presentatie 2 maart 2012
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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
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