Grantee Research Project Results
2004 Progress 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 Health Science Center at San Antonio , Tufts University
Current 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 Period Covered by this Report: September 1, 2003 through August 31, 2004
Project Amount: $524,540
RFA: Drinking Water (2000) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall goal of this proposal is to investigate the infectivity, illness, and immune response to three non-parvum Cryptosporidium species in healthy adult volunteers. In addition, we will examine the infectivity and development of these isolates in an in vitro system (HCT-8 cells) that has shown promise as a surrogate for human infection. These goals have changed to some extent from the original proposal. Specifically, two species will be studied because the third species is not available. The specific objectives of the study are to:
- establish infection in a laboratory host (gnotobiotic pig or other appropriate species) with each of two non-parvum Cryptosporidium species;
- 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;
- and determine the potential for infectivity of two non-parvumCryptosporidium species in healthy adults.
The project is proceeding based on the restated objectives and specific aims.
Progress Summary:
Objective 1
A C. meleagridis isolate (TU1867) and a C. muris isolate (RN66) were previously confirmed for species, and infections were established in gnotobiotic piglets and interferon gamma knockout (GKO) mice, respectively. These species are genetically stable after multiple passages, and there is no indication of a subpopulation or contamination with any other isolate.
Objective 2
C. muris infectivity and growth results were previously reported. C. meleagridis has been cultured in Madin-Darby bovine kidney cell monolayers (laboratory of Dr. Saul Tzipori, Tufts University). Additional studies with C. muris and C. meleagridis in HCT-8 cells are underway.
Objective 3
Five healthy adult volunteers received a single dose of 105C. meleagridis oocysts. Four of the five (80%) volunteers were male. At the time of challenge the oocysts were within 6 weeks of production in the gnotobiotic piglet, and the excystation rate was approximately 49-66 percent. Three of the five volunteers were positive for fecal oocysts by EIA (Cryptosporidium Antigen Detection Microwell ELISA, IVD Research, Inc., Carlsbad, California) and IFA (Crypto Cel, Cell Labs, Inc., Sydney, Australia). Four of the infected volunteers had a diarrheal illness, yielding an 80 percent illness attack rate. Of these four, only two volunteers shed oocysts detectable by EIA or IFA. One volunteer had no unformed stools or symptoms and shed low, but detectable, numbers of oocysts on days 3 and 7. The intervening “negative” oocyst days likely represent oocyst production below the level of test sensitivity.
Diarrheal illness was characterized by an incubation period of 5.3 days (range of 4-7 days) followed by the passage of approximately 8 unformed stools (range of 3-15) in a period of 77 hours (3.2 days; range of 50-105 hours). Total weight of unformed stools during the diarrheal episode ranged from 0.44 to 3.0 kg (mean of 1.2 kg). In three volunteers, diarrhea was accompanied by additional gastrointestinal symptoms. All three experienced increased gas; two reported abdominal pain; one reported fecal urgency; and one reported 1 day of nausea.
Oocyst shedding typically began in conjunction with diarrhea on day 5-post challenge (range, day 3-8) and lasted for about 3.7 days (range, 3-4 days). Little oocyst shedding was noted in any of the volunteers after resolution of diarrhea. One asymptomatic volunteer, however, shed oocysts for 5 days beginning on day 3 post challenge. The total number of oocysts shed per diarrheal episode ranged from (log) 6.41 to 8.65 with two volunteers at the lower end of the range. The asymptomatic oocyst shedder passed 2.8 x 106 oocysts. All volunteers resolved their diarrhea by day 12 and oocyst shedding by day 11 post challenge.
Future Activities:
Objective 1
Infections with C. meleagridis and C. muris have been established in laboratory models. This objective has been completed.
Objective 2
C.muris has been tested in the HCT-8 cell culture and compared to C. parvum and C. hominis. HCT-8 cells are susceptible to C. muris infection and support parasite replication. Experiments to examine the replication of C. muris and C. meleagridis in HCT-8 cells are in progress.
Objective 3
The C. meleagridis and C. muris isolates have been tested in healthy adult volunteers. Both species were documented to result in infection and/or illness. Additional studies will examine the serological reactivity in volunteers to homologous antigens. Manuscripts for these infectivity studies are in preparation.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 28 publications | 17 publications in selected types | All 14 journal articles |
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Type | Citation | ||
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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) |
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Okhuysen PC, Rogers GA, Crisanti A, Spano F, Huang DB, Chappell CL, Tzipori S. Antibody response of healthy adults to recombinant thrombospondin-related adhesive protein of Cryptosporidium 1 after experimental exposure to Cryptosporidium oocysts. Clinical and Diagnostic Laboratory Immunology 2004;11(2):235-238. |
R829180 (2004) |
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Supplemental Keywords:
mucosal immunity, coccidia, water quality, human subjects, animal subjects, non-parvum Cryptosporidium, Cryptosporidium muris, Cryptosporidium melegridis,, 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.