title: The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis.
abstract: 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.
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. kjh52@cam.ac.uk.
authors.forename: Kate J
authors.initials: KJ
authors.lastname: Howell
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. lucy.weinert@gmail.com.
authors.forename: Lucy A
authors.initials: LA
authors.lastname: Weinert
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK. r.chaudhuri@sheffield.ac.uk.
authors.forename: Roy R
authors.initials: RR
authors.lastname: Chaudhuri
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. sl470@cam.ac.uk.
authors.forename: Shi-Lu
authors.initials: SL
authors.lastname: Luan
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. sep34@cam.ac.uk.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Peters
authors.affiliation: Department of Mathematics and Statistics, University of Helsinki, Helsinki, 00100, Finland. jukka.corander@helsinki.fi.
authors.forename: Jukka
authors.initials: J
authors.lastname: Corander
authors.affiliation: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. deh@sanger.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Harris
authors.affiliation: Norwegian Veterinary Institute, N-0106, Oslo, Norway. Oystein.Angen@vetinst.no.
authors.forename: Øystein
authors.initials: Ø
authors.lastname: Angen
authors.affiliation: Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193, Bellaterra, and, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain. Virginia.Aragon@cresa.uab.cat.
authors.forename: Virginia
authors.initials: V
authors.lastname: Aragon
authors.affiliation: Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193, Bellaterra, and, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain. albert.bensaid@cresa.uab.es.
authors.forename: Albert
authors.initials: A
authors.lastname: Bensaid
authors.affiliation: Animal Health and Veterinary Laboratories Agency (AHVLA), Rougham Hill, Bury St Edmunds, Suffolk, IP33 2RX, UK. Susanna.Williamson@ahvla.gsi.gov.uk.
authors.forename: Susanna M
authors.initials: SM
authors.lastname: Williamson
authors.affiliation: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. parkhill@sanger.ac.uk.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Department of Medicine, Section of Paediatrics, Imperial College London, St. Mary's Campus, London, W2 1PG, UK. p.langford@imperial.ac.uk.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Langford
authors.affiliation: The Royal Veterinary College, Hawkshead Campus, Hatfield, AL9 7TA, Hertfordshire, UK. ARycroft@rvc.ac.uk.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Rycroft
authors.affiliation: Faculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. brendan.wren@lshtm.ac.uk.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.affiliation: School of Medicine, University of St. Andrews, St Andrews, KY16 9TF, UK. mh3@sanger.ac.uk.
authors.forename: Matthew T G
authors.initials: MT
authors.lastname: Holden
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. awt1000@cam.ac.uk.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tucker
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. djm47@cam.ac.uk.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
mesh_terms: D000818:Animals; D016679:Genome, Viral; D055106:Genome-Wide Association Study; D044137:Haemophilus parasuis / Q000145:classification / Q000235:genetics / Q000472:pathogenicity*; D011995:Recombination, Genetic; D013552:Swine / Q000821:virology; D014774:Virulence / Q000235:genetics