title: Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus.
abstract: Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.
authors.affiliation: Department of Microbiology, Monash University, Melbourne, Australia Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Melbourne, Australia.
authors.forename: Marina
authors.initials: M
authors.lastname: Harper
authors.affiliation: Department of Microbiology, Monash University, Melbourne, Australia Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Melbourne, Australia.
authors.forename: Marietta
authors.initials: M
authors.lastname: John
authors.affiliation: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Australia.
authors.forename: Conny
authors.initials: C
authors.lastname: Turni
authors.affiliation: Department of Microbiology, Monash University, Melbourne, Australia Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Melbourne, Australia Poultry CRC, University of New England, Armidale, Australia.
authors.forename: Mark
authors.initials: M
authors.lastname: Edmunds
authors.affiliation: Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: St Michael
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Melbourne, Australia.
authors.forename: Ben
authors.initials: B
authors.lastname: Adler
authors.affiliation: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Australia.
authors.forename: P J
authors.initials: PJ
authors.lastname: Blackall
authors.affiliation: Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Canada.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Cox
authors.affiliation: Department of Microbiology, Monash University, Melbourne, Australia john.boyce@monash.edu.
authors.forename: John D
authors.initials: JD
authors.lastname: Boyce
mesh_terms: D000818:Animals; D053898:Biosynthetic Pathways / Q000235:genetics*; D005838:Genotype; D006801:Humans; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010326:Pasteurella Infections / Q000175:diagnosis / Q000382:microbiology; D016979:Pasteurella multocida / Q000145:classification* / Q000235:genetics*; D013997:Time Factors
chemical_list: D008070:Lipopolysaccharides