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Of Mice, Men and Cell Cultures: Cryptosporidium parvum (Genotype 2) Infectivity

Of Mice, Men and Cell Cultures: Cryptosporidium parvum (Genotype 2) Infectivity. 1. What laboratory models are contributing to the assessment of waterborne exposure to Cryptosporidium oocysts? 2. Are the data from various studies comparable?

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Of Mice, Men and Cell Cultures: Cryptosporidium parvum (Genotype 2) Infectivity

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  1. Of Mice, Men and Cell Cultures: Cryptosporidium parvum (Genotype 2) Infectivity

  2. 1. What laboratory models are contributing to the assessment of waterborne exposure to Cryptosporidium oocysts? 2. Are the data from various studies comparable? 3. How can risk assessment modelers use this data in a meaningful way?

  3. Water Industry Criteria: Susceptible (“pathogenic” oocysts) Sensitive (low dose infection) Ease of handling (size, complexity) Rapid analysis High thru-put Cost effective

  4. Objectives of Cryptosporidium studies Efficacy of disinfection methods Oocyst viability and infectivity Prediction of pathogenicity (infection/illness) in humans

  5. Comparison of Models

  6. Are data comparable—between models and among labs? Question being asked Model system Experimental design “Sensitive” parameters How data are expressed

  7. Murine Model Methodology Oral inoculation Outcome measures Incubation Termination Data expressed (per dose) # of infected/# of challenged Infection per mouse=1-4+ scale

  8. 1 Sensitivity of model—detection limit 2 Housing—cross contamination (esp. neonates) Duration of expt—replication time (fewer oocysts, longer time) 3 Assay sensitivity—amt of tissue examined; oocyst detection (stain, IFA, EIA) 4 Definition of parameters 1 2 3 4 Oral inoculation Outcome measures Incubation Termination

  9. Cell Culture Methodology Inoculate with oocysts Plate cells Terminate • Data expressed as: • Infected vs uninfected • # parasites/# host cells or fields • # of foci (cluster of parasites) • Absorbance values

  10. 1 2 3 4 Cell line Susceptibility Growth rate Ratio of oocysts:cells Replication cycle Outcome Measures Definition of parameters Inoculate with oocysts Plate cells Terminate

  11. Detection systems Microtiter plate Slide chambers Visual counting: Nomarski Stain IFA FISH CISH Optical density: Antibody labelling Agarose bands: PCR RT-PCR:

  12. MEN ? How can infectivity data be used in a meaningful way? OR Is there a laboratory model that will predict pathogenicity in humans?

  13. Predictability Paradigm Human Human cells Animal Animal cells

  14. Cryptosporidium Volunteer Model • Strengths: • Assessment of infectivity and illness • Defined healthy population • Highly relevant • Immunologically mature • Mucosal/systemic responses • Limitations: • Host biological variation (outbred) • Limited numbers (stats) • Relevant for sensitive populations?

  15. Laboratory staff: Blue Johnson Han Dang Constance Wang Michael Coletta Sonia Baker Danny Nguyen Marilyn Marshall (AZ) Investigators: Cynthia Chappell, PhD Pablo Okhuysen, MD Herbert DuPont, MD UCRC Nursing staff: Madeline Jewell, RN Julie Rice, RN Nai-Hui Chiu , RN Isolates: Charles Sterling, PhD (IOWA) Karen Snowden, DVM (TAMU) Joseph Crabb, PhD (UCP)

  16. Cryptosporidium Volunteer Study Volunteers selected for: Excellent general health Normal immune status --Normal IgA levels --Normal T-cell subsets HIV negative C. parvum ab status (ELISA) Stool collection (all stools for 14d, 2/wk for 4 wks) Challenged with a single dose Of Cryptosporidium oocysts Physical exam (daily for 14d, 3/wk for 6 wks) Personal health diary, including all GI symptoms

  17. Dose Response: Antibody-negative Volunteers Ref of method: Reed and Muench, Am J Hyg 27:493, 1938

  18. Infectivity and Illness

  19. Dose Response Curves in Neonatal Mice

  20. Infectivity of C. parvum isolates

  21. Cryptosporidium Infection in HCT-8 Cells

  22. UCP IOWA TAMU

  23. Infectivity of C. parvum isolates

  24. Infectivity in Human vs Neonatal Mice and HCT-8 Cells r2 = 0.933 r2 = 0.548

  25. Conclusions • In vitro system: • More practical than murine model • Meets criteria for water industry • HCT-8 is cell line of choice: • Natural target cell (human) • Supports multiple genotypes • Shows high correlation with human genotype 2 ID50’s

  26. Future Studies • Expand study to other G2 and G1 isolates • Compare predictability of HCT-8 with other cell lines • Characterize and define methods (each step) • Compare detection systems (criteria)

  27. Any Questions??

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