title: Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis.
abstract: Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom kjh52@cam.ac.uk.
authors.forename: Kate J
authors.initials: KJ
authors.lastname: Howell
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Peters
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Jinhong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Juan
authors.initials: J
authors.lastname: Hernandez-Garcia
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Lucy A
authors.initials: LA
authors.lastname: Weinert
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Shi-Lu
authors.initials: SL
authors.lastname: Luan
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, Kingdom.
authors.forename: Roy R
authors.initials: RR
authors.lastname: Chaudhuri
authors.affiliation: Norwegian Veterinary Institute, Oslo, Norway.
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, Bellaterra, and Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Aragon
authors.affiliation: Animal and Plant Health Agency (APHA), Suffolk, United Kingdom.
authors.forename: Susanna M
authors.initials: SM
authors.lastname: Williamson
authors.affiliation: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors.forename: Julian
authors.identifier: http://orcid.org/0000-0002-7069-5958
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St. Mary's Campus, London, United Kingdom.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Langford
authors.affiliation: The Royal Veterinary College, Hawkshead Campus, Hatfield, Hertfordshire, United Kingdom.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Rycroft
authors.affiliation: Faculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, Kingdom.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tucker
mesh_terms: D000818:Animals; D016667:Bacterial Capsules / Q000235:genetics; D056426:Genetic Loci; D060005:Genotyping Techniques / Q000379:methods*; D006192:Haemophilus Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary; D044137:Haemophilus parasuis / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000382:microbiology; D013997:Time Factors