Update on Diagnostic Tools for Erysipelothrix Rhusiopathiae Associated Disease in Pigs

ONGOINGTRAINING

Update on diagnostic tools for Erysipelothrix rhusiopathiae associated disease in pigs

Priscilla F. Gerbera and Tanja Opriessniga,b* aThe Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, UK bVeterinary Diagnostic Laboratory, Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA *Corresponding author. Tel.: +44 (0)131 651 9177. E-mail address: [email protected] (T. Opriessnig).

Summary The genus Erysipelothrix at present consists of two major The gram positive bacterium Erysipelothrix spp. has species, E. rhusiopathiae and E. tonsillarum, and two been associated with clinical disease in pigs for more less frequently isolated species, Erysipelothrix sp. strain than 135 years. Despite availability of effective preventive 1 and Erysipelothrix sp. strain 2 (Table 1) (Takahashi et measures and antimicrobials, treatment is often ham- al., 1992; Takahashi et al., 1999). Species other than E. pered by inappropriate diagnostic approaches. This arti- rhusiopathiae are considered to be of low virulence in cle summarizes current knowledge on diagnostic tools pigs (Takahashi et al., 2008) and it has been suggested available for confirmation of Erysipelothrix rhusiopathiae that Erysipelothrix sp. strain 1 is likely to be bovine spe- associated disease in pigs. cific (Hassanein et al., 2001). There are at least 28 serotypes (1a, 1b-26 and N) recognized to date (Table 1). Introduction Among the 15 serotypes of E. rhusiopathiae, serotype Organisms of the genus Erysipelothrix, which are widely 1 (subdivided into serotypes 1a and 1b) and serotype 2 distributed in nature, cause a wide spectrum of diseases are the most important in global pig production (Coutinho in other mammals, reptiles, amphibians, fish and birds, et al., 2011; Opriessnig et al., 2010; To et al., 2012). including erysipelas in pigs and erysipeloid in people Because of the importance of swine erysipelas, inac- (Brooke and Riley, 1999; Wang et al., 2010). Erysip- tivated and attenuated-live vaccines are widely used. elothrix spp. has been isolated not only from various Nevertheless, economic losses due to swine erysipelas mammals and birds independent of disease status but continue to occur (Bender et al., 2009; Imada et al., also from food products such as pork, chicken and sea- 2004; To et al., 2012). The recent introduction and availa- food (Fidalgo et al., 2000; Nakazawa et al., 1998; Wang bility of novel testing technologies with greater analytical et al., 2002). Human infections with Erysipelothrix spp. are usually related to occupational exposure to infected sensitivity may lead to improved evaluation of vaccine animals and products and swine erysipelas continues to compliance and diagnostic sensitivity for diagnosing be an important reason for carcass condemnations at E. rhusiopathiae infections earlier after exposure in the slaughterhouses (Opriessnig et al., 2004; Takahashi et future. al., 2008). The main host for Erysipelothrix spp. is the pig (Opriessnig and Wood, 2012), but this bacterium also Clinical signs causes economic loses in wild and farmed boars, tur- Three clinical presentations are recognized in pigs keys, chickens, emus, calves, sheep and lambs (Brooke (Opriessnig and Wood, 2012). The acute form may be and Riley, 1999). associated substantial morbidity and mortality within

Table 1. Erysipelothrix species and associated serotypes and surface protective antigen (Spa) types. Erysipelothrix species Serotypes Spa type E. rhusiopathiae 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, N SpaA 4, 6, 11, 19, 21 SpaB E. tonsillarum 3, 7, 20, 14, 20, 22, 23, 24, 24, 25, 26 - E. species strain 1 13 - E. species strain 2 18 SpaC

1 2 ONGOINGTRAINING treatment of the pig. treatment ofthe pig. tamination, tissue conditionsandprevious antimicrobial Direct culture canbecomplicatedbyspecimen con triple-sugar iron agar(Fig.3;VickersandBierer, 1958). 1978), butdoesproduce acidandhydrogen sulfidein with catalase,oxidase,methyl red, orindole(Cottral Erysipelothrix hemolysis on bloodagarmedia.Thegenus of partial 1990).Moststrainsinduceanarrowhours (Carter zone bation at37°C(Fig.2),withincreased sizeafter48 small (0.1–0.5mmindiameter)after24hoursofincu On agarmedia,coloniesare clear, circular, andvery blood (CNA)are commonlyused(Benderatal.,2009). colistin-nalidixic acidagarplatescontaining5%sheep ture soyagarcontaining5%sheepbloodor trypticase 30°C and37°C(Brooke andRiley, 1999).Fordirect cul 5°C and44°C,withoptimalgrowth occurringbetween rods andare facultativeanaerobes thatgrow between non-sporulating, non-acid-fast, slender gram-positive Members ofthegenus A. Bacterialisolation of vaccination. could perhapsalsoprovideoneffectiveness information tic specimentofor (Ramirez etal.,2012).Theuseoforalfluidsasdiagnos ease ofthiscollectionmethodandcosteffectiveness purposes haveincreased inthelastyearsdueto fluid samplecollectionsforsurveillanceanddiagnosis also now available (Giménez-Lirola et al., 2013). Oral nucleic acidsandanti- including theuseoforalfluidsfordetectionbacterial to improvediagnosisof ante-mortem its serotype (Benderetal.,2010).More recently, methods for isolatingthisbacteriumandupto8daysdetermine bacteriological culture methodsrequire atleast1-3days characterization of isthetraditionaltoolforfurther now rarely performed, are laboriousandtime-consuming.Serotyping, although and biochemical characteristics; however, the methods rhusiopathiae E. mainly carriedoutbycultivationandidentificationof is provided inTable is 2.Diagnosisoftheerysipelas ofcommonly used diagnosticmethods A summary Diagnosis andcharacterization (Opriessnig andWood, 2012). tis-like lesionssometimesassociatedwithsuddendeath cardiac insufficiency duetoproliferative endocardi reduced growth,to thedevelopmentofarthritis, and infection andoften is characterized by lamenessdue chronic mayfollowacute,subacuteorsubclinial form canalsoremain unnoticed.The Subacute erysipelas numbers of mummies or small litters may be observed. vulvulardischargeandlitterswithincreased parturient less severe. Inbreeding herds, infertility, pre-and post resembles butclinicalsignsare theacuteform typically essentially pregnant Thesubacuteform sowsmayabort. ated with rhomboid skin lesions (Fig. 1). Acutely infected ment or stiff gait and/or sudden death often associ lethargy, depression, inappetence,avoidanceofmove days andischaracterizedbysuddenillnesswithfever, is generally inactive and does not react isgenerallyinactiveand does notreact from tissuesonthebasis ofgrowth Erysipelothrix Erysipelothrix Erysipelothrix Erysipelothrix

spp. strains. Current spp.strains.Current rhusiopathiae spp. antibodies are spp.antibodiesare are non-motile, are non-motile, E. rhusiopathiae infection infection ------

There aremultifocaltodiffuserhomboidskinlesions. Fig. 1. in triple-sugariron agar. CourtesyofDr. J.Bender. Fig. 3. tesy ofDr. J.Bender. colistin-nalidixic acidagar(CNA)after48hoursofincubation.Cour Fig. 2. Pig experimentally infected with Hydrogen sulphide production by Typical colonyappearanceof Erysipelothrix rhusiopathiae Erysipelothrix Erysipelothrix rhusiopathiae. Erysipelothrix Erysipelothrix rhusiopathiae Erysipelothrix on on -

3 ONGOINGTRAINING characterize thebacterium. Table 2. antigen Detection of bacterium Detection oflive characterization Further antibodies Detection of DNA Detection of Application Application Available diagnostictoolstodemonstrate Direct bacterialisolation Serotyping Serotyping electrophoresis (PFGE) Pulsed-field gel immunoassay (FMIA) Fluorescent microbead ELISA amplification (LAM)assay Loop-mediated isothermal Real-time PCR Conventional PCR Immunohistochemistry after enrichmentsteps Indirect bacterialisolation Diagnostic tool Diagnostic tool • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • No interferenceofpriorantimicrobial treatment with Requires anelectrophoresis step. Sensitive andrapid. Requires availabilityofanti-serum. detection. No interferenceofpriorantimicrobial treatment with usefulonchronicParticularly lesionsandskin. Interference ofpriorantimicrobial treatment. isolation. Significantly increased sensitivity compared todirect characterization. Allows ant Laborious andtimeconsuming(>3days). Interference ofpriorantimicrobial treatment. Low sensitivity. characterization. Allows antimicrobialisolate analysis andfurther Laborious andtimeconsuming(>3days). Requires availabilityofanti-serumagainstallserotypes. Time consuming(>3daysafter initialisolation). Requires availabilityofanisolate. Time consuming(>3daysafterinitialisolation). strains. Capable ofdifferentiating vaccinestrainsfrom field Requires availabilityofanisolate. antibodies againstseveralpathogens. Possibility ofmultiplexingforsimultaneousdetection Low cost. over time. Can beusedtodetectandmonitorhumoralresponse Low cost. over time. Can beusedtodetectandmonitorhumoralresponse Could beuseddirectly onthefarm. Cost effective andrequires equipment. onlyrudimentary Sensitive andrapid. technicians. Requires sophisticatedequipmentandexperiencedlab detection. No interferenceofpriorantimicrobial treatment with conventional PCR. No electrophoresis step;therefore fastercompared to Sensitive andrapid. detection. Erysipelothrix i microbial analysis and further isolate microbial isolate analysisandfurther spp. presence and to further andtofurther spp.presence Comments 4 ONGOINGTRAINING additional benefit that several targets can be detected additional benefitthatseveraltargetscanbedetected 1999; Yamazaki, 2006).Real-timePCRassayshavethe 1994; Paletal.,2009;Shimoji1998;Takeshi etal., issues andreducing turn-aroundtime(Makinoetal., eliminating possiblepostamplificationcontamination detection ofthetargetwithinclosedtubethereby assays whichallowsimultaneousamplificationand fied product mustbedistinguishedfrom real-time PCR which require theuseofelectrophoresis todetectampli lation methods(Fig.5),conventionalPCRtechniques sensitive identificationof 2006). AlthoughPCRassaysprovide more rapidand (Makino etal.,1994;Takeshi etal.,1999;Yamazaki, diagnostic laboratories ingly being used in veterinary Polymerase chainreaction (PCR)techniquesare increas (PCR)assays C. Polymerasechainreaction negative (Opriessnigetal.,2010). antigen inskinlesions(Fig.4),whichare oftenculture also beenfoundbeneficialfordetectionof ded tissuehasbeenfounduseful.Specifically, IHChas tion of a causativerole, (IHC)fordetec immunohistochemistry negative caseswhere success ofdemonstrating with antimicrobials whichcan complicatethediagnostic sues from animals that have been previously treated It iscommonfordiagnosticlaboratoriestoreceive tis (IHC)assay B. Immunohistochemistry if sodesired. characterization of the isolates ties and conduct further toconductantimicrobialallows thelaboratory sensitivi agar (Benderetal.,2009).Theavailabilityoftheisolates rates 9.5foldwhencompared todirect culture onblood enrichment stephasbeenshowntoincrease isolation sion mediasupplementedwithserum.Theuseofthis broth, anutrientbroth infu containingbrainandheart A commonlyusedmediumisselective prior toisolationonagarplates(Benderetal.,2009). based enrichmenttechniquesare commonlyutilized To accountforthelowsensitivityofdirect culture, broth Fig. 5. infection. Datarepresent meanvaluesfromsevenpens andareadaptedfromGiménez-Lirola etal.,2013. 0 E. rhusiopathiae Exposure to E. rhusiopathiae Expecteddetection of 1 Isolation 2 Positive DNA(PCR) 3

4 Erysipelothrix in formalin-fixed, paraffin-embed informalin-fixed, 5 Erysipelothrix rhusiopathiae Erysipelothrix 6 Erysipelothrix Erysipelothrix 7 Positive IgM(ELISA) 8 is suspected to play issuspectedtoplay 9 spp.thaniso spp. In culture spp.Inculture 10 Erysipelothrix Erysipelothrix 11 from oral fluid by different methods from day 1 through day 28 after experimental fromoralfluidby differentmethodsfromday1through day28afterexperimental 12 13 ------

14 achieved ( species of this method,identificationanddifferentiation ofdifferent simultaneously in so-called multiplex reactions. By using peroxidase complexmethodcounterstainedwithhematoxylin. teria-like organisms (darkstaining) in the dermis. Streptavidin–biotin– antiserum against Fig. 4. the LAMP technology generally is considered of similar the LAMPtechnologygenerallyisconsidered ofsimilar directly inthefield(Boonhametal.,2014).Inaddition, its usageinlaboratorieswithlimitedresources oreven cent dyesthatintercalate ordirectly labelDNAfacilitating reaction canbeassessedbythenakedeyeviafluores temperaturemal cyclercapableofalternating steps.The a simpleheat-blockwithoutrequiring anexpensivether temperature of 60-65°C which can be achieved by using nucleicacidamplificationataconstant due toisothermal when compared toreal-time PCRassays.Thisismainly requiring lessinstrumentationtoachieveamplification has themainadvantageofbeinglessexpensiveand opathiae strain 1) (Pal et al., 2009). strain 1)(Paletal.,2009). (LAMP) hasbeendescribedfordetectionof Recently, amplificationassay aloop-mediatedisothermal 15 Positive IgG(ELISA) 16 Skin, pig. Immunohistochemical staining using a polyclonal Skin,pig.Immunohistochemicalstainingusingapolyclonal 17 (Yamazaki etal.,2014).TheLAMPtechnology E. rhusiopathiae Erysipelothrix 18 Erysipelothrix rhusiopathiae Erysipelothrix 19 20 21 in a single reaction has been inasinglereaction hasbeen , 22 . tonsillarum E. 23 24

revealing abundantbac 25 26 , and 27 E. rhusi E. Days 28 sp. sp. - - - - 5 ONGOINGTRAINING ferent isolates ofthesameserotype (Wang etal.,2010). resultstory in pathogenicity havebeenshown withdif pigs (OpriessnigandWood, 2012).However, contradic while serotype 2ismore prevalent inchronically affected that serotype 1ismost commonlypresent in acute cases 2 (Wang etal.,2010).Historicallyithasbeen determined In pigs,75-80%ofisolatesare classifiedasserotype 1or byhot-aqueousextraction (Kucsera,1973)(Fig.6). ery gel withtype-specificrabbitanti-seraandantigenrecov through serotyping byusinganagar-gel immunodiffusion mon characterizationtoolfor et al.,2004;Opriessnig2004).Themostcom outbreakerinarians duringerysipelas situations(Imada can providetopigownersandvet usefulinformation Methods ofdifferentiation of isolates E. Characterizationof 2014). cation ofboundreporterfluorophore (Boonhametal., of thebeadidentity(region) andtheotherfor quantifi instrument withtwolasersorLEDs,oneforclassification gent, thebeadsare separatedwithinaflow-cytometer completion ofassayincubationswithadetectionrea multiplexing upto500analytesinasingletest.After significantly reduce background problems andallows mits highlystringentwashingprocedures, which can This bead-based array per coated in a linear surface. that remain inaliquidsuspensionarrayinsteadofbeing antigen iscoupledtocolor-coded paramagneticbeads similar toanindirect ELISA,withthedifference thatthe Lawson etal.,2010;Wagner etal.,2011).Theassayis et al.,2011; Chen et al., 2013;Gundersenet1992; increasingly serology being usedin veterinary (Anderson rola etal.,2012;Giménez-Lirola etal.,2013).FMIAsare available in-houseand commercial ELISAs (Giménez-Li higher sensitivityforearlydetectionwhencompared to oral fluids(Fig.5).TheFIMAhasbeenshowntohavea opathiae (FMIA) wasdevelopedfordetectionofanti- More recently, afluorescent microsphere immuneassay Imada etal.,2003;Sato1998). ofresults (Chinetal.,1992; objective determination time period with large numbersof samples in a short andtheabilitytotest generally haveasimpleformat anti- nosorbent assays(ELISA)havebeendescribedfor Several in-houseandcommercial enzyme-linkedimmu herd status and for monitoring vaccination compliance. rhusiopathiae E. Evaluation ofthehumoralimmuneresponse against applications D. Serology facilitate usageofthismethod. evaluation onfieldsamplesneedstobeconducted rhusiopathiae (Yamazaki etal.2013)hasshowngreat sensitivityon et al.,2014).Althoughthe the targetDNAsequenceare beingutilized(Boonham four tosixprimersrecognizing sixtoeightregions of sensitivity andcomparabletoreal-time PCRassaysas . rhusiopathiae E. IgM and IgG antibody detection in serum and IgMandIgGantibodydetectioninserum grown inenrichmentculture broths, further can be important for determining the the fordetermining canbeimportant antibody detection. These assays antibodydetection.Theseassays Erysipelothrix Erysipelothrix E. rhusiopathiae Erysipelothrix LAMP assay LAMPassay field isolates fieldisolates isolates is isolatesis E. rhusi spp. E. E. ------of precipitationbetweenanantiserumandtheantigenutilized. placed in the well surrounding the antigen. thrix Fig. 6. types (Shenet al.,2010;To andNagai, 2007). sillarum types (Table 1) (To and Nagai, 2007). In contrast, Spa proteins sero havebeenassociatedwithcertain clinical disease(Ingebritson etal.,2010;To etal.,2010). sipelothrix proteinsis one of the bestcharacterized surface of in SpaA,SpaB1,SpaB2,andSpaC(Shenetal.,2010), genesis. To date,theSpaantigen,whichcanbedivided role oftheSpaproteins invaccineprotection andpatho strains isolatedfrom the fieldtissuesandtodetermine (Nagai et al., 2008). Investigating Spa prevalence among discrimination ofthelivevaccinestrainfrom fieldisolates variable region protective inthesurface (spa)geneAfor developed basedonnucleotidesequencingofahyper More recently, anewstrain-typingmethodhasbeen ing methods(Opriessnigetal.,2004;To etal.,2012). the “goldstandard” amongthecurrent DNA-basedtyp field gelelectrophoresis (PFGE)hasbeenconsidered been usedtodifferentiate Although severalmolecularbiologicalmethodshave virulence (Takahashi etal.,1992). that there isnodirect relationship betweenserotype and ered avirulent)strains(Imadaetal.,2004),suggesting tained serotype 2(considered virulent)and7(consid (RFLP), bothE.tonsillarumand analysed byrestriction fragment length polymorphisms E. rhusiopathiae to classify Various molecular typing methods have been applied Takahashi etal.,1992).When tani etal.,2000;Opriessnig2004;Palal,2009; Dunbar andClarridge,III,2000;Imadaetal.,2004;Oka strain 2are recognized (Table 1)(Coutinhoetal.,2011; spp. Antigen is placed in the center wells and the antisera are spp.Antigenisplacedinthecenterwellsandantiseraare Typical platelayoutforserotypedeterminationof isolates were found to contain no detectable Spa isolateswere foundtocontainnodetectable Spa spp. and is associated with protection against spp.andisassociatedwith protection against Erysipelothrix , E . tonsillarum, isolates into species and today isolatesintospeciesandtoday Erysipelothrix Erysipelothrix E . sp.strain1,and E. rhusiopathiae The arrow indicates a line arrow indicates a line spp.,pulsed- strains were strainswere Erysipelo E. ton E con . sp. . sp. Ery ------6 ONGOINGTRAINING over time (Fig. 7) will likely increase in future. It is impor monitoring of andthereforeapproaches willlikelygainimportance nate theuseofantimicrobials infoodanimals,preventive production. Withtheattemptstodecrease or even elimi Erysipelothrix Discussion detection (Shenetal.,2010). rapid, sensitiveandhigh-through putmethodforSpa real-time PCRassayshastheadvantagetoprovide a SpaC (Ingebritsonetal.,2010).Theusageofamultiplex ventional andreal-time PCRassaysforSpaA,SpaBand 1998; Shimojietal.,1999;To andNagai,2007)con and Westernblotting(Imadaetal.,1999;Makino Spa typeofthe the Several methods have been reported to determine immunoassay (FMIA). Fig. 7. FMIA values 0 1 2 3 4 5 6 7 8 9 Cross-sectionalanalysisof n

Erysipelothrix 0 sp. continues to be of importance in pig inpig sp.continuestobeofimportance Erysipelothrix n =17 Erysipelothrix antibodies 1 spp. infection on a herd basis spp. infection on a herd basis n =7 spp.,includingSDS–PAGE Erysipelothrix 2 antibody levels in a vaccinated breeding herd as determined by a fluorescent microbead-based antibodylevelsinavaccinatedbreedingherdasdeterminedbyfluorescentmicrobead-based n =10 3 n =8 - - - tially decreased costandimproved turn-around time. inthe futurewill likely gain importance due to substan and currently notroutinely usedfor investigations tool to characterize viruses in veterinary Actinobacillus bacteria capableofcausingsystemicdiseasesuchas be considered including classical swine fever virusand pathogens thatcouldinducesimilarlesionsalsoneedto evaluated. Inaddition,incasesofclinicaloutbreaks other with usingappropriate additionaltestscanthisbefurther previous infection or an acute/subacute infection. Only ies, antibodiesinresponse toaprevious vaccination,a elothrix test. Forexample,detectionofantibodiesagainst are lookingforandalsoconsiderthelimitationsofeach carefully assesswhichtestwillprovide theanswerthey tant thatdiagnosticians,practitionersandpigowners 4 spp.couldindicatepassively-acquired antibod n =5 sp. and others. Sequencing, a common sp.andothers.Sequencing,acommon 5 n =7 n =1 6 Erysipelothrix Birth parity number parity Birth n =1 7 Erysip spp., spp., - - - 7 ONGOINGTRAINING • • • • • • • • • • • • • • • References Erysipelothrix rhusiopathiae Erysipelothrix Opriessnig, T., 2013.Improving diagnosisof antemortem Giménez-Lirola, L.G.,Xiao,C.T., Zavala,M.,Halbur, P.G., Microbiol Methods91,73-79. antibodies inpigswithknownandunknownexposure. J immunoassays fordetectionofanti- immunoassay andcomparisonwiththree enzyme-linked 2012. Developmentofanovelfluorescent microbead-based Giménez-Lirola, L.G.,Xiao,C.T., Halbur, P.G., Opriessnig,T., 66, 2066-2070. bution insomeAustralianseafoods.ApplEnviron Microbiol methods fordetectionof Fidalgo, S.G.,Wang, Q.,Riley, T.V., 2000.Comparison of 38, 1302-1304. recognition of Dunbar, S.A.,Clarridge, J.E.,III,2000.Potentialerrors in from Brazilianswine.Diagn Microbiol InfectDis69,123-129. ization ofrecent andarchived Moreno, A.M.,2011.Phenotypic andmolecularcharacter Coutinho, T.A., Imada,Y., Barcellos, D.E.,Oliveira,S.J., Press, Ithica,NY. 429-436. microbiology,ized methodsforveterinary University Cornell Cottral GE.1978. elothrix nated withanimmunodominant65kDaantigenfrom usingnitrocellulose impregfor swineerysipelas particles Chin, J.C.,Turner, B., Eamens, G.J., 1992. Serological assay 87-95. of foot-and-mouthdiseasevirus.JImmunolMethods396, the detectionofswineantibodiestonon-structuralproteins M.C., Tsai, H.J.,2013.DevelopmentofaLuminexassayfor Chen, T.H., Lee,F., Lin,Y.L., Pan,C.H.,Shih,C.N.,Lee, 195-196. and Mycology, 5thed.AcademicPress, Springfield,Illinois, R. (ed.),DiagnosticProcedures inVeterinary Microbiology GR.1990. Carter occupational pathogen.JMedMicrobiol 48,789-799. bacteriology, epidemiologyandclinicalmanifestationsofan Brooke, C.J.,Riley, T.V., 1999. Res 186,20-31. nostics: from ELISAtonextgenerationsequencing.Virus J., Tomlinson, J.,Mumford, R.,2014.Methodsinvirusdiag Boonham, N.,Kreuze, J.,Winter, S.,van,d., V, Bergervoet, 17, 1605-1611. elas outbreaks intheUnitedStates.ClinVaccine Immunol samples, andvaccinestrainsfrom sixrecent swine erysip cies isolatesfrom clinicallyaffected pigs,environmental Opriessnig, T., 2010.Characterizationof Bender, K.J., J.S.,Shen,H.G.,Irwin,C.K.,Schwartz, Invest 21,863-868. experimentally andnaturallyinfectedswine.JVet Diagn and enrichmentculture methodsfor Opriessnig, T., 2009.Comparisonofconventionaldirect Bender, J.S.,Kinyon,J.M.,Kariyawasam,S.,Halbur, P.G., 79-88. existing ELISA detection methods. J Immunol Methods 366, and bovinerespiratorysyncytialvirus,withcomparisonto virus 1, parainfluenza 3 virus,bovineviraldiarrhoea virus, tiplex serology assaytodetectantibodiesbovineherpes yer, J.,2011.DevelopmentandevaluationofaLuminexmul Anderson, S.,Wakeley, P., Wibberley, G.,Webster, K.,Saw antigen. JImmunol Methods148,1-8. trays forrapiddetection ofschistosomalcirculating anodic antigen capture enzyme-linkedimmunoassayinmicrotitre schau, K.J.,de,J.N.,Deelder, A.M.,1992. Magneticbead Gundersen, S.G.,Haagensen, I.,Jonassen,T.O., Figen Methods 92,113-121. bacterial, nucleicacid,andantibody detection.JMicrobiol

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rhusiopathiae Erysipelothrix

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8 ONGOINGTRAINING • • • • • • • • Vet MedSci61,1007-1011. strains isolated from tonsils of slaughter pigs in Thailand. J S., 1999.Serotyping andpathogenicityof thon, S.,Jitnupong,W., Yamamoto, K.,Kijima,M.,Furuuchi, Takahashi, T., Sunama,P., Satra,J.,Cholsindhu,N.,Kong Immunol 52,469-478. numerous strainsisolated from extensiveorigins.Microbiol Erysipelothrix Yamamoto, K., Sawada,T., 2008.Ataxonomicstudyon Takahashi, T., Fujisawa,T., Umeno,A.,Kozasa,T., larum resenting alltwenty-three serovars and relatedness among matsu, M.,Sawada,T., Benno,Y., Mitsuoka,T., 1992.DNA Takahashi, T., Fujisawa,T., Tamura, Y., Suzuki,S.,Mura Microbiol 36,86-89. JClin nation assayforrapiddiagnosisofswineerysipelas. Y., 1998.Useofanenrichmentbroth cultivation-PCRcombi Shimoji, Y., Mori,Y., Hyakutake,K.,Sekizaki,T., Yokomizo, protective immunity. InfectImmun67,1646-1651. rhusiopathiae characterization ofaprotective antigenof Shimoji, Y., Mori,Y., Fischetti, V.A., 1999. Immunological 109, 1227-1233. real-time andconventionalPCRassays.JApplMicrobiol reference strainsanddiagnosticsamplesbyspa multiplex protectiveof surface antigen(spa)typesin Shen, H.G.,Bender, J.S.,Opriessnig,T., 2010.Identification nation. ZentralblVeterinarmed B45,407-420. the enzyme-linkedimmunosorbentassayandlatexaggluti of theprotective antigenof Suzuki, T., Yokoyama, A.,Saito,H.,Maehara,N.,1998.Use Sato, H., Yamazaki, Y., Tsuchiya, K., Aoyama, T., Akaba, N., Prev Vet Med104,292-300. Efficient surveillanceofpigpopulationsusingoralfluids. M., Hoffmann, P., J.,2012. E.,Zimmerman, A.,Kurtz, Kurtz, K.J., Main,R.,Johnson,J.K.,Rademacher, C.,Hoogland, Ramirez, A.,Wang, C., Prickett, J.R., Pogranichniy, R., Yoon, . IntJSysBact42,469-473. by DNA-DNA hybridization experiments with byDNA-DNAhybridizationexperimentswith : identificationoftheregion responsible for Erysipelothrix Erysipelothrix

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• • • • • • • • • • in meat and abattoir samples in Western Australia. J Appl in meatandabattoirsamplesWestern Australia.JAppl of 2002. Thedetectionandrecovery Wang, Q.,Fidalgo,S.,Chang,B.J.,Mee,Riley, T.V., opathiae Wang, Q.,Chang,B.J.,Riley, T.V., 2010. 190-198. teins in canine serum. Vet Immunol Immunopathol 140, pro outersurface detection ofantibodiestoB.burgdorferi rescent bead-basedmultiplexassayforthesimultaneous Wagner, B., Freer, H., Rollins, A.,Erb,H.N., 2011. A fluo 133:543-544. JAmVetaid inthediagnosisoferysipelas. MedAssoc Vickers CL,Bierer BW. 1958.Triple sugariron agarasan Clin Vaccine Immunol17,1991-1997. protective immunityagainstchallengewithvariousserovars. Erysipelothrix Immunization withtruncatedrecombinant protein SpaC of To, H.,Someno,S.,Nagai,Koyama,T., Nagano,T., 2010. breaks inJapan.JVet MedSci74,949-953. siopathiae A., Nagano, T., 2012. Characterization of To, H.,Sato,Tazumi, A.,Tsutsumi, N.,Nagai,S.,Iwata, siopathiae protectivethe surface antigenproteins of To, H., Nagai, S., 2007. Genetic and antigenic diversity of animal tissues.JClinMicrobiol 37,4093-4098. Erysipelothrix Ohyama, T., 1999.Direct andrapiddetectionbyPCRof A., Nakanishi,K.,Oguma,Katoh,Y., Sunagawa,H., Takeshi, K.,Makino,S.,Ikeda,T., Takada, N.,Nakashiro, Microbiol 58,362-369. for discriminating Yamazaki, Y., 2006.Amultiplexpolymerasechainreaction Microbiol 92,844-850. simple detectionof amplification assay forrapidand loop-mediated isothermal Baba, Y., Shimoji,Y., Yamazaki, W., 2014.Developmentofa Yamazaki, Y., Oba,E.,Kashiwagi,N.,Sugita,K.,Shiiba, lothrix tonsillarum.JVet DiagnInvest18,384-387. . Vet Microbiol 140,405-417. strainsisolatedfrom recentout swineerysipelas . ClinVaccine Immunol14,813-820. sp.DNAsprepared from bacterialstrainsand rhusiopathiae Erysipelothrix Erysipelothrix strain 715 serovar 18 confers strain715serovar 18confers

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