TurkJVetAnimSci 29(2005)449-454 ©TÜB‹TAK ResearchArticle AvipoxvirusInfectioninQuails M.YavuzGÜLBAHAR DepartmentofPathology,FacultyofVeterinaryMedicine,OndokuzMay›sUniversity,Samsun-TURKEY MehmetÇABALAR DepartmentofVirology,FacultyofVeterinaryMedicine,HarranUniversity,fianl›urfa-TURKEY BanurBOYNUKARA DepartmentofMicrobiology,FacultyofVeterinaryMedicine,YüzüncüY›lUniversity,Van-TURKEY Received:23.10.2003 Abstract: Thepresentstudydescribesclinical,virological,histopathological,immunohistochemicalandelectronmicroscopicfindings ofpoxinfectionencounteredinaquailflock.Lesionsconsistedofsingleormultiplenoduleswithacrustinvariablesizeof grayto yellowordarkbrowndiscolorationonthecomb,eyelids,andtheotherpoorlyfeatheredareasofthebody.Histopathologicalchanges consistedofhyperplasticepitheliumoverlying,withballooningdegenerationofkeratinocytes,manyofwhichhadeosinophilic intracytoplasmicinclusionbodies(Bollingerbodies),andheterophilicinfiltrationsadmixedwithmononuclearcellsextendingi nto dermis.Avipoxvirusinfectionwasconfirmedbypositiveimmunostaininginthecytoplasmofaffectedcellsforpoxvirusantigen, excludednoimmunostainingofinclusionbodies,usingstreptavidin-biotinperoxidasecomplexmethodandbydemonstrationofbrick- shapedvirusparticleswithacentralcoreusingdirectelectronmicroscopy.Typicalthicknessofchorio-allantoicmembranes(C AMs) infectedwithvirus,comparedwithcontrols,wasobserved,andtheharvestedvirusonCAMswasdetectedaspositiveagainstknown fowlpoxvirususinggeldiffusiontest. KeyWords: Avipoxvirus,pox,quail B›ld›rc›nlardaAvipoxvirusEnfeksiyonu Özet: Buçal›flmada,birb›ld›rc›nçiftli¤indekarfl›lafl›lançiçekolgusununklinik,virolojik,histopatolojik,immunohistokimyasalve elektronmikroskopikbulgular›incelendi.Lezyonlaribik,gözkapaklar›,vevücudunk›ls›zbölgelerindede¤iflikbüyüklüklerde,gri-sar› veyakoyukahverengikabuklu,tekyadaçoksay›danodüllerdenibaretti.Histopatolojikolarak,deridekihiperplastikepitelde keratinositlerdebalonumsudejenerasyonvebuhücrelerdeeozinofilikintrasitoplazmikinklüzyoncisimcikleri(Bollingercisimci kleri) belirlendi.Ayr›cadermisekadaruzananmononüklearhücrelerlekar›fl›khaldeheterofilikhücreinfiltrasyonlar›gözlendi.Avipo xvirus enfeksiyonu,streptavidin-biotinperoksidazkompleksmetotkullan›larakyap›lanboyanmalardapoxvirusantijenietkilenenhücrelerin sitoplazmas›ndasaptand›.Elektronmikroskobikincelemedeise,merkezikorlu,tu¤lafleklindekiavipoxviruspartiküllerigörüldü . Viruslaenfektekorio-allantoikmembranlar(CAM)kontrollerlekarfl›laflt›r›ld›¤›ndatipikolarakkal›nlaflm›flt›veCAM’daüretilenvirus, geldiffüzyontestiilebilinenfowlpoxvirusakarfl›pozitifolarakbelirlendi. AnahtarSözcükler: Avipoxvirus,çiçek,b›ld›rc›n Introduction esophagus(diphtheriticform).Significantmortalityhas Avianpoxisanacutecontagiousdiseasecausedby beenobservedinquails,especiallywiththediphtheritic genusAvipoxvirus,includingfowl,turkey,pigeon,canary, forminvolvingtherespiratorytract(3). junco,quail,sparrow,andstarlingpoxvirusesinthefamily Avipoxvirusinfectionisrareinquails;however,the Poxviridae, andhasbeenreportedinawidevarietyof presenceofthediseasehasbeenreportedinvariousquail domesticandwildbirds(1-3).Thehostantigen-related speciesinsomecountries(5-8).Mostofthosestudiesare virusesknowntoaffectavianspeciesareingeneral ofwildbirdsinAmerica.However,therehasbeenno species-specific,butmayinfecthostofotheravianspecies detailedinformationaboutimmunohistochemical (3,4).Itisaslow-spreadingdiseasecharacterizedby detectionofviralantigeninnaturallyoccurringpoxvirus developmentofdiscretenodularproliferativeskinlesions infectioninJapanesequails.Thispaperdescribesclinical, onpoorlyfeatheredpartsofthebody(cutaneousform)or virological,histopathological,immunohistochemicaland fibrino-necroticandproliferativelesionsinmucous electronmicroscopicfindingsofnaturallyoccurring membraneofupperrespiratorytract,mouth,and poxvirusinfectioninaquailflockinVanprovince,Turkey. 449 AvipoxvirusInfectioninQuails MaterialsandMethods omittedandreplacedbyPBSfornegativecontrols.Tissue Birds. Intheyear2000,twentysick(no:8)ordead sectionsknowntoexpressthepoxvirusantigensserved (no:12)quails( Coturnixcoturnixjaponica )were aspositivecontrols. obtainedfromanoutbreakofpoxinfectionencountered Electronmicroscopy. Preparationwascarriedout inanegg-layingquailflockinVanprovince,Turkey. usingtheflotationtechnique.First,freshtissuesamplesas Approximately3000birdsofdifferentageswereinthe perthescabsforinoculationonCAMwerehomogenizedto flockandtheinfectionwasnotencounteredinyoung a40%suspensionindistilledwater.Eachmixturewas quailsorchicksseparatedfromothersickadultbirds. centrifugedfor20minutesat1000g.Acarbon-coated Necropsywasperformedonthebirdseuthanizedunder electronmicroscopegridwasfloatedonadropofthe etheranesthesiaorimmediatelyfollowingdeath.Tissue supernatantfor45minutes,removedandblottedwithfilter specimenswerecollectedfromvariousorgans,thenfixed paper,thennegativelystainedwith1%phosphotungstic in10%neutral-bufferedformalinsolution.Thetissues acidanddried.Thepreparedgridswereexaminedina wereembeddedinparaffin,sectionedat5µmandstained transmissionelectronmicroscope(Jeol100CX-II). withhematoxylinandeosin(H&E).Additionalfresh tissuesfromscabswerecollectedforvirologicalstudies. Results Chickenembryoinoculation. Scabscollectedfrom affectedquailsweretrituratedtomakea10% Clinically,sickbirdsshowedweightloss,decreased suspensioninphosphatebuffersaline(PBS).After eggproductionandimpairedfertility.Mortalityand centrifugationfor30minutesat2000g,thesupernatant morbidityinadultbirdsofthequailflockwere wasinoculatedat0.2mldosesonchorio-allantoic approximately20%and60%respectively.Thedisease membranes(CAMs)of10-day-oldchickenembryos.The wascharacterizedbythecutaneousformcomprising infectedCAMswereharvested7daysafterinoculation. smallmultifocaltocoalescingnodularlesionsinvariable sizeofgraytoyellowordarkbrowndiscolorationon Geldiffusiontest.TheharvestedvirusonCAMswas predominantlyaffectingthecomb,eyelids,andtheother suspendedinPBS,andthenthemixturewascentrifuged poorlyfeatheredareasofthebody.Whenthebrown for20minutesat1000g.Thesupernatantwastested wart-likescabscoatingtheextensivethicklesionswere forprecipitatingpotencyagainstknownpositivefowlpox virusantisera.Thetestwasperformedinplasticpetri removed,avariableamountofcaseousexudatesadmixed dishcontaining0.7percentagarnobleandexaminedfor withbloodwasfound.Manyofaffectedbirdshad lineofprecipitationafter48hours. unilateralorbilateralblindnessduetomildtosevere blepharitisandconjunctivitis(Figure1).Insuchbirds, Immunohistochemistry. Investigationforthe opacityofthecorneaduetokeratitiswasacommon distributionofavianpoxvirusantigenusingstreptavidin- finding.Somebirdshadbrowncrustypapulesatthe biotincomplexmethod(ABC)wasperformedwitha commissureofthebeakandaroundtheexternalnares.In commercialkit(ShandonInc.,Pittsburgh,PA,USA).The threecasesnecropsied,superficialsmallraisedwhite- techniqueincludedanenzyme(protease)pretreatment yellownodularlesionsathardpalateandthebaseof andalldeparaffinizedsectionswerequenchedfor tonguewerealsoobserved,butinvolvementofother endogenousperoxidasewith3%H2O2 inPBSandblocked visceralorganswasnotseeninanybirds. with5%normalgoatserumpriortoexposuretoprimary antisera.Thesectionsthenwereincubatedwithprimary Microscopically,theaffectedskin,featherfolliclesand rabbitpolyclonalantibodiesraisedagainstfowlpoxvirus conjunctivashowedvaryingdegreesofepithelial (PoultryDiseasesResearchandVaccineProduction hyperplasia,coveredbyanecroticmaterial,with Institute,Manisa,Turkey),diluted1:200inPBSfor4 ballooningdegenerationofkeratinocyteswithvacuolated hoursatroomtemperature.Theantiserawereprepared cytoplasmandintraepithelialvesicles.Numerous aspreviouslydescribed(9).Thesectionswereincubated degeneratedandenlargedkeratinocytes,especiallyinthe withthebiotinylatedsecondaryantibody,followedby epitheliumofskinandfeatherfollicles,contained streptavidin-peroxidaseconjugate,andthenvisualized eosinophiliccytoplasmicinclusions(Bollingerbodies)with with3-amino-9-ethylcarbazoleandcounterstainedwith centralpalezone(Figure2).Inthedermisand Mayer’shematoxylin.Theprimaryantibodieswere subconjunctiva,edemaandcongestionwithnecrosisand 450 M.Y.GÜLBAHAR,M.ÇABALAR,B.BOYNUKARA Figure1.Typicalpoxlesionscharacterizedbyred-brownwart-like Figure2. Hyperplasticanddegenerativechangesintheepidermallayer massesonsnoodandcombofaquail.Noteobstructionof coveredbynecroticmaterial.Arrowsindicateintracytoplasmic visioncausedbyacomplicatedeyelesionwithcaseous inclusionbodies.H&E,200X. material. predominantlyheterophilicinflammatorycellinfiltrations CAMswasdetectedaspositiveagainstknownfowlpox admixedwithmononuclearcellswereobserved.Inthe virususinggeldiffusiontest. birdswithcornealopacity,theanteriorchamberand limbuscorneawasinfiltratedwithheterophilleucocytes. Someofthebirdshadfocalmononuclearcellinfiltrations Discussion inthelungs,spleen,liverandproventriculus. Theclinicalsignsandhistologiclesionsobservedinthe Immunolocalizationofpoxvirusantigenseenasa affectedquailsweresimilartothoseinthepreviously diffuseorgranularbrick-redcoloringofthecytoplasm describedcasesofnaturalavipoxvirusinfectioninquails almostconfinedtotheepitheliumofskinandconjunctiva (5-8,10)andotheravianspecies(11-14).Lesion (Figures3,4).Positiveimmunostainingwereobservedin morphologyanddistributionweretypicalofavianpox, thecytoplasmofdegeneratedorexfoliatedcellsinthe withahighprevalenceoflesionsinvolvingtheskinofthe epidermis,featherfollicleandconjunctiva.Viralantigen head,presumablythepoorlyfeatheredareas.Inthe wasidentifiedoccasionallyinthecytoplasmof diphthericform,superficialsmallraisedwhite-yellow macrophagesinthedermisandinthesubconjunctival nodularlesionslimitedonthehardpalateandthebaseof