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?

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 effectiveCost effective

Water Industry Criteria:

Susceptible (“pathogenic” oocysts)Susceptible (“pathogenic” oocysts)

Sensitive (low dose infection)Sensitive (low dose infection)

Ease of handling (size, complexity)Ease of handling (size, complexity)

Rapid analysisRapid analysis

High thru-putHigh thru-put

Prediction of pathogenicity (infection/illness) in humansPrediction of pathogenicity

(infection/illness) in humans

Objectives of Cryptosporidium studies

Efficacy of disinfection methodsEfficacy of disinfection methods

Oocyst viability and infectivityOocyst viability and infectivity

Murine Cell cultur

es

Volunteers

Disinfection studies

Viability/infectivity

Prediction of pathogenicity

+

+

+

+

+

+ +

Criteria Murine Cell culture

SusceptibleSensitiveEase of handlingRapid analysisHigh thru-putCost effective

+/-++/-7+3++/-

+++

2+3++

Comparison of Models

Are data comparable—between models and among

labs?

How data are expressedHow data are expressed

Question being askedQuestion being asked

Model systemModel system

Experimental designExperimental design

“Sensitive” parameters“Sensitive” parameters

Murine Model Methodology

Oral inoculation Incubation Termination

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 Incubation Termination

11 22 33 44

Outcome measures

11Sensitivity of model—detection limitSensitivity of model—detection limit

22Housing—cross contamination (esp. neonates)Housing—cross contamination (esp. neonates)

33Duration of expt—replication time (fewer oocysts, longer time)Duration of expt—replication time (fewer oocysts, longer time)

44Assay 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 cellsPlate cellsInoculate

with oocystsInoculate

with oocysts TerminateTerminate

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 cellsPlate cellsInoculate

with oocystsInoculate

with oocysts TerminateTerminate

11

Cell lineSusceptibilityGrowth rate

Cell lineSusceptibilityGrowth rate

22

Ratio of oocysts:cells

Ratio of oocysts:cells

33

Replication

cycle

Replication

cycle

44

Outcome MeasuresOutcome Measures

Definition of parameters

Detection systemsDetection systemsMicrotiter plateMicrotiter plate

Slide chambersSlide chambers

Visual counting:Nomarski

StainIFA

FISHCISH

Visual counting:Nomarski

StainIFA

FISHCISH

Agarose bands:PCR

RT-PCR:

Agarose bands:PCR

RT-PCR:

Optical density:Antibody labellingOptical density:

Antibody labelling

How can infectivity data be used in a meaningful way?

ORIs there a laboratory model that will

predict pathogenicity in humans?

How can infectivity data be used in a meaningful way?

ORIs there a laboratory model that will

predict pathogenicity in humans?

??MEN

HumanHuman

AnimalAnimalHuman

cellsHuman

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, PhDPablo Okhuysen, MDHerbert DuPont, MD

Investigators:Cynthia Chappell, PhDPablo Okhuysen, MDHerbert 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 JohnsonHan DangConstance WangMichael ColettaSonia BakerDanny NguyenMarilyn Marshall (AZ)

Laboratory staff:Blue JohnsonHan DangConstance WangMichael ColettaSonia BakerDanny NguyenMarilyn Marshall (AZ)

UCRC Nursing staff:Madeline Jewell, RNJulie Rice, RNNai-Hui Chiu , RN

UCRC Nursing staff:Madeline Jewell, RNJulie Rice, RNNai-Hui Chiu , RN

Study design

Oocyst collection

Calf infection

Oocyst purification

CD1 neonatal mouse

HCT-8 cell cultures

Healthy volunteers

Volunteers selected for:Excellent general healthNormal immune status--Normal IgA levels--Normal T-cell subsetsHIV negativeC. parvum ab status (ELISA)

Volunteers selected for:Excellent general healthNormal immune status--Normal IgA levels--Normal T-cell subsetsHIV negativeC. parvum ab status (ELISA)

Challenged with a single doseOf Cryptosporidium oocystsChallenged with a single doseOf 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

0 1 2 3 4 5

Challenge dose (log)

20

30

40

50

60

70

80

90

100

110

Cu

mu

lati

ve %

infe

cted

IOWATAMUUCP

Infectivity and Illness

Human ID50 Illness (%)

UCP 1042 54

IOWA 87 52

TAMU 9 86

CD1 neonatal mouse model

CD1 mice (4-6 days old) orally gavaged with single dose of C. parvum oocysts

Neonates raised by their dams until termination of experiment at 7 days PI

Infection assessed by microscopy of terminal ileum

Dose Response Curves in Neonatal Mice

0 0.5 1 1.5 2 2.5 3

Challenge dose (log)

0

20

40

60

80

100

120

Cu

mu

lati

ve %

infe

ctio

n

IowaTamuUCP

Human ID50

Mouse ID50

UCP 1042 76

IOWA 87 99

TAMU

9 30

Infectivity of C. parvum isolates

HCT-8 (human enterocyte) cell cultures

HCT-8 cells placed in well (1000 cells/mm2)

Grown to 70-80% confluency (approx. 18 hrs)

Oocysts permeabilized with sodium hypochlorite

Oocysts added to cells in 1:1 ratio

Infection developed for 24 hrs

Fixed with cold MEOH

Stained and counted

Cryptosporidium Infection in HCT-8 Cells

C. parvum infectivity in HCT-8 Cells

0

10

20

30

40

50

60

70

% I

nfe

cti

vit

y

Isolates tested in 4 experiments (each in triplicate)

p=<0.01

TAMUUCP IOWA

Infectivity of C. parvum isolates

Human ID50

Mouse ID50% Cell

infection

UCP 1042 76 24.7

IOWA 87 99 35.4

TAMU 9 30 59.5

0.5 1 1.5 2 2.5 3 3.5

Volunteer ID50 (log)

20

30

40

50

60

70

Per

cen

t in

fect

ion

0.5 1 1.5 2 2.5 3 3.5

Volunteer ID50 (log)

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

Mo

use

ID50

(lo

g)

Infectivity in Human vs Neonatal Mice and HCT-8 Cells

r2 = 0.933r2 = 0.933 r2 = 0.548r2 = 0.548

Conclusions

HCT-8 is cell line of choice: Natural target cell (human) Supports multiple genotypes Shows high correlation with human genotype 2 ID50’s

HCT-8 is cell line of choice: Natural target cell (human) Supports multiple genotypes Shows high correlation with human genotype 2 ID50’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) Compare detection systems (criteria)

Expand study to other G2 and G1 isolates Expand study to other G2 and G1 isolates

Compare predictability of HCT-8 with other cell lines

Compare predictability of HCT-8 with other cell lines

Characterize and define methods (each step)

Characterize and define methods (each step)

Any Questions??