The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs and causes devastating losses to the farming industry. Whilst some hyper-virulent isolates have been described, the relationship between genetics and disease outcome has been only partially established. In particular, there is weak correlation between serovar and disease phenotype. We sequenced the genomes of 212 isolates of H. parasuis and have used this to describe the pan-genome and to correlate this with clinical and carrier status, as well as with serotype. RESULTS: Recombination and population structure analyses identified five groups with very high rates of recombination, separated into two clades of H. parasuis with no signs of recombination between them. We used genome-wide association methods including discriminant analysis of principal components (DAPC) and generalised linear modelling (glm) to look for genetic determinants of this population partition, serovar and pathogenicity. We were able to identify genes from the accessory genome that were significantly associated with phenotypes such as potential serovar specific genes including capsule genes, and 48 putative virulence factors that were significantly different between the clinical and non-clinical isolates. We also show that the presence of many previously suggested virulence factors is not an appropriate marker of virulence. CONCLUSIONS: These genes will inform the generation of new molecular diagnostics and vaccines, and refinement of existing typing schemes and show the importance of the accessory genome of a diverse species when investigating the relationship between genotypes and phenotypes.

We thank several people for their valued help with this study, these include: Patrick Blackall of the University of Queensland, Australia for the provision of copies of the Australian reference strain set; Gemma Murray for the use of the R script that allowed us to analyze the COG functional groups of the homology groups; Simon Harris for the useful discussion about Gubbins and the issues surrounding the prediction of recombination; Feifei Shen for assistance in the purification and DNA preparation of the isolates that were sent for sequencing; Brian Hunt, Jon Rogers and Sarah Howie of the Animal Health and Veterinary Laboratories Agency for collection and biochemical profiling of UK isolates; and TJ McKinley for his advice on the statistical analysis. This work was supported by a BPEX PhD studentship and 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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. List of consortium members: University of Cambridge: Duncan J. Maskell, Alexander W. (Dan) Tucker, Sarah E. Peters, Lucy A.Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, §Roy R. Chaudhuri. § Present address: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK. Royal Veterinary College: Andrew N. Rycroft, Gareth A. Maglennon, Dominic Matthews. Imperial College London: Paul R. Langford, Janine T. Bossé, Yanwen Li. London School of Hygiene and Tropical Medicine: Brendan W. Wren, Jon Cuccui, Vanessa S. Terra.

This is the published version, available at BMC Genomics 2014, 15:1179 doi: 10.1186/1471-2164-15-1179