1. 2 Cryptosporidium Volunteer Study Investigators: Cynthia Chappell, PhD Pablo Okhuysen, MD Herbert DuPont, MD Isolates: Charles Sterling, PhD (Iowa)

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Cryptosporidium parvum: Infectivity in Healthy Volunteers and Laboratory Animal Models

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Cryptosporidium Volunteer Study

Investigators:Cynthia Chappell, PhDPablo Okhuysen, MDHerbert DuPont, MD

Isolates:Charles Sterling, PhD (Iowa)Karen Snowden, DVM (TAMU)Joseph Crabb, PhD (UCP)

Laboratory staff:Blue JohnsonHan DangConstance WangMichael ColettaSonia BakerDanny NguyenMarilyn Marshall

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

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Inclusion criteria

Cryptosporidium serum IgM/IgG statusNegative history and physical

ElectrocardiogramChest X-rayStool for occult blood and parasitesBlood chemistry (SMAC)Complete blood countUrinalysis

Pregnancy testHepatitis BsAgHepatitis CImmunoglobulin levelsSyphilis (RPR)HIVT-cell subsets

Pass written examination documenting understanding of study.

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Infant under 2 years in householdElderly person in householdAny contact with immunocompromised persons

Exclusion criteria

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Gender (%):

Male 53.1 Female 46.9

Ethnicity (%):

Caucasian 59.2Hispanic 15.4Black 13.8 Asian 10.0Other 1.5

Volunteer Study Population (n=130)

18-2

5

26-3

0

31-3

5

36-4

0

41-4

5

45-5

0

>50

Years of age

0

10

20

30

40

Per

cen

t

6

Preparation of Challenge Dose

Passaged in calf and purified

Shipped to Houston in potassium dichromate

Tested for adventitious agents (viruses, bacteria)

Adjusted for appropriate number

Delivered via gelatin capsule

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Specifications for oocyst use

Negative microbiology (no viable adventitious agents)

80+ excystation rate

Within 6 weeks of passage

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Study design

Oocyst collection

Calf infection

Oocyst purification

CD1 neonatal mouse

HCT-8 cell cultures

Healthy volunteers

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Isolate HistoriesIOWA isolate:

Collected from calf with diarrhea (Dr. Harley Moon, University of Iowa)Virulent for calves and humans after multiple passages Stable genotype 2 (multilocus analysis)

TAMU isolate:Identified in a foal with severe diarrhea Oocysts collected from stool student who contracted infection at necropsy Amplified in calfStable genotype 2 (multilocus analysis)

UCP isolate: Collected from calf with diarrhea (Maine)Virulent for calves after multiple passages Stable genotype 2 (multilocus analysis)

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Volunteer Study Design

Good general healthNormal immune status

normal IgA levelsnormal T-cell subsets

HIV-negativeC. parvum ab status (ELISA)

Challenged with a single dose of C. parvum oocysts

Volunteers selected for:

Stool collection(all for 14d, 2/wk for 6 wks)

Physical exam(daily for 14d, 2/wk for 6 wks)Personal health diary, including all symptoms

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0 1 2 3 4 5

Challenge dose (log)

20

30

40

50

60

70

80

90

100

110C

um

ula

tive

% in

fect

ed

IOWATAMUUCP

Ref of method: Reed and Muench, Am J Hyg 27:493, 1938

ID50 in Antibody-negative Volunteers

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Diarrhea

51.7

Oocysts

20.7

Neither

27.6

IOWA (n=29)Diarrhea

58.8

Oocysts5.9

Neither

35.3

UCP (n=17)

Diarrhea85.7

Neither14.3

TAMU (n=14)

Clinical Outcome in Volunteers

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0

10

20

30

40

50

60

70

80

90

ILL

NE

SS

AT

TAC

K R

AT

E (

%)

UCP

IOWA

TAMU

Illness Parameters Among C. parvum Isolates

Incubation period (5-9 days)Duration of diarrhea (2.7-4.4 days)Total # unformed stools (6.7-10.6)Total unformed stool weight (798-1278 g; highest with TAMU)

Not significant: p=0.045

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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

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0 0.5 1 1.5 2 2.5 3

Challenge dose (log)

0

20

40

60

80

100

120

Cu

mu

lati

ve

% i

nfe

cti

on

IowaTamuUCP

Dose Response Curves in Neonatal Mice

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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

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Cryptosporidium Infection in HCT-8 Cells

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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

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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 = 0.933 r2 = 0.548

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Conclusions

C. parvum oocysts vary widely in infectivity, even within the same genotype

HCT-8, a human enterocyte cell line, shows a higher correlation with human genotype 2 infectivity than does the neonatal mouse model.

HCT-8 also has the advantage of supporting genotype 1 replication.