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      A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay

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          ABSTRACT

          Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar—designated serovar 16—of A. pleuropneumoniae.

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          Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection.

          Janine Bossé, Paul R. Langford, J. Kroll … (2002)
          Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a highly contagious disease for which there is no effective vaccine. This review considers how adhesins, iron-acquisition factors, capsule and lipopolysaccharide, RTX cytotoxins and other potential future vaccine components contribute to colonisation, to avoidance of host clearance mechanisms and to damage of host tissues.
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            Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis.

            Kate Howell, Sarah E. Peters, Jinhong Wang … (2015)
            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.
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              Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.

              P.J. Blackall, H.L.B.M. Klaasen, H van den Bosch … (2002)
              We report on the re-examination of nine Australian isolates of Actinobacillus pleuropneumoniae that have been previously assigned to serovar 12. In the ring precipitation test, none of the nine isolates reacted with antisera to serovars 1-14 of A. pleuropneumoniae. Antiserum prepared against one of the Australian isolates gave no reaction with any of the 14 recognised serovar reference strains, except serovar 7. This reaction of the HS143 antiserum with serovar 7 antigen could be removed by adsorption with serovar 7 antigen. The adsorbed antiserum remained reactive with HS143 and the other eight Australian isolates. The nine Australian isolates were all shown to express ApxII and ApxIII, found in serovars 2, 4, 6 and 8, as well as the 42kDa outer membrane protein found in all serovars of A. pleuropneumoniae. The nine Australian isolates were found to possess the following toxin associated genes apxIBD, apxIICA, apxIIICA, apxIIIBD and apxIVA. The toxin gene profile of the Australian isolates is typical of A. pleuropneumoniae serovars 2, 4, 6 and 8. On the basis of the serological characterisation results and the toxin gene profiles, we propose that these isolates represent a new serovar of A. pleuropneumoniae--serovar 15--with HS143 being the reference strain for the new serovar.
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                Author and article information

                Contributors
                Brad Fenwick: Role: Editor
                Journal
                Journal ID (nlm-ta): J Clin Microbiol
                Journal ID (iso-abbrev): J. Clin. Microbiol
                Journal ID (hwp): jcm
                Journal ID (pmc): jcm
                Journal ID (publisher-id): JCM
                Title: Journal of Clinical Microbiology
                Publisher: American Society for Microbiology (1752 N St., N.W., Washington, DC )
                ISSN (Print): 0095-1137
                ISSN (Electronic): 1098-660X
                Publication date (Electronic preprint): 4 January 2017
                Publication date (Electronic): 22 February 2017
                Publication date (Print): March 2017
                Publication date PMC-release: 22 February 2017
                Volume: 55
                Issue: 3
                Pages: 902-907
                Affiliations
                [a ]Section of Paediatrics, Department of Medicine, Imperial College London, St. Mary's Campus, London, United Kingdom
                [b ]Department of Microbiology and Infectious Diseases, University of Veterinary Medicine, Budapest, Hungary
                [c ]Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, Montréal, Québec, Canada
                [d ]Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark
                [e ]Veterinary Research Institute, Brno, Czech Republic
                [f ]Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hawkshead Campus, Hatfield, United Kingdom
                University of Tennessee
                Author notes
                Address correspondence to Janine T. Bossé, j.bosse@ 123456imperial.ac.uk , or Paul R. Langford, p.langford@ 123456imperial.ac.uk .

                J.T.B and Y.L. contributed equally to this work.

                Citation Bossé JT, Li Y, Sárközi R, Gottschalk M, Angen Ø, Nedbalcova K, Rycroft AN, Fodor L, Langford PR. 2017. A unique capsule locus in the newly designated Actinobacillus pleuropneumoniae serovar 16 and development of a diagnostic PCR assay. J Clin Microbiol 55:902–907. https://doi.org/10.1128/JCM.02166-16.

                Author information
                http://orcid.org/0000-0002-8491-4779
                http://orcid.org/0000-0002-6368-4724
                Article
                Publisher ID: 02166-16
                DOI: 10.1128/JCM.02166-16
                PMC ID: 5328458
                PubMed ID: 28053219
                SO-VID: d1b95a09-a38d-4031-8927-6f6410dcecd4
                Copyright © Copyright © 2017 Bossé et al.
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                Date received : 26 October 2016
                Date revision requested : 12 November 2016
                Date accepted : 22 December 2016
                Page count
                Figures: 2, Tables: 1, Equations: 0, References: 25, Pages: 6, Words: 4144
                Funding
                Funded by: Hungarian Scientific Research Fund
                Award ID: OTKA 112826
                Award Recipient : László Fodor
                Funded by: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) https://doi.org/10.13039/501100000268
                Award ID: BB/M023052/1
                Award ID: BB/G018553/1
                Award ID: BB/L024209/1
                Award Recipient : Paul R. Langford
                Categories
                Subject: Clinical Veterinary Microbiology
                Custom metadata
                cover-date March 2017

                ScienceOpen disciplines: Microbiology & Virology
                Keywords: actinobacillus pleuropneumoniae,pcr,diagnostics,serovar 16
                Data availability:
                ScienceOpen disciplines: Microbiology & Virology
                Keywords: actinobacillus pleuropneumoniae, pcr, diagnostics, serovar 16

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