SIDiLV Arnal E. coli Virulence Factors - Colibacillosis
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This study aimed to develop and validate qPCR assays to detect major virulence factors involved in swine colibacillosis. Eleven qPCR assays were validated on 54 E. coli strains, showing 100% concordance for 10 factors. The assays were then applied to clinical samples from 4 cases. Case 1 involved oedema disease and qPCR identified F18 and GAD in intestinal samples and an isolated strain. Case 2 involved piglet diarrhea where qPCR detected rotavirus A and different virulence factor profiles in sample pools. Case 3 showed mixed infection but qPCR ruled out oedema disease. Case 4 also ruled out oedema disease based on relative quantification of Stx2e and GAD.
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SIDiLV Arnal E. coli Virulence Factors - Colibacillosis
- 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 jlarnal@exopol.com 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 jlarnal@exopol.com 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 jlarnal@exopol.com
- 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 jlarnal@exopol.com
- 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 jlarnal@exopol.com
- 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