Prevalence, characterization and long term follow up of enterohemorrhagic E. coli and TTSS specific

antibodies in cattle

Maria-Adelheid Joris

Promotoren: Prof. dr. L. De Zutter, Prof. dr. E. Cox, Dr. ir. K. De Reu

Overview

1. Introduction2. Aims3. Results

o Occurrence EHEC in cattleo Genetic diversityo Serum antibody response

4. General conclusions and future perspectives

Introduction

O104:H4

BENIGNE BACTERIA GONE BAD

Introduction

SITUATION

Campylobacteriosis

Salmonellosis

Yersiniosis

VTEC

Toxoplasmosis

0 100000 200000

198252

108614

7595

3573

1259

Foodborne infections Europe, 2009•VTEC cases (2009)

– EU o 3.573 caseso 242 HUSo 51,7% O157

– Belgiumo 110 caseso 14 HUS

Outbreaks, e.g.o Hamburger outbreak, Jack in the box, USA, 1993 (>700, 51, 4)o Spinach outbreak, USA, 2006 (204, 31, 3)o Ice-cream related outbreak, Belgium, 2007 (12, 5, 0)

Introduction

SITUATION

Introduction

• Attaching and effacing lesiono LEE pathogenicity islando Type Three Secretion Systemo EspA, EspB and Tiro Intimin

IntroductionVIRULENCE PROPERTIES

Enterocyte

Bacteria

Intimin

Tir

TTSS

EspB

EspA

IntroductionVIRULENCE PROPERTIES

Production of Verocytotoxinso Bacteriophageo Vtx1 and Vtx2

• Vtx1: 3 subtypes• Vtx2: 7 subtypes

o Gb3 and Gb4 Inhibit protein synthesis cell death

Introduction

• Asymptomatic carriers - Gb3 ?- Abrogated transcytosis VT

• Variable shedding– Intermittent shedding– Colonization terminal rectum – Supershedders

• Persistence on cattle farms

BOVINE RESERVOIR

IntroductionTRANSMISSION TO HUMANS

Anim

al re

serv

oir

Introduction

• Very low infectious dose • YOPI • Clinical outcome:

CLINICAL OUTCOME

EHEC ingested

AIMS

1. Occurrence in beef cattleSlaughterCattle farms

2. Genetic diversityCattle farms

3. Serum antibody responses Cattle farms

ResultsResults

1 Occurrence in beef cattle

SlaughterCattle farms

2 Genetic diversity

Cattle farms

3 Serum antibody responses in cattle

Cattle farms

MethodsO157 NON-O157 (O26, O103, O111 and O145)

Rectal fecal sample (25g)

Enrichment

IMS after 6h

CT-SMAC

Purification

Molecular methods: Serogroup PCR Multiplex virulence PCR

Rectal fecal sample (25g)

Enrichment

Direct plating (6 & 24h) and IMS (24h)

Differential agar medium

Purification

Molecular methods: Serogroup PCR Multiplex virulence PCR

Results1. OCCURRENCE OF EHEC IN CATTLE

Slaughter Cross-sectional

A B

F

C

E

D

G

K

I

H

J

L

L

H

D

Longitudinal

Results1. OCCURRENCE OF EHEC IN CATTLE

  

Male Female Total

N° animals sampled 349 50 399

O26/O103/O111/O145 positive animals 18 6 24

Vtx-negative animals 5 3 8

Vtx-positive animals 13 3 16

N° farms sampled 174 25 199

Results1. OCCURRENCE OF EHEC IN CATTLE

 Serogroup

Virulence determinantsN° of isolates

vtx1 vtx2 eaeA

O26 - - + 3O26 + + + 1O26 + - + 2O26 - + - 1O26 + - - 2O103 - - - 2O103 + - + 2O103 + + + 1O103 - + + 3O103 - + - 1O103 - - + 1O111 + - + 1O111 - + + 1O145 - - + 2O145 - + + 1

Results1. OCCURRENCE OF EHEC IN CATTLE

Slaughter Cross-sectional

A B

F

C

E

D

G

K

I

H

J

L

L

H

D

Longitudinal

Results

1. OCCURRENCE OF EHEC IN CATTLE

HerdSerogroup(s)at the slaughterhouse

Herd Size

Herd typeN° positive animals/total sampled (%)

Serogroup(s)at the herd

A O157 118 Beef-producing farm 4/53(7.5%) O157

B O157 41 Beef-producing farm 2/25(8.0%) O157

C O157 51 Beef-producing farm 0/31(0.0%) n.d.D O157 270 Mixed 7/70(10.0%) O157E O103 15 Beef-producing farm 0/12(0.0%) n.d.

F O103O157 101 Beef-producing farm

9/49 (18.4%)  7/49 (14.3%)2/49 (4.1%)

O103O157

G O103 18 Beef-producing farm 0/15(0.0%) n.d.

H O103 47 Mixed3/31(9.7%)  3/31 (9.7%)1/31 (3.2%)

O103O26

I O103 47 Mixed 3/31(9.7%) O103J O26 152 Mixed 2/59(3.4%) O26K O26 74 Mixed 1/52(1.9%) O26L O26 58 Beef-producing farm 4/36(11.1%) O26

Results1. OCCURRENCE OF EHEC IN CATTLE

Slaughter Cross-sectional

A B

F

C

E

D

G

K

I

H

J

L

Longitudinal

H

L

D

Results1. OCCURRENCE OF EHEC IN CATTLE

 Sampling time points

1 2 3 4 5 6 7 8

Herd D

Animal number

1     O157   O157 O26   O1572                3       O157        4                5       O157 O157 O157    6 O157     O157 O157     O1577     O157         O1578 O157   O157          9 O157   O157          10 O157              

Herd HAnimal number

1             O1112         O26   O1113              4     O26     O26  5         O26    6     O26   O26    7         O26    8              9     O26     O26  

10         O26    

Results1. OCCURRENCE OF EHEC IN CATTLE

Farm

N° of EHEC positive samples/N° of samples tested

Cats Dogs Feed Water Dust

D 1/8 0/0 2/8 0/8 4/8

H 1/7 2/7 2/7 1/7 3/7

L 1/8 0/0 0/8 1/8 2/8

ResultsResults

1 Occurrence in beef cattle

SlaughterCattle farms

2

Genetic diversity

Cattle farms

3 Serum antibody responses in cattle

Cattle farms

Principle PFGE2. Genetic diversity

Each lane = one organism

Bacterial chromosal DNA cleaved with an enzyme

Electrophoresis champerSeperates DNA fragments

2. Genetic diversityo Cross-sectional study

9 cattle farms max. 4 PFGE types/serogroup 10 pulsotypes 7 farm-specific 1 type on 2 farms in close proximity

o Longitudinal study

3 cattle farms max. 3 PFGE types/serogroup 11 pulsotypes 19 isolates: not typeable 10 farm-specific No common pulsotypes with the cross-sectional study

ResultsResultsResults

1 Occurrence in beef cattle

SlaughterCattle farms

2 Genetic diversity

Cattle farms

3 Serum antibody responses in cattleCattle farms

ELISAPrinciple

3. Serum antibody response

Intimin

Cross-sectional study

- Antibodies long-lived ? - Cross-reaction with other

TTSS bacteria ?- Farms not EHEC-negative ?

3. Serum antibody responseLongitudinal study – Animal 1

EspA ~ Intimin, TirEspB

3. Serum antibody responseLongitudinal study – Animal 2

EspAEspB

3. Serum antibody responseLongitudinal study – Animal 3

EspAEspB

3. Serum antibody responseLongitudinal study – Animal 4

EspAEspB

3. Serum antibody response

- Serum antibody response to intimin, Tir and EspA : Short-lived

- Serum antibody response to EspB :

• Persistent

• Assessment EHEC infection status cattle farms

- Combining serum antibody response to EspA and EspB:

• Identify animals with recent or previous EHEC infection

- Need for validation and evaluation

General conclusions

General conclusions- First study in Belgium: EHEC non-O157 - Cattle at slaughter: 3% EHEC non-O157- Within-herd prevalence: 1,9 – 18,4% - EHEC present in cats, dogs and environment

- Common types on 2 farms in close proximity- No common types: cross-sectional and

longitudinal study

- Screening cattle herds- EspB

- Identification EHEC infected cattle - EspA + EspB

General conclusionsEHEC status cattle farms

General conclusions

General discussionFuture perspectives

Intermittent shedding ?- Re-ingestion ? - Excretion < detection limit ?- EHEC resides in GI tract ?

Importance ecological niches ?

???

Acknowledgements

Acknowledgements

Laboratory of ImmunologyProf. Eric CoxDr. Kris Vande Walle

ILVODr. Ir. Koen De ReuDr. Karen Verstraete

Financial supportFOD Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu