Grantee Research Project Results
2000 Progress Report: Infectivity and Virulence of Cryptosporidium Genotype H Oocysts in Healthy Adult Volunteers
EPA Grant Number: R828035Title: Infectivity and Virulence of Cryptosporidium Genotype H Oocysts in Healthy Adult Volunteers
Investigators: Chappell, Cynthia L.
Current Investigators: Chappell, Cynthia L. , Okhuysen, Pablo C. , Widmer, Giovanni , Tzipori, Saul
Institution: The University of Texas Health Science Center at San Antonio
EPA Project Officer: Hahn, Intaek
Project Period: March 1, 2000 through March 1, 2003 (Extended to February 28, 2005)
Project Period Covered by this Report: March 1, 2000 through March 1, 2001
Project Amount: $503,884
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Water , Drinking Water
Objective:
The overall goal of the present study is to generate dose-response curves in healthy volunteers using genotype 1 oocysts and to compare the resulting infectivity, clinical outcomes, and immune responses to previous genotype 2 results. Two different genotype 1 isolates have been proposed for testing. The specific objectives of the study are to: (1) establish the infectious dose (ID50), clinical outcomes and intensity of infection for two Cryptosporidium genotype 1 isolates in seronegative individuals; (2) investigate the antibody and cellular responses in volunteers to genotype 1 isolates; and (3) examine isolates for subtle genetic differences and determine the stability of DNA markers prior to and after passage in humans and gnotobiotic (GNB) pigs.Progress Summary:
Year 1 activities have been directed toward each of the above objectives:
Objective 1. To Determine the Genetic Stability of Isolates Before and after Passage in Humans and GNB Pigs. Two genotype 1 isolates have been collected from HIV-negative donors and amplified in the GNB pig model. Isolate TU502 was obtained from a donor who developed a relatively mild, symptomatic illness with C. parvum. Oocysts were then passaged multiple times and have been shown (multilocus analysis) to exhibit a stable genotype 1 pattern. A second isolate recently has been identified and is in the process of being amplified in GNB pigs and studied for genetic stability. Additional experiments examining microsatellite sequence polymorphism are in progress for each of the isolates. For human passage studies, stool samples positive for C. parvum oocysts were collected following challenge with TU502 (see below). These specimens are being studied for their genetic stability using multiple loci.
Objective 2. To Establish the Infectious Dose and Clinical Outcomes after Challenge with Genotype 1 Isolates. After purification and safety testing for adventitious agents, 100 TU502 (genotype 1) oocysts were ingested by each of four serologically-negative volunteers. Volunteers were monitored daily for 14 days and 3 times per week for a total of 6 weeks. All four volunteers showed clinical and/or parasitological evidence of infection, suggesting that the ID50 for this isolate may be low. A diarrheal illness was noted in all four volunteers with an onset on days 4-5. Duration of the diarrheal episodes ranged from 51-161 hours with a median of 79 hours. Severity of the illness was measured by the number of unformed stools passed during the diarrheal episode and the total weight of those stools. The number of unformed stools ranged from 4-14 (median = 9), and total weight ranged from 358-1,786 grams (median = 638 grams). Two of the four volunteers shed oocysts that were detectable by direct fluorescence antibody and enzyme-labelled immunosorbent assay. Onset of shedding was on days 6 and 7, respectively. One volunteer shed oocysts for 6 days, while the other subject intermittently shed oocysts for 25 days. The total number of oocysts shed during the study was 4.7 X 106 and 1.4 X 108, respectively. All volunteers were asymptomatic by day 12 post-challenge, and oocyst-negative by day 31.
Objective 3. To Examine the Humoral and Cellular Immune Responses to These Isolates. PBMC's and serum samples, as well as saliva samples, have been collected at five time points (pre- and post-challenge) from all four volunteers. Stool samples were collected throughout the study. All samples have been stored at -90°C prior to batch testing.
Future Activities:
The following activities for each objective are planned for the coming year:
Objective 1. To Determine the Genetic Stability of Isolates Before and after Passage in Humans and GNB Pigs. Selected stool samples collected from volunteers during the oocyst shedding period have been sent to Dr. Giovanni Widmer (Tufts University) for genetic polymorphism studies. These experiments are in progress. Additional challenges with TU502 will be monitored for genetic stability after each amplification in pigs and after human infections with detectable oocyst shedding. The same procedure will be carried out with the second genotype 1 isolate.
Objective 2. To Establish the Infectious Dose and Clinical Outcomes after Challenge with Genotype 1 Isolates. Additional volunteers will be challenged with 100 oocysts or with lower dosages. Groups of approximately five volunteers each will be included with each dosage level. A second genotype 1 isolate will be similarly tested.
Objective 3. To Examine the Humoral and Cellular Immune Responses to These Isolates. Experiments designed to study the antibody and cellular responses to homologous and heterologous isolates will be conducted with frozen specimens. This allows batch processing and minimizes variation in antigen preparations and other reagents. Lymphocyte proliferation studies, as well as serum and secretory antibody responses, will be determined.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 19 publications | 10 publications in selected types | All 10 journal articles |
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Dann SM, Okhuysen PC, Salameh BM, Dupont HL, Chappell CL. Fecal antibodies to Cryptosporidium parvum in healthy volunteers. Infection and Immunity 2000;68(9):5068-5074. |
R828035 (2000) |
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Robinson P, Okhuysen PC, Chappell CL, Lewis DE, Shahab I, Lahoti S, White Jr AC. Transforming growth factor beta1 is expressed in the jejunum after experimental Cryptosporidium parvum infection in humans. Infection and Immunity 2000;68(9):5405-5407. |
R828035 (2000) |
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Robinson P, Okhuysen PC, Chappell CL, Lewis DE, Shahab I, Janecki A, White Jr AC. Expression of tumor necrosis factor alpha and interleukin 1beta in jejuna of volunteers after experimental challenge with Cryptosporidium parvum correlates with exposure but not with symptoms. Infection and Immunity 2001;69(2):1172-1174. |
R828035 (2000) |
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Supplemental Keywords:
mucosal immunity, coccidia, water quality, human subjects, animal subjects., RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Water, POLLUTANTS/TOXICS, HUMAN HEALTH, Genetics, Environmental Chemistry, Health Risk Assessment, Exposure, Risk Assessments, Analytical Chemistry, Drinking Water, Microorganisms, Risk Assessment, cryptosporidium parvum oocysts, Safe Drinking Water, monitoring, water quality parameters, exposure and effects, genotoxic biomarkers, gnotobiotic pigs, immune system response, infectious disease, virulence characteristics, treatment, human exposure, coccidia, microbial exposure, water quality, drinking water contaminants, drinking water treatment, infectivity, cellular responses, water treatment, dietary exposure, drinking water system, serologically positive humnas, cryptosporidium, exposure assessment, genetic susceptibilityProgress 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.