Grantee Research Project Results
2002 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, 2001 through August 31, 2002
Project Amount: $524,540
RFA: Drinking Water (2000) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objective of this research project 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 overall objectives have not changed from the original proposal. The specific objectives of the research project are to: (1) establish infection in a laboratory host (gnotobiotic pig or other appropriate species) with each of three 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 its usefulness as an in vitro surrogate for human infections; and (3) determine the potential for infectivity of three non-parvum Cryptosporidium species in healthy adults.
Progress Summary:
Objective 1. Four Cryptosporidium species have been evaluated to select three isolates for volunteer testing. C. meleagridis (designated TU1867) was isolated from a child with diarrhea, and its identity was confirmed. At the time of writing, TU1867 has been passaged 10 times in piglets, 8 times in mice, 4 times in chicks, twice in turkey poults, and once in calves. Infection occurred regardless of the origin of the oocysts, indicating that C. meleagridis was readily transmissible between and among mammalian and avian animal species. The genetic profiles of DNA extracted from the oocysts isolated from these different passages were identical to that as previously reported by others for C. meleagridis; there were no observable changes, as the oocysts were passaged through different hosts and no indication of the presence of a subpopulation. The nucleotide sequence of the SSU rRNA from TU1867 was submitted to GenBank. Clinical signs were observed consistently in infected piglets only with diarrhea and oocyst excretion normally beginning on day 4 or 5 post-inoculation and continuing for 10 to 12 days. Infection in immunosuppressed and interferon gamma knockout (GKO) mice was indistinguishable from that as normally observed in animals infected with C. parvum genotype 2 isolates.
A C. muris isolate (RN66), originally from an animal source, was received from Waterborne, Inc. The isolate has been passaged in mice (Nu/NuBr) and propagates well in this model. In contrast, inoculation of C57BL/6 mice failed to produce an infection. The identity of the isolate has been verified by sequencing a beta-tubulin gene fragment; microsatellite analysis is underway. Oocyst shedding was detectable after about 4 weeks post-inoculation and continued for about 8 weeks. Approximate oocyst yield was about 106/mouse/day from fecal collections. Other unexpected difficulties in the purification process were encountered, due to the fragility and very short "shelf life" (approximately 3 weeks) of these oocysts.
A confirmed C. canis isolate was inoculated into gnotobiotic piglets, immunosuppressed mice, GKO mice, baby gerbils, baby rats, immunosuppressed young rats, young chicken poults, neonatal calf, and finally, an 8-week-old beagle. All attempts to produce an infection were unsuccessful. The purified oocysts used as inoculum also were tested in vitro in MDBK cells to demonstrate that the oocysts were infectious. Because the propagation efforts were unsuccessful, a new C. canis isolate is being sought from a number of veterinary laboratories.
C. felis was recently identified in a domestic cat in Texas and used to inoculate two gnotobiotic piglets, both of which failed to develop an infection. The genus and species of the isolate were confirmed by 18S rRNA PCR-RFLP analysis. The limited number of oocysts precluded the inoculation of additional animal models. An effort is being made to collect more oocysts from the cat for future studies.
Objective 2. A sensitive detection method for each Cryptosporidium species must be optimized for the accurate assessment of infection in cell culture and fecal oocyst quantitation following volunteer challenge. Seeded stool samples or purified oocysts were tested in two versions of the Alexon EIA Kit. C. meleagridis oocysts (seeded stool samples and purified oocysts) were detectable and essentially equal to C. parvum detection levels. In contrast, the same number of C. muris oocysts yielded negative results.
Immunofluorescence assays (IFAs) have been completed on C. meleagridis and C. muris oocysts using the Meridian kit and two other commercial antibodies (Cellabs, Inc.; Waterborne, Inc.). Some fluorescent antibody bound to these oocysts, but the intensity of the signal was well below C. parvum oocysts. A new IFA also has been introduced into the lab and is being evaluated for oocyst quantitation. The technique, fluorescent in situ hybridization (FISH), has been optimized for C. parvum isolates. Probes developed from C. parvum 18s ribosomal RNA sequence have been tested on C. meleagridis and C. muris isolates. To date, FISH assays using C. parvum probes have failed to bind to C. meleagridis or C. muris 18s rRNA, indicating that more specific probes will be required. HCT-8 infectivity experiments have been conducted with C. meleagridis (TU1867) oocysts, but have been hampered by the lack of a sensitive and specific probe for assessing the numbers of developing parasites. However, replicating parasites were visualized in wells inoculated at a 1:1 ratio (oocyst:cell). Currently, we are inoculating the cells with C. muris to examine whether this cell line can support parasite replication. C. meleagridis oocysts (TU1867) also were inoculated onto Madin-Darby bovine kidney cell monolayers (laboratory of Dr. Saul Tzipori, Tufts University) and were compared with the C. parvum genotype 2 isolate (GCH1). C. meleagridis was capable of infecting these cells; however, at low oocyst doses, the infection rate was substantially less for TU1867 than GCH1.
Future Activities:
Infections with C. meleagridis and C. muris have been established in laboratory models. We will continue to screen veterinary populations to identify a C. nis or C. felis isolate. One or both of these isolates will be tested in a variety of laboratory models to establish an infection and source of oocysts for volunteer studies. In addition, cell culture studies will be continued for comparison of isolates for relative infectivity and growth rates. The C. meleagridis and C. muris isolates have been established in laboratory models, characterized for their genetic stability, and will be tested in two groups of healthy adult volunteers.
Journal Articles on this Report : 9 Displayed | Download in RIS Format
| Other project views: | All 28 publications | 17 publications in selected types | All 14 journal articles |
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Alcantara CS, Yang C-H, Steiner TS, Barrett LJ, Lima AAM, Chappell CL, Okhuysen PC, White Jr AC, Guerrant RL. Interleukin-8, tumor necrosis factor-alpha, and lactoferrin in immunocompetent hosts with experimental and Brazilian children with acquired cryptosporidiosis. American Journal of Tropical Medicine and Hygiene 2003;68(3):325-328. |
R829180 (2002) R828035 (2001) |
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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Messner MJ, Chappell CL, Okhuysen PC. Risk assessment for Cryptosporidium: a hierarchical Bayesian analysis of human dose response data. Water Research 2001;35(16):3934-3940. |
R829180 (2002) R828035 (2001) |
Exit Exit |
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Okhuysen PC, Robinson P, Nguyen MT, Nannini EC, Lewis DE, Janecki A, Chappell CL, White Jr AC. Jejunal cytokine response in AIDS patients with chronic cryptosporidiosis and during immune reconstitution. AIDS 2001;15(6):802-804. |
R829180 (2002) |
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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Okhuysen PC, Rich SM, Chappell CL, Grimes KA, Widmer G, Feng X, Tzipori S. Infectivity of a Cryptosporidium parvum isolate of cervine origin for healthy adults and interferon-gamma knockout mice. Journal of Infectious Diseases 2002;185(9):1320-1325. |
R829180 (2002) R828035 (2001) |
Exit Exit |
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Padda RS, Tsai A, Chappell CL, Okhuysen PC. Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene. International Journal for Parasitology 2002;32(2):187-197. |
R829180 (2002) R828035 (2001) |
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Robinson P, Okhuysen PC, Chappell CL, Lewis DE, Shahab I, Lahoti S, White Jr AC. Expression of IL-15 and IL-4 in IFN-gamma-independent control of experimental human Cryptosporidium parvum infection. Cytokine 2001;15(1):39-46. |
R829180 (2002) |
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Teunis PFM, Chappell CL, Okhuysen PC. Cryptosporidium dose-response studies: variation between hosts. Risk Analysis 2002;22(3):475-485. |
R829180 (2002) R828035 (2001) |
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Supplemental Keywords:
mucosal immunity, coccidia, water quality, human subjects, animal subjects., RFA, Health, Scientific Discipline, Water, ENVIRONMENTAL MANAGEMENT, POLLUTANTS/TOXICS, Health Risk Assessment, Risk Assessments, Immunology, Biology, Epidemiology, Microorganisms, Drinking Water, Risk Assessment, immune system response, microbial contamination, treatment, viruses, water quality, water quality parameters, human exposure, monitoring, animal model, parasites, pathogens, virulence characteristics, microbial effects, drinking water contaminants, drinking water regulations, drinking water treatment, water treatment, exposure, coccidia, genetics, waterborne disease, cryptosporidium , cryptosporidium parvum oocysts, genotype distribution, human health effects, cryptosporidium, human health risk, exposure and effectsProgress 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.