Real Time PCR for the detection of E. coli Virulence Factors in Swine

2. Molecular study of virulence factors
applied to porcine colibacillosis.
Arnal J.L.; Chacón G.; Benito A.A.; García-Manrique B.; Sanz C.; Serrano J.D.; Pradas L.
jlarnal@exopol.com

3. Introduction
Fimbriae
F4 F5 F6 F18 F41
Toxins
LT STa STb Stx2e
Suckling piglets diarrhea Oedema disease
Intimine
Constitutional
eae (EPEC)
GAD
Endogenous control
Sampling
DNA isolation
Amplification
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ETEC
Postweaning disease(PWD)
F4 F4
F5
F6
F41
STa
F18
STa
STb Stx2e
F18
LT

4. Our goal
To develop and to validate qPCR assays to detect
main Virulence Factors (VF) involved in swine
colibacilosis and their later implementation in
rutine laboratorial diagnosis.

5. Materials y Methods
54 E. coli strains supplied by other reputated laboratories
ALERE technologies (n=9)
VISAVET (n=18)
IDT biologika (n=26)
ROUND ROBIN TEST (n=20). International interlaboratorial test.
11 qPCR assays (forward+reverse+probe FAM)
with the same thermal profile
Clinical samples:
feces
rectal swabs
small intestine
Techniques used in this study:
Microbiological culture
Dendrogram based on phenotypic characters (metabolism of 11 compounds)
E. coli virulence factors qPCR assays
Clinical samples:
feces
rectal swabs
small intestine
Techniques used in this study:
Microbiological culture
Dendrogram based on phenotypic characters (metabolism of 11 compounds)
E. coli virulence factors qPCR assays
54 E. coli strains supplied by other reputated laboratories
ALERE technologies (n=9)
VISAVET (n=18)
IDT biologika (n=26)
ROUND ROBIN TEST (n=20). International interlaboratorial test.
11 qPCR assays (forward+reverse+probe FAM)
with the same thermal profile

6. Material y Métodos
DNA ISOLATION Amplification
Pretreatment
Colonies
Swabs/Tissues/Feces
4 mm metallic beads
DNA extraction
LabTurbo 48 Compact System
Genomic mini Kit LGD 500
Taigen Bioscience Corporation
7500 FAST RT PCR system
Applied Biosystems
jlarnal@exopol.com

7. Results of validation on strains
54 strains
10 Virulence Factors F18
GAD
Stx2e
100% concordance

8. Results on clinical samples
Case 1: Oedemas disease
Fattening animals.
Compatible signs with oedemas disease: neurological involvement, eyelid oedema,
diarrhea, intestinal edema.
Sample: 4 small intestines.
Microbiological isolation
ID 1: E. coli*** (B-hemolytic)
ID 2: E. coli*** (B-hemolytic)
ID 3: E. coli*** (B-hemolytic)
ID 4: E. coli*** (B-hemolytic)
Dendogram
1 strain
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3 isolations per animal

9. Pool 4SI Strain1
F4 36.9 neg
F5 NR NR
F6 neg neg
F18 22,28 16,58
F41 NR NR
LT 38.04 neg
Sta neg neg
STb neg neg
Stx2e 24,39 18,29
EAE NR neg
GAD 21,1 16,97
qPCR
Virulence Factors
Case 1: Oedemas disease
Sample and Strain show
same pattern of VF
Results on clinical samples

10. Results on clinical samples
Case 2: Suckling piglets diarrhea
Animals older than 15 days.
Diarrhea and deaths in the farm.
Sample: 10 small intestines.
Microbiological isolation
ID 1: E. coli**
ID 2: E. coli**
ID 3: E. coli*** (B-hemolytic)
ID 4: E. coli*** (B-hemolytic)
ID 5: E. coli*** (B-hemolytic)
ID 6:
ID 7: E. coli**
ID 8: E. coli**
ID 9: Enterobacteria**
ID 10: Enterobacteria*
Dendogram
10 different strains
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11. Small gut (1-5) Small gut (6-10) CEPA 3 CEPA 6 CEPA 8 CEPA 9
F4 neg neg neg neg neg neg
F5 neg neg neg neg neg neg
F6 neg neg neg neg neg neg
F18 neg 36,56 neg neg neg neg
F41 neg neg neg neg neg neg
LT neg neg neg neg neg neg
Sta neg neg neg neg neg neg
STb 35,99 neg neg neg neg neg
Stx2e neg neg neg neg neg neg
EAE Neg Neg Neg Neg neg neg
GAD 19,97 23,66 17,89 18,16 20,66 17,79
RVA 29.68 20.24 NR NR NR NR
qPCR
Virulence Factors
Results on clinical samples
Case 2: Suckling piglets diarrhea
Rotavirus A
qPCR POSITIVE
Pools = Strains

12. Results on clinical samples
Case 3: Oedemas disease + PWD
Fattening animals.
Compatible signs with oedemas disease and PWD.
Sample: 4 Small intestines.
Microbiological isolations
SI 1: E. coli*** (B-hemolytic)
SI 2: E. coli*** (B-hemolytic)
SI 3: E. coli*** (B-hemolytic)
SI 4: E. coli*** (B-hemolytic)
Strain B
Strain A
Dendogram
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13. Strain A Strain B 4 SI
F4 18,11 32,56 28,95
F5 neg neg neg
F6 neg neg neg
F18 neg 15,52 21,9
F41 neg neg neg
LT neg 16,25 23,82
Sta 17,98 15,89 21,37
STb 20,89 19,97 22,21
Stx2 neg neg neg
Stx2e neg neg 26,64
EAE neg neg neg
GAD 17,09 16,82 22,19
qPCR
Virulence Factors
Results on clinical samples
Case 3: Oedemas disease + PWD
Stx2e was not found
in isolations
Relative quantification
[F18] = [GAD]
[GAD] x50times > [Stx2e]
No oedemas disease
diagnosed through
microbiological isolations

14. Resultados en muestras clínicas
Case 4: Oedemas disease + PWD
Animals with transition feeding.
Compatible signs with oedemas disease and PWD.
Sample: 1 small intestine.
Microbiological culture
SI 1: E. coli*** (B-hemolytic)
Strain A
Dendogram
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15. S.I. Strain A
F4 neg NR
F5 neg neg
F6 neg neg
F18 21,22 15,87
F41 neg neg
LT 21,95 17,31
Sta 20,28 15,77
STb 23,2 17,43
Stx2e 27,53 neg
EAE neg neg
GAD 21,73 15,79
qPCR
Virulence Factors
Resultados en muestras clínicas
Case 4: Oedemas disease + PWD
Relative quantification in
small intestine
[GAD] x(100) > [Stx2e]
No oedemas disease
diagnosed through
microbiological isolations

16. Conclusions
1. qPCR indeed increases the sensitiviy of the
virulence factors detection allowing us to:
– to analyse directly on clinical samples, individually or
conforming pools, which saves time and cost of diagnosis.
– To detect some VF that we would have missed if had
tested just microbiological isolations.
jlarnal@exopol.com

17. Conclusions
2. qPCR allows the relative quantification of a
particular VF in relation to the total amount
of E. coli cointained in a sample. Thus,
– we could know in advance if a particular VF will be detected in
their respective microbiological isolations.
– we might discard the clinical value of weakly positive VF.
jlarnal@exopol.com

18. Grazie per l'attenzione.
jlarnal@exopol.com