasite was, in fact, infectious for healthy adults. C. meleagridis or C. muris oocysts were ingested by volunteers, who were then followed for clinical and microbiological outcomes over a period of 6 weeks. Infections were established in all of the healthy volunteers receiving C. meleagridis or C. muris oocysts. Illness, however, was associated more often with C. meleagridis than C. muris. In contrast, C. muris persisted as an asymptomatic infection for months, whereas C. meleagridis was self-limited and resolved after 12 days. These results significantly add to the information needed to assess of the risk of infection and illness in the general population. This project has provided information that significantly advances the understanding of Cryptosporidium infectivity in healthy adults and, by generalization, to the larger community.
Objective 1: To Establish Infection in a Laboratory Host (Gnotobiotic Pig or Other Appropriate Species) With Non-parvum Cryptosporidium Species
The original objective was to test three Cryptosporidium species that were not found commonly in human populations. Despite much searching in canine and feline populations, only two parasite species could be identified successfully and passaged in animal hosts. One study of kenneled dogs revealed the presence of C. muris instead of the expected Cryptosporidium species in dogs, C. canis. Species identification was confirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence analysis. Approximately 47 percent of the dogs were infected, and a similar percentage had specific serum IgG. To our knowledge, this represents the first report of naturally-occurring C. muris in dogs (Langer-Curry, et al., in preparation).
A C. muris isolate (RN66), originally from an animal source in Japan, was received from Waterborne, Inc., verified for species by sequence of a beta-tubulin gene fragment, and amplified in Nu/NuBr mice. Approximate oocyst yield was approximately 106/mouse/day from fecal collections. A second species, C. meleagridis (TU1867) was isolated from a human and confirmed by PCR/RFLP and sequence of fragments amplified from the COWP and SSU rRNA genes. The nucleotide sequence of the SSU rRNA from TU1867 was submitted to GenBank. The infection was established in gnotobiotic piglets and interferon gamma knockout mice. In gnotobiotic piglets, diarrhea and oocyst excretion normally began on day 4 or 5 postinoculation and continued for 10 to 12 days. No changes were observed in the genetic profiles of the isolates as they were passaged in animals or humans nor has there been any indication of the presence of a subpopulation of oocysts or of contamination with C. parvum laboratory isolates. Attempts to passage a C. canis and a C. felis isolate in the laboratory failed despite inoculation of several animal species.
To prepare for human infectivity studies, a series of experiments were done to identify reagents for detection of C. muris and C. meleagridis oocysts. A sensitive detection method for each Cryptosporidium species was required for the accurate assessment of infection in cell culture and fecal oocyst quantitation following volunteer challenge. Most current detection methods are based on polyclonal or monoclonal antibody binding to oocyst cell wall constituents. Because information on antibody cross-reactivity among Cryptosporidium species is sparse, a number of assays were conducted. Purified C. meleagridis or C. muris oocysts or seeded stool samples were tested with three types of enzyme immunoassay kits. C. meleagridis oocysts were detectable with two kits. Detection limits were comparable to those with C. parvum oocysts. In contrast, C. muris oocysts was recognized by only one kit (Cryptosporidium Antigen Microwell ELISA, IVD, Carlsbad, CA). In past volunteer studies, immunofluorescence assay (IFA) (Meridian Diagnostics) has been used to quantitate C. parvum oocysts. All IFAs tested with C. parvum, C. hominis, and C. meleagridis were reactive; however, only one antibody (Crypto Cel; Cellabs Pty Ltd, Brookvale, AU) recognized C. muris oocysts.
Objective 2: To Compare Infectivity and Growth (Cycling Times) of Cryptosporidium Species in HCT-8 (Human Enterocyte) Cell Cultures With Infectivity in Volunteers and Further Evaluate its Usefulness as an In Vitro Surrogate for Human Infections
The infectivity of C. meleagridis oocysts (TU1867) also studied in Madin-Darby bovine kidney (MDBK) cell monolayers (laboratory of Dr. Saul Tzipori, Tufts University) and were compared with a C. parvum isolate (GCH1). After 48 hours incubation at 37°C, the number of immunofluorescent-labeled parasites was quantitated using a computer-based video image analysis system. The data demonstrated that C. meleagridis was capable of infecting MDBK cells. Infectivity of TU1867 and GCH1was proportional to the inoculating dose. At 106 oocysts per well, the infectivity between the two isolates was comparable but differed at the lower doses.
Four concentrations of C. muris oocysts representing oocyst:cell ratios of 1:1, 2:1, 3:1, and 4:1 were added to HCT-8 cell cultures and allowed to develop for approximately 18 hours. All concentrations established infection in the cells and were easily detectable. These data confirmed that human enterocytes are susceptible to C. muris infections, a finding that is consistent with recent reports of C. muris infection in immunocompetent and immunocompromised persons. Further, the degree of infectivity was dose dependent. Because longer term studies were warranted and as even the lowest ratio of inoculum could be detected, this concentration was selected for subsequent experiments.
Permeabilized Cryptosporidium oocysts were added to HCT-8 cultures in a ratio of 1:1 and allowed to develop. The number of parasitophorous vacuoles (foci) were assessed in triplicate wells at 12, 24, 36, 48, 60, and 72 hours postinfection. The growth rate for C. muris was compared to C. parvum and C. hominis oocysts. Initial infectivity was indicated by the number of infectious foci at 12 hours postinoculation, a time that is thought to be just prior to the first replication cycle. All three Cryptosporidium species appeared to be roughly equal in their capacity to invade enterocytes as similar numbers of foci were seen at this early stage of infection. C. parvum and C. hominis developed at a similar rate, which increased up to 48 hours and then began to level off. These growth curves are consistent with the expected asexual replication cycles over the first 48 hours of infection followed by a preponderance of sexual development thereafter. In comparison, C. muris developed more slowly over the entire course of the experiment and reached only 67 percent of C. parvum/C.hominis growth rates at 48 hours. These data suggest that the cycle time for C. muris may be longer than the other two species or that merozoite attrition may be higher.
Objective 3: To Determine the Potential for Infectivity of Two Non-parvum Cryptosporidium Species In Healthy Adults
Five volunteers each were challenged with a single inoculum (105 oocysts) of C. meleagridis oocysts and monitored over a period of 6 weeks for the presence of fecal oocysts and clinical manifestations. All five persons had evidence of infection either microbiologically, clinically, or both. Four volunteers experienced diarrhea that lasted from 2 to 4.5 days and produced 3 to 15 unformed stools. One volunteer remained asymptomatic even though oocysts were detected for a number of days. Three (60%) volunteers had fecal oocysts detectable by IFA. All infections were self-limited; oocysts were shed for 3 to 4 days and cleared within 12 days of initial challenge. In one individual, oocyst shedding was in the range of 4.5 x 108, whereas the other two volunteers each shed 2.5 x 106 and 2.8 x 106 oocysts, respectively. These studies firmly establish that healthy adults can become infected and ill from the ingestion of C. meleagridis oocysts. The illness, however, generally was mild and self-limited.
The second isolate examined for infectivity in healthy adults was C. muris. Each of six volunteers was challenged with 105 C. muris oocysts and monitored for 6 weeks for infection and/or illness. All six became infected, and two experienced a diarrheal illness. Total oocysts shed during the study ranged from 6.7 x 106 to 4.1 x 108, and was higher (mean = 2.8 x 108) in volunteers with diarrhea than in asymptomatic shedders (mean = 4.4 x 107). Perhaps the most remarkable finding in this study was the duration of oocyst shedding, and the volunteer monitoring period was extended up to 9 weeks postchallenge. Follow-up fecal examinations at 7 months post challenge on five of the six volunteers revealed that three volunteers were positive for fecal oocysts. All were offered treatment with nitazoxanide (200 mg twice a day for 3 days), and two accepted the drug; both were confirmed to be oocyst-negative by PCR following completion of the regimen. All three persistent shedders have had follow-up physical examinations, which were normal, and none have reported any recurrence of diarrhea or other GI complaints over the past year.
These data establish the susceptibility of healthy humans to C. muris infection. Overall, only two (33%) had a mild diarrheal illness; all others were asymptomatic. C. muris, however, was self-limiting in two cases and resulted in persistent, asymptomatic infections in three persons for 7 months before treatment cleared the infection. Although this Cryptosporidium species does not appear to be a significant cause of illness in healthy persons, the chronicity of the infection may serve as a source of contamination for the water supply and other more sensitive populations.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 28 publications | 17 publications in selected types | All 14 journal articles |
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Akiyoshi DE, Dilo J, Pearson C, Chapman S, Tumwine J, Tzipori S. Characterization of Cryptosporidium meleagridis of human origin passaged through different host species. Infection and Immunity 2003;71(4):1828-1832. |
R829180 (2003) R829180 (Final) |
Exit Exit |
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Chappell CL, Okhuysen PC. Cryptosporidiosis. Current Opinion in Infectious Diseases 2002;15(5):523-527. |
R829180 (2002) R829180 (Final) |
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Huang K, Akiyoshi DE, Feng X, Tzipori S. Development of patent infection in immunosuppressed C57BL/6 mice with a single Cryptosporidium meleagridis oocyst. Journal of Parasitology 2003;89(3):620-622. |
R829180 (2003) R829180 (Final) |
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Kjos SA, Jenkins M, Okhuysen PC, Chappell CL. Evaluation of recombinant oocyst protein CP41 for detection of Cryptosporidium-specific antibodies. Clinical and Diagnostic Laboratory Immunology 2005;12(2):268-272. |
R829180 (2004) R829180 (Final) |
Exit Exit |
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Okhuysen PC, Chappell CL. Cryptosporidium virulence determinants – are we there yet? International Journal for Parasitology 2002;32(5):517-525. |
R829180 (2002) R829180 (Final) R828035 (2001) |
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Supplemental Keywords:
drinking water, water quality, waterborne pathogen, mucosal immunity, Coccidia, human subjects, animal subjects,, RFA, Scientific Discipline, Health, ENVIRONMENTAL MANAGEMENT, Water, POLLUTANTS/TOXICS, Health Risk Assessment, Epidemiology, Risk Assessments, Drinking Water, Biology, Immunology, Microorganisms, Risk Assessment, monitoring, cryptosporidium parvum oocysts, pathogens, microbial contamination, genetics, genotype distribution, human health effects, water quality parameters, waterborne disease, exposure and effects, animal model, drinking water regulations, viruses, exposure, cryptosporidium , immune system response, treatment, virulence characteristics, microbial effects, human exposure, coccidia, parasites, water quality, drinking water contaminants, drinking water treatment, water treatment, cryptosporidium, exposure assessmentProgress 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.