Grantee Research Project Results
2003 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 at Houston , The University of Texas Medical School , 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, 2002 through August 31, 2003
Project Amount: $524,540
RFA: Drinking Water (2000) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall goal of this 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 [human colonic cancer cells]) that has shown promise as a surrogate for human infection. These overall goals have not changed from the original proposal. The specific objectives of the study 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
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 is about 106/mouse/day from fecal collections.
A C. meleagridis isolate (TU1867) from a human source was confirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) and sequence of fragments amplified from the Cryptosporidium oocyst wall sporidium (COWP) and small ribosomal subunit ribonucleic acid (SSU rRNA) genes. The nucleotide sequence of the SSU rRNA from TU1867 was submitted to GenBank. The infection has been established in gnotobiotic piglets and interferon gamma knockout (GKO) mice. In gnotobiotic piglets, diarrhea and oocyst excretion normally begins on day 4 or 5 post-inoculation and continues for 10 to 12 days. There have been no observable changes in this profile as the oocysts were passaged, nor has there been any indication of the presence of a subpopulation or of contamination with C. parvum laboratory isolates.
Previous attempts to passage a C. canis and a C. felis isolate in the laboratory have failed. Additional isolates are being sought for the human challenge study.
Objective 2
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 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 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 post infection. 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 post inoculation, 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 because similar numbers of foci were seen at this early stage of infection. C. parvum and C. hominis developed at similar rates, which increased for 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
Six healthy adult volunteers were challenged with 105 C. muris oocysts. At the time of challenge, the oocysts were within 6 weeks of production in the murine host, and the excystation rate was approximately 83 percent. All six volunteers became infected and were confirmed by immunofluorescent antibody (IFA) testing and acid fast staining of multiple fecal samples. Only two of the infected volunteers (#162 and #165) had a diarrheal illness, yielding a 33-percent illness attack rate. Three other volunteers passed an occasional unformed stool (#166, #167) or typically had a single soft stool per day (#164) without any accompanying gastrointestinal symptoms. One volunteer (#163) had no unformed stools or symptoms, even though he was shedding high numbers of oocysts.
In one (#162) of the two volunteers with diarrhea, the illness was characterized by the passage of 8 unformed stools during 4 days of illness. These illness days consisted of two episodes: days 9 and 10 (total stool weight = 883 g) and days 14 and 15 (total stool weight = 643 g). The volunteer was clinically well on days 11-13. The second volunteer with diarrhea (#165) had a more prolonged course consisting of two distinct episodes. The first episode occurred between days 2-17 and was characterized by the passage of 8 unformed stools (total stool weight = 1,853 g), increased gas production and 2 days of fecal urgency. During days 21-30 increased gas production and occasional abdominal discomfort were noted. The second episode of diarrhea occurred between days 32-45 and was characterized by 9 unformed stools (total stool weight = 1,391 g), gas, abdominal pain, and occasional fecal urgency.
Oocyst shedding was observed in all volunteers and first detected between days 2 and 5 (median, day 3.5). Shedding patterns revealed significant day-to-day variations in each subject, with several days where oocysts were below the limit of detection. The intensity of the infection varied widely among the volunteers. Total oocysts shed during the study ranged from 6.7 x 106 to 4.1 x 108. The mean oocyst excretion in the two volunteers with diarrhea was 2.8 x 108 versus 4.4 x 107 in the four volunteers with mild/no symptoms. The most remarkable finding in this study was the duration of oocyst shedding, and the volunteer monitoring period was extended past the usual 6 weeks to as long as 64 days (9 weeks) post challenge. Volunteers were released from the study only when two or more sequential stools were IFA-negative. One volunteer (#162) withdrew from the study on day 64, when he moved to another state.
Because of the persistence of the infection and the variability of shedding, an attempt has been made to do followup fecal examinations on all volunteers several months post challenge. Five of the six volunteers have been evaluated on followup. Volunteer #163 has been contacted, but has not submitted a followup fecal sample. It should be noted, however, that this volunteer was negative on three samples between days 47 and 52 prior to release from the study. Volunteers #164 (samples on days 215 and 217) and #165 (sample on day 232) were negative on followup. Volunteer #162 was positive for oocysts on day 194 and was treated with nitazoxanide (200 mg bid x 3 days). Additional fecal samples have been requested. Volunteers #166 (samples on days 212 and 214) and #167 (samples on days 215 and 217) were positive on followup. Volunteer #167 has been treated with nitazoxanide and has submitted a post treatment fecal sample. Volunteer #166 has been contacted, but has not submitted a fecal sample nor come to the clinic for treatment. All three persistent shedders have had followup physical examinations, which were normal, and none have reported any recurrence of diarrhea or other gastrointestinal complaints.
Because two subjects challenged with C. muris exhibited persistent oocyst shedding, a pilot study with 105 C. meleagridis oocyst was carried out on one volunteer. The volunteer experienced a single episode of diarrhea beginning on day 4 post challenge, characterized by nine unformed stools (total stool weight = 600 g) over a period of 2.5 days. A total of 2.6 x 106 oocysts were shed on days 4-8 post challenge. The symptoms resolved, and no oocysts were detected in 12 subsequent stool samples. Because the volunteer had a short course and readily cleared the infection, we plan to continue the study and challenge an additional five volunteers.
Future Activities:
Objective 1: Infections with C. meleagridis and C. muris have been established in laboratory models. We will continue to screen animal populations to identify a C. canis or C. felis isolate.
Objective 2: Three Cryptosporidium species have been tested and compared in the HCT-8 cell culture. HCT-8 cells are susceptible to infection and support parasite replication. These studies will be continued and expanded to include other non-parvum isolates as they become available.
Objective 3: The C. meleagridis and C. muris isolates have been tested in healthy adult volunteers. The C. muris testing has been completed; five additional volunteers will receive the C. meleagridis isolate. A third non-parvum species will be tested when it becomes available.
Journal Articles on this Report : 3 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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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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Robinson P, Okhuysen PC, Chappell CL, Weinstock JV, Lewis DE, Actor JK, White Jr AC. Substance P expression correlates with severity of diarrhea in cryptosporidiosis. The Journal of Infectious Diseases 2003;188(2):290-296. |
R829180 (2003) |
Exit Exit |
Supplemental Keywords:
mucosal immunity, coccidian, water quality, human subjects, animal subjects, non-parvum Cryptosporidium, Cryptosporidium, Cryptosporidium parvum, C. muris, C. meleagridis, HCT-8., RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Water, POLLUTANTS/TOXICS, Health Risk Assessment, Epidemiology, Risk Assessments, Biology, Drinking Water, 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.