This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1) and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortium

Beddow, J.

Bossé, J.T.

Chaudhuri, R.R.

Cuccui, J.

Langford, P.R.

Li, Y.

Luan, S.L.

Maglennon, G.A.

Maskell, D.J.

Peters, S.E.

Rycroft, A.N.

Terra, V.S.

Tucker, A.W.

Wang, J.T.

Weinert, L.A.

Williamson, S.M.

Wren, B.W.

Veterinary Record

PubMed

Y. Li

J. T. Bossé

S. M. Williamson

D. J. Maskell

A. W. Tucker

B. W. Wren

A. N. Rycroft

P. R. Langford

Crossref

<i>Actinobacillus pleuropneumoniae</i>
 serovar 8 predominates in England and Wales

White Rose Research Online

Short CommunicationActinobacilluspleuropneumoniaeserovar 8 predominatesin England and WalesY. Li, J. T. Bossé, S. M. Williamson,D. J. Maskell, A. W. Tucker, B. W. Wren,A. N. Rycroft, P. R. Langford, BRADP1TConsortiumActinobacillus pleuropneumoniae is a major cause of pleuropneumo-nia, an acute or chronic lung disease of pigs that causes signifi-cant morbidity, mortality and economic losses in the worldwidepig industry (Bossé and others 2002, Gottschalk 2012). There are15 established serovars of the bacterium determined by the com-position of the capsular polysaccharide (Gottschalk 2012) withanother proposed based on serology alone (Sárközi and others2015). Determining the serovar allows insights into the epidemi-ology of A. pleuropneumoniae, for example, to map outbreaks ofdisease, to identify introductions of serovars not previouslydetected in countries and to assist in the formulation of bacterinvaccines. Serovar prevalence varies from country to country andwith time (Dubreuil and others 2000, Gottschalk 2015). Forexample, currently serovars 5 and 7 dominate in Canada(Gottschalk and Lacouture 2015), whereas until the 1990s, itwas serovar 1 (Gottschalk 2015). In Australia, serovar 15 ishighly prevalent (Turni and others 2014) but is rarely reportedelsewhere, although it has been recently found in Canada(Gottschalk and Lacouture 2014) and possibly also in Japan(Koyama and others 2007).The last prevalence study in the UK analysed A. pleuropneumo-niae clinical isolates collected in England and Wales between theyears 1995 and 2007, with serovar 8 predominant (O’Neill andothers 2010). In that study, serovar was assigned on the basis ofPCR amplification of chromosomal capsule-specific regions;immunological serotyping by slide agglutination having beenshown erroneously to overestimate serovars 3 and 6 and underesti-mate serovar 8 prevalence. Similar underestimation and overesti-mation were also found in a study of Canadian isolates (Gottschalk2015). In this study, the authors sought to determine whether theserotype prevalence of A. pleuropneumoniae in England and Waleshad changed since 2007 and, in particular, whether new serovars,such as 15, were now present. The isolates evaluated were obtainedfrom clinical cases of diseases in pigs due to A. pleuropneumoniae sub-mitted to the Animal Health and Veterinary Laboratories Agency—Weybridge (AHVLA), now the Animal and Plant Health Agency(APHA), or Royal Veterinary College Diagnostic Laboratories.Isolates were collected between 2008 and 2014. For each year, thenumber of isolates (in brackets) is as follows: 2008 (N=14); 2009(N=17); 2010 (N=8); 2011 (N=10); 2012 (N=30); 2013 (N=19)and 2014 (N=15). Bacteria were grown on plates comprising BrainHeart Infusion Agar supplemented with 10 mg/ml nicotinamideadenine dinucleotide. Initially, serotyping was assessed by a multi-plex PCR that can distinguish between serovars 1–3, 5–8, 10 and 12(Bossé and others 2014). The PCR was based on the authors’ ori-ginal serovar 3–6–8 PCR (Zhou and others 2008) but extended tocover all of the serovars reported in England and Wales in theauthors’ 2010 study (O’Neill and others 2010) and those reportedto be prevalent in Europe and North America (Dubreuil and others2000). A serovar 15-specific PCR (Turni and others 2014) was subse-quently used on strains that were untypable by the 1–3, 5–8, 10 and12 multiplex PCR. Genomic DNA purified using the QIAamp DNAMini Kit (Qiagen) or single colonies were used as the source ofDNA template for PCR reactions as previously described (Bossé andothers 2014). In addition to serovar-specific amplicons, the multi-plex PCR amplifies a region of the A. pleuropneumoniae-specificapxIVA gene (Schaller and others 2001, Bossé and others 2014). Allisolates investigated in this study produced an apxIVA ampliconconfirming that they were A. pleuropneumoniae. Comparison of theseroprevalence of A. pleuropneumoniae in England and Wales in thisand the authors’ previous study is shown in Table 1.As in the authors’ 2010 study, serovar 8 was the most pre-dominant in years 2008–2014 in England and Wales, with a com-paratively smaller percentage of serovars 2, 6, 7 and 12. TheEngland and Wales serovar profile contrasts with that found inother European countries. For example, serovar 9/11 predomi-nates in the Czech Republic (Kucerova and others 2005) andserovar 2 in Denmark (Klausen and others 2007), Norway andSweden (Gottschalk 2012). No serovar 3 isolates were found,possibly reflecting the known low virulence of this serotype(Rosendal and others 1985), and that the A. pleuropneumoniaeisolates evaluated were from clinically confirmed cases. FourTABLE 1: Percentage serovar distribution ofA. pleuropneumoniaeisolates in England and Wales-based studiesPercentage of isolates (number of isolates) in range of years indicatedSerovar 1995–2007* 2008–2014†2 3.4 (13) 6.2 (7)3 0.5 (2) –6 10.1 (38) 7.1 (8)7 5.3 (20) 8.0 (9)8 78.0 (295) 71.7 (81)12 2.7 (10) 3.5 (4)NT – 3.5 (4)*O’Neill and others (2010)†Present studyNT, non-typableVeterinary Record (2016) doi: 10.1136/vr.103820Y. Li, BSc, PhD,J. T. Bossé, BSc, MSc, PhD,P. R. Langford, BSc, PhD, FRSB,Section of Paediatrics, Imperial CollegeLondon, St Mary’s Campus, LondonW2 1PG, UKS. M. Williamson, BVetMed, PhD,MRCVS,APHA—Bury St Edmunds, RoughamHill, Bury St Edmunds, Suffolk IP332RX, UKD. J. Maskell, BSc MA PhDFMedSci,A. W. Tucker, BVetMed, PhD,MRCVS,Department of Veterinary Medicine,University of Cambridge,Madingley Road, CambridgeCB3 0ES, UKB. W. Wren, BSc PhD FRCPath,Faculty of Infectious & TropicalDiseases, London School of Hygiene &Tropical Medicine, Keppel Street,London WC1E 7HT, UKA. N. Rycroft, BSc, PhD, FRSB,FRCPath,Department of Pathology and PathogenBiology, Royal Veterinary College,Hawkshead Lane, North Mymms,Hertfordshire AL9 7TA, UKE-mail for correspondence:p.langford@imperial.ac.ukYL and JTB contributed equally.Provenance: Not commissioned;internally peer reviewedAccepted June 19, 2016September 17, 2016 | Veterinary RecordShort Communicationgroup.bmj.com on September 16, 2016 - Published by http://veterinaryrecord.bmj.com/Downloaded from isolates were non-typable (NT) and subsequent PCR testingestablished that these were not serovar 15 (Turni and others2014). Due to their low numbers, the NT isolates were nottested further. The NT isolates could be serovars 4, 9/11, 13, 14,the newly described serovar 16 or variants such that the primersused did not anneal to genomic DNA. Among these, onlyserovar 9 has been previously reported in Great Britain(McDowell and Ball 1994) or Scotland (Anon 2013).In summary, the authors’ results suggest that serovar 8A. pleuropneumoniae remains the predominant serovar causingclinical disease outbreaks in England and Wales, as in theauthors’ previous study. Any vaccine to be used in the UK toprevent disease caused by A. pleuropneumoniae should have acomponent targeting serovar 8 isolates and, for near-maximalcoverage, additionally serovars 2, 6, 7 and 12.AcknowledgementsThis work was supported by a Longer and Larger (LoLa) grantfrom the Biotechnology and Biological Sciences ResearchCouncil (BBSRC grant numbers BB/G020744/1, BB/G019177/1,BB/G019274/1 and BB/G018553/1) and Zoetis (formerly PfizerAnimal Health) awarded to the Bacterial Respiratory Diseases ofPigs-1 Technology (BRaDP1T) Consortium. The BRaDP1TConsortium comprises Duncan J. Maskell, Alexander W. (Dan)Tucker, Sarah E. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang,Shi-Lu Luan, Roy R. Chaudhuri (University of Cambridge;present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Sheffield, Firth Court,Western Bank, Sheffield S10 2TN, UK), Andrew N. Rycroft,Gareth A. Maglennon, Jessica Beddow (Royal VeterinaryCollege); Brendan W. Wren, Jon Cuccui, Vanessa S. Terra (LondonSchool of Hygiene and Tropical Medicine) and Janine T. Bossé,Yanwen Li and Paul R. Langford (Imperial College London). TheDEFRA-funded project “Scanning surveillance for Pig Diseases inEngland and Wales” (ED1200) funded laboratory and staff costsof obtaining the A. pleuropneumoniae isolates at APHA and theauthors are grateful to veterinary and scientific staff at APHAVeterinary Investigation Centres.Competing interests None declared.Open Access This is an Open Access article distributed in accordance with theterms of the Creative Commons Attribution (CC BY 4.0) license, which permits othersto distribute, remix, adapt and build upon this work, for commercial use, provided theoriginal work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ReferencesANON (2013) Animal and Health Veterinary Laboratories Agency EmergingThreats Quarterly Report. Pig Diseases, January–March 2013 (Q1). www.thepigsite.com/articles/4491/ahvla-pig-disease-surveillance-monthly-report-april-2013/. Accessed March 12, 2016BOSSÉ, J. T., JANSON, H., SHEEHAN, B. J., BEDDEK, A. J., RYCROFT, A. N.,KROLL, J. S. & LANGFORD, P. R. (2002) Actinobacillus pleuropneumoniae: patho-biology and pathogenesis of infection. Microbes and Infection 4, 225–235BOSSÉ, J. T., LI, Y., ANGEN, Ø., WEINERT, L. A., CHAUDHURI, R. R., HOLDEN,M. T., WILLIAMSON, S. M., MASKELL, D. J., TUCKER, A. W., WREN, B. W.,RYCROFT, A. N., LANGFORD, P. R. & BRADP1T CONSORTIUM (2014)Multiplex PCR assay for unequivocal differentiation of Actinobacillus pleuropneu-moniae serovars 1 to 3, 5 to 8, 10, and 12. Journal of Clinical Microbiology 52,2380–2385DUBREUIL, J. D., JACQUES, M., MITTAL, K. R. & GOTTSCHALK, M. (2000)Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis andimmunogenicity. Animal Health Research Reviews 1, 73–93GOTTSCHALK, M. (2012) Actinobacillosis. In: Diseases of Swine. 10th edn. Eds. J. J.ZIMMERMAN, A. K. LOCKE, A. RAMIREZ, K. J. SCHWARTZ, G. W.STEVENSON. Ames, Iowa, USA: Wiley-Blackwell, pp 653–659GOTTSCHALK, M. (2015) The challenge of detecting herds sub-clinically infectedwith Actinobacillus pleuropneumoniae. The Veterinary Journal 206, 30–38GOTTSCHALK, M. & LACOUTURE, S. (2014) Actinobacillus pleuropneumoniae sero-types 3, 6, 8 and 15 isolated from diseased pigs in North America. VeterinaryRecord 174, 452–452GOTTSCHALK, M. & LACOUTURE, S. (2015) Distribution of Streptococcus suis(from 2012 to 2014) and Actinobacillus pleuropneumoniae (from 2011 to 2014) sero-types isolated from diseased pigs. Canadian Veterinary Journal 56, 1093–1094KLAUSEN, J., EKEROTH, L., GRØNDAHL-HANSEN, J. & ANDRESEN, L. O.(2007) An indirect enzyme-linked immunosorbent assay for detection of anti-bodies to Actinobacillus pleuropneumoniae serovar 7 in pig serum. Journal ofVeterinary Diagnostic Investigation 19, 244–249KOYAMA, T., TO, H. & NAGAI, S. (2007) Isolation of Actinobacillus pleuropneumo-niae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan.The Journal of Veterinary Medical Science 69, 961–964KUCEROVA, Z., JAGLIC, Z. & ONDRIASOVA, R. (2005) Serotype distribution ofActinobacillus pleuropneumoniae isolated from porcine pleuropneumonia in theCzech Republic during period 2003–2004. Veterinární Medicina 50, 355–360MCDOWELL, S. W. & BALL, H. J. (1994) Serotypes of Actinobacillus pleuropneumo-niae isolated in the British Isles. Veterinary Record 134, 522–523O’NEILL, C., JONES, S. C. P., BOSSÉ, J. T., WATSON, C. M., WILLIAMSON,S. M., RYCROFT, A. N., KROLL, J. S., HARTLEY, H. M. & LANGFORD, P. R.(2010) Prevalence of Actinobacillus pleuropneumoniae serovars in England andWales. Veterinary Record 167, 661–662ROSENDAL, S., BOYD, D. A. & GILBRIDE, K. A. (1985) Comparative virulenceof porcine Haemophilus bacteria. Canadian Journal of Comparative Medicine 49,68–74SÁRKÖZI, R., MAKRAI, L. & FODOR, L. (2015) Identification of a proposed newserovar of Actinobacillus pleuropneumoniae: Serovar 16. Acta Veterinaria Hungarica63, 444–450SCHALLER, A., DJORDJEVIC, S. P., EAMENS, G. J., FORBES, W. A., KUHN, R.,KUHNERT, P., GOTTSCHALK, M., NICOLET, J. & FREY, J. (2001) Identificationand detection of Actinobacillus pleuropneumoniae by PCR based on the geneapxIVA. Veterinary Microbiology 79, 47–62TURNI, C., SINGH, R., SCHEMBRI, M. A. & BLACKALL, P. J. (2014) Evaluationof a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae sero-vars 1, 5, 7, 12 and 15. Letters in Applied Microbiology 59, 362–369ZHOU, L., JONES, S. C. P., ANGEN, Ø., BOSSÉ, J. T., NASH, J. H. E., FREY, J.,ZHOU, R., CHEN, H. C., KROLL, J. S., RYCROFT, A. N. & LANGFORD, P. R.(2008) Multiplex PCR that can distinguish between immunologically cross-reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains. Journal ofClinical Microbiology 46, 800–803Veterinary Record | September 17, 2016Short Communicationgroup.bmj.com on September 16, 2016 - Published by http://veterinaryrecord.bmj.com/Downloaded from predominates in England and Wales serovar 8Actinobacillus pleuropneumoniaeWren, A. N. Rycroft and P. R. LangfordY. Li, J. T. Bossé, S. M. Williamson, D. J. Maskell, A. W. Tucker, B. W.doi: 10.1136/vr.10382020162016 179: 276 originally published online August 16,Veterinary Record  http://veterinaryrecord.bmj.com/content/179/11/276.2Updated information and services can be found at: These include:References #BIBLhttp://veterinaryrecord.bmj.com/content/179/11/276.2This article cites 16 articles, 5 of which you can access for free at: Open Accesshttp://creativecommons.org/licenses/by/4.0/use, provided the original work is properly cited. See: others to distribute, remix, adapt and build upon this work, for commercialthe Creative Commons Attribution (CC BY 4.0) license, which permits This is an Open Access article distributed in accordance with the terms ofserviceEmail alertingbox at the top right corner of the online article. Receive free email alerts when new articles cite this article. Sign up in theCollectionsTopic Articles on similar topics can be found in the following collections  (93)Open accessNoteshttp://group.bmj.com/group/rights-licensing/permissionsTo request permissions go to:http://journals.bmj.com/cgi/reprintformTo order reprints go to:http://group.bmj.com/subscribe/To subscribe to BMJ go to:group.bmj.com on September 16, 2016 - Published by http://veterinaryrecord.bmj.com/Downloaded from 

Actinobacillus pleuropneumoniae serovar 8 predominatesin England and Wales

Li, Y

Bosse, JT

Williamson, SN

Maskell, DJ

Tucker, AW

Wren, BW

Rycroft, AN

Langford, PR

Spiral - Imperial College Digital Repository

1  Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales  *Y. Li, *J. T. Bossé, S. M. Williamson, D. J. Maskell, A. W. Tucker, B. W. Wren, A. N. Rycroft, P. R. Langford; BRADP1T Consortium   Y. Li, BSc PhD J. T. Bossé, BSc, MSc, PhD P. R. Langford, BSc, PhD, FRSB Section of Paediatrics, Imperial College London,  St Mary’s Campus, London, W2 1PG  S. M. Williamson, BVetMed, PhD, MRCVS APHA - Bury St Edmunds, Rougham Hill, Bury St Edmunds, Suffolk,  IP33 2RX  D. J. Maskell, BSc MA PhD FMedSci A. W. Tucker, BVetMed, PhD, MRCVS Department of Veterinary Medicine,  University of Cambridge,  Madingley Road,  Cambridge,  CB3 0ES  B. W. Wren, BSc PhD FRCPath Faculty of Infectious & Tropical Diseases,  London School of Hygiene & Tropical Medicine,  Keppel Street,  London,  WC1E 7HT  A. N. Rycroft, BSc, PhD, FRSB, FRCPath Department of Pathology and Pathogen Biology Royal Veterinary College, Hawkshead Lane,  North Mymms, Hertfordshire, AL9 7TA  Correspondence to Professor Langford, e-mail: p.langford@imperial.ac.uk  * Contributed equally       2   Actinobacillus pleuropneumoniae is a major cause of pleuropneumonia, an acute or chronic lung disease of pigs that causes significant morbidity, mortality and economic losses in the worldwide pig industry (Bossé and others 2002; Gottschalk 2012). There are 15 established serovars of the bacterium determined by the composition of the capsular polysaccharide (Gottschalk 2012) with another proposed based on serology alone (Sárközi and others 2015). Determining the serovar allows insights into the epidemiology of A. pleuropneumoniae, for example, to map outbreaks of disease, identify introductions of serovars not previously detected in countries, and to assist in the formulation of bacterin vaccines. Serovar prevalence varies from country to country and with time (Dubreuil and others 2000; Gottschalk 2015). For example, currently serovars 5 and 7 dominate in Canada (Gottschalk and Lacouture 2015) whereas until the 1990s it was serovar 1 (Gottschalk 2015). In Australia serovar 15 is highly prevalent (Turni and others 2014) but is rarely reported elsewhere, although it has been recently found in Canada (Gottschalk and Lacouture 2014), and possibly also in Japan (Koyama and others 2007).    The last prevalence study in the UK analysed A. pleuropneumoniae clinical isolates collected in England and Wales between the years 1995-2007, with serovar 8 predominant (O'Neill and others 2010). In that study, serovar was assigned on the basis of PCR amplification of chromosomal capsule-specific regions, immunological serotyping by slide agglutination having been shown erroneously to over-estimate serovars 3 and 6, and under-estimate serovar 8 prevalence. Similar under- and over-estimation was also found in a study of Canadian isolates (Gottschalk 2015). In this study, we sought to determine whether the serotype prevalence of A. pleuropneumoniae in England and Wales had changed since 2007 and, in particular, whether new serovars, such as 15, were now present. The isolates evaluated were obtained from clinical cases of diseases in pigs due to A. pleuropneumoniae submitted to the Animal Health and Veterinary Laboratories 3  Agency (AHVLA), now the Animal and Plant Health Agency (APHA), or Royal Veterinary College Diagnostic Laboratories. Isolates were collected between 2008 and 2014. For each year the number of isolates (in brackets) is as follows: 2008 (N=14); 2009 (N=17); 2010 (N=8); 2011 (N=10); 2012 (N=30); 2013 (N=19); 2014 (N=15). Bacteria were grown on plates comprising Brain Heart Infusion agar supplemented with 10 µg/ml NAD. Initially serotyping was assessed by a multiplex PCR that can distinguish between serovars 1-3, 5-8, 10 and 12 (Bossé and others 2014). The PCR was based on our original serovar 3-6-8 PCR (Zhou and others 2008) but extended to cover all of the serovars reported in England and Wales in our 2010 study (O'Neill and others 2010), and those reported to be prevalent in Europe and North America (Dubreuil and others 2000). A serovar 15-specific PCR (Turni and others 2014) was subsequently used on strains that were untypable by the 1-3, 5-8, 10 and 12 multiplex PCR. Genomic DNA purified using the QIAamp DNA mini kit (Qiagen) or single colonies were used as the source of DNA template for PCR reactions as previously described (Bossé and others 2014). In addition to serovar-specific amplicons, the multiplex PCR amplifies a region of the A. pleuropneumoniae-specific apxIVA gene (Schaller and others 2001; Bossé and others 2014). All isolates investigated in this study produced an apxIVA amplicon confirming that they were A. pleuropneumoniae. Comparison of the seroprevalence of A. pleuropneumoniae in England and Wales in this and our previous study is shown in Table 1.    As in our 2010 study, serovar 8 was the most predominant in years 2008-14 in England and Wales, with a comparatively smaller percentage of serovars 2, 6, 7 and 12.  The England and Wales serovar profile contrasts with that found in other European countries. For example, serovar 9/11 predominates in the Czech Republic (Kucerova and others 2005) and serovar 2 in Denmark (Klausen and others 2007), Norway and Sweden (Gottschalk 2012). No serovar 3 isolates were found, possibly reflecting the known low 4  virulence of this serotype (Rosendal and others 1985), and that the A. pleuropneumoniae isolates evaluated were from clinically confirmed cases. Four isolates were non-typable and subsequent PCR testing established that these were not serovar 15 (Turni and others 2014). Due to their low numbers the NT isolates were not tested further. The non-typable isolates could be serovars 4, 9/11, 13, 14, the newly described serovar 16 or variants that the primers used did not anneal to genomic DNA. Among these, only serovar 9 has been previously reported in Great Britain (McDowell and Ball 1994) or Scotland (Anon 2013).    In summary our results suggest that serovar 8 A. pleuropneumoniae remains the predominant serovar causing clinical disease outbreaks in England and Wales, as in our previous study. Any vaccine to be used in the UK to prevent disease caused by A. pleuropneumoniae should have a component targeting serovar 8 isolates and, for near maximal coverage, additionally serovars 2, 6, 7 and 12.      Acknowledgements  This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1), and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. The BRaDP1T Consortium comprises: Duncan J. Maskell, Alexander W. (Dan) Tucker, Sarah E. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, Roy R. Chaudhuri (University of Cambridge; present address for R. Chaudhuri is: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK), Andrew N. Rycroft, Gareth A. Maglennon, Jessica Beddow (Royal Veterinary College); Brendan W. Wren, Jon Cuccui, Vanessa S. Terra (London 5  School of Hygiene and Tropical Medicine); and Janine T. Bossé, Yanwen Li and Paul R. Langford, (Imperial College London).  The DEFRA-funded project “Scanning surveillance for Pig Diseases in England and Wales” (ED1200) funded laboratory and staff costs of obtaining the A. pleuropneumoniae isolates at APHA and we are grateful to veterinary and scientific staff at APHA Veterinary Investigation Centres.   Percentage of isolates (number of isolates)  in range of years indicated  Serovar              1995-2007a                                             2008-2014b   2   3.4      (13)    6.2    (7)   3   0.5        (2)           -   6         10.1      (38)    7.1    (8)   7   5.3     (20)    8.0    (9)   8         78.0  (295)  71.7  (81) 12   2.7       (10)    3.5    (4) NTc          -      3.5    (4)   Table 1. Percentage serovar distribution of A. pleuropneumoniae isolates in England and Wales-based studies.  a O’Neill and others (2010) b present study c NT = Non-typable   Conflicting interests   None   References  ANON (2013) Animal and Health Veterinary Laboratories Agency Emerging Threats  Quarterly Report, Pig Diseases, January-March 2013 (Q1). 6   www.thepigsite.com/articles/4491/ahvla-pig-disease-surveillance-monthly-report-april- 2013/. Accessed March 12, 2016 BOSSÉ, J.T., JANSON, H., SHEEHAN, B.J., BEDDEK, A.J., RYCROFT, A.N., KROLL, J.S. and LANGFORD, P.R. (2002) Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes and Infection 4, 225–235 BOSSÉ, J.T., LI, Y., ANGEN, Ø., WEINERT, L.A., CHAUDHURI, R.R., HOLDEN, M.T., WILLIAMSON, S.M., MASKELL, D.J., TUCKER, A.W., WREN, B.W., RYCROFT, A.N., LANGFORD, P.R., and BRADP1T CONSORTIUM (2014) A multiplex PCR to unequivocally differentiate A. pleuropneumoniae serovars 1-3, 5-8, 10 and 12. Journal of Clinical Microbiology 52, 2380–2385 DUBREUIL, J.D., JACQUES, M., MITTAL, K.R. and GOTTSCHALK, M. (2000) Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity. Animal Health Research Reviews 1, 73–93 GOTTSCHALK, M. (2012) Actinobacillosis. In: Diseases of Swine. 10th edn. Ed(s) J. J. Zimmerman, A. K. Locke, A. Ramirez, K. J. Schwartz and G. W. Stevenson. Wiley-Blackwell, Ames, Iowa, USA. pp 653-659  GOTTSCHALK, M. (2015) The challenge of detecting herds sub-clinically infected with  Actinobacillus pleuropneumoniae. Veterinary Journal 206, 30-38 GOTTSCHALK, M. and LACOUTURE, S. (2014) Actinobacillus pleuropneumoniae  serotypes 3, 6, 8 and 15 isolated from diseased pigs in North America. Veterinary  Record 174, 452–452 GOTTSCHALK, M. and LACOUTURE, S. (2015) Distribution of Streptococcus suis (from  2012 to 2014) and Actinobacillus pleuropneumoniae (from 2011 to 2014) serotypes  isolated from diseased pigs. Canadian Veterinary Journal 56, 1093–1094 KLAUSEN, J., EKEROTH, L., GRØNDAHL-HANSEN, J. and ANDRESEN, L.O. (2007) An indirect enzyme-linked immunosorbent assay for detection of antibodies to 7  Actinobacillus pleuropneumoniae serovar 7 in pig serum. Journal of Veterinary Diagnostic Investigation 19, 244–249 KOYAMA, T., TO, H. and NAGAI, S. (2007) Isolation of Actinobacillus pleuropneumoniae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan. The Journal of Veterinary Medical Science 69, 961–964 KUCEROVA, Z., JAGLIC, Z. and ONDRIASOVA, R. (2005) Serotype distribution of Actinobacillus pleuropneumoniae isolated from porcine pleuropneumonia in the Czech Republic during period 2003-2004. Veterinární Medicina 50, 355-360  MCDOWELL, S. W. & BALL, H. J. (1994) Serotypes of Actinobacillus pleuropneumoniae isolated in the British Isles. Veterinary Record 134, 522-523 O'NEILL, C., JONES, S.C.P., BOSSÉ, J.T., WATSON, C.M., WILLIAMSON, S.M., RYCROFT, A.N., KROLL, J.S., HARTLEY, H.M. and LANGFORD, P.R. (2010) Prevalence of Actinobacillus pleuropneumoniae serovars in England and Wales. Veterinary Record 167, 661–662 ROSENDAL, S., BOYD, D.A. and GILBRIDE, K.A. (1985) Comparative virulence of porcine Haemophilus bacteria. Canadian Journal of Comparative Medicine 49, 68–74 SÁRKÖZI, R., MAKRAI, L. and FODOR, L. (2015) Identification of a proposed new serovar of Actinobacillus pleuropneumoniae: Serovar 16. Acta Veterinaria Hungarica 63, 444–450 SCHALLER, A., DJORDJEVIC, S.P., EAMENS, G.J., FORBES, W.A., KUHN, R.,  KUHNERT, P., GOTTSCHALK, M., NICOLET, J. and FREY, J. (2001) Identification  and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA.  Veterinary Microbiology 79, 47–62 TURNI, C., SINGH, R., SCHEMBRI, M.A. and BLACKALL, P.J. (2014) Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. Letters in Applied Microbiology 59, 362–369 8  ZHOU, L., JONES, S.C.P., ANGEN, Ø., BOSSÉ, J.T., NASH, J.H.E., FREY, J., ZHOU, R., CHEN, H.C., KROLL, J.S., RYCROFT, A.N. and LANGFORD, P.R. (2008) Multiplex PCR that can distinguish between immunologically cross-reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains. Journal of Clinical Microbiology 46, 800–803  

English

Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales

Bossé, J T

Williamson, S M

Maskell, D J

Tucker, A W

Wren, B W

Rycroft, A N

Langford, P R

RVC Research Online

Short CommunicationActinobacilluspleuropneumoniaeserovar 8 predominatesin England and WalesY. Li, J. T. Bossé, S. M. Williamson,D. J. Maskell, A. W. Tucker, B. W. Wren,A. N. Rycroft, P. R. Langford, BRADP1TConsortiumActinobacillus pleuropneumoniae is a major cause of pleuropneumo-nia, an acute or chronic lung disease of pigs that causes signifi-cant morbidity, mortality and economic losses in the worldwidepig industry (Bossé and others 2002, Gottschalk 2012). There are15 established serovars of the bacterium determined by the com-position of the capsular polysaccharide (Gottschalk 2012) withanother proposed based on serology alone (Sárközi and others2015). Determining the serovar allows insights into the epidemi-ology of A. pleuropneumoniae, for example, to map outbreaks ofdisease, to identify introductions of serovars not previouslydetected in countries and to assist in the formulation of bacterinvaccines. Serovar prevalence varies from country to country andwith time (Dubreuil and others 2000, Gottschalk 2015). Forexample, currently serovars 5 and 7 dominate in Canada(Gottschalk and Lacouture 2015), whereas until the 1990s, itwas serovar 1 (Gottschalk 2015). In Australia, serovar 15 ishighly prevalent (Turni and others 2014) but is rarely reportedelsewhere, although it has been recently found in Canada(Gottschalk and Lacouture 2014) and possibly also in Japan(Koyama and others 2007).The last prevalence study in the UK analysed A. pleuropneumo-niae clinical isolates collected in England and Wales between theyears 1995 and 2007, with serovar 8 predominant (O’Neill andothers 2010). In that study, serovar was assigned on the basis ofPCR amplification of chromosomal capsule-specific regions;immunological serotyping by slide agglutination having beenshown erroneously to overestimate serovars 3 and 6 and underesti-mate serovar 8 prevalence. Similar underestimation and overesti-mation were also found in a study of Canadian isolates (Gottschalk2015). In this study, the authors sought to determine whether theserotype prevalence of A. pleuropneumoniae in England and Waleshad changed since 2007 and, in particular, whether new serovars,such as 15, were now present. The isolates evaluated were obtainedfrom clinical cases of diseases in pigs due to A. pleuropneumoniae sub-mitted to the Animal Health and Veterinary Laboratories Agency—Weybridge (AHVLA), now the Animal and Plant Health Agency(APHA), or Royal Veterinary College Diagnostic Laboratories.Isolates were collected between 2008 and 2014. For each year, thenumber of isolates (in brackets) is as follows: 2008 (N=14); 2009(N=17); 2010 (N=8); 2011 (N=10); 2012 (N=30); 2013 (N=19)and 2014 (N=15). Bacteria were grown on plates comprising BrainHeart Infusion Agar supplemented with 10 mg/ml nicotinamideadenine dinucleotide. Initially, serotyping was assessed by a multi-plex PCR that can distinguish between serovars 1–3, 5–8, 10 and 12(Bossé and others 2014). The PCR was based on the authors’ ori-ginal serovar 3–6–8 PCR (Zhou and others 2008) but extended tocover all of the serovars reported in England and Wales in theauthors’ 2010 study (O’Neill and others 2010) and those reportedto be prevalent in Europe and North America (Dubreuil and others2000). A serovar 15-specific PCR (Turni and others 2014) was subse-quently used on strains that were untypable by the 1–3, 5–8, 10 and12 multiplex PCR. Genomic DNA purified using the QIAamp DNAMini Kit (Qiagen) or single colonies were used as the source ofDNA template for PCR reactions as previously described (Bossé andothers 2014). In addition to serovar-specific amplicons, the multi-plex PCR amplifies a region of the A. pleuropneumoniae-specificapxIVA gene (Schaller and others 2001, Bossé and others 2014). Allisolates investigated in this study produced an apxIVA ampliconconfirming that they were A. pleuropneumoniae. Comparison of theseroprevalence of A. pleuropneumoniae in England and Wales in thisand the authors’ previous study is shown in Table 1.As in the authors’ 2010 study, serovar 8 was the most pre-dominant in years 2008–2014 in England and Wales, with a com-paratively smaller percentage of serovars 2, 6, 7 and 12. TheEngland and Wales serovar profile contrasts with that found inother European countries. For example, serovar 9/11 predomi-nates in the Czech Republic (Kucerova and others 2005) andserovar 2 in Denmark (Klausen and others 2007), Norway andSweden (Gottschalk 2012). No serovar 3 isolates were found,possibly reflecting the known low virulence of this serotype(Rosendal and others 1985), and that the A. pleuropneumoniaeisolates evaluated were from clinically confirmed cases. FourTABLE 1: Percentage serovar distribution ofA. pleuropneumoniaeisolates in England and Wales-based studiesPercentage of isolates (number of isolates) in range of years indicatedSerovar 1995–2007* 2008–2014†2 3.4 (13) 6.2 (7)3 0.5 (2) –6 10.1 (38) 7.1 (8)7 5.3 (20) 8.0 (9)8 78.0 (295) 71.7 (81)12 2.7 (10) 3.5 (4)NT – 3.5 (4)*O’Neill and others (2010)†Present studyNT, non-typableVeterinary Record (2016) doi: 10.1136/vr.103820Y. Li, BSc, PhD,J. T. Bossé, BSc, MSc, PhD,P. R. Langford, BSc, PhD, FRSB,Section of Paediatrics, Imperial CollegeLondon, St Mary’s Campus, LondonW2 1PG, UKS. M. Williamson, BVetMed, PhD,MRCVS,APHA—Bury St Edmunds, RoughamHill, Bury St Edmunds, Suffolk IP332RX, UKD. J. Maskell, BSc MA PhDFMedSci,A. W. Tucker, BVetMed, PhD,MRCVS,Department of Veterinary Medicine,University of Cambridge,Madingley Road, CambridgeCB3 0ES, UKB. W. Wren, BSc PhD FRCPath,Faculty of Infectious & TropicalDiseases, London School of Hygiene &Tropical Medicine, Keppel Street,London WC1E 7HT, UKA. N. Rycroft, BSc, PhD, FRSB,FRCPath,Department of Pathology and PathogenBiology, Royal Veterinary College,Hawkshead Lane, North Mymms,Hertfordshire AL9 7TA, UKE-mail for correspondence:p.langford@imperial.ac.ukYL and JTB contributed equally.Provenance: Not commissioned;internally peer reviewedAccepted June 19, 201610.1136/vr.103820 | Veterinary Record | 1 of 2Short Communicationgroup.bmj.com on August 19, 2016 - Published by http://veterinaryrecord.bmj.com/Downloaded from isolates were non-typable (NT) and subsequent PCR testingestablished that these were not serovar 15 (Turni and others2014). Due to their low numbers, the NT isolates were nottested further. The NT isolates could be serovars 4, 9/11, 13, 14,the newly described serovar 16 or variants such that the primersused did not anneal to genomic DNA. Among these, onlyserovar 9 has been previously reported in Great Britain(McDowell and Ball 1994) or Scotland (Anon 2013).In summary, the authors’ results suggest that serovar 8A. pleuropneumoniae remains the predominant serovar causingclinical disease outbreaks in England and Wales, as in theauthors’ previous study. Any vaccine to be used in the UK toprevent disease caused by A. pleuropneumoniae should have acomponent targeting serovar 8 isolates and, for near-maximalcoverage, additionally serovars 2, 6, 7 and 12.AcknowledgementsThis work was supported by a Longer and Larger (LoLa) grantfrom the Biotechnology and Biological Sciences ResearchCouncil (BBSRC grant numbers BB/G020744/1, BB/G019177/1,BB/G019274/1 and BB/G018553/1) and Zoetis (formerly PfizerAnimal Health) awarded to the Bacterial Respiratory Diseases ofPigs-1 Technology (BRaDP1T) Consortium. The BRaDP1TConsortium comprises Duncan J. Maskell, Alexander W. (Dan)Tucker, Sarah E. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang,Shi-Lu Luan, Roy R. Chaudhuri (University of Cambridge;present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Sheffield, Firth Court,Western Bank, Sheffield S10 2TN, UK), Andrew N. Rycroft,Gareth A. Maglennon, Jessica Beddow (Royal VeterinaryCollege); Brendan W. Wren, Jon Cuccui, Vanessa S. Terra (LondonSchool of Hygiene and Tropical Medicine) and Janine T. Bossé,Yanwen Li and Paul R. Langford (Imperial College London). TheDEFRA-funded project “Scanning surveillance for Pig Diseases inEngland and Wales” (ED1200) funded laboratory and staff costsof obtaining the A. pleuropneumoniae isolates at APHA and theauthors are grateful to veterinary and scientific staff at APHAVeterinary Investigation Centres.Competing interests None declared.Open Access This is an Open Access article distributed in accordance with theterms of the Creative Commons Attribution (CC BY 4.0) license, which permits othersto distribute, remix, adapt and build upon this work, for commercial use, provided theoriginal work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ReferencesANON (2013) Animal and Health Veterinary Laboratories Agency EmergingThreats Quarterly Report. Pig Diseases, January–March 2013 (Q1). www.thepigsite.com/articles/4491/ahvla-pig-disease-surveillance-monthly-report-april-2013/. Accessed March 12, 2016BOSSÉ, J. T., JANSON, H., SHEEHAN, B. J., BEDDEK, A. J., RYCROFT, A. N.,KROLL, J. S. & LANGFORD, P. R. (2002) Actinobacillus pleuropneumoniae: patho-biology and pathogenesis of infection. Microbes and Infection 4, 225–235BOSSÉ, J. T., LI, Y., ANGEN, Ø., WEINERT, L. A., CHAUDHURI, R. R., HOLDEN,M. T., WILLIAMSON, S. M., MASKELL, D. J., TUCKER, A. W., WREN, B. W.,RYCROFT, A. N., LANGFORD, P. R. & BRADP1T CONSORTIUM (2014)Multiplex PCR assay for unequivocal differentiation of Actinobacillus pleuropneu-moniae serovars 1 to 3, 5 to 8, 10, and 12. Journal of Clinical Microbiology 52,2380–2385DUBREUIL, J. D., JACQUES, M., MITTAL, K. R. & GOTTSCHALK, M. (2000)Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis andimmunogenicity. Animal Health Research Reviews 1, 73–93GOTTSCHALK, M. (2012) Actinobacillosis. In: Diseases of Swine. 10th edn. Eds. J. J.ZIMMERMAN, A. K. LOCKE, A. RAMIREZ, K. J. SCHWARTZ, G. W.STEVENSON. Ames, Iowa, USA: Wiley-Blackwell, pp 653–659GOTTSCHALK, M. (2015) The challenge of detecting herds sub-clinically infectedwith Actinobacillus pleuropneumoniae. The Veterinary Journal 206, 30–38GOTTSCHALK, M. & LACOUTURE, S. (2014) Actinobacillus pleuropneumoniae sero-types 3, 6, 8 and 15 isolated from diseased pigs in North America. VeterinaryRecord 174, 452–452GOTTSCHALK, M. & LACOUTURE, S. (2015) Distribution of Streptococcus suis(from 2012 to 2014) and Actinobacillus pleuropneumoniae (from 2011 to 2014) sero-types isolated from diseased pigs. Canadian Veterinary Journal 56, 1093–1094KLAUSEN, J., EKEROTH, L., GRØNDAHL-HANSEN, J. & ANDRESEN, L. O.(2007) An indirect enzyme-linked immunosorbent assay for detection of anti-bodies to Actinobacillus pleuropneumoniae serovar 7 in pig serum. Journal ofVeterinary Diagnostic Investigation 19, 244–249KOYAMA, T., TO, H. & NAGAI, S. (2007) Isolation of Actinobacillus pleuropneumo-niae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan.The Journal of Veterinary Medical Science 69, 961–964KUCEROVA, Z., JAGLIC, Z. & ONDRIASOVA, R. (2005) Serotype distribution ofActinobacillus pleuropneumoniae isolated from porcine pleuropneumonia in theCzech Republic during period 2003–2004. Veterinární Medicina 50, 355–360MCDOWELL, S. W. & BALL, H. J. (1994) Serotypes of Actinobacillus pleuropneumo-niae isolated in the British Isles. Veterinary Record 134, 522–523O’NEILL, C., JONES, S. C. P., BOSSÉ, J. T., WATSON, C. M., WILLIAMSON,S. M., RYCROFT, A. N., KROLL, J. S., HARTLEY, H. M. & LANGFORD, P. R.(2010) Prevalence of Actinobacillus pleuropneumoniae serovars in England andWales. Veterinary Record 167, 661–662ROSENDAL, S., BOYD, D. A. & GILBRIDE, K. A. (1985) Comparative virulenceof porcine Haemophilus bacteria. Canadian Journal of Comparative Medicine 49,68–74SÁRKÖZI, R., MAKRAI, L. & FODOR, L. (2015) Identification of a proposed newserovar of Actinobacillus pleuropneumoniae: Serovar 16. Acta Veterinaria Hungarica63, 444–450SCHALLER, A., DJORDJEVIC, S. P., EAMENS, G. J., FORBES, W. A., KUHN, R.,KUHNERT, P., GOTTSCHALK, M., NICOLET, J. & FREY, J. (2001) Identificationand detection of Actinobacillus pleuropneumoniae by PCR based on the geneapxIVA. Veterinary Microbiology 79, 47–62TURNI, C., SINGH, R., SCHEMBRI, M. A. & BLACKALL, P. J. (2014) Evaluationof a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae sero-vars 1, 5, 7, 12 and 15. Letters in Applied Microbiology 59, 362–369ZHOU, L., JONES, S. C. P., ANGEN, Ø., BOSSÉ, J. T., NASH, J. H. E., FREY, J.,ZHOU, R., CHEN, H. C., KROLL, J. S., RYCROFT, A. N. & LANGFORD, P. R.(2008) Multiplex PCR that can distinguish between immunologically cross-reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains. Journal ofClinical Microbiology 46, 800–8032 of 2 | Veterinary Record | 10.1136/vr.103820Short Communicationgroup.bmj.com on August 19, 2016 - Published by http://veterinaryrecord.bmj.com/Downloaded from predominates in England and Wales serovar 8Actinobacillus pleuropneumoniaeWren, A. N. Rycroft and P. R. LangfordY. Li, J. T. Bossé, S. M. Williamson, D. J. Maskell, A. W. Tucker, B. W. published online August 16, 2016Veterinary Record  http://veterinaryrecord.bmj.com/content/early/2016/08/16/vr.103820Updated information and services can be found at: These include:References #BIBLhttp://veterinaryrecord.bmj.com/content/early/2016/08/16/vr.103820This article cites 16 articles, 5 of which you can access for free at: Open Accesshttp://creativecommons.org/licenses/by/4.0/use, provided the original work is properly cited. See: others to distribute, remix, adapt and build upon this work, for commercialthe Creative Commons Attribution (CC BY 4.0) license, which permits This is an Open Access article distributed in accordance with the terms ofserviceEmail alertingbox at the top right corner of the online article. Receive free email alerts when new articles cite this article. Sign up in theCollectionsTopic Articles on similar topics can be found in the following collections  (92)Open accessNoteshttp://group.bmj.com/group/rights-licensing/permissionsTo request permissions go to:http://journals.bmj.com/cgi/reprintformTo order reprints go to:http://group.bmj.com/subscribe/To subscribe to BMJ go to:group.bmj.com on August 19, 2016 - Published by http://veterinaryrecord.bmj.com/Downloaded from 

Bossé, JT

Williamson, SM

BRADP1T Consortium

LSHTM Research Online

Li, Y; Boss, JT; Williamson, SM; Maskell, DJ; Tucker, AW; Wren,BW; Rycroft, AN; Langford, PR; BRADP1T Consortium, (2016)Actinobacillus pleuropneumoniae serovar 8 predominates in Englandand Wales. The Veterinary record, 179 (11). p. 276. ISSN 0042-4900DOI: https://doi.org/10.1136/vr.103820Downloaded from: http://researchonline.lshtm.ac.uk/2782902/DOI: 10.1136/vr.103820Usage GuidelinesPlease refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alterna-tively contact researchonline@lshtm.ac.uk.Available under license: http://creativecommons.org/licenses/by/2.5/Short CommunicationActinobacilluspleuropneumoniaeserovar 8 predominatesin England and WalesY. Li, J. T. Bossé, S. M. Williamson,D. J. Maskell, A. W. Tucker, B. W. Wren,A. N. Rycroft, P. R. Langford, BRADP1TConsortiumActinobacillus pleuropneumoniae is a major cause of pleuropneumo-nia, an acute or chronic lung disease of pigs that causes signiﬁ-cant morbidity, mortality and economic losses in the worldwidepig industry (Bossé and others 2002, Gottschalk 2012). There are15 established serovars of the bacterium determined by the com-position of the capsular polysaccharide (Gottschalk 2012) withanother proposed based on serology alone (Sárközi and others2015). Determining the serovar allows insights into the epidemi-ology of A. pleuropneumoniae, for example, to map outbreaks ofdisease, to identify introductions of serovars not previouslydetected in countries and to assist in the formulation of bacterinvaccines. Serovar prevalence varies from country to country andwith time (Dubreuil and others 2000, Gottschalk 2015). Forexample, currently serovars 5 and 7 dominate in Canada(Gottschalk and Lacouture 2015), whereas until the 1990s, itwas serovar 1 (Gottschalk 2015). In Australia, serovar 15 ishighly prevalent (Turni and others 2014) but is rarely reportedelsewhere, although it has been recently found in Canada(Gottschalk and Lacouture 2014) and possibly also in Japan(Koyama and others 2007).The last prevalence study in the UK analysed A. pleuropneumo-niae clinical isolates collected in England and Wales between theyears 1995 and 2007, with serovar 8 predominant (O’Neill andothers 2010). In that study, serovar was assigned on the basis ofPCR ampliﬁcation of chromosomal capsule-speciﬁc regions;immunological serotyping by slide agglutination having beenshown erroneously to overestimate serovars 3 and 6 and underesti-mate serovar 8 prevalence. Similar underestimation and overesti-mation were also found in a study of Canadian isolates (Gottschalk2015). In this study, the authors sought to determine whether theserotype prevalence of A. pleuropneumoniae in England and Waleshad changed since 2007 and, in particular, whether new serovars,such as 15, were now present. The isolates evaluated were obtainedfrom clinical cases of diseases in pigs due to A. pleuropneumoniae sub-mitted to the Animal Health and Veterinary Laboratories Agency—Weybridge (AHVLA), now the Animal and Plant Health Agency(APHA), or Royal Veterinary College Diagnostic Laboratories.Isolates were collected between 2008 and 2014. For each year, thenumber of isolates (in brackets) is as follows: 2008 (N=14); 2009(N=17); 2010 (N=8); 2011 (N=10); 2012 (N=30); 2013 (N=19)and 2014 (N=15). Bacteria were grown on plates comprising BrainHeart Infusion Agar supplemented with 10 mg/ml nicotinamideadenine dinucleotide. Initially, serotyping was assessed by a multi-plex PCR that can distinguish between serovars 1–3, 5–8, 10 and 12(Bossé and others 2014). The PCR was based on the authors’ ori-ginal serovar 3–6–8 PCR (Zhou and others 2008) but extended tocover all of the serovars reported in England and Wales in theauthors’ 2010 study (O’Neill and others 2010) and those reportedto be prevalent in Europe and North America (Dubreuil and others2000). A serovar 15-speciﬁc PCR (Turni and others 2014) was subse-quently used on strains that were untypable by the 1–3, 5–8, 10 and12 multiplex PCR. Genomic DNA puriﬁed using the QIAamp DNAMini Kit (Qiagen) or single colonies were used as the source ofDNA template for PCR reactions as previously described (Bossé andothers 2014). In addition to serovar-speciﬁc amplicons, the multi-plex PCR ampliﬁes a region of the A. pleuropneumoniae-speciﬁcapxIVA gene (Schaller and others 2001, Bossé and others 2014). Allisolates investigated in this study produced an apxIVA ampliconconﬁrming that they were A. pleuropneumoniae. Comparison of theseroprevalence of A. pleuropneumoniae in England and Wales in thisand the authors’ previous study is shown in Table 1.As in the authors’ 2010 study, serovar 8 was the most pre-dominant in years 2008–2014 in England and Wales, with a com-paratively smaller percentage of serovars 2, 6, 7 and 12. TheEngland and Wales serovar proﬁle contrasts with that found inother European countries. For example, serovar 9/11 predomi-nates in the Czech Republic (Kucerova and others 2005) andserovar 2 in Denmark (Klausen and others 2007), Norway andSweden (Gottschalk 2012). No serovar 3 isolates were found,possibly reﬂecting the known low virulence of this serotype(Rosendal and others 1985), and that the A. pleuropneumoniaeisolates evaluated were from clinically conﬁrmed cases. FourTABLE 1: Percentage serovar distribution ofA. pleuropneumoniaeisolates in England and Wales-based studiesPercentage of isolates (number of isolates) in range of years indicatedSerovar 1995–2007* 2008–2014†2 3.4 (13) 6.2 (7)3 0.5 (2) –6 10.1 (38) 7.1 (8)7 5.3 (20) 8.0 (9)8 78.0 (295) 71.7 (81)12 2.7 (10) 3.5 (4)NT – 3.5 (4)*O’Neill and others (2010)†Present studyNT, non-typableVeterinary Record (2016) doi: 10.1136/vr.103820Y. Li, BSc, PhD,J. T. Bossé, BSc, MSc, PhD,P. R. Langford, BSc, PhD, FRSB,Section of Paediatrics, Imperial CollegeLondon, St Mary’s Campus, LondonW2 1PG, UKS. M. Williamson, BVetMed, PhD,MRCVS,APHA—Bury St Edmunds, RoughamHill, Bury St Edmunds, Suffolk IP332RX, UKD. J. Maskell, BSc MA PhDFMedSci,A. W. Tucker, BVetMed, PhD,MRCVS,Department of Veterinary Medicine,University of Cambridge,Madingley Road, CambridgeCB3 0ES, UKB. W. Wren, BSc PhD FRCPath,Faculty of Infectious & TropicalDiseases, London School of Hygiene &Tropical Medicine, Keppel Street,London WC1E 7HT, UKA. N. Rycroft, BSc, PhD, FRSB,FRCPath,Department of Pathology and PathogenBiology, Royal Veterinary College,Hawkshead Lane, North Mymms,Hertfordshire AL9 7TA, UKE-mail for correspondence:p.langford@imperial.ac.ukYL and JTB contributed equally.Provenance: Not commissioned;internally peer reviewedAccepted June 19, 2016September 17, 2016 | Veterinary RecordShort Communication on 2 November 2018 by guest. Protected by copyright.http://veterinaryrecord.bmj.com/Veterinary Record: first published as 10.1136/vr.103820 on 16 August 2016. Downloaded from isolates were non-typable (NT) and subsequent PCR testingestablished that these were not serovar 15 (Turni and others2014). Due to their low numbers, the NT isolates were nottested further. The NT isolates could be serovars 4, 9/11, 13, 14,the newly described serovar 16 or variants such that the primersused did not anneal to genomic DNA. Among these, onlyserovar 9 has been previously reported in Great Britain(McDowell and Ball 1994) or Scotland (Anon 2013).In summary, the authors’ results suggest that serovar 8A. pleuropneumoniae remains the predominant serovar causingclinical disease outbreaks in England and Wales, as in theauthors’ previous study. Any vaccine to be used in the UK toprevent disease caused by A. pleuropneumoniae should have acomponent targeting serovar 8 isolates and, for near-maximalcoverage, additionally serovars 2, 6, 7 and 12.AcknowledgementsThis work was supported by a Longer and Larger (LoLa) grantfrom the Biotechnology and Biological Sciences ResearchCouncil (BBSRC grant numbers BB/G020744/1, BB/G019177/1,BB/G019274/1 and BB/G018553/1) and Zoetis (formerly PﬁzerAnimal Health) awarded to the Bacterial Respiratory Diseases ofPigs-1 Technology (BRaDP1T) Consortium. The BRaDP1TConsortium comprises Duncan J. Maskell, Alexander W. (Dan)Tucker, Sarah E. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang,Shi-Lu Luan, Roy R. Chaudhuri (University of Cambridge;present address for R. Chaudhuri is Department of MolecularBiology and Biotechnology, University of Shefﬁeld, Firth Court,Western Bank, Shefﬁeld S10 2TN, UK), Andrew N. Rycroft,Gareth A. Maglennon, Jessica Beddow (Royal VeterinaryCollege); Brendan W. Wren, Jon Cuccui, Vanessa S. Terra (LondonSchool of Hygiene and Tropical Medicine) and Janine T. Bossé,Yanwen Li and Paul R. Langford (Imperial College London). TheDEFRA-funded project “Scanning surveillance for Pig Diseases inEngland and Wales” (ED1200) funded laboratory and staff costsof obtaining the A. pleuropneumoniae isolates at APHA and theauthors are grateful to veterinary and scientiﬁc staff at APHAVeterinary Investigation Centres.Competing interests None declared.Open Access This is an Open Access article distributed in accordance with theterms of the Creative Commons Attribution (CC BY 4.0) license, which permits othersto distribute, remix, adapt and build upon this work, for commercial use, provided theoriginal work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ReferencesANON (2013) Animal and Health Veterinary Laboratories Agency EmergingThreats Quarterly Report. Pig Diseases, January–March 2013 (Q1). www.thepigsite.com/articles/4491/ahvla-pig-disease-surveillance-monthly-report-april-2013/. Accessed March 12, 2016BOSSÉ, J. T., JANSON, H., SHEEHAN, B. J., BEDDEK, A. J., RYCROFT, A. N.,KROLL, J. S. & LANGFORD, P. R. (2002) Actinobacillus pleuropneumoniae: patho-biology and pathogenesis of infection. Microbes and Infection 4, 225–235BOSSÉ, J. T., LI, Y., ANGEN, Ø., WEINERT, L. A., CHAUDHURI, R. R., HOLDEN,M. T., WILLIAMSON, S. M., MASKELL, D. J., TUCKER, A. W., WREN, B. W.,RYCROFT, A. N., LANGFORD, P. R. & BRADP1T CONSORTIUM (2014)Multiplex PCR assay for unequivocal differentiation of Actinobacillus pleuropneu-moniae serovars 1 to 3, 5 to 8, 10, and 12. Journal of Clinical Microbiology 52,2380–2385DUBREUIL, J. D., JACQUES, M., MITTAL, K. R. & GOTTSCHALK, M. (2000)Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis andimmunogenicity. Animal Health Research Reviews 1, 73–93GOTTSCHALK, M. (2012) Actinobacillosis. In: Diseases of Swine. 10th edn. Eds. J. J.ZIMMERMAN, A. K. LOCKE, A. RAMIREZ, K. J. SCHWARTZ, G. W.STEVENSON. Ames, Iowa, USA: Wiley-Blackwell, pp 653–659GOTTSCHALK, M. (2015) The challenge of detecting herds sub-clinically infectedwith Actinobacillus pleuropneumoniae. The Veterinary Journal 206, 30–38GOTTSCHALK, M. & LACOUTURE, S. (2014) Actinobacillus pleuropneumoniae sero-types 3, 6, 8 and 15 isolated from diseased pigs in North America. VeterinaryRecord 174, 452–452GOTTSCHALK, M. & LACOUTURE, S. (2015) Distribution of Streptococcus suis(from 2012 to 2014) and Actinobacillus pleuropneumoniae (from 2011 to 2014) sero-types isolated from diseased pigs. Canadian Veterinary Journal 56, 1093–1094KLAUSEN, J., EKEROTH, L., GRØNDAHL-HANSEN, J. & ANDRESEN, L. O.(2007) An indirect enzyme-linked immunosorbent assay for detection of anti-bodies to Actinobacillus pleuropneumoniae serovar 7 in pig serum. Journal ofVeterinary Diagnostic Investigation 19, 244–249KOYAMA, T., TO, H. & NAGAI, S. (2007) Isolation of Actinobacillus pleuropneumo-niae serovar 15-like strain from a ﬁeld case of porcine pleuropneumonia in Japan.The Journal of Veterinary Medical Science 69, 961–964KUCEROVA, Z., JAGLIC, Z. & ONDRIASOVA, R. (2005) Serotype distribution ofActinobacillus pleuropneumoniae isolated from porcine pleuropneumonia in theCzech Republic during period 2003–2004. Veterinární Medicina 50, 355–360MCDOWELL, S. W. & BALL, H. J. (1994) Serotypes of Actinobacillus pleuropneumo-niae isolated in the British Isles. Veterinary Record 134, 522–523O’NEILL, C., JONES, S. C. P., BOSSÉ, J. T., WATSON, C. M., WILLIAMSON,S. M., RYCROFT, A. N., KROLL, J. S., HARTLEY, H. M. & LANGFORD, P. R.(2010) Prevalence of Actinobacillus pleuropneumoniae serovars in England andWales. Veterinary Record 167, 661–662ROSENDAL, S., BOYD, D. A. & GILBRIDE, K. A. (1985) Comparative virulenceof porcine Haemophilus bacteria. Canadian Journal of Comparative Medicine 49,68–74SÁRKÖZI, R., MAKRAI, L. & FODOR, L. (2015) Identiﬁcation of a proposed newserovar of Actinobacillus pleuropneumoniae: Serovar 16. Acta Veterinaria Hungarica63, 444–450SCHALLER, A., DJORDJEVIC, S. P., EAMENS, G. J., FORBES, W. A., KUHN, R.,KUHNERT, P., GOTTSCHALK, M., NICOLET, J. & FREY, J. (2001) Identiﬁcationand detection of Actinobacillus pleuropneumoniae by PCR based on the geneapxIVA. Veterinary Microbiology 79, 47–62TURNI, C., SINGH, R., SCHEMBRI, M. A. & BLACKALL, P. J. (2014) Evaluationof a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae sero-vars 1, 5, 7, 12 and 15. Letters in Applied Microbiology 59, 362–369ZHOU, L., JONES, S. C. P., ANGEN, Ø., BOSSÉ, J. T., NASH, J. H. E., FREY, J.,ZHOU, R., CHEN, H. C., KROLL, J. S., RYCROFT, A. N. & LANGFORD, P. R.(2008) Multiplex PCR that can distinguish between immunologically cross-reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains. Journal ofClinical Microbiology 46, 800–803Veterinary Record | September 17, 2016Short Communication on 2 November 2018 by guest. Protected by copyright.http://veterinaryrecord.bmj.com/Veterinary Record: first published as 10.1136/vr.103820 on 16 August 2016. Downloaded from 

Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales.

https://researchonline.rvc.ac.uk/id/eprint/10483/1/10483.pdf

Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales

Abstract

Similar works

Full text

Available Versions

Crossref

White Rose Research Online

Spiral - Imperial College Digital Repository

RVC Research Online

LSHTM Research Online