Poster Presentation International Pasteurellaceae Conference 2014

A multiplex PCR to unequivocally differentiate Actinobacillus pleuropneumoniae serovars 1-3, 5-8, 10 and 12 (#42)

Janine T. Bosse 1 , Yanwen Li 1 , Oystein Angen 2 , Roy R. Chaudhuri 3 4 , Lucy A. Weinert 3 , Matt T. Holden 5 , Susanna M. Williamson 6 , Duncan J. Maskell 3 , Alexander W. Tucker 3 , Brendan W. Wren 7 , Andrew N. Rycroft 8 , Paul R. Langford 1 , on behalf of the BRaDP1T Consortium 1 3 7 8
  1. Department of Medicine, Imperial College London, London, UK
  2. Department of Bacteriology - Aquatic and Terrestrial Animals, Norwegian Veterinary Institute, Oslo, Norway
  3. Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
  4. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK
  5. The Pathogen Sequencing Unit, The Wellcome Trust Sanger Institute, Cambridge, UK
  6. Animal Health and Veterinary Laboratories Agency (AHVLA), Bury St Edmunds, UK
  7. Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London, UK
  8. Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, UK

Aims: (1) Design a new serovar 3, 6, 8 and apxIV multiplex PCR (mPCR) that eliminates a double banding pattern found in some serovar 6 Actinobacillus pleuropneumoniae (APP) clinical isolates when our original PCR1 is used; and (2) Extend the mPCR to detect serovars 1, 2, 5, 7, 10, and 12.

Methods: Complete capsule loci for APP serovar 1, 2, 3, 5, 6, 7, 10 and 12 strains were obtained from Genbank. Genome sequence data was generated for a double-banding serovar 6 isolate (MIDG2472) and for serovar 8 strain 405. Capsule loci were identified by BLAST and unique sequences were identified for generation of serovar-specific primers. These were combined with an apxIV primer pair in a mPCR whose specificity was tested using an extensive collection of APP and control strains.

Results : Only 3 APP isolates, previously serotyped as serovars 1 and 7, did not give the predicted serovar-specific  amplicon. Two isolates produced only the apxIV amplicon, indicating that they may be one of the serovars not included in the mPCR. The other previously designated serovar 7 isolate amplified serovar 12-specific and apxIV  PCR products.  Only 5/352 APP isolates (all serovar 1) failed to amplify the apxIV band, although the presence of the gene was confirmed using a different set of primers. All other APP isolates amplified the predicted serovar-specific and apxIV amplicons. No amplification products were obtained from other bacterial pig pathogens or commensals.

Conclusion: The new mPCR was highly specific for the 9 serovars of APP tested.

Acknowledgements

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G003203/1), the UK Department for Environment, Food and Rural Affairs and Zoetis awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium.

  1. Zhou L, Jones SC, Angen Ø, Bossé JT, Nash JH, Frey J, Zhou R, Chen HC, Kroll JS, Rycroft AN, and Langford PR. 2008. Multiplex PCR that can distinguish between immunologically cross- reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains. J Clin Microbiol. 46:800-3.