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? 3. How can risk assessment modelers use this data in a meaningful way? 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?

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

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

MurineCell cultures Volunteers Disinfection studies Viability/ infectivity Prediction of pathogenicity

CriteriaMurineCell culture Susceptible Sensitive Ease of handling Rapid analysis High thru-put Cost effective +/- + +/ / Comparison of Models

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

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

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

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

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

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

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

Human Animal Human cells Human cells Animal cells Animal cells Predictability Paradigm

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? 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? Cryptosporidium Volunteer Model

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

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

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

Infectivity and Illness Human ID 50 Illness (%) UCP IOWA8752 TAMU986

Dose Response Curves in Neonatal Mice

Human ID 50 Mouse ID 50 UCP IOWA8799 TAMU930 Infectivity of C. parvum isolates

Cryptosporidium Infection in HCT-8 Cells

TAMUUCPIOWA

Infectivity of C. parvum isolates Human ID 50 Mouse ID 50 % Cell infection UCP IOWA TAMU

Infectivity in Human vs Neonatal Mice and HCT-8 Cells r 2 = r 2 = 0.548

Conclusions HCT-8 is cell line of choice:  Natural target cell (human)  Supports multiple genotypes  Shows high correlation with human genotype 2 ID 50 ’s HCT-8 is cell line of choice:  Natural target cell (human)  Supports multiple genotypes  Shows high correlation with human genotype 2 ID 50 ’s In vitro system:  More practical than murine model  Meets criteria for water industry In vitro system:  More practical than murine model  Meets criteria for water industry

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

Any Questions??