Maria-Adelheid Joris conducted research on the occurrence and genetic diversity of Shiga toxin-producing Escherichia coli (STEC) in Belgian cattle herds. The study found: (1) STEC non-O157 strains were present in 3% of cattle at slaughter, with within-herd prevalence ranging from 1.9-18.4%; (2) STEC strains were isolated from the environment on farms, including cats, dogs, feed, and water; (3) genetic analysis identified unique STEC strains on individual farms. Serum antibody responses in cattle suggested EspB could be used to identify animals with past or present STEC infection. The research provides new insights into

1. Maria-Adelheid Joris
Promotoren: Prof. dr. L. De Zutter, Prof. dr. E. Cox, Dr. ir. K. De Reu

2. 1. Introduction
2. Aims
3. Results
o Occurrence EHEC in cattle
o Genetic diversity
o Serum antibody response
4. General conclusions and future perspectives

3. Introduction
O104:H4
BENIGNE BACTERIA GONE BAD

4. Introduction
SITUATION
198252
108614
7595
3573
1259
0 100000 200000
Campylobacteriosis
Salmonellosis
Yersiniosis
VTEC
Toxoplasmosis
Foodborne infections Europe, 2009
•VTEC cases (2009)
–EU
o3.573 cases
o242 HUS
o51,7% O157
–Belgium
o110 cases
o14 HUS

5. Outbreaks, e.g.
oHamburger outbreak, Jack in the box, USA, 1993 (>700, 51, 4)
oSpinach outbreak, USA, 2006 (204, 31, 3)
oIce-cream related outbreak, Belgium, 2007 (12, 5, 0)
Introduction
SITUATION

6. Introduction
• Attaching and effacing lesion
o LEE pathogenicity island
o Type Three Secretion System
o EspA, EspB and Tir
o Intimin
Introduction
VIRULENCE PROPERTIES

7. Enterocyte
Bacteria
Intimin
Tir
TTSS
EspB
EspA

8. Introduction
VIRULENCE PROPERTIES
 Production of Verocytotoxins
oBacteriophage
oVtx1 and Vtx2
• Vtx1: 3 subtypes
• Vtx2: 7 subtypes
oGb3 and Gb4  Inhibit protein synthesis  cell death

9. Introduction
• Asymptomatic carriers
- Gb3 ?
- Abrogated transcytosis VT
• Variable shedding
– Intermittent shedding
– Colonization terminal rectum
– Supershedders
• Persistence on cattle farms
BOVINE RESERVOIR

10. Introduction
TRANSMISSION TO HUMANS
Animalreservoir

11. Introduction
• Very low infectious dose
• YOPI
• Clinical outcome:
CLINICAL OUTCOME
EHEC ingested

12. AIMS

13. ResultsResults

14. Methods
O157 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

15. Results
1. OCCURRENCE OF EHEC IN CATTLE
Slaughter Cross-sectional
A B
F
C
E
D
G
K
I
H
J
L
L
H
D
Longitudinal

16. Results
1. 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

17. Results
1. OCCURRENCE OF EHEC IN CATTLE
Serogroup
Virulence determinants
N° of isolates
vtx1 vtx2 eaeA
O26 - - + 3
O26 + + + 1
O26 + - + 2
O26 - + - 1
O26 + - - 2
O103 - - - 2
O103 + - + 2
O103 + + + 1
O103 - + + 3
O103 - + - 1
O103 - - + 1
O111 + - + 1
O111 - + + 1
O145 - - + 2
O145 - + + 1

18. Results
1. OCCURRENCE OF EHEC IN CATTLE
Slaughter Cross-sectional
A B
F
C
E
D
G
K
I
H
J
L
L
H
D
Longitudinal

19. Results
1. OCCURRENCE OF EHEC IN CATTLE
Herd
Serogroup(s)
at the
slaughterhouse
Herd
Size
Herd type
N° 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%) O157
E O103 15 Beef-producing farm 0/12(0.0%) n.d.
F
O103
O157
101 Beef-producing farm
9/49 (18.4%)
7/49 (14.3%)
2/49 (4.1%)
O103
O157
G O103 18 Beef-producing farm 0/15(0.0%) n.d.
H O103 47 Mixed
3/31(9.7%)
3/31 (9.7%)
1/31 (3.2%)
O103
O26
I O103 47 Mixed 3/31(9.7%) O103
J O26 152 Mixed 2/59(3.4%) O26
K O26 74 Mixed 1/52(1.9%) O26
L O26 58 Beef-producing farm 4/36(11.1%) O26

20. Results
1. OCCURRENCE OF EHEC IN CATTLE
Slaughter Cross-sectional
A B
F
C
E
D
G
K
I
H
J
L
Longitudinal
H
L
D

21. Results
1. OCCURRENCE OF EHEC IN CATTLE
Sampling time points
1 2 3 4 5 6 7 8
HerdD
Animalnumber
1 O157 O157 O26 O157
2
3 O157
4
5 O157 O157 O157
6 O157 O157 O157 O157
7 O157 O157
8 O157 O157
9 O157 O157
10 O157
HerdH
Animalnumber
1 O111
2 O26 O111
3
4 O26 O26
5 O26
6 O26 O26
7 O26
8
9 O26 O26
10 O26

22. Results
1. 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

23. Results
Results

24. Principle PFGE
2. Genetic diversity
Each lane = one organism
Bacterial chromosal DNA
cleaved with an enzyme
Electrophoresis champer
Seperates DNA fragments

25. 2. Genetic diversity
o 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

26. Results
Results
Results

27. ELISA
Principle

28. 3. Serum antibody response
Intimin
Cross-sectional study
- Antibodies long-lived ?
- Cross-reaction with other
TTSS bacteria ?
- Farms not EHEC-negative ?

29. 3. Serum antibody response
Longitudinal study – Animal 1
EspA ~ Intimin, Tir
EspB

30. 3. Serum antibody response
Longitudinal study – Animal 2
EspA
EspB

31. 3. Serum antibody response
Longitudinal study – Animal 3
EspA
EspB

32. 3. Serum antibody response
Longitudinal study – Animal 4
EspA
EspB

33. 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

34. General conclusions

35. 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

36. General conclusions
EHEC status cattle farms

37. General conclusions

38. General discussion
Future perspectives
Intermittent shedding ?
- Re-ingestion ?
- Excretion < detection limit ?
- EHEC resides in GI tract ?
Importance ecological niches ?

39. Acknowledgements

40. Acknowledgements
Laboratory of Immunology
Prof. Eric Cox
Dr. Kris Vande Walle
ILVO
Dr. Ir. Koen De Reu
Dr. Karen Verstraete
Financial support
FOD Volksgezondheid, Veiligheid van
de Voedselketen
en Leefmilieu