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

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
(No Transcript)
6
Comparison of Models
7
Are data comparablebetween models and among labs?
Question being asked
Model system
Experimental design
Sensitive parameters
How data are expressed
8
Murine Model Methodology
Oral inoculation
Outcome measures
Incubation
Termination
Data expressed (per dose) of infected/ of
challenged Infection per mouse1-4 scale
9
Definition of parameters
1
2
3
4
Oral inoculation
Outcome measures
Incubation
Termination
10
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

11
Definition of parameters
Inoculate with oocysts
Plate cells
Terminate
12
Detection systems
Microtiter plate
Slide chambers
Visual counting Nomarski Stain IFA FISH CISH
Agarose bands PCR RT-PCR
Optical density Antibody labelling
13
How can infectivity data be used in a meaningful
way? OR Is there a laboratory model that will
predict pathogenicity in humans?
14
Predictability Paradigm
Human
Human cells
Animal
Animal cells
15
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?

16
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)
17
(No Transcript)
18
(No Transcript)
19
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
20
Dose Response Antibody-negative Volunteers
Ref of method Reed and Muench, Am J Hyg 27493,
1938
21
Infectivity and Illness
22
(No Transcript)
23
Dose Response Curves in Neonatal Mice
24
Infectivity of C. parvum isolates
25
(No Transcript)
26
Cryptosporidium Infection in HCT-8 Cells
27
TAMU
UCP
IOWA
28
Infectivity of C. parvum isolates
29
Infectivity in Human vs Neonatal Mice and HCT-8
Cells
r2 0.933
r2 0.548
30
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
  ID50s

31
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)

32
Any Questions??