Short Communication

Characterisation of Streptococcus suis isolates from wild boars(Sus scrofa)Veroacutenica Saacutenchez del Rey a Joseacute F Fernaacutendez-Garayzaacutebal ab Gregorio Mentaberre cViacutector Briones d Santiago Laviacuten c Lucas Domiacutenguez a Marcelo Gottschalk eAna Isabel Vela aba Centro de Vigilancia Sanitaria Veterinaria (VISAVET) Universidad Complutense 28040 Madrid Spainb Departamento de Sanidad Animal Facultad de Veterinaria Universidad Complutense 28040 Madrid Spainc Servei drsquoEcopatologia de Fauna Salvatge (SEFAS) Departament de Medicina i Cirurgia Animals Facultat de Veterinagraveria Universitat Autogravenoma deBarcelona 08193 Bellaterra Spaind Centro de Investigacioacuten en Sanidad Animal Instituto Nacional de Investigacioacuten y Tecnologiacutea Agraria y Alimentaria Ministerio de Economia yCompetitividad Valdeolmos 28130 Madrid Spaine Groupe de Recherche sur les Maladies Infectieuses du Porc Faculteacute de Meacutedecine Veacuteteacuterinaire Universiteacute de Montreacuteal 3200 Sicotte St-Hyacinthe QCCanada J2S 2M2

A R T I C L E I N F O

Article historyAccepted 13 March 2014

KeywordsWild boarStreptococcus suisSerotypingMolecular characterisation

A B S T R A C T

Wild boar are widely distributed throughout the Iberian Peninsula and can carry potentially virulent strainsof Streptococcus suis The objective of this study was to determine the prevalence of S suis in wild boarsfrom two large geographical regions of Spain Serotypes 1 2 7 and 9 identified were further geneticallycharacterised by virulence-associated genotyping pulsed-field gel electrophoresis (PFGE) and multilocussequence typing (MLST) to determine the population structure of S suis carried by these animals Strep-tococcus suis was isolated from 391 of the wild boars examined serotype 9 was the most frequentlyisolated (125) followed by serotype 1 (25) Serotype 2 was rarely isolated (03) Eighteen addition-al serotypes were identified indicating wide diversity of this pathogen within the wild boar populationThis heterogeneity was confirmed by PFGE and MLST analyses and the majority of isolates exhibited thevirulence-associated genotype mrp-epf-sly- The results of this study highlight that the carriage of S suisby wild boars is commonplace However MLST data indicate that these isolates are not related to prev-alent clonal complexes ST1 ST16 ST61 and ST87 typically associated with infection of pigs or humansin Europe

copy 2014 Elsevier Ltd All rights reserved

Streptococcus suis is an important pathogen of pigs worldwide(Higgins and Gottschalk 1999) and is associated with sporadic in-fections in other mammalian and avian species (Cruz Colque et al1993 Salasia et al 1994 Gottschalk et al 2007) including humans(Gottschalk et al 2007) Wild boar (Sus scrofa) populations havegrown in many European countries over recent decades (Saez-Royuela and Telleria 1986 Artois et al 2002) increasing the riskof transmission of the shared pathogen S suis to domestic pigsfarmed outdoors (Albina et al 2000) Furthermore Baums et al(2007) found that wild boar carry potentially zoonotic strains ofS suis and several cases of S suis infection in hunters have been re-ported (Halaby et al 2000 Rosenkranz et al 2003 Baums et al2007) The objective of the present study was to determine the prev-alence of S suis in wild boars of two large geographical regions of

Spain where the boar population is dense and to genetically char-acterise the isolates to enhance our understanding of the popula-tion structure of this pathogen

Samples of tonsil (n = 379) nasal cavity (n = 173) or lung (n = 275)were collected from 425 hunted wild boars between 2007 and 2010254 and 171 animals from North-east and Central regions of Spainrespectively Samples were cultured on Columbia-CNA agar (37 degCfor 24ndash48 h under aerobic conditions) A maximum of six coloniessample with a presumptive identification of S suis based on colonymorphology α-haemolytic activity Gram positive staining andabsence of catalase activity were subcultured onto blood agar andidentified by the Rapid ID 32 STREP system (BioMeacuterieux) and PCR(Okwumabua et al 2003) Serotypes 1 (and 14) 2 (and 12) 7 and9 were screened by multiplex PCR (Silva et al 2006) All strains neg-ative for these capsular types were serotyped by co-agglutination(Gottschalk et al 1993) Non-typeable strains were examined forthe presence of capsule by cell surface hydrophobicity (Rosenberget al 1980)

Corresponding author Tel +34 913943709E-mail address avelavetucmes (AI Vela)

httpdxdoiorg101016jtvjl2014030131090-0233copy 2014 Elsevier Ltd All rights reserved

The Veterinary Journal 200 (2014) 464ndash467

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Strains of serotypes 1 2 7 and 9 were further investigated forvirulence-associated factors using pulsed-field gel electrophoresis(PFGE) and multilocus sequence typing (MLST) (Saacutenchez del Rey et al2013) To identify groups of related serotypes the isolates from thisstudy were grouped with all of the isolates present in the S suis da-tabase using the eBURST algorithm1 and software provided by theMLST website Genetic diversity (GD) was calculated as the ratio oftotal number of PFGE patterns to the total number of isolates(Martiacutenez et al 2002)

A total of 320 S suis isolates were recovered from 391 of thewild boars examined Carriage rates of S suis were 437 and 322in the North-eastern and Central regions respectively This detec-tion rate (approximately 40) confirms that the carriage of S suisby wild boars in Spain is common and emphasises the role of thisspecies as a potentially important reservoir of this pathogen Whiletonsils are typically sampled when investigating the presence ofS suis in carrier animals (Martiacutenez et al 2002 Baums et al 2007Wang et al 2013) the nasal cavities have also been targeted(Brisebois et al 1990) In the current study S suis was detected in861 of wild boar tonsil samples while only 96 of animals cul-tured positive from the samples taken from their nasal cavities afinding that clearly highlights the greater sensitivity of tonsillar sitein detecting S suis

Serotypes 1 2 7 or 9 represented the 162 of the isolates iden-tified (Table 1) Isolates of serotype 9 were the most frequently found(n = 40 125) and were recovered from 20 of S suis-positive wildboars This serotype was also the most frequently found in Germany(Baums et al 2007) and is one of the most prevalent of the iso-lates of S suis circulating in pigs in Spain (Vela et al 2003 Blumeet al 2009) Unlike the findings from wild boars in Germany (Baumset al 2007) isolates of serotypes 2 and 7 were rarely detected(Table 1) Almost 84 of isolates could not be typed by multiplexPCR (Silva et al 2006) results similar to those of Baums et al (2007)As these large percentages of non-typeable isolates limit our ap-preciation of the diversity of the S suis population in this speciesall non-typeable isolates were serotyped by co-agglutination atwhich point 18 additional serotypes were identified (Table 1)

This result suggests that there is a wide diversity in the S suispopulation in wild boar in Spain Almost 26 of strains (n = 83) couldnot be serotyped and of these 44 (138) had hydrophobicity values

gt70 indicating they are probably poorly or non-capsulated(Gottschalk et al 2013) A total of 39 isolates (121) did not ag-glutinate with any of the 35 typing antisera In agreement with theresults of previous studies (Hampson et al 1993 Hoa et al 2011)a high percentage of isolates reacted repeatedly with more than oneserotype (Table 1)

Although serotype 2 is considered the most prevalent serotypeassociated with disease in pigs and humans serotypes 1 7 and 9have also been implicated and are highly prevalent in Europe (Smithet al 1999 Wisselink et al 2000 Blume et al 2009) Thus we furthercategorised isolates of these serotypes (n = 52) according to thevirulence-associated genotype PFGE and MLST By PFGE isolates ofserotypes 1 2 7 and 9 displayed 47 different pulsotypes (Fig 1) withmost isolates represented by unique pulsotypes This reflects greatgenetic diversity (GD 094) a finding in line with the fact that S suisisolates were collected from a large number of wild boars over dif-ferent years and in two geographical regions This finding also agreeswith the diversity observed by serotyping (Table 1) and with thegenomic variability previously reported in pig isolates (Vela et al2003 Blume et al 2009 Luque et al 2010) This genetic hetero-geneity was also observed following MLST analysis with S suis iso-lates assigned to 37 new STs (sequence types) (Fig 1 and seeAppendix S1 Supplementary material in the online version atdoi101016jtvjl201403013) Sequence types 214 and 360 were themost frequently detected (77 and 115 respectively) while ST206ST209 ST210 ST212 ST297 and ST396 were represented by two tothree isolates and the remaining STs (784) were singletons

Most isolates of the serotypes 1 2 7 and 9 (962) exhibited thevirulence-associated genotype mrp-epf-sly- and only two geno-types cps1mrp+epf+sly+ and cps9mrp+epf-sly+ The single isolateof serotype 2 identified in the current study did not exhibit eitherthe high prevalence virulence-associated genotype mrp+epf+sly+found in porcine and human clinical isolates (Silva et al 2006Gottschalk et al 2007 Blume et al 2009) or the genotype mrp+epfsly+ found in most serotype 2 isolates in wild boars in Germany(Baums et al 2007) None of the serotype 9 isolates belonged togenotypes mrpepf-sly+ or mrp-epf-sly+ very commonly found inclinical isolates from pigs in Europe (Wisselink et al 2000 Silvaet al 2006 Rehm et al 2007 Blume et al 2009)

Analysis of the allelic profiles of the STs detected in wild boarsrevealed that most did not share any allele with genotypes withinthe ST1 ST16 ST61 and ST87 complexes (see Appendix S1 Supple-mentary material in the online version at doi101016jtvjl201403013) associated with clinical isolates of serotypes 2 and9 from pigs in Spain and other European countries or with humanisolates of serotype 2 (King et al 2002 Rehm et al 2007 Blumeet al 2009 Schultsz et al 2012) Only ST278 and ST280 shared oneallele (genes thrA and recA) with genotypes comprising the ST87 andST61 complexes Therefore despite their prevalence in wild boarsin Spain strains of S suis are genetically distantly related to thecommon clonal complexes associated with porcine or zoonotic in-fection in Europe (see Appendix S1 Supplementary material in theonline version at doi101016jtvjl201403013)

The findings of our study suggest that wild boar in Spain do notrepresent a health risk for pigs or humans at least within the contextof S suis infection The similar testing of wild boar populations inother regions of Europe is recommended to corroborate our find-ings and provide a broader overview of the epidemiology of thispathogen

Conflict of interest statement

None of the authors of this paper has a financial or personal re-lationship with other people or organisations that could inappro-priately influence or bias the content of the paper1 See httpeburstmlstnet

Table 1Serotype distribution of Streptococcus suis isolates recovered from wild boar

Serotype Number ofisolates ()

Number of S suis-positiveboar with this serotype ()

1 8 (25) 3 (18)2 1 (03) 1 (06)7 3 (09) 3 (18)9 40 (125) 33 (200)16 13 (41) 13 (78)31 18 (56) 12 (72)127a 22 (69) 14 (84)9222731a 19 (59) 15 (90)Othersb 49 (153) 44 (265)Other double or triple serotypesc 64 (200) 51 (307)Non-typeabled 83 (259) 63 (380)

a Serotyping repeated three times Confirmed as S suis by PCR (Okwumabua et al2003)

b Includes serotypes 12 3 4 5 6 8 15 20 21 23 26 27 28 29 33 and 34c Isolates other than serotype12 reacting with antisera against two or three dif-

ferent serotypesd Forty-four isolates with hydrophicity values gt70 indicating they are probably

non-encapsulated (Gottschalk et al 2013) Thirty-nine isolates did not agglutinatewith any antisera directed against the 35 serotypes

465V Saacutenchez del Rey et alThe Veterinary Journal 200 (2014) 464ndash467

Similarity PFGE patterns Pulsotype Serotype ST

9 9 9 9

1 1

9

9 9

7 9

2 9

9 9

9 9

9 9

9 9

9 9

7 7

9 9

9 9

9 9

9 9

1 1

1 9 9 9

9 9

9

9 9

9

9 9

1

1 1

280 212 285 279

360 360

359

214 211

297 297

281 362

276 209

277 363

208 283

214 274

212 278

296 286

275 271

206 206

269 214

213 214

360 357

356 358 361 215

214 273

214

212 209

210

210 359

360

360 360

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

7 8

9 10

23 24

4

5 6

15 16

17 18

11 12

13 14

19 20

21 22

41 42

43 44

37 38

39 40

33 34

35 36

25 26

27 28 29 30

31 32

45

46 47

1

2 3

23

24 24

463

5 44

27

370

6 42

13

341

4

272

8 24

31

202

7 19

32

144

4

110

2

819

542

307

22

5

174

15

4

MW (Kbs)

Fig 1 Dendrogram illustrating the genetic relationship based on unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of 50 Streptococcus suisisolates (serotypes 1 2 7 and 9) identified in wild boar (two isolates were not included in the analysis as DNA degradation resulted in a lsquosmearrsquo pattern) The allelic profilesof novel serotypes and the GenBank accession number of novel alleles given in Appendix S1 (Supplementary material in the online version at doi101016jtvjl201403013)

466 V Saacutenchez del Rey et alThe Veterinary Journal 200 (2014) 464ndash467

Acknowledgements

This work was funded by project AGL2009-14303-C02-01 of theSpanish Ministry of Science and Innovation Ms Veroacutenica Saacutenchezis the recipient of a PhD Grant from the Spanish Ministry of Edu-cation and Science

Appendix Supplementary material

Supplementary data to this article can be found online atdoi101016jtvjl201403013

References

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Artois M Depner KR Guberti V Hars J Rossi S 2002 Classical swine fever (hogcholera) in wild boar in Europe Revue Scientifique et Technique (InternationalOffice of Epizootics) 21 287ndash303

Baums CG Verkuhlen GJ Rehm T Silva LM Beyerbach M Pohlmeyer KValentin-Weigand P 2007 Prevalence of Streptococcus suis genotypes in wildboar of Northwestern Germany Applied and Environmental Microbiology 73711ndash717

Blume V Luque I Vela AI Borge C Maldonado A Domiacutenguez L Tarradas CFernaacutendez-Garayzaacutebal JF 2009 Genetic and virulence-phenotypecharacterization of serotypes 2 and 9 of Streptococcus suis swine isolatesInternational Microbiology 12 161ndash166

Brisebois LM Charlebois R Higgins R Nadeau M 1990 Prevalence of Streptococcussuis in four to eight week old clinically healthy piglets Canadian Journal ofVeterinary Research 54 174ndash177

Cruz Colque JI Devriese LA Haesebrouck F 1993 Streptococci and enterococciassociated with tonsils of cattle Letters in Applied Microbiology 16 72ndash74

Gottschalk M Higgins R Boudreau M 1993 Use of polyvalent coagglutinationreagents for serotyping of Streptococcus suis Journal of Clinical Microbiology 312192ndash2194

Gottschalk M Segura M Xu J 2007 Streptococcus suis infections in humans TheChinese experience and the situation in North America Animal Health ResearchReviews 8 29ndash45

Gottschalk M Lacouture S Bonifait L Roy D Fittipaldi N Grenier D 2013Characterization of Streptococcus suis isolates recovered between 2008 and 2011from diseased pigs in Queacutebec Canada Veterinary Microbiology 162 819ndash825

Halaby T Hoitsma E Hupperts R Spanjaard L Luirink M Jacobs J 2000Streptococcus suis meningitis a poacherrsquos risk European Journal of ClinicalMicrobiology and Infectious Diseases 19 943ndash945

Hampson DJ Trott DJ Clarke IL Mwaniki CG Robertson ID 1993 Populationstructure of Australian isolates of Streptococcus suis Journal of ClinicalMicrobiology 31 2895ndash2900

Higgins R Gottschalk M 1999 Streptococcal disease In Straw BE DrsquoAllaire SMengeling WL Taylor DJ (Eds) Diseases of Swine 8th ed Iowa State UniversityPress Ames Iowa USA pp 563ndash578

Hoa NT Chieu TTB Nga TTT Dung NV Campbell J Anh PH Tho HH ChauNVV Bryant JE Hien TT et al 2011 Slaughterhouse pigs are a major reservoirof Streptococcus suis serotype 2 capable of causing human infection in southernVietnam PLoS ONE 6 e17943

King SJ Leigh A Heath PJ Luque I Tarradas C Dowson CG Whatmore AM2002 Development of a multilocus sequence typing scheme for the pig pathogenStreptococcus suis Identification of virulent clones and potential capsular serotypeexchange Journal of Clinical Microbiology 40 3671ndash3680

Luque I Blume V Borge C Vela AI Perea JA Maacuterquez JMFernaacutendez-Garayzaacutebal JF Tarradas C 2010 Genetic analysis of Streptococcussuis isolates recovered from diseased and healthy carrier pigs at different stagesof production on a pig farm The Veterinary Journal 186 396ndash398

Martiacutenez G Harel J Lacouture S Gottschalk M 2002 Genetic diversity ofStreptococcus suis serotypes 2 and 12 isolates recovered from carrier pigs in closedherds Canadian Journal of Veterinary Research 66 240ndash248

Okwumabua O OrsquoConnor M Shull E 2003 A polymerase chain reaction (PCR)assay specific for Streptococcus suis based on the gene encoding the glutamatedehydrogenase FEMS Microbiology Letters 218 79ndash84

Rehm T Baums CG Strommenger B Beyerbach M Valentin-Weigand P GoetheR 2007 Amplified fragment length polymorphism of Streptococcus suis strainscorrelate with their profile of virulence-associated genes and clinical backgroundJournal Applied Microbiology 56 102ndash109

Rosenberg M Gutnick D Rosenberg E 1980 Adherence of bacteria tohydrocarbons A simple method for measuring cell-surface hydrophobicity FEMSMicrobiology Letters 9 29ndash33

Rosenkranz M Elsner HA Stuumlrenburg HJ Weiller C Roumlther J Sobottka I 2003Streptococcus suis meningitis and septicemia contracted from a wild boar inGermany Journal of Neurology 250 869ndash870

Saacutenchez del Rey V Fernaacutendez-Garayzaacutebal JF Briones V Iriso A Domiacutenguez LGottschalk M Vela AI 2013 Genetic analysis of Streptococcus suis isolates fromwild rabbits Veterinary Microbiology 165 483ndash486

Saez-Royuela C Telleria JL 1986 The increased population of the wild boar (Susscrofa L) in Europe Mammal Review 16 97ndash101

Salasia SI Laumlmmler C Devriese LA 1994 Serotypes and putative virulence markersof Streptococcus suis isolates from cats and dogs Research in Veterinary Science57 259ndash261

Schultsz C Jansen E Keijzers W Rothkamp A Duim B Wagenaar JAvan der Ende A 2012 Differences in the population structure of invasiveStreptococcus suis strains isolated from pigs and from humans in The NetherlandsPLoS ONE 7 e33854

Silva LM Baums CG Rehm T Wisselink HJ Goethe R Valentin-Weigand P2006 Virulence-associated gene profiling of Streptococcus suis isolates by PCRVeterinary Microbiology 115 117ndash127

Smith HE Veenbergen V van der Velde J Damman M Wisselink HJ Smits MA1999 The cps genes of Streptococcus suis serotypes 1 2 and 9 Development ofrapid serotype-specific PCR assays Journal of Clinical Microbiology 37 3146ndash3152

Vela AI Goyache J Tarradas C Luque I Mateos A Moreno MA Borge C PereaJA Domiacutenguez L Fernaacutendez-Garayzaacutebal JF 2003 Analysis of genetic diversityof Streptococcus suis clinical isolates from pigs in Spain by pulsed-field gelelectrophoresis Journal of Clinical Microbiology 41 2498ndash2502

Wang K Zhang W Li X Lu C Chen J Fan W Huang B 2013 Characterizationof Streptococcus suis isolates from slaughter swine Current Microbiology 66344ndash349

Wisselink HJ Smith HE Stockhofe-Zurwiedenb N Peperkampc K Vecht U 2000Distribution of capsular types and production of muramidase-released protein(MRP) and extracellular factor (EF) of Streptococcus suis strains isolated fromdiseased pigs in seven European countries Veterinary Microbiology 74 237ndash248

467V Saacutenchez del Rey et alThe Veterinary Journal 200 (2014) 464ndash467