Author Index Presentation Abstracts · Conference of Research Workers in Animal Diseases Page 2 CRWAD 2017 Dedicatee: Katherine M. Kocan, PhD Regents Professor, Veterinary Pathobiology

©ConferenceofResearchWorkersinAnimalDiseases-2017

Author Index &

Presentation Abstracts

98th Conference of Research Workers in Animal Diseases

December 3-5, 2017

Chicago Marriott, Downtown Magnificent Mile Chicago, Illinois

Conference of Research Workers in Animal Diseases

ConferenceofResearchWorkersinAnimalDiseases Page1

ConferenceofResearchWorkersinAnimalDiseases2017Officers

President

PaulS.Morley,DVM,PhD,DACVIMProfessor,ColoradoStateUniversity

Vice-President

ChristopherC.L.Chase,DVM,MS,PhD,DACVMProfessor,SouthDakotaStateUniversity

CouncilMembers

QijingZhang,BVsc,MS,PhDProfessorandAssociateDean,IowaStateUniversityAmeliaR.Woolums,DVM,MVSc,PhD,DACVIM,DACVMProfessor,MississippiStateUniversityM.M.Chengappa,BVSc,MVSc,MS,PhDUniversityDistinguishedProfessor,KansasStateUniversityCharlesJ.Czuprynski,PhDProfessorandDirector,UniversityofWisconsin,Madison,WI

ImmediatePastPresident

LaurelJ.Gershwin,DVM,PhD,DACVMProfessor,UniversityofCaliforniaatDavis

ExecutiveDirector

DavidA.Benfield,DVM,PhDProfessorandAssociateDirector,OhioStateUniversity

ExecutiveAssistant

LorenD.HarperOhioStateUniversity

CRWAD2017 SPONSORS


CRWAD2017Dedicatee:

KatherineM.Kocan,PhDRegentsProfessor,VeterinaryPathobiology

OklahomaStateUniversityKatherineKocan,Ph.D.andOSURegentsProfessorEmerita,recentlyretiredfroma42-yearcareerattheCenterofVeterinary Health Science, Oklahoma State University where her research was focused on ticks and tick-bornediseases.Throughouthercareer,Kocanandherteamandgraduatestudentspublishedover325scientificpaperswhich included defining the role of ticks in transmission of the cattle diseases, anaplasmosis and heartwater,developmentofvaccinesagainstticks,definitionofmolecular interactionsbetweenticksandpathogens,andthedevelopment of a sheepmodel for studying tick transmission of the pathogen that causes human granulocyticanaplasmosis. SheearnedaB.A.degree (1968) fromHiramCollege inHiram,Ohio, anM.S.P.H. (1971) from theUniversityofNorthCarolinaatChapelHillandherPh.D.(1979)fromOklahomaStateUniversity.SheheldtheWalterR.SitlingtonEndowedChairinFoodAnimalResearchfrom1996-2016.KocanwashonoredwiththeDistinguishedAlumniAwardandtheJ.J.TurnerAlumniAchievementAwardsfromHiramCollege(1984,2010).ShewasnamedFellowoftheSocietyforTropicalVeterinaryMedicine(STVM)in2007andwastheDedicateeofthe9thBiennialmeetingofSTVM.ShewasaninvitedspeakerattheItalianSocietyofVeterinarySciencesSpecialSymposiuminPalermo,Sicily(2006)andtheConferenceofResearchWorkersonAnimalDiseases(CRWAD)(2010).SheservedasPresidentofseveralorganizationsincludingSTVM(1993),theOSUChapterofSigmaXi (1997), CRWAD (2003), and theOSU Regents Professors (2006). She served on theOklahoma Center of theAdvancementofScienceandTechnology,HealthResearchCommitteefor20yearsandwastheCommitteeChairfrom1998to2004.Kathyservedasafacultymentortomanystudentsandfaculty,includingNativeAmericansinBiologicalSciences,WomeninScienceandtheNSFAdvanceOSUProgram.ShereceivedtheInnovatoroftheYear“OntheBrink”AwardfromtheJournalRecordin2001andtheOSURegentsDistinguishedResearchAwardin2003.In 2006, Kocan received two Patent Recognition Awards. She was honored with the CVM Beecham Award forResearchExcellencein1986andthePfizerResearchAwardin1996and2010.

CRWAD2017 SPONSORS


ThankYou!

SilverSponsors:GoldSponsors:

BronzeSponsor:

CRWAD2017 AUTHORINDEX


Abdalaal,Hosein.....................P072Abdalla,Amina.......................P042Abdelsalam,Karim..................214,P112Abdi,RetaD............................136,P096Abdo,Zaid..............................21,40Abdul-Cader,M.S....................149Abdul-Careem,M.F.................148,149,P117Abrahante,JuanE...................200Abundo,MichaelC.................P104,210Aburu,GetachewT.A..............192Aceto,Helen...........................76Acharya,Ramchandra............P009Adams,RachaelJ....................23,24,25,P038Afshan,Kiran..........................173Agalarov,Dilgam....................P128Aguillella,VincenteM.............219Agulto,ReginaL......................122,196Ahmad,Karam........................87Ahmed,BegumS....................103Ahmed-Hassan,Hanaa...........148,149Ahn,HeeSeop.........................P061,P119Albers,AmyL..........................23,24,25,26,27Albert,Istvan..........................P066Alcaraz,Antonio.....................219Aldridge,Brian........................13,33,79,80Alekseev,KonstantinP...........P132Aliper,TarasI..........................P132Alkhamis,Moh........................97Allué-Guardia,Anna...............32Al-Mamun,M.A.....................P032Almeida,RaulA.......................P096Alt,David................................P003,P015Alusi,PhyllisM........................P045Alvarez,Julio...........................93Alvarez-Narvaez,Sonsiray......180Amachawadi,RG....................16,17,37,39Amadi,VictorA.......................163Amarasinghe,Aruna...............149Amimo,JoshuaO....................106,164Aminabadi,Peiman................90Anderson,Cameron...............21,22,34Anderson,Gary.......................56,75,187,188,P077,P100,

P118Angelos,JohnA......................122,196,P013Anis,EmanA...........................P071Anklam,Kelly..........................P041Antony,Linto..........................P092Apamaku,MichaelB...............64Apley,MichaelD.....................39,182Aqui-Salces,Milena................158Archer,Holly...........................P141

Areda,Demelash....................53,61,213Arenas-Gamboa,Angela.........191,P004,P006Ariza,Juan-Manuel.................P041Arndt,Emily............................P023,P024Arriaga-Pizano,Lourdes.........P095Arrondo,JoseL.R...................219Arroyo,Luis............................P141Arruda,AndreiaG...................97,P125Arruda,Bailey.........................75Arruda,Paulo..........................75Arthur,TerranceM.................8,88Arzumanyan,Anahit...............P067Auger,Jean-Philippe...............198,235,P022Aulik,Nicole............................28,195Avaliani,Lasha........................P049Awosile,BabafelaB................P037Azimov,Anar..........................P128Aznar,MariaNatalia...............83Babasyan,Susanna.................P083,238,239Babuadze,Giorgi....................P106Bai,Jianfa................................56,75,166,188,P077,P100,

P118Bailey,Michael.......................P091Bakhtawar,Khawaja...............149Bakkar,lutfy...........................P072Bakke,Brock...........................124Bakker,Henkden...................20Baldwin,CynthiaL..................133,134,145Ballash,GregoryA..................24,25,26Bandrick,Meggan...................P135Bannantine,JohnP.................P010,193Barletta,RaulG......................P010Baron,JeromeN.....................83Barr,Bonnie............................72BarreraVaca,MariaS.............74Bartens,Marie-Christine........141Bartlett,PaulC.......................57,58,59,P113Baruch,Joaquin......................179Bastos,ReginaldoG................170,P142Bauermann,FernandoV........111,236Baum,David...........................73,233,234Baumann,MaximilianP.O.....P129Bayles,DarrelO......................190,237,P003,P010Bearson,BradleyL..................P014Beauvais,WendyB.................6,8Becker,Tyler...........................60BedollaAlva,MarioA.............P089Beigel,Brittney.......................50,P126,P017Bekele,SisayG........................101Belk,KeithE............................3,21,22,34,40,88,181Beltrán-Alcrudo,Daniel..........84BenArous,Juliette.................110



Benitez,OscarJ......................59,P113Benítez-Guzmán,Alejandro....P095,P114Benn,Jamie............................P088Berggren,Keith.......................216,219Berghaus,LondaJ...................209,240Berghaus,RoyD.....................180Berke,Olaf..............................47Bernardo,Pauline...................114Bernardo,Theresa..................48Besser,ThomasE....................45Bett,BernardK.B...................66Bewley,JeffreyM....................154,P036Bhushan,Gitanjali..................230Bian,Ting................................217Bicalho,Rodrigo.....................P080Bird,BrianH...........................P042Bird,Ian..................................230Bishop,JeanetteV..................215Bishop,Richard.......................P142Biswas,Debabrata..................162Bittar,JoaoH.J.......................120Blair,Benjamin.......................96Blecha,Frank..........................237Boa,Anaïs...............................P022Boerlin,Patrick.......................165Boettcher,AdelineN..............135,P094Boggiatto,Paola.....................P003,P068Boisvert,Natalie.....................133Booker,CalvinW....................5,22Borca,ManuelV.....................216,219Borg,Sarah.............................P063Boucher,Christina..................21Bowman,AndrewS................P053,P099Boxler,David..........................77Bradley,Andrew.....................105Brandenburg,KennethS........P012Brault,StephanieA.................5,43,181,182Braun,Lyle..............................214Brierley,Ian............................225,226Briggs,RobertE......................253Brocchi,Emillia.......................219Brockmeier,SusanL...............P020Brost,BrianM.........................68Brown,AmandaL...................P012Brown,Emma.........................141Brown,Jennifer......................98Brown,Justin..........................233Brown,WendyC.....................144Brunelle,BrianW...................P014Brunner,Timothy...................P090Buck,Jessica...........................133Buckley,Alexandra.................220

Buist,Sterling.........................118Bujold,AdinaR.......................P019Bukachi,Salome.....................66,P045Burakova,Yulia.......................118Burgess,BrandyA...................76Burton,Brandi........................13Butcher,Marion.....................176Buza,Joram............................99,P066Buza,Teresia..........................P066Byrem,ToddM.......................57,58Byrne,BarbaraA....................P078Byrne,KristenA......................150Cabello,AnaL.C.....................P122Cabezas,AurelioH..................54,55Caldwell,Marc........................7,152Campigotto,Adam.................48Canal,ClaudioW....................252Cano,JeanPaul......................221Capps,KaylenM.....................166Carballo,Matilde....................P127Carrillo,CelenaA....................224,P111CarrisozaUrbina,Jacobo........P089Carroll,Laura..........................P080Carson,Carolee......................5,14,182Carter,Craig...........................42,P018Casas,Eduardo.......................151,P085Casey,Thomas........................91,P014Cattadori,Isabella..................P066Cazer,CaseyL.........................11Cerdeira,Louise......................P137Ceres,Kristina.........................P032Cernicchiaro,Natalia..............179,250Cesar,Fernanda......................P086Chae,Joon-Seok.....................P064Chain,Patrick..........................P001Chaki,SankarP.......................191,P004,P006,P088Chamba,Fabian......................74Chamorro,ManuelF..............250Chandrasekar,Shaswath........124Chandrashekhar,Kshipra.......P124Chang,Hsiao-FenG................212Chang,Yung-Fu.......................P016Chanturia,Gvantsa.................P001Charley,SaraE........................135,P094Chase,Christopher.................P112,214Checkley,SylviaL....................10,P130Chen,Fangzhou......................161Chen,WeiYu...........................117Cheng,Chuanmin...................171Chigerwe,Munashe................61,122,213Chitko-McKown,CarolG........141Cho,Eugene............................P099



Cho,JeongSang......................P021Cho,Ki-Hyun...........................102,P051Choi,Hwan-Won....................P110Choi,HwiYeon.......................P131Choi,InSoo.............................P061,P119,P131Choi,JongChul.......................P131Choi,Kyoung-Seong................P064Chothe,ShubhadaK...............230Chou,Pin-Hsing......................212Christmann,Undine...............P018,P126Christopher-Hennings,Jane...78Chung,Simon.........................212CinoOzuna,AdaGiselle..........118Cino-Ozuna,AdaG.................135Ciriacks,Jennifer.....................195Clack,Herek............................82Clapp,Beata...........................146Clarridge,AvaElainM............P074Clawson,Michael...................203Clifton,Rachel........................65Clothier,KristinA....................196,208Clow,KatieM..........................P141Coatney,John.........................P030,P031Comis,Catherine....................162Conner,JuliaG........................42Conte,CarlosA.......................P076Cook,Gericke.........................87Cook,WalterE........................P088Cook-Webb,Melissa...............P074Costa,RenataG......................P076Costard,Solenne....................181Cousins,MelanieM................104Coussens,PaulM....................138,155,156Couture,VictoriaL..................P036Cox,Madison..........................P041Cramer,MaryC......................P105Credille,BrentonC.................180Crespo,Rocio..........................P074Crossley,Beate.......................61,208,213Cubas-Delgado,Franklin.........2Culhane,Marie.......................93Cull,CharleyA........................179Cullen,Jonah..........................35Cunnick,JoanE.......................135,P094Curtiss,Roy.............................123Czuprynski,CharlesJ..............P012,P062Dai,Lei....................................P039Dam,VuiT..............................113Damani-Yokota,Payal............145Daniels,JoshuaB....................23,30,P038Dassanayake,RohanaP..........211,252Daugherty,Elizabeth..............175

Davenport,KristenA..............68Davern,Alec...........................P090Davidson,KellyE....................P078Davies,PeterR.......................32Davis,A.S...............................231Davis,MargaretA...................45Dean,Chris.............................21Deblais,Loic............................201,P136DeCastro,Cristina..................207Dego,OudessaKerro...............P096Dee,Scott...............................78Deering,Kathleen...................28DeFigueiredo,Paul.................P122DeHart,Jason.........................70DeJong,Jon.............................78Dekkers,JackC.M..................135DeKuiper,JustinL...................155delaArafa,Igor......................219delaTorre,Ana......................P047,P127Delay,Josepha........................98Delgado,MaríadelMar..........P127Delia,Grace............................66deLima,Marcello...................78,236Delmore,RobertJ...................88Denich,Leann.........................P023,P024Deressa,Asefa........................101Deringer,JamesR...................144Dewey,Cate...........................241,P029Dhagat-Mehta,Bakul.............176Dhakal,Santosh......................108,114,125Diamos,Andrew.....................P065Diaz-Goodwin,Zakia...............146Diel,DiegoG...........................78,111,236,P120Digianantonio,Rose...............P081Dillberger-Lawson,Steven......107,P120Ding,Jessica............................P033Dinkel,Kelcey.........................P142Do,HoaT................................113d'Offay,JeanM.......................204,205Donde,BalakoG.....................101Donohue,Kevin......................154Dopfer,Dorte.........................P041Doster,Enrique.......................21,22,88Dritz,Steve.............................39,78Droscha,CaseyJ.....................156,P113Drouillard,JamesS.................16,17,37Dryden,MichaelW.................177Duan,Qiangde........................P057Dubovi,EdwardJ....................205DugumaAbdi,Reta.................P011Dupuis,Laurent......................110Dzavashvili,Giorgi..................P001



Earleywine,Tom.....................P062Eberle,KirstenC.....................P020Eberle,Richard.......................204Eblate,Ernest.........................99,P066Ehiremen,Godsent.................P117Ehricht,Ralf............................137Eissa,Attia..............................P133Ekakoro,JohnE......................4,7Ekiri,Abel................................P042Elaish,Mohamed....................119,210,P099,P104ElEbissy,Eman.......................P133Ellis,John................................121Elmowalid,Gamal...................214ElZowalaty,MohamedE........106Ensermu,DestaB...................136,P096Ensley,Steve...........................P121Eppinger,Mark.......................32Erickson,Nathan....................10Erkkila,Tracy..........................P001Erol,Erdal...............................P018Erskine,RonJ..........................57,58Esperón,Fernando.................P127Espinosa-Castillo,Fernando...P114Estes,Anna.............................P066,99Evason,MichelleD.................P140Faccin,TatianeC.....................236Fakhr,MohamedK.................P073Falkenberg,ShollieM.............211,252Fan,Huihao............................P135Fang,Ying...............................56,75,114,187,188,223,224,

225,226,P077,P100,P111,P118

Farzan,A.................................P023Farzan,Vahab.........................P024Fedorka-Cray,Paula...............P137Feicht,SydneeM....................23,24,25Felippe,Julia...........................P097Fernandes,MaureenH...........111Fernandes,MiriamR..............P137Ferreira,FernandaC...............120Ficht,ThomasA......................P004,P088,P122Fioravante,Paulo....................97Firth,AndrewE.......................225,226Fishbein,Mark........................204Fisman,DavidN......................104Fitzsimmons,Daniel...............P003Flancman,Rebecca.................242Fleming,Damarius..................189,190,237Flynn,Katherine.....................19Foditsh,Carla..........................P080Forde,Taya.............................99Forsberg,Nikki........................247

Fox,Andrew...........................87Frana,TimothyS.....................32Francis,Ore............................P091Freer,Heather........................238,239,P083Frie,MeredithC......................138,156,P113Friendship,RobertM..............98,P023,P024Fry,LindsayM........................170,P142Fujita,Takashi.........................P107Fulton,RobertW....................204,205Furutani,Aina.........................P027Gaire,TaraN...........................12Galligan,David........................2Gamsjaeger,Lisa.....................206Ganta,RomanR......................171,174Garcia,Carolina......................112,245Garcia,Daniel.........................191GarciaGonzalez,DanielG......P004,P006GarridoMantilla,Jorge...........93Gauger,PhillipC.....................P098Ge,Xinna................................217Geary,Emily...........................97Gebhart,Connie.....................158,200Gebrehiwot,Tadesse..............53Gehring,Ronette....................98Gentry-Weeks,Claudia...........43Geornaras,Ifigenia.................34,40Gerlach,S.C...........................P130Ghanem,MohamedM...........P133Ghimire,Shristi.......................108Ghorbani,Amir.......................210,P104Gibson,AmandaJ...................141Giguère,Steeve......................180,209Gilbert,JackA.........................1Gill,Ed....................................P066Gillespie,Alexandria...............133,145Gillespie,Barbara...................136,P011,P096Giménez-Lirola,Luis...............70,73,222,233,234Gisch,Nicolas.........................198Githae,Dedan.........................106Gladue,DouglasP...................216,219Go,HyeonJeong.....................P061,P119Gogin,Andrei..........................P047Gogoladze,Giorgi...................P001Gong,Wenjie..........................118Gonzales,AmandaM.............P065Gorden,Patrick.......................P030,P031Goss,Ryan..............................P126Gottschalk,Marcelo...............198,235,P022Gourapura,R..........................114Gow,SherylP.........................5,10,22,121,182Grady,Shannon......................P090Grant,RickJ............................153



Gray,Darren...........................121Grebennikova,TatianaV........P132Green,LauraE........................65,105Green,Martin.........................105Greenlick,Ashley....................156Greer,AmyL...........................85,86,104,P070GreinerSafi,Amelia................6Gresch,Sara............................74Grinkova,Yelena.....................117Gröhn,YrjöT..........................11,P010,P032Grooters,SusanV...................30Gu,Suna.................................P101Guerra-Maupome,M.............140Gunaskekera,Umanga...........P129Guo,Baoqing..........................220,P121Guo,Rui..................................224,P111Guo,Xin..................................217Guptill,Linn............................P054Gutierrez,Alyssa.....................120GutierrezPabello,JoséÁ........P089Gutman,Sarah........................P054Gwakisa,Paul.........................P066Haan,JisunS...........................32Habing,Greg...........................P081Habitu,Takele........................53Haddad,Monica.....................95Hadi,SyedaA..........................P008Hain,KyleS.............................111Haines,DeborahM.................250Hakobyan,Naira.....................P067Hall,Alexandra.......................169Hamonic,Glenn......................247Hamrick,Brianna....................120Han,Jun..................................217Han,SangHoon.......................P061,P119Hannon,SherryJ....................5,22Hansen,ThomasR..................215Harding,JohnC.S...................241,P029Hariharan,H............................163Harhay,Gregory.....................190Harmon,KarenM...................P098Harre,Shay.............................43Hash,Kody...............................P096Hassan,AhmedO...................109Hassebroek,AnnaM..............52Hau,SamanthaJ.....................32,P020Hause,Ben..............................223Havenga,LourensJ.................120Hazlett,Murray......................P141Headrick,SusanI.....................P096Heaton,MichaelP..................141Heider,LukeC........................P037

Heller,MeeraC......................206Helmy,YosraA.......................201Henao-Diaz,YulyA.................67Hennessy,Sean......................2Henningson,Jamie.................118Hensel,MarthaE....................191,P006Hernández-Medrano,J...........P114HernándezPando,R...............P089Herrmann,JohnA...................62Hesse,Richard........................187,188Hetman,BenjaminM.............15Hill,AshleyE...........................P055HoangThiThu,Ha..................100Hoffman,Carol.......................146Hoffman,Kyle.........................176Hogshead,BradleyT...............108,114Holder,Angela........................141Holinka,Lauren......................216,219Holman,DevinB.....................P014Holzer,KatlynL.......................34,40Honkawa,Kazuyuki................P034Hoogland,Marlin....................95Hoover,EdwardA...................68Horii,Yoichiro.........................P034Horohov,DavidW..................P086Hostetter,JesseM..................193,194Hou,Yixuan............................157Houston,Elizabeth.................222Hoyos-Jaramillo,A..................120Hsieh,Ching-Lin......................P016Hsu,Haoting...........................133Hu,Hui....................................P053Huang,Jiachen.......................112Huang,Jin...............................217Huang,Yanyun.......................248Huarte,Nerea.........................216Hudgens,Edward...................133Hudson,Peter.........................P066Huebner,KateL......................34,40,88Huisman,Dianna....................P082Hunter,Joseph.......................P065Hur,Jin....................................P021Hurley,David..........................120Husmann,Robert...................117Hutchinson,Holden................57,59Hwang,Sunyoung...................P124Ibrahim,P.A.S.B.......................230Iglesias,Irene..........................P047,P127Im,YoungBin.........................147,P002Imanishi,Maho.......................P025Imnadze,Paata.......................P001,P063,P106Inbody,MattH.......................243



Inzana,ThomasJ....................207Isaacson,Richard....................200,P057Ivanek,Renata........................6,8Jaberi-Douraki,Majid.............55,183Jackson,Ron...........................P128Jacob,MeganE.......................81Jaing,CrystalJ.........................224,P111James,Angela.........................87Janecko,Nicol.........................29,89Jang,Guehwan.......................P108Jang,Hyesun...........................119,210,P104Jang,Hyun..............................249,P109Jang,Jinjoo.............................P109Jardine,ClaireM.....................29,89,251Jashiashvili,Tamar..................P063Jayarao,Bhushan....................230,P066Jay-Russell,Michele................90Jennings,Rachel.....................8Jenvey,CaitlinJ.......................193,194Jeon,Byeonghwa....................P124Jeon,JiHyun...........................228Jeong,Chang-Gi......................P101Jeong,JiYun...........................P131Ji,Ju........................................78,233Jiang,Yin.................................74Jin,Duo...................................245Jittapalapong,Sathaporn.......176Johnson,Gayle.......................176Johnson,Ron..........................98Johnson,TimothyA................243Johnson,TimothyJ.................210,P104Johnson,Wendell...................P142Jones,Cassandra....................78Joseph,Asteria.......................P066Joshi,LokR.............................111,236Jung,Chung-Sik......................102,102Jung,Hyeim............................P058Kairu-Wanyoike,Salome........66Kalbfleisch,TheodoreS..........141Kaltenboeck,Bernhard...........137Kamrud,Kurt..........................70Kang,YeongLim.....................P131Kapur,Vivek............................99,P066Karanja,Joan..........................66Karczmarek,Aneta.................P077Karki,AnandB........................P073Karsky,Jenna..........................P028Kartskhia,Natia......................P049Katani,Robab.........................99,P066Kathayat,Dipak......................201Katwal,Pratik.........................P092Kaushik,RadheyS...................P092

Kawabata,Tadahiro................P027Kazwala,RudovickR...............P042KC,Mahesh.............................210,P104Ke,Hanzhong..........................227,P107Kehrli,Marcus........................190Keller,Margo..........................230Kenney,Scott..........................P091Kenworthy,Amanda...............P135KerroDego,Oudessa..............136,P011Kesterson,ClayB....................152Khaitsa,MargaretL................P048Khalaf,Omar...........................P004Khatibi,Masud.......................P128Khatun,Amina........................P101Kilonzo,Christopher...............P042Kim,Beomseok.......................P101Kim,CharnWoon...................P131Kim,DongHwi.........................P119,P061Kim,Hyeon-Cheol...................P064Kim,Hyun-Joong.....................P121Kim,OanhT............................P025Kim,Pil-Won...........................P051Kim,Seong-Hee......................P108Kim,Sharron...........................P038Kim,Suji..................................197Kim,Suk..................................147,P002Kim,WonKyong.....................P021Kim,Won-Il.............................P101Kim,YongHyun.......................P119,P061KimThuy,Oanh......................100Kino,Erina..............................P034Kirkpatrick,Brian....................138Kish,Daniel.............................50Kitts-Morgan,Beth.................P126Klos,Rachel............................28Knapek,KatieJ........................215Knowles,DonaldP..................170,P142Knutson,Todd........................74,161Kolakowski,Jeannine..............141Kolvasov,Denis.......................P047Kolyvushko,Oleksandr...........P120Konnai,Satoru........................144Koziol,Jennifer.......................P058Krakowka,Steven...................108Krawczel,PeterD...................152,153,154,P036Kreuder,Amanda...................P059Kreuger,Kurt..........................10Kropinski,AndrewM..............P019Krull,Adam.............................P030,P031Krunkosky,Madelyn...............229Kuchipudi,Suresh...................230Kuehn,Larry...........................203



Kuhn,Jens...............................225Kukielka,Esther......................84Kull,JessieA...........................154Kulow,Megan.........................P041Kulshreshtha,Vikas................220Kumari,Rashmi......................109Kung'u,MathewM.................P007Kupiec,Caitlin.........................158Kuruppu,Kaushalya................P023,P024Kutz,SusanJ...........................P130Kyriakis,ConstantinosS..........229,P116LaBresh,Joanna......................P091Lacoste,Stacei........................250LaDronka,RebeccaM.............58Lager,Kelly.............................190,220Lai,Ken...................................248Lakin,StevenM......................22,40Lakshmanappa,Y.S................108,125Landgraf,Mariza.....................P137Lang,Yuekun..........................P100Lankin,StevenM....................21Largo,Eneko...........................216,219Larsen,MichelleH..................140Larson,Laurie.........................P093Laskowski,Marek...................86,P070Laurin,Emilie..........................P052Lavagna,Agustina...................235Lawhon,SaraD.......................20,37,46Lawson,Steve.........................236Lechtenberg,KellyF...............179LeCuyer,TessaE.....................45Lee,AmandaR........................153Lee,BeomJoo........................P131Lee,Chang-Hyung...................P051Lee,Chang-Won.....................108,119,210,P099,P104Lee,Changhee........................228,P108,P110Lee,JinJ..................................P043Lee,Jiyoung............................24,25,P079Lee,JoongBok........................P061,P119,P131Lee,Kwang-Nyeong................102,P051Lee,Kyoung-Ki........................P108Lee,Pei-YuA...........................212Lee,SangIn............................77Lee,SangWon........................P061,P119,P131Lee,Sang-Myeong..................P101Lee,Sera.................................227Lee,Seung-Chul......................P110Lee,Seungjun.........................24,25,P079Lee,SoHyun...........................P131Lee,Sunhee............................P110Lee,Won-Jin...........................102Leighton,PatrickA..................251

Leistikow,Devan....................P093Leite,FernandoL.L................200,P057LeonMoreno,IngridM..........46LePage,Lauren......................133Lescun,Timothy.....................P090Levings,RandallL...................132Levy,Michael..........................2Lewis,GentryL.......................71Li,Chengyao...........................P005Li,Ganwu................................P098Li,Jinfeng................................P005Li,Lingling...............................P066Li,Xiangdong..........................237Li,Yanhua...............................225,226,P100,P118Lim,Levina..............................230Lima,Svetlana........................P080Lincopan,Nilton.....................P137Lindahl,Johanna.....................66Lindsay,LeslieR......................251Line,JohnE.............................243Lippolis,JohnD.......................P014,P085Liu,Huitao..............................171,174Liu,Rui....................................P019Liu,Xuming.............................56,75,187,188,P077,P100,

P118Loest,Daleen..........................182Logue,CatherineMary...........201Loneragan,GuyH...................20Looft,Torey............................91,243Lopez,Franklin.......................P075López-Reyes,YahelF..............P095Lorentz,Ben...........................P136Loving,CrystalL......................135,150,P020Lowe,James...........................13,33,79,80,96Loy,JohnDustin.....................203Lu,Nanyan..............................P100Lu,Ti.......................................160,245,P056Luczo,JasminaM....................229Ludemann,Larry.....................202Ludwig,Justin.........................166Lunney,Joan...........................P091Lyashchenko,Konstantin........P008Lyimo,Beatus.........................99,P066Lyons,LeeA............................49Ma,Wenjun............................P100Ma,Xiaoping...........................P039Macaluso,KevinR..................178Machado,Gustavo.................97Machuka,EuniceM................164MacInnes,JanetI....................P019MacKinnon,MelissaC............14MacLachlan,N.James............218



Madera,Rachel......................118Madsen-Bouterse,Sally..........P142Maeroff,JacquelineE.............58Maggioli,MayaraF.................236Magiri,RoyfordB....................248Magtoto,Ronaldo..................222,233Magzamen,Sheryl..................87Mahrous,Ayman....................P072Maichomo,Monica................P045Main,Rodger..........................73,95,233,234Makingi,George.....................P042Mandzyuk,Boguslav...............122,196Manirarora,Jean....................P091Mar,Kaitlin.............................P073Marabella,Ian........................82Markazi,AshleyD...................125Marthaler,Douglas.................74,161Martin,Andimile....................99,P066Martin,JenniferN..................3,34,40Martin,MIchael......................87Martínez-Chavarría,Luary......P114Martínez-López,Beatriz.........83,84Mason,Hugh..........................P065Mathiason,CandaceK............68Mathys,DimitriaA..................23,24,25,26,27,30,P038Mazet,JonnaA.K...................18,P042McAllister,TimA....................22McAnulty,JonathanF.............P012McClenahan,David.................P082McClure,JT............................P037McConnico,RebeccaS...........76McCool-Eye,MaryJane..........87McDaneld,Tara......................203McEwen,Scott........................14McFarland,Kim......................P041McGill,JodiL...........................140,143McVey,WalterR.....................P066Meekins,DavidA....................231Megahed,Ameer....................13,79,80MehdizadehGohari,Iman......165Mellata,Melha.......................38,126,159Mem,Andressa......................P137Mendez,Jonatan....................P087Menshawy,Ahmed.................P134Menteshashvili,Ioseb.............P049Merrill,Caitlin.........................136,P011,P096Metcalf,JessicaL....................34,40,88Meyer,DamienF....................P122Meyer,EllaL...........................243Meyer,Roberty......................51Michel,Pascal.........................251Miller,Kayla............................120

Miller,KristinaM....................P052Miller,LauraC........................189,190,237Milwid,Rachael......................P070,86Minamino,Tomoya................P034Mishin,AlexanderM..............P132Misra,Saurav..........................223Mitchell,Jeffery......................175Mitchell,MontessaM............P115Mittal,SureshK......................109Mkrtchyan,Hovhannes..........P044,P067Mmbaga,Blandina.................99Mohamed,Abdelrahman.......P115Mohamed,Hussein................101MohamedAbdul-Cader,MS...148,P117Mohammed,Rasha................P133Molinaro,Antonio..................207Mollenkopf,DixieF.................23,24,25,26,27,30,P038,

P079Monaghan,Emma..................105Montanye,Mary.....................P041Monte,DanielF......................P137Monterubianesi,Mariela........83Montes,Fernando..................P047Moon,JaYoung......................P021Mooney,MarkH....................121Moore,Alison.........................P141Moore,George.......................P054,P090Mora,JuanC...........................73MoraDíaz,JuanCarlos...........234Morales,JessicaY...................P075Morales-Aguilar,Aldo.............P095MoralesSalinas,Elizabeth......P089Moreno,MiguelÁngel............P127Morgan,Joey..........................50Mørk,Torill.............................P013Morley,PaulS.........................3,5,21,22,34,40,43,76,88,

181,182Moroni,Paolo.........................6Morozov,Igor.........................232Morrison,Robert....................97Mosha,FaustaS.F..................99,P066Mosher,BrittanyA.................68Mou,KathyT..........................P020Moxley,RodneyA...................P056,71Mueller,Adam........................200Muellner,Petra......................97Mugisha,Lawrence................P048Muirhead,Sarah.....................P040Müller,Elke............................137Muller,HansC........................16,17Mulvey,MichaelR..................29Muñoz-Vargas,LohendyM....P081



Munuo,Lydia..........................P066Murphey,Kelly.......................P126Murphy,SeanC......................170Murray,SarahA......................37Murtaugh,Michael.................161Musser,Robert.......................P062Mutwiri,George.....................248Muwonge,Adrian...................53Mwatondo,AthmanJ.............P007Nader,Paul.............................P126Nagaraja,KakambiV..............163,P136Nagaraja,T.G.........................37,39,71,92,166,P077Nagy,Eva................................P117,149Naidoo,Vinny.........................44Nakatake,Shingo....................P027Nally,Jarlath...........................P015Nandre,RahulM....................P057Natradze,Ioseb......................P106Navarro-Gonzalez,Nora.........90Nazki,Salik..............................P101Neely,Megan.........................P141Neill,JohnD............................252Nelson,EricA.........................78,111,107,P028,P084,P120Ng,Siew..................................246Ng,Victoria.............................169Ngo,HoaT..............................P025Ngunjiri,JohnM.....................119,210,P099,P104Nguyen,DangH......................113Nguyen,HangT.T..................P123Nguyen,ThoD........................113,P103Nicholson,TracyL...................32,199,P020Nickell,Jason..........................179Niederwerder,Megan............78,P111Nietfeld,Jerome.....................118Nieva,Jose..............................216,219Ninidze,Lena..........................P049Nisar,Muhammad..................163,P136Nissly,Ruth.............................230Njenga,Kariuki.......................P007Njeru,Ian................................66Njuguna,JoyceN....................164Noh,Yun-Hee.........................P110Nolan,LisaK...........................201Noll,Lance..............................P077,P100Norby,Bo................................20,57,58,59,60Norman,KeriN.......................20,37,46,P075Noroy,Christophe..................P122Norton,Natalie.......................120Noyes,NoelleR......................21,22Nyamwaya,Doris....................66Nymo,IngebjørgH.................P013Oakes,MichaelJ.....................P050

Oakey,ClarkK.........................P073Obradovic,MilanR.................246O'Connor,Annette.................9,35Odemuyiwa,SolomonO.........P115,P048Odoi,Agricola.........................41,42,44Ogden,NicholasH..................251Ogunrinu,OJ..........................17Oguttu,JamesW....................41,44Oh,MyungWhan...................197O'Hara,Bruce.........................154O'HaraRuiz,Marilyn...............49Ohta,Naomi...........................20Okafor,ChikaC.......................4,7Okagawa,Tomohiro...............144Okda,Faten............................111Okoth,Edward........................106,164Oliver,S.P................................P096Ollivett,TheresaL...................P033,P105Olsen,Renee..........................202Olsen,Steven..........................P003,P068Olson,Bernard........................82Ontiri,Enoch...........................66Opiyo,StephenO...................P081Orinde,AustineB...................P007Osgood,Nathaniel..................10Osorio,Fernando....................116Osorio-Gallardo,Tzayhri.........P114Osoro,Eric..............................P007Osterrieder,Nikolaus.............238O'Sullivan,TerriL....................47,85,86,98,241,P029,P070Oswald,RobertE....................P016Ou,Bingming..........................245Ouyang,Ben...........................43Pabilonia,KristyL...................76Paim,FrancineC.....................P053Palmer,Michael......................165Palmer,MitchV......................140Palomares,RobertoA.............120,P116Palomino-Tapia,Victor...........149Pang,Jinji................................P039Pang,Michelle........................10Pannone,Stephen..................43Panzenhagen,PedroH.N........P076Park,Bae-Keun.......................P064Park,ByungJoo.......................P061,P119Park,JieYeun.........................P098Park,Jinho..............................P064Park,SeungYong....................P061,P119,P131Parker,JenniferK...................21,22,34,40,88Parmley,E.J...........................14Parrillo,Matthew...................P069,P102Pascual,DavidW....................146



Pasternak,J.Alex....................246,247Pastrana-Negron,Norimar.....P115Pasupuleti,Vijai......................P077Patrick,Kristin........................P122Patterson,Gilbert...................78Patterson,Laura.....................90Patyk,Kelly.............................87Paudel,Sandhya.....................P086Paul,Goodluck........................P042Paul,NarayanC......................P060,P074,P076Pavlovic,Nada........................P040Pearl,DavidL..........................14,15,29,47,89,251Peddireddi,Lalitha..................75,187,188,P100,P118Peisker,Madlen......................137Pelle,Roger............................164Peng,Mengfei........................162Pereira,RichardV...................P078,P080Perez,AndresM.....................97,P047Perkins,Gillian........................238Perri,AmandaM....................241,P029Petruzzi,BrianaL....................207Pham,ThaiQ..........................P025PhamQuang,Thai..................100Phebus,Randall......................166Phophi,Lufuno.......................44Pierce,StevenJ.......................60Pieters,Maria.........................P026Pighetti,GinaM......................152,153,154,P036,Pilch,HannahE.......................P062Pillatzki,Angela......................107,P084,P120Piñeyro,PabloE......................222,P098Pires,AldaF.A........................90Pithua,Patrick........................P048,176Plummer,Paul........................P030,P031,P039,P059Pluske,John............................39Pogranichniy,RomanM.........P054Poljak,Zvonimir......................48,85,86,P023,P024,P029,

P070,P132Pollakova,Jana.......................P063Poonsuk,Korakrit...................70,73,233,234Porlsen,Elizabeth...................56,187,188Poulsen,Elizabeth..................P077,P100Poulsen,Keith.........................195Powell,EllisJ...........................135,P085,P094Prescott,JohnF......................165Prosser,Diann........................87Proudfoot,KatyL....................154Ptak,ChristopherP.................P016Pugh,RobertaA......................37Punyapornwithaya,V.............P129Purdy,KevinJ.........................65,105Qekwana,DanielN.................44

Qekwana,NeneneD...............41Qian,Winchell........................10Qu,Bingming..........................112Raabis,SarahM......................P033Rademacher,Chris.................233Raev,SergeiA.........................P132Rahman,Kh.Shamsur............137Rahman,Md.S.......................103Rajashekara,Gireesh..............201,P124,P136Ramamoorthy,Sheela............107,P028,P084,P120Rambo,Zachary......................200Ramirez-Medina,E.................216,219Ransburgh,Russell.................P077,P100Rathnaiah,Govardhan............P010Redding,Laurel.......................2Reddy,P.G.............................P115Redweik,GrahamA.J.............38Regalado,M.H........................P095Register,KarenB....................211Reicks,Darwin........................221Reid-Smith,Richard................15,29,89Reinhardt,TimothyA.............P014,P085Remfry,SarahE......................39Rendahl,Aaron.......................200Renter,DavidG......................179,P077Rentsch,Dennis......................P066Renu,Sankar...........................108,114,125Renukaradhya,G....................108,125,P091Reppert,EmilyJ......................250Resende,Talita.......................158Reyes-Velez,Julian.................31,P037Reynolds,KristenJ..................98Rhea,Meridith........................P055Rice-Ficht,Allison...................P088Richardson,Jodi.....................51Richt,JuergenA......................231,232Ridpath,JuliaF.......................252Risatti,Guillermo....................216Rius,AgustinG........................P036Riwa,Carolyn..........................99Roberson,Jerry.......................P126Robinson,LaceyA...................250Rodrigues,DaliaP...................P076Rodriguez,Adriana.................120Rodriguez-Lecompte,J.C........31,P037Rogers,AnnaT........................81Roland,KennethL..................P065Rollins,Alicia...........................238Rossano,Mary........................72Rossi,Gianluigi.......................62Rossow,Stephanie.................221Rotolo,Marisa........................95



Rovira,Pablo..........................21,181Rowland,Bob.........................78Rowland,RaymondR..............135,P111Roy,David...............................198,235Ruddell,Brandon....................P059Ruggiero,VickieJ....................59,P113Ruiz,Jamie..............................21Ruiz,NinoshakalyF.................108Ruple,Audrey.........................52Rusconi,Brigida......................32Ryu,SangryeolRyu.................P124Saab,MathewE......................P037Sacco,RandyE........................P014Sachse,Konrad.......................137Sadeghieh,Tara......................169Sahin,Orhan...........................P039Saif,LindaJ.............................157,167,P053Salaheen,Serajus...................162Salam,Sameerul.....................P101Salzbrenner,Holly..................P082Sammel,Mary........................2Samson,Lyimo.......................P066Samuel,James........................191Sanchez,Javier.......................31,P037SanchezRomano,Javier.........P013Sanderson,MichaelW............54,55,92Sang,Yongming......................237,P091Sanhueza,Juan.......................97Sargeant,JanM......................35,36,104,169Sargent,Ron...........................250Sargsyan,Lilit..........................P046Sarmento,Luciana..................73,234Sarmiento-Silva,RosaE..........P095Sasaki,Yosuke........................P027,P034Sayedahmed,EkramyE..........109Scallan,Elaine.........................3Scaria,Joy...............................P136Schilling,Megan.....................P066Schissler,Jennifer...................43Schleining,Jennifer.................P059Schmidt,JohnW.....................88Schnabel,ChristianeL............238,239,P083Schnee,Christiane..................137Schneider,LieselG.................71Schubert,Evelyn.....................137Schultz,Kelly...........................P041Schultz,Ronald.......................P093Scoles,GlenA.........................144Scott,H.Morgan....................12,16,17,20,37,46,P075Sebola,Dikeledi......................41Sechrist,EmilyK.....................24,25,26,P079Seger,HannahL......................92

Segura,Mariela......................235Sellers,Laura..........................12Selvaraj,Ramesh....................125Severson,Emily......................195Seyidov,Tural.........................P128ShaanLakshmanappa,Y.........114Shah,DevendraH...................P060,P074,P076Shahzad,Sammuel.................175,176Shang,Pengcheng..................P111,114,223,224,226Shanmugasundaram,R...........125Shapiro,Michael.....................6Sharif,Shayan.........................149Sharma,S.................................163Shatek,Madison.....................P082Shearer,Jan............................P030,P031Shen,Zhenyu..........................175Shepherd,Frances..................161Shi,Jishu.................................118Shi,Xiaorong...........................39,166,P077Shikhiyev,Mazahir.................P128Shim,Soojin............................147,P002Shirima,Gabriel......................P066Shivley,Jacob.........................51Shrestha,S.P..........................P009Shure,Andrew........................P019Sidamonidze,Ketevan............P001Siebert,LydiaJ........................P036Siler,Julie................................P080Sill,Miranda............................230Silveira,Simone......................252Singer,Randall........................P026Singh,Ameet..........................242Singh,Gagandeep...................107,P028,P084,P120Singh,Kritika...........................P039Singh,Pankaj..........................107,P028Singrey,Aaron........................78Skinner,BrandonM................81Skjerve,Eystein......................53Sligar,SteveG.........................117Slovis,NathanM.....................76Smith,AmandaM...................P125Smith,DavidR........................51,63,71Smith,Edward........................105Smith,Gary.............................2Smith,Jacqueline....................42Smith,RebeccaL....................49,62Smith,Tim..............................134Smith,Woutrina.....................P042Smola,ChristopherCarl..........P126Snijder,Eric.............................225Snyder,EmilyR.......................180Sobraske,Jacob......................214,P112



Sockett,Donald......................28Sodergrath,Travis..................87Soh,SangHee.........................147,P002Song,ChangSeon...................P061,P119,P131Sordillo,Lorraine....................60Sørensen,KarenK..................P013South,David...........................87Spangler,Dawn......................50Spence,Kelsey........................85Sporer,KellyR.B....................138Spronk,Gordon......................78Sreevatsan,Srinand................P008Srivastava,Vishal....................P124Stabel,JudithR.......................193,194,P010Stacey,Deborah.....................47,48Stahl,Chase............................P118Stanley,Sarah.........................120Steffen,David.........................116Steinman,Margaret...............P018Stenkamp-Strahm,Chloe........87Stich,RogerW........................175,176StillBrooks,Kelly....................P039Stoffel,Ryan...........................176Stomeo,Francesca.................P066Stone,BradC..........................170Stone,Elizabeth......................48Stoute,Simone.......................208Stover,SusanM......................P055Straka,Kelly............................175Stranahan,LaurenW..............P006Strand,ElizabethB.................7Stromberg,ZacharyR.............159Strouss,Samantha..................72Stuever,DavidB.....................23,24,25Stull,JasonW.........................P125Sturos,Matthew.....................221Suen,Garret...........................P041Sukhiashvili,Roena.................P063Suleymanova,Chichak............P128Sullivan,Mazeika....................26Sumaye,RobertD...................P042Sun,Haiyan.............................116Sun,Yaxuan............................95Sundberg,Paul.......................78Sung,Dajung...........................P109Susta,Leonardo......................243Swirski,AlexandraL................47Sylte,MatthewJ.....................243Symonds,Dan.........................26Szymanski,Christine...............185Tabashsum,Zajeba.................162Tabatabai,LouisaB.................253

Taboada,EduardoN...............15,89Tadeo,JoséLuis......................P127Talaat,AdelM........................124Tang,Yizhi...............................P039,P040Tark,DongSeob.....................P021Tatum,FredM........................253Taxis,TasiaM.........................151Taylor,Daniel..........................3Taylor,EthanA.......................16Taylor,Sandra.........................P090Telfer,JaniceC........................133,134,145Temeeyasen,Gun...................222Tevdoradze,Tea.....................P001Thacker,TylerC......................P014Thakur,KrishnaK....................P052Thomas,KevinM....................88Thomas,Milton......................P092Thompson,Dana....................175Threadgill,Deborah................46Thumbi,SM............................P007Timsit,Edouard......................184To,ThanhL.............................113,P103Toepfer,Jeri............................P135Tokach,MichaelD..................39Tokach,Mike..........................78Tomaselli,Matilde..................P130Tomlinson,JoyE.....................P097Tompkins,S.M.......................229,P116Tonui,Triza.............................P066Torremorell,Montserrat........82,93Torrison,Jerry.........................200Toth,Mindy............................202Totton,Sarah..........................35Tran,HangT.T.......................P123TranNhu,Duong....................100Trinh,TrungT.........................P123Trujillo,JessieD......................231,232Tryland,Morten.....................196,P013Tsai,Chuan-FuM....................212Tsai,Yun-LongD.....................212Tu,Changchun........................118Tucker,Strahan......................P052Tuggle,ChrisK........................P094Tuggle,ChristopherK.............135,150Tuo,Wenbin...........................P087Ueti,Massaro.........................144,P142Ugarte-Ruiz,María.................P127Uprety,Tirth...........................P092Urbaniak,Kinga......................231,232Urdaneta,Jose........................120Urushadze,Lela......................P106Uzal,FranciscoA.....................P055



ValerisChacin,RobertJ...........P026Valley,Ann..............................28VanBibber-Krueger,C.L.........16,17,37VanCampen,Hana.................215VanderLey,Brian...................77Vanderstichel,Raphaël...........P052VanderWaal,Kimberley..........97VandeWalle,Gerlinde...........P097vanGeelen,AlbertG..............115,220Vannucci,Fabio......................74,158,200,221VanWormer,Elizabeth...........P042Varley,LisaM.........................P094Vasquez,Erika........................200Vats,Ashutosh........................141Vaughn,Jacqueline.................136,P011,P096Velasco-Villa,Andres..............P106Verkhovsky,OlegA.................P132Verma,Ashutosh....................50,P017,P018,P126Versillé,Nicolas......................110Vilalta,Carles..........................97Vimonish,Rubikah..................P142Vinasco-Torres,Javier.............20,37,P075ViscosaBauermann,F............78Vlasova,AnastasiaN..............P053Vogt,Luke...............................43Vogt,NadineA........................29,89Volkova,VictoriyaV................11,12,54,55,183Voronov,Andriy.....................P084Vrentas,CatherineE...............P003Vrentas,Cathy........................P068Vu,Hiep..................................116Waack,Ursula.........................199,P020Waddell,LisaA.......................169Wagner,Bettina.....................238,239,P083,P097Wainaina,Martin....................66Waldner,Cheryl......................10Walker,Angela.......................202Walker,TateBradley...............P096Wall,Savannah.......................120Walz,PaulH............................252Wang,Chengming..................172Wang,Chong..........................9,95,233Wang,Hongin.........................146Wang,Hwa-TangT.................212Wang,Jiawei...........................172Wang,Lihua............................118Wang,Min..............................63Wang,Qiuhong.......................157Wang,Tao..............................P111Wang,Thomas........................232Wang,Wenjing.......................P005Wang,Yin...............................56,187,188,P077,P100

Wang,Ying..............................171Wang,Yuanyuan.....................69,P050Wanjala,Kennedy...................P045Wannemuehler,M.J..............159Warnick,Lorin........................P080Waters,W.Ray.......................140,142,P008Watson,RobertO...................P122Watts,Christina......................238Webb,Brett............................107,P120,P084Wedasinghe,Nihal.................P129Weese,J.S..............................242,P141Wei,Lanjing............................171,174Weinroth,MargaretD............21,22,40,88Weissend,CarlaJ....................34,40Welch,Michael.......................P098Welcome,FrancisL.................6Wells,ScottJ..........................69,P050Wemette,Michelle.................6,P083Werling,Dirk...........................141Wheeler,TommyL.................88White,BradJ..........................92Wick,Joyce.............................250Wijeratne,Asela.....................P081Wilde,Shyra...........................P065Wilkes,Rebecca......................P071Williams,H.E..........................39Williams,Michele...................P081Williams,Simon......................105Willingham-Lane,J.M............209Wills,RobertW.......................63Wilson,HeatherL...................246,247,248Wilson-Welder,Jenny............P015Wimer,Christine....................238Winkelman,Nathan................200Wisener,LeeV........................36Wisnieski,Lauren...................60Wittum,ThomasE..................23,24,25,26,27,30,P038,

P079,P125Woerner,DaleR.....................88Wojcechowskyj,J.A...............P122Wölfel,Roman........................P063Wolking,DavidJ.....................P042Wood,Paul.............................P018Woodruff,KimberlyA.............51Woodworth,Jason.................78Wooldridge,Linda..................P091Workman,AspenM................77,116,141,203Wu,Zuowei............................243,P039,P040Xia,Jing...................................P039Xia,Lining...............................P039Xia,Tian..................................82Xiao,Nan................................112



Xiao,Zhengguo.......................P087Xu,Changyun..........................P039Xu,Meng-xian........................245Xu,SophiaBingling.................110Yaeger,Michael......................P059Yan,Xingyu.............................114,P111Yang,Bin.................................56Yang,Hanchun........................217Yang,Heng..............................P005Yang,Hua................................88Yang,My.................................82Yang,Xiang.............................21Yang,Xin.................................P005Yang,Xinghong.......................146Yeske,Paul..............................P026Yim-im,Wannarat..................186Yingst,SamuelL......................P058Yirsaw,AlehegneW................134,145Yoo,Dongwan........................227,P107Yoo,HanSang.........................147,197,P002Yoon,Hachung.......................102,P051Yoon,In-Joong........................P110Yoon,Kyoung-Jin....................187,220,P121Youmans,BonnieP.................P104,210Young,AlanJ...........................244Yu,Do-Hyeon..........................P064Yuan,Chaohui........................9Yuan,Fangfeng.......................75,188Yuan,Jian................................P121Yuzhakov,AntonG.................P132Zaberezhny,AlexeiD..............P132Zadoks,Ruth...........................31Zagmutt,Francisco.................181Zarling,Darrick.......................82Zeineldin,Mohamed..............13,33Zhang,Guoquan.....................176,P133Zhang,Hewei..........................P077,P100Zhang,Jianqiang.....................187,233,P098Zhang,Jilei..............................172Zhang,Michael.......................175Zhang,Qijing...........................243,P039,P040Zhang,Qingzhan.....................P107Zhang,Shuping.......................175Zhang,Weiping.......................112,160,245,P056,P057Zheng,Wanglong....................56,187,188,P100Zhgenti,Ekaterine..................P001Zholobko,Oksana...................P084Zhou,Lei.................................217Zhu,Guoqiang........................245Zimmerli,MandyK.................253Zimmerman,Brian..................26Zimmerman,Jeffrey...............70,73,78,95,233,234

Zinniel,DeniseK.....................P010Zorn,Chelsea..........................176Zuckermann,FedericoA.........117

CRWAD2017 ABSTRACTS


1-Invisibleinfluence:themicrobiomeandhumanhealthJ.A.Gilbert;DepartmentofSurgery,UniversityofChicago,Chicago,IL,[email protected]:CouncilKeynote,12/3/2017,5:30PMThehumanmicrobiomeisquicklybeingrecognizedasadynamicpartofthehumanecosystem,andresearchisstartingtodemonstratethat using ecology to understand this ecosystem has profound benefits for patient wellness. The immune system controls ourinteractionwiththemicrobialworld,andyetthemicrobialcommunitiesinourbodiesarecentraltomodulatingtheimmuneresponse.Changesinthehumanmicrobiomehavesubstantial influenceonatopy,neurologicaldisorders,metabolicdisorders,andarangeofcomplexconditionsanddiseasestates.Wewilldiscussevidenceofthesemechanismsofinteractionandhowwehavestartedtodisturbthedelicatebalanceoftheimmune-microbeequilibrium,impactingthedevelopmentandfunctionofourimmunesystems.Centraltothisdisturbanceisthedistancewehaveplacedbetweenourchildrenandthemicrobialworld,whichhasbeendemonstratedtohaveasubstantialinfluenceontheirphysiological,immunological,neurologicalandevenendocrinologicaldevelopment.Wearenowabletosignificantlyreducecowsmilkallergyininfantsthroughactivemanipulationofthegastrointestinalmicrobiota.Wecanalsoreducesurgicalinfectionsbyfeedingthemicrobiome,preventingvirulenceactivation,andreducesepsisbyusingthemicrobiometostimulateimmuneactivation.Applyingnewstrategiesto identifyhowthemicrobialecosystemcorrelateswithdiseasesstatesandtreatmentefficacythroughMicrobiome-WideAssociationStudies(MWAS)isalteringthetrajectoryofprecisionmedicine,andprovidinganewframeworkforfacilitatingpatientcare.2-ComparisonoftwomethodsforcollectingantibioticusedataonsmalldairyfarmsL.Redding1,F.Cubas-Delgado2,M.Sammel3,G.Smith1,D.Galligan1,M.Levy3,S.Hennessy3. 1ClinicalStudies,SchoolofVeterinaryMedicine,UniversityofPennsylvania,KennettSquare,PA,USA,2SchoolofVeterinaryMedicine,UniversidadNacionaldeCajamarca,Cajamarca, Peru, 3Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA, [email protected]:AntimicrobialDrugUse,Room1,12/4/2017,9:00AMAntibioticsarecommonlyusedinfood-producinganimals.Theycanimproveanimalhealthandproductivity,buttheirusemayalsorepresent a public health threat. Very little is known about antibiotic use on small farms in lower/middle income countries. Tounderstandantibioticuseonthesefarmsandpromotethejudicioususeofthesedrugs,pharmacoepidemiologicdataarenecessary.However,acquiringsuchdatacanbedifficult,asfarmersareoftenilliterate(andthereforecannotparticipateinwrittensurveysorkeeptreatmentrecords),antibioticscanbeobtainedover-the-counter(inwhichcasenoprescriptionsaregenerated)andmonitoringand surveillance systems for drug use are often non-existent. The goal of this study was to compare twomethods of acquiringpharmacoepidemiologicdatapertainingtoantibioticsthatarewell-adaptedtofarmsinlower-middleincomecountries:self-reportandthecollectionofdiscardeddrugpackaging.Aconveniencesampleof20farmersinCajamarca,Peru,participatedinthestudy.Farmersplaceddiscardedantibioticpackaginginbinsforsixmonths.Attheendofthesix-monthperiod,farmerswereinterviewedandaskedto recall the antibiotic usage that occurred on their farm over the past month and past six months. Self-reported data werequantitativelyandqualitativelycomparedtothebincontentscollectedinthelastmonthandprevioussixmonths.Agreementbetweenthebinsandself-reportwasrelativelypoorforboththequantityandtypesofantibioticsused.Thebinsappearedtoperformbetterthanself-reportwhenbottlesandmLsofantibioticsweremeasured,whileself-reportappearedtoperformbetterforintra-mammaryinfusions.Thebinsalsoappearedtoperformbetterwhendatapertainingtoanextendedtimeperiod(sixmonths)werecollected.Theresultsofthisstudywillprovideguidancetoinvestigatorsseekingtocollectpharmacoepidemiologicdatainsimilarenvironments.

CRWAD2017 ABSTRACTS


3-AnassessmentofveterinaryprescriptionpracticesandfactorsinfluencingusageofantimicrobialdrugsD.Taylor1,J.Martin2,K.E.Belk2,P.S.Morley3,E.Scallan1.1ColoradoIntegratedFoodSafetyCenterofExcellence,ColoradoSchoolofPublicHealth,Denver,CO,USA,2Dept.ofAnimalSciences,ColoradoStateUniversity,FortCollins,CO,USA,3Dept.ofClinicalSciences,ColoradoStateUniversity,FortCollins,CO,[email protected]:AntimicrobialDrugUse,Room1,12/4/20179:15AMPurpose:Themitigationofantimicrobialresistant(AMR)microorganismsisarguablyoneofthemostimportantchallengesfacingpublichealth.Althoughsignificantstridesaimedtoreducetheuseofclinicallyimportantantimicrobialdrugs(AMD)havecommenced,thereisa lackof critical information regardingveterinaryprescriptionpractices, veterinaryperceptionsofantimicrobialusepoliciesandmotivating factors influencingAMDusage.AMR isacomplex issue involvingaplethoraofdynamicsocial,politicalandeconomicalfactors.GiventheevolvinglandscapesurroundinguseofAMDinanimals,webelievethatgenerationofaccurateusagedataandanassessmentofveterinaryperceptionsandattitudesregardingAMDuseisimperativeforthedirectionofdevelopmentofeffectivetoolsand trainings aimed at mitigating AMR.Methods: During 2016-2017, we developed and launched a study to assess veterinaryprescription practices, perceptions and factors influencing usage of antimicrobial drugs among veterinarians who prescribeantimicrobialdrugs(AMD)tobeefcattle,dairycattle,swineandpoultry.Veterinariansweresurveyedusinganonlinesurvey,whichincluded demographic questions, disease scenarios and attitude questions. Disease scenarios were created for feedlot cattle,backgroundingcattle,dairycattle,swineandpoultry,andveterinarianswereaskedtoprovidetreatmentrecommendationsbasedonthesescenarios.Thestudyalsoincludedwillingparticipantsmaintainingaprescriptiondiaryforsix-weeksinordertovalidatesurveyresponses and gain insight intoAMDusepractices outsideof the survey’s scope.Results:Wehave collected181 responses fromproductionanimalveterinariansandarecurrentlyanalyzingdata.Conclusions:Withthisdata,wewillbeabletoassesscurrenttrainingandeducationalgaps,whichwillleadtothedevelopmentofeffectivetrainingsotherresourcesforveterinariansaimedatpreventingantibiotic resistance and promoting antibiotic stewardship. Additionally, our results provide baseline information on prescribingpracticesandcreateavalidatedtoolforcollectingfuturedataonantimicrobialuse.4-Across-sectionalstudyofthedeterminantsofantimicrobialusepracticesofveterinarycliniciansataUSveterinaryteaching

hospitalJ.E. Ekakoro, C.C.Okafor. Department of Biomedical andDiagnostic Sciences, College of VeterinaryMedicine at theUniversity ofTennessee,Knoxville,TN,[email protected]:AntimicrobialDrugUse,Room1,12/4/20179:30AMObjectives—ToidentifyfactorsinfluencingcliniciandecisionstobeginusingantimicrobialsaswellasthechoiceofantimicrobialsusedatTheUniversityofTennesseeVeterinaryMedicalCenter(UTVMC);toevaluatethepractices,perceptions,opinionsandconcernsofveterinarycliniciansatUTVMCconcerningantimicrobialuse,antimicrobialstewardship,andantimicrobialresistance(AMR).Design—Acrosssectional study. Sample—62veterinaryclinicians. Procedures—Surveysoftwarewasusedtosendaquestionnaire to121eligibleparticipants,whereallwereUTVMCfacultywithclinicalappointmentsandhouseofficers.Cumulativelogitmodelswerefittedto investigateassociationsbetweencategoricalexplanatoryvariablesandordinal responsevariables. Results—A response rateof51.24%wasachieved.Ofthe62respondents,47(75.81%)reportedthatbacteriologicalcultureandantimicrobialsusceptibilitytestresults were extremely important in their antimicrobial prescription decision-making. Thirty-two (51.61%) respondents believedantimicrobialsarebeingover-prescribed.Thecephalosporinclasswasthemostpreferredantimicrobialclass.Fromthemultivariablecumulative logitmodel,yearofgraduationfromveterinaryschool (P=0.034)andclinicians’primarypatient load(P=0.009)weresignificantlyassociatedwithclinicians’degreeofconcernaboutAMR.Conclusionsandclinicalrelevance—ThefindingssuggestaneedformoreawarenessaboutAMRamongveterinaryclinicians.Improvementsinantimicrobialstewardshipareneeded,especiallyamongveterinaryclinicianswhograduatedafter1999.EducationalpracticesthattargetmodificationofantimicrobialprescriptionpracticesofveterinaryclinicianswouldlikelyimproveaGoodStewardshipPractice(GSP)mindset.GSPisimportantinprolongingtheefficacyofcurrentlyavailableantimicrobialdrugs.

CRWAD2017 ABSTRACTS


5-AntimicrobialdruguseinCanadianbeefcattle:extent,indications,andriskfactorsforuse

S.Brault1,S.Hannon2,S.Gow3,C.Carson4,C.Booker2,P.S.Morley1.1ClinicalSciences,ColoradoStateUniversityCollegeofVeterinaryMedicineandBiomedical Sciences, FortCollins,CO,USA, 2FeedlotHealthManagementServices,Okotoks,AB,Canada, 3Center forFoodborne,Environmental,andZoonoticDiseases,PublicHealthAgencyofCanada,Saskatoon,SK,Canada,4CenterforFoodborne,Environmental,andZoonoticDiseases,PublicHealthAgencyofCanada,Guelph,ON,[email protected]:AntimicrobialDrugUse,Room1,12/4/20179:45AMAntimicrobialdrugs(AMDs)areroutinelyusedinbeefcattleproductionsystems.TheuseofAMDsinfood-producinganimalsisunderscrutinyduetothepotentialforpromotionofantimicrobialresistance.AcomprehensiveunderstandingofthetypesofAMDsused,extentofuse,andthemostcommonindicationsforuseinthebeefcattleindustryisanimportantprecursortomeaningfulassessmentoftheassociatedpublichealthrisk. Inthisstudy,detaileddataregardingAMDusein2,639,970cattlefrom36feedlotsinwesternCanadawerecollectedover4years(2008-2012).DataobtainedincludedthetypeofAMDsused,dosageandrouteofadministration,reasonforuse,daysonfeedattimeofuse,weightattimeofuse,anddemographiccharacteristicsoftheanimal(sex,ageatfeedlotarrival,andassessedlevelofriskforrespiratorydiseaseatfeedlotarrival).Highriskcattlecomprised39.3%ofthecattle,while60.7%werelowrisk.Parenteralantimicrobialswereadministeredmetaphylacticallyto70%ofthecattleandtherapeuticallyto9.6%ofthecattle. The most common indication for metaphylactic and therapeutic parenteral AMD use was undifferentiated fever/bovinerespiratory disease (65.5% of treatments). The relative risk of use of any parenteral AMD in cattle deemed to be at high risk forrespiratorydiseasewas1.4times(95%CI1.398-1.401)thatofcattledeemedtobeatlowriskforrespiratorydisease.6-AqualitativestudyofNewYorkStatedairyfarmers’perceptionsregardingantibioticuseandresistanceindairycattleM.Wemette1,W.Beauvais1,A.GreinerSafi2,M.Shapiro2,F.Welcome3,P.Moroni3,R.Ivanek1.1DepartmentofPopulationMedicineandDiagnosticSciences,CollegeofVeterinaryMedicine,CornellUniversity,Ithaca,NY,USA,2DepartmentofCommunication,Collegeof Agriculture and Life Sciences, Cornell University, Ithaca, NY, USA, 3QualityMilk Production Services, Department of PopulationMedicineandDiagnosticSciences,CollegeofVeterinaryMedicine,CornellUniversity,Ithaca,NY,[email protected]:AntimicrobialDrugUse,Room1,12/4/201710:00AMThe Food and Drug Administration (FDA) recognizes the overuse of antibiotics in animal agriculture as a potential contributor toantibiotic resistance. To curboveruse, the FDAhas implemented regulations topromote the judicioususeofmedically importantantibioticsinagriculture.FewstudieshaveinvestigatedtheattitudesofU.S.dairyfarmers,whomakeregularantibioticusedecisions,towardsantibioticuse.This lackofknowledgehindersdevelopmentofeffective farm-level interventions to improveantibioticusepractices.Theobjectiveofthisongoingqualitativestudyistoinvestigatetheknowledge,attitudes,andbehaviorsofNYSdairyfarmersconcerningantibioticuseandresistance incattle.Semi-structured in-person interviewsarebeingconductedwith farmersselectedthroughpurposivesamplingandanalyzedusingthematicanalysis.Ofthe25plannedinterviews,12havebeenconductedandsubjectedtopreliminaryanalysis.Major topics addressed includedbasic knowledgeof antibioticuseandantibiotic resistance, adherence tojudicious antibiotic use practices, and veterinarians as a source of information on antibiotic use which could be useful in futurecommunicationefforts.Mostparticipantswereabletoaccuratelydefinethetermantibioticanddescribeantibioticresistance,thoughtherewerevaryingdegreesofconcernabouton-farmantibioticresistance.Preliminaryresultssuggestthatfarmersperceivetheiruseofantibioticsasprudent.Despitethisperceptionofjudicioususe,inthecourseoftheinterviewparticipantsalsoreferredtoinstanceswhenantibioticswerenotproperlyusedorwereuncertainofhowproperlyothersontheirfarmweremanagingcattletreatedwithantibiotics.Mostreportedconsultingtheirveterinarianwhentheyhadquestionsaboutantibioticuse.TheseresultssuggestthatNYSdairyfarmersperceivetheiruseofantibioticsasjudiciousandwell-manageddespitedescribingincidentsthatsuggestotherwiseandthusmaylackmotivationtochangetheirpractices.Targetingfarmers’perceptionswitheducationalmessagescouldbeapromisingstrategytooptimizeantibioticuseindairyfarming.

CRWAD2017 ABSTRACTS


7-Antimicrobialusepractices,andperceptionsofcattleproducersinTennesseeregardingantimicrobialuse:aqualitativestudyJ.E. Ekakoro1,M. Caldwell2, E.B. Strand1, C.C. Okafor1. 1Department of Biomedical and Diagnostic Sciences, College of VeterinaryMedicine at theUniversity of Tennessee, Knoxville, TN,USA, 2Departmentof LargeAnimal Clinical Sciences, CollegeofVeterinaryMedicineattheUniversityofTennessee,Knoxville,TN,[email protected]:AntimicrobialResistance–2,Room1,12/4/201710:45AMObjective—To determine the most common drivers for using antimicrobials and the perceptions of Tennessee cattle producersregarding implementing judicious practices towards antimicrobial use (AMU) in cattle. Design—A qualitative study. Samplepopulation—Cattleproducerswithcharacteristicsrelevanttothestudywerepurposivelyselected. Procedures—Atotalof6focusgroupmeetingswereconductedinEast,MiddleandWestTennessee.Ofthe6focusgroups,5werebeefproducersandtheotherwasa groupof dairy producers. Eachbeef producer focus grouphad6 to 9participantswhile thedairy producer focus grouphad14participants.Asemi-structuredinterviewguidewasutilized.Eachfocusgroupwasaudioandvideorecorded.Thematicanalysiswasperformedtoidentifyemergingthemes.Results—SeveralmajorthemesemergedwhichcattleproducersconsideredtodriveAMU.Thesecommonthemeswereeconomicfactors,veterinarianconsultation,producer’sself-perceivedlevelofknowledgeandexperience,animalwelfare,aggressivemarketingbypharmaceuticalcompanies,peersupport fromotherproducers,diseaseepidemiologyandoutcomes,managementfactors(cattleproductionsystem),efficacyoftheantimicrobialdrug,consumerpressure,andpromotingfoodsecurity.Mostproducersperceive that theVeterinaryFeedDirective (VFD)would leadto increaseduseof injectableantimicrobialagentsbyproducers.Also,mostproducersthinkthatveterinariansneedtobetrainedonhowtowriteVFDprescriptions.Theproducersalso suggested that more education for cattle producers on prudent use of antimicrobials is needed. Conclusions and clinicalrelevance—SeveralfactorsdrivetheuseofantimicrobialsamongcattleproducersinTennessee.Producer’sexperience,strongsocialconnectionandpeersupportamongcattleproducersemergedasimportantdriversofAMUacrossallthe6focusgroups.ContinuingeducationonprudentuseofantimicrobialsandwritingofVFDprescriptionsisneededforproducersandveterinarians,respectively.8-AmathematicalmodeltoexplorepotentialreductionsinantibioticresistanceinfeedlotsW.B.Beauvais1,R. Jennings1,T.Arthur2,R. Ivanek1. 1CornellUniversityCollegeofVeterinaryMedicine, Ithaca,NY,USA,2U.S.MeatAnimalResearchCenter,ClayCenter,NE,[email protected]:AntimicrobialResistance–2,Room1,12/4/201711:00AMThisstudyexploredthequantitativeimpactofreducingormodifyingantibioticuseonfeedlotsintermsoftheleveloffecalsheddingof antibiotic resistant bacteria and the level of antibiotic resistant bacteria in the feedlot environment. Tetracycline resistance inEscherichiacoliwasusedasamodelsystem.Anoveldeterministic,differenceequationmodelwasdevelopedtoinvestigatethelevelof antibiotic-resistant bacteria in (1) cattle feces and (2) the feedlot pen floor. The model accounts for: (1) direct and indirecttransmissionofbacteria;(2)excretionofantibioticsintotheenvironment;and(3)selectivepressureofantibioticsonbacteriainboththecattlegutandtheenvironment.Comparedantibiotictreatmentprotocolsincludedthemasstreatmentofcattleforshippingfeverfollowingarrivalatthefeedlotvs.nomasstreatment.MonteCarlosimulationwasusedtoexploretheimpactofuncertaintyontheresults.GeneticAlgorithmforRuleSetProduction(GARP)wasusedtoexplorethresholds inparametervaluesthatwouldresult insimilar or different levels of resistance between the tested treatment protocols. Preliminary results indicated a relatively largeproportionoftetracycline-resistantE.coliinfecesincattleleavingforslaughter(56%)evenwhennoantibioticswereusediftherewasahighinitiallevelofresistantE.coliintheenvironmentatstocking.ThemasstreatmentprotocolwouldincreasetheproportionofresistantE.colito73%.Therewasawideuncertaintyrangeduetouncertainparametersbut,interestingly,75%ofsimulationsresultedinanabsolutedifferencebetweenthetreatmentprotocolsof less than10%.GARP identified theparameterspacewherereducedantibioticusemayhaveanegligibleimpactonantibioticresistanceinabatchoffeedlotanimals.Inconclusion,themodeldemonstratesthatimposingrelativelylargereductionsinantibioticuseonindividualfeedlotbatchesmaynotnecessarilyreduceantibioticresistancetoa largeextent, ifothercontrolmeasuresarenot implemented.Thesefindingswillbediscussed inrelationtoobservationalandexperimentaldataintheliterature.

CRWAD2017 ABSTRACTS


9-BayesianlatenthierarchicalmodelfordetectingMICcreepC.Yuan,A.O'Connor,C.Wang.IowaStateUniversity,Ames,IA,[email protected]:AntimicrobialResistance–2,Room1,12/4/201711:15AMAspublichealthofficialsseektoproperlyinterpretthevolumesofdatageneratedbysurveillanceprogramssuchastheUSNationalAntimicrobialResistanceMonitoringScheme(NARMS),thereisacriticalneedtodevelopnew/alternativeapproachestothestatisticalanalysisofantimicrobialresistance(AMR)datathatwilldetectdevelopmentofresistanceinatimelymannerandenablethequickimplementationofmitigationmeasures.AMRdataarecollectedthroughsurveillanceprogramsanddescribetheconcentrationofanantibioticatwhichanorganismceasestogrowandproliferatei.e.aminimuminhibitoryconcentration(MIC).Forstatisticalanalysis,proportionofbacteriaintheresistantcategoryisusedasanindicatorofchangesinresistance.ThecentralhypothesisofourprojectisthatstatisticalanalysisbasedonMICbreakpoints,whilesimpletoconduct,doesnotfacilitatetimelydetectionofchangesinresistance.Themajorissuewithstatisticalanalysisbasedonproportioninthebreakpointcategories,isthattheaverageMICcanbeincreasinglongbeforechangesintheproportionabovethethresholdarestatisticallydetectablei.e.MICcreep.TheobjectivewiththeprojectwastodevelopthestatisticalmethodsthatfacilitatetimelydetectionofMICcreep.WedevelopedastatisticalmodeltodetectMICcreepandtestthehypothesisthatthenumberofyearsrequiredtodetectMICcreepusingaBayesianlatentclasshierarchicalmodelisless thanan analysis basedon theMICbreakpoint-based categories analysis. For this projectwedemonstrated themethodusingNARMShumandataforSalmonella.WefoundthatMICvaluesfornon-resistantcategorywerestatisticallysignificantincreasingfrom1996to2014forTyphimuriumserotypetestedonchloramphenicolantibiotic,whilenosignificancewasdetectedontheproportionofnon-resistantcategory.Alsoourproposedpair-wisecomparisonforMICvaluesbetweenconsecutiveyearscouldmimictheobservedMICtrendreasonablywell.Thisanalysisenablesmoretimelydetectionofemergingresistance.Theimpactwillbe,thatpublichealthofficialscanimplementtargetedantimicrobialstewardshipprogramssooner.10-Agent-basedandhybridmodelsofantimicrobialuseandtransmissionofantimicrobialresistanceinawesternCanadianfeedlot

throughtheproductionofretailhamburgerC.Waldner1,M.Pang1,K.Kreuger1,N.Erickson1,S.Gow2,S.Checkley3,W.Qian1,N.Osgood1.1UniversityofSaskatchewan,Saskatoon,SK, Canada, 2Public Health Agency of Canada, Saskatoon, SK, Canada, 3University of Calgary, Calgary, AB, [email protected]:AntimicrobialResistance–2,Room1,12/4/201711:30AMAntimicrobialresistance(AMR)threatenstheeffectivetreatmentofanever-increasingrangeofinfectionsinbothhumanandanimals.Asaresult,antimicrobialuse(AMU)inlivestockproductionisundereverincreasingscrutiny.FieldresearchtocontrolthedevelopmentandspreadofAMRwithinthelivestockindustry,andfromthelivestockindustrytohumansislimitedbyexpenseandthecomplexityofthesystemsunderstudy.Betterunderstandingiscriticaltolimitthespreadofexistingresistancegenesandreduceemergenceofnewresistancetypes.Agent-basedmodels(ABMs)andhybridormultimethodmodelscanprovideaneffectiveoptionforsummarizingandvisualizingourunderstandingofsuchcomplexsystems.Thesemodelsactaslearningtoolsandifadequatelyinformedbygooddata,canprovideanoptiontofacilitateourreasoningaboutthehypotheticalsystem-wideconsequencesofpolicyandmanagementchanges.Theobjectiveofthepresentstudywastodevelopanagent-basedmodelthatsimulatesthemovementofcattlethroughaWestern Canadian feedlot and examine the impact of restrictingAMU formetaphylaxis and prophylaxis onAMR. Themodelwasdevelopedthroughconsultationwithindustryandincludeslocaldataondiseaseincidenceandantimicrobialuse.ModelinputsthatwerenotdirectlyavailablefromexistingdatasuchastherateofAMRselectionandofwaningAMRfollowingthecessationofuse,aswellastheprobabilityofresistancespreadwereestimatedusingmodelcalibrationtoolsreferencingexistingtimeseriesAMRdata.TofurtherexplorethepotentialimpactsofchangesinfeedlotAMUontheprevalenceofAMRinretailmeat,theoutputsgeneratedbythis model were also exported to a linked hybrid agent-based/discreet events model of a typical processing plant. The netcontamination in the hybrid processing plantmodelwas calibrated to reflect recovery rates of genericE. coli in retail hamburgergenerated by the CIPARS national surveillance program. Both models are continually evolving based on discussion with industrypartnersandwillbeusedtobetterunderstandtheoccurrenceandtransmissionofAMRwithinthebeefindustry.

CRWAD2017 ABSTRACTS


11-Behavior pattern sensitivity analysis identifies interventions to reduce enteric antimicrobial resistance in beef steers fedchlortetracycline

C.L.Cazer1,V.V.Volkova2,Y.T.Gröhn1.1PopulationMedicineandDiagnosticSciences,CornellUniversityCollegeofVeterinaryMedicine,Ithaca,NY,USA,2InstituteofComputationalComparativeMedicine,DepartmentofDiagnosticMedicine/Pathobiology,KansasStateUniversityCollegeofVeterinaryMedicine,Manhattan,KS,[email protected]:AntimicrobialResistance–2,Room1,12/4/201711:45AMAntimicrobialresistantentericbacteriainlivestockcancontaminatemeatduringslaughterandprocessingandthenpotentiallyinfectconsumers.Eachuseofantimicrobialsinlivestockcanincreasetheprevalenceofresistantentericbacteria,whichmayremainelevatedafterendingantimicrobial therapy. Inorder to study thebehaviorof resistantbacteriaduringandafterantimicrobial therapy,wedevelopedamathematicalmodelofentericEscherichiacolipopulationsinbeefcattlefedchlortetracycline(CTC)for28daysat350mgperheadperdayandasubsequent60-dayCTC-freeperiod.Weusedbehaviorpatternsensitivityanalysistoidentifymodelparametersassociatedwith thebehaviorandvariationof resistantbacteria,whichmay reveal leveragepoints toalter the systemand reduceresistance prevalence. The model included 29 random parameters related to E. coli population dynamics, chlortetracyclinepharmacokinetics and pharmacodynamics. The resistant bacteria exhibited three behaviors during the 88 day simulation period:increasingtoequilibrium,decreasingtoequilibrium,andatemporalincreaseduringantimicrobialtherapyfollowedbydecreasingtoequilibrium.Thebehaviorpatternsensitivityanalysisappliesregressionanalysistomeasuretheassociationbetweenparametersandbehaviorpatternmeasures(e.g. inflectionpointsandequilibriums). Inthissystemdynamicsmodel,theprevalenceofresistance iningested bacteria, bacterial inflow and outflow rates, resistance fitness cost, and resistance gene transfer rate were consistentlyassociatedwithequilibriumlevelsafterendingchlortetracyclinetherapy.Thissuggeststhatinterventionsaimedattheentericbacterialpopulations,suchasprobioticsanddiet,maybeeffectiveatreducingtheprevalenceoftetracyclineresistantbacteriainbeefcattle.Inaddition,CTCminimuminhibitoryconcentrationanditspharmacokineticparametersofdegradationrate,antimicrobialadsorptiontodigesta,andvolumeofthelargeintestinecontentweresignificantlyassociatedwithresistancepatternsduringtherapy;howeverthesearenoteasilymanipulatedtodecreaseresistantbacteria.12-Age-dependencyofantimicrobialresistanceinfecalbacteriaofanimals-ascopingreviewT.N.Gaire1,H.M.Scott2,L.Sellers1,V.V.Volkova1.1Dept.ofDiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,USA,2Dept.ofVeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,USA.,[email protected]:AntimicrobialResistance–3,Room1,12/4/20172:00PMThephenomenonofadecreaseinantimicrobialresistance(AMR)infecalbacteriawithageinfoodanimalshavebeennotedinfieldstudies.Factorscontributingtothesedynamicsareunknown,butifdeterminedcansignalnewavenuesforAMRmitigation.Wehaveconductedascopingreviewtosummarizetheextent,range,andnatureofresearchactivityanddataonthequestion:“Isenteric/fecalantimicrobialresistancepredictablyshiftsaccordingtothehostageinanimals?”PertinentliteraturepublishedbyMarch2017forallanimals except humanwas retrieved using a similar search string from two databases (PubMed.gov andWeb of ScienceTM CoreCollection)without filtering the publication date or language. Studies reporting longitudinal sampling of an animal cohort or age-equalizedproductiongroupandstudiesreportingcross-sectionalsamplingofdifferentagegroupswithinonefood-animalproductionsystemorcatchmentareaofaveterinarypracticewereretained.Fortythreearticleswithrelevantdataforfoodanimalswereidentified.TheoutcomesstudiedwerephenotypicAMR(25ofthe43studies),AMRgenequantities(1study),orboth(17studies).Thedatahavecomefromthelongitudinalorcohort(24)andcross-sectional(10)studies,andexperimentaltrails(9).Thirtytwoofthestudieswereincattleandswine;theseshowedthatthedeclineinabundanceorprevalenceofresistantfecalbacteriaincattleandswinewithage(75%ofthe32studies)hasbeenreportedsincethe1970s.Itisunclearwhetherthephenomenonisrelatedtoorpre-datesantimicrobialdruguse.Inathirdofthesestudies,theage-dependencywasobservedaccidentally,asitconfoundedtheimpactonAMRofotherfactorssuchasantimicrobialdruguse.SynthesisofthedatasuggestedthephenomenonofadecreaseinfecalAMRwithageisobservedirrespectivelyofgeographiclocationinswineandruminants,butdifferentage-dependentAMRdynamicsmayexistinpoultry.

CRWAD2017 ABSTRACTS


13-Impacts of peri-natal antibiotic administration on gutmicrobiota composition and antibiotic resistance gene prevalence inpiglets

M.Zeineldin,B.Burton,A.Megahed,B.Aldridge,J.Lowe.IntegratedFoodAnimalManagementSystems,DepartmentofVeterinaryClinicalMedicine,IllinoisUniversityatUrbana-Champaign,Urbana,IL,[email protected]:AntimicrobialResistance–3,Room1,12/4/20172:15PMThe swine gastrointestinalmicrobiota is comprised of a diverse and complexmicrobial population that coexists in a coordinated,complex mucosal ecosystem that contributes to host mucosal health. It is important to understand how common managementpractices, such as antimicrobial administration, might affect this complex ecosystem. While the antibiotic resistance profiles ofpathogensandopportunisticbacteriaculturedhavebeencharacterized,theAntimicrobialResistanceGenes(ARG)fromthewholegutmicrobiotahavereceivedfarlessattention.Theobjectiveofthisstudywastounderstandtheimpactofperi-natalantimicrobialsongrowth,mortality,fecalmicrobiotacompositionandARGprevalence.Forty-eightlitterswereblockedtooneofsixtreatments(N=8).Withinlitter,allpigsreceivedthesametreatment.Pigswereweighedandtreatmentsadministeredat24hoursofage,afterlittershavebeenbalancedforsize.Treatmentswereasfollows:Control(saline1cc),Tulathromycin(2.5mg/kgIM),CeftiofurCrystallinefreeacid(5.0mg/kgIM),Ceftiofurhydrochloride(5mg/kgIM),Oxytetracycline(22mg/kgIM)andProcainePenicillinG(33,000units/kgIM).Twopigsperlitterwereindividuallyidentifiedanddeepfecalswabswerecollectedatdays0(priortotreatment),5,10,15and20.Highthroughput,nextgenerationsequencingwasusedtoassessmicrobialdiversityandthepresenceofARGs(TetO,TetW,TetC,Sul1,Sul11,blactxandermB).Preliminaryanalysisshowsthat,whileantimicrobialtreatmenthadnoeffectonindividualweightgain,ormortality,itwasassociatedwithsignificantchangesintheabundanceofARGs,andthecompositionandprogressionofthefecalmicrobiota.Interestingly,thedurationandextentoftheobservedchangeswerecontingentontheclassofantimicrobialadministered.Thedataalsoindicatesthatperinatalantimicrobialadministrationincreasestheprevalenceofantibiotic-resistantbacteriaandARG.Incombination,theseresultsraiseimportantquestionsregardingthepracticeofperinatalantimicrobialadministrationandonthedesignofantimicrobialstewardshipprogramsintheswineindustry.14-ComparisonofannualandregionalvariationidentifiedusingvariousmultidrugresistanceclassificationmetricsforE.colifrom

chickenabattoirsurveillanceinCanadaM.C.MacKinnon1,D.Pearl1,C.Carson2,E.J.Parmley2,S.McEwen1.1PopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2Canadian Integrated Program for Antimicrobial Resistance Surveillance, Public Health Agency of Canada, Guelph, ON, [email protected]:AntimicrobialResistance–3,Room1,12/4/20172:30PMThestudyobjectivesweretodescribethemostcommonresistancepatternsingenericE.coliisolatesfromchickencecalsamplesanddeterminetheimpactofusingdifferentmultidrugresistance(MDR)classificationmetricsforanalysisofannualandregionalvariationinMDR.From2006-2015,therewere1598E.coliisolatesfromchickencecalsamplescollectedatabattoirsfortheCanadianIntegratedProgram forAntimicrobialResistanceSurveillance.ThreeMDRclassificationmetricswereused:MDR-drug,MDR if the isolatewasresistantto≥3ofthe13antimicrobialsincludedinthestudy;MDR-cat,MDRifitwasresistantto≥3ofthe9antimicrobialscategories;andMDR-class,MDRifitwasresistantto≥3ofthe6antimicrobialclasses.Themostfrequentresistancepatternsoverall,andbyyearandregionwereextracted.ForeachMDRmetric,mixed logisticregressionmodels,which includedrandominterceptsforabattoir,werefittedtoanalyzetheassociationbetweentheprevalenceofMDR,andyearandregion. Interactioneffectsbetweenyearandregionwereevaluated.Overall,andinallyearsandregions,pansusceptiblewasthemostcommonsusceptibilitypattern.Resistancepatternsthatincluded3rdgenerationcephalosporinsandβ-lactamswithβ-lactamaseinhibitorswerecommon;however,thosethatincludedquinoloneswereuncommon.TheprevalenceofMDRwaslowestwithMDR-classandhighestwithMDR-drug.Basedonmodelsfittedwithindividualfixedeffects,significantannualvariationintheprevalenceofMDRwasidentifiedwithMDR-drugandMDR-classmodels,butsignificantregionalvariationwasidentifiedforallthreeMDRmetricmodels.SignificantinteractioneffectsbetweenyearandregionwereidentifiedwiththeMDR-drugandMDR-catmultivariablemixedlogisticregressionmodels.BoththeprevalenceofMDRandinterpretationoftheassociationbetweentheprevalenceofMDR,andyearandregiondiffereddependingontheMDRmetricused.TheseresultsaresupportiveofthepreviousconcernsthatcautionmustbetakenwhencomparingMDRresultsbetweenstudies.GlobalconsensusisneededfortheoptimalMDRmetricfornon-clinicalentericbacteriasurveillance.

CRWAD2017 ABSTRACTS


15-AssessingthetransmissiondynamicsofantimicrobialresistantSalmonellaheidelberginCanadianpoultryproductionusingdraftgenomesequencedata

B.M.Hetman1,E.N.Taboada2,R.Reid-Smith3,D.L.Pearl1.1OVCUniversityofGuelph,Guelph,ON,Canada,2PublicHealthAgencyofCanada,Lethbridge,AB,Canada,3PublicHealthAgencyofCanada,Guelph,ON,[email protected]:AntimicrobialResistance–3,Room1,12/4/20172:45PMAntimicrobialresistance(AMR)iscriticallyimportanttohumanhealthduetothedifficultyoftreatingresistantbacterialinfections.ThegovernmentofCanada’sAMR-genomicsresearchdevelopment initiativewascommissionedtodevelopagreaterunderstandingofhow food production systems contribute to the development of AMR and ultimately impact human health.SalmonellaHeidelberg(SH)isazoonoticpathogencommonlyisolatedfrompoultryinNorthAmericaandhasbeenimplicatedinseveralrecent outbreaks across Canada and the United States. For the present study, we selected SH isolates from federal surveillanceprogramsatpoultryfarm,abattoirandretaillevelsinOntario,Canadain2013,andcharacterizedthembywholegenomesequencing(WGS).UsinganovelSHsingle-nucleotidevariantbased(SNV)typingassay,weexaminedthegeneticdiversitypresentinthebacterialcore genome to assess the underlying population structure of SH circulating in the Canadian food chain. Intra-genomic diversitymeasuredbySNVdifferencesinthecoregenomesofSHclusteredinthesameestablishmentwasfoundtobegreaterthantheinter-genomicdiversityatthislevel.Thiseffectwasmorepronouncedatthefarmandabattoirlevelsthanattheretaillevel,wherethetrendwasreversed.AnalysisoftheWGSdatauncoveredgeneticdeterminantsofAMR,providingamolecularbasisforresultsofphenotypictesting,andidentifying38%ofisolatesasmultidrug-resistant.Weidentifiedatotalof15chromosomalandmobileAMRdeterminantsthatincludedCMY-13andCMY-59,whichencodeextended-spectrumbeta-lactamases.Finally,weidentifiedatotalofsevenplasmidrepliconelementsinourdataset,witharangeof1-4repliconspresentperisolate.Resultsfromouranalysisdemonstratetheabilitytoextractmeaningfulinformationfromdraftgenomesequencedata,enhancingourunderstandingofthemolecularbasisforAMRandplasmidscirculatinginenvironmentsrelatedtofoodproduction.However,challengesconcerningsensitivitythresholds,clustering,andplasmididentificationwillstillneedtobeaddressedbeforetheoptimalusageofWGSfordiseasesurveillancecanbeachieved.16-EffectsofincreasingconcentrationsofdietaryzinconlevelsofantimicrobialresistanceamongfecalEscherichiacoliinfeedlot

cattleE.A. Taylor1, R.G. Amachawadi2, C.L. Van Bibber-Krueger3, H.C. Muller3, J.S. Drouillard3, H.M. Scott1. 1Department of VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,USA,2DepartmentofClinicalSciences,KansasStateUniversity,Manhattan,KS,USA,3DepartmentofAnimalSciences&Industry,KansasStateUniversity,Manhattan,KS,[email protected]:AntimicrobialResistance–3,Room1,12/4/20173:00PMAsantimicrobialresistancecontinuestobeatopicof increasingconcerninpublichealth,moreresearchisbeingdoneinsearchofalternativestoantibioticsanddevelopingstewardshiptechniquestopromoteresponsibleusageofantibioticsinanimalagriculture.Onepartofthoseeffortsissupplementingincreasinglevelsofdietaryelements,suchascopperandzinc,beyondnutrientrequirementsincattlediets.However,muchremainsunknownonhowthesemetalsinteractwiththebacterialpopulationsthatcolonizethegutoftheseanimals.Weconductedastudytoevaluatetheimpactofincreasedlevelsofzincsupplementationincattlefeedontheprevalenceof antimicrobial resistance among fecal Escherichia coli. Twenty-four pens of 10 cattle were supplemented with increasingconcentrationsofZincat0,30,60or90ppmintheirfeed.FecalsampleswerespiralplatedontoplainMacConkeyagar,MacConkeyagarwithceftriaxone(4µL/mL),andMacConkeyagarwithtetracycline(16µL/mL)followedbycolonycountingbasedonphenotypicqualities.BothPoissonandnegativebinomialregressionwereusedtomodelroundedlog10CFUcountsofbacteriaonplain,aswellasantibioticsupplemented,platestoaccountfortheover-inflatedzerocountsonceftriaxonemedia.TherewerenosignificantdifferencesobservedamongE.coliisolatedonanyofthethreemediatypesbasedonthemainmodeleffectsassessing0,30,60,and90ppmofzinc supplementation.However, thereweresignificantdifferences in thezero-inflatedcomponents (logit).Thispoints toavaryingprobabilitythatsampleassaysexceededthelimitofdetection(LOD)basedonsupplementation.Ourresultssuggestincreasingzincconcentrationsincattlefeeddoesnotdirectlyimpactthequantitativeexpressionofresistancetotetracyclineandceftriaxone.Whileourresultsdonotsuggestasignificantdifference,itisimportanttocontinuetoevaluatehowincreasingdietaryzincconcentrationsinteractwithotherentericbacteriainthesepopulationssincehigherconcentrationsofzincincattlefeedappeartonegativelyimpactappetite.

CRWAD2017 ABSTRACTS


17-EffectsofintermittentfeedingoftylosinphosphateduringthefinishingperiodonantimicrobialresistanceofEnterococcusspp.infeedlotsteers

O.J.Ogunrinu1,H.C.Muller2,C.L.VanBibber-Krueger3,R.G.Amachawadi4,H.M.Scott1,J.S.Drouillard3.1VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,USA,2AnimalSciencesandIndustry,KansaStateUniversity,Manhattan,KS,USA,3AnimalSciencesand Industry, Kansas State University, Manhattan, KS, USA, 4Clinical Sciences, Kansas State University, Manhattan, KS, [email protected]:AntimicrobialResistance–3,Room1,12/4/20173:15PMTheuseofantibioticsinfoodanimalshasbeenidentifiedasapotentialhealthhazardtothehumanpopulationduetodevelopment,expansionandspreadofantibioticresistantbacteria.Theobjectiveofthisstudywastoevaluatetheeffectof intermittent-versuscontinuous-administrationoftylosinphosphateontheantibioticresistanceprofileofEnterococcusspp.insteers,aswellasmeasuringitseffectontheincidenceandseverityofliverabscesses.Steers(n=312)wereblockedbysourceandweightandrandomlyassignedtooneof3studygroups:acontinuouslyfedtylosingroup,anintermittentlyfedtylosingroup,andanegativecontrolgroup.Groupsweredividedintopensof13animalseach.Fecalsampleswererandomlycollectedfrom8animalswithineachpenondays0,20and118.Onegramofeachstoolsamplewasdilutedinphosphatebufferedsaline(PBS),thenspiral-platedontoplainm-Enterococcus(ME)agarplates,erythromycin(8µg/ml)-infusedMEagar(MEEry)platesandtetracycline(16µg/ml)-infusedMEagar(METet)plates.Theplateswereincubatedat42oCfor48hrs,afterwhichthecolonyformingunits(CFU)wereestimated.TheLog10CFUforeachsampleonplainME,MEEryandMETetplateswerecalculatedandcompared.Multi-levelmixedlinearregressionwasperformedusingStata®12.1(StataCorp.,CollegeStation,TX).Therewasasignificantperiodeffect(P<0.01)whentheEnterococcusspp.Log10CFUonplainMEagarwascomparedwiththatofMEEry,implyingamajorincreaseintheproportionofenterococciresistanttoerythromycin.Overall,therewasnodifferenceinEnterococcusresistanceprofileacrossthetreatmentgroupsateachofthedifferenttimepointsinthestudy.Thissuggeststhattheprimarydeterminantofincreasingantibioticresistanceduringthefeedingperiodisnottheconcurrentfeedingregimen but rather the feeding environment itself, which presumably reflects cumulative burdens of resistance associated withhistoricallyadministeredantibiotics.Thisfindinghas important implicationsfortemperingexpectationsforreductions inresistancefollowingchangesinpolicy,prescribingandusepatternsinthefuture.18-Veterinaryepidemiology:expandingsolutionsforglobalhealthproblems

J.A.K. Mazet1, P. REDICT Consortium2. 1One Health Institute, University of California, Davis, Davis, CA, USA,www.consortium.predict.global,Davis,CA,[email protected]:PopulationHealthKeynotes,Room1,12/4/20174:00PMThedevastatinglossoflifeassociatedwiththeWestAfricaEbolavirusoutbreakrevealedtheurgentneedforincreasedanimalandpublic health sector capacity strengthening for zoonotic pathogen detection, identification, and surveillance. A collaborativetransdisciplinary team, led by veterinary epidemiologists, has developed a targeted and adaptive wildlife and human pathogensurveillanceapproachaimedatdevelopingandoperationalizingstrategiestoreducezoonoticpathogenspillover,amplification,andspread. It includes the introduction of new technologies, as well as the application of cutting-edge molecular techniques andinformationmanagementtools,torealizeanintegrated,globalapproachtoemerginginfectiousdiseases.Asaresult,theteamhasadvancedOneHealthcapacity inmorethan30countries inemerging infectiousdiseasehotspotregions.Environmental,host,andbehavioraldataarecollected,andsamplesassayedforthepresenceofpotentialzoonoses.Inadditiontodetectingapproximately200knownviruses,wehaveidentifiedmorethan800previouslyundetectedviruses.Bycombiningthesediscoverieswithdataonhuman-wildlife contact and potential pathogenicity, we are assessing risk to inform mitigation strategies. Focusing our work whereenvironments,humanbehaviors,andmarketsystemsarechanginginwaysthatareconducivetothespilloverofvirusesamonghosts,welocateareasposingthehighestriskforexposure;detectandbettercharacterizepathogensofepidemicandpandemicpotential;identifysignificantanimalreservoirsandamplificationhostsofviruses;ascertainthepotentialofvirus-spilloverintoothernon-typicalhosts,suchaslivestockorcompanionanimals;gainagreaterunderstandingofhigh-riskhumanbehavioralactivities;improvediseasesurveillanceandlaboratorycapacitiesthroughworkforcedevelopmentinlinewithGlobalHealthSecurityAgendapriorities;andprovideinformationneeded toefficientlydesign intervention strategies thatpromotewildlife conservationand targetdiseaseemergence,amplification,andspread.

CRWAD2017 ABSTRACTS


19-Biosecurityatequineevents-realityandscienceK.Flynn;AnimalHealthBranch,CADeptofFoodandAgriculture,Sacramento,CA,[email protected]:PopulationHealthKeynotes,Room1,12/4/20174:45PMEquineeventspose aunique risk fordisease agent introductionand transmission. Equine athletes in all disciplines incur stressoffrequenttransportationlocally,nationallyandinternationally.Manyequineeventgroundsandfacilitydesignsallowexhibitorseasy,direct,accesstocompetition/exhibitionareas.Additional risk factorsofcompetitions includethecomminglingofhorsesofvaryinghealthstatus,closestablingofhorses,concentrationofanimalsandhumansonthepremises,frequencyofmovementsonthepremises,andvariousroutinemanagementpracticesoftheeventfacility.Humanandanimalhealthprofessionalsroutinelyperformbiosecuritypracticestodecreaseriskofdiseasetransmission(i.e.,cleaninganddisinfectionofequipmentbetweenuse,disposingofneedlesandsyringes after single use, andwashing hands and boots); however, these simple actions are not currently embraced as standardpracticesbymembersoftheequineindustry.Humanathletesgenerallyusecommonsensewhencompeting.Anathletewithafeverandnasaldischargeisnotlikelytocompeteinanathleticeventandusedtissuesaredisposedof,notsharedamongotherathletes.Incontrast,anowner/trainermay loada febrileequineathletewithmildnasaldischargeontoa trailerwithseveralotherhorses fortransporttoavenueforcompetition,andwithoutathoughtwhilecompeting,wipethenostrilsofmultiplecompetitionhorseswiththe same nose rag as used on the horse with a fever and nasal discharge. Although scientific studies substantiate the diseasetransmission risksposedbyclinically illhumans invarious settings (i.e.,hospitals, clinics,homes), there is limiteddataquantifyinginfectiousdiseaserisksspecificallyassociatedwithpracticesatequineevents(i.e.,useofsharedcrossties,useofsharedfencerailfortyinghorses,theuseofminimally-disinfectedstalls,orthepotentialdiseasetransmissionriskstoahorseinanovercrowded,warm-uparena).Opportunitiesforqualitativeandquantitativeresearchtosubstantiateequinebiosecurityrecommendations/principlesandtoenhanceoutreachandeducationprevail.20-PopulationdynamicsofSalmonellaentericainresponsetoantibioticuseinTexasfeedlotcattleN. Ohta1, K.N. Norman2, B. Norby3, S.D. Lawhon4, J. Vinasco4, H. Bakker5, G.H. Loneragan6, H. Scott4. 1Department of VeterinaryPathobiology, Texas A&M University, College Station, TX, USA, 2Department of Veterinary Integrativee Biosciences, Texas A&MUniversity,CollegeStation,TX,USA,3MichiganStateUniversity,EastLansing,MI,USA,4TexasA&MUniversity,CollegeStation,TX,USA,5UniversityofGeorgia,Griffin,GA,USA,6TexasTechUniversity,Lubbock,TX,[email protected]:PopulationHealthKeynotes,Room1,12/4/20175:30PMCeftiofur,athird-generationcephalosporin,candirectlyselectforgenesencodingresistancetoceftriaxone,oneofthefewantibioticchoicesforhumanpediatricsalmonellosis.Weconductedarandomizedcontrolledlongitudinaltrialtoassesstheeffectsofinjectableceftiofurversusin-feedchlortetracyclineonthetemporaldynamicsofantibiotic-resistantSalmonellainfeedlotcattle.Tworeplicatesof8pens(total176steers)receivedoneof4differentregimens.Alloroneoutof11steersweretreatedwithceftiofurcrystalline-freeacid(CCFA)onday0in8pens,withhalfofthesepenslaterreceiving5-daypulsesofchlortetracycline(CTC)fromday4today20.WeattemptedtoisolateSalmonellafromindividualsteerfecalsamplesonDays0,4,8,14,20,and26(n=1040).Antibioticsusceptibilityofisolateswasanalyzedviamicrobrothdilution.Serotypeswereestimatedbywhole-genomesequencing.Onday0,themeanSalmonellaprevalencewasapproximately75%andthevastmajorityofisolateswerepansusceptibletoapanelof14antibiotics.TreatmentwithCCFAalone,orCTCalone,orCCFAandCTCincombinationreducedtheoverallprevalenceandthequantityofSalmonella;however,thesetreatmentsincreasedtheproportionofmulti-drugresistant(MDR)Salmonellafromday4throughday26.OnlytheSalmonellaserotypesMbandaka,Give,Reading,Kentucky,Montevideo,andAnatumweredetected.All isolatesofS.ReadingwereextensivelyMDR,suggestingastrongcorrelationbetweenserotypeandresistance.OurstudydemonstratesthattheselectionpressureofCCFAandCTCduringthefeedingperiodcontributestodynamicpopulationchangesamongantibioticsusceptibleandresistantSalmonellaintheintestinaltractofcattle.

CRWAD2017 ABSTRACTS


21-Metagenomicinvestigationsofantimicrobialresistanceinbeef,pork,andbroilerproductionsystemsM.D.Weinroth1,S.M.Lankin2,N.R.Noyes3,X.Yang4,P.Rovira1,E.Doster3,C.Dean3,J.K.Parker2,C.Anderson2,Z.Abdo3,C.Boucher5,J.Ruiz5,K.E.Belk1,P.S.Morley2. 1DepartmentofAnimalSciences,ColoradoStateUniversity,FortCollins,CO,USA,2DepartmentofClinicalSciences,ColoradoStateUniversity,FortCollins,CO,USA,3DepartmentofMicrobiology,Immunology,andPathology,ColoradoStateUniversity,FortCollins,CO,USA,4DepartmentofAnimalSciences,UniversityofCalifornia,Davis,Davis,CA,USA,5DepartmentofComputerandInformationScienceandEngineering,UniversityofFlorida,Gainesville,FL,[email protected]:AntimicrobialResistance–4,Room1,12/5/20179:00AMAntimicrobialdruguseinproductionoffoodanimalsisrecognizedasaglobalpublichealthconcernbasedonconcernsthatresistantbacteriacouldbepresentinfoodproducts,andthiscouldinturnhavenegativeimpactsonpublichealth.Antimicrobialdrugsareusedinanimalagriculturefortreatmentandpreventionofdiseaseandtoimprovefeedefficiency;however,differentantibioticsaremorecommonlyutilizedindifferentspecies.Inordertoaddresspublichealthconcernsrelatedtoantibioticresistance,informationisneededtounderstandtheimpactofantibioticusageinfoodanimals.Theobjectiveofthisstudywastousetargetedshotgunmetagenomicsequencingtocharacterizeandcontrastthefecalresistomeofmarketcattle,pig,andchickenfeces.Sixtycompositefecalsampleswerecollectedfromeachproductionsystem(20samplesfromfivepensofcattle,20samplesfromfivechickenhouses,and20samplesfromfivehogbarns).MetagenomicDNAwasextractedfromthefecalsamplesandacustomizedbait-pulldownsystem(Agilent,SureSelectXT)wasusedtobuildlibrariestargetingAMRgenesequences.TheselibrariesweresequencedusingtheIlluminaHiSeqplatform.RawsequenceswereanalyzedusingtheAMR++bioinformaticspipelineandMEGAResdatabaseofAMRgenesequences.Hitstoresistancegeneaccessionswerecharacterizedhierarchicallybyclass,mechanism,andgroupandwerecomparedamongthedifferentproductionsystems.Additionally,theresistomesfromeachproductionsystemweredescribedinrelationtoTheWorldHealthOrganization’slistofCritically ImportantAntimicrobials forHumanMedicine.Beefcattleshedresistancegenesprimarilyassociatedwith tetracyclineresistance,followedbytheMLSclass.Thiswassimilartotheswineoperationthathadthemosthitstotetracyclineresistancebacteria,thoughintermsofrelativeabundance,swineoperationshadahighpercentageofaminiglycosideresistance.BroilerproductionhadthemostevenratioofMLStotetracyclineresistancegeneswhencomparedtotheothersystems.Thesedatashowantibioticresistancegenesvarybylivestockproductionsystem.22-IncorporatingtraditionalbacterialculturemethodsandmetagenomicsequencingtoevaluateantimicrobialuseandresistanceinbeeffeedlotproductionE.Doster1,J.K.Parker2,C.A.Anderson2,S.M.Lakin2,N.R.Noyes2,M.Weinroth3,C.W.Booker4,S.J.Hannon4,S.P.Gow5,T.A.McAllister6,K.E.Belk3,P.S.Morley2.1Microbiology,Immunology,andPathologyDept.,ColoradoStateUniversity,FortCollins,CO,USA,2Dept.ofClinicalSciences,ColoradoStateUniversity,FortCollins,CO,USA,3Dept.ofAnimalSciences,ColoradoStateUniversity,FortCollins,CO,USA,4FeedlotHealthManagementServices,Okotoks,AB,Canada,5CentreforFood-borne,EnvironmentalZoonoticInfectiousDiseases,PublicHealthAgencyofCanada,UniversityofSaskatoon,Saskatoon,SK,Canada,6Microbiology, Immunology,andPathologyDept.,AgricultureandAgri-FoodCanadaResearchCentre,Lethbridge,AB,[email protected]:AntimicrobialResistance–4,Room1,12/5/20179:15AMThe increasingprevalenceofantimicrobialresistance(AMR) isaglobalpublichealthconcern,and iscommonlyhypothesizedtobe“driven”byantimicrobialuse(AMU)inhumansandfoodproducinganimals.Traditionally,studiesofAMRuseaerobicculturetostudyjustafewbacterialspeciesfromacomplexbacterialcommunity(themicrobiome),andresultscandifferdependingonthespeciesunderstudy.However,advancementsinhigh-throughputsequencingcanbeusedtoprovideaholisticperspectiveintoAMRecologybysequencingDNAfromtheentiremicrobiome,includingthe“population”ofresistancegenes(theresistome).Inthisstudyweusedmetagenomicsequencingtoanalyzethemicrobiomeandresistomeinfecescollectedduringapreviouslypublished3-yearlongitudinalstudy of Canadian beef feedlot operations. The goal of the previous study was to investigate the effect of AMU practices onsusceptibilitypatternsofnon-type-specificEscherichiacoliandMannheimiahaemolytica.Pensofcattlewererandomlyselectedforinclusionintothestudyandpooledfecalsampleswerecollectedfromthepenfloorwhencattlearrivedtothefeedlotandataseconddateduringthefeedingperiod.AllAMU,includingparenteraltreatmentsandin-feedexposures,wasrecordedandsummarizedusinganimaldefineddailydose(ADD).PenlevelAMUwascalculatedasthesumofADDsforallcattlehousedinapen.Asubsetof42penswasrandomlyselectedforinclusioninthepresentstudy,basedoncategorizationofwhenthesecondsetoffecalsampleswasobtained:<100daysafterarrivalatthefeedlot(n=21)and>100days(n=21.)Ourresultscharacterizethemicrobiomeandresistomeecologyinbeeffeedlotoperations;further,wewereabletomakeauniquecomparisonofstudiesinvestigatingtheimpactofAMUonAMRwhenusingtraditionalaerobicculturemethodsincomparisontonewermethodsusingshotgunmetagenomicsequencing.

CRWAD2017 ABSTRACTS


23-CarbapenemresistantEnterobacteriaceaepresentinwastewatertreatmentplanteffluentandnearbysurfacewatersintheUSD.A.Mathys1,D.F.Mollenkopf1,S.M.Feicht1,R.J.Adams1,A.L.Albers1,D.B.Stuever1,J.B.Daniels2,T.E.Wittum1.1VeterinaryPreventiveMedicine, Ohio State University, Columbus, OH, USA, 2Veterinary Clinical Science, Ohio State University, Columbus, OH, [email protected]:AntimicrobialResistance–4,Room1,12/5/20179:30:AMCREareacriticallyimportantthreattothepublichealth,andhavebeenidentifiedasanurgentthreatbytheCDCandWHO.CREarerarebuthighlyresistantorganismsmostcommonlyassociatedwithhospitalizedpatients.MetropolitanWWTPsfilterwaterfromlargegeographicareaswhichoftenincludehospitals,andcanserveasmaintenancereservoirsforCRE.However,littleisknownaboutthepotential impact of theseWWTP CRE on the local surfacewater. If CRE are present in the downstream surfacewater, theymayultimatelydisseminatetointensively-managedanimalagriculturefacilities.Weobtainedone-litersamplesfromtheeffluentandboththeupstreamanddownstreamsurfacewaterfrom50WWTPsthroughouttheUnitedStates.Sampleswerevacuumfilteredusingaseriesofsterilefiltersculminatingina0.45µmporesizefilter.Allfilterswereincubatedovernightwith100mlofMacConkeybrothmodifiedwith0.5µg/mlofmeropenemand70µg/mlofzincsulfate,theninoculatedontosimilarlyenrichedMacConkeyagartoidentifycarbapenem-resistantphenotypes.IsolatesthenunderwentCarbaNPtestingtoconfirmcarbapenemaseproductionandwerespeciesidentifiedusingMALDI-TOFmassspectrometry.Ofthe50WWTPs,26werefromlargemetropolitanareas,while24camefromsmalltownswithpopulations lessthan10,000.Ourresults indicatethatsurfacewater intheUS isroutinelycontaminatedwithclinicallyimportant,hospitalassociatedCRE.Thisamajorconcernforpublichealthandagriculture,becauseintroductionofCREintointensivelymanagedagriculturalenvironmentscouldleadtoamplificationandfoodbornedisseminationtolargepopulations.24-Recoveryofcarbapenemase-producingEnterobacteriaceaefromwasteandsurfacewatersD.F.Mollenkopf1,D.A.Mathys1,S.M.Feicht1,A.L.Albers1,E.K.Sechrist1,G.A.Ballash1,R.J.Adams1,D.B.Stuever1,S.Lee2,J.Lee2,T.E.Wittum1. 1VeterinaryPreventiveMedicine,Ohio StateUniversity, Columbus,OH,USA, 2EnvironmentalHealth Sciences,Ohio StateUniversity,Columbus,OH,[email protected]:AntimicrobialResistance–4,Room1,12/5/20179:45:AMCarbapenemase-producingEnterobacteriaceae(CRE)aremigratingbeyondhumanhealthcarereservoirs.Whilecolonizedpatientsorhospital staff canmoveCRE into the local community,medical facility effluentwastewater can serve as an overlooked vehicle todisseminate CRE into the environment. Healthcare-associated wastewater enters the sanitary sewer system and is received at awastewatertreatmentplant(WWTP)whereitisreclaimedanddischargedintothelocalwaterway.ToassessthecontributionofCREinurbanwastewatertothereceivingriver’sbacterialpopulation,wecollectedforty-fourweeklyonelitersamplesofWWTPinfluentandeffluentandsurfacewaterfrombothanupstreamandtwolocationsdownstreamofthedischargepoint.Onthedayofcollection,sampleswerevacuum-filteredthroughmultipleporesizefilterswithfinal filtrationusinga0.45μmporefiltertocapturebacterialcolonies.Resulting filterswere incubatedovernight in100mlMacConkeybrothsupplementedwith0.5μg/mlmeropenemand70μg/mlzincsulfate.TheselectivebrothwasasepticallyinoculatedtoMacConkeyagarsupplementedwith0.5μg/mlmeropenemand70μg/mlzincsulfate.Afterincubation,uptothreeisolateswereselectedfromeachplatewithpreferencegiventolactosepositivecolonies.TheresultingisolatesweretestedforcarbapenemaseproductionusingtheCarbaNPtestwithpositiveisolatesspeciatedbyMALDI-TOF.Overone-third(34%)oftherecoveredisolateswithmobilecarbapenemasegeneswererecoveredfrominfluentwater,followedbytheeffluent(17%),downstream(11%),andwaydownstream(12%)locations.Only4%oftherecoveredCREwerefoundintheupstreamwatersuggestingthatthesehighlyresistantbacteriaareenteringthelocalwatershedviawastewaterandpresentapublichealththreat.

CRWAD2017 ABSTRACTS


25-Characterizationofplasmidmediatedcarbapenemase-producingEnterobacteriaceaefromwasteandsurfacewatersD.F.Mollenkopf1,D.A.Mathys1,S.M.Feicht1,A.L.Albers1,E.K.Sechrist1,G.A.Ballash1,R.J.Adams1,D.B.Stuever1,S.Lee2,J.Lee2,T.E.Wittum1. 1VeterinaryPreventiveMedicine,OhioStateUniversity,Columbus,OH,USA, 2EnvironmentalHealthSciences,OhioStateUniversity,Columbus,OH,[email protected]:AntimicrobialResistance–4,Room1,12/5/201710:00AMWastewater from large medical centers routinely transports carbapenemase-producing Enterobacteriaceae (CRE) to wastewatertreatmentplantswheretheyaremaintainedandultimatelydischargedintotheenvironmentinsurfacewater,posinganimportantpublic health risk.We characterizedover 400non-chromosomallymediated carbapenemase-bearing isolates recovered frombothinfluentandeffluentwastewateratalocalwastewatertreatmentplant(WWTP)whichreceiveseffluentfrommultiplemetropolitanhospitalsandfromsurfacewatercollectedbothupstreamanddownstreamoftheWWTPdischarge.IdentifiedbyMALDI-TOF,bacterialspeciesnotassociatedwithchromosomalcarbapenemasegenecarriagewereassessedusingconventionalPCRandSangersequencingforthepredominantcarbapenemresistancegene,blaKPC.blaKPC-negativeisolateswerecharacterizedusingwholegenomesequencing.Todate,overhalfoftheplasmidmediatedCREisolateshavebeenidentifiedasEnterobactersp.(58%),followedbyKlebsiellasp.(17%),Escherichiacoli(12%),Citrobactersp.(7%),andRaoultellasp.(5%).blaKPCisthedominantcarbapenemasegene,detectedin84%oftheseisolateswithmostharboringtheblaKPC-2gene.However,weidentifiedblaKPCinonly26%ofE.coliand64%ofRaoultellaisolates.WhileWWTPreclamationdoesappear to reduce theprevalenceofCREbetween the influentandeffluent flows, thedownstreamcollectionaremorereflectiveoftheeffluentCREprevalencethantheprevalenceofCREdetectedupstreamofthedischargeandposesanimportantrisktothedownstreamwatershed.26-RecoveryofcarbapenemresistantEnterobacteriaceaefromwildlife,agriculture,andtheenvironmentintheSciotoRiver,Ohio

watershedG.Ballash1,D.Mollenkopf1,D.Mathys1,A.Albers1,E.Sechrist1,D.Symonds2,B.Zimmerman2,M.Sullivan2,T.Wittum1.1VeterinaryPreventiveMedicine,TheOhioStateUniversity,Columbus,OH,USA,2SchoolofEnvironmentalandNaturalResources,TheOhioStateUniversity,Columbus,OH,[email protected]:AntimicrobialResistance–5,Room1,12/5/201710:45AMCarbapenem-resistantEnterobacteriaceae (CRE)area significantpublichealthconcern.Currently, thesebacteriaare isolated fromindividualswithrecenthealthcareexposure,buttheirprevalenceintheenvironmentandanimalsisunknown.Herewecollectedsoiland crop samples,wildlife anddairy cattle fecal samples, and fish andbird vent swabs fromwithin the SciotoRiverwatershed, awatershed inColumbus,OHthatreceivestreatedwastewateroriginatingfromamajormedicalcenter.Sampleswerescreenedforreducedsusceptibilitytomeropenemandcarbapenemaseproduction.Carbapenemase-producingisolateswereclassifiedusingMALDI-TOF and subjected to PCR and gene sequencing, if the isolate was suspected to carry a transferable carbapenemase gene. Theprevalenceofcarbapenemaseproducingisolateswas8.33%(3/36)ofwildlifeisolates,26%(8/50)ofcropisolates,15.2%(10/66)ofsoilisolates,11.2%(36/322)ofdairycattle isolates,34.5%(10/29)ofbird isolatesand16.2%(73/450)offish isolates.Carbapenemase-producing isolates from the soil, crops, dairy cattle, wildlife and birds were primarily species with chromosomally-encodedcarbapenemasegenesconferringintrinsicresistancesuchasStenotrophomonassp.andPseudomonasotitidis.However,12fishisolatesweremembersofEnterobacteriaceaesuspectedtocarrytransferablecarbapenemasegenesonmobileplasmids.Thesefindingssuggestthatcarbapenem-resistantEnterobacteriaceaehavespreadbeyondthehealthcaresettingandintotheenvironment,likelyasaresultofwaterbornetransmission.Thepublichealthsignificanceofthesefindingsisnotyetfullyunderstood.

CRWAD2017 ABSTRACTS


27-TheefficacyofpulsedelectricfieldtoreduceantimicrobialresistantbacteriainwastewaterA.Albers,D.Mollenkopf,D.Mathys,T.Wittum.DepartmentofVeterinaryPreventativeMedicine,TheOhioStateUniversity,Columbus,OH,[email protected]:AntimicrobialResistance–5,Room1,12/5/201711:00AMAntimicrobialresistantbacteriaareofconcerninbothhumanandveterinarymedicine.PulsedElectricFieldusesanelectriccurrenttolysebacterialcellsandiscurrentlyusedtoreducewastewatercoliformcountsinseveralwastewatertreatmentplants.OurobjectivesweretodeterminetheefficacyofPulsedElectricField (PEF)asan interventiontobeused inhighriskenvironmentstoreducetheamount of viable carbapenemase-producing bacteria in raw influent before entering the wastewater treatment plant. UntreatedinfluentsampleswerecollectedfromawastewatertreatmentplantservingColumbus,Ohiotwiceweeklyfor24weeks.Thesampleswere strained to remove any large particles prior to treatment. A small aliquot of samplewas assessed for conductivity, pH, andturbidity.AftertreatmentwithPEF,a100µlaliquotfrombothinfluentandtreatedsampleswasspreadplatedontobothCHROMagar,andpetrifilmstoquantifythereductionofE.coli,coliforms,Pseudomonassp.,andAcinetobactersp.Theresultsindicateapproximatelya one log reduction of both coliforms and carbapenemase-producing bacteria following treatment. We observed an additionalreductionofcoliformsandcarbapenemase-producingbacteriabyusingalongertreatmenttime.TheseresultssuggestthepotentialforPEFtreatmenttoreducethedischargeofcarbapenemase-producingbacteriafromhighriskenvironmentssuchashospitalICUsintowastewaterflowsandultimatelyintotheenvironment.28-Multidrugresistant(MDR)Salmonellaheidelberg:anemergingprobleminthedairyindustryN.Aulik1,D.Sockett1,K.Deering1,A.Valley2,R.Klos3.1WisconsinVeterinaryDiagnosticLaboratory,Madison,WI,USA,2WisconsinStateLaboratoryofHygiene,Madison,WI,USA,3WisconsinDivisionofPublicHealth,Madison,WI,[email protected]:AntimicrobialResistance–5,Room1,12/5/201711:15AMA multi-agency outbreak investigation was initiated when bovine isolates of Salmonella enterica subspecies enterica serotypeHeidelberg were confirmed to share the same molecular finger print and antibiotic resistance profile as isolates obtained fromindividualswhobecameillafterbuyingcalves.Reviewoflaboratorydatarevealedthatinlate2015,theWisconsinVeterinaryDiagnosticLaboratorystartedreceivingdiagnosticsamplesfromdairybullcalves(lessthan3weeksofage)thatdiedwithinhoursofexhibitingsymptomsorwerefounddeadwithoutpriorsymptoms.Someofthecalveshaddiarrheaandafever(>40oC),butmanycalvesdidnot.MDRSalmonellaHeidelbergwasisolatedfrommultipleorgansconsistentwithbacteremia.AsofAugust2,2017,46confirmedhumancasesofMDRSalmonellaHeidelberghavebeenreportedtotheCenterforDiseaseControlandPrevention(CDC)occurringinresidentsof14states.Fourteen(30%)peoplehavebeenhospitalized.Two-thirdsofpeoplereportedhavingcontactwithdairybullcalvesorothercattlejustpriortoonsetofsymptoms.MorethanhalfoftheillnessesinWisconsinoccurredinchildrenunder18yearsofage.Wholegenomesequencingofhuman,bovineandenvironmental isolatesalsoconfirmedthatthesestrainsarehighlyrelated.AsofAugust24,2017,theWVDLhasisolatedMDRSalmonellaHeidelbergfrom34uniquepremiseslocatedin5states(IN,SD,MN,MOandWI).Themajorityofthelivestockoperations(80%)werelocatedinWisconsinwithatleasttwo-thirdsoftheisolatessubmittedbydairybeefoperationsthatexperiencedhighdeathloss(25-65%).Thisorganismisonlysusceptibletogentamicinandtherefore,therearenorecommendedantimicrobialdrugseffectiveforuseincattle,sotreatmentissupportivecare.ControlofMDRSalmonellaHeidelbergrelies on proper cleaning and disinfection. The WVDL and other state and federal authorities continue to collaborate on thisinvestigationandworktobetterunderstandthedistributionandfactorsaffectingspreadofthisorganism.

CRWAD2017 ABSTRACTS


29-EpidemiologyofantimicrobialresistantgenericEscherichiacoliamongfree-livingCanadageeseinOntario,CanadaN.A.Vogt1,D.L.Pearl1,M.R.Mulvey2,N.Janecko3,R.Reid-Smith1,C.M.Jardine4.1DepartmentofPopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2PublicHealthAgency,NationalMicrobiologyLaboratory,Winnipeg,MB,Canada,3PublicHealthAgency,CentreforFoodborne,EnvironmentalandZoonoticInfectiousDiseases,Guelph,ON,Canada,4DepartmentofPathobiology,UniversityofGuelph,Guelph,ON,[email protected]:AntimicrobialResistance–5,Room1,12/5/201711:30AMCanadageesehavepreviouslybeenidentifiedaspotentialreservoirsandtransmittersofantimicrobialresistantbacteria.Thereareconcernsthatthesebirdsmaytransmitantimicrobialresistantbacteriafromonegeographiclocationtoanotherbecauseoftheirhighlevelofmobilityassociatedwithforagingaswellasmigration.OurstudyobjectivewastoexaminetheprevalenceandpatternsofcarriageofantimicrobialresistantgenericE.coliamongCanadageeseinsouthernOntario.FecalswabswereobtainedfromlivebirdsinparksinGuelph,OntariofromMaythroughOctober,2016.Escherichiacoliisolatesweretestedforsusceptibilityto14antimicrobialsusing theCanadian IntegratedProgramforAntimicrobialResistanceSurveillance (CIPARS)panel.Escherichiacoli isolateswerealsoscreenedforphenotypicresistancetocolistinusingselectivemedia,andPCRwasusedtodetectmcr-1andmcr-2.Usingmultilevellogisticregressionwitharandominterceptforflock,the impactofseasonandageclass(adultvs. juvenile)wereexaminedforthefollowingoutcomes:colistinresistance,andresistanceto≥1classand≥2classesofantimicrobialsontheCIPARSpanelinE.coliisolatesfromgeese.Escherichiacoliwasisolatedfrom73.0%offecalsamples.BasedontheCIPARSpanelwith8classesofantimicrobials,3.8%ofE.coliisolateswereresistantto≥1classofantimicrobials,and1.7%wereresistantto≥2classes.Resistancetocriticallyimportantantimicrobialsforhumanmedicinewasalsoidentified,includingresistancetoamoxicillin&clavulanicacid(0.42%),cefoxitin(0.42%),ceftriaxone (0.42%),andcolistin (6.0%).AllE.coli isolateswithphenotypic resistancetocolistinweremcr-1andmcr-2negative.AsignificantassociationwasonlynotedbetweenseasonandtheprevalenceofcolistinresistanceinE.coliwithpeakprevalenceoccurringinlatesummer.TheprevalenceofCanadageesesheddingantimicrobialresistantE.coliisgenerallylow,butcanreachrelativelyhighlevelsduringspecificperiods.Longtermstudiesareneededtounderstandwhetherapeakincolistinnotedduringthelatesummerreflectedaseasonaleffectorasporadicevent.30-SequencecharacterizationofanIncHI2plasmidwithanencodedcarbapenemase-geneS.V.Grooters,J.Daniels,D.Mollenkopf,D.Mathys,T.Wittum.VeterinaryPreventiveMedicine,TheOhioStateUniversity,Columbus,OH,[email protected]:AntimicrobialResistance–5,Room1,12/5/201711:45AMEpidemiologyfrequentlyutilizeswholegenomesequencingforcomparativegenomics.Nextgenerationsequencingtechnologieshavevaryingapplicationsforthispurpose.WeattemptedtoutilizeWGSsequencedatatocharacterizeabacterialplasmidencodingblaKPC-4. Initially,pairedend Illumina readsof thewholebacterialgenome, includingbothchromosomeandplasmidDNA,wereused forsequenceanalysis.However,assemblyofthisshort-readsequencingfailedtolocatethegeneresponsibleforcarbapenem-resistanceon the same contiguous sequence as plasmid origin. Bioinformatics tools that differentiate plasmid from chromosome (e.g.PlasmidSPAdes)dosobyvariationsinsequencecoverage.Whenchromosomeandplasmidsequencecoveragearesimilar,orplasmidsizeisrelativelylarge,thenitmaybedifficultorimpossibletofullyseparatechromosomalandplasmidDNA.Inthissituation,long-readsequencingtechnologymaybeamoreeffectivetool.Long-readPacBiosequencingiscostlierandrequiresadditionalattentiontodetailinDNAisolation,buthastheabilitytofullydistinguishchromosomalandplasmidDNA.MinIONtechnologyalsoproduceslongreadsandmaybemoreaccessibletomanypotentialusers.WhenweusedSPAdessoftwaretoassemblethetrimmedIlluminareads,scaffoldscontainingblakpc-4andthemarkersforplasmidincompatibilitygroupscouldnotbefullyassembledtoaccuratelyannotatetheplasmid.Similar results were obtainedwhen Illumina readswere combinedwithMinion reads for assembly. However, PacBio sequencingproducedasinglelinearcontigandtwocircularcontigsthatfullyrepresentedthebacterialchromosomeandtwoplasmids,allowingfull characterizationof the IncHI2 plasmid. Identifying the genes responsible for antimicrobial resistance, and fully annotating theplasmidsthatmaytransferthesegenesrequiresnotonlynextgenerationsequencingtools,butathoroughworkingknowledgeoftheapplicationofbioinformaticstoolsaswell.

CRWAD2017 ABSTRACTS


31-UseofwholegenomesequencetoidentifynewsequencetypesofStreptococcusuberisfromdairycattleJ.ReyesVelez,C1A5V21,J.Rodriguez-Lecompte1,J.Sanchez1,R.Zadoks2.1UPEI,Charlottetown,PE,Canada,2InstituteofBiodiversity,UniversityofGlasgow,Charlottetown,[email protected]:AntimicrobialResistance–1,Room2,12/4/20179:00AMTheprimaryobjectiveofthisstudywastodeterminethegeneticvariationofStreptococcusuberis(S.uberis)isolates,throughwholegenomesequencing(WGS)usingMultilocussequencetyping(MLST)technique,recoveredfromlactatingcowsindairyherdsfromtheAtlantic regionof Canada. The secondaryobjectivewas todetermine thephenotypic and genotypic antimicrobial resistant (AMR)characteristicsoftheseisolates.WholegenomeswereanalysedthroughthebacterialAnalysisPipelinewiththeMLST-1.6toolandtheBacterialIsolateGenomeSequenceDatabaseusingaSevenhousekeepingMLSTscheme.Sixtytwoisolateswererecoveredfrom16herdsdistributedinthreeAtlanticCanadianprovinces:NewBrunswick(14.5%),NovaScotia(48.3%),andPrinceEdwardIsland(37.1%).Forty-fivepoint fivepercentof theS.uberis isolateswere recovered fromhealthy lactational samples (Lactational andpre-dry-offsamples),37.1%fromcowsthathadclinicalmastitis (2and5weeksafterarecordedclinicalmastitisevent),and17.4%fromcowsbetweenpost-calving(0and14daysinmilk).Atotalof62S.uberisgenomeswereclassifiedinto34differentsequencetypes,while52genomeswere classified into 26 unique new ST, and the remaining 10 genomeswere classified into existing STs in the pubMLSTdatabase.Atotalof13isolatesweregroupedwithinthreespecificclonalcomplexes.Isolatesfrompost-mastitissamplescontainedthemajorityuniqueSTs(10STs),followedbyhealthyLactationalsamples(9STs),andpost-calvingsamples(5STs).TheST-857waspresentinallthreeprovinces.AlargemajorityofSTswerefoundonlyonceacrossthedairyherds;however,ST-851,ST-855,ST-864,andST-866werepresentmorethanonceinthesameherdandindifferentcows.SpecificSTsshowedphenotypicresistancetoatleastoneoftheeightantimicrobialstested,andexhibitedmultipleAMRandvirulencegenesacrossallSTs.TheresultsofthisstudyindicatedthatS.uberis isageneticallydiversepathogen,withnotclearradialgroups.Moreover,differentresistanceandvirulencemarkerswereconservedacrossthepopulationofSTswithnospecificpatternonpathogenicitypotential.32-GenotypicdifferencesbetweenLA-MRSAST5andMRSAST5fromhumanswithnoswinecontactS.J.Hau1,A.Allué-Guardia2,B.Rusconi2,J.S.Haan3,P.R.Davies4,T.S.Frana5,M.Eppinger2,T.L.Nicholson6.1VeterinaryMicrobiologyandPreventativeMedicine,IowaStateUniversity,Ames,IA,USA,2SouthTexasCenterforEmergingInfectiousDiseases,UniversityofTexasatSanAntonio,SanAntonio,TX,USA,3EntericsUnit-InfectiousDiseaseLab,MinnesotaDepartmentofHealth,StPaul,MN,USA,4DepartmentofVeterinaryPopulationMedicine,UniversityofMinnesota,StPaul,MN,USA,5DepartmentofVeterinaryDiagnosticandProductionAnimalMedicine, IowaStateUniversity,Ames, IA,USA, 6AgriculturalResearchService,NationalAnimalDiseaseCenter,Ames,IA,[email protected]:AntimicrobialResistance–1,Room2,12/4/20179:15AMPurpose:Livestock-associatedmethicillin-resistantS.aureus(LA-MRSA)hascausedpublichealthconcernsstemmingfromthepotentialthatlivestockareareservoirofMRSAoutsideofhospitalsettingsandLA-MRSAisolatesharbortransmissibleantimicrobialresistance(AMR)genesorvirulencefactorsharboredonmobilegeneticelements(MGEs).ReportsinEuropeidentifiedsequencetype(ST)398asthepredominantlineageofMRSAinswine,whilelineagesdetectedintheU.S.includeST398,ST9,andST5.LA-MRSAST398isolatesarelesscapableofhuman-to-humantransmissionandhavefewervirulencefactorsthanMRSAisolatesfromhumans.LA-MRSAST5isolateselevatedpublichealthconcernsbecause,unlikeMRSAST398andST9,theST5lineageisagloballydisseminatedandsuccessfullineageinhumans.ToaddresstheLA-MRSAST5publichealthconcerns,weevaluatedthegeneticrelatednessofswineassociatedandclinicalMRSAST5isolatesandcomparedtheMGEsofLA-MRSAST5andclinicalMRSAST5isolates.Methods:Draftgenomesofswine-associated(n=82)andclinicalMRSAST5isolates(n=71)wereusedforsinglenucleotidepolymorphism(SNP)detectionandphylogeneticanalysisandcomparisonofAMRgenesandvirulencefactors.Results:LA-MRSAST5werephylogeneticallydistinctfromclinicalMRSAST5.LA-MRSAST5wereclonalwithinproductionsystems,allowingtracebackusingfarmspecificSNPprofiles.ClinicalMRSAST5isolateshadahigherdegreeofgenomeplasticitythanLA-MRSAST5isolates.SNPanalysiswassupportedbyMGEanalysis,whichshowedLA-MRSAandclinicalMRSAST5isolatesharbordifferentAMRgenesandvirulencefactors.Conclusions:Here,wedeterminedLA-MRSAandclinicalMRSAST5isolatesweredistinctpopulationsoftheST5lineage, indicatingLA-MRSAST5maynotposethesameriskasMRSAST5isolatesfoundwithinthehospitalsetting.ScreeningoftheaccessorygenomerevealeddifferencesinAMRgenesandtheprevalenceofvirulencefactors.Combined,ourdataindicategeneticexchangebetweenthesepopulationsisunlikelyandLA-MRSAST5arenotcontributingtotheburdenofMRSAinhealthcaresettingsinregionswithouthighdensityswineproduction.

CRWAD2017 ABSTRACTS


33-MicrobialshiftsintheswinenasalmicrobiotainresponsetotheparenteralantimicrobialadministrationM.Zeineldin,B.Aldridge,J.Lowe.IntegratedFoodAnimalManagementSystems,DepartmentofVeterinaryClinicalMedicine,IllinoisUniversityatUrbana-Champaign,Urbana,IL,[email protected]:AntimicrobialResistance–1,Room2,12/4/20179:30AMThereisagrowingevidencethatmucosalmicrobialpopulationscontributesignificantlytolocalandsystemicandimmunedefenses.Whileantimicrobialsarecost-effectivetoolsforthepreventionandtreatmentofinfectiousdisease,thecontinuousadministrationofantimicrobials has been widely criticized with the increase of antimicrobial-resistant bacteria and dysbiosis of the beneficialmicrobiotas.Thepurposeof thisstudywastocharacterizethe impactofparenteralantibioticsadministrationonthecomposition,diversityandfunctionalprofilesoftheresidentnasalmicrobiotainpigs.Fiveantimicrobialtreatmentgroups,eachconsistingoffour,eight-week old piglets, were administered one of the antimicrobials: Tulathromycin (TUL), Ceftiofur Crystalline free acid (CCFA),Ceftiofur hydrochloride (CHC), orOxytetracycline (OTC) at label dose and routeor ProcainePenicillinG (PPG) at 33,000IU/kg, IM.Individualnasalswabswerecollectedimmediatelybeforeantimicrobialadministration(control=day0),andagainondays1,3,7,and14afterdosing.GenomicDNAwasextracted,andtheV1-V3regionof16SrRNAgenewasamplifiedandsequencedusing IlluminaMiseq-basedsequencing.Acrossallsamples,themostpredominantphylawereFirmicutes,proteobacteriaandBacteroidetes.While,themostpredominantgenerawereMoraxella,Clostridium,Streptococcus,CalothrixandPrevotella.AshiftintherelativeabundanceofseveralmicrobialgeneraandfunctionalprofileswereobservedafteradministrationofTUL,CCFAandCHC.Onlyminoralterationsinmicrobiota relative abundancewere notedwith the administration ofOTC and PPG.Discriminant analysis showed a pronounced,antimicrobial-dependentshiftinthecompositionofnasalmicrobiotaovertimefromday0.Basedonourresults,exposuretoasingle,parenteraldoseofanantimicrobialhasasignificantimpactonthecompositionandfunctionofnasalmicrobiota.Understandingthehealthimpactoftheseimportantantimicrobial-inducedchangestothemucosalmicrobiotawillbeacriticalstepinoptimizingtheuseofantimicrobialsinhealthmanagementprogramsintheswineindustry.34-CharacterizationandprevalenceofSalmonellaenterica inthefecesoffeedlotcattlefedrationswithandwithoutTylosinor

TylosinalternativesC.J. Weissend, K.L. Holzer, K.L. Huebner, J.L. Metcalf, I. Geornaras, J.K. Parker, C. Anderson, K.E. Belk, P.S. Morley, J.N. Martin.ColoradoStateUniversity,FortCollins,CO,[email protected]:AntimicrobialResistance–1,Room2,12/4/20179:45AMThere is increasingpressure toreducetheuseofantimicrobialdrugs inanimalproductionbecauseofconcernsabout theharmfulimpactstheymayhaveonthepromotionofantimicrobialresistance.TylosinphosphateisamacrolidecurrentlyusedinNorthAmericaforthereductionandpreventionofliverabscessesinfeedlotcattle.Theremovaloftylosinfromcattlefeedingcouldhavesignificanteconomicandfoodsafety impacts. In lightofthis,ablinded,randomized,controlledfieldtrialwasconductedtoevaluateeffectoftylosinandtylosinalternativesonthemicrobialpopulationsoffecesfromfeedlotcattle.Steersandheifers(n=5,481hdhousedin40pensatacommercialfeedyardinTexas)wererandomlyassignedtooneoffourtreatmentgroups:1)Finishingrationwithtylosin(90mg/hd/d) (Tyl); 2) Finishing rationwithout tylosin (NTyl); 3) Finishing rationwithout tylosin,butwithanessentialoil (EsOil);or4)Finishing rationwithout tylosinbutwitha yeast fermentationproduct (18g/hd/d) (SCP). Compositepen-floor fecal sampleswerecollected from each pen at the time of placement and just prior to harvest. Detection and isolation of Salmonella entericaweredetermined through enriched culture methods. Across all treatment groups the prevalence of Salmonella enterica was 85% atplacementand95%atharvest.Therewasnochange(P>0.05)inprevalenceofSalmonellafromthetimeofplacementtoharvest,norwasthereadifference(P>0.05)inprevalencebetweenthetreatmentgroupsateitherplacementorharvest.Isolatesarecurrentlyundergoingcharacterizationandassessmentofantimicrobialsusceptibility.Asfurtherinvestigationoftylosinalternativescontinues,understandingtheimpactontherelatedmicrobialpopulationswillaidinimprovingtheirefficacyaswellasassuringthesafetyofthebeefsupply.

CRWAD2017 ABSTRACTS


35-ThereportingcharacteristicsofbovinerespiratorydiseaseantibiotictrialspublishedpriortoandfollowingpublicationoftheREFLECTstatement

S.Totton1,J.Cullen2,J.Sargeant3,A.O'Connor2.1noaffiliation,nostreetaddress,ON,Canada,2IowaStateUniversity,Ames,IA,USA,3UniversityofGuelph,Guelph,ON,[email protected]:AntimicrobialResistance–1,Room2,12/4/201710:15AMOurobjectivewastodeterminetheprevalenceofREFLECTStatementreportingitems1to19withrespecttocontrolledtrialspublishedbetween1970and2017examiningthecomparativeefficacyofFDA-registeredantimicrobialsagainstnaturallyacquiredBRD(bovinerespiratorydisease)inCanadianand/orUSweanedbeefcalves.WesearchedMEDLINE®andCABI(CABAbstracts®andGlobalHealth®)(Web of ScienceTM) in April 2017 and screened 2327 references. Two reviewers independently assessed the reporting of the 19REFLECT items.Ninety-five referenceswereeligible for thestudy.Fifty-three (79%)of67studiespublishedbefore2010andall28(100%) papers published after 2010 reported using a random allocationmethod in either the title, abstract ormethods section.However, 8 studies published prior to 2010 and 7 studies published after 2010 reported the term “systematic randomization” orvariationsofthisterm,whichisnottruerandomization.ThereportingoftheotherREFLECTitems(apartfromitem10.3(whoassignedstudyunitstotheinterventions),item13(theflowofstudyunitsthroughthestudy),item18(multiplicity)anditem19(adverseeffects))showedanincreasedproportionofstudiesreportingtheitemssubsequenttothepublicationoftheREFLECTStatement.Thereportingofrecommendeditemsinresearchreports inthisbodyofworkisgenerally improvinghoweverthereisalsoevidenceofconfusionaboutwhat constitutes a random allocation procedure, and this suggest an educational need. As this study is observational, thisprecludesconcludingthatthepublicationoftheREFLECTStatementwasthecauseofthistrend.36-Alternativestoantibioticuseinbeefcattle:ascopingreviewL.V.Wisener,J.M.Sargeant.PopulationMedicine,UniversityofGuelph,Guelph,ON,[email protected]:AlternativestoAntibiotics,Room2,12/4/201710:45AMThepurposeofthisscopingreviewwastoidentifyandcharacterizetheevidencepertainingtoalternativestoon-farmuseofantibioticsforhealthconditionsinbeef.Expertopinionsobtainedfromthebeefindustry,governmentrepresentatives,andacademicsinformedthesearchandcitationinclusioncriteria.Searchstringsincludedspecifictermsforpopulationandinterventionsintheformofhealthproductsormanagementpractices.DatabasessearchedforEnglishpublishedandunpublishedcitationssince1990includedWebofScience,Medline,PubMed,Agricola,andABI/INFORM.Twopersonsindependentlyreviewedallcitationswithdisagreementsresolvedby consensus. Only citations that described an intervention and health outcome in the title or abstract were included forcharacterization.Basedonthefulltext,includedstudieswerecharacterizedbystudyandpopulationtype,intervention,comparatorgroup,healthoutcomesandotheroutcomesmeasured,studysize,countryorregionofstudyandpublicationyear.Thedatabasesearchidentified13,553uniquecitationsforeligibilityscreening.Ofthese,1,074(8%)wereincludedforcharacterization.ThemajorityofthestudiestookplaceintheUSA,Canada,orWesternEurope(91%).Thereweretwiceasmanyfieldtrialsinexperimentalpopulationsvs.commercial populations. Only 17 studies described a health outcome for a non-antibiotic treatment or management practiceinterventiongroupdirectlycomparedtoanantibiotictreatmentgroup.Themostcommoninterventiontypeswerenon-antibioticfeedormilk replacer additives followed by vaccination of calves or dams then feed types and feeding schedules. This scoping reviewidentifiednumerousspecificinterventionsthatareavailableforevaluationofefficacythroughfurtherknowledgesyntheses.Althoughawidevarietyofinterventionproductsandmanagementpracticeshavebeenstudied,veryfewstudieshavedirectlycomparedtheseinterventionstotreatmentgroupsthatreceivedantibiotics,representingagapintheliterature.

CRWAD2017 ABSTRACTS


37-Zincandmentholasalternativestoantibiotics:impactsonEnterococcusspp.resistanceinfeedercattleS.A.Murray1,K.N.Norman2,S.D.Lawhon1,R.G.Amchawadi3, J.Vinasco1,R.A.Pugh1, J.S.Drouillard4,C.L.VanBibber-Krueger4,T.G.Nagaraja3,H.M.Scott1.1VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,USA,2VeterinaryIntegrativeBiosciences,TexasA&MUniversity, College Station, TX,USA, 3DiagnosticMedicine/Pathobiology, Kansas StateUniversity,Manhattan, KS,USA,4AnimalSciences&Industry,KansasStateUniversity,Manhattan,KS,[email protected]:AlternativestoAntibiotics,Room2,12/4/201711:00AMRisks to public health relating to the worldwide rise of antimicrobial resistance have necessitated research on alternative feedsupplements, including heavymetals and essential oils.We aimed tomeasure the impact of two antibiotic alternatives, zinc andmenthol,onantimicrobialresistanceamongcommensalentericbacteriaoffeedercattle.A2x2factorialdesignwasemployedwith80individuallyhousedsteersrandomlyassignedtooneoffourtreatments:1)acontroldietformulatedtoNRCguidelines(30ppmofzinc),2) a supranutritional zinc (ZN) diet (300 ppm), 3) a menthol (MENTHOL) supplemented diet (0.3%) and, 4) a ZN * MENTHOLsupplementeddiet.Feceswerecollectedeverysevendaysduring thestudyperiodwhichconsistedofa sevendayacclimatizationperiod,followedbya21-dayfeedingtrial,andfinallya14-daywashoutperiod.Fecalsuspensionsfromdays0and21wereplatedontom-EnterococcusagarasplainandalsosupplementedwitherythromycinandtetracyclineatCLSIbreakpointconcentrations for thepurposeofquantifyingEnterococcusspp.Inaddition,twoisolateswereanalyzedforphenotypicresistanceviatheTREKSensititre®systemusingCMV3AGPFplates.Mixedlinearstatisticalmodelswereusedtomodellog10CFUofbacteriagrownonplainagar,aswellastherelativeandabsolutecountsgrownonantibioticsupplementedplates,againstthe3-wayfactorialeffectsofZN,MENTHOL,andday(0,21).Mixedlogisticmodelswereconstructedtolookatsingleresistancephenotypesasdependentbinaryvariables,withrepeatswithinanimalincludedasarandomeffect.TheZNgroup(aloneandincombinationwithtreatmentday)exhibitedasignificantdecreaseinthedifferenceinlog10CFUofEnterococcusspp,grownonplainanderythromycinsupplementedagar.Consistently,ZNshowedasignificantincreaseinerythromycinresistantphenotypesaccordingtoSensititre®.Conversely,MENTHOLincombinationwithdayhada significant increase in the difference for tetracycline plate log10 CFU, but did notmaintain decreased resistance for tetracyclineresistantphenotypes.38-CombinedtreatmentsofprobioticsandattenuatedSalmonellavaccineinchickenselicitedenhancedhumoralresponseagainst

bacterialantigensG.A.J.Redweik,M.Mellata.FoodScienceandHumanNutrition;InterdepartmentalMicrobiology,IowaStateUniversity,Ames,IA,[email protected]:AlternativestoAntibiotics,Room2,12/4/201711:15AMPurpose:Antibioticresistanceisamajorpublicconcerntothepoultryindustry,whichstronglyimpactstheeconomyofmostdevelopedcountries. Two common enteric pathogens that can infect chickens and thereafter transmit to humans include Salmonella andEscherichiacoli.Increasinggovernmentregulationsofantibioticutilizationinpoultrypressureresearcherstodevelopalternativemeansofpreventingbacterialinfections.Previously,wefoundthatspecificpathogen-freechickensorallysupplementedwithprobioticsandrecombinantattenuatedSalmonellavaccine(RASV)χ9373(pYA3337)treatmentshadenhancedbactericidalabilityoftheirseraagainstnumerousavianpathogenicEscherichiacoli(APEC)serotypes.Theobjectiveofthisstudywastoevaluatethehumoralresponsesagainstsome conserved surface bacterial antigens to correlate observed killing ability with systemic (IgY) and mucosal (IgA) immunity.Methods:Bloodseraandcecacontentswerepreviouslycollectedfromnon-treatedortreated(probioticsand/orRASV)five-week-oldchickens.SeraIgYresponsewasevaluatedin10individualserumsamplespergroup,whereasIgAresponsewasevaluatedinpooledsupernatantsof5chickencecacontentsfromeachgroup.ELISAwasperformedagainstbacterialantigens,E.colisiderophorereceptors(IroN,IutA)andSalmonellaLPStoassessbroad-protectivepotential.Results:Chickensgivenprobiotics,RASV,orbothallhadpositiveseraIgYresponsestoIroN,IutA,andLPS.However,onlyserafromchickensfedwithcombinedtreatmentshadasignificant(P<0.05)IgY response to LPS compared to chickenswith no treatments. Among treatment groups tested, only RASV yielded a positive IgAresponseagainstLPS.Conclusions:Though individual treatmentofprobioticsorRASVelicitedan immuneresponse, thecombinedtreatmentselicitedthestrongestantibodyresponse,suggestingapotentialtopreventinfectionsandcolonizationfrombacteriasuchasE.coliandSalmonellainchickensandthereforetheirtransmissiontohumans.

CRWAD2017 ABSTRACTS


39-FecalprevalenceandantimicrobialsusceptibilitiesofSalmonellaentericaandCampylobacterspp.inpigletssupplementedwithprobiotics

S.E.Remfry1,R.G.Amachawadi1,X.Shi2,H.E.Williams3,M.D.Tokach3,S.Dritz2,J.Pluske4,M.Apley1,T.G.Nagaraja2.1ClinicalSciences,Kansas State University College of Veterinary Medicine, Manhattan, KS, USA, 2Diagnostic Medicine/Pathobiology, Kansas StateUniversityCollegeofVeterinaryMedicine,Manhattan,KS,USA,3DepartmentofAnimalSciences&Industry,KansasStateUniversity,Manhattan,KS,USA,4SchoolofVeterinaryandLifeSciences,MurdochUniversity,Perth,[email protected]:AlternativestoAntibiotics,Room2,12/4/201711:30AMProbiotics,whichcanbeneficiallyaffectthehostanimalbyimprovingthemicrobialbalanceinthegut,arewidelyusedinswinediets.Probioticspromotethegrowthandpersistenceofselectivespeciesorgroupsofbacteria inthegutandalterpHandfermentationproducts.Theresultingbalancedgutmicrobiomeislikelytoimpact,directlyorindirectly,theprevalenceofpathogens.Theimpactofprobioticsontheprevalenceandantimicrobialresistance(AMR)offoodbornepathogensinswineisanunexploredarea.Therefore,weconductedastudytoassesstheprevalenceandAMRprofilesofSalmonellaandCampylobacter,whichareshedinthefecesofpigletsfeddietssupplementedwithprobiotics.Thestudyconsistedof300weanedpigletswith5pigletsperpen,whichwererandomlyallocatedtosixtreatmentgroups;controldiet,dietswiththeadditionof1of2commerciallyavailableprobiotics(BioPlus2B;PoultryStarME)orchlortetracycline(CTC;22mg/kgBW)and2moredietsthatwereacombinationofeachprobioticandCTC.Fecalsampleswerecollectedfrom3pigletsperpenondays0(pre-treatment),7,14,21,28(treatment),35,and42(post-treatment)fortheisolationofCampylobacterandSalmonella.TheoverallprevalenceofCampylobacterspp.andSalmonellawere21.6%(273/1,260)and6.6%(84/1,260), respectively.TheprevalenceofC.hyointestinalisandC.coliwere17.7%(224/1,260)and3.8%(49/1,260), respectively.TherewasnoprobioticorCTCeffects (P>0.05)on theprevalenceofCampylobacteror Salmonella.However, the treatmentandsampling phase interactionwas significant (P = 0.03). The prevalencewas higher in the treatment phase (31.4%;P = 0.02)whencomparedtopre-andpost-treatmentphases.Basedonthisstudy,thetwoprobioticproducts,testedaloneorincombinationwithCTC,hadnoeffectsonfecalsheddingofSalmonellaorCampylobacterspp.FurtherstudiesarebeingdonetostudyboththephenotypicandgenotypicdifferencesamongSalmonellaandCampylobacterstrainsisolatedinthestudy.40-EffectofaSaccharomycescerevisiaefermentationproductonliverabscessesinbeefcattleK.L.Huebner1,J.N.Martin2,C.J.Weissend2,K.L.Holzer2,S.M.Lakin1,M.D.Weinroth2,Z.Abdo3,J.L.Metcalf2,I.Geornaras2,I.Geornaras2,J.K.Parker1,P.S.Morley1,K.E.Belk2. 1ClinicalSciences,ColoradoStateUniversity,FortCollins,CO,USA,2AnimalSciences,ColoradoStateUniversity,FortCollins,CO,USA,3Microbiology,Immunology,andPathology,ColoradoStateUniversity,FortCollins,CO,[email protected]:AlternativestoAntibiotics,Room2,12/4/201711:45AMLiverabscessesare the leadingcause for livercondemnation inbeefcattle, resulting insignificant financial losses.Asconcerns forantimicrobialresistancerise,thebeefindustryischallengedwithfindingalternativestoantimicrobialdrugsforthepreventionofliverabscesses.ArandomizedcontrolledfieldtrialwasconductedtoassesstheefficacyofaSaccharomycescerevisiaefermentationproduct(SCFP)forpreventionofliverabscesses;thestudywasconductedinasystemforproductionofnatural-brandedbeefproducts.Atotalof4,689commercialsteerswererandomlyallocatedintotwotreatmentgroups:acontrolgroup(n=14pens)wasfedacorn-baserationwhile treatedcattle (n=14pens)weresupplementedwithSCFP(18g/head/day).Prior toharvest,composite fecalandsoilsampleswerecollectedfrompens.Atslaughter,liverabscesseswerescoredand5abscessedliversperpenwerecollected.Microbialcommunitiesandtheresistomeofpenfecalsampleswereassessedusing16SrRNAregiongenesequencingandshotgunmetagenomicsequencing.Overall,abscessprevalencewas38.5%(95%CI37.0–39.9),andwasnotdifferentbetweentreatmentgroups(P=0.82).Therewerenosignificantdifferencesinthemicrobialcommunitiesbetweentreatmentgroupsinliverabscesses,fecesorsoil(P=0.62and0.98,0.97respectively).Liverabscesseswerediverselypolymicrobial,astherewere333uniquetaxaidentified.Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria,andFusobacteriawerethemostcommonphyla.Althoughnotassociatedwithtreatment,therewasanegativecorrelationintheratiooffecalFirmicutes:Bacteroidetesandtheliverabscessprevalence.Overall,34uniqueresistancemechanisms to antibiotics, biocides, andmetals were detected in feces, and resistome composition was not differentbetweentreatmentgroups.

CRWAD2017 ABSTRACTS


41-AntimicrobialresistanceamongStaphylococcusspp.fromcatspresentedataveterinaryteachinghospitalinSouthAfricaJ.W.Oguttu1,N.D.Qekwana2,D.Sebola3,A.Odoi4.1AgricultureandAnimalHealth,UniversityofSouthAfrica,Johannesburg,SouthAfrica, 2DepartmentofParaclinicalSciences,SectionVeterinaryPublicHealth,UniversityofPretoria, FacultyofVeterinaryScience,Pretoria, South Africa, 3Department of Paraclinical Sciences, Section Veterinary Public Health,, University of Pretoria, , Faculty ofVeterinary Science, Pretoria, South Africa, 4Biomedical and Diagnostic Sciences, University of Tennessee, College of VeterinaryMedicine,Knoxville,TN,[email protected]:CompanionAnimalEpidemiology–1,Room2,12/4/20172:00PMPurpose:We identified Staphylococcus spp. isolated from samples from cats presented at a teaching hospital, their antimicrobialresistanceprofiles andpredictors of infection.Methods: Twohundred and sixteen (n=216) records submitted to thebacteriologylaboratoryofateachinghospitalbetween2007and2012wereincludedinthestudy.ChisquareandFisher’sexacttestswereusedtoassess simple associations between antimicrobial resistance and age group, sex, breed and specimen type. Adjusted associationsbetweenStaphylococcusinfectionandagegroup,breed,sexandspecimentypewereassessedusinglogisticregression.Significancewassetatp<0.05.Results:Ofthe216samples included inthestudy,17.6%(38/216)werepositiveforStaphylococcus.Ofthese,CoagulasePositiveStaphylococcus(CoPS)isolatescomprised11.1%(24/216),ofwhich7.4%(16/216)wereS.pseudintermedius,3.2%(7/216)wereS.aureusand0.5%(1/216)wereS.delphini.CoagulaseNegativeStaphylococcus(CoNS)madeup1.9%(4/216)oftheisolates,andconsistedofequalnumbersofS.felisandS.simulans,(0.9%;2/216).Atotalof63%(24/38)isolateswereresistanttoatleastoneantimicrobial agent, and15.8%exhibitedmultidrug resistance (MDR).Resistance to clindamycinwasexhibitedby34.2%(13/25)isolatesfollowedbyampicillin32.4%(2/26),lincospectin31.6%(12/26)andpenicillin-G29.0%(11/27).Multidrugresistancewasmore commonamongS.aureus28.6% (2/7) compared toS. pseudintermedius (12.5%;2/16). Theoddsof testingpositive forStaphylococcusspp.infectionsweresignificantlyhigheramongearcanal(p=0.0002)andskinsamples(p<0.0001)thanurinesamples.Conclusions:CoPSwerethepredominantspeciesisolatedfromcats.Althoughantimicrobialresistancelevelsamongisolatesfromcatspresentedatthehospitalwerenotashighashasbeenobservedinotherspecies,thereisevidencethatcatscarryisolatesthatareresistanttoseveralgroupsofantimicrobials.Thisisaseriouspublichealthconcernandcallsforlargerprimarybasestudiestofurtherassesstheextentofantimicrobialresistanceandassociatedriskfactorsincats.42-Temporaltrends&predictorsofantimicrobialresistanceamongStaphylococcusspp.isolatedfromcaninesamplessubmitted

toadiagnosticlaboratoryA.Odoi1,J.G.Conner1,J.Smith2,C.Carter2.1Biomedical&DiagnosticScience,UniversityofTennessee,Knoxville,TN,USA,2UniversityofKentucky,Lexington,KY,[email protected]:CompanionAnimalEpidemiology–1,Room2,12/4/20172:15PMPurpose: The objective of this study was to investigate temporal patterns and predictors of antimicrobial resistance amongStaphylococcus spp. isolated from canine samples submitted to a diagnostic laboratory between 1993 and 2009. Methods:Retrospectivedataof4,971Staphylococcusisolateswereusedtodeterminetheresistanceto16antimicrobialscovering8drugclasses.Temporal trends over the 16 year study period were determined for each antimicrobial using the Cochran-Armitage trend test.Predictors ofStaphylococci resistance to antimicrobialswere evaluated using logistic regressionmodels.Results: A total of 68.1%(3387/4971)Staphylococcus isolateswereS. intermedius, 18.3% (907/4971)wereCoagulasenegativeStaphylococcus (CoNS), 7.5%(375/4971)wereS.aureus, 5.83% (290/4971)wereS.hyicus, andS. schleiferi subsp. coagulanscomprised0.24% (12/4971)of theisolates.Thepercentageofantimicrobialresistant(AMR)andmultidrugresistant(MDR)isolateswere64.2%and18.8%,respectively.ThehighestlevelsofAMRwereseeninS.aureus(70.7%;265/375),CoNS(66.4%;602/907),andS.intermedius(64.7%;2192/3387)while the lowest levelsofAMRwereobserved inS.hyicus (44.1%;128/290)andS. schleiferi subsp.coagulans (33.3%;4/12).AMRshowed a significant (p=0.011) decreasing temporal trend while no significant (p=0.062) temporal trend was observed forMDR.Significanttemporaltrendswereseenamong11ofthe16antimicrobialscovering6ofthe8drugclassesassessed.Isolatesshowedsignificant increasing temporal trends in resistance to β-lactams, Aminoglycosides, Lincosamide, and Enrofloxacin. By contrast,SulfonamidesandTetracyclinesbothshowedsignificantdecreasingtemporaltrends.SignificantpredictorsofbothAMRandMDRwereStaphylococcus spp., geographic region, and sample source.Conclusions: Continuedmonitoringof antimicrobial resistance amongStaphylococcusspp.iswarrantedsincesignificantincreasingtemporaltrendswereseenamongroutinelyusedantimicrobials.Futureevaluationsshouldalsoconsiderhowregionalfactorscontributetoresistancepatterns.

CRWAD2017 ABSTRACTS


43-Prevalenceofmethicillin-resistantStaphylococcuspseudintermediusisolationindogstreatedataveterinaryteachinghospitalB.Ouyang1,S.Brault1,C.Gentry-Weeks2,S.Pannone1,L.Vogt2,S.Harre2,P.S.Morley1,J.Schissler1.1ClinicalSciences,ColoradoStateUniversityCollegeofVeterinaryMedicineandBiomedicalSciences,FortCollins,CO,USA,2Microbiology,Immunology,andPathology,ColoradoStateUniversityCollegeofVeterinaryMedicineandBiomedicalSciences,FortCollins,CO,[email protected]:CompanionAnimalEpidemiology–1,Room2,12/4/20172:30PMStaphylococcuspseudintermedius is a commensal organism in dogs and anopportunistic pathogen. The emergenceofmethicillin-resistant S. intermedius (MRSP) strains is of significant concern, as these are resistant to all beta-lactam antimicrobial drugs. TheprevalenceofMRSPcolonizationindogswithvaryingriskfactorsforinfection,aswellassheddingbeforeandaftercareinaclinic,hasnotbeenwelldescribed.TheobjectiveofthisstudywastodeterminetheprevalenceofMRSPisolationfromcommoncolonizationsitesinpopulationsofdogswithdifferentriskfactorsforinfectionseenattheColoradoStateVeterinaryTeachingHospital(CSU-VTH)beforeandaftercare.Atotalof243dogspresentedtotheCSU-VTHCommunityPractice,Dermatology,andSurgicalOncologyserviceswereenrolledinthisstudy,andswabswereobtainedfromcommoncolonizationsitesforS.pseudintermediusatenrollmentandatafollow-up appointment whenever possible. Enriched cultures, polymerase chain reaction, and matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry were performed to detect MRSP. Owners completed a standardizedquestionnaire,andmedicalrecordswereexamined.At initialenrollment,9/243(3.7%)dogswereMRSPculture-positiveand7/155(4.5%)wereculture-positiveatfollow-up;therewasnosignificantdifferenceintheproportionsofdogsMRSPpositiveatadmissionandonfollow-up,eitheroverallorwhenstratifiedbyservice.DermatologypatientsweremorelikelytobeculturepositiveforMRSPthanotherpatientgroups(OR12.0,95%CI0.7-217.7).PrecautionstakentomitigatespreadofMRSPinahospitalsettingshouldbemore stringent forpatients seenbydermatology services, anddermatologypatients shouldbeconsidered tobeathigher risk foradversesequelaesuchasMRSPsurgicalsiteinfections.44-OccurrenceandantimicrobialresistancepatternsofE.colifromdogswithurinarytractinfectionattheveterinaryacademic

hospitalinSouthAfrica.D.N.Qekwana1,L.Phophi1,V.Naidoo2,J.W.Oguttu3,A.Odoi4.1DepartmentofParaclinicalSciences,UniversityofPretoria,Pretoria,South Africa, 2University of Pretoria Biomedical Research Centre, University of Pretoria, Pretoria, South Africa, 3Department ofAgricultureandAnimalHealth,Universityof SouthAfrica, Florida, SouthAfrica, 4Biomedical andDiagnostic Sciences,UniversityofTennessee,knoxville,TN,[email protected]:CompanionAnimalEpidemiology–1,Room2,12/4/20172:45PMPurpose: This study investigated theprevalenceandantimicrobial resistancepatternsofE. coli isolates fromdogspresentedwithurinarytractinfectionsataveterinaryacademichospitalinSouthAfrica.Methods:ThestudyusessecondarylaboratorydataofcanineurinarytractinfectionspresentedattheacademicveterinaryhospitalbetweenJanuary2007andDecember2012.E.coliisolatesweresubjectedtoantimicrobialsusceptibilitytestingagainstapanelof15drugsusingtheKirby-Bauermethod.Factor-specificproportionsofE.colipositiveandantimicrobialresistantisolatesaswellastheir95%confidenceintervalswerecomputedbyage,sex,breedandyear.TheCochran-ArmitagetrendtestwasusedtoinvestigatetemporaltrendsandlogisticregressionmodelswereusedtoinvestigatepredictorsofinfectionsandantimicrobialresistanceamongE.coliisolates.Results:Atotalof22.3%(168/755)ofurinesamplestestedpositiveforE.coli.Therewasasignificant(p=0.0004)decreaseintheproportionofE.colicasesbetween2007and2012.E.coliisolateshadhigher levelsof resistance topenicillin-G (99.4%), lincomycin (100%), tylosine (95.0%), cephalothin (83.7%),amoxycillin (70.1),doxycycline (67.5), lincospectin (63.4%) and lower levels of resistance to enrofloxacin (16.2%), orbifloxacin (21.0%), trimethoprim-sulphamethoxazole(24.7%),chloramphenicol(24.58%).Almostall (98.2%,164/167)E.coli isolatesweremultidrugresistant(MDR),while, 11.38% (19/167)wereextensivedrug resistant (XDR) and2.4% (4/167)werepandrug resistant (PDR).E. coli infection andantimicrobialresistancewerenotassociatedwithage,sexordogbreed.Conclusions:ThisstudyshowsthatalthoughcasesofE.coliurinarytractinfectionsamongdogspresentedattheacademicveterinaryhospitalshowsadecreasingtrend,theproportionofisolatesresistanttotheantimicrobialagentsroutinelytestedisquitehigh.Ofconcern,isthehighlevelsofMDRisolatesandthepresenceofXDRandPDRisolates.ThereisaneedtoemphasizeprudentuseofantimicrobialsdrugsintreatmentofE.colirelatedurinarytractinfectionsindogs.

CRWAD2017 ABSTRACTS


45-BacterialandpatientfactorsinpersistentandrecurrentEscherichiacoliurinarytractinfectionindogsT.E.LeCuyer1,M.A.Davis2,T.E.Besser1.1VeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA,USA,2PaulG.AllenSchoolforGlobalAnimalHealth,WashingtonStateUniversity,Pullman,WA,[email protected]:CompanionAnimalEpidemiology–1,Room2,12/4/20173:00PMRecurrenturinarytract infections(UTI)areaclinicalchallengeforsmallanimalpractitioners.Escherichiacoli isthemostfrequentlyisolatedorganismfromdogswithmultipleurinarytractinfections.LiteratureonrecurrentUTIindogsislimiteddespitethefactthatitis an important clinical problem and an indication for prescription of antimicrobials, sometimes for prolonged treatment periods.WecollectedE.coliisolatedfromcanineurinesamplesandassociatedclinicaldatafromfiveveterinarydiagnosticlaboratoriesbetweenMarch2015andOctober2016toassessbacterialandpatientfactorsassociatedwithpersistentorrecurrenturinarytractinfectionduringthestudyperiod.Atotalof173dogshadasingleurinesamplepositiveforE.coliduringthestudyperiodwhile42dogshadatleasttwoindependenturineculturespositiveforE.coli.Ofthese42dogs,26hadevidenceofpersistentUTIwithisolatesofthesamemultilocussequencetype(MLST)isolatedfromallurinesamples.Theprevalenceoftheextraintestinalvirulence-associatedgenespapC,papA,kpsMTII,fyuA,iutA,afa/draBC,ireA,andsfa/focdidnotdifferintheisolatesfrompersistentinfectionscomparedtothosefromsingleinfections.However,theE.coliisolatedfromthe16dogswithrecurrentUTI,infectedbydifferentMLSTtypesduringeachUTIevent,had significantly lower prevalences ofpapC, papA,and sfa/foc, aswell as fewer virulence genes per isolate overall (MannWhitneyUtest,p=0.004)comparedtosingleUTIisolates.ThemultipleUTIgroupasawholehadahighercancerprevalence(16.7%)thandogsinthesingleUTIgroup(3.8%,Chi-squaretestofindependence,p=0.005),butprevalenceofotherpotentialUTIriskssuchasimmunosuppressivetherapyandneurologicdiseasewassimilarbetweenthetwogroups.Weconcludethatwhilepersistenturinarytract infections in dogs are not associated with any of the urovirulence genes assessed here, patients with recurrent infectioncharacterizedbydifferentMLSTstrainsseemtobesusceptibletoinfectionbyE.colistrainswithfewervirulence-associatedgenes,indicatingthatthesepatientsmayhaveimpairedurinarytractimmunity.46-GenotypicandphenotypicanalysisofSalmonellaentericaisolatedfrompatientsatanequinereferralhospitalI.M. Leon Moreno1, K. Norman2, S. Lawhon3, D. Threadgill3, H.M. Scott3. 1Department of Veterinary Pathobiology, Texas A&MUniversity,Bryan,TX,USA,2DepartmentofVeterinaryIntegrativeBiosciences,TexasA&MUniversity,TexasA&MUniversity,TX,USA,3DepartmentofVeterinaryPathobiology,TexasA&MUniversity,TexasA&MUniversity,TX,[email protected]:CompanionAnimalEpidemiology–1,Room2,12/4/20173:15PMSalmonellosisisoneofthemaincausesoflifethreateningcolitisinhorses.Multidrug-resistanceamongSalmonellaserotypesassociatedwithclinicaldiseaseinbothhorsesandhumanshasbeenreported.TheaimofthisstudywastoevaluatetheproportionalmorbidityofSalmonellaenterica serotypesandtheirantimicrobial resistance(AMR)fromhorsesadmittedtoareferralhospital inthesouthernUnited States, usingWhole Genome Sequencing (WGS). Salmonella strains (n=255)were obtained from patients admitted to thehospital between 2007 and 2015. The AMR phenotype was determined using the Sensititre® system.WGS was used to validateserotypesandtoidentifytheAMRgenotype.SequencinglibrarieswerepreparedusingtheIlluminaNexteraXTkitandsequencedonthe IlluminaMiSeqplatform. Phylogenetic outbreak analyseswereperformed in Parsnp and FigTree. Themost common serotyperecoveredwasNewport(18%),followedbyAnatum(14.1%)andBraenderup(11.4%).AsymptomatichorsesweremoreassociatedwithS.BraenderupanddigestiveversussepticemiawithS.Typhimurium.Mostoftheisolateswerepansusceptible(n=219;85.9%),10(3.9%)wereresistanttooneortwoclassesofantimicrobials,while25(10.2%)weremultidrugresistant(≥3classes).Betalactamase(bla)genes such as blaTEM-1, ESBLs (blaSHV-12 and blaCTX-M-27) and AmpC (blaCMY-2) were detected. Isolates with reduced susceptibility tociprofloxacinwerecarryingtheplasmid-mediatedquinoloneresistancegene(qnrB)oraac(6')-Ib-cr.Additionally,resistancegenesforgentamicin,streptomycin,folatepathwayinhibitors,phenicol,tetracycline,andmacrolideswereidentified.ThemainplasmidtypewastheconjugativeplasmidI1(10%),thatoftencarriesESBLgenes.Phylogeneticanalysesonthemainserotypesrevealedrelatedtemporalandgeographicaloutbreakclusters.ThepresenceofmultipleAMRgenesinSalmonellacouldlimitthetreatmentofinvasivediseasesinhorsesandremainapotentialhazardtopublichealth.UnderstandingtheepidemiologyofSalmonellainhorsesadmittedtoreferralhospitalsisimportantfortheprevention,control,andtreatmentofsalmonellosis.

CRWAD2017 ABSTRACTS


47-AssessingthesuitabilityofaNorthAmericancompanionanimalpoisoncontrolcentreasanoveldatasourceforsurveillanceA.L. Swirski1, D.L. Pearl1, O. Berke1, T. O'Sullivan1, D. Stacey2. 1PopulationMedicine, University of Guelph, Guelph, ON, Canada,2ComputerScience,UniversityofGuelph,Guelph,ON,[email protected]:CompanionAnimalEpidemiology–2,Room2,12/5/20179:00AMPurpose:Theobjectivesofourstudyweretodeterminethespatialdistributionofcallstoapoisoncontrolhotlineconcerningaccidentalingestionofpoisonsincompanionanimals,identifyanyspatial“gaps”insurveillance,andexaminefactors(e.g.,season,animalfactors)thatmaybiastheresultsofquantitativemethodsusedforsurveillance.Methods:DataconcerningcallstothehotlinewerecollectedfromtheAmericanSocietyforthePreventionofCrueltytoAnimal’sAnTOXdatabasefromJanuary1,2005toDecember31,2014,inclusive. Population size and demographics were gathered from the 2010 U.S. census and the American Community Survey,respectively.Choroplethmapswereusedtoexaminethedistributionofreportingtothehotlineatthecounty-levelandidentifyany“holes”insurveillance.Spatialscanstatisticswereemployedtoidentifyifgapsinreportingwereclusteredorrandomlydistributed,andtodetectanypredictableclustersofhighreportingrates.WealsofitmultilevelPoissonregressionmodels,toaccountforclusteringwithincountyandstate,toidentifyfactorsrelatedtopredictablechangesincallvolumeorreporting,whichmaybiastheresultsofquantitativeaberrationdetectionmethods.Results:Throughoutthestudyperiod,over40%ofcountiesreportedatleastonecalltothehotlineeachyear,withthemajorityofcallscomingfromtheNortheast.Conversely,therewasalarge“hole”incoverageintheMidwestandSoutheaststates.Thelocationofthemostlikelyhighandlowriskclusterswererelativelystablethroughoutthestudyperiodandwereassociatedwithsocioeconomicstatus(SES),asthemostlikelyhighriskclusterswereidentifiedinareasofhighSES.SimilarresultswereidentifiedusingmultivariableanalysisasindicatorsofhighSESwerefoundtobepositivelyassociatedwithratesofcalls to thehotlineat thecounty-level.Conclusions: The results fromtheseanalyseswill allowus todetermine thesuitabilityandmannerinwhichtousetheAnTOXdatabase’scalldataaspartofaquantitativesurveillancesystemforcompanionanimalpoisoningsintheUnitedStates.48-UsingbigdataandmachinelearningtoanalyzecatweightA.Campigotto1,Z.Poljak1,E.Stone2,D.Stacey3,T.Bernardo1.1PopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2ClinicalMedicine,UniversityofGuelph,Guelph,ON,Canada,3ComputerScience,UniversityofGuelph,Guelph,ON,[email protected]:CompanionAnimalEpidemiology–2,Room2,12/5/20179:15AMPurpose:Withtherisingconcernofpetobesity,knowingthebodyweightdevelopmentforcatsprovidesimportanthealthinformationonweightmanagementforownersandveterinarians.Theuseofelectronicmedicalrecordstoexaminedatafrommanyanimalsinaclinicalsettingcanbeusedtofillthegapsinknowledgeregardinghealthparameterssuchasweight.Thus,theobjectiveofthisstudywastodeterminetheimpactofbreed,reproductivestatus,decadeandgenderonthebodyweightateachyearofageincats.Methods:Aretrospectivecohortstudywasperformedusingbodyweightdatagatheredfromdomesticfelinesfrom3972uniqueveterinaryclinicsintheUnitedStatesandCanadafrom1981to2016throughelectronicmanagementrecords(Avimark,Cornerstone, Impromed,orIntravet)madeavailable foranalysis fromaveterinarydiagnosticcompany(IDEXXLaboratories, Inc). Initially,bodyweight fromallfelinesrecordedintheelectronicmedicalrecords(n=19,416,753)wereincludedinthestudypopulationandexaminedusingdescriptivestatistics. Linear regression, calculated through ordinary least squares as well as stochastic gradient descent, was used to assessassociationbetweenbodyweightandage,breed,gender,decadeofmeasurementandtheirinteractions.Accuracyofpredictionswasevaluated on a validation dataset. Descriptive statistics and linear regressionmodels and predictionswere created using Python.Results:Breed,gender,reproductionstatus,andageallimpactedtheaveragebodyweightofacat.Thepredictiveabilityofthelinearmodelcreatedusingstochasticgradientdescentandordinaryleastsquareswascomparable.Conclusions:Thisstudydemonstratesthattheuseofbigdatacouldhelptofillinknowledgegapsincompanionanimalhealth,providingfurtherevidencetodiscussionwithownersregardingissuessuchasweightmanagement.

CRWAD2017 ABSTRACTS


49-ObservationalstudytodetermineriskfactorsassociatedwithtickexposureindogsandtheirownerswithinChampaignandPiattcountiesofIllinois

L.A.Lyons,R.L.Smith,M.O'HaraRuiz.Pathobiology,UniversityofIllinoisatUrbana-Champaign,Champaign,IL,[email protected]:CompanionAnimalEpidemiology–2,Room2,12/5/20179:30AMInIllinois,thenumberofreportedcasesofhumanillnessfromthefourmostcommontick-bornediseases(TBDs),allofwhichalsoafflictdogs,increasedten-foldbetween1990-2013.Currently,thereisinsufficientinformationonhow,where,andwhypeopleanddogsareexposedtoticks.Ourstudyaimistodetermineriskfactorsassociatedwithexposuretoticksindogsandtheirowners.Wearecurrentlyconductinga15month,3-layered,observationalstudyconcluding in July2018.First,asurveyof thegeneralpopulation isusedtodeterminetherelationshipbetweendogownershipandobservationofticksonhumanfamilymembers.Second,asurveyofidentifieddogowners includesquestions todetermineconnectionsbetween individual characteristics,activities,andobservationof ticksonhumanandcaninefamilymembers.Third,movementtrackingandhome-basedtickcollectionisdoneondogsandownersselectedfromthe2ndsurveytodetermineassociationsbetweenenvironmentalfactors,movement-relatedactivities,andobservationofticks.The3rdlayerparticipantsareplacedbyrandomizedblockdesignintothreegroups(n=20)andeachblockismonitoredduringoneofthreeseparatetimeperiods.Currently,259participantshavecompletedsurveyone,145havecompletedsurveytwo,and8familiesareenrolledinthe3rdlayer.Sofar,dogownershipissignificantlyassociatedwithobservingticksonhumanfamilymembersduringpastyear (OR=3.21,p=0.0001).Observationof ticksondogs issignificantlyassociatedwithwalkingdogs inparks (OR=1.846,p=0.001),travelingwithdogs(OR=1.59,p=0.009),andticksonhumanfamilymembers(OR=3.65,p=0.008).Urbanizedlandcoveraroundhomesisnegativelyassociatedwithobservingticksondogs(OR=0.07,p=0.016).Therelationshipbetweenticksonfamilymembersanddogownershipwillbeanalyzedafterthe1stblockofdatacollectioniscompleted,inNovember2017.WhileTBDshavebeenrelativelyrareinthepast,webelieveduetoincreasednumbersofvectorticks,morepeopleandtheirdogsarebeingexposed,andtheexposurehappensclosetohome.Ourstudywillprovideasystematic,robustandscientificallysoundassessmentofthis.50-LeptospirosisinshelterdogsandcatsinthetristateareaofKentucky,Tennessee,andVirginiaD.Kish1,B.Beigel1,J.Morgan1,D.Spangler1,A.Verma2.1LincolnMemorialUniversityCollegeofVeterinaryMedicine,Harrogate,TN,USA, 2Center of Infectious, Zoonotic, and Vector Borne Diseases, Lincoln Memorial University College of Veterinary Medicine,Harrogate,TN,[email protected]:CompanionAnimalEpidemiology–2,Room2,12/5/20179:45AMPurpose:Leptospirosis,aworldwidezoonoticinfectionthataffectsdogsandmanyothermammalianspecies,includingman,iscausedby infection with pathogenic members of the genus Leptospira. Pathogenic leptospires live in the proximal renal tubules ofasymptomatic carrier animals and are shed in the urine. Virtually any mammalian species can act as asymptomatic reservoir,characterizedbychronic renalcarriageandsheddingofahost-adapted leptospiral serovar.Environmentalcontaminationby thesechronicsheddersresultsinacquisitionofinfectionbysusceptibleanimals.Methods:Inthisstudy,weinvestigatedifclinicallynormalshelterdogsandcatsharborleptospiresintheirkidneysbyscreeningurinesamplesforthepresenceofLeptospirasppusingahighlysensitivelipl-32basedTaqManqPCR.Additionally,wemeasuredLeptospira-specificserumantibodiesusingmicroscopicagglutinationtest(MAT)totestcorrelationbetweenseroprevelanceandurinaryshedding.Results:Theresultsshowedthatapproximately17%ofthe133shelterdogsandcatsscreenedbyqPCRwerepositiveforleptospiralDNAinurineand21ofthe145animalsscreenedwithMAThadatiterlevelgreaterthan1:100acrossfivetestedLeptospiraserovars.Nineteenanimalsinthestudyshowedpositiveresultsfor both qPCR andMAT. Twelve animalswere positivewith qPCR but notwithMAT. Conclusion: These findings have significantimplicationsregardinganimalandpublichealthintheareaandpossiblyoutsidewheretheseanimalsmaybeadopted.

CRWAD2017 ABSTRACTS


51-TransectionasasafeandfastermeanstoreleasethesuspensoryligamentduringcanineovariohysterectomyJ.Richardson1,J.Shivley1,D.R.Smith2,R.Meyer1,K.A.Woodruff1.1ClinicalSciences,MississippiStateUniversityCollegeofVeterinaryMedicine, Mississippi State, MS, USA, 2Pathobiology and Population Medicine, Mississippi State University College of VeterinaryMedicine,MississippiState,MS,[email protected]:CompanionAnimalEpidemiology–2,Room2,12/5/201710:00AMBreakingdownthesuspensoryligamentusingdigitalstrumming(DS)iswidelyaccepted,taught,andpublishedinveterinarysurgerytextbookswhendescribing the canineovariohysterectomyprocedure. Sharp transection (ST)of the suspensory ligamenthasbeendevelopedto,presumably,decreasesurgerytimeandperi-operativepain,butthereislittleresearchavailabletoconfirmtheefficiencyandsafetyoftheprocedure.Thisisaprospective,longitudinalpilotstudy.Thehypothesesinvestigatedrelatedtosuspensoryligamentrelease are: ST of the suspensory ligament is a faster technique to elevate the ovarian pedicle from the canine abdomen; STwilldecreaseintra-operativenociception;STwillbeassafeasDSwhencomparingintra-operativecomplicationrates.Thirtyadultfemaledogswere randomlyassigned to theSTorDSgroup.Nociceptionwasassessed throughbaselinemeasurementsofpre-and intra-operativeheartrate,specificallyduringmanipulationofthesuspensory ligament.PainwasassessedusingtheGlasgowPainScore.Efficiencywasmeasuredthroughtotalsurgicaltimeandthetimetorupturethesuspensoryligamentasanisolatedevent.Safetywasmeasuredbyobservingepisodesofacutehemorrhageintra-operatively.Alphawassetat0.1forthepilotstudy.Therewasnosignificantdifferenceinpost-operativepainscoresbetweenSTandDS.Nointra-orpost-operativecomplicationsoccurred.Timeevaluatedduringsuspensoryligamentreleasewasmeasuredasbeinglessthanoneminuteorbetween1to2minutes.TheSTgrouphadsignificantlyloweroddsoftakinglessthanoneminutewhencomparedtotheDSgroup(p-value=.004).Thechangeinheartrate(HR)wasmeasuredasincreaseinHRfromthetimetheproperligamentwasclampedtothepeakHRduringsuspensoryligamentmanipulation.ThemeandifferenceinHRwassignificantlylowerforSTthenDSgroup(p-value=.06).WeconcludedthatSTprovideslessintensenociceptiveinputwhencomparedtoDS.STwhenviewedasanisolatedeventisfasterthanDS.STappearedtobeequallyassafeasDS.52-AsystematicreviewoftheliteraturepertainingtohealthoutcomesinwildandcaptivewolfpopulationsworldwideA.M.Hassebroek,A.Ruple.DepartmentofComparativePathobiology,CollegeofVeterinaryMedicine,PurdueUniversity,Lafayette,IN,[email protected]:EpidemiologyandAnimalHealth,Room2,12/5/201710:45AMThediseasesthataffectwolvesarediverseandtheimpactsvaried.Diseaseinwolvescanaffectecosystem,livestock,andhumanhealth,giveinsightintothegeneticbackgroundofdomesticateddogs,andassistinconservationplanningandprojectimplementation.Asakeystonepredator,wolveshavealargeimpactonthehealthanddiversityofthesurroundinghabitatanddiseasesthatimpactwolfsurvivalwill,inturn,affecttheentireecosystem.Infectiousandparasiticdiseasescarriedbywolvescanbetransmittedtootherwildlifespecies,livestock,andhumans.Also,conservationandre-populationeffortsmaybeimpactedbythepresenceofheritablecongenitaldefectsanditisthereforeimportanttoreporttheprevalenceoftheiroccurrencewithinwolfpopulations.Systematicreviewmethodswere utilized to identify published reports of health outcomes in either grey (canis lupus) or red (canis rufus) wolf populationsworldwide.Searchtermsincludedintheliteratureretrievalpertainedtobothwolfpopulationsandtermsrelatedtomultiplehealthoutcomesincludinginfectious,parasitic,neoplastic,congenital,andmetabolicdiseases.Relevantdatawereextractedfrommanuscriptsincludedinthereviewafterimplementationofatwo-steprelevancescreeningprocess.Atotalof3718articlesmettheinitialsearchrequirementsand301articleswereretainedafterthefirstrelevancescreening.Healthoutcomeswerestratifiedbasedonspeciesandpopulationtype.Themajorityofhealthoutcomesdescribedintheliteratureconcernedparasiticinfections;otheroutcomesdescribedinclude infectious diseases, neoplasia, congenital disorders, trauma and others. To the authors’ knowledge, this is the firstcomprehensivesystematicreviewofallhealthoutcomesinwolfpopulations.

CRWAD2017 ABSTRACTS


53-Clustereddataanalysisinepidemiologicstudies:demonstrationofapplicationofmultilevelmixedmodelforbinaryoutcomeusingdatafrombovinetuberculosisstudyinresourcepoorsettings

T.Habitu1,E.Skjerve2,T.Gebrehiwot3,A.Muwonge2,D.Areda4.1EpidemiologyandBiostatistics,NorwegianUniversityofLifeSciences,Aos,Norway,2EpidemiologyandBiostatistics,Ullevålsveien72,0454Oslo,NorwegianUniversityofLifeSciences,Aos,Norway,3CollegeofVeterinaryMedicine,MekelleUniversity,Mekelle,P.O.Box:231,Ethiopia,4SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,3121TupperHall,Davis,CA95616,CA,[email protected]:EpidemiologyandAnimalHealth,Room2,12/5/201711:00AMPurpose:Dataanalysisinvolvingmanyepidemiologicstudieswithhierarchicalstructureoftenfailtoconsiderclusteringeffectduetostudydesign,andthisoftenleadstoerroneousestimatesandbiasedinference.Aimofthisstudywastodemonstratetheusefulnessofmultilevelmixedmodel foranalysisofclustereddata involvingbinaryoutcomeusingdata frombovine tuberculosis (bTB)studyconducted inresourcepoorsettings,where farmunitsandpopulationsettlementarehighlyclustered.Methods:Acrosssectionalmulti-stagesamplingtechniqueinvolvingrandomselectionofanimalswascarriedout.BovineTB(bTB)wasdiagnosedbyacomparativeintradermaltuberculinskintest.Atotalof1357cattlefrom28herdsand10villageswereexamined.Therewerethreelevelsindatahierarchy:county/village(level3),herd(level2)andindividualanimal(level1).AmultilevellogisticmixedeffectmodelusingRstatisticalcomputingsoftwarewasusedtoanalysisthedata.Randomeffectswereintroducedtorepresentclusteringatlevels2(farm)and3(village)..Similarly,standardlogisticregressionmodel(thatignoresclustering)wascomputedtocomparetheresultsfrommultilevelmodeling.Results:MultilevellogisticmixedmodelidentifiedbreedandhygienicconditionofcattlebarnassignificantpredictorsofriskofexposuretobTB.Exoticbreedandcross-bredcattleweremorelikelytobepositiveforbTB((OR=8.92;95%CI=[2.61,30.44];OR=4.99;95%CI=[1.37,18.21],respectively)comparedtoindigenouslocalbreed.TheoddsofbeinginfectedbybTBwashigher(OR=2.34;95%CI=[1.16,4.71])amongcattlelivinginahousewithoccasionalremovalofdungcomparedtothoselivinginahousewithregularremovalofdung.Intraclasscorrelationcoefficient(ICC)forcounty/villagewas4.41%;whereasICCforherdwas14.1%.Conversely,inadditiontobreedandbarnhygiene,standardlogisticregressionerroneouslyidentifiedanimalsourceandhousetypeassignificantriskfactors.Conclusions: This study highlights the importance of selecting the right statistical model in epidemiologic studies that involvehierarchicaldesign.54-Amodeloffoot-and-mouthdiseasetransmission,detection,andinterventionstrategieswithinaU.S.beeffeedlotA.H.Cabezas,M.W.Sanderson,V.V.Volkova.DepartmentofDiagnosticPathobiology,KansasStateUniversity,Manhattan,KS,[email protected]:EpidemiologyandAnimalHealth,Room2,12/5/201711:15AMMathematicalmodeling isa tool torepresentapotentialepidemicof infectiousdiseasessuchas footandmouthdisease(FMD) indisease-freeareas.MostpublishedmodelsfocusonfarmtofarmFMDtransmissionandrepresentthefarmasahomogenousunit.WedevelopedamodelofFMDtransmissiondynamicsinaU.S.beeffeedlotwithtypicallayout,management,andanimaldemographics.We used the model to estimate the time to detection depending on the surveillance sensitivity, and to evaluate the outbreakprogressiondependingonpost-detectioninterventionssuchastargetedcullingstrategiesandanimalmovementrestrictionsinsidethefeedlot.ThisisastochasticSLIR(susceptible-latent-infectious-recovered)modelnestedinameta-populationofhomepensandhospitalpensinthefeedlot.ThemodesofFMDtransmissionwithin-penandbetween-penweremodeledexplicitly.Afeedlotof24,000cattledistributedin120penswith200headperpen,and2hospitalpenswasmodeled.Basedonactiveobservationalsurveillancebyfeedlotemployeeswemodeledadetectionthresholdof3%prevalenceofclinicalFMDcattleintheindexpen.Fourpost-detectioninterventionscenariosweremodeled-S1:nointervention;S2:stoppinghospital-pencattlemixingondayofdetection;S3:S2andcullinganimalsinthepenssurroundingtheindexpenondayofdetection;S4:S2andcullingcattleinhomepensthatreceivedanimalsfromhospitalpenswithin 7 days prior to detection.Detection occurred at amedian of day 8 after FMD introduction to the feedlot.Under S1,simulations showed that the outbreak took 75-90 days to fade-out and all penswere infected by amedian of 47 days post FMDintroduction.ThedailyincidencedecreasedunderS2andS3buttheoutbreakcontinued.TheoutbreakwascontrolledunderS4butrequiredcullingof~50%ofthefeedlotpopulation.Targetedcullinginterventionsmaybechallengingtoimplementduetothehumanlaborrequirementsandanimalwelfarecomplications,anditsefficacyisdependentonthetimetodetection.Slowingtheoutbreakmayallowtimeforotherstrategiessuchasvaccination;thiswillbeassessedinfuturework.

CRWAD2017 ABSTRACTS


55-Anexpertopinionsurveyoffoot-and-mouthdiseasenaturalhistoryandclinicalmanifestationsinbeefcattleA.H.Cabezas1,M.W.Sanderson1,M.Jaberi-Douraki2,V.V.Volkova1.1DepartmentofDiagnosticPathobiology,KansasStateUniversity,Manhattan,KS,USA,2DepartmentofMathematics,KansasStateUniversity,Manhattan,KS,[email protected]:EpidemiologyandAnimalHealth,Room2,12/5/201711:30AMAFootandMouthDiseaseoutbreakinanFMD-freecountrywillhavesevereconsequencesforthelivestockindustry.Simulationmodelsarepowerfultoolstorepresentandanalyzepotentialoutbreaks.However,modelparameterizationischallengingduetodeficienciesandgapsindatageneratedbycontrolledinvivoFMDexperiments.Wedevelopedaquestionnairetocollectexpertopinionaboutkeyparametersofpotentialnaturalhistory,transmissibility,andclinicalmanifestationofFMDininfectedimmunologically-naïvecattleoftypicalU.S.beeffeedlots.Theexperts(virologists,epidemiologists,modelers,practicingveterinarians,andgovernmentalveterinaryofficers)withexperienceworkingonFMDinendemicsettingsoronFMDoutbreaksinnon-endemicsettingswereinvited.Quantitativeestimates were requested for high and low virulence FMD strains for the duration of infectiousness and clinical disease stages,proportionofanimalsexhibitingspecificclinicalsigns,reductioninfeedconsumption,andaqualitativedescriptionoftransmissibility.Twenty-seven experts agreed to participate and survey completion ratewas 56%. Survey results indicated themedian estimateddurationoflatentandinfectiousperiodsincattleinfectedbyhighvirulencestrainsrangedfrom1to7and3to10days,respectively.For subclinical, incubation, and clinical period themedian estimated duration ranged from 0.5 to 4, 2 to 7, and 3.5 to 10 days,respectively.ExpertestimatesoftheprobabilityofexposedcattledevelopingclinicalFMDvariedforhighvirulence(40-100%)andlowvirulence(10-90%)strains.Themediandurationofreductioninfeedconsumptioninclinicalcattleinfectedbyhighvirulencestrainsrangedfrom2to8.5days.ExpertsindicatedthevirusstrainandintrinsiccharacteristicsofthehostmightaffectFMDprogressionandanimalinfectiousness.SurveydatawillimprovetherobustnessofFMDepidemicpredictionsinnon-endemicsettings,whencombinedwithexperimentalandoutbreakinvestigationdata.Moreover,surveydatawillbeusefulforassessingthelikelihoodofearlydetectionandrapidresponsetoanoutbreak.56-Developmentandvalidationofamultiplexreal-timePCRassayforthedetectionanddifferentiationofFMDVandSVVstrainsY.Wang1,B.Yang2,W.Zheng1,E.Porlsen1,X.Liu1,Y.Fang1,G.Anderson1,J.Bai1.1KansasStateVeterinaryDiagnosticLab,KansasStateUniversity,Manhattan,KS,USA,2LanzhouVeterinaryResearchInstitute,Lanzhou,[email protected]:EpidemiologyandAnimalHealth,Room2,12/5/201711:45AMPurpose:Foot-and-mouthdiseasevirus(FMDV)andSenecaValleyvirus1(SVV-1)arenon-enveloped,single-stranded,positive-senseRNAvirusesbelongingtothefamilyofPicornaviridae.FMDVischaracterizedbytheappearanceofvesiclesonthefeetandmouthoncloven-hoofedanimalspeciesincludingcattleandpigs,whicharemajorfoodanimalsinmanycountries.Itcauseshighfever,blistersonmouthandfoot,weightloss,reducedmilkproduction,andsometimescanbefatal.Itisanextremelycontagiousdisease,andcanbetransmitted inanumberofways,mainlythroughdirectcontact,andaerosoltransmission.TheSVV-1,unknowntill2002,couldresultinsimilarclinicalsymptomsinpigsthatareindistinguishablefromthatcausedbyFMDV.Wehavedevelopedamultiplexreal-time(r)reverse-transcription(RT)PCRassay(rRT-PCR)forFMDVandSVV-1detectionanddifferentiation.Methods:Twosetsofprimersandprobesweredesigned,targetingonthe5’-UTRand3DpolymerasegeneofSVV-1respectively.TheUSDANAHLNdiagnosticassaywasapplied forFMDVdetection.The3setsofprimersandprobesweremultiplexed,andanalyticallyanalyzedusing10-foldserialdilutionsofaclonedpartialgenomeofSVV-1andasmallpieceofFMDV.Results:Thelimitofdetection(LOD)forbothSVV-1andFMDVwere10copiesperreaction,whichcorrespondstoCt37.1forSVV-1,andCt38.4forFMDV.Correlationcoefficientswere0.995and0.997,andPCRamplificationefficiencieswere96.2%and99.5%,respectively,forSVV-1andFMDV.Conclusions:TheassaywillenablerapiddetectionanddifferentiationofSVV-1andFMDV.

CRWAD2017 ABSTRACTS


57-RiskofnewinfectionswithbovineleukemiavirusonMichigandairyherdsH. Hutchinson1, B. Norby2, T. Byrem3, R. Erskine2, P. Bartlett2. 1Comparative Medicine and Integrative Biology, Michigan StateUniversity,Haslett,MI,USA,2LargeAnimalClinicalSciences,MichiganStateUniversity,Haslett,MI,USA,3AntelBiosystems,Lansing,MI,[email protected]:HealthinCattlePopulations–1,Room3,12/4/20179:00AMBovine leukemia virus is a retrovirus which infects lymphocytes of cattle. Approximately 30-40% of infected cows will develop aconditionofpersistent lymphocytosisand less than5%willdevelopmalignant lymphosarcoma. Ina2010 studyconductedbyourresearchgroup,usingmilkELISAsandherdprofiling,itwasestimatedthat87%(97/112)ofdairyherdsinMichiganwereinfected,withherdprevalencerangingbetween0and76%.Analysisofthisstudyshowedthatasherdprevalenceincreased,milkproductionandcowlongevity decreased. Risk factor analysis indicated needle reuse, absence of fly control, gouge dehorning, and increased dry cowinjectionswereassociatedwithincreasedherdprevalence.In2012,cowsseronegativein2010wereretestedin108herds(range1-25).Usingtheseresults,theobjectiveofthisanalysiswastoexaminetheseroconversionriskamongMichigandairyherdsandhowitwasinfluencedbyherd-andcow-levelfactors.Inthetimebetweenindividualretests(range:567-770d),26.6%(350/1316)ofstudycowswithin82%(89/108)of retestedherdsseroconverted.AmongtheherdsconsideredtobeBLV-negative in2010,73%(11/15)remainedseronegative.Herd-levelanalysisshowedthattheproportionofaherd’sstudycowsthatseroconvertedrangedbetween0and100%(mean32.9%;median27.9%).Toanalyzetheherd-levelriskofseroconversion,countmodelingwasusedtoaccountforthevariationinthenumberofcowsretestedperherd.Theprobabilityofanindividualcowseroconvertingwasexaminedusinglogisticregressionmodelsthatcontrolledforthehierarchicalstructureofthedata.Theresultsofthesemodelsshowsignificantvariationsamongherdswithherdprevalencebeingpositivelyassociatedwithseroconversion.Atthecow-level,lactationnumberandthenumberofdaysbetweentestingdidnotsignificantlyaffecttheprobabilityofseroconversion.58-Impactofbovineleukemiavirusoncow-levelsomaticcellcountsasanindicatorofmammaryinflammationR.M.LaDronka1, J.E.Maeroff1,B.Norby1,T.M.Byrem2,R.J.Erskine1,P.C.Bartlett1. 1LargeAnimalClinicalSciences,MichiganStateUniverstiy,EastLansing,MI,USA,2AntelBioSystems,Lansing,MI,[email protected]:HealthinCattlePopulations–1,Room3,12/4/20179:15AMBovineleukemiavirusisaretrovirusofcattlewhichhasbeendemonstratedtoaltertheimmunefunctionofinfectedcattle.Themajorityofstudies-especiallythoseinthelastdecade-haveconsistentlyfoundthatBLVinfectionisnegativelyassociatedwithmilkproductionandcowlongevity.Onepossibleexplanationforthisfindingisthatalteredimmunefunctionrendersinfectedcattlemoresusceptibleto infectious diseases such as mastitis. In clinical practice, an elevated somatic cell count is used as a measure of mammaryinflammation,indicatingsubclinicalorclinicalmastitis.TheobjectiveofthisstudyistoestimatetheeffectofBLVinfectiononudderhealthbasedonSCCdata. Herdswereenrolledwiththeassistanceof localDHIorganizations.Asampleof40cowsperherdweretestedforanti-BLVantibodiesbymilkELISA,andwithin-herdprevalencewasestimatedforeachherd.Atotalof103herds(4,120cows)wereenrolledfrom11states.Atleastonecowtestedpositivein94%ofherds.Themeanwithin-herdprevalencewas46.5%.Herdproductionrecords,includingSCCdata,werecollectedelectronicallyforalltestedcows.AnewcaseofmammaryinflammationwasdefinedasacowhavingaSCC≥200,000cells/mLonthecurrentDHItestandaSCCof<200,000cells/mLontheprevioustest.InapreliminaryanalysisoftheSCCdata,anaverageof8.7milktestsfor387cowsin9herdswereevaluated.37.1%ofBLVnegativecowsand 45.5% of BLV positive cows had at least 1 new case ofmammary inflammation. Using PROC GLIMMIX in SAS with a binarydistribution,controllingforthenumberoftestdaysthatwereevaluatedforeachcowandincludingherdasarandomeffect,ELISA-positivecowswere1.64(1.016-2.661)timesmorelikelythantheirnegativeherdmatestohaveat leastonenewcaseofmammaryinflammation.TheincreaseinmammaryinflammationinBLVpositivecowssupportsthehypothesisthatdecreasedmilkproductioninBLVpositivecowsisatleastpartlyduetoincreasedratesofsubclinicalorclinicalmastitis.

CRWAD2017 ABSTRACTS


59-SelectiveremovalofhighproviralloadcowstocontrolbovineleukemiavirusindairyherdsV.J. Ruggiero1, O.J. Benitez1, H. Hutchinson1, B.Norby2, P.C. Bartlett2. 1ComparativeMedicine and Integrative Biology, College ofVeterinaryMedicine,Michigan State University, East Lansing,MI, USA, 2Department of Large Animal Clinical Sciences, College ofVeterinaryMedicine,MichiganStateUniversity,EastLansing,MI,[email protected]:HealthinCattlePopulations–1,Room3,12/4/20179:30AMBovineleukemiavirus(BLV),aretrovirusofcattle,isoftenpresentathighprevalenceinUSdairyherds(>40%),resultingindecreasedmilkefficiencyandanimalhealth.CurrentBLVcontrolprogramsfocusonreducingtransmissionby improvingmedicalhygieneandreducing prevalence by recommending removal of infected cows as identified by ELISA antibody testing. However, these controlprogramshavehadmixedresultsandcantakedecadestobesuccessful.Furthermore,aggressiveprogramstoremove(cull)allinfectedanimals are not economically feasiblewhen prevalence is high. A different approach to BLV control is therefore needed in high-prevalencedairyherds.Proviralload(PVL)hasbeenusedasameasureofinfectiousnessincontrolandtreatmentprogramsformanyretroviruses. We hypothesize that selective removal of cows with high PVL will reduce BLV prevalence by reducing within-herdtransmission.ThreedairyherdswithhighinitialBLVprevalence(65%,64%,and58%)wereenrolledinaBLVcontrolprogramfocusedonselectiveremovalofhighPVLcows.BloodfromBLV-ELISApositivecowswasanalyzedforPVLandlymphocytecount.Herdmanagerswereprovidedthisdataforeachcowandaskedtoconsiderthisinformationwhenmakingcullingdecisions.In1.5yearsofenrollment,BLVprevalencedecreasedsignificantlyintwoherds-from64%to30%andfrom58%to44%.HerdBLVprevalenceremainedstableinthe remainingherdover a one-year enrollmentperiod. Thedecrease in prevalence in the3herds togetherwas significant at p <0.0000001bytheMHchi-squaretestfortrend.Overall,thisevidencesupportsthisapproachtoBLVcontrolandholdsthepotentialforuseinBLVcontrolprograms.60-PredictivemodelingforearlylactationdiseasesintransitiondairycattleL.Wisnieski,B.Norby,S.J.Pierce,T.Becker,L.Sordillo.MichiganStateUniversity,EastLansing,MI,[email protected]:HealthinCattlePopulations–1,Room3,12/4/20179:45AMTransitioncowdiseasessuchasmastitis,metritis,andketosis,cannegativelyimpactanimalwelfareandreducedairyherdprofitability.Disease incidence has remained relatively stable over time despitemonitoring andmanagement efforts of the transition period.Typically,dairycattlediseaseriskismonitoredbyassessingmultiplefactors,includingcertainbiomarker-testresults,healthrecords,feedintake,andmilkproduction.However,thesefactors,whichareusedtomakeherdmanagementdecisions,areoftenreviewedseparatelywithouttheconsiderationofthecorrelationortherelationshipbetweenthem.Predictivemodeling,whichusesdatatopredict future outcomes, is amethod to combine themost predictive factors and their interactions efficiently in one calculation.Another issuewith currentmonitoringprocedures for transition cowhealth is that risk factorsareoften identified too late in thelactationcycletoimplementproactiveinterventionsthatwouldreducediseaseincidenceinthatparticularcohort.Thereisaneedtodetectcattleatriskfordiseaseinearlylactationbeginningatdry-off,sothatproactiveinterventionsmaybeapplied.Ourobjectivewastobuildapredictivemodelatdry-offforearlylactationdiseasesusingmulti-levellogisticregression.Oursampleincludedapproximately280cowsfromfiveMichiganfarms.Variablestestedformodelinclusionincludedbiomarkersformetabolicstress,andothercovariatesincludingparity,season,anddiseasehistory.First,thenumberofvariablesincludedinmodelselectionwasreducedusingsignificancelevel(p<0.25) inbivariablelogisticregressionanalyseswithtransitioncowdiseaseastheoutcome.Then,weimplementedforwardselectionproceduresusingAkaikeInformationCriteria(AIC).Forourfinalpredictivemodel,weevaluatedpredictivevalueviareceiveroperatingcharacteristic(ROC)curveanalysisandgoodness-of-fitbytheHosmer-Lemeshowtestanddiagnosticgraphs.Thenextstepistotesttheeffectivenessofourmodelfordiseasepredictioninotherherds.

CRWAD2017 ABSTRACTS


61-Bovineherpesvirustype-4amongpostpartumdairycowsinCalifornia:riskfactoranalysisusingmultilevelmixedmodelD.Areda1,M.Chigerwe2,B.Crossley3.1SchoolofVeterianryMedicine,UniversityofCalifornia,,3121TupperHall,Davis,CA95616,CA,USA,2SchoolofVeterianryMedicine,UniversityofCalifornia,Davis,CA,USA,3SchoolofVeterianryMedicine,CaliforniaAnimalHealthandFoodSafetyLaboratory,ThurmanLab,Davis,CA95616,CA,[email protected]:HealthinCattlePopulations–1,Room3,12/4/201710:00AMPurpose:Bovineherpesvirus-4knowntobeassociatedwitharangeofinfectionsanddiseasesincludingabortion,metritis,vaginitis,enteritis,andpneumonia.InCaliforniaonlyafewstudieswerecarriedoutonBoHV-4statusindairycows.AimsofthisstudyweretodetermineaprevalenceandriskfactorsforBoHV-4infectionamongpostpartumdairycowsintwocounties:TulareandYolo.Methods:Across-sectional study involvingmulti-stagesampling techniquewasused.Uterine/vaginaldischargesampleswerecollected fromcowswithpost-partummetritisandthosewithoutmetritis(controls).SamplesweretestedforBoHV-4andotherco-infectingvirusesusingreal-timePCR.Datawereanalyzedusingmulti-levellogisticmixedeffectmodel.Onehundredandforty-eightcowswereenrolledfrom11dairyfarms.Results:PrevalenceofBoHV-4infectionwas22.3%(33/148),whilepost-partummetritiswas33.8%(48/142).StrongassociationwasfoundbetweenBoHV-4infectionandlactationnumber,lactationstageandpost-partummetritis.TheoddsofbeingpositiveforBoHV-4infectionwere6.47times(95%CI;1.17,35.92;P<0.05)and6.79times(95%CI;1.19,38.55;P<0.05)higherforcowsinthe4thand5thlactation,respectively,comparedtocowsinthe1stlactation.Bovineherpesvirusinfectionwas8.27timesmorelikely(95%CI;1.43,47.94;P<0.05)amongcowsinearlystageoflactation(0-120days)comparedtothoseinlatelactation(>240days).Cowswithpost-partummetritiswere4.51times(95%CI;1.27,16.02;P<0.05)morelikelytotestpositiveforBoHV-4infectioncomparedtothosewithoutpost-partummetritis.Conclusions:ThisstudydemonstratedstrongassociationofBoHV-4withmultiparityandearlystageoflactation.SignificantassociationofBoHV-4infectionwithpost-partummetritissuggeststheneedforimplementingvirusinclusivetreatmentregime.62-Ratesofco-infectionforthreeendemicdiseasesinUSdairyherdsG.Rossi1,J.A.Herrmann2,R.L.Smith1.1Pathobiology,UniversityofIllinoisCollegeofVeterinaryMedicine,Urbana,IL,USA,2VeterinaryClinicalMedicine,UniversityofIllinoisCollegeofVeterinaryMedicine,Urbana,IL,[email protected]:HealthinCattlePopulations–2,Room3,12/4/201710:45AMPurpose:DairyfarmsintheUSoftenstrugglecontrollingmultipleendemicdiseasessimultaneously.Despitethis,manydiseasecontrolstudies focusoneachdisease individually.One reason for thismightbe the lackof informationon interactionsbetweenendemicdiseases.Weconductedapilotcohortstudytodeterminetheratesofco-infectionwiththreeendemicpathogensincommercialdairyfarms:BovineLeukosisVirus(BLV),Neosoporacaninum (NC),andMycobacteriumaviumsubsp.paratuberculosis (MAP). Methods:TwocommercialdairyherdsintheupperMidwestwereenrolled,and50animalsfromeachwererandomlyselectedforparticipation,withstratificationacrossthefirstthreeparities.SerumsampleswerecollectedeverytwomonthsfromallanimalsinthestudybetweenJanuaryandJuly2017(atotalof4samplings),andallsamplesweretestedforeachofthethreepathogens;culledanimalswerereplacedwithanimalsrandomlyselectedfromthesamelactation.Results:Intotal,54animalsweresamplefromfarmMand59animalsweresampledfromfarmB.SeroprevalenceofMAPandNCwerelowonbothfarms(MAP=0.02andNC=0onfarmM;MAP=0.03andNC=0.07onfarmB).SeroprevalenceofBLVwaslowonfarmM(0.04)buthighonfarmB(0.73).Co-infectionprevalencewas0forallpathogenpairsonfarmM,butonfarmBoneanimalwaspositiveforbothBLVandMAPandtwoanimalswerepositiveforbothBLVandNC.Conclusion:Ourdatashowednoindicationofasignificantincreaseinco-infectionriskoverexpectedbasedonFisher’sexacttests.Co-infectionratesweretoolowtoanalyzetheimpactofco-infectiononproductionparameters.However,furtherstudiesarerequired,includingmorefarms,aspowerwaslowduetothelowprevalenceofmostpathogens.

CRWAD2017 ABSTRACTS


63-WhatistheeffectofeliminatingpneumoniaincalvespriortoweaningonnetincomeoftheUScow-calfindustry?M.Wang,R.W.Wills,D.R.Smith.DepartmentofPathobiologyandPopulationMedicine,CollegeofVeterinaryMedicine,MississippiStateUniversity,Starkville,MS,[email protected]:HealthinCattlePopulations–2,Room3,12/4/201711:00AMPurpose:ToinvestigatethedifferenceinnetincomebetweenaUSbeefcow-calfsystemeitherwithorwithoutpneumoniainbeefcalvespriortoweaning.Methods:Cattlemarketdatafrom1990to2016wereusedtocreateequationstosimulatetheannualnetincomeofbeefcow-calfindustryovera110yearperiod.ParametervaluesforsimulationsweredrawnfromUSDAandpeer-reviewedpapers.AsystemdynamicsmodelwasdevelopedbyusingVensimandseveralscenariosweredesigned:1)thecurrentsituationwithpneumoniainbeefcalvespriortoweaning;2)eliminationofpneumoniawithoutanycost;2)eliminatepneumoniawithanannualcostpercowof$10,$20,$30,$40,and$50,respectively.Thesimulationresultswerevalidatedtoseewhetherthemodelrepresentedtheactualbehaviorofthebeefcattlecow-calfsystem.Results:TheoscillationinUSbeefcowinventory,feedercattlevalue,andnetincomeper cow followed the classically described 10-year cattle cycle. The cumulative industry net income decreased in the scenario ofeliminatingpneumoniawithoutanycostcomparedtothecurrentsituationwithpneumonia.Asmoremoneywasspenttoremovepneumonia,thebeefcowinventoryreducedwhichincreasedfeedercattlevalueoveryears,andannualnationalnetincomeincreasedwithlessfluctuationcomparedtothesituationwithpneumoniaintheindustry.Conclusions:Currently,beefcow-calfproducersnotexperiencingpneumoniaintheircalveshaveaneconomicadvantageoverproducerswithpneumonia.Affectedproducersbearthecostofthedisease,butnon-affectedproducersbenefitfromrelativelyhighermarketpricesbecauseoffewercalvesinthemarket.Assumingnochangeindemand,eliminatingpre-weaningpneumoniawithoutcostmaybenefitpreviouslyaffectedproducers,buttheadditionalsupplyofbeefreducescalfmarketpricesforallsothatnetincometotheindustrydoesnotchange.Withincreasedcoststoeliminatepneumonia,thedecreasednumberofcowsandsupplyofcalvesintothemarketresultsinanincreasedcattlevalue,whichmaybenefittotalnetincomerelativetothecurrentsituationwithpneumoniaincalvespriortoweaning.64-Aneconomicdecisiontoolforcow-calfproducersinKansasunderriskofbovineanaplasmosisM.B.Apamaku.AgriculturalEconomics,KansasStateUniversity,Manhattan,KS,[email protected]:HealthinCattlePopulations–2,Room3,12/4/201711:15AMProduction losses due to bovine anaplasmosis impact cow-calf producer returns. Available prevalence estimates for the US, andanecdotalreportsinKansaspointtoanexpandingdistribution.IntheUS,bovineanaplasmosisisnon-reportableandendemic.Intheabsenceoffederalcontrolprograms,cow-calfproducerslackobjectiveriskmanagementoptions.WedevelopatargetMOTADmodeltoassessnetreturnsfor3anaplasmosismanagementoptions-‘DoNothing’,‘Test/Treat’and‘Vaccinate’.Using5-yearprevalencedata(K-StateVet.DiagnosticLab;2010/2014)and5-yearenterprisebudgetandincomedata(KansasFarmManagementAssoc.),netreturnsforrepresentativecow-calffarms(maximumexpectedreturns/cow)wereassessedatvariedprevalencerates,underriskscenariosof‘DoNothing’,‘Test/Treat’,or‘Vaccinate’.Preliminaryresultsindicatethatproducersoptto‘Test/Treat’(netreturn/cow=$234),or‘Donothing’ (net returns/cow=$234),comparedwith ‘Vaccinate’ (net return/cow=$222).Underourmodelspecifications, sensitivityanalyses(varyingincomedeviation)resultedinnochangeinexpectednetreturn-farmersdonotexperienceachangeinexpectedreturnbyvaryingrisk.Modelevaluationisongoing,torefineconstraintsbyexpertopinion,andtoincorporateotherenterprisesbeyondcow-calf. Well-specified models representing realistic producer behavior would provide an objective template for cow-calf riskmanagementforoptimalbiosecurity.

CRWAD2017 ABSTRACTS


65-UnderstandingreservoirsitesofFusobacteriumnecrophoruminsheepandtheirimportanceforovinefootrotR.Clifton,K.J.Purdy,L.E.Green.SchoolofLifeSciences,UniversityofWarwick,Coventry,[email protected]:HealthinCattlePopulations–2,Room3,12/4/201711:30AMPurpose:Footrotisthemostimportantcauseoflamenessinsheepandresultsinsignificanteconomiclossesforsheepproducers.Itisaninfectiousdisease,causedbyDichelobacternodosus.Fusobacteriumnecrophorumisasecondarypathogenthatincreasesfootrotseverity. It isassumedthatF.necrophorum iswidespread in theenvironmentof sheep;however, therehavebeenno longitudinalstudies investigatingreservoirsitesofF.necrophorum insheep.Thepurposeofthisstudywasto identifysitesofpersistenceofF.necrophorum,andtodeterminetheirrelevanceforfootrot.Methods:Agroupof40sheepwereexaminedweeklyfor20weeksduringSpring2015.Footrotlesionswerescored,andfootswabs,mouthswabsandfaecalsamplesweretaken.Samplesofsoilandgrasswerecollectedweeklyfromlowandhighuseareasofthepasture.Allenvironmentalsamples,plussamplesfrom30sheep,wereanalysedusing quantitative PCR and multiple locus variable number tandem repeat analysis (MLVA). Non-parametric tests were used forstatistical analyses.Results:F. necrophorumwasdetected in 0.9%of soil samples and0%of grass samples. Faecal sheddingofF.necrophorumoccurred in3sheepfor1to4consecutiveweeks.F.necrophorumwasdetected inthemouthsof8sheepfor1to6consecutiveweeks,andonthefeetof20sheepfor1to12consecutiveweeks.Medianlog10(load+1)ofF.necrophorumwashigheronfeetwithfootrot(5.38,IQR3.87-6.26)comparedtoonhealthyfeet(2.76,IQR2.37-3.57).UsingsurvivalanalysisF.necrophorumwasmore likelytopersistonfeetwithfootrot,whilsthealthyfeetwereonlytransientlypositive.MLVAindicatedthattwostrainsofF.necrophorumwerepresentonfeetwithintheflock,andthesetwostrainsofF.necrophorumwerealsodetected inmouthsand infaecesforupto3consecutiveweeks.Conclusions:Contrarytopriorassumption,theenvironmentwasnotasignificantreservoirofF.necrophorum; insteadF.necrophorum persisted in sheep,primarilyon feetwith footrot.Mouthsand faeceswerean intermittentreservoirforthestrainsofF.necrophoruminvolvedinfootrot,andmayfacilitatepersistenceandtransmissionintheabsenceofdisease.66-SeroepidemiologyofBrucellaspp.inhumansandlivestockinnorthernKenya:opportunitiesforOneHealthinterventionsB.K.B.Bett,Sr.1,S.Kairu-Wanyoike2,D.Nyamwaya1,M.Wainaina1,J.Lindahl1,E.Ontiri3,S.Bukachi4,I.Njeru5,J.Karanja5,G.Delia1.1Animal and Human Health, ILRI, Nairobi, Kenya, 2Epidemiology, Department of Veterinary Services, Nairobi, Kenya, 3SustainableLivestockSystems,ILRI,Nairobi,Kenya,4GenderandAgricanStudies,UniversityofNairobi,Nairobi,Kenya,5DiseaseSurveillanceandResponse,MinistryofHealth,Nairobi,[email protected]:HealthinCattlePopulations–2,Room3,12/4/201711:45AMBrucellosisisabacterialzoonosiswithconsiderablemorbidityratesinlivestockandhumans.Weimplementedacross-sectionalstudyinGarissaandTanaRiverCounties,Kenyatodeterminetheprevalence,riskfactorsandthedistributionofthediseaseinpeopleandlivestockathouseholdandvillagelevels.Foursiteswereused-twoinpastoralareasinGarissaCountyandtheothersinirrigatedandpastoralareasinTanaRiverCounty.Ahouseholdwasaunitofanalysisandpeopleweresampledinalltheseareaswhilelivestockcouldonlybe sampled inTanaRiverCounty. Serumsampleswereobtained fromselected livestockandpeople from randomly selectedhouseholdsandscreenedforanti-BrucellaIgGantibodiesusingELISA.Datawereanalyzedusingahierarchicalrandomeffectslogisticregressionmodel.Theoverallseroprevalencesinlivestock(n=2,017)andhumans(n=1022)were3.47%(95%CI:2.72–4.36%)and35.81%(95%CI:32.87–38.84),respectively.Livestockfromthepastoralareashadsignificantlyhigherseroprevalence(9.09%;95%CI:5.90–13.13)comparedtothosefromtheirrigatedarea(2.89%;95%CI:2.11–3.86).Asimilartrendwasobservedinhumanswherebrucellosisseroprevalenceinthepastoralandirrigatedareaswere43.92%(95%CI:40.27–47.63)and16.38%(95%CI:12.33–21.13),respectively.UsingresultsfromTanaRiverCounty,theoddsofexposureinhumanswere3.40(95%CI:1.63–7.06)timeshigherinhouseholdsthathadatleastoneseropositiveanimalcomparedtothosethatdidnot.Forlivestockdata,therewasahighercorrelationinBrucellaspp.seropositivityattheherd(InternalCorrelationCoefficient[ICC]=0.39)comparedtothevillagelevel(ICC=0.18).AsimilarobservationwasobservedonthehumandatawherethehouseholdandvillageICCestimatesbeing0.33and0.21,respectively.HumanandanimalBrucellaspp.seroprevalencesclusteredmoreatthehouseholdthanvillagelevels.WeconcludethatOneHealthinterventionscanbeusedsuccessfullytoidentifyinfectedherds/householdsespeciallyifinformationonthelocationsofhumancasesreportedinlocalhealthcenterscanbeusedforrisk-basedsurveillance.

CRWAD2017 ABSTRACTS


67-AsmallsteptowardSchwabe'svisionforpreventivemedicine?Y.A.Henao-Diaz.VeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversity,Ames,IA,[email protected]:DiagnosticTesting–1,Room3,12/4/20172:00PMIn 1982, Calvin Schwabe urged veterinary preventive medicine to become "a form of on-going on-farm research, based uponsurveillanceandemphasizingthefactthatdiagnosis⋯ resemblesaresearchprocess." PROBLEM:Schwabe'svisionhasneverbeenrealizedbecauseroutinesurveillanceusingindividualanimalsamples(serum)istoodifficultandexpensive.Oralfluid(OF)isaneasilycollected,welfare-friendly,population-basedspecimencompatiblewithSchwabe'sgoalof"on-goingon-farmresearch",butOFoftencontainfeed,feces,orothercontaminantsthatmayaffectpipettingaccuracyand/ortestperformance.Samplequalityisanissuethatmustberesolved.Filtrationorcentrifugationofsamplesarenotcompatiblewithhigh-throughputlaboratories.However,treatmentofsampleswith"coagulants"(chemicalsusedtoclarifyliquidmatrices)isanoptionthathasnotbeenexploredpreviously.Tofollowthisline of investigation, we evaluated the effect of chitosan-based clarification of oral fluids on the detection of PRRSV antibody.METHODS:OFsamplesofknownstatusweregeneratedbyvaccinatingpigs(n=17)withaPRRSVMLVvaccine.Individualpigsampleswere collected from day post vaccination -7 to 42 and subdivided into 4 aliquots. Each aliquot was treated with one coagulantformulation(A,B,C)withthe4thaliquotservingasanuntreatedcontrol(NC).AllsamplesweretestedbyPRRSVOFELISAimmediatelyaftertreatment(daypost-treatmentDPT0).Sampleswerethenheldat4°Candre-testedonDPTs2,4,6,and14.RESULTS:AmongallDPTs,nodifferencewasdetectedintheproportionofpositivePRRSVantibodysamplesamongtreatments(Cochran'sQ,p>0.05).ArepeatedmeasuresmultiplecomparisonanalysiswithTukeyadjustmentfoundnosignificantdifferenceinELISAS/Presponsesbetweentreatments by storage time. CONCLUSIONS: Clarification oral fluids using chitosan-based formulations did not affect the PRRSVantibodyELISAresultseitherimmediatelyorovertime(DPT).Theresultssuggestedthatchitosan(orothercoagulants)couldimproveoralfluidhandlingcharacteristicswithoutaffectingantibodytestingresults.Thisdirectionholdspotential.68-ModelingthesheddingofprionsindeersalivainthefaceofimperfectdetectionK.A.Davenport1,B.A.Mosher2,B.M.Brost3,C.K.Mathiason1,E.A.Hoover1. 1PrionResearchCenter,ColoradoStateUniversity,FortCollins, CO, USA, 2Fish, Wildlife and Conservation Biology Department, Colorado State University, Fort Collins, CO, USA, 3MarineMammalLaboratory,AlaskaFisheriesScienceCenter,NationalOceanicandAtmosphericAdministration,Seattle,WA,[email protected]:DiagnosticTesting–1,Room3,12/4/20172:15PMChronicwastingdisease(CWD)isanemerginginfectiousdiseaseofcervidswithunknownpotentialtoinfecthumansandlivestock.CWDspreadsefficientlyamongcervidpopulations,butthemechanismofhorizontaltransmissionremainsundefined.Infecteddeershedprionsinsaliva,urineandfeces,whichlikelycontaminatetheenvironmentandestablisharesilientreservoirofpathogensfortheinfectionof naïve cervids. To complicate the studyof prion shedding and transmission, the specificity and sensitivity of detectionmethodsare imperfect inexcreta.Wesoughtananalyticalapproach thatcouldwithstand imperfect sensitivityandspecificityandenableustoexaminetherelationshipbetweenprionsheddingandcharacteristicsoftheinfecteddeer.Wehavecombinedacutting-edgepriondetectionmethod(real-timequaking-inducedconversion,RT-QuIC)withoccupancymodeling,anapproachborrowedfromecology.Ouroccupancymodelenabledustoaccountforbothfalsepositiveandfalsenegativedetectionerrorsinouranalysisof200longitudinalsalivasamplesfrom45deer.Therefore,wewereabletodiscriminatethesheddingofprionsinsalivaandthedetectionofprions in saliva, a distinction crucial to understanding the role of prion shedding in disease transmission and for diagnosis. Wedemonstratedthatassaysensitivityandspecificitywereindeedimperfect,andwewereabletodrawseveralconclusionspertinenttoCWDbiologyfromouranalyses:(1)sheddingofprionsinsalivaincreaseswithtimepost-inoculation,butiscommonthroughoutthepre-clinicalphaseofdisease;(2)sheddingpropensityisinfluencedneitherbysexnorbythegeneticsusceptibilityofthedeertoCWD;and(3)thesourceofprion-containinginoculumusedtoinfectdeeraffectsthelikelihoodofprionsheddinginsaliva-oralinoculationofdeerwithCWD(+)salivaresultedin2.94timesthelikelihoodofprionsheddinginsalivacomparedtoinoculationwithCWD(+)brain.TheseresultsarepertinenttothetransmissiondynamicsofCWD.Moreover,ourapproachisapplicabletootherdiagnosticassayswithimperfectdetection.

CRWAD2017 ABSTRACTS


69- Identifyingwithin-hostmultipleinfectionsofparatuberculosisusingwholegenomesequencingdatatoimproveprecisionintransmissionanalysis

Y.wang1,S.J.Wells2.1GraduatePrograminBioinformaticsandComputationalBiology,UniversityofMinnesota,SaintPaul,MN,USA,2VeterinaryPopulationMedicine,CollegeofVeterinaryMedicine,UniversityofMinnesota,SaintPaul,MN,[email protected]:DiagnosticTesting–1,Room3,12/4/20172:30PMParatuberculosis,(orJohne’sdisease(JD))isachronic,nontreatablemycobacterialdiseaseresultinginweightloss,diarrhea,anddeath,in cattle and other ruminants. Studies have shown that at least 90% of U.S. dairy operations have JD infected cattle. High herdprevalence, an extensive latent period of infection (1.5 years), and persistence in farm environments have resulted in a complexsituationwhere a cow can be infectedwithmultiple strains on farm. For thesemultiple infection cases, phylogenetic trees oftenproduce misleading results in disease transmission analysis, leading to confusion in understanding transmission.To solve this problem, we developed a method that uses whole genome sequence (WGS) alignments ofMycobacterium aviumsubspecies paratuberculosis (MAP) isolated from cattle feces to calculate strain concentration for multiple infected samples. Asdemonstratedinpreviousresearch,sharedSNP(s)betweenanimalscouldbeconsideredasevidenceoftransmissioninslowevolvingmycobacterium.Hence,webelieve that sharingmultipleevolutionarypathswithothers suggest thatan individualmayhavebeeninfectedbymultiple individuals.Ourmethodutilizedacombinationofdistance-basedphylogenetictreesandNon-NegativeMatrixFactorization(NMF),aMachineLearningtechnique,toconstructareferencepanelofpossibleancestralstrainscirculatinginthelocalenvironmentandcalculatesmixturecoefficientsforstrainconcentrationinmixedinfectedsamples.Testedon106samplesfrom5MNdairyherds,ourmethodidentified~10%animalswithmultiple infectionsofJD.Recognizingthesecaseshaveresulted instructuralchangesofphylogenetictreeintransmissionanalysis.Toourknowledge,thisworkrepresentsthefirstattempttouseWGSdatatocomputationally quantify multiple MAP infections from a transmission perspective. Use of this method will greatly improveunderstandingofheterogeneityofstraininfectivityandprovideusefulinformationtocattleproducersofbreedingfarmsandheiferraiserstoidentifyhighlyinfectiousanimalsandtracktransmissionroutesinlocalfarmenvironments.70-Moleculartechnologyinforeignanimaldisease(FAD)antibodyassaydevelopmentK.Poonsuk,500101,L.Giménez-Lirola1,K.Kamrud2,J.DeHart3,J.Zimmerman1.1VeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversity,Ames,IA,USA,2SyntheticGenomicsVaccinesInc.,LaJolla,CA,USA,3SyntheticGenomicsInc.,LaJolla,CA,[email protected]:DiagnosticTesting–1,Room3,12/4/20172:45PMINTRODUCTIONExperiencehasshownthatimportationcontrolswillnotalwaysstopthemovementofFADsviaanimals,animalby-products,and/orfeedstuffs.WhenanFADentersNorthAmerica,massivenumbersofsampleswillneedtobetested.BothPCR-andantibody-basedassayshavearoleinthiseffort;eachhaslimitations,butonecompensatesfortheweaknessoftheother.Mostrecenteffort in assay development has focused on PCR technology, in part because antibody assay development requiresworkingwithspecimensofknownFADantibodystatus.HereinwedescribeanapproachforantibodyassaydevelopmentthateliminatestheneedtohandlepathogensorsamplesfromFAD-infectedanimals.METHODSAntibodyassaydevelopmentrequires:1)antigentobeusedintheassayand2)samplesofknownantibodystatustoguidetheoptimizationofthetest.Antigensarereadilycreatedusingmoleculartechnology,e.g.,anAfricanswinefeverELISAwasbasedonarecombinantp30proteincreatedbycloningpolyproteingenep30intoanEscherichiacoliplasmid(Giménez-Lirolaetal.,2016).FADantibody-negativesamplesarereadilyaccessible,butcreatingantibody-positivesamplesoutsideofBSL-3facilitieshasbeenimpossible.Inpreviouswork,safe,ASFVantibody-positivespecimens(serum,oralfluid)werecreatedbyinoculatingpigs(n=17)witharepliconparticleexpressingtheASFVp30gene.ForthedevelopmentofaFMDV3ABCELISA(workinprogress),anRNAvector(SyntheticGenomics®,LaJolla,CA)designedtostimulatetheproductionofFMDV3ABCantibodywillbeused.CONCLUSIONSTheuseofmoleculartechnologyinantibodyassaydevelopmentremovestheneedforinfectingpigswithFADs,therebyeliminatingtheneedforbiocontainmentfacilitiesandgreatlyreducingdevelopmentcosts.Thisapproachwillallowustodevelopaprototype3ABCoralfluidantibodyassay.

CRWAD2017 ABSTRACTS


71-ComparisonofthreedetectionmethodsusedtoidentifyenterohemorrhagicEscherichiacolisamplesofbovineoriginL.G.Schneider1,G.L.Lewis2,T.G.Nagaraja3,R.A.Moxley2,D.R.Smith1.1CollegeofVeterinaryMedicine,MississippiStateUniversity,MississippiState,MS,USA,2SchoolofVeterinaryMedicineandBiomedicalScience,UniversityofNebraska-Lincoln,Lincoln,NE,USA,3CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,[email protected]:DiagnosticTesting–1,Room3,12/4/20173:00PMPurpose:SevenserogroupsofenterohemorrhagicEscherichiacoli(EHEC-7)arepathogensofpublichealthinterest.Cattlepopulationsserveasareservoirfortheseorganisms,andbeefproductsareimplicatedasamajorsourceoffoodborneillness.TheobjectiveofthisstudywastocompareresultsfromthreeEHECdetectionmethodsforeachserogroupofEHEC-7.Methods:Fecal(n=1,357)andhide(n=699)sampleswerecollectedandtestedforEHEC-7inparallelbythreemethods:enrichedculture,NeoSEEK™STECDetectionandIdentificationtest(NS),andmultiplexquantitativepolymerasechainreaction(mqPCR).Three-wayagreementwasdeterminedusingFleiss’Kappastatistic. Inaddition, two-waycomparisonsweremadeusingCohen’sKappaandMcNemar’sChisquare.Results:TheprobabilityofdetectingEHEC-7infecalsampleswas10.0%,14.4%,and18.9%forculture,NS,andmqPCR,respectively.TheprobabilityofdetectingEHEC-7inhidesamplesasdeterminedbyculture,NS,andmqPCRwas2.0%,7.2%,and19.0%,respectively.Infecalsamples,Fleiss’KappastatisticsforthethreedetectionmethodsrevealedmoderateagreementforEHECO157(Κ=0.49),fairagreementforEHECO26(Κ=0.40)andO103(Κ=0.37),slightagreementforthedetectionofEHECO45(Κ=0.12),O111(Κ=0.11),andO145(Κ=0.11),andpooragreement for EHECO121 (Κ=0.03). In hides samples, Fleiss’ Kappa statistics indicatedmoderate for the detection of EHECO111(Κ=0.52)andO157(Κ=0.50),fairagreementforEHECO26(Κ=0.37)andO103(Κ=0.26),slightagreementforEHECO45(Κ=0.16),andpooragreementforEHECO121(Κ=-0.005)andO145(Κ=-0.005).Conclusions:Resultsofthisstudyindicatethatagreementbetweenculture,mqPCR,andNSvariesby serogroup,but is similarbetweensample types. Inconclusion,nogold-standardEHECdetectionmethod exists, and further work is needed to determine which EHEC detection methodology should be utilized to increase theprobabilitytocorrectlyclassifysamplesofbovineorigin.72-Assessingtheuseofacommerciallyavailablestall-sideserumamyloidAtestfordiagnosingsepsisinequineneonatesS.Strouss1,B.Barr2,M.Rossano1.1Animal&FoodSciences,TheUniversityofKentucky,Lexington,KY,USA,2RoodandRiddleEquineHospital,Lexington,KY,[email protected]:DiagnosticTesting–1,Room3,12/4/20173:15PMTheobjectiveofthisstudywastoassessmeasuringserumamyloidA(SAA)usingacommerciallyavailablestall-sidelateralflowkittodiagnosesepsisinneonateslessthanoneweekofage.Theclinicalrecordsof124neonateslessthanoneweekofageadmittedtotheintensivecareunitof thesameequinehospital from2014-2016wereanalyzedretrospectively.Theneonatesweregrouped into9categories based on primary diagnosis: colitis, contracted limbs, enteritis, hypoxic-ischemic encephalopathy (HIE), pneumonia,prematurebirth,sepsis,weakfoalandnormal.Theattendingveterinariansdeterminedprimarydiagnosisafterobtainingthepatient’shistory, performing a physical examination, and interpreting bloodwork results. Bloodwas obtained for each neonate by jugularvenipunctureorajugularcatheteruponadmissionorimmediatelyafterbirthatthehospital.TheserumwastestedusingtheStableLabEquineBloodAnalysisKit.ThedataobtainedforthisstudywereanalyzedusingtheWilcoxonranksumtestinSAS.Ap-valueof<0.05wasdeemedsignificant.ThemedianSAAconcentrationforthenormal,prematurebirth,contractedlimbs,weakfoalandHIEgroupswerewithinthetestmanufacturer’snormalrangesforneonates(0-20mg/L).Themedianconcentrationforthecolitisgroupindicatedpossibleinfection(20-100mg/L).Themedianconcentrationforthesepsis,enteritis,andpneumoniagroupsindicatedinfection(>100mg/L).TherewasasignificantdifferencebetweentheSAAconcentrationsfortheprematurebirth,contractedlimbs,weakfoal,HIEandnormalfoalgroupswhencomparedtothesepsisgroup.TherewasnosignificantdifferencebetweentheSAAconcentrationsfortheenteritisandpneumoniagroupswhencomparedtothesepsisgroup.Theresultsofthisstudysuggestthatacommerciallyavailablestall-sidelateralflowkitisahelpfultooltobeusedindiagnosingsepsis,enteritisandpneumoniainneonateslessthanoneweekofage.Additionaldiagnostictestingwouldberequiredtodifferentiatesepsisfromenteritisandpneumonia.

CRWAD2017 ABSTRACTS


73 -Adaptationof theporcineepidemicdiarrheavirus (PEDV) fluorescent focusneutralization (FFN)assaytoahigh-throughputformatusingimagingcytometry

L.Sarmento, J.C.Mora,K.Poonsuk,R.Main,D.Baum,J.Zimmerman,L.Gimenez-Lirola. DepartmentofVeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversity,Ames,IA,[email protected]:DiagnosticTesting–2,Room3,12/5/20179:00AMSinceitsfirstreportintheUS(April2013),PEDVhasspreadaggressivelythroughoutfarmsaffecting36states.Lastyearalone(2016),1092confirmedcaseswerereported(www.aphis.usda.gov/animal-health/secd)totheUSDA.Becauseofitshighlycontagiousnatureanditsconsequentialeconomiclosses,effortshavebeenmadetodevelopdiagnostictestsabletoaccuratelydiagnoseand/ormonitorPEDVincommercialswinefarms,e.g.,ELISA,RT-PCR,andimmunofluorescence(IFA)assays.Currently,thetestsdescribedtodetectPEDV neutralizing antibodies in pigs are the serum-virus neutralization (SVN) and fluorescent focus neutralization (FFN) assays.Althoughbothprovidehighspecificity,theyaretime-consumingandlabor intensive.Most importantly,theseassaysare inherentlyvariableduetothesubjectivenatureinwhichantibodytitersaredetermined.Inthepresentstudy,wedescribetheadaptationofFFNtoahigh-throughputvirusreductionneutralizationtest(HTNT)usingimagingcytometry(SpectraMaxi3xandSoftMaxPro6.5).Resultsbasedontestingofserumsamplesofprecisely-knownPEDVstatusshowedthatapercentageofvirusneutralizationcut-offof≥85%providedadiagnosticsensitivityof95%andspecificityof98%.Furthermore,resultsshowedfourclearadvantagesovertraditionalFFNassays: 1) fluorescence reading of a 96-well plate is fast (3-4minutes); 2) the use of imaging cytometry eliminates the eye strainassociated with reading plates under amicroscope; 3) reliance on data generated through imaging cytometry eliminates humanoperator-dependentvariationinplatereadingsandmakesdeterminationofvirusneutralizationstatusmoreconsistentandrepeatable;and4)digital imagescapturedby the softwareareavailable to resolveunexpected resultsandsharewithclients.Webelieve thisapproachisbroadlyapplicabletoavarietyofpathogens.74-NewduplexrealtimeRT-PCRtodetectPorcineTeschovirusandSapelovirusinUSpigsM.S.BarreraVaca1, F.Chamba2, T.Knutson1, Y. Jiang1, S.Gresch1, F.Vannucci1,D.Marthaler3. 1VeterinaryDiagnostic Laboratory,UniversityofMinnesota,StPaul,MN,USA,2VeterinaryPopulationMedicine,UniversityofMinnesota,StPaul,MN,USA,3CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,[email protected]:DiagnosticTesting–2,Room3,12/5/20179:15AMPurpose:Wedeveloped,optimizedandvalidatedanewduplexrealtimeRT-PCRtodetectPorcineTeschovirusandSapelovirus,andassessedthedistributionofbothvirusesinswinediagnosticsamples.Methods:Analyticalspecificitywasassessedusingapanelof54porcinepathogens, includingPorcineEnterovirusG.Finally, inter-and intra-assay repeatabilitywasevaluatedtesting10-foldserialdilutionsofbothvirusisolates,5and10timesrespectively.DiagnosticsensitivityandspecificitywasevaluatedusingaconventionalRT-PCRdescribedbefore.DistributionofPTVandPSVindiagnosticsampleswasassessedusing231sampleswithin108submissionsfrom the University of Minnesota Veterinary Diagnostic Laboratory (UMN-VDL). Submissions included environmental, fecal, andintestinalsamplesfrompigsofdifferentagesandUSstates.Results:Intheoptimizationphase,wetested7setofprimersandprobes,6 RT-PCR kits, 4 primers and probes concentrations, 2 sample volumes, 2 final volumes, 2 internal controls concentrations and 4fluorescentdyes,yielding94combinationsfortesting.ThefinalPTVandPSVprimerandprobesetstargetsthe5’UTRregionofeachvirus.Limitofdetectionwas1.0x10-6and1.0x10-810-foldserialdilutionsforPTVandPSVrespectively.Coefficientofvariationforbothviruseswaslessthan9.2%and1.9%forinter-andintra-assayrepeatability.Whilethediagnosticsensitivitywas100%forbothviruses,thediagnosticspecificitywas88%and60%forPTVandPSVrespectivelyandincomparisontotheconventionalPCRwhichhasalowerlimitofdetection.Finally,weassessedthedistributionofPTVandPSVin108submissionsfrom15USstates,whichidentifiedPTVandPSVin82%(89/108)and72%(78/108)ofthesubmissionsfromsuckling,nurseryandfinishingpigs.Seventypercent(76/108)ofthesubmissionstestedpositiveforbothviruses.Conclusions:OurstudyshowedthatournewPCRisspecifictobothvirusesandmoresensitivethanthereportedconventionalPCR.WefoundthatPTVandPSVwerewidelydetectedinthetestedsubmissions.Finally,co-infectionsofPTVandPSVwerecommoninthissubsetofsubmissions.

CRWAD2017 ABSTRACTS


75-DevelopmentandvalidationofrealtimeRT-PCRassayforthedetectionofatypicalporcinepestivirusF.Yuan1,X.Liu1,J.Bai1,G.Anderson1,Y.Fang1,B.Arruda2,P.Arruda2,L.Peddireddi1.1KansasStateVeterinaryDiagnosticLaboratory,KansasStateUniversity,Manhattan,KS,USA,2IowaStateUniversity,Ames,IA,[email protected]:DiagnosticTesting–2,Room3,12/5/20179:30AMAtypicalPorcinePestivirus(APPV)wasreportedastheetiologicagentfortypeA-IIcongenitaltremorinnewbornpiglets.AsensitiveandreliablePCR-baseddiagnostictestiscriticalforaccuratedetectionofAPPV.Wedevelopedaquantitativereal-timeRT-PCR(qRT-PCR)assayforreliabledetectionofallAPPVstrains.Theassaydesignalsoincludedaswine18SrRNAinternalcontroltomonitorPCRinhibitions. A positive control plasmid containing APPV-target region was constructed and a serial of 10-fold dilutions of in vitrotranscriptsobtainedfromtheplasmidwasusedtodeterminelimitofdetection(LOD).Individual18SrRNAandAPPVqRT-PCRassayswereoptimizedseparatelyandthencombinedintoaduplexassay.Theindividualandduplexassayshadcorrelationcoefficientsof0.997andPCRamplificationefficienciesof91-92%.ComparisonofdetectionlimitandanalyticalsensitivitybetweenassaysindicatednoinhibitionofPCRsensitivity,whenbothassaysarecombined.ThedetectionlimitforAPPVtarget,basedonanalyticalsensitivity,is~12copiesperreaction,whichcorrespondstoaCtof~38forbothindividualandduplexreactions.Assayspecificitywasverifiedusingnucleicacids(NA)ofothercloselyrelatedpestivirusesandtheNAfromclinicalsamplespositiveforothercommonswinepathogens.Nocrossreactivitywasobserved.Datafromsixindependentruns,including5replicatesofthreeclinicalsampleswiththreeCtranges,were utilized to assess inter-assay repeatability and intra-assay reproducibility. This analysis demonstrated intra-and inter-assaycoefficientsofvariationof0.57%and1.46%,respectively,withaPCRefficiencyof102.13%.Screeningof758porcineclinicalsamplesfromKansasStateVeterinaryDiagnosticLabidentified110APPV-positive(Ct≤38)samples,suggesting14.52%prevalenceofAPPVintheUSswineherds.Amongthesampletypestested,oralfluidhadloweraverageCtcomparedtoothers.DetectionofAPPVpositivescasesfrompostweaningandgrowerpigpopulationssuggestsAPPVpersistentinfections.Furtherstudiesareneededtosupportthisspeculation.76-EvaluationofarapidteststripfordetectionofSalmonellaentericainequinefecalsamplesB.A.Burgess1,K.L.Pabilonia2,R.S.McConnico3,H.Aceto4,N.M.Slovis5,P.S.Morley2.1PopulationHealth,UniversityofGeorgia,Athens,GA, USA, 2Colorado State University, Fort Collins, CO, USA, 3Louisiana State University, Baton Rouge, LA, USA, 4University ofPennsylvania,KennettSquare,PA,USA,5HagyardEquineMedicalInstitute,Lexington,KY,[email protected]:DiagnosticTesting–2,Room3,12/5/20179:45AMPurpose: Salmonella enterica is one of the most commonly reported organisms associated with epidemic disease at veterinaryhospitals, significantly impactinganimalmorbidityandmortalityandadverselyaffecting thepublic’sperceptionof thesehospitals.ComprehensivescreeningandrapiddetectionofS.entericainrelevantsamplesisessentialforeffectiveinfectioncontrolinhigh-riskpopulations.TheobjectiveofthisprojectwastoestimatethesensitivityandspecificityofacommerciallyavailablerapidteststripfordetectionofSalmonellaincomparisontooptimizedenrichedaerobicculturesandacommercialPCRkitwhenusedtotestfecalsamplesviastate-of-the-art,latentclass(“no-gold-standard”)analyticalmethods.Methods:One-gramoffecesfromequinepatients(n=569)weretestedusing6techniques:1)tetrathionatebroth(43C,18hrs)ontoXLT-4agar(43C,18hrs);2)tetrathionatebroth(43C,18hrs)ontohektoenentericagar(36C,18hrs);3)selenitebroth(36C,18hrs)ontohektoenentericagar(36C,18hrs);4)selenitebroth(36C,18hrs)ontoXLT-4agar(43C,18hrs),5)rapidteststrip,and6)PCR.Results:Ingeneral,theproportionofpositivesampleswasgreaterwhentestingwiththecommerciallyavailablerapidtestthanwithanyothermethodevaluated.However,onapersamplebasis,PCRwasthemostsensitivemethod,followedbymethodsusingtetrathionatebrothforenrichment,therapidteststrip,andthenthosemethodsusingselenitebrothforenrichment.Conclusions:ThisprojectdemonstratestheimportancethattestselectionmayhaveondetectionofSalmonellainfecalsamples.Thisstudyalsoshowsthatcommerciallyavailablerapiddetectionsystemsmaybevaluablewhenmanaginganimalfacilities,howeverusersshouldbepreparedtomanagefalsepositivetestresults.

CRWAD2017 ABSTRACTS


77-DetectionofBovineViralDiarrheaVirusinstableflies(Stomoxyscalcitrans)followingconsumptionofbloodfrompersistentlyinfectedcattle

B.VanderLey1,S.Lee1,A.Workman2,D.Boxler3.1VeterinaryMedicineandBiomedicalSciences,UniversityofNebraska-Lincoln,ClayCenter,NE,USA,2Genetics,Breeding,andAnimalHealthResearchUnit,UnitedStatesDepartmentofAgriculture,ClayCenter,NE,USA,3WestCentralResearchandExtensionCenter,UniversityofNebraska-Lincoln,NorthPlatte,NE,[email protected]:DiagnosticTesting–2,Room3,12/5/201710:00AMPurpose:Bovineviraldiarrheavirus(BVDV)isanimportantpathogenofcattlearoundtheworld.Effectivecontrolcentersondetectingcattlepersistentlyinfected(PI)withBVDVandremovingthemfromherds.Currently,detectingPIcattlerequiresresourceintensiveindividualanimalsampling.Consequently,routinesurveillanceforBVDVisnotcommonlyconducted.DevelopingmethodstoclassifytheBVDVstatusataherdlevelwithouttheneedtohandlethecattlewouldremovemanyobstaclestocontrollingBVDV.Theobjectiveofourresearchwastodeterminethefeasibilityofusingbloodfeedinginsects,inthiscasestableflies,asasamplingmodalitytodetectBVDVinaherd.WehypothesizedthatBVDVwouldbedetectableinstablefliesthathadfedonbloodfromPIcalves.Methods:Totestthehypothesis,bloodfromPIcalveswasfedtostablefliesobtainedfromaresearchbreedingcolony.BloodfreeofBVDVwasfedconcurrentlytoagroupofstablefliesthatservedasnegativecontrols.LimitsofdetectionweretestedbydilutingBVDV-fedflieswithBVDV-freefliesatthefollowingratios(BVDV-fed:totalnumberofflies):100:100,40:100,20:100,10:100,1:100.Eachofthesepoolswas constructed from stable flies that had been fed 1, 2, 3, or 4 days priorwith blood from PI calves. Pools of stable flieswerehomogenizedandRNAwasextractedforreversetranscriptasepolymerasechainreaction(rtPCR)analysis.Results:Intotal,a4dayby5dilutionlevelmatrixofsampleswasproduced.ThefollowingsampleswereanalyzedusingrtPCR:samplesateachdaylevelof100:100dilution,samplesateachdaylevelof1:100dilution,day1sampleof40:100dilution,day2sampleof20:100,andday3sampleof10:100.Withtheexceptionofdays2,3,and4ofthe1:100dilution,allstableflypoolsgeneratedpositivertPCRresults.Conclusions:WhilesignificantresearchisstillrequiredtorefineandvalidateuseofstablefliesasasamplingtoolforBVDVdetectionataherdlevel,ourresultsdemonstratethatBVDVcanbedetectedinstablefliesanumberofdaysfollowingingestionofBVDVandevenwhenonlyafewofthefliespresenthavefedonBVDVinfectedblood.78-EvaluationofthesurvivalofviralpathogensincontaminatedfeedingredientsusingtransboundaryshipmentmodelsS.Dee1,F.ViscosaBauermann2,M.Niederwerder3,A.Singrey2,M.deLima2,G.Patterson4,S.Dritz3,M.Tokach3,J.Woodworth3,C.Jones3, J. DeJong1, G. Spronk1, J. Christopher-Hennings2, J. Ji5, J. Zimmerman5, B. Rowland3, E. Nelson2, P. Sundberg6, D. Diel2.1PipestoneVeterinary Services, Pipestone,MN,USA, 2SouthDakota StateUniversity,Brookings, SD,USA, 3Kansas StateUniversity,Manhattan, KS, USA, 4Lincoln Memorial University, Harrogate, TN, USA, 5Iowa State University, Ames, IA, USA, 6Swine HealthInformationCenter,Ames,IA,[email protected]:DisinfectionandBiosecurity,Room3,12/5/201710:45AMPurpose: This studyevaluated survivalof importantviralpathogensof swineor their surrogates in contaminated feed ingredientsduringsimulatedtransboundarytransportation.Methods:Basedonglobalsignificance,11viruseswereselected,includingFootandMouthDiseaseVirus(FMDV),ClassicalSwineFeverVirus(CSFV),AfricanSwineFeverVirus(ASFV),InfluenzaAVirusofSwine(IAV-S),Pseudorabiesvirus(PRV),NipahVirus(NiV),PorcineReproductiveandRespiratorySyndromeVirus(PRRSV),SwineVesicularDiseaseVirus (SVDV),VesicularStomatitisVirus (VSV),PorcineCircovirus type2 (PCV2)andVesicularExanthemaofSwineVirus (VESV).TomodelthesurvivalofFMDV,CSFV,PRV,NiV,SVDVandVESV,surrogateviruseswithsimilarphysicalpropertiesandstabilitywereused,andthoseconsistedofSenecavirusA(SVA)forFMDV,BovineViralDiarrheaVirus(BVDV)forCSFV,BovineHerpesvirusType1(BHV-1)forPRV,CanineDistemperVirus(CDV)forNiV,PorcineSapelovirus(PSV)forSVDVandFelineCalicivirus(FCV)forVESV.Remainingassessmentsinvolvedtheactualpathogen.Controlsincludedcompletefeed(positiveandnegativecontrols)andstockviruspositivecontrols(virusonly,nofeedmatrix).VirussurvivalwasevaluatedusingeitheraTrans-PacificorTrans-Atlantictransboundarymodel,involvingrepresentativefeedingredients,transporttimesandenvironmentalconditions,withsamplestestedbyPCR,VIand/orswinebioassay.Results:Selectviruses(SVA,FCV,BHV-1,PRRSV,PSV,ASFVandPCV2)maintainedinfectivityduringtransport,whileothers(BVDV,VSV,CDVandIAV-S)didnot.Survivalwasmaximizediningredientssuchasconventionalsoybeanmeal,lysinehydrochloride,cholinechloride,andvitaminD.Conclusions:Theseresultsdemonstratesurvivalofcertainvirusesinspecificfeedingredients(“high-riskcombinations”)underconditionssimulatingtransportbetweencountries.ThisworksupportspreviouslypublisheddataonthesurvivalofPorcineEpidemicDiarrheaVirusinfeedandprovidesfurtherevidenceindicatingthatcontaminatedfeedingredientsmayserveasriskfactorsforforeignanimaldiseases.

CRWAD2017 ABSTRACTS


79-EvaluationofthedecontaminationpowerofaqueousozoneonvariousSalmonellatyphimuriumcontaminatedsurfacesA.Megahed,B.Aldridge,J.Lowe.DepartmentofVeterinaryClinicalMedicine,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,[email protected]:DisinfectionandBiosecurity,Room3,12/5/201711:00AMSalmonellaearewidespreadamonganimalsandconsideredoneofthemostreportedzoonosisworldwide.Ahighreactivityandlowproductionofharmful residuesmakeozone (O3)anattractivealternativedisinfectant for improving farmhygieneandbiosecurity.Accordingly,ourobjectiveswereto(1)characterizethekillingcapacityofaqueousO3atdifferentoperationalconditionsonattenuatedSalmonellaTyphimuriumandS.choleraesuis(aSTC)contaminateddifferentsurfaces(plastic,nylon,rubber,andwood);(2)determinetheeffectsofsequentialwashingtodecontaminatesurfacesfromthehighsalmonellaload.Inacrossoverdesign,14strips(7.5X2.5cm)ofeachmaterialwererandomlyassignedbetween3groups,treatment(n=6),positivecontrol(n=6),andnegativecontrol(n=2).Thestripsweresoakedindairycattlesterilefecesinoculatedwith106-107microbesofaSTCfor60minutes.ThestripswerefirstexposedtoaqueousO3at4or9ppmfor4minutes.Followingsoaking,treatmentstripsweresequentiallywashed(5-serialwash)with20ccofaqueousO3atconcentrationof4ppmfortwominutesexposureeach.Followingeachozoneexposure,quantitativebacterialcultureswereperformedusing3M™Petrifilm™rapidaerobiccountplate(RAC)andplatereader.Plasticsurfacesweremosteffectivelydecontaminated.With4minutesexposure,anO3concentrationof4ppmreducedaSTCcountsby5.1-log10 (P≤0.001)and9ppmresultedinnodetectableaSTC.Onnylonandrubbersurfaces,4minutesof9ppmO3reducedaSTCcountsby5.3-log10(P≤0.001)and4.7-log10(P≤0.001),respectively.Onwood,aqueousO3didnotshowasignificantreductioninaSTCcounts.Onnylon,rubber,andwoodsurfaces,thesecond,third,andfourthwashingcycles,respectively,resultedinnon-detectablelevelsofaSTC.Weconcludethatexposureto9ppmofaqueousO3for4minutesexposureisaneffectivemeanstoclearthesmoothsurfacesofahighSalmonellaload.OurfindingsstronglyindicatethatasequentialwashingsystemisneededtoeffectivelydecontaminatecomplexsurfaceswithahighSalmonellaload.80-MicrobialkillingcapacityofaqueousandgaseousozoneondifferentsurfacescontaminatedwithmanureA.Megahed,B.Aldridge,J.Lowe.DepartmentofVeterinaryClinicalMedicine,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,[email protected]:DisinfectionandBiosecurity,Room3,12/5/201711:15AMAhighreactivityand lowproductionofharmful residuesmakeozone(O3)anattractivealternativedisinfectant for improving farmhygieneandbiosecurity,andforaidinginthepreventionofinfectiousdiseases.Toexplorethepracticalpotentialofusingozonewedesigned a study to (1) characterize themicrobial killing capacity of aqueous and gaseousO3 onmanure-based pathogens (MBP)associatedwithvariousmaterials(plastic,metal,nylon,rubber,andwood);(2)determinetheeffectofmicrobial loadonthekillingcapacityofaqueousO3.Inacrossoverdesign,14strips(7.5X2.5cm)ofeachmaterialwererandomlyassignedbetween3groups,treatment(n=6),positivecontrol(n=6),andnegativecontrol(n=2).Thematerialstripsweresoakedindiluteddairycattlemanureharboringaninoculumlevelof106-107microbes,for60minutes.ThetreatmentstripswereexposedtoaqueousO3of2,4,and9ppm,andgaseousozoneof1and9ppmfor2,4,and8minutesexposure.Followingozoneexposure,quantitativebacterialcultureswereperformedoneachstripusing3M™Petrifilm™rapidaerobiccountplate(RAC)andplatereader.Onsmoothsurfaces(plasticandmetal)aqueousO3,at4ppmorgreater,reducedMBPtoasafelevel(>5-log10reduction)within2minutes.Onthesamesurfaces,gaseousozone,at9ppmfor4minutes,inactivated3.3-log10ofMBP.AqueousO3of9ppmwasalsoeffectiveinreducingMBPtoasafelevelonnylonandrubbersurfaces.Onmorecomplexsurfaces(e.g.wood)bothaqueousandgaseousO3,atupto9ppm,didnotreduceMBPtoasafelevel.Overall,thebacterialloadwasastrongpredictorforreductionincellcounts(P<0.0001,R2=0.72).WeconcludethataqueousO3of4and9ppmfor2minutesareefficient inreducingMBPtoasafe levelonsmooth,andmoderatelyroughsurfaces,respectively.However,O3aloneisnotanadequatemeansofcontrollingbacterialpopulationsoncomplexsurfaces.

CRWAD2017 ABSTRACTS


81-BiofilmformationandsusceptibilityofEscherichiacoliO157:H7fromgoatstodisinfectantsinvitroandinfootbathsB.M. Skinner, A.T. Rogers, M.E. Jacob. Population Health and Pathobiology, North Carolina State University, Raleigh, NC, [email protected]:DisinfectionandBiosecurity,Room3,12/5/201711:30AMEscherichiacoliO157:H7(O157)isahumanpathogenthatcancauseseverefoodbornediseases.Naturallyexistinginruminants,O157mayspreadthroughthefoodchainorbydirectcontact.FootbathuseinagritourismfarmsettingsmayserveasaneffectivemeasuretocontrolO157contamination;however,thetypeofdisinfectant,thepresenceoforganicmatter,andthepotentialofbacteriatoformbiofilmsonfootbathsmayaffecttheirefficacy.OurobjectivesweretodeterminethesusceptibilityofO157towardsdisinfectantsanddeterminethebiofilmpotentialofO157isolatesbothinvitroandinfootbaths.Theminimuminhibitoryconcentration(MIC)forO157strains isolated fromNorth Carolina goatswere determined using laboratory-grade disinfectants. ThemedianMICswere 20 ppmhydrogenperoxide,320ppmsodiumhypochlorite,625ppmglutaraldehyde,3.2ppmDDAC,and2500ppmphenol.Therewaslittlevariabilitybetweenisolates.Duringtime-killassays,twicetheMICofhydrogenperoxidereducedgrowthby3logCFU/mLin24hours,sodiumhypochloriteandglutaraldehydekilledallbacteriawithin20minutes,andDDACandphenolkilledallbacteriawithin4hours.SevendifferentstrainsofO157wereevaluatedfortheirabilitytogrowbiofilmsonplasticfor24,48,and72hoursat22°C,37°C,and42°C.Crystalvioletassayswereperformedtodeterminebiofilmbiomass.ThebiofilmswerealsosonicatedandplatedtoestablishCFUcounts.Variationsinbiofilmdensitywereobservedbetweenisolates,incubationtimes,andincubationtemperatures.Ourinvitrodatasupports disinfectant efficacy in killingO157and the ability ofO157 to formbiofilmonplastic in a strain, time, and temperaturedependentmanner.Futurestudieswillevaluatetheeffectivenessofcommercialdisinfectants(CloroxBleach,0.26%;VirexII,0.034%;Virkon-S,1%)atkillingO157infootbathswithandwithoutfecalcontaminationandthebiofilm-formingabilityofO157onfootbathsurfacestoidentifytheefficacyoffootbathuse.82 - Airborne inactivation of porcine reproductive and respiratory syndrome virus (PRRSv) by a packed bed dielectric barrier

dischargenon-thermalplasmaT.Xia1,M.Yang2, I.Marabella3,B.Olson3,D.Zarling3,M.Torremorell2,H.Clack1. 1Civil&EnvironmentalEngineering,UniversityofMichigan,ANNARBOR,MI,USA,2VeterinaryPopulationMedicine,UniversityofMinnesota,St.Paul,MN,USA,3MechanicalEngineering,UniversityofMinnesota,Minneapolis,MN,[email protected]:DisinfectionandBiosecurity,Room3,12/5/201711:45AMPorcineReproductiveandRespiratorySyndromevirus(PRRSv)hasbeendetectedinairmorethan9kmdownwindofinfectedswine.ApplyingHEPAfiltrationtoventilationairsuppliedtohogbarnsinvolvesstructuralretrofitstobuildingsthatcanbecostly,inadditiontotheperiodicreplacementofusedfilters.Non-thermalplasmas(NTPs)areelectricaldischargescomprisedofreactiveradicalsandexcited species that inactivate viruses and bacteria. Our previous experiments using a packed bed non-thermal plasma reactordemonstratedeffectiveinactivationofbacteriophageMS2asafunctionofappliedvoltageandpower,rangingfromlessthanone-loginactivationat20kVandafewwattstogreaterthantwo-loginactivationat30kV.ThepresentstudyexaminedtheeffectivenessofthesamereactorininactivatingaerosolizedPRRSv.APRRSvsolutioncontaining~105TCID50/mlwasaerosolizedatarateof3ml/minbyanair-jetnebulizerandintroducedintoairflowsof5or12cfmfollowedbyNTPexposureinthereactor.Twinimpingersupstreamanddownstreamofthereactorcollectedsamplesofthevirus-ladenairflow.SubsequentTCID50assayandquantitativepolymerasechain reaction (qPCR)analysesof thecollectedsamplesdeterminedthepre-andpost-treatmentabundanceof infectivePRRSv (inTCID50/ml)ascomparedwiththeabundanceoftheviralgenome(qPCR),whetherinfectiveorrenderedinactivebyNTPexposure.Anopticalparticlesizermeasuredupstreamanddownstreamaerosolsizedistributions,givingestimatesofaerosolfiltrationbythereactor.TheresultsshowedthatPRRSvwasinactivatedtoasimilardegreeasMS2atthesameconditions,withthe1.3-loginactivationofPRRSvachieved at 20 kV and 12 cfmair flow rate.Differential pressure across the reactorwasminimal compared toHEPA filters and aconsumer-grade ozone filter reduced residual ozone concentrations down to levels commensurate with the ambient laboratoryenvironment.TheresultsdemonstratethepotentialofproperlyoptimizedNTPsforpreventinginfiltrationofPRRSvintohogbarnswithventilationair.

CRWAD2017 ABSTRACTS


83-SpatialandtemporalnetworkanalysisofswinemovementsinArgentinafrom2011to2016J.N.Baron1,M.Aznar2,M.Monterubianesi3,B.Martinez-Lopez1.1CenterforAnimalDiseaseModellingandSurveillance,DeparmentofMedicineandEpidemiology,SchoolofVeterinaryMedicine,UCDavis,Davis,CA,USA,2GrupodeEpidemiologíayMedicinaPreventiva,InstitutoNacionaldeTecnologíaAgropecuaria(INTA),Hurlingham,Argentina,3ServicioNacionaldeSanidadyCalidadAgroalimentariadelaRepúblicaArgentina(SENASA),BuenosAires,[email protected]:ModelingandNetworkAnalysis,Room4,12/4/20179:00AMPurpose:Theaimofthisstudyistoevaluatethespatio-temporalcharacteristicsofswinemovementnetworkinArgentinafrom2011to 2016, using network analysis and graph theorymethodologies, aswell as graphic representation.Methods: Firstwe created adirectednetwork,plottingfarmsandmarketsasnodesandindividualmovementsasedges.Wethencalculatedmeasuresofdegreecentrality,closeness,andbetweenness foreachmonth, to identifyand locatemonthsandfarmsof interest inregardstoboththepotentialintroductionanddistributionofdisease.WenextidentifiedcommunitiesofstronglyinterconnectedfarmsandmarketsusingInfomapandWalktrapalgorithms.Results:Thenetworkconsistsofapproximately19,000nodes(19.5%ofthecountry'sfarms)and135,500movements.Onaveragetherewere1,883movementspermonthoverameandistanceof143km(89miles).Themeannumberofpigstransportedineachmovementwas44,withanincreasingtrendovertheyears.ThemaindestinationandsourceprovinceswereBuenosAires(39,122incomingand40,878exitingmovements),Córdoba(37,457and44,274)andSantaFe(30,869and26,523).Theseprovincesrepresent36.7%offarmsbutconcentrated79.3%ofincomingand82.2%ofoutgoingmovementsduringthatperiod.ThedepartmentsgeneratingthelargestnumberofmovementswereCarmenAreco,Caseros,GeneralLópez,MarcosJuárezandRíoCuarto,whichwereinvolvedin20%ofallmovementswhilstonlycontaining3%offarms.Conclusions:Theresultsofthisworkwillservetoimprovedecision-makingandfacilitatetheprioritizationofsurveillanceandcontrolmeasuresforbothendemicandemergingswinediseasesinthecountry.84-Modelingthelive-pigtradenetworkinGeorgia:ImplicationsfordiseasepreventionandcontrolE.Kukielka1,B.Martínez-López1,D.Beltrán-Alcrudo2.1CenterforAnimalDiseaseModelingandSurveillance,UCDavis,Davis,CA,USA,2FoodandAgricultureOrganization,Budapest,[email protected]:ModelingandNetworkAnalysis,Room4,12/4/20179:15AMPurpose:Livepigtradepatterns,driversandcharacteristics,particularlyinbackyardpredominantsystems,remainlargelyunexploreddespitetheirimportantcontributiontothespreadofinfectiousdiseasesintheswineindustry.Abetterunderstandingofthepigtradedynamicscaninformtheimplementationofrisk-basedandmorecost-effectivepreventionandcontrolprogramsforswinediseases.Methods:Inthisstudy,asemi-structuredquestionnaireelaboratedbyFAOandimplementedto487farmerswasusedtocollectdataregardingbasiccharacteristicsaboutpigdemographicsandlive-pigtradeamongvillagesinthecountryofGeorgia,whereveryscarceinformationisavailable.Socialnetworkanalysisandexponentialrandomgraphmodelswereusedtobetterunderstandthestructure,contactpatternsandmaindriversforpigtradeinthecountry.Results:Resultsindicaterelativelyinfrequent(atotalof599shipmentsinoneyear)andgeographicallylocalized(medianEuclideandistancebetweenshipments=6.08km;IQR=0-13.88km)pigmovementsinthestudiedregions.Themainfactorscontributingtolive-pigtrademovementsamongvillageswerebeingfromthesameregion(i.e.,localtrade),usageofamiddlemanoraliveanimalmarkettotradelivepigsbyatleastonefarmerinthevillage,andhavingalargenumberofpigfarmersinthevillage.Conclusions:Theidentifiedvillages’characteristicsandstructuralnetworkpropertiescouldbeusedtoinformthedesignofmorecost-effectivesurveillancesystemsinacountrywhichpigindustrywasrecentlydevastatedbyAfricanswinefeverepidemicsandwherebackyardproductionsystemsarepredominant.

CRWAD2017 ABSTRACTS


85-Descriptionofhorsedemographicsandmovementsduringthe2015competitionseasoninOntario,CanadaK.Spence,T.O'Sullivan,Z.Poljak,A.Greer.PopulationMedicine,UniversityofGuelph,Guelph,ON,[email protected]:ModelingandNetworkAnalysis,Room4,12/4/20179:30AMTheCanadianequineindustryconsistsofadiversegroupofhorses,rangingfromcompetitiontocompanionanimals.Ashorsestraveltoattendvariousshows, trainingactivities,or to receiveveterinarycare,opportunities fordirectcontactwithnewhorsesat theirdestination are created. InOntario, Canada, limiteddata exists todescribe thesemovements and contacts, therefore limitingourunderstanding of the potential for disease spread amonghorses as they travel. The objectives of this studywere to describe thedemographiccharacteristicsofhorsesenrolledinalongitudinalstudyinOntario,Canada,andtocharacterizetheirmovementsovera7-monthperiod.Twohundredandtwenty-twohorseownerscompletedaninitialquestionnairetoprovidedemographicinformationfor570horses.Horseownersthatcompletedtheinitialquestionnairedocumentedthetravelpatternsoftheirhorse(s)usinganonlinequestionnaireeachmonthfromMaytoNovember,2015.Theprimarydisciplineofparticipatinghorsesincludedcompetition(63.3%),leisure(33.3%),andracing(3.2%).Duringthe7-monthperiod,therewere3001unidirectionalmovementsofhorsesbetweenfacilities.Reasonsforhorsemovementson/offafacilityovertheentirestudyperiodincludedattendingacompetition(38.7%),leisureactivities(18.8%),andattendingatrainingclinic(7.5%).ThecharacterizationofhorsemovementpatternsinOntarioprovidesvaluableinsightintotheconnectivityofhorseswithinthepopulation,andcanlendsupportindescribingthepotentialfordiseasespreadbetweenhorsefacilities.86-QuantifyingtheheterogeneityincontactpatternswithinanOntarioequinefacility:apilotstudyR.Milwid1,T.L.O'Sullivan1,Z.Poljak1,M.Laskowski2,A.L.Greer1. 1PopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2MathematicsandStatistics,YorkUniversity,Toronto,ON,[email protected]:ModelingandNetworkAnalysis,Room4,12/4/20179:45AMThe assumption of homogenous mixing is often used to define the effective contact rate in disease transmission models. Thisassumptionhasthepotentialtoover-orunder-estimatethecontactsthatoccurinapopulation,potentiallyresultingininappropriatemodelinterpretations.Onemethodofcorrectingforthisoversimplificationistousecontactnetworkstoinformthecontactrate.Theobjectiveofthisstudywastoquantifycontactpatternsusingradiofrequencyidentification(RFID)loggerswithinanequinefacility.The7-daystudytookplaceinNovember2016onanequinefacilityinSouthwesternOntario.Thetagswereattachedtothehorses’halters(N=9)andkeptwiththehorsesatalltimes.Facilitystaffcarriedouttheusualfacilityscheduleforeachparticipatinghorsei.e.turnout,feeding,andtraining.When2horses,wearingloggers,camewithin2metersofeachother,theloggersrecordedtheIDofthehorsesandthetimeofthecontactevent.ThedatawereanalyzedusingtheigraphpackageinRtodescribethecontactnetworksthatoccurredoneachstudyday.Ananalysisincludingnetworkmetricssuchascentrality,wasconducted,toassessthegeneralizabilityandconsistencyofthecollecteddata.Furthermore,thenetworkswereanalyzedtoseeiftheyconformedtotheassumptionofhomogenousmixing.Thenodaldegreerangedfrom1to8withanaverageof4.8contacts, indicatingthatatnotimeduringtheweekwereanyhorsesisolated.TheJaccarddifferenceindexindicatedthatthemostsimilardayshad~74%ofidenticalcontacts(ICs)andtheleastsimilardaysshared~53%ofICs.Themajorityofcontactsoccurredbetweenhorseswhowerehousedinthesamebarnorsharedapasture,withtheremainderofthecontactsoccurringduetothestochasticityinthedailyschedules.Heatmapsofthenetworksshowthatneitherthedailynetworks,northecumulativestaticnetworkconformtothehomogenousmixingassumption.Theheterogeneityin the resulting contact patterns can be used to parameterize disease transmission models in order to obtain a more robustunderstandingofthepotentialfordiseasespread.

CRWAD2017 ABSTRACTS


87-UsinggeospatialmethodstomeasuretheriskofenvironmentalpersistenceofavianinfluenzainSouthCarolinaC.Stenkamp-Strahm1,K.Ahmad2,T.Sodergrath2,D.South2,S.Magzamen2,M.McCool-Eye3,A.Fox3,K.Patyk3,A.James3,G.Cook3,D.Prosser4, M. Martin5. 1Clinical Sciences, Colorado State University, Fort Collins, CO, USA, 2Environmental and Radiological HealthSciences, Colorado StateUniversity, Fort Collins, CO,USA, 3USDA-APHIS, Veterinary Services, Center for Epidemiology andAnimalHealth, Fort Collins, CO, USA, 4USGS Patuxent Wildlife Research Center, Beltsville, MD, USA, 5Clemson Livestock Poultry Health,Columbia,SC,[email protected]:ModelingandNetworkAnalysis,Room4,12/4/201710:00AMAvianinfluenza(AI)isahighlycontagiousvirusaffectingwildbirdsanddomesticatedpoultry.Inadditiontoexposurefromwildbirds,outbreaks in domestic poultry can be initiated and propagated by exposure to virus surviving in the environment near poultryoperations.ThisstudyaimedtodefineareasofSouthCarolinaatheightenedrisk forenvironmentalpresenceof theAIvirususinggeospatialmethods.EnvironmentalcovariatesknowntoinfluenceAIsurvivalweredeterminedbasedonpublishedstudies.DataonthedistributionofthesevariableswithinSouthCarolinaweredownloadedfrompubliclyavailablesources.Allcovariatelayersweremappedata1-kmresolutionforecologicaltimeperiods(e.g.breeding,fallmigration,winter,springmigration)andweightedbasedontheirinfluenceforvirussurvivability.EnvironmentalsuitabilitymapswerecreatedusingtheselayersandESRIArcGIS10.4softwarewith thePredictiveAnalysis tool.After classifying suitabilitymapvaluesbasedonWorldOrganization forAnimalHealth (OIE) riskassessmentguidelines,<1%ofthe1-kmgeographicareasshowedahighriskofAIpersistenceinthefourtimeperiodsassessed.Ahighernumberofgeographicareasshowedeithermoderateorlowrisk(1-2%and17-19%,respectively),withahigherpercentageofriskpresent inwinterandspringmigration.WhenfarmdensitydatawerecombinedwithAIsuitabilitymaps,therewasaverylowpercentageoflocationswheremoderateorhighenvironmentalriskco-locatedwithlow,moderate,orhighfarmdensityareas(0.001-0.120%ofareasbasedontimeperiod).Theseresultsmaybeusedtoguidefuturebiosecurityandemergencypreparednesseffortsthataimtomitigateand/orquellagriculturaloutbreaksofAIwithinthestateofSouthCarolina.88-ThecharacterizationofretailgroundbeefmicrobiomeK.M.Thomas1,M.D.Weinroth1,E.Doster2,K.L.Huebner2,J.K.Parker2,J.L.Metcalf1,D.R.Woerner1,R.J.Delmore1,H.Yang1,T.M.Arthur3,J.W. Schmidt3, T.L.Wheeler3, P.S.Morley2, K.E. Belk1. 1Animal Sciences, Colorado State University, Fort Collins, CO, USA, 2ClinicalSciences,ColoradoStateUniversity,FortCollins,CO,USA,3MeatAnimalResearchCenter,USDA-AgriculturalResearchCenter,ClayCenter,NE,[email protected]:EcologyandManagementofFoodborneAgents,Room4,12/4/201710:45AMPurpose:Groundbeefisareservoirforavarietyofmicrobialspecies,includingbutnotlimitedtospoilageorganismsandpathogenicfoodbornebacteria,suchasSTECs,E.ColiO157:H7,andSalmonellaenterica.Theseorganismsdonotfunctionindependently,butrathercoinhabit thespacewithothermicrobialspecies,makingupthemicrobiome.Characterizingandunderstandingthemicrobiomeofgroundbeefwillprovideusefulinformationforfuturestudiesinvolvinggroundbeefmicrobialcommunities.Theobjectivesofthisstudyweretoinvestigateandcharacterizethemicrobiomeofretailgroundbeefproductsreadilyavailabletoconsumers.Methods:Priortosamplecollection,apilotstudyconsistingof10unassociatedgroundbeefsamples,5naturaland5conventional,wereprocessedforDNAisolationfollowing72hourspost-collectiontomimicconsumerbehavior.qPCRwasperformedtoquantifytotalprokaryoticandeukaryoticDNA.qPCRresultsindicatedthatthemeatrinsateprotocolusedinthisstudysuccessfullyisolatedenoughprokaryoticDNAtobeusedindownstreamsequencing.Followingthepilotstudy,atotalof100unassociatedgroundbeefsamplesofbothconventionallyraisedandnaturallyraisedcattle(n=50),werecollectedfrompopularretailchainsthroughoutthecityofFortCollins,CO.DNAwasextractedandisolatedfromeachsamplebeforebeingsubjectedto16SrRNAsequencing.Results:16Sdataanalysisisongoingandthemicrobiomeofbothtypesofgroundbeefsampleswerecharacterized.Followingcharacterization,differencesinthemicrobiomeofnaturalandconventionalgroundbeefwillbeanalyzedusingRstatisticalprogramming.Conclusions:Resultsofthisstudywillaidfutureendeavorsininvestigatingthesafetyofthebeefsupplychain.

CRWAD2017 ABSTRACTS


89-EpidemiologyofCampylobacteramongfree-livingCanadageeseinOntario,CanadaN.A.Vogt1,D.L.Pearl1,E.N.Taboada2,N.Janecko3,R.Reid-Smith1,C.M.Jardine4.1DepartmentofPopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2PublicHealthAgency,NationalMicrobiologyLaboratory,Lethbridge,AB,Canada,3PublicHealthAgency,CentreforFoodborne,EnvironmentalandZoonoticInfectiousDiseases,Guelph,ON,Canada,4DepartmentofPathobiology,UniversityofGuelph,Guelph,ON,[email protected]:EcologyandManagementofFoodborneAgents,Room4,12/4/201711:00AMZoonoticpathogensofpublichealthimportance,suchasCampylobacter,havebeenisolatedfromCanadageeseintheUnitedStates.Duetothehighlymobilenatureofthesebirdsduringmigrationandfeeding,aswellastheirfrequentinteractionswithhumansanddomesticanimals,thereareconcernsthatthesebirdsmaybeasourceofpotentiallyharmfulmicroorganisms.Ourprimaryobjectivewas toexamine theprevalenceandpatternsof carriageofCampylobacteramongCanadageese in southernOntario.A secondaryobjectivewastoassessthepatternsanddiversityofCampylobactergenotypesindifferentflocksofCanadageesebasedoncomparativegenomicfingerprintingusinga40-geneassay(CGF40).FecalswabswereobtainedfromlivebirdsinurbanrecreationalareasinGuelph,Ontario fromMay through October, 2016. Standard microbiological techniques were used to isolate Campylobacter. The CGF40Campylobacter genotypewas determinedby the binary pattern of 40marker genes identified using PCR.Usingmultilevel logisticregressionwitharandominterceptforflock,theimpactofseasonandageclass(adultvs.juvenile)wereexaminedfortheisolationofCampylobacter. Campylobacter was isolated from 9.3% of goose samples. The overall prevalences of C. jejuni and unspeciatedCampylobacterwere7.3%and2.0%, respectively.Asignificantassociationwasonly identifiedbetweenseasonandCampylobacterisolation,withpeakprevalenceoccurringinthefall.Inaddition,unspeciatedCampylobacterwasonlyisolatedfromfallsamples.Flockwasasignificantrandomeffectinourmodels,suggestingthatobservationsfromthesameflockwerenotindependent.Atotalof7CGF40patternswereidentifiedamongCampylobacterpositivebirds.InflockswithmorethanoneCampylobacterpositivebird,mediansimilarity scoresamongCGF40patterns fromeachbirdwereat least85% for4outof5 flocks.The remaining flockhadamediansimilarityscoreof60%.ThefallpeakinCampylobacterprevalence,aswellastheidentificationofadditionalCampylobacterspeciesmayberelatedtoincreasedbirdmobilityfollowingthenestingseasoninMayandJune.90-PrevalenceandrisksfactorsofCampylobacterinlivestockonsmall-scale,diversifiedfarmsinCaliforniaL.Patterson1,A.F.A.Pires1,N.Navarro-Gonzalez2,P.Aminabadi2,M.Jay-Russell2.1PopulationHealthandReproduction,UniversityofCaliforniaDavis,Davis,CA,USA,2WesternCenterforFoodSafety,UniversityofCaliforniaDavis,Davis,CA,[email protected]:EcologyandManagementofFoodborneAgents,Room4,12/4/201711:15AMTheincreasingpopularityofsmall-scalefarmsreflectsgrowingconsumerinterestinlocalandsustainablefoodproduction,includinghumanely-producedmeatproductsandeggs.However,livestockharborfoodbornepathogensthatcancausemajorhumanillness.TheobjectiveofthisstudywastoassessriskfactorsandtheprevalenceofCampylobacterspp.inlivestockraisedonsmall-scalediversifiedfarms.Twenty-onefarmsinNorthernCaliforniawereenrolledinthiscross-sectionalstudy.Seventeen(81.0%)werediversifiedfarmsand4raisedonlylivestock.Elevenraisedmorethanonelivestockspecies(52.4%)and18(85.7%)keptpoultry.Campylobacterspp.wasfound on 61.9% (13/21) of the farms at least once during the study. The overallCampylobacter prevalencewas 10.7% (109/999)including isolates from poultry (10.9%), swine (10.5%), cattle (15.9%), goats (7.5%), and sheep (8.6%). Twenty-nine of the posiveCampylobactersampleswereC.coliandfifty-fourwereC. jejuni.Campylobacter jejuni,an importantfoodbornepathogenthatcancauseseverehumanillnessandsequelae,wasfoundinalllivestockspecies.Multilevellogisticmodels,withfarmasarandomeffect,wereusedtoassesstheassociationbetweenfarmmanagementpracticesandCampylobacterspp.presence.Thisstudyhighlightstheneedtoassesspotentialfoodsafetyrisksassociatedwithsmall-scale,diversifiedfarms.Findingswillprovidescale-appropriatefoodsafetymetricsandrecommendationsforriskreductiontosmall-scalediversifiedfarms.

CRWAD2017 ABSTRACTS


91-PoultryintestinalmucusmodulatesCampylobacterjejunigeneexpressionT.Looft,T.Casey.FSEP,USDA-ARS-NADC,Ames,IA,[email protected]:EcologyandManagementofFoodborneAgents,Room4,12/4/201711:30AMCampylobacter jejuni isan importanthumanpathogen,causingupto400million infectionsayearworld-wide.Poultry isanaturalreservoirofC.jejuni,colonizingandresidingwithintheintestinalmucuswithoutcausingdisease.Mucuscolonizationisanessentialstep in C. jejuni colonization and previous studies suggest that host intestinal mucus impacts Campylobacter function.InthisstudywecharacterizetheglobaltranscriptomeofC.jejunigrownonhostmucusisolatedfromavian(chickenorturkey)andmammalian(cow,pig,orsheep)sources.C.jejuniNCTC11168wasgrownfor24hoursondefinedmediasupplementedwithorwithout0.5%wt/volofeachhostmucus.FollowingRNAisolation,directionalRNAlibrariesweresequenced,andmappedtothereferencegenome.AvianandmammalianmucussourcesdifferentiallyimpactgeneexpressioninwaysthatmayreflectCampylobacter’sabilityto colonize different animal intestinal tracts. Differentially expressed genes between the avian andmammalianmucus-containingmedia,includedtheup-regulationofchemotactic,oxidativestressresponse,andironacquisitiongenesintheavianmucusmedia.Non-coding antisense RNAswere associatewith differentially expressed genes between avian andmammalianmucus, andmay be inresponsetoenvironmentalcues.ThesedatasuggestthatC.jejunialtersitsgeneexpressioninthepresenceofavianmucusinsuchawaythatpromotesintestinalcolonization.UnderstandinghowC.jejuniinteractswithinthehost-intestinalenvironmentwillprovideinsightsintohowC.jejunihasadaptedtocolonizingdifferentintestinalenvironments. 92-NonpathogenicE.colifecalsheddingandenvironmentalsurvivalinweanedHolsteincalvesandfeedlotsteersH.L.Seger1,M.W.Sanderson1,B.J.White2,T.G.Nagaraja1. 1DMP,KansasStateUniversityCollegeofVetMed,Manhattan,KS,USA,2ClinicalSciences,KansasStateUniversityCollegeofVetMed,Manhattan,KS,[email protected]:EcologyandManagementofFoodborneAgents,Room4,12/4/201711:45AMShigatoxin-producingEscherichiacoli(STEC)isamajorfoodbornepathogenthatresidesinthehindgutofcattleandisshedintheirfeces,butsheddingandtransmissiondynamics incattlearenotwelldefined.Anon-pathogenicE.colimodelwouldallowstudyoftransmission parameters outside a BSL-2 facility. Our objective was to provide preliminary validation of shedding duration andconcentrationforanonpathogenicE.coliinweanedHolsteincalvesandfeedlotsteersintwoseparateexperiments.Inexperiment1,fourHolsteincalveswereorallyinoculatedoncedailyforfiveconsecutivedayswith109colonyformingunits(CFU)ofE.coliO28:H43maderesistanttonaladixicacid(50µg/ml)andrifampicin(50µg/ml).Fecal,oral,hide,pensurface,andwatersampleswerecollecteddailyforsixdaysafterthefirstinoculationandthenthreetimesaweekforfourweeks.Inexperiment2,fiverandomlyselectedfeedlotsteersfromapenof70steerswere inoculatedwith109CFUofnalidixicacid-andrifampicin-resistantE.coliO28:H43dailyforfiveconsecutivedays.Fecal,oral,andhidesamplesfromallsteersandpensurface,feed,andwatersampleswerecollectedweeklyforsixweeks. Sampleswere spiral platedonMacConkey agar supplementedwith naladixic acid (50µg/ml) and rifampicin (50µg/ml) toquantifytheconcentrationofE.coli.Samplesnegativebyspiralplatingwereenrichedandplatedtodetectpresenceoftheinoculatedstrain.Establishedsheddingwasnotedinbothgroups.Thepeakfecalsheddingconcentrationforthefirsttrialwas5.8logCFU/gattheendofthetrialperiodandpositivesamplesweredetectedinallsampletypes.Forthesecondtrial,transmissionoccurredfromtheinoculatedsteerstothecohortsandwholepenprevalencepeaked14daysafterinitialinoculation.Peakindividualfecalconcentrationwas3.5logCFU/gonday14afterthefirstinoculationforthefeedlotsteers.Theinoculatedstrainwasdetectedinhide,oral,feedandpen surface samples during the trial. The fecal shedding concentration and duration provide data for amodeling framework fortransmissiondynamicswiththepotentialtoexploreinterventionsinthecontrolofpre-harvestSTEC.

CRWAD2017 ABSTRACTS


93-Novelapproachesforinfluenzasurveillanceinswinebreedingherds.J.GarridoMantilla,J.Alvarez,M.Culhane,M.Torremorell.UniversityofMinnesota,CollegeofVeterinaryMedicine,StPaul,MN,[email protected]:HealthinSwinePopulations,Room4,12/4/20172:00PMSurveillanceofinfluenzaAvirus(IAV)iscentraltothecontrolofinfluenzainpigsandthepreventionofzoonoticinfections.However,routinely and readily detecting influenza infections can be challenging specially in endemically infected herds. In this study, wecomparednovelsamplingstrategiestoassessthebeststrategyfordetectingandisolatingIAVinswinebreedingherds.Thestrategiesevaluatedwerenasalswabs(NS),nasalwipes(NW),oropharyngealswabs(OS),oralfluids(OF),surfaceswipes(SW),sowudderskinwipes (UW), environmental particle depositionwipes (EPD) and air. Samplingwas conducted in piglets prior to weaning or theirenvironmentin6breedingherds.AllsamplesweretestedbyIAVmatrixgenerRT-PCRandresultsconsideredpositiveifctvaluewas<35.AsubsetofrRT-PCRpositivesampleswereculturedforvirusisolationusingMDCKcells.TheoptimumsampletypeforIAVdetectionwasidentifiedusingMcNemartest(p<0.05).IAVwasdetectedin4outof6breedingherds.Outofthe40samplescollectedinpigletsinIAVpositiveherds,78%(31/40)ofOS,55%(22/40)ofNSand53%(21/40)ofNWwererRT-PCRpositive(p=0.012).78%(31/40)ofUWwerepositivecomparedwith60%(24/40)ofSWand86%(6/7)ofOF(p=0.035).IAVwasdetectedin100%(40/40)ofairsamplescomparedwith88%(35/40)ofEPD.IAVwasisolatedfrom80%(40/50)OS,43%(23/54)NS,68%(41/60)NW,35%(9/26)SW,75%(18/24)UW,20%(1/5)OF,26%(7/27)EPDand33%(9/27)airpositivesamples.UseofUWandOSandtoalesserextentOFandSWsignificantlyincreasedthelikelihoodofdetectingIAVbyrRT-PCRcomparedtoNS(baselinecategory)(p<0.01).Inthisstudy,OSwastheoptimumsampletypeforbothdetectingandisolatingIAVfrompigs.UWoflactatingsowswasalsoasensitivemethodtoidentifypositivelittersandyieldedviableIAV.CollectionofOFwaslimitedgiventhereluctanceofpigletstochewontheropesanddidnotyieldaviableisolate.SamplingtheenvironmentalsoappearedtobeagoodapproachtodetectIAVsinceIAVwasreadilydetectedfromsurfacesandairbutyieldedfewerisolatesthanOSandUW.Thisstudyprovidesnewinformationonsamplingapproachestodetectinfluenzainbreedingherds.95-Surveillance...is"30"stilltherightanswer?M.Rotolo1,C.Wang2,M.Haddad2,Y.Sun2,M.Hoogland3,R.Main2, J.Zimmerman2. 1VeterinaryDiagnosticandProductionAnimalMedicine, Iowa State University, Ames, IA, USA, 2Iowa State University, Ames, IA, USA, 3Smithfield Foods, Algona, IA, [email protected]:HealthinSwinePopulations,Room4,12/4/20172:30PMSincethe1980's,swinesurveillancehasbeenbasedontheassumptionthatsubjectsareindependentofeachother("hypergeometricdistribution").Underthisassumption,itcanbeshownthat30samplesfromapopulationaresufficienttoachievea95%probabilityofdetection,if10%ofthepopulationisinfected.Inswinesurveillance,"30"hasbecomethestandard.However,farmshavechangeddramatically since the 1980's, which raises the question: does this assumption hold in contemporary production systems?MethodsandResults:ArecentstudymadeitpossibletoevaluatethespatiotemporalpatternsassociatedwiththespreadofPRRSV.Oralfluidswerecollectedfromeveryoccupiedpen(108pens;~25pigsperpen)in3commercialwean-to-finishbarnsononefinishingsitefor8weeksforatotalof972OFsamples.SampleswerecompletelyrandomizedandthentestedforPRRSVbyRT-rtPCR.Thereafter,thedatawereanalyzed for spatial autocorrelationusing thresholddistanceas the spatialweightmatrix.Moran’s I, aquantitativemeasureofspatialautocorrelation(calculatedusingGeoDa1.10)showedpositiveglobalspatialautocorrelationinthedistributionofPRRSVwithinbarns:thesubjectswerenotindependent.Discussion:TheRT-rtPCRdatashowedthatPRRSVmovedfrompen-to-pen,with the result that positive pens clustered together (positive spatial autocorrelation). Tobler was the first to codify spatialautocorrelationinhisFirstLawofGeography:“everythingisrelatedtoeverythingelse,butnearthingsaremorerelatedthandistantthings.” This simple, intuitive concept has huge implications for the way we conduct disease surveillance because it violates theassumption of hypergeometric distribution. Spatial autocorrelation probably appeared as the swine industry evolved in size andstructureovertheprevious20years.Thatis,astheindustryshiftedfromsmall,outdoorherdstolarger,confinedpopulations.Oursurveillancemethodshavenotkeptpacewithchangesintheindustry.Inparticular,thepresenceofspatialautocorrelationsignalstheneedtoreevaluateandexplorenewsurveillancemethodologiesthataccountforpositivespatialautocorrelation.

CRWAD2017 ABSTRACTS


96-DescribingthecullsowandcullhogmarketnetworksintheUS:apilotproject.B.Blair,J.Lowe.IntegratedFoodAnimalMedicineSystems,DepartmentofVeterinaryClinicalMedicine,UniversityofIllinois,CollegeofVeterinaryMedicine,Urbana,IL,[email protected]:HealthinSwinePopulations,Room4,12/4/20172:45PMCurrently,littleobjectivedataisavailabledescribingthemovementsofcullsowsandcullpigsthroughmarketchannelsandthediseasetransmission risk theymaypose. This investigation setout todetermine if it is possible to collect data todescribe the scopeandcomplexityofmovementswithincullmarketchannels.PremiseIDtagswherecollectedfromallanimalsmovingthroughoneharvestplantoveraone-weekperiodinthespringof2017.Allpremisetagswerematchedwiththeirfinalcollectionpoint.ThepremiseID’swerecrossreferencewithapublicdatabasetoobtainorigininformationforeachtag.Thisallowedterminalmarket,finalcollectionpointandpointoforiginofcullstoberecorded.90.4%ofallcullsmovingthroughtheplantduringtheone-weekperiodwereidentified.DemonstratingthatcapturingpremiseIDsattheplantyieldsenoughdatatoallowforreasonableconclusions.Cullsoriginatedfromfarmsin21statesandCanada,andshippedfromcollectionpointsin7statesandCanada.Cullstraveledamedianstraight-linedistanceof1057km, identifying thenationalscopeof thecullmarket.Of theculls identified,86%enteredtheterminalmarket froma finalshippingpointthatwasincloseproximity,lessthan240kmtothesourcefarm.Theremaining14%traveledmorethan240kmtothefinalpointofshipmentwith2.5%travelingdistances5timeslargerbetweenfarmandshippingpoint,thentheydidfromcollectpointtoterminalmarket.Thesedatasuggestthatbetween2.5-14%ofcullsaremovedbetweenmultiplecollectionpointspriortoarrivingattheterminalmarket,allowingthemtoserveasvectorsofdiseasetransmissionandspreadthroughoutthecullmarket.97-Epidemiologicaldynamicsofporcinereproductiveandrespiratorysyndrome(PRRS)inU.S.sowfarmsA.M.Perez1,A.Arruda2,M.Alkhamis1,C.Vilalta1,J.Sanhueza1,E.Geary1,P.Fioravante1,G.Machado1,P.Muellner3,K.VanderWaal1,R.Morrison1.1UniversityofMinnesota,StPaul,MN,USA,2OhioStateUniversity,Columbus,OH,USA,3EpiInteractive,Wellington,[email protected]:HealthinSwinePopulations,Room4,12/4/20173:00PMPurpose:Porcinereproductiveandrespiratorysyndrome(PRRS)isanon-reportablediseasethatcausesfar-reachingfinanciallossestotheU.S.swineindustry.TheDr.Morrison’sSwineHealthMonitoringProgram(MSHMP)isaninitiativethatfacilitatesproducersandveterinariansvoluntarilysharingPRRSstatusdataonaweeklybasis.TheMSHMPcurrentlycollectsdataforalmosthalfofthesowpopulation of the country. The objective of the MSHMP is to contribute to the understanding, in quantitative terms, of PRRSepidemiologicaldynamicswiththeultimateobjectiveofsupportingdiseasepreventionandcontrolintheU.S.Methods:Anumberofanalyticalmethods have been applied to farm level data routinely collected in theMSHMP, including time series analysis, clusterdetectiontechniques,andgeneticanalysisofvirussequences.Results:UseofthosemethodshashelpedtheU.S.swineindustrytoquantifythecyclicalpatternsofPRRS,todescribetheimpactthatemergingpathogenshavehadonthatpattern,toidentifythenatureandextentatwhichenvironmentalfactors,e.g.precipitationorlandcover,influencePRRSrisk,toidentifyPRRSvirusemergingstrains,andtoassesstheinfluencethatvoluntaryreportinghasondiseasecontrol.Conclusions:ResultsfromthestudiesthatwillbepresentedhereprovideimportantinsightsintoPRRSepidemiologythathelptocreatethefoundationsforanearreal-timepredictionofdiseaserisk,and,ultimately,willcontributetosupportthepreventionandcontrolof,arguably,oneofthemostdevastatingdiseasesaffectingtheNorthAmericanswineindustry.Theworkalsodemonstrateshowdifferentapproachestoanalyzeandvisualizethedatamayhelptoaddvaluetotheroutinecollectionofsurveillancedataandcansupportinfectiousanimaldiseasecontrol.

CRWAD2017 ABSTRACTS


98-BioavailabilityofketoprofenwhencompoundedwithirondextranforuseinnursingpigletsK.J.Reynolds1,R. Johnson2,R.M.Friendship1, J.Brown3, J.Delay4,R.Gehring5,T.L.O'Sullivan1. 1PopulationMedicine,UniversityofGuelph,Guelph,ON,Canada, 2Biomedical Sciences,UniversityofGuelph,Guelph,ON,Canada, 3Prarie SwineCentre,UniversityofSaskatchewan, Saskatoon, SK, Canada, 4Pathobiology,University ofGuelph,Guelph,ON, Canada, 5College of VeterinaryMedicine,KansasStateUniversity,Manhattan,KS,[email protected]:HealthinSwinePopulations,Room4,12/4/20173:15PMTheCanadianCodeofPracticeforCareandHandlingofPigsstatesthatitisarequirementforpigstoreceiveanalgesiatocontrolpost-proceduralpain.Medicationoptionsarelimitedtonon-steroidalanti-inflammatorydrugs(NSAIDs)suchasketoprofen.Pigletsreceiveirondextran(ID)topreventanemiaasstandardpracticeinNorthAmerica.ThepracticeofcompoundingdrugsisdiscouragedinCanada.However,thereisevidencethatsomeproducerscombineIDwithNSAIDs,toadministerasasingleinjection,tominimizepiglethandlingand labor. It is important to know if this practice of compounding provides effective pain control, as drug mixing can lead topharmaceuticaldruginteractionsandapotentialalterationofdrugbioavailabilityandpharmacokinetics.Commercialpiglets,3-4daysofage(9male,9female),wereenrolledandindividuallyhoused,fedandmonitored.Pigletswereadministered1of3treatmentsviaintramuscularinjection:(a)ketoprofen(Anafen®,MerialCanada),(b)ketoprofenmixedwithID(Dexafer200®,Vetoquinol),or(c)IDalone.Fifteenserialbloodsampleswerecollectedfromeachpigletviaindwellingjugularcatheters,andtheplasmawasanalyzedforS-andR-ketoprofenenantiomerlevelsbymassspectrometry.Standardpharmacokinetic(PK)analyseswerecompletedforR-andS-ketoprofenenantiomersandcomparisonsofparametermeans(t-test)betweentreatments(a)and(b)(±SD)showedthattherewasnodifferenceforS-ketoprofen:Cmax(7631.25±1070.35and6665.00±740.27,respectively,P=0.05),AUClast(35102.30±16188.15and25636.10±6940.08respectively,P=0.15)orAUC0-infinity(35869.66±16048.31and26151.96±6881.39respectively,P=0.14).ThesamePKparametersforR-ketoprofenwerenotdifferent(allP>0.05).ThelackofstatisticaldifferenceinthePKparameterssupportsthatthebioavailabilityofketoprofenisnotalteredbytheprocessofcompoundingwiththeID.However,giventhesmallsamplesizeandpotentialforbiologicalsignificance,paincontrolefficacystudiesareneededbeforeanyfurtherrecommendationsforthecontinuedpracticeofcompoundingthese2drugscanbemade.99-ConfirmationofzoonoticanthraxoutbreaksinhumansinTanzania,2016-2017F.S.F.Mosha1,C.Riwa1,E.Eblate2,B.Lyimo2,R.Katani3,A.Martin2,A.Estes4,T.Forde5,B.Mmbaga6,J.Buza2,V.Kapur4.1NationalHealthLaboratory,MinistryofHealth,DaresSalaam,Tanzania,UnitedRepublicof,2NelsonMandelaAfricanInstituteofScienceandTechnology,Arusha,Tanzania,UnitedRepublicof,3DepartmentofAnimalScience,PennsylvaniaStateUniversity,Pennsylvania,PA,USA,4TheHuckInstitutesoftheLifeSciences,PennsylvaniaStateUniversity,Pennsylvania,PA,USA,5InstituteofBiodiversity,AnimalHealthandComparativeMedicine,UniversityofGlasgow,Glasgow,UK,6KilimanjaroClinicalResearchInstitute,Moshi,Tanzania,[email protected]:EcologyofInfectiousAgents–1,Room4,12/5/20179:00AMPurpose:BacillusanthracisinfectionisrareinTanzaniahowever;therehavebeenreportedsporadiccasesfromtheNorthernzoneoftheCountry.Intheperiodof2016to2017,theMinistryofHealthTanzaniareceivedreportsofcasesofsuspectedAnthraxfromthreeregionsofMwanza,KilimanjaroandArusha.Thecaseswererelatedto theslaughteringandeatingofsickdomesticanimals.Mosthumancaseswereprecededbyfewmonthsofsuddenandunexplaineddeathsoflivestock.Methods:Atotalof53patientsmetthecase definition, however the laboratory received a total of 21 samples. Due to lack ofmolecular capacity at the National HealthLaboratoryQualityAssuranceandTrainingCenter(NHLQATC)toconfirmtheAnthraxcases,thesamplesweresenttotwolaboratoriesatdifferenttimes;theTanzaniaVeterinaryLaboratory(TVL)inDaresSalaamandtheNelsonMandelaAfricanInstituteforScienceandTechnology (NM-AIST) laboratory in Arusha.We evaluated the Turn Around Time (TAT) and logistical challenges to transport thesamplestothelaboratories.Results:Amajorityofthesamples(81%)originatedfromArushaandKilimanjaro.TheaverageTATwas3weekswhenallthesamplesweretransportedfromtheregionstotheNHLQATCandlatertoTVL.TwentyfourpercentofthesamplesweretestedatNM-AIST,transporteddirectlyfromKilimanjarotothelaboratorywithanaverageTATof10days.Overall,31%ofthesamples testedpositive forAnthrax.Conclusions:Despite the facts that,majority of patients presentedwith typical symptomsofAnthrax,thepositivityratewaslow,whichcouldbeduetoprolongedtimefortransportingandprocessingofthesamples.TheTAThasbeenconsiderably improved through strengtheningof theNM-AIST laboratory that investigatesglobalhealth,emerging infectiousdiseases,andfoodsafetyimplicationsofbushmeatconsumptioninTanzania.ThisalsoreducedtherisksandlogisticalchallengesoftransportingthespecimenfromArushaandKilimanjarotoDaresSalaam.MeasuresarecurrentlyinplacenowtoenableNM-AISTtocontinue to support confirmation of human Anthrax cases in the Northern Zone of Tanzania to allow immediate control of theoutbreaks.

CRWAD2017 ABSTRACTS


100-GeographicepidemiologyofanthraxinVietnamT.PhamQuang1,H.HoangThiThu1,D.TranNhu1,O.KimThuy2.1Epidemiology,NationalInstituteofHygieneandEpidemiology,HaNoi,VietNam,2USCDCofficeinHanoi,HaNoi,[email protected]:EcologyofInfectiousAgents–1,Room4,12/5/20179:15AMBackground:Thereisaworldwidedistributionofanthrax.Livestockvaccination,antibiotictreatment,andquarantineregulationsleadtoastrongdeclineinanthraxinfectionsindomesticlivestock.InVietnam,anthraxisaneglecteddiseasewhichstillendemicandoccurssporadically.There isnoroutinesurveillance in livestocksohumansurveillance isbased foralloutbreakresponse forbothhumanmedicine and veterinary. Purpose: to determine the detailed geographical distribution and epidemiology of anthrax in Vietnam.Methods:Aretrospectiveepidemiologicalinvestigationwasconducted.Datafrom1997to2016wasusedfortrendanalysis.WeeklyepidemicreportswereusedtoobtaintheGPSlocationandperformGPSmodel.Results:Before2011,Anthraxmainlyoccurredin9provincesintheborderareaswithChina,Laos,Cambodia.Eachyear,somedozensofcasesoccurredmostlyinnorthernregionbutalsoincentralandsouthernpartVietnam.In2011,anoutbreakofanthraxinLaiChauprovincewasreportedwith25-30peopleaffected,andshortlyafter,additionalcaseswerereportedfromotherprovinceslikeDienBienandHaGiang.Thecaseswerealltracedbacktoeatingandhandlingmeatfromsickcattle.Priortothis,anoutbreakoffoodpoisoningcausebyanthraxinthenorthernmountainousregionofHaGiangwasalsoreportedin2008.After2011tillpresent,Anthraxstilloccurredinthoseprovinceswithsmallerscaleandonlyhappenedinthenorthernregion.Increaseoflivestockmarkets,cattleproductionandtradinginthoseprovincesmaycontributetothediseasesituation.Conclusions:AnthraxisstillendemicdiseaseinmountainousareaofVietnam.Gastrointestinalsymptomsarehighinhumancases,althoughtherehavebeenreportsofcutaneouscases;andrelativelyconcentratedinthenorth.Furtherstudyusing a ‘One Health’ approach, where veterinary and medical public health experts are working together is needed to improveunderstandingofdiseases’distributionandtransmission,andproposesolutionswhicharebothsustainableandacceptable101-Rabiesoutbreakamonglivestockinapastoralistcommunityandlackofpostexposurevaccineseekingbehavior,southern

EthiopiaB.G.Donde1,S.G.Bekele1,H.Mohamed1,A.Deressa2.1AnimalandRangeScience,BuleHoraUniversity,BuleHora,Ethiopia,2EthiopianPublicHealthInstitute,AddisAbaba,[email protected]:EcologyofInfectiousAgents–1,Room4,12/5/20179:30AMPurpose:RabiesstillposesasignificanthealthproblemthroughoutmostofAfricancountries,wherethemajorityofthecasesresultfromdogbitesandsituationinmarginalizedpastoralcommunitieshasnotbeenwelldocumented.wereportrabiesoutbreakcaseinvillagefamilylivestockinmarginalizedpastoralistcommunityinEthiopia,forappropriateinterventionstrategyMethods:InSeptember2015,rabidwildfoxenteredthePastoralistvillageandbitedomesticdog.Thevictimdoghadturnedrabidafterfourmonthsandbitelivestock,andrabiesoutbreakwasoccurredinafamilylivestock.Consequently;onebull,onelactatingcow,onecalf,twodonkeysandoneheiferweredied.Theheadofheiferwasremovedandtransportedwithin24hourstorabiesreferrallaboratoryofEthiopianPublicHealthInstituteinAddisAbaba.Results:ThesamplewasconfirmedasstrongpositiveforlyssavirusantigenbyDirectFluorescentAnti-BodyTest.ThiswasfirstconfirmedcasefromsouthernEthiopianPastoralists.Familyexposedtocasesdidnotseekforpostexposurevaccine,rathertreatedbytraditionalhealeruntiltheywereconvincedtoreceivepostexposurevaccineathealthcenter.Occurrenceofrabiescasesacrossthedistrictwasalsoreportedbyveterinaryandhealthofficers.Lossoflivestockduetooutbreakfrustratedfamily,as the resultof livestock lossand fearofhuman rabies. Lowawarenessof familyabout importanceofpostexposurevaccinewasobserved.Conclusions:Integratedinterventionstrategy,includingwildlifeandneedforimmediateresponsefromlocalgovernmentwasrecommended.

CRWAD2017 ABSTRACTS


102-BigdatamachinelearningmodelforassessingriskofhighlypathogenicavianinfluenzaoutbreakonpoultryfarmsinKoreaH.Yoon,K.-N.Lee,W.-J.Lee,K.-H.Cho,C.-S.Jung,C.-S.Jung.VeterinaryEpidemiologyDivision,AnimalandPlantQuarantineAgency,Gimcheon,Korea,[email protected]:EcologyofInfectiousAgents–1,Room4,12/5/20179:45AMPurpose:Amachinelearningbasedmodelwasdevelopedaimingatassessingriskofhighlypathogenicavianinfluenza(HPAI)outbreakonpoultryfarms.Methods:DatawereprovidedbyKoreaanimalhealthintegratedsystem(KAHIS)underbigdatascheme.Results:Trainingmodelincluded34,401recordsonmovementforlivestock-relatedvehiclesinassociationwith137HPAIconfirmedchickenorduckfarmsduringperiodfromJanuarytoNovember2015.DuringHPAIepidemicin2016/2017,thisHPAIoutbreakriskassessmentmodelwasappliedtoassessspreadofdiseasefrom419HPAIoutbreakfarmsthroughmovementoflivestockrelatedvehicles.Riskassessmentwasreleasedfor4,061poultryfarms.Riskwascategorizedintofourlevelsand8.6%offarmswereassignedtothehighestlevel ‘severe’,0.8%was ‘warning’, theother1.2%was ‘alert’and the rest90.6%was ‘attention’.To farmswith ‘severe’ risk level,intensivecontrolmeasureswereimplementedtopreventfurtheroutbreak.Theoverallpositivepredictivevaluewasapproximately12%.Conclusions:ThisbigdatabasedriskassessmentmodelplayedanactivepartduringtheepidemicofHPAIin2016/2017inKorea.103-BrucellosisinsmallruminantsinMymensingh,BangladeshM.S.Rahman,B.S.Ahmed.Medicine,BangladeshAgriculturalUniversity,Mymensingh,[email protected]:EcologyofInfectiousAgents–1,Room4,12/5/201710:00AMPurpose:Rose Bengal test (RBT) andMAb based blocking Enzyme-Linked Immunosorbent Assay (MAb-ELISA) were performed todeterminethetrueprevalenceofbrucellosiswithlargenumberrandomlycollectedserumsamplefromgoatandsheepinMymensingh,Bangladesh. Methods: A survey plan was designed in both longitudinal and cross-sectional dimension covering all upazilla ofMymensinghdistrict,Bangladesh.Bloodsampleswerecollectedfromrandomlyselectednativegoatandsheep.About5mlbloodwascollectedfromjugularveinofeachoftheselectedgoatandsheep,1710and746,respectively,inseparatesterilizedtesttubesandkeptinrefrigeratorovernight.Thetesttubewasfurtherrefrigeratedat4-8°Covernight.Thentheserumwascentrifugedat2500rpmfor8-10mintoobtainclearserafreefrombloodcells.Finally,seraweretransferredintoasterilizedeppendorftubeandstoredat−20°Cuntilused.Results:Theprevalenceofcaprineandovinebrucellosiswasestimatedtobe1.67%whereas1.52%and2.01%ingoatsandsheep respectively. Sheepwas insignificantly1.33 timesmoreprevalent (95%CI,0.7 to2.52) thangoats.Theareawiseprevalencewassignificantly(p<0.05;95%CI,1to54.75)7.01timeshigherinMymensinghsadarupazillathanDhubaura.Theprevalenceofbrucellosiswasfoundtobesignificantly(p<0.01)associatedwiththegenderandageofthesmallruminants.Femalegoats(1.96%)3.57times(p<0.05;95%CI,1.07to11.95)andinsheep(2.83%)7.28times(p<0.05;95%CI,0.95to55.66)weremoreprevalenttobrucellosisthanmale(0.51%ingoatand0.4%insheep).Adultsabove2years(3.84%)weresignificantly(p<0.01)higherthanyoung(0.96%)between1and2years.Adultgoats(3.52%)weresignificantly(p<0.01;95%CI,1.37to9.81)3.67timesandsheep(4.5%)weresignificantly(p<0.05;95%CI,1.16to23.20)5.18timesmoresusceptiblethanmaletobeinfectedwithbrucellosis.Conclusions:ThenegativeresultofMAbbasedblockingELISAincaseRBTpositivesamplesindicatethattheprevalenceofbrucellosisinsmallruminantsinBangladeshisverylow.

CRWAD2017 ABSTRACTS


104-UsingadynamicinfectiousdiseasemodeltoexaminemultipletransmissionpathwaysforcampylobacteriosisM.M.Cousins1,D.N.Fisman2,J.Sargeant1,A.L.Greer1.1PopulationMedicine,OntarioVeterinaryCollege,UniversityofGuelph,Guelph,ON, Canada, 2Epidemiology, Dalla Lana School of Public Health, University of Toronto, Toronto, ON, [email protected]:EcologyofInfectiousAgents–2,Room4,12/5/201710:45AMCampylobacterarebacteria thataffectbothhumansandanimals.Therearemultiplepathways throughwhichCampylobacter canspread including both direct and indirect transmission pathways. The human incidence of Campylobacteriosis exhibits strongseasonalitysuggestingthatenvironmentalconditionsmayinfluencediseaserisk.Significantmathematicalmodelingworkconductedtodatehasfocusedonmodellingtheon-farmtransmissioncycleoffood-bornepathogenssuchasCampylobacter.Significantlylessattention has been focused on examining the role of environmental reservoirs of Campylobacter on the occurrence of disease inhumans. The objectives of this study are 1) to extend a simple SEIR compartment model for Campylobacteriosis in humans toincorporateanenvironmentalreservoircompartment(W)andalivestockreservoircompartment(L),and2)tousetheextendedmodeltoexaminetheimpactofseasonalforcingactingupontheenvironmental(W)andlivestock(L)reservoirs.Wewillusethemodeltocalculate the basic reproductive number, epidemic growth rate, and final outbreak size and examine how these differentmodeloutcomesvarydependingontherelativecontributionsofthedifferentdiseasetransmissionpathwaysunderconsideration.UsingadatasetofhumanCampylobactercasesandfarm-levelprevalencefromtheprovinceofOntario,Canadabetween2007and2013,wehopetoclarifytherelativeimportanceofenvironmentaland/orlivestockpathogenreservoirscomparedtodirectperson-to-persontransmissionaswell asdevelopabetterunderstandingof the roleof seasonality.Understanding the relative contributionsof thedifferenttransmissionpathwaysisanimportantsteptowardsdevelopinganimprovedunderstandingofhowbesttoimplementdiseasecontrolandpreventionstrategies.105-ThecomplexityanddynamicsofthebovinemammaryglandmicrobiomeK.Purdy1,M.Green2,A.Bradley2,E.Smith3,E.Monaghan3,S.Williams3,L.Green3. 1SchoolofLifeSciences,UniversityofWarwick,Covnetry, UK, 2School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK, 3School of Life Sciences,UniversityofWarwick,Coventry,[email protected]:EcologyofInfectiousAgents–2,Room4,12/5/201711:00AMPurpose:Mastitis,themosteconomicallyimportantendemicdiseaseofdairycattle,iscausedbyawiderangeofbacterialpathogens.Itisnowclearthatamicrobiomeinhabitsthebovinemammarygland(MG)andtherelationshipbetweenthesemicrobialcommunitiesand their hosts is critical to understanding andmanagingmastitis.Methods: Here we present the results of a large (200 cows),prospectivelongitudinalstudy(fromdryingoffuntil4weeksafterparturition)ofthebovineMGmicrobiomeandreportchangesinthemicrobiome inquartersaffectedandunaffectedby intra-mammary infections.Results:Dataon the relationshipbetween thehostimmune response (asmeasured bymilk somatic cell count) and bacterial load, alongwithmicrobiome diversity and dynamics inindividualMGquartersovertimewillbepresented.LatentclassanalysisofMGquartersproducedsignificantclasseswithdistinctSCCdynamics.AnalysisofmicrobiomeswithinandbetweentheselatentclassesshowamultilayeredcomplexitytobovineMGmicrobiomes.Analysis ofmicrobiomes before the development of sub-clinical and clinicalmastitis again suggest complexity in the relationshipbetweenthemicrobiomeandMGhealthanddisease.Conclusions:WecriticallyassesstherelationshipbetweenthebovinehostanditsMGmicrobiomeinthecontextofhowthismightalterourunderstandingandmanagementofmastitis.

CRWAD2017 ABSTRACTS


106-ViralmetagenomicsanalysisidentifypathogensofzoonoticpotentialinasymptomaticpigsinEastAfricaJ.O.Amimo1,D.Githae2,M.E.ElZowalaty3,E.Okoth4.1AnimalScience,UniversityofNairobi,Nairobi,Kenya,2BiosciecesofEasternandCentralAfrica(BecA),InternationalLivestockResearchInstitute(ILRI),Nairobi,Kenya,3BiomedicalResearchCenter,QatarUniversity,Doha,Qatar,4Biosciences,InternationalLivestockResearchInstitute(ILRI),Nairobi,[email protected]:EcologyofInfectiousAgents–2,Room4,12/5/201711:15AMPigsharboravarietyofvirusesthatarecloselyrelatedtohumanvirusesandaresuspectedtohavezoonoticpotential.Littleisknownaboutthepresenceofvirusesinsmallholderfarmswherepigsareinclosecontactwithhumansandwildlife.Thisstudyprovidesinsightintoviralcommunitiesandtheprevalenceandcharacteristicsofentericviralco-infectionsinsmallholderpigsinEastAfrica.Sequence-independentamplificationandhighthroughputsequencingwereappliedtothemetagenomicsanalysisofvirusesinfecescollectedfrom asymptomatic pigs. A total of 47,213 de novo-assembled contigswere constructed and comparedwith sequences from theGenBankdatabase.Blastxsearchresultsrevealedthat1039contigs(>200nt)wererelatedtoviralsequencesintheGenBankdatabase.Of the 1039 contigs, 612were not assigned to any viral taxa because they had little similarity to known viral genomic or proteinsequences,while427contigshadahighlevelofsequencesimilaritytoknownvirusesandwereassignedtoviraltaxa.Themostfrequentcontigs related to mammalian viruses resembling members of the viral genera Astrovirus, Rotavirus, Bocavirus, Circovirus, andKobuvirus. Other less abundant contigswere related tomembers of the genera Sapelovirus, Pasivirus, Posavirus, Teschovirus andPicobirnavirus.ThisisthefirstreportonthediversityofthefecalviromeofpigpopulationsinEastAfrica.Thefindingsofthepresentstudyhelptoelucidatetheetiologyofdiarrhealdiseasesinpigsandidentifypotentialzoonoticandemergingvirusesintheregion.Furtherinvestigationsarerequiredtocomparetheincidenceofthesevirusesinhealthyanddiseasedpigsinordertobetterelucidatetheirpathogenicrole.107-RapidresponsevaccineagainsttheporcineepidemicdiarrheavirusG.Singh1,P.Singh1,A.Pillatzki2,E.Nelson2,B.Webb3,S.Dillberger-Lawson3,S.Ramamoorthy1.1MicrobiologicalSciences,NorthDakotaStateUniversity,Fargo,ND,USA,2AnimalDiseaseResearchandDiagnosticLaboratory,SouthDakotaStateUniversity,Brookings,SD,USA,3VeterinaryDiagnosticLaboratory,NorthDakotaStateUniversity,Fargo,ND,[email protected]:VaccinesandVaccinology–1,Room5,12/4/20179:15AMThenumberof newly emerging infections, especially those causedbyRNA viruses, has increased rapidly in the last twodecades.Vaccinesareacriticalpartofoutbreak/pandemicpreparednessplans.With the recent increase innewlyemerging infections, theemphasisandinterestinimprovingtechnologyforepidemicorrapidresponsevaccineshasalsogrown.Epidemicvaccinesdifferfromconventionalvaccinesintherequirementforeaseandrapidityofdevelopmentanddeployment.Theemergence,rapidspreadandenormouseconomicdamagecausedbyporcineepidemicdiarrheavirus (PEDV) in2013, in theU.S., representsa typicaloutbreakscenarioinvolvinganemerginginfectiousagent.UsingPEDVasamodelforrapidresponsevaccinedevelopment,wedisruptedtheintegrityoftheviralgenomicRNAorreplicativeability,withoutalteringviralstructureorantigenicity,togeneratethevaccinevirus.Thedevelopedcandidatecombinedthesafetyandefficacyadvantagesof inactivatedandattenuatedvaccines, respectively.Whentestedinnaivepiglets,vaccinatedpigletsmountedstrongspike-proteinspecificbindingantibodyresponses,andvirusneutralizationtiters.Postchallengeviralsheddingandintestinallesions,asassessedbyhistopathology,wereundetectableinvaccinatedpigs,whilethecontrolpigsshowedclearevidenceofviralreplication.Thus,therapid-responsevaccineelicitedcompleteprotectionagainstviralreplicationandclinicaldisease.Thevaccineviruswasnotdetectedinfecalsamplesorintheintestinaltissuesofvaccinatedpigs,priortochallenge,indicatingthattheprocessusedhadahighsafetymargin.Insummary,therapidresponsevaccinedevelopmentmethoddescribedhassignificantadvantagesintermsofefficacy,safetyandrapidnessofdevelopment;andisbroadlyapplicabletootherRNAviruses,inoutbreakorpandemicsituations.

CRWAD2017 ABSTRACTS


108 - Chitosan delivery of inactivated influenza vaccine improves heterologous protection by enhancing antibody and cellularimmuneresponsesinpigs

S.Dhakal1,S.Renu1,S.Ghimire1,Y.S. Lakshmanappa1,B.T.Hogshead1,N.F.Ruiz1,S.Krakowka2,C.Lee1,G.J.Renukaradhya1. 1FoodAnimal Health Research Program, Department of Veterinary PreventiveMedicine, The Ohio State University,Wooster, OH, USA,2DepartmentofVeterinaryBiosciences,TheOhioStateUniversity,Columbus,OH,[email protected]:VaccinesandVaccinology–1,Room5,12/4/20179:30AMPurpose:Currentlyusedinactivatedinfluenzavaccinesinpigsbyintramuscularrouteprovidehomologousprotection,butthemuchrequiredheterologousprotectionagainstconstantlyevolvingvirusesislimited;becausetheyfailtoinduceadequatemucosalhumoralandcellularimmuneresponsesintherespiratorytract.AnovelvaccinedeliveryplatformusingmucoadhesivechitosannanoparticlescontaininginactivatedSwIAVadministeredthroughintranasalroutehasthepotentialtoelicitstrongmucosalimmuneresponseinpigs.Thus,inthisstudyweevaluatedtheimmuneresponsesandcross-protectiveefficacyofintranasalchitosanencapsulatedinactivatedSwIAVvaccineinpigs.Methods:Inactivated/killedSwIAVH1N2(δ-lineage)antigens(KAg)wereencapsulatedinchitosanpolymerbasednanoparticles(CNPs-KAg).Influenzaantibodyfree4-5weeksoldpigswereprime-boostvaccinatedintranasalasmistandchallengedwith a zoonotic and virulent heterologous SwIAVH1N1 (γ-lineage). Results:Pigs vaccinatedwith CNPs-KAg (~200nm) significantlyincreasedthespecificIgGantibodyinserumandIgAantibodyresponseintherespiratorytractsamples(nasalswab,BALfluidandlunglysate)againsthomologous(H1N2),heterologous(H1N1)andheterosubtypic(H3N2)SwIAV,comparedtosolubleKAgvaccinatedpigs.Atday35post-vaccination(pre-challenge)increasedtrendsinthefrequencyofcytotoxicTlymphocytes,antigenspecificlymphocyteproliferationindexandrecallIFN-γsecretionbyrestimulatedPBMCswereobservedinCNPs-KAgcomparedtocontrolKAgvaccinatedpigs. At day post-challenge (DPC) 6,macroscopic andmicroscopic pneumonic lesionswere reduced in CNPs-KAg vaccinated pigs.Importantly,thereplicatinginfectiousSwIAVtitersinnasalswabatDPC4andBALfluidatDPC6weresignificantlyreducedinCNPs-KAg(butnotKAg)vaccinatedcomparedtomock-challengepigs.Conclusion:TheintranasalchitosanSwIAVnanovaccineelicitsstrongmucosalantibodyresponseandreducesvirusloadintherespiratorytractofpigs.Futurestudiesareaimedatfurtherimprovingthecell-mediatedimmuneresponsetoachieveenhancedbreadthofcross-protection.109-LongevityofadenovirusvectorimmunityanditsimplicationforannualimmunizationE.E. Sayedahmed, A.O. Hassan, R. Kumari, S.K. Mittal. Comparative Pathobiology, Purdue University, West Lafayette, IN, [email protected]:VaccinesandVaccinology–1,Room5,12/4/20179:45AMAdenovirus(AdV)vector-basedvaccinesinduceexcellenthumoralandcell-mediatedimmuneresponsesduetotheadjuvant-likeeffectofAdVinstimulatingtheinnateimmunesystemthroughbothToll-likereceptor(TLR)-dependentandTLR-independentpathways.AdVvector-basedvaccineshaveelucidatedexcellentpotentialinbothanimalmodelsandclinicaltrials.ThedevelopmentofAdV-specificneutralizingantibodies,hasbeenconsideredapotentialconcernforAdVvector-basedvaccineefficacy.ThevectorimmunitycouldbeduetoanaturalinfectionwithaparticularAdVorfollowingimmunizationwithanAdVvectoredvaccine.Earlierinanexperimentalanimalstudy,wehavedemonstratedthatanimalshavingthevirusneutralizationtiterbelow500canbeeffectivelyimmunizedwiththesameAdVvector.TheobjectiveofthisinvestigationwastodeterminewhetherannualvaccinationwithanAdVvector-basedvaccinewillbepossibleduetothedeclineinAdVneutralizingantibodytitersbelow500withinayear.Inanelaborativestudy,naïveandhumanAdVtypeC5(HAdV-C5)-primedmiceweremock-inoculated(withPBS)orinoculatedi.m.with108p.f.u.ofeitherHAd-GFP[HAdV-C5vector expressing green fluorescent protein (GFP)] or BAd-GFP [bovineAdV type 3 (BAdV-3) vector expressingGFP] tomimic theconditionsforthefirstinoculationwithanAdVvector-basedvaccine.At1,3,6,and10monthspost-HAd-GFPinoculationanimalswerevaccinatedi.m.with108p.f.u.ofHAd-H5HA[HAdV-C5vectorexpressingH5N1hemagglutinin(HA)].Similarly,at1,3,6,and10monthspost-BAd-GFPinoculation,animalswerevaccinatedi.m.with108p.f.u.ofBAd-H5HA(BAdV-3vectorexpressingH5N1HA).Therewasa significant continualdecline in vector immunity titerswith time, leading to significant continual rises in the levelsofHA-specifichumoralandcell-mediatedimmuneresponseswithtime.FollowingchallengewithanantigenicallyheterologousH5N1virus,thelevelofprotectionalsoimprovedwiththereductionsinvectorimmunitytiters.TheseresultsindicatethattheannualimmunizationwithAdVvector-basedvaccineswillbeeffectiveduetodeclineinvectorimmunity

CRWAD2017 ABSTRACTS


110-Waterinoiladjuvantselectionfortheformulationofone-shotsafebacterialvaccinesforswineS.Xu1,J.BenArous2,N.Versillé2,L.Dupuis2.1SeppicInc,Fairfield,NJ,USA,2SeppicSA,Paris,[email protected]:VaccinesandVaccinology–1,Room5,12/4/201710:00AMIntroduction:Oilemulsionadjuvantsareextensivelyusedinswineinactivatedvaccines.Classicaloilbasedswinevaccinesconsistsofoil-in-water emulsion,orwater-in-oil-in-waterdoubleemulsion. Theseadjuvants inducea strong short term response,butusuallyrequiretwoinjectionsforalongtermprotection.One-shotvaccinescanbeachievedwithwater-in-oiladjuvants.Hereweshowthatanadaptedwater-in-oiladjuvantallowsone-shotvaccineagainstActinobacilluspleuropneumoniae(APP)withastrongantigenicloadreductioncomparedtooil-in-waterformulation.Methods:APPS2vaccineswereformulatedwith2differentwater-in-oiladjuvants(WO1andWO2).3APPconcentrationswereformulatedforeachadjuvant:100%(2.25108CFU/ml),10%,1%.Injectabilityofallvaccineswasassessed.806-weekoldpigswere randomlyseparated in7 testgroupsand1non-adjuvantedcontrolgroup.AtD0,3groupsreceived1mlofWO1basedvaccinesat100%,10%and1%,3groupsreceived1mlofWO2basedvaccinesat100%,10%and1%,1group received 2 ml of oil-in-water classical vaccine at 100% antigen concentration. Body temperature and local reactions weremeasured4and24hafterinjection,andcarcassqualitywasassessedatD120.BloodsamplesweretakenatD0,28,80and120,andIgG1andIgG2titersagainstAPPweremeasuredbyELISA.Results:Waterinoilvaccinesdidnotinducebodytemperatureincrease,localorgeneral reactionafterthe injection.With100%antigenic load,bothWO1andWO2 inducedsignificantlystrongerhumoralresponsestoAPPthanclassicalOWvaccine,butalsoinducedlocalreactions.With10%ofantigenicload,1mlinjectionofWO2basedvaccinedidnot induce injectionsite lesions,and inducedahumoral responsesimilar to the injectionof2mlofOWbasedvaccinecontaining100%antigenic load.Conclusions:Water-in-oiladjuvantsareeffectiveformulationsforswinevaccination.However,theselectionofanadaptedadjuvantandantigenicloadiscriticaltoavoidlesionsatslaughter.Water-in-oiladjuvantsallowtheformulationofone-shotvaccinesforpigswithlimitedamountofantigen,withlowpyrogenicityandlowrisksofanaphylactoidshocks.111-PassiveimmunityagainstporcineepidemicdiarrheavirusfollowingimmunizationofpregnantgiltswitharecombinantOrf

virusvectorL.R.Joshi,F.Okda,M.H.Fernandes,K.S.Hain,F.V.Bauermann,E.A.Nelson,D.G.Diel.VeterinaryandBiomedicalSciences,SouthDakotaStateUniversity,Brookings,SD,[email protected]:VaccinesandVaccinology–2,Room5,12/4/201710:45AMPorcine epidemic diarrhea virus (PEDV) causes acute diarrhea leading to highmorbidity andmortality in neonatal piglets. PassivematernalimmunitytransferredfromsowstopigletsthroughmilkandcolostrumiscriticalforprotectionofneonatalpigletsagainstPEDV.HereweevaluatedtheabilityofarecombinantOrfvirus(ORFV)expressingthefull-lengthspike(S)proteinofPEDV(ORFV-PEDV-S)toinducelactogenicimmunityinpregnantgilts.ThreedosesoftheORFV-PEDV-SweregiventotwogroupsofPEDVnegativepregnantsowswiththelastdosebeingadministeredtwoweekspriortofarrowing.Oneofthe2groupsimmunizedwithORFV-PEDV-ScandidatewasalsoexposedtolivePEDVorallyon31daypostimmunization.Serologicalresponsesinducedbyimmunizationwereassessedinserum,colostrumandmilkofimmunizedgilts.Additionally,transferofantibodiesfromimmunizedgiltstotheirpigletswasevaluatedbymeasuringPEDVspecificantibodiesintheserumofpiglets.TheprotectiveefficacyoftheORFV-PEDV-SwasevaluatedfollowingchallengeinfectionofthepigletswithPEDV.PEDV-specificIgG,IgAandneutralizingantibodyresponsesweredetectedinbothORFV-PEDV-SimmunizedgiltsandinORFV-PEDV-Simmunized+PEDV-exposedgilts.Neutralizingantibodies,spikespecificIgGandIgAweredetectedintheserumofpigletsborntoimmunizedgiltsindicatingsuccessfulpassivetransferofantibodiesthroughmilkandcolostrum.When challenged with PEDV piglets born to immunized gilts showed a marked reduction in overall morbidity and mortality incomparisontocontrolpigletsborntonon-immunizedgilts.PigletsborntogiltsthatreceivedORFV-PEDV-SfollowedbyexposuretoPEDVshowedhigherneutralizingantibodyresponsesandreducedclinicalscoreswhencomparedtopigletsborntogiltsimmunizedwithORFV-PEDV-Salone.ThisstudydemonstratesthepotentialofORFVvirusasavaccinedeliveryplatformcapableofelicitingpassiveimmunityagainstPEDV,animportantpathogenofswine.

CRWAD2017 ABSTRACTS


112 - Immunogenicity characterization of intradermally immunized enterotoxigenic Escherichia coli (ETEC) subunit vaccinecandidates

C. Garcia, B. Qu, J. Huang, N. Xiao,W. Zhang. Diagnostic Medicine/ Pathobiology, Kansas State University, Manhattan, KS, [email protected]:VaccinesandVaccinology–2,Room5,12/4/201711:00AMEnterotoxigenic Escherichia coli (ETEC) are themost common bacterial cause of diarrhea. ETEC bacterial adherence to the smallintestineepithelialcellsanddeliveryofenterotoxinscausediarrheainhumansandanimalsthatleadstowaterydiarrheaanddeaths.Currently,therearenovaccineslicensedforthisdisease.Ithasbeendemonstratedthattoxoidfusion3xSTaN12S-dmLT,adhesinMEFACFA/I/II/IV, and toxoid-adhesin MEFA CFA-3xSTaN12S-dmLT induced neutralizing antitoxin and/or anti-adhesin antibodies inintraperitoneally (IP) or subcutaneously (SC) immunizedmice, or intramuscularly (IM) immunized pigs, suggesting these antigenspotentialcandidacyfordevelopmentofETECsubunitvaccines.However,theseantigenshavenotbeenexaminedinintradermal(ID)route,a routeperhaps ismoresuitable forhumanvaccineadministration. In this study,we ID immunizedmicewith toxoid fusion3xSTaN12S-dmLT, the CFAMEFA, alone or combined, toxoid-adhesinMEFA CFA-3xSTaN12S-dmLT, and characterized antigen-specificantibodyresponses.DatashowedthatmiceIDimmunizedwiththetoxoidfusionantigendevelopedanti-LTandanti-STaantibodies,andmiceimmunizedwiththeCFAMEFAdevelopedantibodyresponsestoallsevenadhesins(CFA/I,CS1-CS6).Inaddition,miceco-administeredwiththetoxoidfusionandtheCFAMEFA,orwithtoxoid-adhesinMEFACFA-3xSTaN12S-dmLTdevelopedantibodiestobothtoxinsandallsevenadhesins.AntibodyneutralizationstudiesoftheserumsamplesoftheimmunizedmiceshowedinducedantibodiesneutralizedenterotoxicityofLTandSTaand/orinhibitedadherenceofETECorE.colibacteriaproducinganyofthesesevenadhesins.These data confirmed immunogenicity of these ETEC subunit vaccine target antigens and provide useful information for vaccinedevelopmentagainstETECdiarrhea.113-GeographicandtemporaldistributionofHPAIA/H5N1inVietnamin2015-2016andefficacyofvaccinesT.L. To, T.D. Nguyen, H.T. Do, V.T. Dam, D.H. Nguyen. National Center for Veterinary Diagnosis, Hanoi, Viet [email protected]:VaccinesandVaccinology–2,Room5,12/4/201711:15AMAvianinfluenza,reportedforthefirsttimeinVietnaminlate2003andearly2004,wascausedbyhighlypathogenicavianinfluenzaA(H5N1)viruses.Infectionsweremainlydetectedinbackyardchickensanddomesticducks.TheevolutionofthevirushasbroughtnewviruscladesinVietnam.During2015-2016,samplesofchickens,ducksandquailsindifferentprovincesofVietnamweretakenandscreenedforAIviruses.TheresultsshowedthattheAIvirusesdetectedinVietnaminthisperiodweremainlytwosub-clades:Sub-clade 2.3.4.4 distributed mainly in the North and Central and sub-clade 2.3.2.1c mainly in the South provinces. The challengeexperiments in chickens and ducks were performed using sub-clade 2.3.4.4A. The results indicated that Navet-vifluvac vaccine,producedlocally,providedaprotectionin80%ofvaccinatedchickensand100%ofvaccinatedducks;whileRe-5vaccineprovided70%and100%ofprotection,respectively.Theresultsofanotherexperimentusingclade2.3.2.1cvirusshowedthatNavet-vifluvacvaccineprovidedaprotectionfor68.96%ofvaccinatedchickens;Re-6vaccinefor76.67%andRe-5for96.67%.Allvaccineswereeffectiveinreducingtheamountofvirusshedfromvaccinatedchickensandducksincomparisionwithnon-vaccinatedbirds.

CRWAD2017 ABSTRACTS


114-Concurrentbutconsecutivevaccinationofmodifiedlivetype1andtype2PRRSVprovidesbetterprotectioninnurserypigsY.ShaanLakshmanappa1,P.Shang2,S.Renu1,S.Dhakal1,B.Hogshead1,P.Bernardo1,X.Yan2,Y.Fang2,R.Gourapura1.1FoodAnimalHealthResearchProgram,DepartmentofPreventiveVeterinaryMedicine,TheOhioStateUniversity,Wooster,OH,USA,2DepartmentofDiagnosticMedicineandPathobiology,KansasStateUniversity,,Manhattan,KS,[email protected]:VaccinesandVaccinology–2,Room5,12/4/201711:30AMPorcinereproductiveandrespiratorysyndromevirus(PRRSV)causessignificanteconomiclosstotheswineindustryworldwide.Thecurrentavailablevaccinesdonotprovidesufficientheterologousprotection.Inthisstudy,thelevelofprotectionagainstbothPRRSVgenotypes following concurrent vaccination was evaluated. Conventional 4-5 weeks old PRRSV free pigs (n=12 per group) werevaccinatedwithmodifiedlivevirus(MLV)strainsofbothtype1andtype2PRRSV.Thetype1andtype2MLVswereadministeredeitherincombinationonthesameday(group1)or3daysapart(group2,type1MLVfollowedbytype2MLV).Atday42,halfofthepigspergroup(n=6)werechallengedwithhomologoustype1ortype2PRRSV.Thepigexperimentwasterminatedat10dayspostchallenge.QuantitativeRT-PCR(qRT-PCR)resultshowedthattype1PRRSVRNAwasdetectablefromday3-42ingroup2pigs,whileonlylowleveloftype1PRRSVRNAwasdetectedfromday28-42ingroup1pigs.Thetype2PRRSVRNAlevelswerecomparableanddetectedfromday7-42inbothgroupsofpigs.Afterchallenge,themeanviralloadoftype1PRRSVisloweringroup2pigsthanthatofgroup1pigs,whilenoreplicatingtype2viruswasdetectedinbothgroupsofpigs.InTBLNthatrestimulatedwiththerespectivechallengevirus,thetestforrecalledlymphocytesresponseshowedenhancedIFN-γsecretingT-helper/memoryandcytotoxicTlymphocytesinbothpiggroups. In stimulated PBMCs, only T-helper/memory cells were IFN-γ positive in type II virus challenged animals. In conclusion,vaccinationofpigswithbothPRRSVgenotypesat3daysapart(type1MLVfollowedbytype2MLV)providesbetterprotectionandclearanceofviralinfectionthanthosepigsvaccinatedsimultaneouslywithbothtype1andtype2MLVs.115-Livevirusimmunization(LVI)witharecent1-7-4PRRSVisolateelicitsbroadprotectionagainstPRRSVchallengeinfinishingage

swineA.G.VanGeelen.VirusandPriondiseasesResearchUnit,USDA-ARS,Ames,IA,[email protected]:VaccinesandVaccinology–2,Room5,12/4/201711:45AMPurpose:PRRSisthemosteconomicallyimportantswinediseaseintheUnitedStatesandinmanycountries.Livevirusimmunization(LVI)isbasedonimmunizinghealthyswinewithserumfromapiginfectedwitharecentlocalPRRSVisolateaffectingthatfarmorareaandhasbeenfrequentlyutilizedwithreportedsuccessinimmunizingreplacementgilts,suggestingitconfersbetterprotectionthancommercialMLVPRRSvaccines.However,recentfieldreportssuggestthatLVIisnotasefficaciousasitoncewas,implyingachangeinhowPRRSVinteractswiththepig.MaterialsandMethods:ToevaluatetheefficacyofLVIwithcontemporary1-7-4strains,60finishingageswinewereused inthisstudyanddivided into7groups.Threegroupswerechallengedwith2mlserumI.M. fromanNADC34infectedpig,acurrent1-7-4strain.EachoftheseLVIgroupswereagainchallengedaftersixweeksintranasallywith5x10E4TCID50ofeitherNADC34(homologouschallenge),NADC36,acloserelative(98.4%homologouswholegenome),orSDSU73,aknownmoderatelypathogenic1-4-4strainthatis83.2%homologouswithNADC34.Eachofthesegroupswerecomparedwithanequivalentbutnaïvegroupandevaluatedforpyrexia,ADG,viralload,lunglesionsandvirusneutralizationtiter.Results:LVIwithNADC34protectedallthreeLVIgroupsagainsthomologousorheterologouschallengebyloweringviralloadbyalmostthreelogs,byeliminatingnegativeeffectsonADGofallthreevirusesandbyelicitingbroadlyneutralizingcalculatedantibodytitersofgreaterthan100at50%inhibitionin5outof8pigsinthehomologouschallengegroup,5out8intheNADC36group,and7outof9intheSDSU73group.Conclusions:LVIwithNADC34inducesequivalentneutralizationandprotectionpergroupfromchallengewiththeNADC34,NADC36orheterologousvirusSDSU73.Whenseraofindividualpigswereexaminedbywesternblotting,individuallydistinctreactivitypatternsagainstviralproteinswereobservedbetweenpigsinagroup,suggestingindividuallydistinctanti-PRRSVantibodyresponses.Futureworkaimstoelucidateifapredictivevaluecanbegleanedfromthesepatternsforefficacyofprotectionagainstfuturechallenge.

CRWAD2017 ABSTRACTS


116 – Development of a broadly protectivemodified-live virus vaccine candidate against porcine reproductive and respiratorysyndromevirus

H.Sun1,F.Osorio1,A.Workman1,D.Steffen2,H.Vu3.1UniversityofNebraska,Lincoln,NE,USA,2U.S.MeatAnimalResearchCenter,ClayCenter,NE,USA,3UniveristyofNebraska,Lincoln,NE,[email protected]:VaccinesandVaccinology–3,Room5,12/4/20174:00PMLive-attenuatedvaccines(LAVs)arewidelyusedtoprotectpigsagainstporcinereproductiveandrespiratorysyndromevirus(PRRSV).However,currentPRRSVLAVsdonotconferadequatelevelsofheterologousprotection,presumablyduetothesubstantialgeneticvariationamongPRRSV isolatescirculating in the field.Toovercomethisgeneticvariationchallenge,werecentlygenerateda fullysynthetic PRRSV strain (designated PRRSV-CON) containing a consensus genomic sequence of type 2 PRRSV. We demonstratedpreviouslythatthePRRSV-CONiscapableofconferringunprecedentedlevelsofheterologousprotection.However,thePRRSV-CONpassage1(P1)ishighlyvirulentandtherefore,isnotsuitabletobeusedasavaccineinpigs.Inthepresentstudy,weattenuatedthePRRSV-CONbycontinuouslypassagingthevirusinMARC-145,anon-naturalhostcellline.After90passagesinMARC-145cells,thePRRSV-CON P90 genome contains 43 nucleotide substitutions, resulting in 27 amino acid changes. Using a young pigmodel, wedemonstratedthatthePRRSV-CONP90issuccessfullyattenuated,asevidencedbythesignificantlyreducedviralloadsinserumandtissues and the absence of lung lesion in the infected pigs.Most importantly, the PRRSV-CON P90 confers comparable levels ofheterologousprotectionasdoes thePRRSV-CONP1. It also induces similar levelof innateandadaptive immune responsesas theparentalvirusdoes.Insummary,thePRRSV-CONP90isanexcellentcandidatefordevelopmentofthenextgenerationofPRRSVLAVvaccinewith improved levelsofheterologousprotection.KeywordsPorcinereproductiveandrespiratorysyndromevirus(PRRSV);Live-attenuatedvaccine(LAV);Heterologousprotection;Innateimmunity;Virus-specificinterferon-γsecretingcells117-Intranasalvaccinationofswinewithnanodisc-incorporatedPRRSvirusenvelopeglycoproteinsprovidesprotectiveimmunity

againsthomologousvirusinfectionF.A.Zuckermann1,W.Chen1,Y.Grinkova2,R.Husmann1,S.G.Sligar2.1DepartmentofPathobiology,UniversityofIllinois,Urbana,IL,USA,2DepartmentofBiochemistry,UniversityofIllinois,Urbana,IL,[email protected]:VaccinesandVaccinology–3,Room5,12/4/20174:15PMIntranasalimmunizationagainstviralrespiratoryinfectionsisknowntoprovideasuperiorlevelofprotectiveimmunitywhencomparedtovaccinesdeliveredbyotherroutes.Weinvestigatedtheefficacyofanovelporcinereproductiveandrespiratorysyndromevirus(PRRSV)subunitvaccinebasedonnanodisc(ND)technology.NDparticlesaresoluble,stable,andreproduciblyprepareddiscoidshapednanoscalestructuresthatcontainadiscretelipidbilayerboundbytwoamphipathicscaffoldproteins.BecauseNDparticlespermitthefunctional reconstitution of membrane/envelope proteins, we incorporated the envelope glycoproteins of PRRS virions into NDs(PRRSVglyco-ND)andtestedtheirimmunogenictyandabilitytoconferprotectiveimmunityto3-week-oldpigs.ThePRRSVglyco-NDvaccinewasadjuvantedwithawholecell lysateof saprophyticMycobacteriaandwasadministered intranasally twiceata2-weekinterval.Sixteendaysaftertheboostervaccinationtheanimalswerechallengedintranasallywith2x104TCID50ofvirulent lineage1North American PRRSV, of the same strain used to prepare the vaccine. Two groups of animals were similarly vaccinated usinginactivatedwholevirionswithorwithouttheMycobacterialadjuvant.Thepresenceofanti-GP5IgAinthelunglavagewasevidentatthetimeofeuthanasia(13daysafterviruschallenge)toagreaterextentinPRRSVglyco-NDvaccinatedswineascomparedtoswinevaccinatedwithinactivatedvirions.Thesubstantialextentofgross-lungpathologyobservedinmock-vaccinatedandvirus-challengedpigsatthetimeofeuthanasia(>85%oflunginvolvement),wassignificantlyreducedinthePRRSVglyco-NDvaccinatedpigsto<30%,andwassuperiortothereductionobservedinthelungsofanimalsvaccinatedwithintactvirionswithorwithouttheMycobacterialadjuvant.TheseresultsprovideencouragingevidencethatanefficaciousintranasalND-basedsub-unitvaccineagainstPRRSVisfeasible.

CRWAD2017 ABSTRACTS


118-Developmentofaone-doseclassicalswinefeversubunitvaccine:antigentitration,onsetanddurationofimmunityR.Madera1,L.Wang1,W.Gong2,Y.Burakova1,S.Buist1, J.Nietfeld3,J.Henningson3,A.CinoOzuna3,C.Tu2,J.Shi1.1DepartmentofAnatomyandPhysiology,KansasStateUniversity,Manhattan,KS,USA,2AcademyofMilitaryMedicalSciences, InstituteofMilitaryVeterinaryMedicine,Changchun,China,3DepartmentofDiagnosticMedicineandPathobiology,KansasStateUniversity,Manhattan,KS,[email protected]:VaccinesandVaccinology–3,Room5,12/4/20174:30PMPurpose:Classicalswinefever(CSF)isahighlycontagious,multi-systemicandhemorrhagicswineviraldiseasecausinglargeeconomiclossesworldwide.Fordecades,liveattenuatedvaccinesormodifiedlivevaccines(MLV),commonlyderivedfromtheCSFvirus(CSFV)C-strainhavebeencommonlyusedtocontrolthediseaseinCSF-endemiccountries.However,tocompletelyeradicatethedisease,apotent,safeandnon-infectiousCSFvaccineshouldbeeasilyaccessibleandavailable.Theaimofthisstudyistodevelopacost-effective,noninfectiousCSFsubunitvaccinethatcanelicitrapidandlonglastingimmunity.Methods:WerecentlyreportedonthedevelopmentofaCSFE2subunitvaccineformulatedinoil-in-waterbasedadjuvant,KNBE2,whichcanconferprotectionagainstCSFwithasingledose(PMID:27612954).Tofurthercharacterizetheefficacyofthisnovelvaccine,wedeterminedtheminimumE2antigenneededforprotection,theonsetanddurationofimmunityinducedbyKNB-E2.Results:SwinevaccinationandchallengeexperimentsshowedthataKNB-E2dosewithminimal25µgofrecombinantCSFVglycoproteinE2perdosecanprotectpigsagainstCSFVchallenge.OurresultsalsoshowedthatKNB-E2-mediatedprotectionisasearlyastwoweekspostvaccination.Furthermore,KNB-E2immunizedpigsexhibitedimmunityagainstdiseaseatleastfourmonthspostvaccination.Conclusions:ThesubunitvaccineKNB-E2confersprotectionagainstCSFV challenge. A single dose of this experimental vaccine withminimal antigen concentration gives fast acting and long lastingprotectionfromCSFdisease.119-InductionofmucosalimmunityandbroadenedantibodyresponsebyliveattenuatedinfluenzavaccinecandidateinchickensH. Jang, J. Ngunjiri, M. Elaish, C.-W. Lee. Food Animal Health Research Program, The Ohio state university, Wooster, OH, [email protected]:VaccinesandVaccinology–3,Room5,12/4/20174:45PMThecurrentstrategiesforcontrolofavianinfluenza(AI)inpoultryhavenotbeensuccessfulduetothecomplicatedecologyoftheAIvirusandthenarrowprotectionspectrumofcurrentlyavailablevaccines.Thereisaneedfornewvaccinesandvaccinationapproachesthatcaninducebroadlyprotectiveandlong-lastingimmunityagainstdivergentAIviruses.Livevaccinesareknowntoprovidebroadlyprotectiveimmunitybydirectlystimulatingthelocalmucosal,cellular,andhumoralimmuneresponses.Ourpreviousstudiesshowedthatpc4-LAIV,avariantofAIvirusthatencodesatruncatednon-structuralprotein1,hasthepotentialforuseasalive-attenuatedinfluenza vaccine (LAIV) in chickens. Pc4-LAIV was shown to induce type I interferon related signaling, acceleration of serumhemagglutination-inhibition antibody response, and heterologous protection in 1-day-old and 3-week-old birds. In this study, theadvantageoustraitsofpc4-LAIVwerefurtherinvestigatedfocusingonthemucosalimmuneresponseandthequalityofantibodyintermsofcross-reactivityandavidity.Weusedtearorserumsamplescollectedfromchickensvaccinatedat1-dayor3-weeksofagetodetermine the level ofmucosal antibody response, reactivity of serum to heterologous antigen, and avidity of serum antibodies.Comparedtowholevirusinactivatedvaccine,pc4-LAIVwasabletoinduceahigherlevelofmucosalIgAresponseandserumantibodieswithastrongerreactivitytoheterologousantigen.Moreover,inbirdsprimedwithpc4-LAIVandboostedwiththeinactivatedvaccine,therewerenotonlyhighlevelsofmucosalIgAantibodiesbutalsoasynergisticinductionofserumantibodieswithenhancedcross-reactivity.Thisstudydemonstratedfurtherthatpc4-LAIVisapromisingvaccinecandidatethatisabletoelicitbroadlyreactivemucosalandsystemicantibodyresponsesandsynergizewiththeinactivatedvaccineinaprime-boostapproach.

CRWAD2017 ABSTRACTS


120-EffectofinjectabletracemineralsgivenatthetimeofprimingvaccinationonthesystemicandmucosalantibodyresponsetoBHV1,BRSVandPI3Vinyoungdairycalves

A.Hoyos-Jaramillo1,J.H.J.Bittar1,S.Stanley1,B.Hamrick2,A.Gutierrez3,K.Miller4,S.Wall5,N.Norton6,F.C.Ferreira7,J.Urdaneta8,A.Rodriguez1,D.Hurley1, L.J.Havenga9, R. Palomares1. 1PopulationHealth,University ofGeorgia,Athens,GA,USA, 2Poultry Science,University of Georgia, Athens, GA, USA, 3Biological Science, University of Georgia, Athens, GA, USA, 4Animal and Dairy Science,UniversityofGeorgia,Athens,GA,USA,5WarnellSchoolofForestryandNaturalResources,UniversityofGeorgia,Athens,GA,USA,6LargeAnimalMedicine,UniversityofGeorgia,Athens,GA,USA,7AnimalSciences,UniversityofFlorida,Gainesville,FL,USA,8GeorgiaGwinnettCollege,Lawrenceville,GA,USA,9Multimin,FortCollins,CO,[email protected]:VaccinesandVaccinology–3,Room5,12/4/20175:00PMIntranasal(IN)vaccinationisapromisingtoolforimmuneprimingyoungcalveswithmaternalantibodiestopreventbovinerespiratorydisease (BRD).Administrationof injectable traceminerals (ITM)at the timeofmodified-livevirus (MLV)vaccinationhas shown toenhancetheproductionofserumneutralizingantibodies(SNA)andcellmediatedimmunity(CMI)againstrespiratoryvirusesincattle.OurobjectivewastodeterminetheeffectsofITMadministrationatthetimeofvaccinationonSNAtiterstoBHV1,BRSV,andPI3VandmucosalBHV1 specific IgA levels innasal secretions followingMLV INprimingvaccinationof youngdairy calves. Sixtydairy calvesreceived2mLofINMLVvaccine(Inforce3®)at3-4weeksofageandwererandomlyassignedtooneoftwogroups(ITMandControl,n=30pergroup).AtthetimeofvaccinationITMreceived1mlofMultimin-90®(containingSe,Cu,ZnandMn),whilecontrol1mlofsalineSQ.Bloodandnasalsecretionsampleswerecollectedondays-21,0(vaccination)7,14,21,28and42relativetothedayofvaccination. Serum was assessed for SNA titers against BHV1, BRSV and PI3V. Nasal secretions were used for BHV1 IgA titermeasurement.TitersofSNAandBHV1IgAwerecomparedbetweengroupsusingtwo-samplet-testandovertimewithingroupusingrepeatedmeasuresanalysisofSAS®.TherewasanotabledecayinSNAtitersinbothgroupsduringtheexperimentalperiod.SignificantdifferenceswerenotobservedinSNAtiterstoBHV1,BRSV,andPI3Vbetweengroups.However,ITMcalvestendedtohavegreaterSNAtiterstoBRSVondays14(P=0.16)and28(P=0.14)thancontrolcalves.TherewasasignificantincreaseinBHV1IgAtitersinbothgroupsondays14(P<0.05)and21(P<0.0001)comparedtoday0.Further,nosignificancedifferenceswereobservedforBHV1IgAbetween groups during the study. Incidence of BRDwas higher in control group (13.3%; 4/30) compared to ITM calves (0/30). Inconclusion,theuseof ITMconcurrentlywith INMLVvaccination inyoungdairycalvestendedtoenhancetheantibodyproductionagainst BRSV diminishing its decay rate, and reduced the incidence of BRD, which may be associated with improved circulatingprotectiveantibodyandCMI.121 - Proteomic analysis of local disease-sparing responses to bovine respiratory syncytial virus in intranasally vaccinated and

challengedcalvesJ.Ellis1,D.Gray2,S.Gow1,M.H.Mooney3.1UniversityofSaskatchewan,Saskatoon,SK,Canada,2Queen'sUniversityBelfast,Belfast,UK,3QueensUniveristyBelfast,Belfast,[email protected]:VaccinesandVaccinology–3,Room5,12/4/20175:15PMBovineandhumanrespiratorysyncytialviruses(BRSV,HRSV)areprimarycausesofpneumoniaincalvesandchildren,respectively.Parenteralandintranasal(IN)vaccinesconferprotectionagainstBRSVinfection,whichisassociatedwithsystemicandlocalantibodyandcellularimmuneresponses.TobetterunderstandtheresponseengenderedbyINvaccination,3-8dayoldcalvesreceivedasinglecomponentBRSVvaccine,a“3-way”vaccine(BRSV,BHV-1,BPIV-3),orplacebo,IN,andwerechallengedviaaerosolizationofBRSV42days later. Both groups of BRSV-vaccinated calves had significantly less pulmonary lesions and significantly higher arterial PO2concentrationscomparedtocontrols.2D-DIGE(24cm,pH3-10NL)proteomicanalysisofpharyngealtonsillysates(n=8/gp)indicatedadifferential proteome response among the groups. Principal component analysis (PCA) of 864 detected protein spots revealedclusteringoftreatmentgroups(20.41%R2Xvariation-PC1andPC2).Threeplaceboand2vaccinatedcalvesthatrequiredeuthanasiaondays6and7postchallengewereseparatedfromothercalveswithinthePCAscoresplot.VaccinatedcalvesthatoverlappedonthePCAplotwiththeplacebogrouphadlowerserumIgGpost-challenge,suggestingthatalterationstothepharyngealtonsilproteomeindicateprotectiveimmunity.Seventy-sixproteinspotsweresignificantlydifferent(ANOVA,p<0.05)betweenvaccinatedandplacebogroups,and105betweenanimalseuthanizedearlyversusonday8.Ofthese,67proteinspotswereselectedbasedonFDRtesting(q<0.2) forMALDI identification.Thesedata indicatedifferential responses in thepharyngeal tonsilproteomewhichcorrelateswithvaccinationanddisease-sparing.

CRWAD2017 ABSTRACTS


122 - Intranasal recombinant Moraxella bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovinekeratoconjunctivitis(pinkeye)

J.Angelos,R.L.Agulto,B.Mandzyuk,M.Chigerwe.Dept.ofMedicineandEpidemiology,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,CA,[email protected]:VaccinesandVaccinology–4,Room5,12/5/20179:00AMInfectiousbovinekeratoconjunctivitis (IBK;pinkeye) is themostcommoneyediseaseofcattleandefforts todevelopeffective IBKvaccineshavehistoricallyfocusedonparenteraladministrationofMoraxellabovisantigens.VariablesuccessatpreventingIBKwithnativeorrecombinantMbovispilin-orcytotoxin-basedvaccinesarereported,however,resultsaremixedandeffortstoimproveIBKvaccineshaverecentlyfocusedonthedevelopmentofintranasalvaccinesthatboostocularimmuneresponsestoMbovisantigens.ToevaluateefficacyofanexperimentalintranasalrecombinantMboviscytotoxinsubunitvaccinetopreventnaturallyoccurringIBK,arandomizedcontrolledfieldtrialwasconductedduringsummer2016overa16weekperiodinaherdof175northernCaliforniabeefsteers.CattlewithoutevidenceofactiveIBKwerevaccinatedintranasallywitheitherrecombinantMboviscytotoxinadjuvantedwithpolyacrylicacid(vaccinegroup)oradjuvantalone(controlgroup)ondays0and21andexaminedonceweeklyfor16weekstodocumentoccurrence,progression,and,ifneeded,treatmentofIBKbyanexaminerwhowasblindedtothegroupassignments.Tearandserumcytotoxinneutralizingantibodyresponses,tearandserumantigenspecificIgG,andtearantigenspecificIgAresponsesweremeasuredintear/bloodsamplescollectedonstudydays0,42,and112.Changesfromd0-d42andd0-d112intheseimmuneresponsevariableswerecalculated.Whilethecumulativeproportionofulceratedanimalswassimilarbetweengroups,overallcumulativecornealulcersurfaceareameasurementsofvaccinateswere lower thancontrols,andvaccinates requiredsignificantly fewernon-steroidalanti-inflammatorydrugtreatments.Additionally,significantlydifferentchangesintearcytotoxinneutralizingresponses,tearIgA,andtearIgGweremeasuredatthed0-d42andd0-d112intervalsamongstthevaccineandcontrolgroups.ResultssuggestedthatthisintranasalvaccinereducedocularinjuryandneedfortreatmentinsteersaffectedwithnaturallyoccurringIBK.AdditionalstudiesareneededtodetermineiffurtherimprovementstothisvaccinecanenhanceprotectionagainstIBK.123-VaccinedesigntosafelyinduceprotectiveimmunityoflongdurationR.Curtiss.UniversityofFlordia,Gainsville,FL,[email protected]:VaccinesandVaccinology–4,Room5,12/5/20179:15AMSurvivalafterpathogeninfectioninvariablyinduceslife-longimmunitytoprecludere-infectionwiththatpathogen.Vaccinestopreventinfections in the absence of associated morbidity and mortality was and continues to be a preferred/civilized way to deal withpathogens.Althoughmanyinfectiousdiseaseshavebeeneliminatedorarewellcontrolledbyvaccines,therearemanybacterial,viral,fungalandparasitepathogensthatarerefractorytocontrolbyvaccines.Liveattenuatedvaccinesaregenerallysuperiortosubunitandkilledvaccinesinelicitingprotectiveimmunityoflongduration.However,manypathogenshavedevisedmultiplemeanstoevadeorresistnaturalhostdefensestoenhancetheirsuccessatinvasionandcolonizationand/ormeanstonotelicitinductionofinnateand/oracquiredimmunitiesand/ormeanstosuppressinductionofimmunityand/ormeanstosubvertimmunitybyelicitingnonprotectiveimmune responses.Thus,unlessoneunderstandsandeliminates theseevasions,generatinga successful livevaccine isdifficult.Asecond problem diminishing effectiveness of live vaccines has persisted since Pasteur’s pioneering studies to attenuate severalpathogensbyrepetitivepassage.Thus,manyvaccinesdevelopedbyaccumulationofattenuatingmutationseitherintentionallyorbyrepetitivepassageleadstodecreasedabilityofvaccinestocopewithhostdefensesand/orinvadeand/orreplicateand/orpersistinvivo.Thus,asadequatesafetyandattenuationtoprecludecausationofdiseasesymptomsisachievedthereisaconcomitantlossinimmunogenicity. We have solved these problems by constructing live Salmonella vaccine vectors with elimination ofimmunosuppressingattributesandwithenhancedabilitiestocontentwithhostdefensebarriersandthatdisplayregulateddelayedattenuation,antigensynthesisandlysisinvivotomaximizeinductionofinnateandacquireprotectiveimmunitieswhilebeingtotallysafeandbiologicallycontained.WethushaveorareintheprocessofevaluatingrecombinantattenuatedSalmonellavaccines(RASVs)toprotectpoultryagainstC.perfringens,C.jejuni,EimeriasppandavianpathogenicE.coli.

CRWAD2017 ABSTRACTS


124-ChickenimmunizationusingpolyanhydridenanovaccinesA.M.Talaat,S.Chandrasekar,B.Bakke.UniversityofWisconsin-Madison,Madison,WI,[email protected]:VaccinesandVaccinology–4,Room5,12/5/20179:30AMAccordingto2015USagriculturestatistics,thecombinedvalueofproductionandsalesfrombroilers,eggs,turkeys,andchickswas$48.1billion.TheeconomicsuccessofthepoultryindustryintheUSAandworldwidehingesonextensiveuseofvaccinestocontrolbacterialandviralinfections.Unfortunately,traditionalvaccinesdonotprovidesufficientprotectionagainstemerginginfectionsandmostofthemarenotstableunderfieldconditions.Recently,weutilizedsynthetic,biodegradablepolyanhydridenanoparticles(PAN)to improveefficiency anddeliveryof protective antigens in chickens. Polyanhydridenanoparticles are approved foruse inhumanimmunizationandhaveahighsafetyprofile.Inourhands,weexaminedthefateofPANsinembryonatedchickeneggsandchickens.Fortunately,nountowardeffectswereobservedinembryosorchicksallowingthefeasibleimmunizationbothinovoorinlivebirds.We also deciphered the immunogenicity of avian influenza virus (AIV) proteins encapsulatedwithin PANs to develop a protectivevaccineagainstoutbreaksofLowPathogenicAvianInfluenza(LPAI).ElectronmicroscopyanalysisindicatedourabilitytoprepareaninactivatedbutintactvirionsreadyforPANencapsulation(sizesavg,165nm).Moreimportantly,whenchickswereimmunizedwithAIV-PAN, a robust hemagglutination inhibition (HI) titers were raised from one single immunization within the first 7 days postimmunizationwhichstayedupto14dayspostimmunization.Basedonviralreplicationtiters,allbirdsimmunizedwithnanovaccineswereprotected.Similarly,wearepreparingaPANconstructagainstInfectiousBronchitisVirus(IBV),anotherhighlypathogenicthreattothepoultryindustry,toensurethatourtechnologycouldbeappliedagainstmajorthreatstothepoultryindustry.Overall,currentexperiments provided very encouraging results which could significantly improve poultry immunization programs in the USA andworldwide.ExperiencesgainedfrombothAIV-PANandIBV-PANwillbesharedwithconferenceparticipantstohighlightthepotentialuseofPANasaplatformtechnologytoimmunizeagainstmostpoultrypathogens.125-MucoadhesivechitosannanovaccineoraldeliveryagainstSalmonellainpoultryS.Renu1,A.D.Markazi2,S.Dhakal1,Y.S.Lakshmanappa1,R.Shanmugasundaram2,R.Selvaraj2,G.J.Renukaradhya1.1FoodAnimalHealthResearchProgram,DepartmentofVeterinaryPreventiveMedicine,TheOhioStateUniversity,OARDC,Wooster,OH,USA,2DepartmentofAnimalSciences,TheOhioStateUniversity,OARDC,Wooster,OH,[email protected]:VaccinesandVaccinology–4,Room5,12/5/20179:45AMPurpose: Salmonellosis in poultry remains amajor health problem in the United States and globally. Significant economic lossesreportedthroughmortalityandpoorgrowthofSalmonellaenteritidis(S.enteritidis)infectedchicken.AlsotheothermajorconcernisthepublichealthhazardthroughSalmonellafoodpoisoningbyconsumptionofcontaminatedmeatandeggofpoultry.CurrentlyusedSalmonellavaccinesarenoteffectiveincombatingthediseaseproblem.Therefore,thereisanurgentneedtodevelopaneffectivevaccine,especiallyapotentoralSalmonellakilledorsubunitvaccinewhichelicitsrobustlocalmucosalimmunityintheintestinestomitigatebothsufferinganddiseasetransmission.Biodegradableandbiocompatiblepolymersareprovenvehiclesforvaccinedelivery.Methods: We prepared novel Salmonella candidate vaccines containing highly immunogenic protein antigens, outer membraneproteins(OMPs)andflagellarprotein,whichareentrappedinchitosannanoparticlesandalsosurfacedecoratedwithflagellarprotein(OMPs-F-CSNPs).Results:ThephysicochemicalandbiocompatibilitypropertiesofOMPs-F-CSNPssuchasparticlesizedistribution,surfacemorphology,proteinloadingefficiency,pHstabilityandtoxicityanalyseswereperformed.LikehowtheliveSalmonellatargettheileumPeyer’spatches(PPs)Mcellsofchicken,ourfluorescentlabelledOMPs-F-CSNPswereshowntotargettoileumPPsbyexvivoandinvivostudies.Interestingly,twomonthsoldlayerchickensvaccinatedorallywithOMPs-F-CSNPsinducedsignificantlyhigherOMPs-specificintestinalIgA(butnotsystemicIgG)response,associatedwithsignificantproliferationofantigenspecificlymphocytes.Furthermore,OMPs-F-CSNPsinducedsignificantlyhighertoll-likereceptor(TLR)-4,TLR-2andIFN-γcytokinemRNAexpressioninthececaltonsilsofvaccinatedbirds.Conclusions:ourpilotstudyinbirdsforthefirsttimedemonstratedtargetingOMPs-F-CSNPsoralSalmonellavaccinetointestinesandinducedsignificantlyhigherspecificantibodyandcell-mediatedimmuneresponses.

CRWAD2017 ABSTRACTS


126-OurvaccinestrategiestoprovidebroadprotectionagainstEscherichiacoliandSalmonellainpoultryM.Mellata.IowaStateUniversity,Ames,IA,[email protected]:VaccinesandVaccinology–4,Room5,12/5/201710:00AMBacterialinfections,suchascolibacillosisandsalmonellosis,arethreatstothepoultryindustryandhumanhealth.AvianpathogenicEscherichiacoli(APEC)causescolibacillosisinchickensresultingineconomiclossbecauseoftreatment,condemnationofproducts,anddeath. Likewise,Salmonella isamajorproblem in thepoultry industrydue to its impacton chickenhealthand foodbornehumandisease.Treatmentwithantibioticsoftenfailduetoantibiotic-resistanceinbacteriaandwithincreasedregulationonuseofantibioticstopreventinfections,alternativessuchasvaccinesareneededtoimprovepoultryhealthandwelfare.Developingpreventionstrategiesagainstthesepathogensarechallengingduetotheirantibioticresistanceandantigenicdiversity.MypresentationwilloverviewthedifferentvaccinestrategieswedevelopedtoprovidebroadprotectionagainstmultipleserotypesofE.coliandSalmonellainchickens.Theseincludeevaluationofbacterialantigensfortheirvaccinepotential,developmentandevaluationofmulti-antigenandattenuatedSalmonella vaccines for broad protection againstmultiple serotypes of APEC and Salmonella. Host responses to vaccinationweremeasuredusingELISA,RT-qPCRandhigh-throughput sequencing.Protectionwas testedusingboth in vitro (splenocyteand serumbactericidalassays)andinvivo(bacterialchallenges)againstmultipleserotypesofE.coliandSalmonella.Mostvaccineselicitedstrongimmuneresponses,buttheydifferdependingonthevaccineformulationadministered.TestingdifferentAPECandSalmonellastrains,wehavefoundlimitedtostrongprotectiondependingonthechallengestrain,dose,androute.Overall,ourvaccinestrategieshavethepotential to increase poultry heath, welfare, and food production and safety. Future work is still needed to optimize vaccineformulationsandincreasevaccineprotectivenessagainstAPECandSalmonellainchickens.132 - Number and location of activation induced cytidine deaminase (AID) ‘hotspot sequences’ in germline variable genes of

veterinaryandlaboratoryspeciesR.L.Levings.VeterinaryServices,USDA-APHIS,Ames,IA,[email protected]:ImmunologicalResponses–1,Room6,12/4/20179:00AMGenerationofantibody(Ab)diversityisaccomplishedthroughfivemechanisms:Heavy/Lightchaincombination,Variable-(Diversity)-Joininggenesegmentcombination,junctionalbaseaddition/deletion,geneconversion,andsomatichypermutation(SHM).Differentspecieshavebeenshowntousethemechanismstovaryingdegrees.Complementaritydeterminingregions(CDRs)aremorehighlymutatedthanframeworkregions(FRs)inthefinal,affinity-maturedAb.Genesequence‘hotspots’fortheenzymeprimarilyresponsiblefor SHM (activation induced cytidine deaminase, AID) have been identified -WRCY or its complement RGYW. Specieswith fewervariable(V)genes(e.g.,bovidaerelativetomuridaeorhominidae)mightbeexpectedtohaveVgenesegmentscontainingmoreAIDhotspots (particularly in CDRs), so as to ‘preposition’ the genes for SHM. Germline V sequences identified and annotated in theImMunoGeneTics(IMGT)databasewereanalyzedforAIDhotspotnumbersinCDRandFRregions(anddiversitygenes).Examplesoffunctionalgermlinesequencesfromsixclassesofvertebrateswereselected.Speciesincludedlaboratoryandfarmedanimals.Althoughnumbersofsequencesstudiedwerenotlarge,generalobservationscouldbemade.ManyhotspotswereobservedinFRs.Therelativefrequencyofhotspots(hotspots/bases inregion) in individualgeneCDRswasoftenzero,butrangedtohighervaluesthanforFRs.Strandpreference(senseornon-sense,orWRCYvs.RGYW)wasobservedandvariedwithspeciesandregion.NoclearpatternofAIDhotspotnumberorlocationwasobservedtobeassociatedwithphylogenyordiversificationstrategy(whenknown).However,‘outlier’speciesandgeneswerenoted.Theresultsindicatethatcautionshouldbeexercisedingeneralizingorextrapolatingbetweenspeciesregarding Ab gene sequences. They also reinforce the importance of conducting sequence studies including germline, fetal Blymphocytes, andmatureB-cells acrossmany species. Informationon theoriginandmechanismsofAbdiversityhas relevance tovaccinationstrategiesincludingthechoiceofantigenandadjuvant.

CRWAD2017 ABSTRACTS


133 - ClassificationofWC1genes inSus scrofaandevaluationof individual SRCRdomain affinity forMycobacteriumbovis andLeptospira

L. Le Page1, H. Hsu2, J. Buck1, N. Boisvert1, A. Gillespie1, E. Hudgens1, C.L. Baldwin1, J.C. Telfer1. 1Veterinary and Animal Sciences,University of Massachusetts Amherst, Amherst, MA, USA, 2University of Maryland School of Medicine, Baltimore, MD, [email protected]:ImmunologicalResponses–1,Room6,12/4/20179:15AMWC1,amemberofthegroupBScavengerReceptorCysteineRich(SRCR)superfamily,isfoundinthegenomesofmostmammalsandbirds,andisexpressedexclusivelyonγδTcellsinruminants.BovineWC1moleculescontainuptoelevenextracellularSRCRdomains,organizedintheSRCRdomainpatternofa1-[b2-c3-d4-e5-d6]-[b7-c8-d9-e10-d’11],wherethealphabetdesignationsindicatehomologybetweengenesandacrossspecies.Previously,wecharacterized13distinctgenesincattle(WC1-1toWC1-13).WehaveshownthatWC1-3,butnotWC1-4,expressingγδTcellsrespondtoLeptospira.ThisiscorrelatedwithdirectWC1-3,butnotWC1-4,bindingtoLeptospiraviaitsSRCRdomains.BecauseWC1+γδTcellssharearestrictionintheirγδTCR,andWC1isabletofunctionasaco-receptor,wehypothesizethatWC1co-ligationwiththeTCRplaysthedeterminingrole inactivationofWC1+γδTcellsbypathogens.Swinebelongtothesameorderascattle,Artiodactyl,andalsohaveWC1+γδTcells.WehaveshownthatWC1+γδTcellsincattlerespondtoLeptospiraandMycobacteria,twopathogensthatcaninfectswine.WC1isalsocloselyrelatedtothePRRSVreceptorCD163A.TherearetwopredictedWC1proteinsannotatedinthecurrentporcinegenomeassembly,oneofwhichinitiallyappearedtobeanassemblyerrorasitcontainedanN-terminald1SRCRdomaininsteadofanN-terminala1SRCRdomain.Priortothisstudy,therewasnocDNAevidence to confirm either of the genes in the assembly, and only one full-length cDNA transcript (ppwc1) had been successfullyamplified.Using5’/3’RACEPCRandRT-PCR,wehaveobtainedcDNAevidenceforsevenWC1geneswiththeSRCRdomainpatternsofa1-[b-c-d-e-d’]ord1-[b-c-d-e-d’].Throughbacterialpull-downassays,wehaveshownthatmultipleSRCRdomainsfromdifferentWC1genesbindtovaccinestrainLeptospiraspp,andfreshlygrownPasteurandDanishstrainsofMycobacteriumbovis.ClassificationofWC1genes,andtheirroleintheinteractionofγδTcellswithpathogensrelevanttoswine,willallowthesecellstoberecruitedinnextgenerationvaccinestopathogensthathavesignificantnegativeeconomicimpact.134-CharacterizationoftheWC1multigenicfamilyofγδTcellco-receptorsandpatternrecognitionreceptorsingoatsA.W.Yirsaw1,J.Telfer1,T.Smith2,C.Baldwin1.1Veterinaryandveterinaryscience,UniversityofMassachusetts,Amherst,MA,USA,2USDA-ARS,ClayCenter,NE,[email protected]:ImmunologicalResponses–1,Room6,12/4/20179:30AMγδTcellscanrepresentupto75%ofthebloodlymphocytesin“γδTcellhigh”speciessuchasruminants.Theyareoftenthefirstcellsto respond to infection.WC1molecules are transmembrane glycoproteinswithmultiple Scavenger Receptor Cysteine-rich (SRCR)domains thatactas signaling co-receptorsaswell aspattern recognition receptorsbindingbacterialpathogens; theyareuniquelyexpressedonγδTcells.WC1isamultigenicfamilyincattlewith13genescodingfor138SRCRdomainsforbindingvariouspathogens.ThehypothesisandrationaleforthisworkisthatgoatWC1salsowillbecodedbyamultigenefamilywithsomegeneshavingahighdegreeof identitywithbovineWC1genes,whileotherswillbeunique,sincesomepathogensaresharedbygoatsandcattlewhileothersareuniquetogoats.Inthepresentstudy,wedefinedgoatWC1genenumbersandstructuresbygenomeannotationandcDNAevidenceusingbothSangerandPacBiosequencing.SanClementegoatgenomewassequencedbyPacBiobyTimSmithandDerekBickhart,ARSUSDA.Boer goatbloodwasused toobtainboth cDNAandadditional gDNAevidenceof annotatedWC1genes.Weannotated16completecaprineWC1genesand8partials.IngoatssevendifferentWC1structureswereidentified,unlikethethreeonlythatexist incattle.Alsoingoatsunusual intracytoplasmictailsequenceswereobtainedbyRT-PCRandthesearenotfoundincattle;theyrepresentedsplicevariantsthatcouldaffectintracytoplasmicsignalingandthusactivationofγδTcellsfollowingbindingofpathogens.

CRWAD2017 ABSTRACTS


135 - Preliminary evidence for CD3+ cell generation in pigs with severe combined immunodeficiency (SCID): a leaky causativemutationwithintheArtemisgene

A.N.Boettcher1,A.G.Cino-Ozuna2,C.L.Loving3,J.E.Cunnick1,E.J.Powell3,R.R.R.Rowland4,S.E.Charley1,J.C.M.Dekkers1,C.K.Tuggle1.1Iowa State University, Ames, IA, USA, 2Kansas State University, Manhattan, KS, USA, 3USDA-ARS, Ames, IA, USA, 4Kansas StateUniversity,Manhattan,IA,[email protected]:ImmunologicalResponses–1,Room6,12/4/20179:45AMSeverecombinedimmunodeficiency(SCID)isdefinedasalackoffunctionalTandBcells.Insomecases,mutantgenesencodeproteinsinvolvedintheprocessofvariable(V),diversity(D),andjoining(J)recombinationthatretainpartialactivity,andarethereforeclassifiedashypomorphs.HypomorphicmutationswithinRAG1andArtemishavebeenstudiedinmouseandhumanpopulationsandhavebeenassociatedwiththeproductionofTcells,Tcelllymphomas,andOmenn’ssyndrome.HereweshowpreliminaryevidenceforCD3+cellsdevelopinginswineaffectedbynaturallyoccurringSCID.WehaveidentifiedbothanArtemisallelewithaspicesitemutationwithinintron8oftheArtemisgene(ART16),aswellasanothermutationwithinexon10thatcodesforaprematurestopcodonwhich ispredictedtocompletelyabolishfunction(ART12).PaststudieshaveshownthatbonemarrowtransplantedART16/16pigsdevelopedTcell lymphomasthatwerederivedfromthehost.Additionally,earlierworkfoundevidenceofCD3+cells in IlealPeyer’spatches,lymphnodes, thymus, spleen,and tonsils fromyoungSCIDpigs, and thishasbeenconfirmed recently foranART16/16animal. Incirculation,CD3+cellshavebeenfoundinART16/16aswellasART12/16pigs,althoughtheydonotpersistforlongperiodsoftime.Necropsyof3montholdSCIDpigsrevealedthepresenceofCD3+cellswithinflow-gatedlymphocytesofthespleenandlymphnodes.TheseCD3+cellsaregamma/deltaTCRneg,andhaveabnormally lowexpression levelsofCD4andCD8,withsomecells showingaCD4+CD8+phenotype.MolecularinvestigationsareongoingtodeterminethemechanismofdevelopmentofthesecellsintheSCIDenvironment.136-StaphylococcusaureuscellsurfaceproteinsextractionandevaluationofimmunogenicityR.D.Abdi,D.Ensermu,J.Vaughn,C.Merrill,B.Gillespie,O.KerroDego.AnimalScience,UniversityofTennessee,Knoxville,TN,[email protected]:ImmunologicalResponses–1,Room6,12/4/201710:00AMStaphylococcusaureus isthemostprevalentzoonoticpathogenandanotoriousantimicrobialresistantsuperbugbothinveterinaryandmedicalhealth.Itisoneofthemajorcausativeagentofmastitisindairyindustry.S.aureushasseveralcellsurfaceproteinsthathavemultiple functions including immune evasion and pathogenesis. Some of these cell surface proteins are good candidates todevelopeffectivevaccineagainstS.aureus.ThereisnoeffectivevaccineagainstS.aureusmastitis,whichcanbeduetostrainvariation.Little is knownwhethermultiple strains of S. aureus isolates from cases of bovinemastitis express similar conserved cell surfaceproteins.Moreover,theimmunogenicityofcellsurfaceproteinsofmulti-strainsarenotfullycharacterized.TheaimofthisstudywastoextractandevaluateimmunogenicityofcellsurfaceproteinsofgeneticallydistinctS.aureusisolates.Weevaluatedgeneticdiversityof239S.aureus isolatesfromcasesofbovinemastitisbypulsedfieldgelelectrophoresis(PFGE)andfoundninegeneticallydistinctclones.Subsequently,weextractedcellsurfaceproteinsfromeachclonebythreedifferentmethodsincludingtreatingcellswithanionicdetergent(1%cholicacid),withhexadecane,orthroughbiotinylationandultimatelyelutedfromNeutrAvidinAgarosecolumn.TheextractedsurfaceproteinswereevaluatedusingSDS-PAGEforproteinbandsimilarityandWesternblotforimmunogenicity.OurSDS-PAGEresultsshowedthatthethreeextractionmethodsprovidedcomparablenumberofproteinsbands(17-25bands).Thewesternblot result showed10conserved immunodominant surfaceproteinsacrossall9geneticallydistinct strains.Of10 immunodominatproteins,5proteins(18,22,30,48and110KDa)arestronglyreactedtoconvalescentserumfromacowinfectedwithS.aureus.Theidentityof these immunodominatproteinsarecurrentlybeingevaluatedby sequencing.Weconcluded thatdespitedifferences innumberofproteinbandsbothonSDS-PAGEandWesternblotallthethree-extractionmethodsprovided10similarimmunodominatsurfaceproteinsthatcanbeusedtodevelopeffectivevaccineagainstS.aureusmastitis.

CRWAD2017 ABSTRACTS


137-AnovelpeptidemicroarrayELISAdetectsspecies-specificantibodiesagainstChlamydiainruminantseraK.Rahman1,C.Schnee2,B.Kaltenboeck1,E.Müller3,M.Peisker2,E.Schubert2,R.Ehricht3,K.Sachse2.1Pathobiology,AuburnUniversity,Auburn,AL,USA,2MolecularPathogenesis,FederalResearchInstituteforAnimalHealth,Jena,Germany,3AlereTechnologiesGmbH,Jena,[email protected]:BovineImmunology–1,Room6,12/4/201710:45AMManyChlamydiaspeciescauseasymptomatictoclinicaldiseasesinlive-stockanimals.Speciesortype-specificserologicalassaysforchlamydiaearenotavailableduetohighcross-reactivityamongChlamydiaspp.Previously,weidentifiedhighlyimmunodominantB-cellepitopesofallchlamydialspeciesforuseaspeptideELISAantigens.Here,weused52peptideantigensofthe11chlamydialspeciesin a microarray peptide ELISA platform. Peptides were covalently linked to the 3-D epoxy surface of themicroarray, and boundantibodies were detected by precipitation of the horseradish peroxidase substrate. The peptide microarray was validated usinghyperimmuneseraforeachofthe11Chlamydiaspp.raisedinmiceby3×intranasalinfection.Confirmingspecificity,eachofthe11Chlamydia species-specific serum pools reacted strongly with homologous Chlamydia species-specific peptides and did not showreactivitywithanypeptideantigenoftheremainingChlamydiaspp.Subsequently,serafromcalves,cowsandsheepwithknownhistoryofnaturalChlamydiainfectionswereexamined.C.pecorum-specificmicroarraypeptideantigensreactedstronglywithserafromcalvesor cowsexposed toC.pecorum anddidnot showreactivitywith sera fromcalves thatwerenotexposed toC.pecorum. TheseC.pecorum-specificreactivitiesinmicroarrayformatareconsistentwithC.pecorumelementarybodyELISAsandPCR-confirmedexposureofthecattletoC.pecorum.ThespecifichumoralresponseofsheepagainstC.abortusaftervaccinationwithC.abortusvaccineswasdeterminedagainstabackgroundofantibodiesagainstC.pecorum,theendemicchlamydialspeciesinruminants.Inbothsheepandcattle sporadic, butdistinct antibody responses againstother chlamydial species suchasC. felis,C. suis, andC.pneumoniaeweredetected.Theparallelmulti-antigenpeptidemicroarrayELISAproveninthisstudysimultaneouslytracksantibodiesagainstdifferentchlamydial species with unprecedented serodiagnostic specificity. It has the potential to enhance our understanding of antibodyresponsesbydefiningnotonlyasinglequantitativeresponsebutalsothepatternofthisresponse.138-CharacterizingresponsesofimmunecellsubsetsfromM.paratuberculosis(MAP)testpositiveandtestnegativecowsfrom

commercialherdstoMAPantigenstimulationinvitroM.C.Frie1,P.M.Coussens2,K.R.B.Sporer2,B.Kirkpatrick3.1CellandMolecularBiologyProgram,MichiganStateUniversity,EastLansing,MI,USA,2DepartmentofAnimalScience,MichiganStateUniversity,EastLansing,MI,USA,3DepartmentofAnimalScience,UniversityofWisconsin-Madison,Madison,WI,[email protected]:BovineImmunology–1,Room6,12/4/201711:00AMJohne’s disease (JD) is a chronic wasting disease of ruminants caused by infection with Mycobacterium avium subspeciesparatuberculosis(MAP).Recentestimatessuggestthatover50%ofUSdairyfarmsareMAP-contaminatedandasmanyas91%ofdairyherds could be infected.MAPhas beenparticularly resistent to control and eradication efforts,which are hamperedby a lack ofapprovedvaccinesandapoorunderstandingofprotectiveimmuneresponses.AnothermajorobstacletoJDandMAPmanagementisthatJDisdifficulttodetectinmanyanimals,inpartduetovariableimmunityagainstMAP.OurgoalistoimproveknowledgeofimmuneresponsestoMAP,identifypossiblecorrelatesofprotection,andtoeventuallycorelatethesefactorswithgeneticdifferencesbetweencows. Inthepresentstudy,samplegroupsconsistedofMAPtestpositiveandMAPtestnegativecowsfrom8commercialherds inMichigan.Peripheralbloodmononuclearcells(PBMCs)from154MAPtestnegativeand96age-matchedMAPtestpositivecowswerestimulatedwithMAPantigensinvitro.Followingstimulation,subsetsofCD4+,CD8+andγδTcellsandBcellswereexaminedusingflowcytometrywithCD25(IL-2receptor)expressionasanactivationmarker. IncomparingMAPtestpositivetoMAPtestnegativecows,MAPstimulationsignificantlyincreasedCD25expressiononCD4+CD45R0+Tcells,γδ+MHCII+andγδ+MHCII-TcellsandSIgM+Bcells.WhilecleardifferencesweredetectedbetweenMAPtestpositiveandMAPtestnegativecows,wewerealsoabletodetectanumberofMAPtestnegativecowswithsubsetsofPBMCsthatapparentlyrespondedtoMAPantigensbyupregulationofCD25.TheseresultshighlightthedifficultyindistinguishingJDpositiveandnegativecowsincommercialsettingswherepriorexposuretoMAPislikely.ItisnotyetknownifthetestnegativerespondercowswereintheearlystagesofMAPinfectionorhadbeenexposedtoMAPandweresuccessfulinclearingorcontrollingthepathogen.

CRWAD2017 ABSTRACTS


140-CharacterizationofbovinegammadeltaT-cellphenotypefollowingM.bovisinfectionM.Guerra-Maupome1, J.McGill2,W.Waters3,M.Palmer4,M.Larsen5.1DiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,USA,2DiagnosticMedicine/Pathobiology,CollegeVeterinaryMedicineKansasStateUniversity,Manhattan,KS,USA,3InfectiousbacterialDiseasesResearchUnit,,NationalAnimalDiseaseCenter,AgriculturalResearchService,USDA,Ames, IA,USA,4Infectious bacterial Diseases ResearchUnit,National AnimalDisease Center, Agricultural Research Service,USDA, Ames, IA,USA,5Microbiology&Immunology,AlbertEinsteinCollegeofMedicine,Bronx,NY,[email protected]:BovineImmunology–1,Room6,12/4/201711:30AMBovine tuberculosis caused byMycobacterium bovis is a globally significant veterinary health problem. γδ T cells are known toparticipate intheimmunecontrolofmycobacterial infections.Data inhumanandnon-humanprimatessuggestthatmycobacterialinfectionregulatesmemory/effectorphenotypeandadaptiveimmunefunctionsofmycobacterium-responsiveγδTcells.Todate,theimpact of both age andM. bovis infection on bovine γδ T cellsmemory/effector phenotype remains unknown. In this study,weaddressedtheagedependentchangesofcirculatingγδTcells,analyzedthefunctionalandphenotypicaldifferencesinmemorymarker(CD45RO, CD45R), activationmarker (CD27, KLRG1) and chemokine receptor (CD62L, CCR7, CCR9, CXCR3) expression ofM.bovis-specificγδTcells,andevaluatedfunctionalresponsesofM.bovis-specificγδTcellsfollowingM.bovisBacilleCalmette-Guerin(BCG)vaccinationorvirulentM.bovisinfection.PhenotypicanalysisofγδTcellsinperipheralblood,lungsandpulmonarylymphnodesofM.bovis infectedcattle indicatedthatγδTcellshavephenotypicdifferencesbasedontheexpressionofCD27moleculesthatsuggestdistinctfunctionalproperties.HereweshowthatmostperipheralγδTcellsdisplayedaCD27+phenotypeindependentofage.Recallresponses after in vitro stimulation with mycobacterial antigens showed that expression of CD27 is critical for the expansion ofperipheral IFN-γ-producingγδT-cells.Collectively,suchCD27+subsetmaycomposetheadaptiveγδTcellscellcompartment,withspecificityformycobacterialantigens.Furthermore,ourstudyshowedthatγδTcellsfromneonatalcalvesarefunctionallyenhancedbyM.bovisBCGmucosalvaccinationandsuggestsanimportantroleforthisT-cellsubsetinacquiredimmunityconferredbyM.bovisBCGvaccinationatthesiteofinfection.TheuniqueabilityofγδTcellstomountarobustresponseduringmycobacterialinfectionssupports thatvaccine-elicitedγδTcell immunitymightprovebeneficial.Therefore,definingcorrelatesofprotectionbasedon thispopulationcanacceleratethedevelopmentofnovelvaccinesagainstTB.141-Developmentandcharacterizationofabovinemonocyte-derivedmacrophagecelllineC.G.Chitko-McKown1,M.-C.Bartens2,A.J.Gibson2,A.Holder2,A.Vats3,E.Brown2,J.Kolakowski2,A.M.Workman1,M.P.Heaton1,T.S.Kalbfleisch4,D.Werling2.1Genetics,Breeding,&AnimalHealthResearchUnit,U.S.MeatAnimalResearchCenter,ClayCenter,NE,USA,2Department of Pathobiology & Population Sciences, Royal Veterinary College, Hatfield, UK, 3Animal Biotechnology Center, ICAR-National Dairy Research Institute, Karnal (HR)-132001, India, 4University of Louisville, Louiville, KY, [email protected]:BovineImmunology–1,Room6,12/4/201711:45AMMonocytescirculateintheblood,andlaterdifferentiateintomacrophagesinthetissues.Theyarecomponentsoftheinnatearmoftheimmuneresponseandareoneofthefirst linesofdefenseagaininvadingpathogens.However,theyalsoserveashostcellsforintracellular pathogens such asMycobacteria, Brucella, and Salmonella. Becausemonocytes represent only a small percentage ofcirculatingleukocytes,harvestingsufficientquantitiesofthemforinvitroexperimentscanbecostly,timeconsuming,andcanvaryamongcattle.Therefore,itwasourobjectivetodevelopamonocyte-derivedmacrophagecelllinethatcouldbeutilizedforstudiesinvolvingmacrophages.Wholebloodfromacross-bredsteerwascollected intosyringescontainingEDTAasananticoagulant,andmonocytesobtainedusingdensitygradientcentrifugation.Themonocyteswerepurifiedfromtheperipheralbloodmononuclearcellfractionbyadherence.AfterextendedcultureinRPMI1640mediasupplementedwith10%FBS,apopulationemergedspontaneouslythatproliferatedincultureandcouldbeeasilydetachedfromthetissueculturevesselsusingtryspin-EDTA.Theseproliferatingcellsweretestedforcellsurfacedeterminantsindicativeofmonocyte/macrophagelineage,aswellasbactericidalandphagocyticactivity.Furthermore,thecelllineandwholebloodfromthedonorsteerweresubjectedtowholegenomesequencinginordertodeterminehowtheyalignwiththebovinewholegenomebuild,andtoidentifyanygeneticmutations.Thisbovinemonocyte-derivedmacrophagecell linehasbeenpassagedover25times,morphologicallyresemblesmacrophages inculture,expressestheCDmarkersCD14/16,C172a,CD11bamongstothers,isphagocyticandbactericidal(byROSandNOproduction),producesIFNinresponsetoTLR/RLRligands,andappearstofallintotheM2macrophagecategory.Alignmentofthegeneticsequenceofthecelllineison-going.Thereisadearthofbovinemacrophagecelllinesavailabletoveterinaryresearchers,andtherelativeeaseofcultureshouldrenderthiscelllineausefultoolfortheinvitrostudyofnumerousmacrophage-trophicpathogens.

CRWAD2017 ABSTRACTS


142 - Immunology of bovine tuberculosis: perspectives on one health approaches and defining correlates of protection versusinfection

W.R.Waters.NationalAnimalDiseaseCenter,Ames,IA,[email protected]:ImmunologyMini-Symposium–1,Room6,12/4/20172:15PMTuberculosis(TB),primarilyduetoMycobacteriumtuberculosisinhumansandMycobacteriumbovisincattle,isanexemplarymodeloftheOneHealthConcept.ThehumanTBvaccine,M.bovisbacilleCalmette-Guerin(BCG),wasfirstproveneffectiveincattlepriortouseinhumans.Recentexperimentaltrialswithcattlehavedemonstratedthat:(1)selectattenuatedM.bovismutantsprovidesimilartoimprovedefficacyasBCG,(2)subunitvaccinesmaybeusedtoaugmentimmunityelicitedbyBCGincattle,and(3)differentiationofinfectedfromvaccinatedanimals(DIVA)isfeasibleincattleusingbothinvitro(IFN-γreleaseassays,developedforuseincattleandnowusedwidely in both humans and cattle) and in vivo (skin test, developed for use in cattle prior to use in humans)methods.Experimentalinfection/vaccineefficacystudieswithcattlehavealsodemonstratedacorrelationofvaccine-elicitedcentralmemoryTcell(TCM)responseswithprotectionuponsubsequentchallengewithM.bovis.Specifically,higherfrequenciesofTCMproducingIFN-γ/TNF-α/IL-2 early after infection are associatedwith vaccine-elicited protection and bacterial arrest by the host. Using RNA-seq,numerousTh17-associatedcytokinegenes(includingIL-17A, IL-17F, IL-22, IL-19,andIL-27)areup-regulated>9fold inresponsetopurifiedproteinderivativestimulationofPBMCfromM.bovis-infectedcattle,demonstratingarobustinductionofthesecytokinesintuberculous cattle. Protective vaccines (i.e., BCG and immune-enhancing BCGmutants) also elicit IL-17A, IL-17F, IL-22, and IL-27responsesasmeasuredbyrt-PCRincattle.Moreimportantly,reducedIL-17Aresponsesbyvaccinatesascomparedtonon-vaccinatesat2.5weeksafterM.bovisaerosolchallengecorrelatewithreducedmycobacterial(antigen)burdenandlesionseverity.ThesefindingsfurthercharacterizethenatureofprotectiveTCMresponsesanddemonstrateacorrelationofmycobacterialburdenonthe levelofeffectorCMIresponsesearlyafterM.bovisinfection.143-BovinegammadeltaTcellsparticipateinlocalimmunitytoMycobacteriumbovisinfectionJ.L.McGill.DiagnosticMedicineandPathobiology,KansasStateUniversity,Manhattan,KS,[email protected]:ImmunologyMini-Symposium–1,Room6,12/4/20173:00PMMycobacteriumbovisisamemberoftheM.tuberculosis(TB)complexandthecausativeagentofTBincattleandzoonoticinfectionsinpeople.Manyreportshaveanalyzed immuneresponses toTB infectionorvaccinationbymeasuringthesystemicresponse,e.g.circulating immune cell populations and their cytokine responses. However, systemic immune responses are not an accuraterepresentation of the response that occurs in lesion sites. Results from humans and animalmodels suggest that the fate of theMycobacterium-infectedhostisdeterminedveryearlyinthecourseofdisease,likelydictatedbyinitialhost-pathogeninteractionsatthesiteofinfection.Therefore,characterizingtheimmuneresponseinvivoatthesiteofmycobacterialgranulomaformationiscrucialtoourabilitytocontroldisease.γδTcellsrespondtoTBinhumansandanimalsandareamongstthefirstimmunecellstoarriveatthesiteofM.bovisinfectioninthelungs.γδTcellsarethoughttobecriticalinskewingtheimmuneresponsetowardsTh1duringearlyM.bovisinfection;however,studyingtheirfunctioninlesionsiteshasremainedchallenging.Ourlaboratoryisemployingbothinvitroandin vivo experimental approaches to elucidate the role of γδ T cells in local immunity toM. bovis infection. Through the use oftranscriptionalprofiling,invitroγδTcellco-cultureexperimentsandinsituanalysisoftissuesfromM.bovisinfectedanimals,wehavedetermined that γδ T cells are a significant source of inflammatory chemokines and cytokines in the developingM. bovis lesion.TogetherourresultssuggestacriticalroleforγδTcellsintheestablishmentandmaintenanceofTBgranulomas.Futurestudieswillbeaimedatidentifyingapproachesformoreeffectivelyengagingthisversatilecellpopulationinvaccine-inducedprotectionfromTB.

CRWAD2017 ABSTRACTS


144-ImmunoinhibitoryreceptorsPD-1andLAG-3contributetoT-cellexhaustionduringAnaplasmamarginaleinfection.W.C.Brown1,T.Okagawa2,S.Konnai2,J.R.Deringer1,M.Ueti3,G.A.Scoles3.1VeterinaryMicrobiologyandPathology,WashingtonStateUniversity, Pullman, WA, USA, 2Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 3Animal DiseaseResearchUnit,ARS,USDA,Pullman,WA,[email protected]:ImmunologyMini-Symposium–2,Room6,12/4/20174:00PMSeveralpublishedstudiesfromourlabhavedocumentedtheinductionofanexhaustedCD4T-cellresponse,specificforAnaplasmamarginaleantigensinducedbypriorimmunization,followingeitherneedleortick-borneinfectionwiththisbacterialpathogen.Thisexhausted phenotype, characterized by significantly reduced or absent A. marginale-specific T-cell proliferation and cytokineproductionandT-celldeletion,waspartiallyreversedwhenthepersistentinfectionwasclearedbytetracyclinetherapy,supportingamechanisticroleofhighantigenloadinsuppression.Furthermore,inductionofT-cellexhaustionrequiredthepresenceofthespecificpriming T-cell epitope on the infecting bacteria, suggesting T-cell receptor engagement by the immunization-primed T cells wasinvolved.WelatershowedthatinductionofTcellimmunoinhibitoryreceptorsprogrammeddeath-1(PD-1)andlymphocyteactivationgene-3(LAG-3)wereupregulatedonallTcellsubsetsfollowinginfection,withhighestpercentagesofPD-1+LAG-3+doublepositiveTcellsoccurringatthepeakofinfection,concurrentwiththerapidlossofA.marginale-specificCD4Tcellresponses.TheligandPD-L1wasalsosignificantlyupregulatedonCD14+antigenpresentingcells,followingthesamekineticsofexpression.Finally,invitroantibodyblockadeofdualPD-1-PD-L1andLAG-3-MHCclassIIreceptor-ligandinteractionsresultedinpartial,butsignificant,restorationoftheantigen-specificT-cellresponse.TheseresultssupportthecontributionofPD-1andLAG-3receptor-ligandinteractionsininhibitingtheA.marginaleantigeninduced-specificCD4T-cellrecallresponsefollowingA.marginaleinfectionincattle.145-WC1hybridpathogenrecognitionreceptorsandsignalingco-receptorsdirectimmuneresponsesbybovinegammadeltaTcells

topathogensC.L.Baldwin,J.Telfer,A.Yirsaw,P.Damani-Yokota,A.Gillespie.VeterinaryandAnimalSciences,UniversityofMassachusetts,Amherst,MA,[email protected]:ImmunologyMini-Symposium–2,Room6,12/4/20174:30PMCellsoftheimmunesystemrecognizedisease-causingpathogensandrespondinamannertostoptheinfection.Whileweknowhowconventionalcellsoftheimmunesystemdothis,forsomenon-conventionalcellssuchasγδTcellsthisprocessislessclear.BovineγδTcellsbear lineage-specific transmembraneglycoproteinsknownasWC1.Wehaveshownthattheyarecodedforbyamultigenicfamilyandthesemoleculesfunctionbothaspatternrecognitionreceptors(PRR)andsignalingco-receptorsforcellularactivation.Forexample,asubpopulationknownasWC1.1+γδTcellsrespondearlyandbeforeCD4TcellsfollowingvaccinationagainstLeptospiraborgpetersenii serovarharjo andproduce interferon-γ. They continue to respond in in vitro recall responses formonths followingvaccinationwhileothersubpopulationsofγδTcellsthatexpressadifferentsetoftheWC1genesdonot.WehaveshownthatWC1moleculesonγδTcellsthatrespondtoLeptospirabindthebacteriaandthattheWC1moleculesareessentialformaximalγδTcellresponses.Thus,wehypothesizethattheWC1familymembersexpressedbyaparticularcellwilldetermineitsabilitytorespondtoaninfection.TotestthishypothesiswehaveevaluatedthedistributionofWC1geneexpressionamongindividualγδTcellclonesthatwereculturedwithleptospiraandfoundthatthemajorityexpressWC1moleculesthatbindleptospira.WeproposethatvaccinescouldbedesignedtoactivateγδTcellsthroughengagementoftheWC1co-receptorwiththeTcellreceptortostimulaterapidresponsesbyinterferon-γ-producingcellsthatcouldinfluencethedevelopmentofaTh1CD4Tcellresponse.

CRWAD2017 ABSTRACTS


146-AlternativeimmunemechanismsforprotectionagainstbrucellosisD.W. Pascual, X. Yang, H. Wang, Z. Diaz-Goodwin, C. Hoffman, B. Clapp. Dept. of Infect. Dis. & Immunol., University of Florida,Gainesville,FL,[email protected]:ImmunologyMini-Symposium–2,Room6,12/4/20175:00PMBrucellosis produces a systemic disease that can cause fetal abortion in livestock. This disease remains a global health problemimpactinglivestockandhumans.Althoughlivestockvaccinesforbrucellosisareavailable,theseperformsuboptimallywithanefficacyof60-70%.Notably,Brucellainfectionsprimarilyoccurfollowingamucosalexposure,yetfewstudieshaveconsideredmucosalaspectsof Brucella's pathogenesis, let alone vaccination. To enable further study ofmucosal vaccines for brucellosis, genetically definedmutantsweredevelopedforbothB.melitensisandB.abortus.Whengivenbytheoralornasalroutes,exquisiteprotectionagainstpulmonaryBrucellachallengewasconferredwithnearlycompleteprotectionof the lungsandspleen. Interestingly, thesemutantselicitedstrongIFN-γ+andpolyfunctionalCD8+Tcells.AlthoughIFN-γ+andpolyfunctionalCD4+Tcellswereelicited,thesewerenottothelevelsproducedforCD8+Tcells.Infact,protectionwasindeedCD8+Tcell-dependent.TheseresultscontrastedwiththeCD4+Tcells inducedbyconventional livestockvaccines, inwhichCD8+Tcellswereweakly induced.Thedifferences inthetypesofTcellselicitedwerenotdependentontherouteofvaccination,butmorerelatedtothevaccinecomposition.Anothersalientfeatureofthesemutantswas their ability toelicit residentmemoryT cells in the lungswhichwere important forprotection, andperhapsabatingsystemicbrucellaedissemination.Currentworkisdeterminingifsimilarresponsesareinducedincalvesandpigs.Developmentofnewvaccines and vaccination regimens that incorporate aspects formucosal protection can help improve prevention of brucellosis inlivestock.WorkissupportedbyR01AI-123244,R03AI-128123,andUSDA-NIFA2013-01165.147-Inductionoftoll-likereceptor8expressioninTHP-1cellsstimulatedwithBrucellaabortusrecombinantproteins,PsDandPmGY.Im1,S.Shim1,S.Soh1,S.Kim2,H.Yoo1.1DepartmentofInfectiousDiseases,CollegeofVeterinaryMedicine,SeoulNationalUniversity,Seoul,Korea,Republicof,2CollegeofVeterinaryMedicine,GyeongsangNationalUniversity,Jinju,Korea,[email protected]:ImmunologicalResponses–2,Room6,12/5/20179:00AMBrucellaspp.isacausativeagentofbrucellosiswhichisazoonoticpathogenthatmainlycausesabortionandlossinmilkproductionindomestic animals and serious symptoms in human. Early detection and removal of the intracellular bacteria have been receivingattentionbecausethesebacteriainvadeandsurviveinmacrophagesthroughtheirabilitytomodulatehostcellfunction.Moreover,understandingofBrucellapathogenicitycouldbecrucialtopreventandcontrolbrucellosis.TLRsareoneofthemajorcompoundsthatplay importantroles inactivationof innate immunityandacquired immunity inhostafter infection.TLR4 isawell-knownreceptorwhichisrelatedtocellapoptosisduringtheinfectionwhileTLR8mediatedsignalsinhibitthecellapoptosisinTHP-1cells.Inthisstudy,therefore,humanmacrophages(THP-1cells)werestimulatedwithtwoBrucellaabortusrecombinantproteins,30SribosomalproteinS4(PsD)and50SribosomalproteinL33(PmG)atdifferenttimeintervals(2hrs,6hrs,12hrsand24hrs).ExpressionofTLRsinTHP-1cellswasanalyzedusingrealtimePCRafterstimulationwiththoseproteins.OftheTLRs,TLR8wasproducedintime-dependentmannerafterstimulationwiththetworecombinantproteinsuntil24hrs.TheseresultssuggestthatthetwoB.abortusproteins,PsDandPmG,inhibitedcellapoptosisinTHP-1cellsintime-dependentmanner.Therefore,thesetwoproteinsaseffectiveantigencandidatesmayprovidebetterunderstandingsofBrucellapathogenicity.ThisworkwassupportedbyKHIDI(No.HI16C2130),theBK21PLUSprogramandtheRIVS,SeoulNat’lUniversity,RepublicofKorea.

CRWAD2017 ABSTRACTS


148 -Singlestranded (ss) ribonucleicacids (RNA)-mediatedantiviral responseagainst infectious laryngotracheitisvirus infectioncorrelatingwithmacrophagenumbersandtheexpressionofpro-inflammatorymediators

M.S.Abdul-Cader1,H.Ahmed-Hassan2,M.Abdul-Careem1.1UniversityofCalgary,UniversityofCalgary,Calgary,AB,Canada,2ZoonosesDepartment,FacultyofVeterinaryMedicine,CairoUniversity,Giza12211,[email protected]:ImmunologicalResponses–2,Room6,12/5/20179:15AMNucleicacidssuchassinglestranded(ss)ribonucleicacids(RNA)arerecognizedbytoll-likereceptor(TLR)7inchickensandknowntoelicitprotectiveresponsesagainstinfectiousbursaldiseasevirusinfection.However,theinformationonthemechanismsofprotectionaswellasssRNA-mediatedantiviralresponseagainstotheravianvirusesarescarce.TheobjectiveofthestudywastodeterminetheantiviraleffectinovodeliveredssRNA,againstinfectiouslaryngotracheitisvirus(ILTV)infection.WefoundthatwhenssRNAisdeliveredat embryo day (ED)18 in ovo and subsequently challenged with ILTV at day 1 post-hatch ssRNA reduces ILTV in cloacal andoropharyngealswabsassociatedwithmacrophagerecruitmentinrespiratoryandgastrointestinaltissues.Invitro,weshowedthatnitricoxide(NO)andinterleukin(IL)1βoriginatedfrommacrophagescorrelatewithantiviralresponseagainstILTVreplication.ThisstudyprovidesinsightsintothemechanismsofssRNA-mediatedantiviralresponse,particularlyagainstILTVinfectioninavianspecies.149-InovodeliveredCpGDNAincreasesinnateandadaptiveimmunecellsinmultiplebodysystemspost-hatchcorrelatingwith

lowerinfectiouslaryngotracheitisvirusinfectionM.S.Abdul-Cader1,A.Amarasinghe1,V.Palomino-Tapia1,H.Ahmed-Hassan2,K.Bakhtawar1,E.Nagy3,S.Sharif4,M.Abdul-Careem1.1DepartmentofEcosystemandPublicHealth,UniversityofCalgary,Calgary,AB,Canada,2ZoonosesDepartment,FacultyofVeterinaryMedicine,CairoUniversity,Giza12211,Egypt,3DepartmentofPathobiology,UniversityofGuelph,Guelph,ON,Canada,42DepartmentofPathobiology,UniversityofGuelph,Guelph,ON,[email protected]:ImmunologicalResponses–2,Room6,12/5/20179:30AMCytosine-guanosinedeoxynucleotides(CpG)DNAcanbedeliveredinovoatembryoday(ED)18forthestimulationoftoll-likereceptor(TLR)21signalingpathwaythatultimatelyprotectschickensagainstanumberofbacterialandviral infections.There isadearthofinformationunderstandingthemechanismsofprotection inducedby inovodeliveredCpGDNA.Theobjectiveof thestudywastodeterminemacrophages,BcellsandTcellsubsetsinmultiplebodysystems(respiratory,gastrointestinalandimmunesystems)asanindicatorofcell-mediatedimmuneresponsespost-hatchfollowing inovodeliveryofCpGDNA.Wefoundincreasedrecruitmentsofmacrophages in organs of these body systems post-hatch following in ovo delivery of CpG DNA. Although B cells, cluster ofdifferentiation(CD)4+andCD8+Tcellswereincreasedinlungsandimmunesystemorgans,thesecellswerenotquantifiablefromthetracheaandsomegastrointestinalorgansimmediatelyfollowingthehatch.Incorrelatingwiththecellularresponses,wealsofoundthatwhenCpGDNAisdeliveredinovoandsubsequentlyinfectedwithinfectiouslaryngotracheitisvirus(ILTV)post-hatch,CpGDNAreducesILTVinfectionpotentiallydecreasingtransmission.ThisstudyprovidesinsightsintothemechanismsofhostresponseselicitedfollowinginovodeliveryofCpGDNAinavianspecies.

CRWAD2017 ABSTRACTS


150-Toleratedvstrained:porcinemonocyteresponsestobeta-glucanK.A.Byrne1,C.K.Tuggle2,C.L.Loving1.1NADCUSDA-ARS,Ames,IA,USA,2DepartmentofAnimalScience,IowaStateUniversity,Ames,IA,[email protected]:ImmunologicalResponses–2,Room6,12/5/20179:45AMβ-glucanfrombrewer’syeast(S.cerevisiae)isfrequentlyincorporatedintothedietofagriculturalanimalswiththeintentofimprovinghealthandproductionparameters.However,themechanismandbenefitsoffeedingS.cerevisiaeβ-glucantoproductionanimalshasyettobeelucidated.Recentworkinmiceandhumanshasshownthatpriorexposuretoβ-glucanfromC.albicans(CaBG)caninduceepigenetic reprograming in monocytes resulting in enhanced responses to heterologous agonists. This process, termed trainedimmunity,istheoppositeofimmunetoleranceinwhichpriorexposuretolipopolysaccharide(LPS)reducestheabilityofmonocytestorespondtofutureLPSstimulation.Tounderstandtheabilityofβ-glucantoinduceatrainedortolerantphenotypeinswine,primaryporcinemonocyteswerestimulatedwithβ-glucansfromvaryingsources,LPS(tolerizationcontrol),orremainedunstimulated.After24hprimarystimulation,agonists(media,LPS,orvariousβ-glucans)wereremovedandthecellswererestedfor5dinmediaalone,andthenrestimulatedwithLPStodeterminetrainedortolerantphenotype,asindicatedbyadecreaseorincreaseincytokineproductionrelativetounstimulatedcontrols.AfterLPSrestimulation,themedia-primedcellsproducedtheproinflammatorycytokinesTNF-αandIL-1β(naïvephenotype),whiletheLPSprimedcellsdidnot(classicalendotoxintolerance).Primingwiththesolubleβ-glucan,laminarin(L. digitata), and restimulating with LPS did not significantly alter cytokine production compared to media stimulated controls.Monocytes primedwith β-glucan fromS. cerevisiae (zymosan) exhibited a tolerant phenotype (decreased cytokineproduction) toheterologous stimulationwith LPS, alongwith decreasedmitochondrial respiration and glycolysis.However, CaBGprimedporcinemonocytes for increasedcytokineproductionafter LPS stimulation,an indicatorof trained immunity.Thesedata indicate that thesource of β-glucan drives monocytes to enter one of two very different states, trained or tolerized, and will be an importantconsiderationforinfluencingswinehealth.151-CirculatingtransferRNAfragmentsinwhitebloodcellsassociatedwithantibodyresponsetobovineleukemiavirusincattleE.Casas,T.M.Taxis.RuminantDiseasesandImmunologyResearchUnit,USDA,ARS,NationalAnimalDiseaseCenter,Ames,IA,[email protected]:ImmunologicalResponses–2,Room6,12/5/201710:00AMBovine leukemia virus (BLV) causes abnormal immune function and immunosuppression, affecting cattle health and productivityworldwide.TransferRNAfragments(tRFs)areknowntobeinvolvedininhibitionofgeneexpressionandhavebeenassociatedwithstressandimmuneresponse,tumorgrowth,andviralinfection.TheobjectiveofthisstudywastoidentifytRFsassociatedwithantibodyresponsetoBLVinfemaleHolsteincattle.Serumfrom14animalswascollectedtoestablishIgGreactivitytoBLVbyELISA.Sevenanimalswereseropositive (positivegroup)and7wereseronegative (negativegroup) forBLVexposure.Leukocytes fromeachanimalwerecollectedandtRFswereextractedforsequencing.tRF5GlnCTG,tRF5GlnTTG,andtRF5HisGTG,weresignificantlydifferentbetweenseropositiveandseronegativegroups (P<0.0067). Inall cases thepositivegrouphada lowernumberofnormalizedsequences for tRF5swhencomparedtothenegativegroup.ResultsuggeststhattRF5scouldpotentiallybeusedasbiomarkerstoestablishexposureofcattletoBLV.

CRWAD2017 ABSTRACTS


152-EvaluationofsingleversuspairedcalfhousingonacquiredimmunityC.B.Kesterson1,P.D.Krawczel1,M.Caldwell2,G.M.Pighetti1.1AnimalScience,UniversityofTennessee,Knoxville,Knoxville,TN,USA,2UniveristyofTennesseeCollegeofVeterinaryMedicine,Knoxville,TN,[email protected]:BovineImmunology–2,Room6,12/5/201710:45AMDairycalvestypicallyhavebeenhousedseparatelyforthefirst6-8weeksof lifetominimizetheriskofdiseasetransmission.Withadvancesincalfmanagementthatlimitthisrisk,anopportunityexiststoevaluatetheinfluenceofgreatersocializationandcalf-contactonimmunity.Ourobjectivewastodeterminetheeffectofpairvsindividualhousingofcalvesonhumoralandcellmediatedimmunity.Calveswithsuccessfulpassivetransferofimmunoglobulinsfromcolostrumfeeding(STP≥5.5g/dL)wereblockedbysexandbirthdateandenrolledintopaired(n=28)orindividual(n=14)housingby5d(±1.4d)ofage.Calfpairingwasimplementedbycombiningtwoindividualpens;onecalfwasusedfordatacollectionandtheothertoestablishthetreatment.Milkreplacer(protein26:fat20)wasfedtwicedaily(3L/feeding),andgrainandwaterwereprovidedadlibitum.Humoralimmunitywasevaluatedbyinoculatingcalveswitha1mLinjectionofkeyholelimpethemocyanin(KLH;0.1mg),Quil-Aadjuvant(0.5mg)andpyrogen-freesalineat7d.Anotherinjectionatd21containedKLH(0.1mg),Quil-A(0.5mg),andheat-killedCandidaalbicans(CA,2x106cells)inpyrogen-freesaline.KLHantibodyconcentrations inseracollectedon0,7,and14dafter injectionswillbeanalyzedbyELISA.Cellmediated immunitywastestedbytriplicateintradermalinjectionsofCA(2x106cells)andsalineintheneckond28.Skinfoldthicknessmeasurementsviacaliperswereconducted0,6,24,48,72,and96hpost injections.Astimulation indexwascalculatedbymeanCAresponseoversaline injectionresponse.AMIXEDmodelwasusedinSAS9.4toevaluatetheeffectsofcalf,housingtreatments,time,injectionresponse,sex,andallinteractions.Thestimulationindexwasgreaterat24hacrossbothhousingtypesrelativeto0and6h(P<0.001),butwassimilarto48,72,and96hpost injection.Maximumskin thickness fromCA injectionswashigher inmales than females (P<0.005),despiteblockingcalvesbysex.ThissuggeststhatcellmediatedimmunitytoCAwassimilarregardlessoftreatmentinhealthy,well-managedcalves.AntibodyresponsestoKLHarecurrentlybeingevaluated.153-Evaluatingtheinteractionofstockingdensityandheatstressonchangesincell-mediatedandhumoralimmunityA.R.Lee1,P.D.Krawczel1,R.J.Grant2,G.M.Pighetti1.1AnimalScience,UniversityofTennesseeKnoxville,Knoxville,TN,USA,2WilliamH.MinerAgriculturalResearchInstitute,Chazy,NY,[email protected]:BovineImmunology–2,Room6,12/5/201711:00AMPhysiologicadaptationstosingleandmultiplestressorscaninfluencethedevelopmentofcellmediatedandhumoralimmunity.Theobjective of this studywas to evaluate delayed type hypersensitivity (DTH) reactions to heat killedCandida albicans (HKCA) andantibodygenerationtokeyholelimpethemocyanin(KLH)indairycowshousedunderfourtreatments:control(onebedandfeedingspacepercowwithheatabatement),asingleinducedstressor(7fewerbedsandfeedingspacesthancowsornoheatabatement),andmultipleinducedstressors(7fewerbedsandfeedingspacethancowsandnoheatabatement).OnemLintramuscularneckinjectionscontainingKLH(0.1mg),Quil-Aadjuvant(0.5mg),HKCA(strainCS5314,2*106cells)andsterilenon-pyrogenicsalinewereadministeredthedayafterstudyinitiation.Serawascollectedondays0(baseline),7,and14post-administrationforfutureevaluationofhumoralimmunitytoKLH.Delayedtypehypersensitivitywasevaluatedondays7and14post-intramuscular injections.Cows(n=64)wereinjectedwith0.1mLHKCA(day7:2*106cells;day14:4*106cells)andsterilenon-pyrogenicsalineintradermallyinsixdistinctspotsontheneck(3each).At0,24,48,72,and96hafterHKCAadministration,allsixinjectionsiteswereevaluatedusingcalipers.InjectionsiteswereevaluateduntilDTHresponsedecreased.AstimulationindexwascalculatedbymeanHKCAreactionsiteresponseoversalineinjectionreactionsiteresponse.TheMIXEDprocedureofSAS(SAS9.4,Cary,NC)wasusedtoevaluatetheeffectsofcow,treatment,dayofinjection(7or14),timeafterinjection(0,24,48,72,or96h),andall2wayinteractions.Althoughthestimulationindexwasequalamongalltreatments,DTHreactionsweregreaterat24h(1.15±0.02)thanat0h(1.02±0.02,P<0.01),andequalto48(1.08±0.02),72(1.04±0.08),and96(1.02±0.23)hpostHKCAadministration.Delayedtypehypersensitivityat24hindicatedlimitedcell-mediatedimmuneresponsetothisdoseofHKCAinallcows,regardlessoftreatment.Thissuggeststheadditionofsingleormultipleinducedstressorshaslimitedimpactoncell-mediatedimmunitytolowdosesofantigenamongdairycattle.

CRWAD2017 ABSTRACTS


154-EffectsofacutelyingandsleepdeprivationonmetabolismandimmunityofHolsteindairycowsJ.A.Kull1,P.Krawczel1,K.Proudfoot2,J.Bewley3,B.O'Hara3,K.Donohue3,G.Pighetti1.1UniversityofTennessee,Knoxville,TN,USA,2TheOhioStateUniversity,Columbus,OH,USA,3UniversityofKentucky,Lexington,KY,[email protected]:BovineImmunology–2,Room6,12/5/201711:15AMTheobjectiveofthestudywastodeterminetheeffectsofsleepandlyingdeprivationonmetabolismandimmunityofdairycows.Datawerecollectedfrom8multi-and4primiparouscows(DIM=199±44(mean±SD);dayspregnant=77±30).Eachcowwasexposedtotwo24hbaselineperiods(d-1)followedbytwo24htreatmentperiods(d0)usingacrossoverdesign:1)sleepdeprivationachievedbynoiseorphysical contactand2) lyingdeprivation imposedbyawoodengridplacedon thepen floor.A2dacclimationperiodoccurredbeforeeachbaselineperiod,witha12dwashoutperiodbetweentreatments.Baselineandtreatmentperiodswereimposedfrom 2100 to 2059 h. Cows were housed in individual boxstalls during the acclimation period, d -1 and d 0. NEFA and glucoseconcentrationsweremeasuredat0300,0900,1500,and2100hond-1and0.Functionalactivityofbloodleukocyteswasassessedat2100hond-1and0.Bloodsampleswereseparatedintotwoaliquots(5mLeach);onesamplewasstimulatedwithLPS(5mg/mL),andonewasnotstimulated(saline).Frombothsamples,theexpressionofTNF-α,IL-1βandIL-6mRNAgenerationwasmeasuredviaRT-qPCR.DatawereanalyzedusingamixedmodelinSASincludingfixedeffectsoftreatment(sleepandlyingdeprivation),day(d-1and0),samplingtimeandtheirinteractionwithsignificantmaineffectsseparatedusingaPDIFFstatement(P≤0.05).NEFAandglucosevariedbytimeofday(P≤0.03),butwerenotaffectedbytreatmentorday(P≥0.05).IL-1βandTNF-αwerehigherond0,comparedtod-1forbothtreatments(day:P=0.04andP=0.004,respectively).Whennotstimulated,therewasatendencyforlyingdeprivedcowstonaturallyproducemoreIL-1βond0,comparedtosleepdeprivedcows(day:P=0.24andtrt:P=0.08).IL-6concentrationdidnotdifferonanyday(P>0.05).Toconclude,wefoundnoeffectofdayortreatmentonNEFAorglucose,suggestingshiftsinenergybalancedidnotoccurwhencowsaresleeporlyingdeprivedforashortperiod.However,regardlessofstimulation,bothsleepandlyingdeprivationelicitedanimmuneresponse,andmayposehealthriskslongterm.Thus,energyandotherresourcesmaybeallocatedawayfromactivitiessuchasmilkproduction.155-ThepotentialroleofTh17-likeimmuneresponsesinJohne'sdiseasetestpositivecowsJ.L.DeKuiper,P.M.Coussens.DepartmentofAnimalScience,MichiganStateUniversity,EastLansing,MI,[email protected]:BovineImmunology–2,Room6,12/5/201711:30AMJohne’sdisease(JD)isachronicgastrointestinaldisorderofruminantscausedbyMycobacteriumaviumsubspeciesparatuberculosis(MAP).ControlofJDhasprovendifficultduetoalackofapprovedvaccines,poorsensitivityindiagnosticassays,andalackofknowledgeconcerning correlates of protection. Our research focuses on understanding immune responses to MAP, defining correlates ofprotection,andimprovingdiagnosticassays.LaterstagesofJDappeartocoincidewithaclassicalTh2-likeimmuneresponse.DefiningtheimportanceofaclassicalTh1-likeimmuneresponseinJDhasbeenmoredifficult.Thishasraisedthepossibilitythatnon-classicalresponses,suchasaTh17-likeresponse,mightbeofimportanceinMAPimmunity.Indeed,mRNAsencodingthecytokinesIL-23andIL-17aaresignificantlyelevatedininPBMCsfromMAPtestpositivecowsrelativetoPBMCsfromMAPtestnegativecowsfollowingstimulationwithMAPantigens.BothIL-23andIL-17aproductionhavebeenassociatedwithTh17-likeresponses.Th17cellsarealsodefinedbysurfaceexpressionofIL-23receptor(IL-23R).TodeterminetherelativeprevalenceofpotentialTh17cellsinPBMCsfromMAPtestpositiveandMAPtestnegativecows,PBMCswereisolatedandanalyzedbyimmunostainingandflowcytometry.SurfacestainingforT-celltype(CD4,CD8,TCR1(ΥδTcell))andeitherIL-23RorintracellularIL-17awasperformedafteran18-hourincubationwithorwithoutMAP.FreshPBMCsfromMAPtestpositivecows(n=6)containedasignificantlyhigherproportionofIL23RpositivecellsingatedCD4+cellsthaningatedCD4+inPBMCsfromMAPtestnegativecows(n=6)(p<0.05)whentreatedwithMAP.IntracellularstainingforIL-17aafterMAPantigenstimulationrevealedahigherproportionofIL-17apositivecellsinPBMCsfromMAPtestpositivecows(n=3)thaninPBMCsfromMAPtestnegativecows(n=3)(p<0.01)whentreatedwithMAP.ThisdatasuggeststhatTh17cellsmayindeedplayaroleinimmuneresponsestoMAPinfectionanddevelopmentorcontrolofJD.FutureworkwillfocusonspecificcelltypesproducingIL-17ainresponsetoMAPantigensandonthepotentialroleofTh17-likecellsinMAPinfectedtissues.

CRWAD2017 ABSTRACTS


156-Reducedantigen-specificandtotalIgMinplasmafromcattleinfectedwithbovineleukemiavirusM.C.Frie1,A.Greenlick2,C.Droscha3,P.M.Coussens2.1CellandMolecularBiologyProgram,MichiganStateUniversity,EastLansing,MI, USA, 2Department of Animal Science,Michigan StateUniversity, East Lansing,MI, USA, 3AntelBio, Northstar Select Sires, EastLansing,MI,[email protected]:BovineImmunology–2,Room6,12/5/201711:45AMBovineleukemiavirus(BLV)isadeltaretrovirus,similarinstructuretohumanTcellleukemiavirus,typeI.BLVspecificallytargetsBcellsoftheimmunesystem,buthasalsobeenfoundinothercelltypes,suchasmammaryepithelialcells.BLVisprevalentintheUSdairyindustrywithover83%ofherdsand40%ofallcowspositiveforinfection.AlthoughpersistentlymphocytosisiscommoninBLVinfectedcows,lessthan5%ofinfectedcowswilldevelopBLVassociatedleukemiaorlymphoma.NewresearchclearlydemonstratesthatBLVinfectedcowsproduce lessmilkandhaveshorter lifespansthanBLVnegativeherdmates,costingtheUSdairy industryover$500millionannually.BLVinfectionhassignificanteffectsonmanyaspectsofbovineimmunity.WehighlightourrecentworkdemonstratingthedeleteriouseffectsofBLVinfectiononimmuneresponsestoroutinevaccinationsandtonovelantigens,suchaskeyholelimpethemocyanin(KLH).WepresentdatademonstratingthatBLVinfectionreducesoverallantigenspecificIgMinbovineplasmapriortoandfollowinginoculation.ReducedIgMreactiveagainstspecificboostervaccinecomponentswasobservedonday0ofarecentstudyandcontinuedthroughouta60-dayperiod.Similarly,KLHantigen-specificIgMisinitiallylowerinplasmaofBLVinfectedcows,relativetouninfectedcows.Thiscontinuesthroughouttheprimaryresponse.KLH-specific IgMlevelsdoappeartorecoveraftersecondaryantigenexposure.Further,wedemonstrate thatBLV infectiongenerally reduces totalplasma IgM levels (natural IgMandantigen-specific).ReducedIgMlevelsobservedinplasmaofBLVinfectedcowswereconsistentwithreducedexpressionofIGJmRNAinsortedBcells.AstheJchainisessentialforassemblyofsecretedpentamericIgM,reducedIGJgeneexpressioncouldpartiallyaccountforBLVinducedreductionintotalplasmaIgM.157-Twotraffickingsignalingmotifsattheendofthespikeproteinofporcineepidemicdiarrheavirusarevirulencedeterminants

inpigsQ.Wang,Y.Hou,L.Saif.TheOhioStateUniversity,Wooster,OH,[email protected]:PathobiologyofEntericPathogens–1,Room7,12/4/20179:00AMPorcineepidemicdiarrheavirus(PEDV)causeshighmortalityinneonatalpigs,butviralgeneticfactorscontributingtopathogenicityarenotwellidentified.Aprematureterminated(ΔEVFEKVHVQ)spike(S)proteinwasidentifiedinthe120thandhigherpassagelevelsofthecellculture-attenuatedPEDVstrainPC22A.SimilarSproteinswerealsoreportedforseveralVerocell-attenuatedPEDVstrainsormildfieldvariants.Withoutaffectinganyknownvirusneutralizingepitopes,thesePEDVvariantslosepartialYXXFand/orKXHXXmotifs. These twomotifs are intracellular trafficking signals critical for retaining S proteins in PEDV assembly sites, the ER-Golgiintermediatecompartments.Toinvestigatewhetherthesemotifsarevirulencedeterminants,wegeneratedthreerecombinantviruseswiththesingleordoublemotif-deletionsbyintroducingstopcodonsoranaminoacidsubstitutionintotheinfectiouscloneofvirulentPC22Astrain(icPC22A):1)icPC22A-S2Δ10aa(ΔYEVFEKVHVQ);2)icPC22A-S2Δ5aa(ΔKVHVQ);and3)icPC22A-Y1378A(inactivatedmotifAEVF).Weorallyinoculated(100PFU/pig)5-day-oldgnotobioticpigs(n=4-5pergroup)witheachmutant,andvirulenticPC22A.Twopigs weremock inoculatedwith cell culturemedium.Within 52 hours post-inoculation (hpi), all PEDV-infected piglets developeddiarrhea.However,pigletsinoculatedwithicPC22A-S2Δ10aahadasignificantlylowerrate(50%)ofseverediarrhea,shedsignificantlylowertitersof infectiousvirus in feces,anddisplayedmilder intestinalvillousatrophyby52hpi thanpigs intheotherthreevirus-inoculatedgroups.InVerocells,icPC22A-S2Δ10aaandicPC22A-Y1378AreplicatedtosignificantlylowertitersbutformedsignificantlylargerplaquesthanicPC22AandicPC22A-S2Δ5aa.TheseresultssuggestthatthetwomotifsattheendoftheSproteinarevirulencedeterminantsofPEDVinpigs.ThelossofthemotifslikelyresultsinmoreSproteinsonthecellsurface,triggeringmorecell-to-cellmembranefusiontoformlargersyncytia,butfewer infectiousvirusparticlesassembled.Detailedbiological functionsofthesetwomotifsinPEDVreplicationareunderinvestigation.

CRWAD2017 ABSTRACTS


158-EvaluationofmouseenteroidsasainvitromodelforLawsoniaintracellularisinfectionT.Resende,C.Kupiec,F.Vannucci,M.aqui-Salces,C.Gebhart.UniversityofMinnesota,SaintPaul,MN,[email protected]:PathobiologyofEntericPathogens–1,Room7,12/4/20179:15AMThe investigationofhost-pathogen interactions iscomplexandusually reliesonanimalmodelsor invitrocultures.Duetovariousconcerns,theuseofanimalmodelshasdeclinedinfavorofinvitrocultures.Buttraditionalinvitrocellcultureslackthearchitecturaldetailsthatareseeninvivo.Organoids,tridimentionalinvitrocultures,havetheabilitytofunctionasthetissueoforigin,tobeself-renewed and self-organized. Hence, organoids represent a promising model to investigate host-pathogen interactions. Lawsoniaintracellularis,amicroaerophilic and obligate intracellular bacterium, is the causative agent of proliferative enteropathy (PE). ThepathogenesisofPEincludesproliferationofintestinalcellsofaffectedanimals,andthusisresponsibleforeconomiclossesinswineproductionworldwide.AlthoughtraditionalcellcultureshavebeenusedasinvitromodelsforL.intracellularisgrowthandinfection,theydonotreplicatethecellularproliferationthatisthehallmarkofPE.Smallintestinalorganoids,alsonamedenteroids,representapromisinginvitromodeltostudythepathogenesisofPE.TheobjectiveofthisstudywastoevaluatemouseenteroidsasamodelforL. intracellularis infection.Mouse enteroidswere acclimated to the L. intracellularis culture conditions, and then infectedwith L.intracellularis. Changes in morphology were monitored daily for 8 days and immunohistochemistry was used to evaluate L.intracellularis infectionat5dayspost-infection. Infectedenteroidsdevelopedsimilarly tonon-infectedcontrolsover the8daysofinfection.ImmunocytochemistryrevealedpositivestainingforL.intracellularisinthecytoplasmofinfectedcells,thoughthequantityandlocationoftheintracellularbacteriadifferedsomewhatfromthoseobservedinvivo.ThesepreliminaryresultsdemonstratethatLawsonia-infectedenteroidsmaybeasuitableexperimentalmodelforstudyingPEpathogenesis.159-HostinflammatoryresponsetooralinoculationwithenterohemorrhagicEscherichiacoliinadefinedmicrobiotamousemodelZ.R.Stromberg1,M.J.Wannemuehler2,M.Mellata1.1DepartmentofFoodScienceandHumanNutrition,IowaStateUniversity,Ames,IA,USA,2DepartmentofVeterinaryMicrobiologyandPreventiveMedicine,IowaStateUniversity,Ames,IA,[email protected]:PathobiologyofEntericPathogens–1,Room7,12/4/20179:30AMEnterohemorrhagicEscherichiacoli(EHEC)colonizesthegastrointestinal(GI)tractandcausesbloodydiarrheainhumans.ToovercomeE.colicolonizationresistance,conventionally-reared(CONV-R)mousemodelsareeithertreatedwithantibioticsorfedabnormaldiets;alternatively,germ-freemiceareused.Previously,wereportedanalternativemodelusingdefinedmicrobiotamiceconsistingofthealtered Schaedler flora (ASF) that resulted in significantly (P < 0.05) higher levels of EHEC in the feces andGI tract contents, andincreasedGIinflammationasrevealedbyProSense680imaginginEHEC-infectedASFcomparedwithCONV-Rmice.Theobjectiveofthis studywas to determinehost inflammatory genesmodulated in response to EHEC infection inASFmice. Colon sectionswerecollectedfromuninfectedorEHEC-infectedbygavageC3H/HeNmiceofbothsexesharboringtheASF.TotalRNAfromcolonsections(n=3/group)wasisolatedandsubmittedfortargetedgeneexpressionprofilingusingamouseinflammatorypanel(Qiagen)withathresholdPvalue<0.05andfoldchange(FC)cutoffs<0.5and>1.5.Atday28post-infection,EHEChadamodesteffectongeneexpressioninthecolonofASFmicebydifferentiallymodulatingfourinflammatorygenes(Inhba,Ido1,CD36,andAdipoq).InhibinbetaA (Inhba) is a subunit of activin which activates signaling pathways mediating inflammation and immunity and was significantlyincreased in EHEC-infected colons (FC = 2.4). Interestingly, a tryptophan catabolic enzyme (Ido1) that has antimicrobial effects,immunoregulationfunctions,andisabiomarkerforinflammatoryboweldiseasewassignificantlyupregulated(FC=1.9).Inaddition,CD36 which is a scavenger receptor involved in phagocytosis, toll-like receptor signaling, and lipid metabolism was significantlydownregulated(FC=0.3),andAdipoqalsoinvolvedinlipidmetabolismwassignificantlydownregulated(FC=0.1).Thesechangesingeneexpressioninvolvedintryptophancatabolismandlipidmetabolismhavebeenlinkedtoobesityandothermetabolicdisorders.TheseresultsindicatethatEHECcanmodulatehostinflammatoryresponsesandmetabolicprocessesinASFmice.

CRWAD2017 ABSTRACTS


160-MappingimmunodominantandneutralizingepitopesofenterotoxigenicEscherichiacoli(ETEC)F18adhesinsubunitFedFT.Lu,W.Zhang.DiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,[email protected]:PathobiologyofEntericPathogens–1,Room7,12/4/20179:45AMPurpose:Porcinepost-weaningdiarrhea(PWD)remainsoneofthemostimportantswinediseasesglobally.EnterotoxigenicEscherichiacoli (ETEC)bacteriaexpressingK88orF18fimbriaarethepredominantcausesofPWD.However,unlikeK88fimbria,F18fimbria isunabletoinduceneutralizingantibodiesagainstF18-fimbrialETECdiarrhea.RecentstudiesindicatedthatapeptidefromF18minoradhesinsubunit(FedF)inducedantibodiesinhibitingF18fimbriaeadherence.WehypothesizethatimmunodominantandneutralizingepitopesfromFedFcanbeusedforthedevelopmentofabroadlyprotectivevaccineagainstPWD.Methods:We insilico identifiedepitopesfromF18minorsubunitFedF,geneticallyfusedeachepitopetoacarrierprotein,andscreenedthemwithanti-F18antiserumforimmunodominantepitopes.Furthermore,weimmunizedmicewitheachepitopefusion,examinedmouseserumsamplesforanti-F18IgGantibodyresponse,andthenmeasuredantibodyadherenceinhibitionactivityagainstF18fimbrialETECwildtypestrain8516andporcinecell line IPEC-J1.Results:Datashowedthatatotalof7epitopeswere identified.Amongtheseepitopes, INSSASSAQV,IPSSSGTLTCQAGT,AQTYPLSSGD,PNQNDMPSSNandQPDATGSWYDshowedstrongerreactivitywithanti-F18antisera.Datafrommouseimmunizationshowedthatepitopefusionsdevelopedvarious levelsofanti-F18antibodyresponses.Antibodyadherence inhibitionassayexhibitedmouseserumantibodieswithdifferentneutralizationactivity.EpitopeLGTGKTNTTQMinducedalowtiterofantibodieswith weak neutralizing activity, whereas epitope NESQWGQQSQ induced a low titer of anti-F18 IgG antibodies but with greatneutralizingactivityagainstF18+ETECstrain8516.Incontrast,antibodiesderivedfromepitopeIPSSSGTLTCQAGTandQPDATGSWYDhad greater anti-F18 IgG titers and also greater neutralization activity against F18 fimbrial adherence. Conclusions: These resultssuggestedIPSSSGTLTCQAGTandQPDATGSWYDepitopesaretheF18representativeantigensforPWDvaccines.161-VariabilityinporcineRVBandRVCVP7proteinsillustratespotentiallyimportantimmunologicalsitesF.Shepherd1,F.Chen1,T.Knutson1,M.Murtaugh1,D.Marthaler2.1UniversityofMinnesota,St.Paul,MN,USA,2KansasStateUniversity,Manhattan,KS,[email protected]:PathobiologyofEntericPathogens–1,Room7,12/4/201710:00AMRotavirusBandC(RVBandRVC)areprominentgastrointestinalpathogensinswinepopulationsthroughouttheworld.SinceRVBandRVCaredifficulttoadapttocellculture,subunitvaccinesmayprovideasolutiontoprotectpigletsfromtheseinfections.Theobjectiveofthisstudywasto identifyvariableregionsoftheRVBandRVCVP7proteinstodetermine immunologicalsites insilico todesignsubunitvaccines.NucleotidesequencesofRVB(n=174)andRVC(n=369)oftheVP7fromswinesamplesoriginatingfromtheUnitedStatesandCanada in2009-2017wereassignedgenotypesbyBLAST (NCBI).MUSCLEalignmentsof theVP7 forRVBandRVCwerecreated,andvariabilitywasdeterminedbycalculatingthepercentofaminoacidsdifferingfromtheconsensusaminoacidateachresidue location. Variable regionswere visualized using Circlize in R. To identify functionally important variable regions, antigenicepitopeswerepredictedusingEPCESanddN/dSanalysiswasperformedusingFEL,FUBAR,SLAC,andMEMEavailableinDataMonkey.TheRVBstrainsbelongedto11Ggenotypes,whiletherewere5GgenotypesidentifiedinRVC.Therewere43and7residuesinRVBandRVC,respectively,thatwereconsideredhighlyvariable,asdefinedbymorethan30%ofstrainsdifferingfromtheconsensusresidueand thepresenceof3ormoreaminoacid functional groups.Overall, 4 codons inRVBwere consideredhighly variable,positivelyselected,andhadhighpredictedantigenicity.InRVC,onlyoneofthe7hypervariableregionsalsoshowedpositiveselection,butnonewerepredictedepitopes.Interestingly,dN/dSanalysishighlightedadditionalresiduesundergoingepisodicpositiveselectionwithinRVBandRVC,indicatedpotentialmarkeddifferencesbetweengenotypesorlineagesofVP7.ThelowerpresenceofvariablecodonsinRVCmayalsosuggestthatRVCislessdiverseoverallthanRVBwithrespecttoaminoacidfunctionaldiversity.Inconclusion,weidentifiedsiteswithinRVBandRVCVP7thatmayplayanimportantroleinimmuneescapeandshouldbeconsideredwhendesigningsubunitvaccinestrains.ThisworkalsoprovidesframeworkforsimilaranalysesusingVP4,theotherrotavirusoutercapsidprotein.

CRWAD2017 ABSTRACTS


162-CompetitiveexclusionofCampylobacterjejunibylinoleicacidoverproducingLactobacilluscaseiZ.Tabashsum,M.Peng,S.Salaheen,C.Comis,D.Biswas.DepartmentofAnimalandAvianSciences,UniversityofMaryland,CollegePark,MD,[email protected]:PathobiologyofEntericPathogens–2,Room7,12/4/201710:45AMPurpose:Campylobacterjejuni(CJ)isazoonoticpathogenandcauses>14%offoodborneinfectionsinUSyearly.Probiotics,prebioticsand/orcombinationofbothknownassynbiotic,haveemergedaspromisingalternativeapproachto treat infectionparticularlybyantibioticresistantentericpathogens.Lactobacilluscasei(LC)isconsideredasprobioticwhichproducesdifferentbioactivecompoundsandeffectuallyworksagainstpathogens. Inpresenceofprebioticpeanut flour (0.5%), LCproduces increasedamountofbioactivecompoundsandlinoleicacidisoneofthemosteffectivemetaboliteswhichcontrolthegrowthofdifferentpathogens.Inourlab,wehavegeneticallymodifiedLCbyoverexpressingmcra(linoleicacidproducing)geneandnamedLC-CLAandevaluatedtheeffectofLC-CLAonCJ-hostcellinteraction.Methods:CJwasco-culturedwithLC(with/withoutpeanutflour),LC-CLAandalsogrowninpresenceoftheircellfreeculturesupernatants(CFCSs)inDulbecco'sModifiedEagle'smediumforgrowthinhibitionassay.CelladhesionandinvasionabilityofCJwasassayedfortheabovementionedconditionsinchickenmacrophage(HD-11)andmammalian(HeLa)cellline.Physiologicalproperties related tovirulencewereexamined invitroandalterationofvirulentgeneexpressionswereevaluatedbyqPCR.Results:LC-CLAandLCwithpeanutflourreducedgrowthofCJwithin24handexcludedcompletelyby48hcomparedtoLCreducedgrowth(>2logCFU/mLat72h)butcouldnotexclude.CFCSsofLC-CLAreducedgrowthofCJmostefficiently(>2logCFU/mLat48hand>4logCFU/mLat72h),followedbyCFCSsofLCwithorwithoutpeanut.LC-CLAco-cultureandCFCSsconditionsreducedadhesion and invasion efficiency of CJ numerically if not statistically significantly compared to LC alone. Injured cell ratio,hydrophobicity,auto-aggregationandvirulentgeneexpressionwerealsoalteredinpresenceofCFCSsofLC-CLA.Conclusions:Thesefindingssuggestthatinsteadofusingexpensivepeanutoranyotherprebioticlikefeedadditivesandavoidtheirnegativeimpact,LC-CLAcanbeanalternativeinreducingCJsurvival,colonizationandalteringitsvirulencepropertiesinpoultry.163-MolecularepidemiologyofCampylobacterupsaliensisisolatedfromdogsinGrenadaM.Nisar1,V.A.Amadi2,H.Hariharan2,R.Sharma2,K.V.Nagaraja1.1DepartmentofVeterinaryandBiomedicalSciences,CollegeofVeterinaryMedicine,UniversityofMinnesota,MN,USA,2DepartmentofPathobiology,SchoolofVeterinaryMedicine,St.George’s,Grenada,[email protected]:PathobiologyofEntericPathogens–2,Room7,12/4/201711:00AMPurpose: The purpose of this study was to characterize Campylobacter jejuni and Campylobacter upsaliensis isolates from dogs.Materials andMethods:DNA samples from Sixty-seven suspected Campylobacter isolates were included for analysis by PCR forconfirmationandspeciationofCampylobacter.Inthis58CampylobacterstrainsthatincludedthreeisolatesofC.jejuniandfiftyfiveisolatesofC.upsaliensiswereanalyzedbyMulti-locusSequenceTyping(MLST)byamplifyingsevenhousekeepinggenestodeterminegeneticdiversityamongtheseisolatesinCampylobacterspecies.Results:Outofthese67samples,3wereconfirmedasC.jejuni,55asC.upsaliensisand4asamixedinfectionofC.jejuniandC.upsaliensisbymultiplexPCR.Among3strainsofC.jejuniand55strainsofC.upsaliensis,17differentsequencetypes(STs)wereidentified.AllthethreestrainsofC.jejuniwereidentifiedasST-2304while49C.upsaliensisstrainswereidentifiedasnewSTs.ThepredominantsequencetypeinC.upsaliensiswasST-213(n=7)followedbyST-204andST-207(n=6),ST-206(n=5),ST-15(n=4)andST-214,ST-208,ST-210,ST-218(n=3).TwooftheisolatesweretypedasST-216andST-217whileoneisolateasST-205-,ST-209,ST-211,ST-212andST-219.Conclusions:CampylobacterspeciesarecirculatingindogsandthemostcommonsequencetypeST-2034forC.jejuniandST-213,ST-204,ST-207,ST-206forC.upsaliensis.Thepresenceoftheseagentmayposerisktopublichealth.

CRWAD2017 ABSTRACTS


164-CharacterizationofdiversenovelporcineastrovirusesinEastAfricansmallholderpigletsJ.O.Amimo1,J.N.Njuguna2,R.Pelle2,E.M.Machuka2,E.Okoth3.1AnimalScience,UniversityofNairobi,Nairobi,Kenya,2BiosciecesofEasternandCentralAfrica(BecA),InternationalLivestockResearchInstitute(ILRI),Nairobi,Kenya,3Biosciences,InternationalLivestockResearchInstitute(ILRI),Nairobi,[email protected]:PathobiologyofEntericPathogens–2,Room7,12/4/201711:15AMAstroviruses(AstV)iswidelydistributedandisassociatedwithgastroenteritisinhumanandanimals.Itsprevalenceamongpigswithorwithoutdiarrheaisreportedtobehigh;however,ourknowledgeofthediversityandepidemiologyofAstVinEastAfricaislimited.ThecurrentstudywasconductedtogeneticallycharacterizeastrovirusesinasymptomaticsmallholderpigletsinwesternKenyaandeasternUgandausingviralmetagenomicsapproach.Twentyfour(24)sampleswererandomlyselectedfromatotalof446pigletsagedbelow6 months that was initially collected to study rotaviruses distribution and diversity in the same region. Sequence-independentamplificationandhighthroughputsequencingwereappliedtothemetagenomicsanalysisofvirusesinsampleselected.Thirteen(13)out of the 24 samples analyzed had contigs with high identity to mamastroviruses. Phylogenetic analysis of the detectedmamastrovirusesrevealedgeneticheterogeneitywithfourdistinctgeneticlineagesofporcineastrovirus(PoAstV)detected(PoAstV2,PoAstV3PoAstV4andPoAstV5).NinefecalsampleswerehavingcontigsthatwerenotassignedtoanygeneticlineageofknownAstVintheGenBank. In-depthcharacterizationof5strainswithcomplete(ornearly full)genomerevealeddiversenucleotidesequenceidentities (49-96%)with knownPoAstV strains, indicating novel types or genotypes of PoAstV. This study concluded that geneticdiversityamongPoAstVstrainsreportedheremaypresentsachallengefordiseaseprevention,developmentofaccuratediagnostictools and even vaccine development. These findings provide new insights into the molecular epidemiology and prevalence ofastroviruses inEastAfricanSwinepopulation.Furtherresearchand investigation intothepathogenesisofAstVwouldbenefitbothveterinaryandhumanmedicine165-TheroleoftargetmembranesialicacidresiduesinbindingtheClostridiumperfringensNetFtoxintohostcellsI.MehdizadehGohari1,M.Palmer2,P.Boerlin1,J.F.Prescott1.1Pathobiology,UniversityofGuelph,Guelph,ON,Canada,2Chemistry,UniversityofWaterloo,Waterloo,ON,[email protected]:PathobiologyofEntericPathogens–2,Room7,12/4/201711:30AMClostridiumperfringenstypeA-associatedentericdiseaseinfoalsanddogsisnotwellcharacterizedbutourgrouphasidentifiedatoxinrelatedtotheLeukocidin/Hemolysinsuperfamilyassociatedwithdiseaseinthesespecies,andhavedesignateditNetF.NetFhasbeenimplicatedastheprimaryvirulencefactoroffoalandcaninenecrotizingenteritis.AminoacidsequenceofNetFpreviouslysuggestedthatthistoxinbelongstothebeta-poreformingtoxinfamily.Anosmoticprotectionassayusingpolyethyleneglycol(PEG)ofdifferentmolecularsizessuggeststhatNetFformsporesinthecellmembranewithafunctionaldiameterofapproximately4-5nm.ElectronmicroscopicobservationofrNetF-RBCconfirmsthatNetFtoxinisabletooligomerizeonthelipidbilayerofcellmembranesandformpores,asdoothermembersofthissuperfamily.However,themodeofactionofNetFisnotfullyunderstood.IdentifyingthecellularreceptorsinvolvedinNetF-hostcellinteractionisanimportantfirststepinunderstandingitsmodeofaction.Previously,EquineOvarian(EO)cellswereshowntobethecellsmostsusceptibletoNetF..Todeterminethechemicalnatureofthehostcellsurfacereceptor(s)for NetF, a series of preliminary experiments were conducted. EO cells treated with sodium periodate to remove cell surfacecarbohydrateswererenderednon-susceptibletoNetF,suggestingthatcarbohydratesareimportantintheNetF-hostcellinteraction.TofurtherelucidatethecarbohydratecompositionofNetFreceptors, theEOcellswereexposedtodifferentcarbohydratebindinglectins.Sialicacidbindinglectin(Triticumvulgaris)wasfoundtoinhibitthecytotoxicityofNetF.Sialidasetreatment,andneutralizationofNetFcytotoxicitywithsialicacid,furtherindicatedthatsialicacidplaysacriticalroleinbindingofNetFtoEOcellsurface.Furtherworkisinprogresstoidentifythehostcellsurfacereceptor(s)ofNetF.

CRWAD2017 ABSTRACTS


166-DevelopmentandvalidationofmultiplexPCRassaystoidentifyserogroupsofnontop-7Shigatoxin-producingEscherichiacolianddeterminetheirprevalenceincattlefeces

K.M.Capps1,J.Ludwig1,X.Shi1,J.Bai2,R.Phebus3,T.Nagaraja1.1DiagnosticMedicine/Pathobiology,KansasStateUniversityCollegeofVeterinaryMedicine,Manhattan, KS, USA, 2Veterinary Diagnostic Laboratory, Kansas StateUniversity,Manhattan, KS, USA, 3FoodScienceInstitute,KansasStateUniversity,Manhattan,KS,[email protected]:PathobiologyofEntericPathogens–2,Room7,12/4/201711:45AMShigatoxin-producingEscherichiacoli(STEC)arepathogensthatinducearangeofhostresponsesfrommilddiarrheatohemorrhagiccolitisandhemolyticuremicsyndrome.SevenserogroupsthatincludeO26,O45,O103,O111,O121,O145andO157,oftencalled‘top-7’,causemosthumanSTECinfections.Cattleareamajorreservoirofthetop-7STEC.Theorganismsresideinthehindgutandareshedinthefeces,whichisamajorsourceoffoodandwatercontaminations.Cattleharborasmanyas113additionalSTECserogroups,andsomehavebeenassociatedwithhuman infections.Conventionally, serogroupingofE. coli is donebyagglutination reactionswithserogroup-specificantisera.ThefirstobjectivewastodevelopandvalidateelevenmultiplexPCR(mPCR)assaystargetingserogroup-specificgenestobeusedasasettodetect113serogroupsofSTEC.ThesecondobjectivewastodevelopandvalidateamPCRassaytodetectthemajor‘nontop-7’serogroups,anddeterminetheirprevalenceincommercialfeedlotcattlefeces.ElevenmPCRassaysweredevelopedtargetingserogroup-specificgenes(wzx,wzy,gnd,andwbdA)andvalidatedtodetect113serogroupsof‘non-top-7’STEC.Atotalof359strainsofSTECisolatedfromseveralfeedlots,whichwerePCR-negativeforthetop-7STEC,weresubjectedtotheelevenmPCRassays.SerogroupsO168(29.8%),O109(17.5%),O131(8.1%),O2(7.0%),O171(4.2%),andO74(3.6%)werethedominantSTECincattlefeces.A6-plexPCRassaytargetingthewzxgenewasdevelopedtodetectthesixmajornontop-7STEC(O2,O74,O109,O131,O168,andO171).Theassaywasvalidatedbyspikingcattlefecesnegativeforthemajornontop-7STECwithpurecultures.Usingthisassay,atotalof911pen-floorfecalsamplesoriginatingfromthreecommercialfeedlotsintheMidwestwereanalyzedtodeterminetheirprevalence.TheprevalenceofthesixserogroupsincattlefeceswasO109(88.9%),O171(82.0%),O168(70.6%),O2(51.5%),O74(18.6%), and O131 (2.0%). The PCR-based assay is a relatively simple method to identify serogroups compared to conventionalserogrouping.Identifyingserogroupsotherthanthetop-7aidsindeterminingtheirprevalenceandpotentialriskforhumanillness.167-Enteropathogenichumanandanimalcaliciviruses:pathogenesisandhostco-factorinteractionsL.J.Saif.OhioStateUniversity,Wooster,OH,[email protected]:PathobiologyofEntericPathogensKeynote/VectorborneDisease,Room7,12/4/20172:00PMCaliciviruses causevariousdiseases in their respectivehosts,butonlyenteropathogenic calicivirus infections [norovirus (NoV)andsapovirus(SaV)]arereportedinhumans.GapsremaininourunderstandingofthepathogenesisofNoVandSaVinfectionsinhumansandanimals, includingtargetcells infectedanddiarrheamechanisms.Thisreviewwill focusonNoVandSaV infections,bothhost-specificandhumanNoV(HNoV)strains,indomesticanimalsandhumans.Ingnotobiotic(Gn)pigsinfectedwithporcineSaVandGnandconventionalcalvesinfectedwithbovineNoV,virusesreplicatedprimarilyinsmallintestinalepithelialcells(IEC),withantigenalsoinunidentifiedcellsinthegutlaminapropria,andwithIEClesions,diarrheaandfecalvirusshedding.InhumansinfectedwithNoVs,IEClesions,barrierdysfunctionandmalabsorptionoccurred,butantigenwasdetectedinfrequently,eveninthelaminapropria.Likecalves,nodefinitiveevidenceofvirusreplicationversusantigenuptakeintoAPCswaspresented.SimilarpatternsofintestinalinfectionhavebeenrecapitulatedinHNoVorallyinoculatedGnpigsandcalves,butnotinIVinoculatedchimpswhereantigenwasonlyinAPCs,includingBcells.Howeverforacuteentericinfectionsthatresolvequickly,earlyassessmentofintestinallesionsandantigeninIECs(shedrapidlyfromvilli)iscritical.PrioradaptationofporcineSaVtocellculturein1988,therecentlyreportedreplicationofHNoVinBcells and the groundbreaking adaptation ofmultiple strains of HNoVs to human enteroids have identified essential or enhancingcofactorsforentericcalicivirusreplicationinvitro.Theyinclude:1)HBGAs,2)bileacids,3)gutcommensals,and4)cholesterolinhibitors(invivo). Investigationofhowcofactors,age, immunestatusandvirusstrain influenceNoVandSaV infections(cell types infected,diarrheamechanisms,etc) inthehostspeciesandinrelevantanimaldiseasemodels, iscriticalfor improvedunderstandingofviralpathogenesis,controlstrategiesandoptimizedcellculturesystems.

CRWAD2017 ABSTRACTS


169-Ascopingreviewofimportationandpredictivemodelsforvector-bornedisease,pathogensand/orvectorsthatexistgloballyT.Sadeghieh1,V.Ng2,L.A.Waddell2,A.Hall1,J.Sargeant1.1UniversityofGuelph,Guelph,ON,Canada,2PublicHealthAgencyofCanada,Guelph,ON,[email protected]:PathobiologyofEntericPathogensKeynote/VectorborneDisease,Room7,12/4/20173:00PMPurpose:Weconductedascopingreviewtocompileandcharacterizedocumentsdescribingpredictiveand importationmodelsofvector-bornediseases,pathogens,reservoirsand/orvectorsthatexistglobally.OursecondaryobjectivewastosummarizemodelswithafocusonCanadaand/orthenorthernUnitedStates,aswellasthosewhichincorporatedtheimpactofclimatechange.Methods:Aliteraturesearchwasconductedtoidentifypublicationspublishedbetween1999and2016throughasearchoffivescientificdatabasesusingrelevantkeywords.Relevancescreeninganddatacharacterizationwereperformedbytworeviewersusingpretestedforms.Thedatawerecleaned,andanalyzedusingdescriptivestatistics.Results:Thesearchinitiallyidentified19710uniquearticles,reports,andconferenceabstracts.Thiswasreducedto513relevantdocumentsaftertherelevancescreening.Preliminaryresultsof288of513articlesshowthatthereweremainlypredictivemodels(86%),ratherthanimportationmodel(2%),orthosewhichincorporatedboth(12%).Themajorityofmodelswerestatistical(64%),ratherthanmathematical(36%).Twentytwopercent(22%)focusedonNorthAmericaasalocation,with95%ofthosearticlesfocusingontheUnitedStatesand27%onCanada.Only30%ofthemodelsincorporatedtheimpactsofclimatechange.Conclusions:Thespreadandintroductionofvector-bornediseasesintoCanadaandtheUnitedStatesisexpectedtooccurunderclimatechange.Modelscanbeuseful inpredictingwhenandwherefuturedistributionofvector-bornediseases thataffectanimalsandhumansmayoccur.This reviewwill informresearcherswheregapsmayexist in the literatureonpredictiveandimportationmodelsofvector-bornediseases.Additionally,thisworkwillprovideaframeworkforcreatingthesemodelsspecifictoCanadaandtheUnitedStates.170-Genegun-deliveredDNAimmunizationofcattleinduceshumoralandcell-mediatedimmuneresponsesagainsttheTheileria

parvapolymorphicimmunodominantmoleculeL.M.Fry1,B.C.Stone2,R.G.Bastos3,D.P.Knowles, Jr.1,S.C.Murphy2.1AnimalDiseaseResearchUnit,USDA-ARS,Pullman,WA,USA,2CenterforEmergingandRe-emergingInfectiousDiseases,UniversityofWashington,Seattle,WA,USA,3VeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA,[email protected]:VectorborneandParasiticDisease,Room7,12/3/20176:30PMEastCoastFever(ECF),causedbythetick-borneapicomplexanparasite,Theileriaparva,killsoveramillioncattleeachyear insub-SaharanAfrica.Protectiveimmunityiscomprisedofhumoralresponsestosporozoitesandcell-mediatedresponsestoparasite-infectedlymphocytes. Significant parasite genetic complexity and strain variation, pronounced immunodominance of the cellular immuneresponse,anddiversityofbovineMHClocihaveprecludeddevelopmentofatraditionalT.parvasubunitvaccinewithpopulation-wideefficacy.Onepotential solution ismulti-antigen immunizationbyparticle-mediatedepidermaldelivery (PMED,alsoknownasgenegun),anapproachintendedtoachievesimultaneousintradermalinoculationoflargenumbersofDNA-encodedantigens.Thismethodhasshownpromiseinviralvaccinestudiesinmice,primates,pigs,andhumans,andinmalariavaccinesininmice,buthasneverbeenappliedtocattle.Inthisstudy,weutilizedtheT.parvapolymorphicimmunodominantmolecule(PIM)antigentooptimizeandtestPMED DNA immunization in eight Holstein steers. Following a series of immunizations, 7/8 steers developed significant anti-PIMantibody responses and cell-mediated immune responses to T. parva-infected cells. These results demonstrate that PMED DNAimmunizationisapromisingvaccineplatformforT.parvaandothercomplexpathogensincattle.

CRWAD2017 ABSTRACTS


171-AgeneticsystemforcreatingtargetedmutationstodisruptandrestoregenesinEhrlichiachaffeensisthatisbroadlyapplicabletootherobligatebacteria

Y.Wang,L.Wei,H.Liu,C.Cheng,R.R.Ganta.DepartmentofDiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,[email protected]:VectorborneandParasiticDisease,Room7,12/4/20174:00PMObligateintracellularbacteria(obligates)belongingtotheorderRickettsialesandChlamydialesareresponsibleforcausingdiseasesinhundreds of millions of people worldwide. Lack of an efficient system for targeted mutagenesis in obligates remains a majorimpedimentinunderstandingmicrobialpathogenesisandindefiningthefunctionalsignificanceofmanygenes.Challengesincreatingtargetedmutationsmaybeattributedtotheessentialnatureofageneselectedformutagenesis,intracellularreplicationdependenceandthelackofmethodstosupportextracellulargrowth.Despitethesuccessingeneratingmanyrandommutationsusingtransposonmutagenesis,andhavingalimitedsuccessofcreatingtargetedmutationsinrickettsialandchlamydialpathogens,presentlyamethodthatworkswellincreatingtargetedmutationsinspecificgenesofinterestfollowedbycomplementationremainsproblematicfortheobligatepathogensandisalsoahighlysought-aftergoal.WehavefilledthismajormethodologicaldeficiencybydevelopingprotocolstogeneratestabletargetedmutationsbyallelicexchangemethodinEhrlichiachaffeensis,anobligateintracellulartick-bornebacteriumresponsibleforthedisease,humanmonocyticehrlichiosis.TargetedmutationsinE.chaffeensiswerecreatedtonotonlytodisrupttwogenes,butalsotorestoretheintactgenebyanotherallelicexchangemutation,whichresultedintherestoredtranscriptionfromtheinactivatedgenefromitsownpromoter.Weexpectthatthemethodsdevelopedarebroadlyapplicabletootherobligateintracellularbacteriatoroutinelyperformtargetedmutationstoenablestudiesfocusedonstructure-functionanalysesofbacterialproteins,host-pathogeninteractionsandindevelopingvaccines.172-Experimentaltransfusion-inducedBabesiacanisinfection:pathogenicityanddynamicsofparasitemiainaBeagledogmodelC.Wang1,J.Wang2,J.Zhang2.1AuburnUniversity,Auburn,AL,USA,2YangzhouUniversity,Yangzhou,[email protected]:VectorborneandParasiticDisease,Room7,12/4/20174:15PMPurpose:Caninebabesiosisisasignificanttick-bornediseasecausedbyvariousspeciesoftheprotozoangenusBabesia.Althoughitoccursworldwide,thepathogenicityofBabesiaspp.hasnotbeenexploredinacaninemodel.Methods:.ThisstudyaimstoinvestigatethepathogenicityandkineticsofparasitemiaofBabesiacanis inaBeagledogmodelusingbloodsmear,CBC,biochemicalprofile,quantitativePCRandhistology.AtotalofsixbeagledogsweretransfusedwithbloodfromB.canis-infecteddogswhileanothersixdogsservedasnegativecontrol.Spleenwas removed inhalfof these infectedandcontroldogs, respectively.Results:All infecteddogsshowedfever,anemia,depression,anorexia,weightloss,aswellasreducedWBC,RBCandplatelet.DogswhosespleenwereremoveddemonstratedsignificantlylowerWBC,RBCandplateletthantheinfecteddogswithspleenandcontroldogs,andtwoofthreedogswithoutspleendiedthreeweeksfollowingB.canisinfection.B.canisdisappearedinthewholebloodofoneinfecteddogonday-102followinginfection,andotherinfecteddogswereconsistentlypositiveoverthis3-monthstudy.TheB.caniscopynumbervariedgreatlyfrom1.2x107to9permlwholeblood.Histologicalexaminationindicatedneutrophilinfiltrationinlungandencephalitisintheinfecteddogs.Conclusions:ThisisthefirstB.canisinfectionmodelindog,andthisexperimentaltransfusion-inducedmodelindicatedthatB.canisishighlypathogenicfordogsandthedogswithoutspleenaremoresusceptibletotheB.canisinfection.

CRWAD2017 ABSTRACTS


173-Evaluationofimmuno-andcopro-diagnostictechniquesagainstFasciolahepaticainfectioninsmallruminants,PakistanK.Afshan.AnimalSciences,Quaid-i-AzamUniversityIslamabad,Islamabad,[email protected]:VectorborneandParasiticDisease,Room7,12/4/20174:30PMPurpose:Parasitismisamajorconstrainttolivestockproductionallovertheworld.Amonghelminths,fasciolosiscauseshugeeconomiclossesinlivestockindustryintermofmorbidity,mortalityandcostoftreatments.Theobjectiveofpresentstudywastodeterminetheprevalence of Fasciola hepatica infection in small ruminants by using indirect ELISA and sedimentation techniques to make acomparisonbetweenbothtechniquesforrapiddiagnosis.ToimprovethediagnosticefficacywefirsttimeevaluatedthecommerciallyavailablebovineF.hepaticaELISAkitforthedetectionofFasciolaIgGinsmallruminants.Methods:TheFasciolaIgGantibodiestestandfecaleggcountwasperformedon1200serumandfecalsamples.TheassociationofFasciolainfectionwithage,sexandbreedofanimalsweredetermined. To checkdiagnostic efficacy 54 animal serawithother parasitic infections, 58healthy controls, and92positivecontrolserewereanalysed.Results:Theresultofcurrentstudyindicateddiagnosticaccuracyoftest95.6%,whilesensitivityand specificity of this assay for small ruminantswas 97.83% and 93.75% respectively. Sedimentation techniquewas used as goldstandard.Theresultsrecordedhigherinfectionrateforsheep39.2%withindirectELISA,while4.08%ingoats.Theinfectionrateingoatswas 5.01%with sedimentation technique and 28.43% in sheep. The result showed significant (p<0.05) association betweenFasciola infectionandbreedsofanimals,whilenosignificant(p>0.05)associationforsexandagegroupsofgoats.Theresultsfromcomparison of both techniques showed that 5.5% of the animal positive for Fasciola IgG indirect ELISA test had no egg count.Conclusions: The study concluded that immunodiagnostic tests are more sensitive and specific for early diagnostic purposes ascomparedtofecalanalysis.However,thecombinationofbothimmuno-andcopro-diagnostictestswasveryhelpfulfordemonstratingthestatusofF.hepaticainfection.Furthermore,ithasbeenrecommendedthatimmunodiagnostictestscouldbehelpfulinlarge-scaleepidemiologicalstudiestoadaptcontrolstrategiesinordertomitigatetheinfection.174-Monovalentcation/sodium:protonantiporterproteinsofEhrlichiachaffeensisL.Wei,H.Liu,R.Ganta.DepartmentofDiagnosticMedicine/Pathobiology,CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,[email protected]:VectorborneandParasiticDisease,Room7,12/4/20174:45PMAnaplasmataceaefamilyrickettsialbacteriaaremostlyvector-transmittedpathogenscausingimportantdiseasesinseveralvertebrates,including humans, canines, and ruminants. Ehrlichia chaffeensis, a tick-transmitted intraphagosomal rickettsial bacterium, is thecausativeagentofhumanmonocyticehrlichiosis(HME).Littleisknownabouthowthisandotherrelatedrickettsialorganismsareabletoresideandreplicatewithinanacidifiedphagosomeenvironment.Similarly,itisunclearhowtheinfectiousformofthebacteriummaintains homeostasis in the extracellular milieu where the pH is about 7.35-7.45, prior to its infection to a naïve host cell.Sodium/cation:protonantiportersareintegralmembraneproteinsreportedfromawiderangeofspecies.TheyexchangesodiumorothermonovalentcationsagainstprotonsacrossaplasmamembraneinmaintainingthecytoplasmicpHofacell.WerecentlydescribedamutationwithintheEch_0379geneofE.chaffeensiswhich ispredictedtoencodeforaNa+/H+antiporterprotein.Themutationcaused the attenuated growth of the organism in vertebrate hosts, resulting in a reduced level of the bacterial presence in thecirculation. In this study, we evaluated the antiporter protein genes of E. chaffeensis. E. chaffeensis genome contains 10 codingsequences encoding polypeptides which may form 6 functional antiporter proteins. To define their function, a sodium sensitiveEscherichia coli strain (EP432, antiporter protein genemutant) is used for the functionally complement of E. chaffeensis putativeantiportergeneswith their respectivepromoters. TheEP432 straingrowth is restoredwhencomplementedwithall 6 genesofE.chaffeensisundertheacidicpH,whileEch_0379andEch_0179complementedalsoatneutralandbasicpH.ComplementationoftheotherfourgenesatneutralandbasicpHmadeEP432moresensitivetosodium.ThisisthefirstdescriptionofantiporterproteinsofE.chaffeensis,whichmayplayacriticalroleinregulatingthebacterialhomeostasiswithinaphagosomeofaninfectedhostcellandaftertheirreleaseintothecell-freeenvironment.

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175-PreliminarydevelopmentofaPCR-basedassaypaneltoscreentickscollectedfromelkinMissouriS.Shahzad1,Z.Shen2,K.Straka3,E.Daugherty2,D.Thompson4,M.Zhang1,J.Mitchell2,S.Zhang2,R.W.Stich1.1VeterinaryPathobiology,UniversityofMissouri,columbia,MO,USA,2VMDL,UniversityofMissouri,columbia,MO,USA,3MissouriDepartmentofConservation,Missouri Department of Conservation, columbia,MO, USA, 4veterinary pathobiology, University ofMissouri, columbia,MO, [email protected]:VectorborneandParasiticDisease,Room7,12/4/20175:00PMElk(Cervuselaphus)werereintroducedtoSoutheasternMissourifromKentuckyin2011.EhrlichiachaffeensishassincebeendetectedinanaturallyinfectedbullelkinMissouri,whichwassubmittedtotheUniversityofMissouriVeterinaryMedicalDiagnosticLaboratoryforpost-mortemevaluation.However,tothebestofourknowledge,furtherinvestigationsofticksthatparasitizeelkinMissourihavenotbeen reported.Theobjectiveof thisprojectwas to identifycollected fromNovember,2015 throughMarch,2016 from26elkindigenousinMissouri,andtoapanelofPCR-basedassaystoscreentheseticksforbacterialpathogens.DichotomouskeyswereusedtoidentifynymphalandadultstageAmblyommaamericanum,Ixodesscapularis,IxodesminorandDermacentoralbipictus.Previouslyvalidateduniversalprimersetswereselectedfordetectionoftick-borneAnaplasmataceae,RickettsiaceaeandBorreliaburgdorferi,andincorporatedintandemintoasyntheticgeneblock,whichwasthenclonedintoaplasmidtoserveasapositivecontrolthatwasused to optimize PCR parameters for each primer set and to compare different tick fixation and template preparationmethods.OptimizedPCRconditionsincluded3.0,2.5and4.0mMMgCl2;0.12,0.16and0.2units/ulofTaqpolymerase;and0.6,0.3and0.8uMofprimersfortheAnaplasmataceae16SrDNA,RickettsiaceaeOMPAgeneandB.burgdorferiOSPAgene,respectively.Templatewasisolatedfromindividualadultticksornymphspooledaccordingtohostandcollectiondate,forscreeningwithconventionalorreal-timePCR,bothofwhicharecurrentlyunderway.ThisPCRarrayisexpectedtoprovidearelativelyconvenient,reliableapproachthatcanbeadaptedtoscreensamplesforabroadrangeofagentsassociatedwithticksandseekstoprovideabetterunderstandingofticksandpathogenstheyharborinMissouri.176-Developmentofabovinemodeltoidentifytickmoleculestargetedbyimmuneseraassociatedwithreducedperformanceof

DermacentorandersoniticksK.Hoffman1,C.Zorn2,S.Shahzad1,S.Jittapalapong3,G.Zhang1,G.Johnson1,P.Pithua1,R.Stoffel1,B.Dhagat-Mehta1,M.Butcher1,R.W.Stich1.1VeterinaryPathobiology,UniversityofMissouri,Columbia,MO,USA,2CollegeofVeterinaryMedicine,UniversityofMissouri,Columbia,MO,USA,3CollegeofVeterinaryTechnology,KasetsartUniversity,Bangkok,[email protected]:VectorborneandParasiticDisease,Room7,12/4/20175:15PMPreviousstudieshaveshownthatimmunizationofdogsandcattlewithticksalivaryglandormidgutextractsresultedindistincteffectsupon tick feeding and fecundity performance parameters. However, these studies involved tick-hostmodel systems that are notendemictotheUSAorthatarenotdirectlyrelevanttoagriculture.Theobjectiveforthisstudywastodevelopanapproachtoidentifyantigensassociatedwithspecificallydecreasedperformanceofticksfedonimmunizedcattle,whichcouldthenbeadaptedtoworkfocusedonticksandpathogensendemictotheUSA.Theworkinghypothesisunderlyingthisprojectisthatantigensspecifictoticksalivary glands ormidgut are differentially recognized by host immune sera associatedwith reduced tick feeding or performanceparameters,respectively.Apilotstudywasconducted,inwhich20female-malepairsofDermacentorandersonitickswerefedondairycalvesbeforeandafter immunizationwithtickmidgutorsalivaryglandhomogenates,andtick feedingandfecundityperformanceparametersweremeasuredforeachtickgroup.Feedingandfecundityperformanceparameterswerereducedinbothgroups.SDS-PAGEandtwo-dimensional(2-D)gelelectrophoresiswereusedtoresolvetickmidgutorsalivaryglandhomogenateproteins,whichwere then transferred to immunoblots that were developedwith sera frommidgut-immune or salivary gland-immune calves. Asexpected, theseWestern blots confirmed that immunizationwith different homogenates induced antibodies reactive to differentproteins.Immuneserafromdifferenthostsimmunizedthesamehomogenateswerereactivewiththesameproteins.Workisunderwaytoidentifyproteinsuniquelyreactivetoimmuneseraassociatedwithreductionofspecifictickperformanceparameters,andtoadaptthissystemtoincludeacquisition,maintenanceandtransmissionofatick-bornepathogen.

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177-TheoryandapplicationofmodernfleacontrolM.W.Dryden.KansasStateUniversity,Manhatten,KS,[email protected]:VectorborneandParasiticDiseaseKeynotes,Room7,12/5/20179:00AMThecatflea,Ctenocephalidesfelisisthemostcommonexternalparasiteofdomesticateddogsandcatsinmostareasoftheworld.Inthe1980sitbecameapparentthatfleaswerebecomingincreasingresistanttomostoftheinsecticidesusedatthattimeandthatourknowledgeofthebiologyandepidemiologyofthisfleaspecieswaseitherincorrectorcompletelylacking.Considerableresearchwasundertaken over the next 2 decades that revolutionized our understanding of the biology and epidemiology this flea. Of primeimportancewasthediscoveryofthepermanentecto-parasiticnatureofreproducingC.felisalongwithworkdefiningonandoff-hostlongevity,eggproduction,bloodconsumption,larvaeandpupaedevelopment,importanceofmicroclimatesandhostattractionamongothers.Thisnewknowledgethenusheredinaveritableavalancheofnewinsecticidesandinsectgrowthregulatorstocombatthisfleaspecies.Astheserevolutionarynewproductswerebeingdevelopednewwaystoassesstheirperformanceandquantifyresistanceselectionhadtobeinvented.Areasofresearchbeganintoassessingovicidalandlarvicidalactivity,decaycurvepharmacokineticsoftopicalandsystemicinsecticides,reproductivebreakpointsandresidualspeedofkillofresidualadulticides,fleabiomassandgenderstructureofimmaturelife-stages.Additionally,thesenewdevelopmentsbroughtintoquestionlongheldpositionsontherelationshipoffleas,fleabiting/feedingandfleaallergydermatitis.Laboratoryandfieldinvestigationshavecompletelychangedourunderstandingof how toeffectively control flea allergydermatitis andpruritus in flea infestedpets. This seminarwill highlight threedecadesofresearchadvancementsthatdramaticallychangedourunderstandingofC.felisanditscontrol.178-ChangingparadigmsofrickettsialdiseasesK.R.Macaluso.DepartmentofPathobiologcalSciences,LouisianaStateUniversity,BatonRouge,LA,[email protected]:VectorborneandParasiticDiseaseKeynotes,Room7,12/5/20179:45AMTheincreaseinreportedrickettsialinfectionsgloballycoincideswiththediscoveryofunfamiliararthropodvectors,newlyrecognizedrickettsialpathogens,anddocumentedtransmissionpotentialofwhathavebeenconsidered tobe rickettsial symbionts.Thus, thetransmissibilityofrickettsiae,vectorialcapacity,andtheclassificationofrickettsialpathogenscanbeconsideredvariablescontributingtoemergingrickettsial infections.Anemergingflea-bornerickettsiosis,causedbyRickettsiafelis, isanexampleofanunderstudiedpathogen.MultipleR. felis genotypes, combinedwith thediversityof potential vectors and transmissionmechanisms, results in auniquetransmissioncyclewithcomplexvector-pathogeninteractions.Foremergingandre-emergingflea-andtick-bornerickettsialagents,recentstudieshaveidentifiednovelaspectsoftransmissionbiology,ecology,andepidemiologyofrickettsialdiseases.

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179 - Evaluation of diagnostic tools for Bovine Respiratory Disease in calves challengedwithBovine Rhinotracheitis Virus andMannheimiahaemolytica

J.Baruch1,C.A.Cull2,N.Cernicchiaro1,J.Nickell3,K.F.Lechtenberg2,D.G.Renter1.1DepartmentofDiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,USA,2VeterinaryandBiomedicalResearchCenter,Manhattan,KS,USA,3AnimalHealth,Merck,DeSoto,KS,[email protected]:AntimicrobialResistance–6,Room7,12/5/201710:45AMPurpose:Bovinerespiratorydisease(BRD)isoneofthemostcostlydiseasestothebeefindustry;itischaracterizedbyhighmorbidity,mortality,andproductionloses.Ante-mortemdiagnosticsarechallengingasindicatedbythepoordiagnosticperformanceofcommonclinicalapproaches (approximately60%sensitivityand specificity). Theobjectiveof this studywas toevaluate theperformanceofchute-sidediagnostic tools for thedetectionofBRDusingachallengemodelwith InfectiousBovineRhinotracheitisVirus (IBR)andMannheimia haemolytica (Mh). Methods: Thirty Holstein steers were inoculated intranasally with IBR on study day 0, andintrabronchiallywithMhonstudyday6.Duringa13-dayperiod,whisperstethoscope(WS),chute-sidebloodleukocytedifferential(CBLD)andpulseoximetry(PO)wereusedondays0,1,2,4,6,6.5,7,7.5,9,11,and13,clinicalillnessscoreswererecordeddailyandthoracicultrasoundwasmeasuredondays0,6,7,9,11and13.Cattlewereeuthanizedandlungconsolidationdatawererecordedondays6,7,9,11and13.Datawereanalyzedusinggeneralizedlinearmixedmodels.Results:Thechallengemodelrepresentedclinicalsignsandlesionstypicalof IBRandMhinfection.Statisticallysignificantdifferencesbystudydaywereobservedforalldiagnostics.BeforeMhinoculation,mostcattlewereassignedclinicalillnessscoresfromnormal(0)tomoderate(2);however,afterMhinoculation,moderate,severe(3)andmoribund(4)scoreswererecorded.Oxygensaturationsignificantlydecreased12to24hafterMhinoculationcomparedtolevelsobservedondays0,1,2and4.Whisperstethoscopescoresweresignificantlylowerondaysbefore(days0,1,2and4)versusthedaysafterMhinoculation(7,7.5,9and11).Averagelungconsolidationwas1.9%(0.9%)onday6and55%(7.7%)onday10.Conclusions:Resultsfromthesediagnostictoolswereconsistentwithpathologicalandclinicaldiseaseprogressionfindings.Subjectivemeasures such as clinical illness scores could be combinedwith objective tools, such as,WS, POor CBLD, contributingtowardsanimprovedapproachtoBRDdiagnosis.180-Epidemiologyofmulti-drugresistantMannheimiahaemolyticaisolatedformhigh-riskstockercattleE.R. Snyder1, B.C. Credille1, S. Alvarez-Narvaez2, R.D. Berghaus1, S. Giguère2. 1Food Animal Health and Management Program,Department of Population Health, University of Georgia, Athens, GA, USA, 2Department of Large AnimalMedicine, University ofGeorgia,Athens,GA,[email protected]:AntimicrobialResistance–6,Room7,12/5/201711:45AMThebacterialpathogenMannheimiahaemolytica(Mh)isamajorcomponentofthebovinerespiratorydiseasecomplex.Antimicrobialresistance in this pathogen is becoming a serious concern as diagnostic labs are reporting an increased prevalence ofmulti-drugresistantisolates.Documentingtheresistancegenespresentinthispathogenisanimportantstepinunderstandingtheepidemiologyofmulti-drugresistantMh.Furthermore,itisessentialthatweunderstandwaysthatresistancemaybegeneratedoracquired,andhowantimicrobialusemightinfluencethis.Deepnasopharyngealswabs(NPS)werecollectedfrom169high-risk,salebarnoriginbullandsteercalvesatarrivaltoacentralGeorgiastockerfacility.Calveswereprocessedfollowingstandardindustryprotocolandreceivedametaphylacticdoseoftheantimicrobialdrugtulathromycin.AsecondNPSwascollectedfromthesamecalves10to14dayslater.AllNPSweresubmittedforculture.ForsamplesculturepositiveforMh,amaximumofthreecoloniesfromeachsampleweresubculturedandsubmittedforantimicrobialsusceptibilitytestingviaKirby-Bauerdiskdiffusion.Of169calvessampled,22wereMhpositiveatbothtime points. DNA was extracted and whole genome sequencing was performed on these “matched” isolates. Fifty isolates weresequencedintotalusingIlluminaNextSeq;multipleisolatesfromeachcalfatagiventimepointwereonlysequencediftheyhadauniquesusceptibilityprofile.AfterprocessingoftheIlluminadata,phylogenetictreesandSNPmatriceswereconstructedusingtheprogramsNDTreeandCSIPhylogeny,toillustratethephylogeneticrelationshipsbetweenisolatescollectedateachtimepoint.Theisolate assemblies were then aligned using NCBI BLAST against resistance genes documented in the Comprehensive AntibioticResistanceDatabase(CARD)andtheMicrobialEcologyGroupResistanceDatabase(MEGARes).Thelocationofeachresistancegenewas identified and mapped. Mutations resulting in amino acid changes were identified in genes involved in fluoroquinolonesusceptibility.Agreementbetweengenotypeandresistancephenotypeiscurrentlyunderanalysis.

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181-Associationbetweenantimicrobialuseandantimicrobialresistanceinbacteriaisolatedfromfecesorrespiratorysecretionsoffeedlotcattle:asystematicreviewandmeta-analysis

P.Rovira1,S.A.Brault2,S.Costard3,F.Zagmutt3,P.S.Morley2,K.E.Belk1.1AnimalSciences,ColoradoStateUniversity,FortCollins,CO,USA,2ClinicalSciences,ColoradoStateUniversityCollegeofVeterinaryMedicineandBiomedicalSciences,FortCollins,CO,USA,3EpiXAnalytics,FortCollins,CO,[email protected]:AntimicrobialResistance–6,Room7,12/5/201711:00AMTheobjectiveofthisstudywastoassesswhetherantimicrobialuse(AMU)infeedlotcattleisassociatedwithantimicrobialresistance(AMR) inbacteria isolatedfromcattlefecesorrespiratorysecretions.A literaturesearchresultedin344uniquepublications,32ofwhichwereselectedafterevaluationby2independentreviewers.Escherichiacoliwasthemostcommonbacteriumstudied,followedbyEnterococcus spp.,Salmonella enterica, Campylobacter spp., andMannheimiahaemolytica. Themost frequently studied targetbacteria/antimicrobialexposurecombinationswereE.coli/tetracyclinesandEnterococcus/macrolides.Dataextractedfrom11studiesthatreportedtheproportionofisolatesresistanttoantimicrobialsincontrolandexposedgroupswereanalyzedinarandom-effectmeta-analysiswith3covariates(exposure-defineddailydoses,cumulativedaysofantimicrobialexposurebeforesampling,andtimebetweenlastexposureandcollectionofsamples)toestimaterelativerisk(RR)ofAMRassociatedwithAMU.Overall, isolatesfromcattleexposedtoanytypeofantimicrobialwere2.3times(95%confidenceinterval1.1-4.6)aslikelytoexhibitAMRtoanytypeofdrug as isolates recovered from unexposed animals. However, the relationship was weaker when some specific combinations ofantimicrobials and bacteria were examined: E. coli isolates from cattle exposed to tetracyclines were 1.7 times (1.1 - 2.5) andEnterococcus isolates fromcattleexposed tomacrolideswere1.8 times (0.5 -6.6)as likely to showAMR tohomologousdrugsasunexposedcattle.Conversely,astudyresearchingflorfenicolexposuresonresistanceinE.colifoundaveryhighlikelihoodofrecoveringresistant bacteria when compared to unexposed cattle (RR=25.0; 1.5 - 415.7). When pooling studies for meta-analysis, carefulconsiderationmustbegiventotheimpactofcomparingstudiesexaminingdisparateantimicrobialsandbacteria.Meta-analysisshouldbeperformedonstudiesrestrictedtospecificantimicrobial/bacterialcombinationstoavoidinappropriateamalgamationofRRresultsthatareactuallyquantifyingdifferentmechanismsofAMR.182-Ionophoreuseinfoodanimalproductionanditsimpactonhumanhealthintermsofantimicrobialresistance:ascopingreviewD. Loest1,S.A. Brault2, S. Gow3, P.S.Morley2,M.D. Apley4, C. Carson1. 1Canadian Integrated Program for Antimicrobial ResistanceSurveillance,PublicHealthAgencyofCanada,Guelph,ON,Canada, 2MicrobialEcologyGroup,ColoradoStateUniversityCollegeofVeterinary Medicine and Biomedical Sciences, Fort Collins, CO, USA, 3Canadian Integrated Program for Antimicrobial ResistanceSurveillance,PublicHealthAgencyofCanada,Saskatoon,SK,Canada,4ClinicalSciences,KansasStateUniversity,Manhattan,KS,[email protected]:AntimicrobialResistance–6,Room7,12/5/201711:15AMIonophores are widely used in food animal production, primarily for coccidiosis control, feed efficiency, and growth promotion.Antimicrobialusecanleadtoantimicrobialresistance(AMR),theincreaseofwhichhasputantimicrobialuseinfoodanimalproductionunderscrutiny.Todate,thegeneralconsensushasbeenthationophoresposelittleornopublichealththreatintermsofAMRbecauseoftheiruniquemodeofaction,lackofevidenceofgenesencodingresistance,andthefactthattheyarenotusedtherapeuticallyinhumans.However,therecentfindingofputativeplasmid-mediatedelevatedminimuminhibitoryconcentrations(MIC)ofnarasininEnterococcusfaeciumhaschallengedpreviouslyheldassumptions.Toexaminethisissuefurther,ascopingreviewwasperformedtoaddress whether use of ionophores in food animals has an impact on AMR affecting human health. English language literature,publishedfrom1990,wassearchedon5databases.Titleandabstractscreening,fulltextreview,anddataextractionwerecompletedby 2 reviewers,with a 3rd reviewer resolving disagreements. Database searches resulted in 2553 publications,with 35 ultimatelyincludedinthestudy.Monensinwasthemoststudiedionophore(n=18),andpoultrythemostexaminedcommodity(n=23).Themostcommon organism examined was E. faecalis (n=10), followed by Clostridium perfringens (n=9), and E. faecium (n=7). In general,documentationofbacterialresistanceto ionophoreswasuncommon.However,the lackofastandardizedmethodfordeterminingresistance(i.e.,standardMICbreakpointinterpretivecriteria)isproblematic.Manyquestionsremainunansweredregardingionophoreuseinfoodanimals,anditspossibleimpactonhumanhealththroughAMR.Thisscopingreviewisthefirstpartofalargerreviewthatwillalsoconsiderionophoreusefortreatmentinhumans,impactonanimalhealthintermsofAMR,impactonpathogenprevalence,aswellastheevidenceconcerningthebenefitsofionophoreuseinfoodanimalproduction.

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183-AnalyzingantimicrobialminimuminhibitoryconcentrationdistributionswithoutinterpretivebreakpointsV. Volkova1,M. Jaberi-Douraki2. 1Department of DiagnosticMedicine/Pathobiology, College of VeterinaryMedicine, Kansas StateUniversity,Manhattan,KS,USA,2DepartmentofMathematics,KansasStateUniversity,Manhattan,KS,[email protected]:AntimicrobialResistance–6,Room7,12/5/201711:30AMToassessphenotypicbacterialantimicrobialresistance(AMR)inahostpopulation,asampleofisolatesofthetargetbacterialspeciesisobtained.Individualisolate’ssusceptibilitytoanantimicrobialismeasuredasthedrug’sminimuminhibitoryconcentration(MIC)forthebacterialculturegrowth.Acrosstheisolatesinthesample,MICvaluesoccuratdifferentrelativefrequencies.Often,theMICrelativefrequency distribution is categorized based on the MIC interpretive breakpoints for the bacterial species and drug (i.e., clinicalbreakpointsorepidemiologicalcut-offvalues).Thisleadstolossesinanalyticalpowerandflexibility.WehavedevelopedapproachestoanalyzefullMICfrequencydistributions,enablingdetectionofpopulation-leveldynamicsinphenotypicAMR.ThisincludesstatisticalteststhatarenonparametricorrobusttohandlevaryingshapesoftheMICfrequencydistributions,aswellasmathematicalmodelstodetailco-dependenciesofAMRtodifferentantimicrobialdrugclasses.Wehaveappliedthedevelopedapproachestoanalyzethedatagenerated by the U.S. National Antimicrobial ResistanceMonitoring System (NARMS) and our field studies. For example, using anonparametrictestacrossthebacterialspeciesandproductsintheNARMS1996-2013data,weshowedtheanalysisusingtheclinicalbreakpointbasedinterpretationmissedsignificantchangesin54%oftheyear-to-yearMICdistributioncomparisonsand71%oftheslaughter-to-retailwithin-yearcomparisons.UsingarobusttestfortheNARMSdata,wefoundthattheground/trimmedmeatsamplingvs.cecalcontentssamplinginthecattleprocessingplantsin2013and2014yieldedstatisticallynon-equivalentestimatesofAMRtosomedrugclassesinSalmonellaentericainthecattlepopulations.EmployinganothernonparametricapproachfortheNARMS1996-2013data,weidentifiedhowtheMICfrequencydistributionchangesovertheyearsvariedamongindividualdrugswithinadrugclassandbetween classes.Usingmathematicalmodelingon theNARMSdata,wearenowdetailing themulti drug classAMRpatternsbetweenbacterialspeciesinmajorfood-animalproductionsystems.184-Upperandlowerrespiratorytractmicrobiotasinfeedlotcattle:theircomposition,relationshipandpotentialroleinrespiratory

healthE.Timsit.ProductionAnimalHealth,UniversityofCalgary,FacultyofVeterinaryMedicine,Calgary,AB,[email protected]:RespiratoryDiseaseKeynote/BacterialPathogenesisKeynote,Room8,12/4/20179:00AMBacterialbronchopneumonia(BP) isoneofthemost importanthealthproblemsinthebeef industry.BeefcattleofallagescanbeaffectedwithBP;however,theyaremostlikelytobeaffectedduringthefirst50daysafterentranceintoafeedlotbecausetheyareexposedtoawiderangeofpathogens(duetocommingling)concurrentwithvariousstressors(e.g.weaningandtransportation),whichcansuppresstheirimmunesystem.ImportantbacterialpathogensassociatedwithBPincludeMannheimiahaemolytica,Pasteurellamultocida, Histophilus somni andMycoplasma bovis. For these pathogens, colonization of the upper respiratory tract (URT) is anecessaryfirststepbeforecausinglowerrespiratorytract(LRT)infection.Inhibitionofthisfirststepofpathogenesisbytheresidentmicrobiota, a process also called “colonization resistance”, might therefore be of the upmost importance to respiratory health.Furthermore,ifapathogenhascolonizedtheURTmucosalsurface,itmightbebeneficialtoboththemicrobialcommunityandthehostthatthesepathogensarekeptatbay,preventingtheirovergrowth,inflammationandsubsequentspreadtothelungs.Inthelastfewyears,evidence for the roles thatbacterial communities in theURThave inpreventing respiratorypathogens fromestablishinganinfectionhasaccumulated.Thispresentationwillreview(i)thecompositionoftheURTandLRTmicrobiotasandtheirrelationshipand(ii) describe the potential role that the URTmicrobiota plays in respiratory health.Microbiota-based intervention (i.e. intranasalprobiotics)forthepreventionofBPwillalsobediscussed.

CRWAD2017 ABSTRACTS


185-ExpecttheunexpectedwithCampylobacterjejuniC.Szymanski.ComplexCarbohydrateResearchCenter,UniversityofGeorgia,Athens,GA,[email protected]:RespiratoryDiseaseKeynote/BacterialPathogenesisKeynote,Room8,12/4/20179:45AMCampylobacter jejuni is a common gastrointestinal pathogen in humans and is typically acquired through the consumption ofcontaminatedpoultryproductsat infectiousdosesas lowas500bacterial cells. In contrast, theorganism ispartof thenativegutmicrobiomeinmostanimalsandcanbefound,particularlyinbirds,atlevelsupto109colonyformingunitspergramofcecalmaterial.Campylobactersarenotoriousasubiquitouspathogensandwererecentlyidentifiedin85%ofstoolscollectedfrominfants<1yearin8 low resource countries. But arguably, C. jejuni has become even better known in the field of glycobiology for the diverseglycoconjugatesitsynthesizes.Thisisalldonewithagenomesizeapproximatelyone-thirdthatofE.coliandinanorganismthatisunabletocatabolisecarbohydratesduetoalackofglucokinaseandphosphofructokinaseenzymesfromtheEmbden-Meyerhof-Parnasglycolysis pathway. So,with the exception of a catabolic fucose pathway in some isolates,C. jejuni strains are asaccharolytic andsynthesize all their carbohydratesdenovo. It is thereforenot surprising thatC. jejunihasbecomea rich resourceof enzymes forglycobiologiststodiscover,butdifficulttoimaginehowsuchanorganismcanthriveandoutcompeteothermicrobesthatpossessamuchgreatercapacityfornutrientacquisitionandcatabolism,inadditiontoanarsenalofvirulencefactorsthatprotectagainsthostandfellowmicrobes.ThispresentationwilldescribemechanismsthatC.jejunihasdevelopedtosurviveinthehostileenvironmentofthegastrointestinaltract.186-ComparisonofPRRSVisolationfromclinicalsamplesusingMARC-145andZMACcelllinesW.Yim-im.VeterinaryMicrobiologyandPreventiveMedicine,Iowastateuniversity,Ames,IA,[email protected]:RespiratoryDiseaseinSwine,Room8,12/4/201710:45AMDuetohighrateofgeneticandantigenicdiversityofPRRSV,thecommercialvaccinesarenotalwayseffectiveagainstfieldisolates.PRRSVvirusisolation(VI)isoftenrequestedforproducingautogenousvaccines.However,PRRSVVIsuccessrateintheconventionalcelllineMARC-145isfrustratinglylow.TheobjectivesofthisstudyweretocomparePRRSVVIefficiencyinZMACandMARCcellsandinvestigateifZMAC-derivedPRRSVisolatesgrowinMARCcells.146typeIIand19typeIPRRSVPCR-positiveclinicalspecimenswithvariousCTvalueswereselectedforthisstudy.Theseincluded53serum(CT11.7-35.2),56lung(13-36.6),and37oralfluid(OF,21.9-36)samplesfortypeIIPRRSV,and3serum(18.4-30.1),8lung(16.4-24.2),and8OF(31-36.8)samplesfortypeIPRRSV.Allsampleswereinoculated intobothcell lines forahead-to-headcomparison.43 type II and3 type IPRRSV isolatesobtained inZMACcellswereinoculatedintoMARCcellstoevaluatetheirgrowth.Among109typeIIserumandlungsamples,61.5%and27.5%wereVIpositiveinZMACandMARCcells,respectively.ThesuccessratesofisolatingPRRSVwithdifferentCTvalueswere:97.2%and50%(CT<20),62.2%and29.7%(CT20-25),45%and5%(CT25-30),and0%and0%(CT30-37)inZMACandMARCcells,respectively.Among11typeIserumandlungsamples,72.7%and45.5%wereVIpositiveinZMACandMARCcells,respectively.Only47%oftypeIIPRRSVisolatesobtainedinZMACcellsgrewinMARCcells.When3typeIPRRSVisolatesobtainedinZMACcellswereinoculatedintoMARCcells,1grewand2didnotgrow.PRRSVwasnotisolatedfromanyOFsamplesevaluatedinthisstudyregardlessofusingZMACorMARCcells.However,successrateofPRRSVVIfromserumandlungsampleswithCT<30usingZMACcellswassignificantlyhigherthanthatusingMARCcells.ForserumandlungsampleswithCT>30,PRRSVVIsuccessratewasverylowineithercellline.ThisstudyclearlydemonstratesthatusingZMACcellscouldsignificantlyimprovesuccessratesofPRRSVVIfromclinicalsamples,whichwillservetheneedsofproducingautogenousvaccinesforpreventionandcontrolofPRRS.ItisnoteworthythatnotallPRRSVisolatesobtainedinZMACcellswillgrowinMARCcells.

CRWAD2017 ABSTRACTS


187-Developmentofaluminexmultiplexassayforthedetectionanddifferentiationoftype2PRRSvfieldstrainsandthefourU.S.vaccinestrains.

Y.Wang1,W.Zheng1,E.Porlsen1,X.Liu1,J.Zhang2,K.-J.Yoon2,L.Peddireddi1,R.Hesse1,Y.Fang1,G.Anderson1.1KansasStateVeterinaryDiagnostic Lab, Kansas State University, Manhattan, KS, USA, 2Veterinary Diagnostic Lab, Iowa State University, Ames, IA, [email protected]:RespiratoryDiseaseinSwine,Room8,12/4/201711:00AMPurpose: Since its emergence inNorthAmerica in the late1980s, PRRSVhasundergone constant changes andnewvariantshaveevolvedupontime,andthatmakesmoleculardiagnosisverychallenging.Therearefourtype2PRRSvaccines,IngelvacMLV,IngelvacATP,FosteraandPrimePac,havebeenusedintheUS.Differentiatingvaccinestrainsfromthefieldstrainsisimportanttoguideandimprovevaccineapplications.Duetothatthevaccinestrainsareverysimilartosomeofthefieldstrains,itisdifficulttodifferentiatefromthefieldstrains.CurrentlythemostusedmethodofvaccinedifferentiationisbyORF5sequencing,whichisexpensiveandtimeconsuming.TheLuminexxTAGassayisabead-basednucleicaciddetectionthathypotheticallycananalyzemorethan100differentnucleotidesequencesinasinglereaction.Inthisstudy,aLuminex-basedmultiplexassaywasdevelopedtodetectthevastmajorityoftype2PRRSVfieldstrains,atthesametimetodifferentiatethefourvaccinestrainsthathavebeenusedintheUS.Methods:Acollectionof694fullornear-fullgenomesofNorthAmericantype2PRRSVstrainswereanalyzed.TwopairsofprimerstargetingtheMandNgenesweredesignedfordetection.Thecoverageforeachsetis85.4%and91.2%,respectively,withacombinedcoverageof98.1%,byaninsilicoanalysis.FourpairsofprimerstargetinginNSP2genesofvaccinestrainsweredesignedfordifferentiation.Results:TestingonanumberoffieldstrainsandthefourvaccinestrainsindicatedthattheassaydetectedallPRRSstrainsandidentifiedeachofthefourvaccinestrainscorrectly.Theanalyticalsensitivitywasperformedby10-foldserialdilutionsofthreevaccinestrains(MLV,ATPandFostera)andaclonedgenomicpieceofPrimePac.Toevaluatethelimitofdetections(LODs),real-time(r)reverse-transcription(RT)PCRassays(rRT-PCR)werealsousedforcomparison.TheLODsoftheLuminexassaywereequivalenttoCt35.8byrRT-PCRforMLV,Ct33.2forATP,Ct31.2forFostera,andCt36.1forPrimePac,whicharesimilartothatgeneratedbyrRT-PCRs.Conclusions:TheLuminexassaywillallowdetectionforemergingvariantsofPRRSVanddifferentiationfromvaccinestrains.188-GeneticdiversityandmoleculardetectionassaydevelopmentforPCV3andPCV2strainsY.Wang,F.Yuan,W.Zheng,E.Porlsen,X.Liu,L.Peddireddi,R.Hesse,Y.Fang,G.Anderson,J.Bai.KansasStateVeterinaryDiagnosticLab,KansasStateUniversity,Manhattan,KS,[email protected]:RespiratoryDiseaseinSwine,Room8,12/4/201711:15AMPurpose:PCV2isacausalagentofporcinecircovirusdiseases(PCVD),includingpostweaningmultisystemicwastingsyndrome(PMWS),porcinedermatitisandnephropathysyndrome(PDNS),porcinerespiratorydiseasescomplex(PRDC),entericdiseaseandreproductivefailure. In2015,PCV3was identified intheUS,andaccumulatedevidences indicated itcouldcausePDNS,reproductivefailureandmultisystemicinflammation.Methods:Completegenomesequencesof33PCV3strainswerecollectedfromNCBIandanalyzedwith31fullgenomessequencedinourlab.SeveralatypicalcircovirusidentifiedinbeefandporkshowedhighidentitytoPCV3especiallyinthereplicaseregionwerealsoanalyzed.The64PCV3strainsshareaminimalof97.3%nucleotideidentitiesatthefull-genomelevel,and97.6%and96.2%for thereplicaseandcapsidgenerespectively. Incontrast,PCV2strainshaveundergonesignificantgenomicchanges,andevolvedintodifferentgenotypesincludingPCV2a,2band2d.Analysisof1,907PCV2fullornear-fullgenomesequencesfromNCBIindicatedthattheminimalnucleotideidentitywasonly76.2%.OnesetofprimersandprobeweredesignedinthecapsidgeneforPCV3with100%coverage;and2setsofprimersandprobes,targetedintheORF3andORF1regionsofPCV2,weredesignedforPCV3andPCV2detections.ThecoverageforeachPCV2setis90.5%and94.5%respectivelywithacombinedcoverageof98.9%.The3setsofprimersandprobesweremultiplexed,andanalyzedusinga10-foldserialtemplatedilutions.Results:Theanalyticallimitof detection (LOD) for both PCV2 and PCV3were 10 copies per reaction. Correlation coefficientswere 0.997 and 0.995, and PCRamplificationefficiencieswere103.1%and102.7%,respectively,forPCV3andPCV2.DiagnosticLODwithclinicalsampleswereCt37.0andCt36,withcorrelationcoefficientsof0.996and0.994,andPCRamplificationefficienciesof95.5%and91.6%,respectively,forPCV3andPCV2.Conclusions:Testingon717pigdiagnosticsamplesidentified156(21.76%)PCV3positives(Ct<37).ThePCV2assaycorrectlydetected40positiveand30negativediagnosticsamplesthatwerepreviouslytestedwithadifferentPCRassay.

CRWAD2017 ABSTRACTS


189-EffectofmiRNAandtRNAgeneexpressiononthehomeostaticstatusofpigsinfectedwithhighlypathogenicPRRSVD.Fleming,L.C.Miller.VPDRU,USDA-ARS-NADC,Ames,IA,[email protected]:RespiratoryDiseaseinSwine,Room8,12/4/201711:30AMIthasbeenestablished that reducedsusceptibility toporcine reproductiveand respiratory syndromevirus (PRRSV)hasagenomiccomponent.Thiscomponent,however, isamulti-facetedcompositionofcodingandnon-codinggeneticelements that functionasregulatorsofimmunefunction.Ourstudyfocusesonthesmallnon-coding(sncRNA)sideofthisresponseinpigsbecauseofemergenceofvarioussncRNAsshowntoplayimportantrolesinthehumanviralimmunity.AmongthesesncRNAsarethemicroRNA(miRNA)andtransfer RNA (tRNA)molecules.Our study looks at changes in expression of these sncRNAs to produce information on how genefunctioninthepigcanbecomedysregulatedandsubsequentlyrespondtothevirus.TheobjectiveofthestudyistoidentifydifferencesinmiRNAandtRNAgeneexpressionbetweenhealthyandhighlypathogenicPRRSVchallengedpigs.ThestudywasconductedusingtotalRNAextractedfrompigwholebloodtakenfromatotalof24pigssplitintoeithercontrol(shaminoculation)orinfectedpigsat1,3,and8dayspostinfection.Sequencingofthesamplesproduced100bpsingleendlibrariesfortranscriptomicanalysisofsncRNAgeneexpression. The results indicated statistically significant changes in sncRNAexpressionweredependenton timeand treatment, inwhich,miRNA expressionwas variablewhile tRNA expression declined steadily post-infection. The results of this study highlightschangesinsncRNAexpressionthathavethepotentialtounlocknewtargetsforunderstandingtheeffectofPRRSVonpighomeostasis.190-ComparisonofthetranscriptomeresponsewithinthetracheobronchiallymphnodefollowinginfectionwithPRRSV,PCV2,or

IAVL.C.Miller1,D.Fleming1,G.Harhay2,D.Bayles3,M.Kehrli4,K.Lager1.1VPDRU,USDA-ARS-NADC,Ames,IA,USA,2USDA-ARS-MARC,ClayCenter,NE,USA,3IBDRU,USDA-ARS-NADC,Ames,IA,USA,4USDA-ARS-NADC,Ames,IA,[email protected]:RespiratoryDiseaseinSwine,Room8,12/4/201711:45AMPorcinereproductiveandrespiratorysyndromevirus (PRRSV) isamajorrespiratorypathogenofswinethathasbecomeextremelycostlytotheswineindustryworldwide,oftencausinglossesinproductionandanimallifeduetotheireaseofspread.However,theintracellularchangesthatoccurinpigsfollowingviralrespiratoryinfectionsarestillscantilyunderstoodforPRRSV,aswellas,otherviralrespiratoryinfections.TheaimofthisstudywastoacquireabetterunderstandingofPRRSdiseasebycomparinggeneexpressionchangesthatoccurintracheobronchiallymphnodesofpigsinfectedwitheitherporcinereproductiveandrespiratorysyndromevirus(PRRSV),porcinecircovirustype2(PCV2),orswineinfluenzavirus(IAV)infections.ThestudyidentifiedandcomparedgeneexpressionchangesintheTBLNof16pigsfollowinginfectionbyPRRSV,PCV2,IAV,orshaminoculation.TotalRNAwaspooledforeachgroupandtime-point(1,3,6,and14DPI)tomake16libraries,foranalysisbyDigitalGeneExpressionTagProfiling(DGETP).Thedataunderwentstandard filtering to generate a list of sequence tag raw counts that were then analyzed usingmultidimensional and differentialexpressionstatisticaltests.TheresultsshowedthatPRRSV,IAVandPCV-2viralinfectionsfollowedaclinicalcourseinthepigstypicalofexperimentalinfectionofyoungpigswiththeseviruses.Geneexpressionresultsechoedthiscourse,aswellas,uncoveredgenesrelatedtosharedanduniquehostimmuneresponsestothe3viruses.Bytestingandobservingthehostresponsetootherrespiratoryviruses,ourstudyhaselucidatedsimilaritiesanddifferencesthatcanassistindevelopmentofvaccinesandtherapeuticsthatshortenorpreventachronicPRRSVinfection.

CRWAD2017 ABSTRACTS


191-IntratrachealinoculationoftheguineapigasanimprovedaerosolmodelforBrucellamelitensisinfectionM.Hensel,D.Garcia,S.Chaki,J.Samuel,A.Arenas-Gamboa.TexasA&MUniversity,CollegeStation,TX,[email protected]:BacterialDiseasesinCattle,Room8,12/4/20172:00PMBrucellamelitensisisanobligate,intracellulargram-negativebacteriawithaworldwidedistributioninhumansandanimalsthatcausesanacutediseasecharacterizedbyfever,malaise,andanorexia.B.melitensisisconsideredthemostvirulentoftheBrucellaspecies,andaneedexistsforanimprovedlaboratoryanimalmodelofinfectionthatmimicsnaturaltransmissionanddisease.Themousemodelhasbeenextensivelyused;however,micedonotdevelopfeverwithanyrouteofinoculationandrequirehighaerosoldosestodevelopsystemicinfection.Similartohumans,guineapigsdevelopsystemicandfeversecondarytoinfection.ThePennCenturyMicroSprayerAerosolizerwasusedtoinoculateguineapigswith16MB.melitensis.Thisrouteofaerosoltransmissionhasadvantagesoveraerosolchambersbecauseitallowsfortargeteddeliveryofaknownquantityofbacteriaintotheupperrespiratorytract.GuineapigsweredividedintosevendosegroupsandinoculatedwithsterilePBSor16MB.melitensisat101to103(lowdose)or106to108CFU(highdose)andwerethenmonitoredfor30daysforfever,weightloss,malaise,andrespiratorychanges.Inthelowdosegroup,noneoftheanimalsdevelopedanyclinicalsignsassociatedwithinfectionincludingfever,andonly1animalinthe103groupdevelopedmacroscopicevidenceofsystemicinfection.Intheabsenceofclinicalsigns,bacteriawererecoveredfromthespleen(avg2.5logs),liver(avg1.3logs), lung,cervical lymphnode (cln), tracheobronchial lymphnode (tbln),anduterus indicatingsystemicdisease. In thehighdosegroup, fever developed in a dose dependentmanner on day 17 at 106, day 16 at 107, and day 15 at 108. Systemic disease wascharacterizedbysplenomegaly,lymphadenomegaly,hepatitis,andembolicpneumonia.Bacteriawererecoveredfromthelung,liver,spleen,tbln,cln,anduterusanddemonstrateddosedependentmeanincreasesinCFU.ThemeanCFUinthespleenwas3.7logsat106,4.9logsat107,and5.5logsat108.Guineapigsdevelopfeveranddosedependentsignsofsystemicinfection,whichmimicsthehumanmanifestationofdiseaseandshouldbeconsideredanimprovedanimalmodelforpathogenesisorvaccinestudies.192-SeroprevalenceofcattlebrucellosisslaughteredinabattoiranditsoccupationalrisksG.T.A. Aburu, Sr.. Bacterial serology, National animal health diagnostic and investigation center, Sebeta, [email protected]:BacterialDiseasesinCattle,Room8,12/4/20172:15PMBrucellosisisoneofthemajorzoonoticdiseasesprevalentthroughouttheworld.Across-sectionalstudywasconductedtoassesstheprevalenceofbovinebrucellosisoncattleslaughteredatabattoirsinDebreZeit.Theobjectivesofthestudyweretodeterminetheseroprevalenceofbovinebrucellosisincattleslaughteredandtoevaluatethepotentialriskfactorsthatpredisposeabattoirworkerstopossibleinfection.Atotalof1250cattlepreparedforslaughteringweresubjectedinthestudy.72questionnaireswereadministeredtoworkerstoassesstheoccupationalrisks.Overallseroprevalenceof0.8%and0.5%wererevealedusingRBTandCFT,respectively.Theresultsofthequestionnairesurveyanalysishand/fingercuts,lacerationandworkingwithbarehandswerefoundtobethemainoccupational risk factors associated with bovine brucellosis infection for abattoir workers. Chi-square tests showed significantassociationbetweenworkexperienceandfrequencyoflaceration.Therefore,basedontheresultsthattheseroprevalenceofbovinebrucellosis inslaughteredcattleand itssignificant risk factorsofourstudyweconcludethatdespite the lowprevalenceofbovinebrucellosisbutstillitconstitutesanoccupationalhazardtoabattoirworkersnotusingproperpersonalprotectiveequipments.

CRWAD2017 ABSTRACTS


193-DiseasestateinfluencesthepresenceofmacrophagesandMycobacteriumaviumsubsp.paratuberculosisinbovineintestinaltissue

C.J.Jenvey1,J.M.Hostetter2,J.P.Bannantine1,J.R.Stabel1.1USDA-ARS-NADC,Ames,IA,USA,2DepartmentofVeterinaryPathology,CollegeofVeterinaryMedicine,IowaStateUniversity,Ames,IA,[email protected]:BacterialDiseasesinCattle,Room8,12/4/20172:30PMJohne’sdiseaseisanentericdiseasecausedbytheintracellularpathogenMycobacteriumaviumsubsp.paratuberculosis(MAP).Upontranslocationfromthelumenofthesmallintestine,mycobacteriahavetheabilitytothwartinnatedefensemechanismsandpersistwithinthemacrophage.ThisstudyaimedtocorrelatethepresenceofmacrophagesandMAPinbovinemid-ilealtissuewithstageofinfection.Immunofluorescent(IF)labelingwasperformedonfrozenbovinemid-ilealintestinaltissuecollectedfrom28Holsteindairycows.Macrophageswerelabeledusingamonoclonalanti-macrophagesurfaceantigen(AM-3K)andMAPwaslabeledusingapolyclonalrabbitheat-killedMAPantigen,with IF labelingvisualizedusingaconfocalmicroscope. Imagingsoftwarewasusedtoquantify thesurfaceareaandintensityofIFlabeling.Thepresenceofmacrophageswithinthemid-ilealtissuesectionswashigherforclinicalcows,followedbysubclinicalcowsandthenuninfectedcontrolcows.Macrophageswerepresentthroughouttheintestinaltissueinclinicalcows,includingtheinnermusclelayer,submucosa,cryptandvilliends,whilepresenceofmacrophagesinbothsubclinicalandcontrolcows were limited to the submucosa and inner muscle layer. Clinical cows also demonstrated significantly higher MAP SA andmacrophageandMAPco-localizationSA,whencomparedtosubclinicalcows,andwaspresentwithinthesubmucosaandcryptlaminapropria, progressing into the villi ends in some clinical cows. Our findings indicate that number of macrophages increases withprogressionofdisease,however,asignificantnumberofthemacrophagespresentarenotassociatedwithMAP.Thissuggeststhatalthough the bovine innate immune system is sufficiently stimulated to recruit macrophages in response to MAP invasion, themacrophagesofclinicallyinfectedcowsareultimatelyunabletoclearMAP,resultingindiseaseprogressionandclinicalpresentation.194-RelationshipbetweenthepathologyofbovineintestinaltissueandcurrentdiagnostictestsforJohne'sdiseaseC.Jenvey1,J.M.Hostetter2,J.R.Stabel1.1USDA-ARS-NADC,Ames,IA,USA,2DepartmentofVeterinaryPathology,CollegeofVeterinaryMedicine,IowaStateUniversity,Ames,IA,[email protected]:BacterialDiseasesinCattle,Room8,12/4/20172:45PMJohne’s disease is an enteric disease caused by the intracellular pathogenMycobacterium avium subsp. paratuberculosis (MAP).Recently,weobserved increasednumbersofmacrophages inmid-ileal intestinal tissueofcowsnaturally infectedwithMAP,whencompared touninfectedcontrols. This studyaimed todeterminewhether thepresenceofmacrophagesandMAPcorrelatedwithcommonmethodsusedforthediagnosisofMAPinfection.DiagnostictestsassessedwereELISA,IFN-γassay,RT-PCR(fecalandtissue),andhistological classification. Immunofluorescent (IF) labelingwasperformedon frozenbovinemid-ileal intestinal tissuecollectedfrom28Holsteindairycows.Macrophageswerelabeledusingamonoclonalanti-macrophagesurfaceantigen(AM-3K)andMAPwaslabeled using a polyclonal rabbit heat-killedMAP antigen. Confocalmicroscopy imaging softwarewas used to quantify IF labelingsurface area (SA). Linear and non-linear regressionmodelswere developed for statistically significant correlations. Simple logisticregressionmodelswereusedtodeterminethepredictiveprobabilityofallstatisticallysignificantcategoricaldependentvariables.AnincreaseinmacrophageSAwasdemonstratedintissuesectionsfrombothsubclinicallyandclinicallyinfectedcows,whencomparedtouninfectedcontrols.SignificantcorrelationswereobservedbetweenELISAS/Pratios,andtissuePCR,withbothmacrophageSAandMAPSA.Inaddition,ELISAS/PratioswerecorrelatedwithmacrophageandMAPco-localizationSA.MacrophageandMAPSAwasnotcorrelatedwithIFN-γassayorfecalPCR.Thepredictedprobabilityofcowstatusshiftingfromcontroltosubclinical,andfromsubclinicaltoclinicalwassignificantlyincreasedwithanincreaseinmacrophageSA.Ourfindingsindicatethatante-mortemserumELISAcouldbeusedasapredictorofmacrophageandMAPpresenceinthetargettissueforinfection.Additionally,macrophageSAcouldinturnbeusedasapredictorofcowstatus.Themodelspresented inthisstudyprovidedefinitive informationontheactivepathogenesisofdiseaseandhowthisisreflectedinante-mortemandpost-mortemdiagnostictests.

CRWAD2017 ABSTRACTS


195-Non-pathogenicbacteriaintheudderandtheireffectonsomaticcellcountE.Severson,J.Ciriacks,K.Poulsen,N.Aulik.WisconsinVeterinaryDiagnosticLab(WVDL),Madison,WI,[email protected]:BacterialDiseasesinCattle,Room8,12/4/20173:00PMThepurposeofthisstudywastoassesstheimpactofnon-pathogenicbacteriasuchasnon-aureusStaphylococcusonindividualcowsomaticcellcounts(SCC)incomparisontomastiticpathogens.Agroupof76Holsteincowsweresampledbi-monthlyoverthecourseof12weeks.Multiparouscows,<300days-in-milkwereselectedandgroupedintohigh,medium,orlowSCCgroupsbasedonmonthlymilksamples.Quarter-levelsampleswerecollectedandcultureswereset-uponblood,eosinmethyleneblue,TKT,andMycoplasmaagars.AllgrowthwasenumeratedandcolonieswereidentifiedbyMALDI-TOFMS.Aftermicrobialcolonizationwasidentifiedforeachsample,theyweregivenaspeciesrichnessscore(SRS)basedoneachnon-pathogenthatwasidentified.AtotalSRSwasgivenforeachmonthandthesamplewas furthercategorized forpresenceofpathogenicbacteria.ThetotalSRS for thoseeachmonthwas thencomparedtotheindividualSCCcollectedimmediatelyfollowingthetwosamplecollectiondates.Separatelinearregressionmodelswerefitforeachofthethree,twoweekperiods.TotalSRSwascomparedtotheSCCfromthatmonthwithlogSCCastheresponse(y)variableandtotalSRSasthepredictor(x)variable.Separatelineswerefitforpathogenicandnon-pathogeniccowswithineachmonthandtheselineswerecomparedusingstatisticalsoftware(R).Theresultsshowedwhenthereismorediversityinthemilksample(highSRS),theSCCincreasesaswell,regardlessofwhetherapathogenispresentintheudder.ThisindicatesthatwhenacowiscategorizedasahavingahighSCC,shemaynotactuallybeinfectedwithapathogen,whichcontradictscurrentdogma.Thisfindinghaspotentialforimmediateimpactfortreatmentandcullingdecisionsonthedairy.Inconclusion,thisstudyconfirmedthatahighSCCisnotalwaysassociated with intramammary infection and current treatment protocols based on high SCC alone may need to be adjusted toaccommodatehigherSCCcowsthatdonothaveaninfection.196-MoraxellabovoculipilinsequencesimilarityamongstgeographicallydiverseisolatesJ.Angelos1,K.A.Clothier2,R.L.Agulto1,B.Mandzyuk1,M.Tryland3.1Dept.ofMedicineandEpidemiology,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis,CA,USA,2Dept.ofPathology,MicrobiologyandImmunology,SchoolofVeterinaryMedicine,Universityof California, Davis, CA, USA, 3Dept. of Arctic and Marine Biology, UiT - Arctic University of Norway, Tromsø, [email protected]:BacterialDiseasesinCattle,Room8,12/4/20173:15PMTheetiologicagentofinfectiousbovinekeratoconjunctivitis(IBK;pinkeye)haslongbeenconsideredtobeMoraxellabovis,however,MoraxellabovoculiisnowfrequentlyidentifiedineyeswabsfromIBK-affectedcattle.AlthoughacausalassociationbetweenMbovoculiandIBKhasnotyetbeenproven,therecentreleaseofacommercialMbovoculibacterinintheUnitedStatessuggeststhatvaccinemanufacturerspossesschallengemodelsforIBKassociatedwithMbovoculi.LikeMbovis,MbovoculiencodesanRTXtoxin(cytotoxin);inMbovisthiscytotoxinisrequiredforpathogenesis.Alongwithcytotoxin,thepathogenesisofMbovisrequirestheexpressionofcellsurfacepilithatarenecessaryforadherencetothecornealepithelium.SevendifferentpilusserogroupswerepreviouslydescribedinMbovis,andpilindiversityhasbeenlinkedtolackofefficacyofsomeMbovisvaccines.Toinvestigateifthereissimilarityamongstpilingenes/deducedaminoacidsequencesfromgeographicallydiverseMbovoculi,oligonucleotideprimersforasuspectedpilingeneweredesignedbasedonthepreviouslypublishedwholegenomesequences(WGS)ofMbovoculi.PrimerswerethenusedtoamplifygenomicDNAextractedfromMbovoculiisolatedfromIBK-affectedcattleintheWesternUSAduring2002andbetween2005-2017andtheresultingampliconsweresequenced.ThededucedaminoacidsequencesoftheMbovoculipilingenewasnearly identicalacross85isolatestestedandalignedwellwithpilinsequencesestablishedfromthepublishedWGSofMbovoculifromIBK-affectedcattleinthemid-WesternandEasternUnitedStates.ThiscommonpilinsequencewasnotablylesssimilartopilinsequencesderivedfromthepublishedWGSofMbovoculithathadbeenisolatedfromnasopharyngealswabscollectedfromIBKasymptomaticcattle.TheamplifiedpilingenewasconservedamongstgeographicallydiverseMbovoculi,however,therearedifferencesamongstpilingenesbetweenMbovoculifromIBK-affectedandIBKasymptomaticcattle.AdditionalresearchisnecessarytofurthercharacterizetherolethatMbovoculiandMbovoculipilinhasinIBKpathogenesis.

CRWAD2017 ABSTRACTS


197-InsilicoanalysisofActinobacilluspleuropneumoniaeexotoxins,ApxIA,-IIA,-IIIAand-IVAM.Oh,S.Kim,H.Yoo.Departmentof InfectiousDiseases,CollegeofVeterinaryMedicine,SeoulNationalUniversity,Seoul,Korea,[email protected]:BacterialDiseasePathogenesis,Room8,12/4/20174:00PMActinobacillus pleuropneumoniae(APP) is themajor etiological agent causing porcine pleuropneumoniae, a highly contagious andsevererespiratorydiseaseofswineposinggreateconomicalthreatinswinerearingindustryworld-wide.Ofthevirulencefactors,agroupofApxAstructuralexotoxinsisreportedascloselyrelatedimmunogenicdeterminantsinpathogenicityofthediseasethatcanalsobepotentialdiagnosticmarkersorevenvaccinecandidates.TheAPPtoxinsarecharacterizedbythe4differentRTXexotoxins(ApxIA,ApxIIA,ApxIIIA,andApxIVA),whichuponinfection,triggertheadaptiveimmunityinswinetoinduceantibodyproduction.Basedonthebioinformaticsanalysis,antigenicepitopeswerepredictedcomputationallytomanufacturethepartialrecombinantproteins.PhysiochemicalpropertiesofApxAexotoxinswerecomputedbydifferentweb-basedtools.GenomeNetandDompredserverswereusedtoidentifytheproteindomainswithintheApxAstructuraltoxinsandpredictthedomainboundaries.ThreedimensionalstructuresofApxAexotoxinswerepredictedbytheIterativeThreadingASSEmblyRefinement(I-TASSER)program.Predicted3-Dmodelswerefurther validated by ProSA program. Antigenic propensity was tested by the Kolaskar and Tongaonkar Antigenicity analysis fromImmuneEpitopeDatabase(IEDB).Basedonthe3-DmodellingofApxAtoxinsandtherespectiveproteindomainswithin,structuralandfunctionalcharacterizationswereattemptedthatmightaidinunveilingthepathogenicmechanismsofAPPinfection.Inrecentstudies,theinsilicobioinformaticsapproachisconsideredasastandardandpromisingmethodthatdeliversinvaluableinsightstoclinicaltrialsespecially indesigningspecific immunogenicepitopes. In thisstudy, insilicocharacterizationsofApxAexotoxinswereexecutedbyseveralweb-basedbioinformaticstoolstodiscoverthediagnosticagentsagainsttheAPPinfection.ThisworkwassupportedbyQIA(No.Z-1543081-2016-17-02),BK21PLUSandRIVS,SeoulNationalUniversity,Seoul,Korea.198-TheStreptococcussuisserotype2surfacelipoproteins,butnotthelipoteichoicacids,areimportantactivatorsoftheinnate

immuneresponseJ.-P.Auger1,N.Gisch2,D.Roy1,M.Gottschalk1.1FacultyofVeterinaryMedicine,UniversityofMontreal,Saint-Hyacinthe,QC,Canada,2Division of Bioanalytical Chemistry, Leibniz-Center for Medicine and Biosciences, Borstel, [email protected]:BacterialDiseasePathogenesis,Room8,12/4/20174:15PMStreptococcussuisserotype2isanimportantporcinebacterialpathogenandzoonoticagentresponsibleforsuddendeath,septicshock,andmeningitis.Alongsidepeptidoglycan,lipoteichoicacids(LTAs)aremajorconstituentsoftheGram-positivebacterialcellwallthatwerepreviouslysuggestedtocontributetotheS.suisvirulence.Moreover,lipoproteins(LPs),whichareoftenco-purifiedwithLTAs,aresurfaceproteinsthathavebeendescribedascontributorstothevirulenceofcertainpathogenichumanstreptococci.However,theimmunostimulatorypropertiesofS.suisLTAs,takingintoaccountthepotentialpresenceofco-purifiedLPs,havenotbeeninvestigatedindetail.Herein,LTApreparationspurifiedfromS.suisserotype2strainsofdifferingoriginsandvirulencewereusedtostimulatemurinedendriticcells(DCs),whichareinnateimmunecellsimplicatedintheresponseagainstS.suisinfection.ResultsdemonstratedthatLTApreparationsfromS.suisstrainsinducedimportantanddose-dependentlevelsofthepro-inflammatorymediatorsTNF,IL-6,CCL3,andCXCL1fromDCs.Inordertoevaluatetheroleofco-purifiedLPs,preparationsweretreatedwithH2O2,whichoxidizesLPstorender them biologically inactive. Treatment resulted in a near complete abolition of pro-inflammatory mediator production,suggestingthatco-purifiedLPsarethemainsourceofDCactivation.Inaccordance,Toll-likereceptor(TLR)2-deficiency,whichisthemainreceptorinvolvedinrecognitionofbacterialLPs,resultedinacompleteabrogationofcytokineproduction.Furthermore,LTAsisolated from isogenic S. suismutants deficient for the prolipoprotein diacylglyceryl transferase (Lgt), an enzyme involved in LPprocessing required for recognition by TLR2, did not induce pro-inflammatory mediators from DCs. In conclusion, this studydemonstrates that the S. suis LPs are almost exclusively responsible for pro-inflammatory mediator production by DCs throughactivation of TLR2, unlike the LTAs, which possess little immunostimulatory properties themselves. Future studies include thecharacterizationoftheroleofLPsintheS.suispathogenesisandinfection.

CRWAD2017 ABSTRACTS


199-Sub-MICconcentrationsofcommonlyusedantibioticsinthelivestockindustryeffectformationofStreptococcussuisbiofilmsU.Waack,T.Nicholson.USDA,Ames,IA,[email protected]:BacterialDiseasePathogenesis,Room8,12/4/20174:30PMRespiratorydisease,includinginfectionscausedbytheGrampositivebacteriaStreptococcussuis,isagreateconomicburdentotheswineindustryduetotreatment,morbidity,andmortalitycosts.S.suiscancolonizetheupperrespiratorytractofswineandcanspreadsystematically.Diseaseisusuallycharacterizedbyfever, lameness, lesions,andmeningitis.Standardtreatmentformanyproducerswhenfacedwithapigshowingsignsofanyrespiratorydiseaseistotreatallpigsinthesamepenorbarnasasymptomaticpigwiththeappropriateantibioticsbyaddingtheantibiotictofoodorwater.Sub-minimalinhibitoryconcentrations(sub-MIC)ofantibioticscanalterbacterialgeneexpressionandsubsequentphenotype,impactingand/oralteringtheabilityofbacteriatocolonize,causedisease,andpersistwithintherespiratorytract.WehypothesizedthatS.suisstrainspresentintheupperrespiratorytractwilldemonstrateachangeinbiofilmformationwhensubjectedtosub-MICsofantibiotics.WedeterminedthebasalbiofilmformationofseveralclinicalstrainsofS.suiswithvariousdegreesofvirulence.S.suisISU2912showedconsistentstaticbiofilmformationusingastandardcrystalviolet assay. Using, a 96-well plate MIC protocol, we determined the MIC for each antibiotic for ISU2912. Several antibioticsdemonstrated an increase in biofilm formation at sub-MICs of antibiotics compared to the control. Future work includes furthercharacterizationofthebiofilmsformedundersub-MICconcentrationsusingaflow-cellbiofilmassay.Collectively,ourdatawillinformusaboutthecollateraleffectsofantibioticexposuretheinductionofbiofilmformationofS.suis.200 - Matrix metalloproteinase-7 and other molecules involved in cellular proliferation and inflammation are associated with

lawsoniaintracellularisinfectioninpigsF.L.L.Leite1,E.Vasquez1,F.Vannucci1,J.E.Abrahante2,A.Rendahl1,J.Torrison1,A.Mueller3,N.Winkelman3,C.Gebhart1,Z.Rambo4,R.Isaacson1.1UniversityofMinnesota,StPaul,MN,USA,2UniversityofMinnesota,Minneapolis,MN,USA,3SwineServicesUnlimitedInc,Rice,MN,USA,4ZinproCorporation,EdenPrairie,MN,[email protected]:BacterialDiseasePathogenesis,Room8,12/4/20174:45PMLawsoniaintracellulariscausesporcineproliferativeenteropathy(PPE).Thisisanentericdiseasecharacterizedbythickeningofthewallof the terminal ileumdue toenterocytehyperplasiawhich leads todecreasedanimalproductionanddiarrhea.Themechanismofenterocyte hyperplasia and the hostmucosal immune response remain largely unknown. In this study, we investigated the hostresponsetoL.intracellularisinfectionbyperformingRNAseqfromsamplesofintestinalmucosa.Groupsofsixinfectedandnon-infectedanimalswereeuthanizedat14,21and28dayspostchallenge.Thepeakofinfectionwasobservedat21dayspostchallenge,whenthegreatestnumberofanimalshadseverelesionsandgreaterbacterialburden,asmeasuredbyH&Estainandimmunohistochemistry,respectively.Atthistimepoint,therewere22differentiallyexpressedgenes(DEG)comparinginfectedandnon-infectedanimalsand494DEGcomparinganimalswithsevereandmoderatelesionstothosewithmild lesions.SeveraloftheDEGwereassociatedwithinflammationandcellularproliferation.TheseincludeMMP7,OSM,IL1BandTGM2.Aclassicinflammatoryresponse,however,isnotobservedsincecellularinfiltrationisnotacharacteristicofPPElesions.Thisstudyforthefirsttimedemonstratestheup-regulationofgenesinvolvedincellularproliferationandinflammatoryresponseswithPPEandshouldbestudiedfurthertobetterunderstandthehostresponsetoL.intracellularisinfection.

CRWAD2017 ABSTRACTS


201-NovelsmallmoleculecompoundswithantimicrobialactivitiesagainstavianpathogenicEscherichiacoliD.Kathayat1,Y.Helmy1,L.Deblais2,C.Logue3,L.Nolan4,G.Rajashekara1.1Dept.ofVeterinaryPreventiveMedicine,TheOhioStateUniversity, Wooster, OH, USA, 2Dept. of Plant Pathology, The Ohio State University, Wooster, OH, USA, 3Dept. of VeterinaryMicrobiologyandPreventiveMedicine,IowaStateUniversity,Ames,IA,USA,4CollegeofVeterinaryMedicine,UniversityofGeorgia,Athens,GA,[email protected]:BacterialDiseasePathogenesis,Room8,12/4/20175:00PMAvianpathogenicE.coli(APEC)isoneofthemostcommonbacterialpathogensaffectingchickens,turkeys,andotheravianspecies.Itcausesmultipleextraintestinaldiseaseswhichsubsequentlyresultsinsubstantialeconomiclossestothepoultryindustryworldwide.Currently,APECinfectionsarecontrolledbyantimicrobialsmedicationandvaccination.However,theemergenceofmulti-drugresistantAPEC serotypesand limitedvaccineefficaciesnecessitates theneed fornovelAPECcontrol approaches.Here,we screenedapre-selectedenrichedsmallmolecules(SMs)librarycontaining4,182SMsforidentificationofnovelnarrowspectrumanti-APECSMgrowthinhibitorswithnovelmechanismsofaction.Atotalof41SMswereidentifiedinhibitingthegrowthofAPECO78at100µM.Amongthem, 11 bactericidal compoundswere selected for further studies. These selected 11 SMswere effective againstmultiple APECserotypes(O1,O2,O8,O15,O18,O35,O109,andO115).Sixof11SMsexhibitednarrow-spectralactivityspecificonlytoAPECserotypesandaffected1-3testedcommensal/probioticbacteria(n=12).ExceptSM11,otherSMswereleasttoxic(<10%)toCaco-2epithelialandHD11macrophagecellsat200μM.SevenoftheseSMswereleasthemolytic(<10%)tochickenredbloodcellsat200μM.All11SMssignificantly(P<0.01)reducedtheintracellularsurvivalofAPECO78,O1,andO2inCaco-2,HD11,andTHP-1cellsatconcentrationsrangingfrom1Xto2XofMICs.TreatmentofAPECO78atlethal(2XMBC)andsub-lethal(0.75XMIC)concentrationofSMsrevealednoresistant colonies. Preliminary studies revealed the SMs efficacy against APEC pre-formed biofilm and synergistic interactionwithconventional antimicrobials.Under invivo evaluationof SMs inwaxmoth (Galleriamellonella) larvae, SMs treatment significantly(P<0.0001)extendedthesurvivalofinfectedlarvae(exceptSM8)andsignificantly(P<0.05)reducedtheAPECloadinsidethelarvae(exceptSM8andSM9).OurfuturestudieswillfocusoninvestigatingefficaciesoftheseSMsininfectedchickensandelucidationoftheirmechanismsofaction.202-EvaluationofclinicalsignsasearlyhumaneendpointsinthehamstervaccinationchallengepotencymodelforLeptospiraA.Walker, R. Olsen,M. Toth, L. Ludemann. Center for Veterinary Biologics, National Centers for Animal Health, Ames, IA, [email protected]:BacterialDiseasePathogenesis,Room8,12/4/20175:15PMThehamsterhasbeenananimalofchoiceforLeptospiraresearch,andregulatorypotencytestsforleptospiralvaccineshavehistoricallybeenperformedbythecodifiedhamstervaccination-challengeassaysdescribedinTitle9,CodeofFederalRegulations(9CFR),Parts113.101-113.104intheUnitedStates.DeathormoribundityistheendpointforthecodifiedregulatorytestsinaccordancewiththeCenterforVeterinaryBiologic’s(CVB)Notice12-12.Theuseofclinicalsignsasearlyhumaneendpointsforregulatorypotencytestingwould be a boon to animal welfare if they correlated to current endpoints, but information on their predictive power is largelyanecdotal.Here,therelationshipbetweenthreeclinicalsigns(epistaxis,hematuria,andneurologicataxia)todeathormoribunditywasmeasured in the regulatory testing of Leptospira (L.) serogroups Canicola, Icterohaemorrhagiae, Pomona, and Grippotyphosa.Specifically, vaccinates (n=10/serial), challenge controls (n=10), and back-titration hamsters (n=20) were inoculated with virulentLeptospira. Presence of clinical signs or death/moribundity was evaluated and recorded twice daily for each hamster. A positivecorrelationwascountedifahamstersuccumbedtodiseasewithin24hoursofobservationofasymptom,andanegativecorrelationwascountediftheanimaldidnotsuccumbtodiseasewithin24hoursofobservationofthesymptom.Survivingsymptomatichamsterscouldhavereoccuringsymptomscountedafterthe24hourobservationalperiodended.Atotalof1283symptomswereobservedin1,661 hamsters. Themost commonly observed symptomswere epistaxis and hematuria at 80% and 13% of observed symptomsrespectively,buttheirpresencedidnotcorrelatetodeathfor62.5%ofepistaxisoccurrencesand17.4%ofhematuriaoccurrences.Neurologic ataxia constituted only 7% of all observed symptoms; and among those occurences, itwas not predictive of death ormoribundity3.3%ofthetime.Notably,noclinicalsignswereobservedin31.6%ofhamstermortalities.Clinicalsignsmaybepredictiveofcertainstagesofleptospirosisprogression,buttheycannotpositivelypredictlossoflife.

CRWAD2017 ABSTRACTS


203-Molecularandepidemiologicalcharacterizationofarespiratorydiseaseoutbreakinpre-weanedbeefcalvesassociatedwithbovinecoronavirus

A.M.Workman1,T.McDaneld1,L.Kuehn1,M.Clawson1,J.Loy2.1Genetics,Breeding,andAnimalHealth,USDA,ARS,USMeatAnimalResearchCenter,ClayCenter,NE,USA,2NebraskaVeterinaryDiagnosticCenter,SchoolofVeterinaryMedicineandBiomedicalSciences,UniversityofNebraskaLincoln,Lincoln,NE,[email protected]:RespiratoryDiseaseinCattle,Room8,12/5/20179:00AMBovinecoronavirus(BCV)isassociatedwithrespiratorytractinfectionsincattleofallages;however,atemporalstudytoevaluatetheeffectofBCV immunityonvirus sheddingandbovine respiratorydisease (BRD) incidence inpre-weanedbeefcalveshasnotbeenreported.Thus,wereporthereaprospectivestudyinthreeherdsofcrossbredbeefcalves(n=817)withendemicBCV.Serialbloodsamplesformeasurementofserumanti-BCVantibodytitersandnasalswabsfordetectionofBCVandotherviralandbacterialBRDpathogensbyreal-timePCRmethodswerecollectedfromallcalvesorsubsetsofcalvesatpredeterminedtimesfrombirththroughweaning.Thecalvesweremonitored forBRDandthose thatdevelopedrespiratorydiseaseweresampledandtested forcommonrespiratorypathogens.Twohundredforty-eightofthe817studycalves(30.4%)weretreatedforBRDpriortoweaning;246ofthosewerefromasingleherdinvolvedintwomasstreatmentevents.MoleculardiagnostictestingrevealedthatBCVsheddingoccurredinconjunctionwith the pre-weaning BRD outbreaks in that herd. However, between herd analyses revealed that levels of passively(maternal)oractivelyacquiredanti-BCVantibodiesdidnotassociatewiththeincidenceofpre-weaningBRDorBCVshedding.Thus,toaccountforpotentiallyconfoundingfactorsthatcouldhaveinfluencedBRDdevelopmentintheseherds,sequencingandphylogeneticanalysisoftheBCVstrainscirculatingineachherd,andtheprevalenceandrelativeabundanceofbacteriainthenasopharynxofsickandapparentlyhealthycattlewerealsoevaluated.MycoplasmaspeciesandHistophilussomniwereidentifiedinhighabundanceinthenasopharynxofcattlewithBRDbutnotinapparentlyhealthyanimals.OurresultsindicatethatBCVinfectionwasassociatedwithbothsubclinicalandclinicaldisease;howeverserumanti-BCVantibodytierswerenotassociatedwithdiseaseincidence.Co-infectionwithMycoplasmasp.andHistophilussomnilikelycontributedtothediseaseoutbreakcharacterizedinthisstudy.204-Bovineherpesvirustype1(BHV-1):comparativeanalysisandphylogeneticrelationshipbetweentherespiratory(BHV-1.1)and

genital(BHV-1.2b)strainsofthevirusJ.M.d'Offay1,M.Fishbein2,R.Eberle1,R.W.Fulton1.1VetPathobiology,OklahomaStateUniversity,Stillwater,OK,USA,2PlantBiology,Ecology,andEvolution,OklahomaStateUniversity,Stillwater,OK,[email protected]:RespiratoryDiseaseinCattle,Room8,12/5/20179:15AMPurpose:Bovineherpesvirussubtype1.1 (BHV-1.1)causes infectiousbovinerhinotracheitis (IBR).Thediseasewas first reported inCaliforniaandColoradofeedlotcattleintheearly1950s.Becauseofitscloseantigenicrelationshiptothegenitalsubtype(BHV-1.2b),itwaspostulatedthattherespiratoryIBRvirushademergedfromBHV-1.2bthatcausedinfectiouspustularvulvovaginitis(IPV)incattle.Usingcompletegenomeanalysis,wecomparedthephenotypicandevolutionaryphylogeneticrelationshipbetween4genitaland7respiratoryBHV-1isolatesofBHV-1.Methods:ViralDNAweresequencedusingIlluminawholegenomesequencing.ThecompleteviralgenomeswereanalyzedandcomparedusingtheCLCMainWorkbenchprograms.TheDNAsequencefor43concatenatedgenesfromall11viruseswerealignedandcomparedphylogeneticallyusing theGeneiousandBEASTprograms.Results:Onaverage,a97.5%geneticidentitywasnotedbetweenthegenomesoftherespiratoryandgenitalstrains.Comparingthehomologousproteinsencodedby the respiratoryversus thegenital strains,wenotedahighdegreeof conservationwithAAsimilarities ranging from94 -100%.Interestingly,wefoundthat14ofthe16mostdiversehomologousviralproteinsareinvolvedinvirionegressfrominfectedcells.TheseincludedpUL31andpUL34 that formthenuclearegresscomplexduringprimaryenvelopment,andpUS3 thatphosphorylates thiscomplexduringde-envelopment.Thephylogeny showed twowell-supported, longdivergedclades,oneconsistingof the4genitalisolatesandtheothercontainingtheremainingrespiratoryisolates.Themediandivergenceageforthese2cladeswas1094yearsonaverage(779-1497years).Conclusions:Becausethevirusdiverged1000ago,itishighlyunlikelythatthemorevirulentIBRvirusevolvedforlessvirulentgenitalstrain.ItismorelikelythatIBRvirusevolvedfromcattlealreadylatentlyinfectedwitha(lessvirulent?)BHV-1.1.Itisreasonabletoassumethatselectivepressure,broughtaboutbyfeedlotpracticesinthelate40’sandearly50’s,favoredmutantswithanincreasedabilitytobetransmittedbyaerosol,resultinginenhancedviralvirulence.

CRWAD2017 ABSTRACTS


205-Isolationofanaturallyoccurringvaccine/wild-typerecombinantbovineherpesvirustype1(BHV-1)fromanabortedbovinefetus

J.M.d'Offay1,R.W.Fulton1,E.J.Dubovi2.1VetPathobiology,OklahomaStateUniversity,Stillwater,OK,USA,2AnimalHealthDiagnosticCenter,CornellUniversity,Ithaca,NY,[email protected]:RespiratoryDiseaseinCattle,Room8,12/5/20179:30AMPurpose:Bovineherpesvirustype1(BHV-1)causesvariousdiseasesyndromesincattleincludingbovinerespiratorydisease(BRD)andabortions.RecentstudiesinvolvingwholegenomesequencinghaveallowedustodeterminethedifferentBHV-1genotypesassociatedwiththesediseasesyndromes.ThepurposeofthisstudywastoexpandthisinvestigationusingBHV-1virusesisolateddecadesagofromabortionandBRDcases.Methods:Wesequenced6archivedBHV-1viruses isolated in1978at theAnimalHealthDiagnosticCenteratCornell,NY.Threewereisolatedfromabortedbovinefetusesand3fromcattlewithBRD.TheviruseswerepropagatedonMDBK cells and viral DNA isolated for sequencing using Illuminawhole genome sequencing. The 6 complete viral genomeswereanalyzed and compared to the genomes of different vaccine viruses andwild-type BHV-1 strains using the CLCMainWorkbenchprograms.Results:Wholegenomesequencingidentified3ofthevirusesasBHV-1vaccineviruses.OnewasisolatedfromanabortedfetusandtwofromBRDcases.Thegenomesofthese3vaccinevirusesareuniqueinthattheyshareanumberofuniqueSNPsandlackaUS2gene.Twootherviruses,onefetalandonerespiratoryisolate,containeduniqueSNPsthatidentifiedthemaswild-typeBHV-1viruses.The sixthvirus,a fetal isolate,wasa recombinantviruswithgenomic sequencesderived frombothwild-typeandvaccineviruses.Theuniquelong(UL)sequenceofthatrecombinantvirusmatchedthewild-typevirusgenomeexceptforaregionencompasingthegenesUL13throughUL19whichcontained5SNPsonlypresentinthevaccinevirusgenome.Theuniqueshort(US)regionoftherecombinantvirusmatchedtheUSregionofthevaccinevirusgenomewithitsdeletedUS2gene.Conclusions:Althoughithasbeenpostulatedtooccur,thisisthefirstconfirmatoryevidencethatanattenuatedBHV-1vaccineviruscanrecombinewiththewild-typeBHV-1virusundernaturalconditionsandcausedisease.Thestudyalsoconfirmspreviouslyreportedobservationsthat infectionofcattlewitheitherwild-typeBHV-1orattenuatedBHV-1vaccinevirusescanresultinbovineabortionandBRDincattle.206-Evaluationofiodidesupplementationtodecreaserespiratorydiseaseinpre-weaneddairycalvesM.C. Heller1, L. Gamsjaeger2. 1Veterinary Medicine and Epidemiology, University of California Davis, Davis, CA, USA, 2VeterinaryMedicineTeachingHospital,UniversityofCaliforniaDavis,Davis,CA,[email protected]:RespiratoryDiseaseinCattle,Room8,12/5/20179:45AMInnateairwaydefensesarecrucialtopreservingthehealthofbovinelungs.Stress,coupledwithviralinfections,compromisesnaturalhostdefensesandallowbacteriatoreachthelowerairwaysandcausepneumonia.Innatedefensesincludeairwaymucusandciliatedepithelium,thattrapandphysicallyremovepathogensfromtherespiratorytract,andsecretedantimicrobialpeptidesthatneutralizepathogens.Augmenting innatemucosaldefensemechanismswith iodine iseffectiveatkillingbovinebacterialandviralrespiratorypathogens in vitro. The overall goal of this studywas to determinewhether NaI treatment could diminish respiratory disease ordecreaseantimicrobialtreatmentsinpre-weaneddairycalves.Pre-weaneddairycalves(n=428)atamixedsourcecalfranchwereandrandomlyassignedtotreatmentorcontrolgroupsat20(±2)daysofage.ThetreatmentgroupwasadministeredNaIorallyatDay0andDay4.CalvesreceivedarespiratoryscoreandlungultrasoundscoreatenrollmentandatDay7.Illness,treatmentdataandweaningweightswereharvestedfromanon-farmreal-timedatacollectionsystem(HealthSum).Contrarytoexpectations,treatedcalveshadworserespiratoryandultrasoundscoresonDay7,andslightlymoretreatmenteventsoverthestudyperiod.Differencesbetweengroupsweresmallbutstatisticallysignificant.Wehypothesizethatiodidemayhaveunintendedeffectsonnormalrespiratoryflora,creatingmoreadvantageousconditionsforpathogens.

CRWAD2017 ABSTRACTS


207-CharacterizationofpolymicrobialbiofilmformationbyPasteurellamultocidaandHistophilussomniinvitroandduringbovinerespiratorydisease

B.L.Petruzzi1,T.J. Inzana1,A.Molinaro2,C.DeCastro3. 1BiomedicalandVeterinarySciences,VirginiaMarylandRegionalCollegeofVeterinary Medicine, Blacksburg, VA, USA, 2Department of Chemical Sciences, Università di Napoli Federico II, Naponi, Italy,3DepartmentofAgriculture,UniversitàdiNapoliFedericoII,Napoli,[email protected]:RespiratoryDiseaseinCattle,Room8,12/5/201710:00AMHistophilus somni and Pasteurella multocida cause bovine respiratory disease (BRD) and systemic infections in cattle. Followingrespiratory infectionofcalveswithH.somni,P.multocida isalsooften isolatedfromthe lowerrespiratorytract.BecauseH.somninormallyformsabiofilmduringBRD,P.multocidamayco-existwithH.somniinapolymicrobialbiofilm.Wesoughttoexaminetheinteractivenatureof the two speciesduringbiofilm formation in vitro and in vivo.H. somniwasgrownasabiofilm for3days,P.multocida added, and the culture incubated for 2 additional days. Interactions between the two species in the biofilm werecharacterized and quantified by fluorescence in situ hybridization (FISH), and the biofilm matrix of each species examined byfluorescently-taggedlectins(FTL),confocalscanninglasermicroscopy,crystalvioletstaining,andchemicalassays.Bacterialinteractionswere determined by auto-aggregation and biofilmmorphology. Exopolysaccharide (EPS) produced during biofilm formation by P.multocidawaspurifiedandanalyzedbygaschromatography-massspectrometry.FISHandFTLwereusedtoshowthatP.multocidaandH.somniwereevenlydistributedintheinvitrobiofilm,andbothspeciescontributedtothepolymicrobialbiofilmmatrix.COMSTATz-stackimageanalysisrevealedthattheaveragebiomassandbiofilmthickness,andthetotalcarbohydrateandproteincontentofthebiofilm,weregreatestwhenbothspecieswerepresent.Polymicrobialbacterialsuspensionsauto-aggregatedfasterthansinglespeciessuspensions,suggestingphysicalinteractionsbetweenthetwospecies.EncapsulatedP.multocida isolatesnotcapableofformingabiofilmstillformedapolymicrobialbiofilmwithH.somni,butonlytheEPSofH.somnicouldbedetectedbyFTLstainingofbovinetissuesfromwhichbothspecieswereisolated.Bacteriawithinabiofilmaremorequiescentthanduringplanktonicgrowthandinducelessofaninflammatoryresponse,indicatingencapsulatedP.multocidamaytakeadvantageoftheH.somnibiofilmtopersistinthehostduringlesssevere,butmorechronic,BRD.TheseresultsmayhaveimportantimplicationsforthemanagementofBRD.208-Validationofareal-timePCRassayforAvibacteriumparagallinaruminclinicalsamplesduringanoutbreakinvestigationK.A.Clothier1, S. Stoute2,B.Crossley1. 1CaliforniaAnimalHealth&FoodSafetyLab,UniversityofCalifornia,Davis,Davis,CA,USA,2CaliforniaAnimalHealth&FoodSafetyLab,Turlock,UniversityofCalifornia,Davis,Davis,CA,[email protected]:RespiratoryDisease,Room8,12/5/201710:45AMPurpose:Avibacteriumparagallinarum,thecauseofInfectiousCoryza,causesrespiratorysignsinchickensandexacerbatesdiseasecausedbyotherpathogens. Recently, strainsofA. paragallinarum havebeenassociatedwithmore severediseaseoutbreaks andmortalityinaffectedbirdpopulations.Duetoitsfastidiousnature,needforspecializedgrowthconditions,andnegativebiochemicaltestprofiles,thisorganismisdifficulttorecoverandidentify,particularlyfromlocationswhicharecolonizedbybacterialflora.StandardPCRmethodshavebeenusedtoconfirmtheidentityofthisbacterium,butthesemethodsarelabor-intensiveandlesssensitivethanreal-timePCRandnotfeasibleforhigh-throughputtestingnecessarytoinvestigatelarge-scalerespiratorydiseaseoutbreaksinpoultryoperations.Thepurposeofthepresentstudywastovalidateareal-timePCRmethodfordetectionofA.paragallinarumthatcanbeperformeddirectlyonsinusswabsamplesandtoevaluateitsutilityinoutbreakinvestigations.Methods:PrimerandprobesequencestargetingtheHPG-2regionwhichhasbeenfoundtobespecificforA.paragallinarumwereusedforPCRreactions,whichincludedaninternalamplificationcontroltoidentifyPCRinhibitionleadingtofalsenegativeresults.Clinicalsamplescollectedfromchonae(livebirds)orinfraorbitalsinuses(collectedatnecropsy)wererinsedin1.0mlPBSandextractedforPCR.Results:ThePCRassaydetectedfourA.paragallinarum(ATCC29945andthreeclinical)isolateswithaLODof10cfu/mlandaPCRefficiencyof98.6%.Cross-reactionwas not detected with 33 non-A. paragallinarum, closely-related Pasteurellaceae including A. volantium, A. gallinarum, andAvibacterium sp. The assay was able to detect A. paragallinarum in 65 clinical samples with 100% agreement compared with aconventionalPCRassay,including38samplesthatwerealsopositivebyculture;noproductwasdetectedfrom34samplesfrombirdswithoutrespiratorysymptoms.Conclusions:ThepresentstudydescribesausefulassayfacilitatingrapidandaccuratedetectionofA.paragallinaruminoutbreakinvestigationsandroutinemonitoring.

CRWAD2017 ABSTRACTS


209-EffectsofmacrolideandrifampinresistanceoninvitrogrowthofRhodococcusequiJ.M.Willingham-Lane,S.Giguère,L.J.Berghaus.LargeAnimalMedicine,UniversityofGeorgia,Athens,GA,[email protected]:RespiratoryDisease,Room8,12/5/201711:00AMRhodococcusequiistheleadingcauseofseverepneumoniainfoals.Therecommendedtreatmentisdualantimicrobialtherapywithamacrolideandrifampin.WidespreadmacrolideandrifampinresistanceinR.equiisolatesisamajoremergingproblemwithresistantisolatesbeingculturedfromupto40%offoalsatsomefarms.WehaverecentlydemonstratedthatmacrolideresistanceinR.equiisconferredbythemethylasegeneerm(46)encodedbyamobileelement,whilerifampinresistanceisduetoamutationinthebetasubunitof theRNApolymerase (rpoB)gene.Theobjectiveof this studywas todetermine theeffectofmacrolideand/or rifampinresistanceoninvitrogrowthofR.equi.Foreachstrain,triplicatebacterialgrowthcurvesweregeneratedinbrainheartinfusion(BHI)andminimalmedia(MM)usinganautomatedplatereader.Thegrowthofwildtypemacrolide-andrifampin-resistantisolates(n=30)wassimilartothatofsusceptibleisolates(n=30)inBHI.However,thegrowthoftheresistantisolateswassignificantlydelayedandtheirgrowthratesignificantlyreducedinMM.Todetermineifimpairedgrowthwasconferredbyacquisitionofmacrolideorrifampinresistance,differentrpoBmutationswerecreated insusceptibleparentstrainsofR.equi (n=6)andthemobileelementconferringmacrolideresistancewasinsertedinthesame6strains.Insertionofthemobileelementconferringmacrolideresistancehadminimaleffectoninvitrogrowth.However,2of6rpoBmutationscausedasignificantdelayingrowthanddecreasedgrowthrateinBHIwhile5of6rpoBmutationsresultedinasignificantdelayingrowthinMM.Inconclusion,thegrowthofmacrolide-andrifampin-resistantR.equiisdelayedundernutrientrestrictionandthisdelayistheresultofresistancetorifampin.NotallrpoBmutationsconferringrifampinresistanceaffectinvitrogrowthtothesameextent.210-UnderstandingtherespiratorymicrobiomeofcommercialturkeysandchickenlayersJ.M.Ngunjiri1,M.C.Abundo1,H.Jang1,M.Elaish1,M.KC1,A.Ghorbani1,B.P.Youmans2,T.J.Johnson2,C.-W.Lee1.1FoodAnimalHealthResearch Program, TheOhio StateUniversity,WOOSTER,OH,USA, 2Department of Veterinary PreventiveMedicine, University ofMinnesota,St.Paul,MN,[email protected]:RespiratoryDisease,Room8,12/5/201711:15AMViralandbacterialinfectionsoftherespiratorytractoccurthroughthemucosalsurfacesthatarecolonizedwithcomplexcommunitiesof resident microorganisms (microbiome). To enhance our understanding of the contributions of respiratory microbiome in theestablishmentofdiseaseincommercialpoultry,itisimportanttodefinethebaselinerespiratorymicrobiomeofhealthyflocks.Inthisstudy,twoclinicallyhealthycommercialflocksofturkeysandchickenlayersweresampledatdifferentagestoharvestbacteriafromnasalsinusesandtrachea.BacterialcommunityprofilingwasconductedthroughNGSsequencingoftheV4regionofthe16SribosomalRNAgeneanddownstreambioinformaticsanalysis.Taxonomicdiversityofrespiratorybacteriawasgenerallyinfluencedbytheirhabitat(sinusvstrachea),birdage,andbirdspecies.Bacterialcolonizationofthetracheaappearedtobetransientandtherewasnoclearcorrelationbetweenbeta-diversityandage.However,therewasaclearage-dependentclusteringofsinusmicrobiomefromindividualbirds,andbetweenchickensandturkeys.Threeindicesofalphadiversity(Chao1,ObservedOTUs,andphylogeneticdistances)showedthatspeciesrichnessinthesinuswassignificantlyhigherinchickenscomparedtoturkeys(p<0.05).However,onlytheChao1indexshowedasignificantdifferenceintrachealmicrobiomesbetweenthetwotypesofbirds.Severaldifferencesbetweenthepresenceofdifferentbacteriataxainchickensandturkeyswereobserved.Notably,inthesinus,therewasanearlycolonizationandpersistenceofhighlevelsofLactobacillusinchickensandStaphylococcussciuriinturkeys.AlthoughStaphylococcussciurihasnotbeenassociatedwithpoultrydisease,ithasbeenimplicatedinseveralhumandiseases.Ourcomparativestudyhasdemonstratedthattheestablishmentofrespiratorymicrobiomeinclinicallynormalcommercialflocksishighlyinfluencedbythepoultryspeciesamongotherfactors.Dataobtainedfromthecurrentstudywillbeastartingpointforfuturestudiestoidentifyhowtherespiratorymicrobiomeshiftspriorto,andduring,theestablishmentofviralrespiratorydiseases.

CRWAD2017 ABSTRACTS


211-AntimicrobialactivityofbovineNK-lysin-derivedpeptidesonMycoplasmabovisR.P.Dassanayake,S.M.Falkenberg,K.B.Register.USDA,Ames,IA,[email protected]:RespiratoryDisease,Room8,12/5/201711:30AMAntimicrobialpeptides(AMPs)areadiversegroupofmoleculeswhichplayanimportantroleintheinnateimmuneresponseinvariousorganisms,includingcattle.BovineNK-lysins,atypeofAMP,havebeenpredominantlyfoundinthegranulesofcytotoxicT-lymphocytesandNK-cells.CollectiveresultsfromourlabandothershavepreviouslyreportedantimicrobialactivityofbovineNK-lysinsonvariousbacterialpathogens,includingseveralinvolvedinbovinerespiratorydiseasecomplex(BRDC)incattle;however,suchstudiesareyettobeperformedwithoneimportantcontributortotheBRDC,Mycoplasmabovis.Therefore,thegoalofthisstudywastoassesstheantimicrobialactivityofbovineNK-lysinsonM.bovis.Syntheticpeptidescorrespondingtothefunctionalregionofhelices2and3ofthebovineNK-lysinsNK1,NK2A,NK2BandNK2CwereassessedforkillingactivityontwoM.bovisbovineisolates.Amongfourpeptidestested,NK2A-derivedpeptideshowedthehighestantimicrobialactivity,whileNK1-derivedpeptideshowedthe leastantimicrobialactivityagainstbothM.bovisisolatesasdeterminedbyabacterialkillingassay.FlowcytometricanalysisofNK-lysin-treatedM.bovisafterstainingwithalive/deadbacterialviabilityindicator(Syto9/propidiumiodide)suggesteddamagetothecellmembranebasedonstainingofnuclearmaterialwithinthecells.ElectronmicroscopicexaminationofM.bovistreatedwithNK-lysinpeptidesconfirmeddamage to the cell membrane. The results of this study suggest that bovine NK-lysins in general, and NK2A in particular, showantimicrobialactivityagainstM.bovisbydirectlycausingdamagetothecellmembrane.Takentogether,findingsinthisstudyalongwithpreviousstudies,wecannowconcludethatbovineNK2AishighlyeffectiveagainstmostbacterialpathogensinvolvedinBRDC.212-Afield-deployableautomaticnucleicacidextractionandinsulatedisothermalRT-PCRsystemforsensitiveon-sitedetectionof

avianinfluenzaAvirusS. Chung, C.-F.M. Tsai, P.-H. Chou, Y.-L.D. Tsai, H.-F.G. Chang, H.-T.T. Wang, P.-Y.A. Lee. GeneReach USA, Lexington, MA, [email protected]:RespiratoryDisease,Room8,12/5/201711:45AMPurpose:AvianinfluenzaAviruses(AIAV)isanimportantavianpathogenworldwide.On-siteidentificationofAIAVfacilitatestimelydiseasemanagementandcontrol.A field-deployablePCR system,POCKITTMcombo, includesdevices forautomaticNAextraction(tacoTMmini)andinsulatedisothermalPCR(iiPCR)(POCKITTM)hadbeenavailable.PerformanceofanIAVRT-iiPCR(Mgene)onthissystemforAIAVdetectionwasevaluated.Methods:AllsampleswereprovidedbyNationalCenterforVeterinaryDiagnosis,DepartmentofAnimalHealth;andVirologyDepartment,NationalInstituteofVeterinaryResearch,Vietnam.Forextractiontest,26AIAV-positiveoropharyngealswabs(OS)and8AIAV(H5N1/16A59)-spikedtissues(brain,lungandspleen;BLS)weresubjectedtotacoTMDNA/RNAExtraction Kit and RNeasyMini Kit (Qiagen) simultaneously. Six BLS and six OSwere repeated for reproducibility. AIAV RNAwasquantifiedbyapublishedqRT-PCR.ForRT-iiPCRevaluation,AIAVH5N1/16A59andaH7N9wereusedinsensitivityandH3,H4,H5,H6,H9andH10subtypesininclusivitytest.PerformancecomparisonoftheRT-iiPCRandqRT-PCRwasbyparalleltestingof50OSandBLSNApreparedbytacoTMmini.Results:BasedonCtvaluesofAIAVRNAtheNAextracts,tacoTMminiandthereferencemethodhadexcellentagreement-regressioncoefficientsof0.997withtissuesand0.956withswabs.CtsofCV%<2.5%wereobservedwithallsamples,exceptfortwoOS(3.70,7.77%),indicatinggreatreproducibility.IAVRT-iiPCRhaddetectionendpoints(fold-dilution)closetoqRT-PCR(106and105forH5N1,and107and105forH7N9,respectively)andcoulddetectalldifferent IAVsubtypes.Contingencyanalysis(kappatest)showsthat38werepositiveand9werenegativeinbothPCRtests,whilethreewereqRT-PCRnegative/RT-iiPCRpositive,resultingin94%agreement(κ=0.82).Conclusions:ThetacoTMminisystemhadgreatefficiency/reproducibilityfortwomostcommonaviansampletypesandthePOCKITTMsystemhadperformancecomparabletothereferenceqRT-PCR.Thisfield-deployablesystemhasbeendeployedatopenpoultrymarketsforsurveillanceapplication.Resultswillbepresentedattheconference.

CRWAD2017 ABSTRACTS


213-PhylogeneticanalysisofBovineherpesvirus-4isolatesfromdairycowsinCaliforniarevealedhighergeneticdiversityandnovelgenotypes

D.Areda1,M.Chigerwe1,B.Crossley2.1SchoolofVeterianryMedicine,UniversityofCalifornia,Davis,CA,USA,2CaliforniaAnimalHealthandFoodSafetyLaboratory,UniversityofCalifornia,Davis,CA,[email protected]:ViralPathogenesis–1,Room9,12/4/20179:00AMPurpose:ToexplaingeneticcharacteristicsoffieldisolatesofBoHV-4fromdairycowsinCaliforniaandidentifydominantgenotypesthatareassociatedwithpostpartummetritis.Methods:thestudywasconductedinYolo(Davis)andTularedairyfarms.Uterine/vaginalsampleswerecollectedfrompostpartummetritisinfectedcowsforPCRdetectionandsubsequentisolationofthevirusanditsDNA.Sequenceanalysisof10fieldisolatesofBoHV4wasperformedusingstandardPCRproceduresalongwithaPath-IDMultiplexqPCRmastermix(LifeTechnology,Carlsbad,CA).Sequencingtargetedthreegenesfromtheviralgenome(ORF3-encodingthymidinekinase;ORF8-encodingglycoproteinB,andORF22-encodingglycoproteinH).DatawasanalyzedusingGeneious10.2.3.Results:Sixsinglenucleotidepolymorphisms(SNPs)acrosstheTKgenealignmentseparatedtheBoHV4isolatesintotwodistinctgroups:TKGenotype1andTKGenotype2.AtranslationoftheTKgenealignmentshowedtwoaminoaciddifferencesbetweenthetwogenotypes.MembersofTKGenotype1werecloselyrelatedtopreviouslydescribedAmerican(DN599-S49773)andEuropean(Movar-AB035516)strains.TKGenotype2 isolatesweremorecloselyrelatedtoMGA514(EU244699)strain.NucleotideandaminoaciddifferencesacrossthegBgeneweremorepronounced.Thesecorrespondedto22aminoacidsubstitutionsand1aminoacidindel.PhylogeneticanalysisofthegBgenerevealedthreedistinctgroups:gBGenotype1,gBGenotype2andgBGenotype3.ContrarytotheTKgene,theevolutionaryrelationshipsbasedonthegBgeneanalysisshowedthatthethreestrainsD1609492-1.6,D1611430-2.48,andD1611430-1.88weremorecloselyrelatedtoFMV-09(KC999113)strainthantheyweretotheotherfieldstrainsorreferencestrains(DN-599and66-p-347(AF318573).TKgenotype2andgBGenotype1weredominantgenotypesinvolvedinpostpartummetritisindairycowsinCalifornia.Conclusions:Thereportingofhighergeneticdiversitysuggeststhepossibilityofinfectionwithmultiplegenotypes.FurtherinvestigationisneededtodeterminespecificroleofBoHV-4genotypesinpathogenesisofBoHV-4infectionsandpostpartummetritis.214-Theindirecteffectofbovineviraldiarrheavirus(BVDV)onmacrophageinflammatoryfunctionandlymphocyteapoptosisK.Abdelsalam1,G.Elmowalid2,L.Braun1,J.Sobraske1,C.Chase1.1VeterinaryandBiomedicalSciences,SouthDakotaStateUniversity,Brookings,SD,USA,2collegeofveterinarymedicine,ZagazigUniversity,Zagazig,[email protected]:ViralPathogenesis–1,Room9,12/4/20179:15AMPurpose:Inthecurrentstudy,weareconcernedwithinvestigatingthepossiblemechanismsofimmunedysfunctioninducedbyBVDVMethods:Wehypothesized that the infectedmacrophagewith high virulent strain of BVDV could secreteharmfulmediators andsubstancesthatmightdisrupttheneighboringmacrophagesaswellasspecificlymphocytesendingupwithimmunedysfunction.Toexamineourhypothesis,Weinfectedbovinemonocytederivedmacrophage(MDM)withlowandhighvirulentstrainsofBVDVandwecomparedtheeffectofinfectedMDMsupernatantonmacrophagesurfacemarkerexpression,phagocyticactivity,bactericidalactivityaswellasnitricoxide(NO)production.WealsostudiedtheeffectofBVDV-infectedMDMsupernatantontheinductionofapoptosisinepithelialcellsaswellaslymphocytes.Weinvestigatedtheapoptosis-relatedcytokineprofileoftheBVDV-infectedmacrophagesaswellasthepossiblesecretedviralproteinsusingBVDVspecificantibodytostudythepossiblemechanismsofindirectapoptoticeffectofBVDVonlymphocytes.Results:WeshowedthatonlysupernatantfrominfectedMDMwithhighvirulentstrainofBVDVsignificantlydecreasedmacrophagephagocyticactivity,CD14andMHCIIsurfacemarkersexpressionaswellasmacrophagebactericidalactivitybutnotthelowvirulentBVDVstrain.Interestingly,highvirulentBVDVstrainhadadirecteffectonNOproductionbutnotthroughitsinfectedMDMsupernatant(theindirecteffect).Wehavealsofoundthatneitherviralproteinsnorapoptosisrelatedcytokinesseemtoplayanyrole in inductionof lymphoidapoptosis.Conclusion:OurdatasuggestthatBVDVhasan indirecteffectonmacrophagegeneral inflammatory functions that could be related to its inhibitory effect on surfacemarkers expression.We also showed theimportanceof infectedmacrophagewithhighvirulentBVDVinepithelialapoptosisand lymphocytedepletion.Takentogether,ourresults shed the lighton the importanceofbothvirulenceand inflammatorymediators inBVDVpathogenesis.Further studiesarerequiredtodeterminetheidentityandmechanismofactionofthesefactorspresentinthesupernatantoftheinfectedmacrophages.

CRWAD2017 ABSTRACTS


215-ImmunologicalgeneexpressionchangesinthefetalthymusaftermaternalinfectionwithbovineviraldiarrheavirusK.J.Knapek1, J.V.Bishop2,H.VanCampen1,T.R.Hansen2. 1DepartmentsofBiomedicalScienceandMicrobiology, ImmunologyandPathology,ColoradoStateUniversity,FortCollins,CO,USA,2DepartmentofBiomedicalScience,ColoradoStateUniversity,FortCollins,CO,[email protected]:ViralPathogenesis–1,Room9,12/4/20179:30AMBovineviraldiarrheavirus(BVDV)infectionofbovinefetusesinthefirst125daysofpregnancyresultsinpersistentlyinfected(PI)cattle.PIcattlearethemainsourceofBVDV infections inpopulationscausingsignificanteconomic lossestothe industryworldwide.Theimmunemechanismsthatleadtothe“immunotolerant”stateofPIsarenotwelldefined.Wehypothesizedthatfollowinganactivatedinnateresponse,adefectintheadaptiveimmuneresponseinPIfetusespreventsviralclearance.ToclarifythestepsoftheadaptiveimmuneresponseaffectedbyfetalPI,pregnantheiferswereinoculatedwithBVDVonday75ofgestation.TotalRNAwasextractedfromuninfected control andPI fetal thymuses at days 89, 97, and190of gestation.Genes important in T cell differentiation anddevelopmentincludinglow-molecular-weightprotein2(LMP2),CD4andCD8werequantifiedbyqRT-PCR.CD8andCD4mRNAsweresignificantlydecreased(P≤0.05)inPIfetalthymusonday89,97and190ofgestation.LMP2,asubunitinthe20Sproteasomecorewhichprocessesforeignproteinstopeptides,transcriptsweresignificantlydecreased(P≤0.05)atdays97and190inPIcomparedtocontrolfetalthymus.Antigenprocessing,antigenpresentationanddecreasedexpressionofCD8andCD4onTcellsmaybeimpairedinPIfetusescontributingtoimmunotoleranceandincompleteviralclearance.Inaddition,impairedantigenprocessingmayexplainthesusceptibility of PIs to postnatal secondary infections. Supported in part by USDA-AFRI Grant #2008-35204-04652 and W3112ReproductioninDomesticRuminantsHATCHproject#1011648fromtheUSDANationalInstituteofFoodandAgriculture.216-Understandingthediverserolesofviroporinactivityofclassicalswinefevervirusproteinp7M.Borca1,E.Largo2,N.Huarte3,L.Holinka1,K.Berggren1,E.Ramirez-Medina1,G.Risatti4,J.Nieva3,D.P.Gladue1.1PlumIslandAnimalDiseaseCenter,UnitedStatesDepartmentofAgriculture,Greenport,NY,USA,2BiophysicsUnitandBiochemistryandMolecularBiologyDepartment,UniversityoftheBasqueCountry,Bilbao,Spain,3BiophysicsUnit(CSIC-UPV/EHU)andBiochemistryandMolecularBiologyDepartment,UniversityoftheBasqueCountry,Bilbao,Spain,4PathobiologyandVeterinaryScience,UniversityofConnecticut,Storrs,CT,[email protected]:ViralPathogenesis–1,Room9,12/4/20179:45AMPurpose:Classicalswinefever(CSF)isahighlycontagiousoftenlethaldiseaseofswine.Theetiologicalagent,CSFvirus(CSFV),isasmallenveloped virus with a positive single-stranded RNA genome, classified as a member of the genus Pestivirus within the familyFlaviviridae.Weinvestigatedtheroleoftheviralencodedputativeviroporin;non-structuralproteinp7.Methods:Minimalmembranemodelsystems,andaseriesofpeptidestoevaluatetheporeactivityandstructureofdifferentpeptidesinp7.Identificationofhostproteinsthatinteractwithp7wasdeterminedbyyeast-twohybrid,andfurtherconfirmedbyduallabelconfocalmicroscopy.Results:Viroporinscompriseafamilyofproteinsthatplaysignificantanddiverserolesduringthereplicationcycleofmanyviruses.Wehavedeterminedthatp7containstwohydrophobicstretchesofaminoacidsthatformtransmembranehelicesthatareinterruptedbyashortchargedsegmentthatformacytosolic loop.WehaveshowninaminimalmodelsystemthattheC-terminaltransmembraneregionofp7 iscapableofpermeabilizingmembranes,and forminga functionalpore.Theprecedingcystosolic loop is required forregulatingporeactivity,andisrequiredforinhibitionbyviroporindrugssuchasamantadineandverapamil.Byusingacombinationofoverlappingpeptidesandtargetedmutagenesisweidentifiedtheminimalstructureforporeformation,andtheresiduescriticalforregulationandfunction.Wehaveidentifiedthefirstcellularproteintointeractwithviralproteinp7,CAMLG,acalciummodulatingligandprotein,capableofmodulatingcellularcalciumconcentration.ThebindingsiteforCAMLGhasbeenidentifiedinthecystosolicloopofp7,andiscriticalforregulatingp7poreactivity.Conclusions:Wehavedeterminedthatnon-structuralproteinp7isafunctionalclassIIviroporin,andtheminimalporeformingdomainrequiredforviroporinactivity.Criticalresiduesrequiredforbothregulationandfunctionofp7havebeenestablished.HostbindingfactorCAMLGhasbeendetermined,andmappedtothecystolicloop,andiscriticalforregulatingp7poreactivity

CRWAD2017 ABSTRACTS


217-TheoutbreakofatypicalporcinepestivirusintheNorthChinaandthegenomiccharacteristicsofnovelChinesestrainsT. Bian, J. Huang, X. Ge, J. Han, X. Guo, H. Yang, L. Zhou. Veterinary Medicine, China Agricultural University, Beijing, [email protected]:ViralPathogenesis–1,Room9,12/4/201710:00AMAtypicalporcinepestivirus(APPV),anoveldiscoveredandgeneticallydistinctpestivirus,whichisregardedtorelatewithcongenitaltremor(CT)ofnewbornpiglets,hasbeendetectedinmanycountriesincludingtheUnitedStates,Germany,theNetherlands,Spain,AustriaandmostrecentlyinChina.Inthisstudy,fivepigfarmsfromfivedifferentprovincesofChina,withtheCTinnewbornpigletswere investigatedtodetecttheAPPVemerged intheherd.ThemorbidityofAPPV infection inthesefivefarmswerevariousfromsporadicappearanceofCTpiglets,deliveredbyonlyoneortwosows,upto70%newbornpigletsfrom80%sowsofherdweresufferedwithCTinthefarm.Eventhoughttherewasnomortalitydirectlyrelatedwiththisdiseaseintheoutbreakfarms,thedailybodyweightgainwasreduced.AllthesefivecaseswereconfirmedasAPPVpositiveinserasamplesofaffectedsoworinCNSsamplesfromshakingpigletsbyRT-PCR.AndthegenomicsequenceofanovelemergedChineseAPPVstrain,namedasHBtl1701,wassequencedbyusingRT-PCRwith7pairsofprimers.ThewholegenomeofHBtl1701was11,556ntinlength,whichshareshighestidentityof93%withtheAmericanstrain000515,and88%withDutchstrainNL1Farm1,88.1%-88.4%withGermanstrainsAPPV_GER_01andBavariaS5-9,88.0%withAustrianstrainAUT-2016_C,87.6%withAmericanstrain ISDVDL2014016573,butonly83.0%-83.1%withnovel isolatedChinesestrainsGD1andGD2,atthenucleotidelevel.PhylogeneticanalysisshowedthatHBtl1701wasclusteredinthesamebranchwiththeAmericanstrain000515,butdifferentiatedfromthebranchwiththeChinesestrainsGD1andGD2,whichindicatesthatthereisobviousdiversityamongdifferentChineseAPPVisolate,evenintheearlyoutbreakstage.Inconclusion,thisisthenovelreportofAPPVintheNorthChinaandthegenomicsequenceofHBtl1701isgeneticallydiversefromotherChinesestrains,indicatingthattheChinesecirculatingstrainsmighthavedifferentorigins.218-Bluetonguepathogenesis:anevolvingstoryfromTheilertoclimatechangeN.MacLachlan.UniversityofCalifornia,Davis,CA,[email protected]:VirologyKeynote/ViralPathogenesis,Room9,12/4/201710:45AMBluetongue(BT)isaninsect-transmitteddiseaseofruminantlivestockandwildlife,andtheonlyOIE(former)ListADiseaseendemicinNorthAmerica.BluetongueisprincipallyadiseaseofEuropeanbreedsofsheep,SouthAmericancamelids(sporadically),andcertainwildlifenotablywhite-taileddeer.InitialstudiestocharacterizethepathogenesisofBTvirus(BTV)infectionincludedstudiesinfetalandadultcattletodeterminethemaximumdurationofviremia,acriticalparameterforthesafeand logicalregulationoftradeoflivestock.Cattleserveassubclinicalamplifiersofthevirus,thustradeofcattleisespeciallyimpactedfromBTV-endemicareasoftheworldsuchastheUnitedStates.ThesestudiesestablishedthatpersistentinfectiondidnotoccurafterexperimentalornaturalBTVinfectionofbovinefetuses,ratherfetusesinfectedinearlygestationhadsevereteratogenicdefects.Infectionofpostnatalcattlecanleadtoprolongedbutnotpersistentviremiaasaconsequenceofinfectionofalltypesofbloodcells,butinfectionoferythrocytesisespeciallycriticalinallowingthevirustoavoidimmuneclearanceandalsofacilitatesinfectionofthehematophagousmidges(Culicoidesspecies)thatserveasbiologicalvectorsofBTVandrelatedviruses.TheOIECodenowreflectstheconclusionthatinfectiousviremiadoesnotexceed60daysinBTV-infectedruminantlivestock.OtherstudieshavefocusedonthemechanismofdiseaseinBTV-infectedruminants,confirmingthatsevereBTofsheepisaviralhemorrhagicfever(VHF)analogoustohumanVHFsuchasEbola.Specifically,thereleaseofvariouspro inflammatoryandvasoactivemediators fromdendriticcellsandothermononuclearphagocytes leadstoendothelialretraction,vascularleakageandcatastrophichypovolemicshock.Thus,itisthevirusinducedcytokinestormandnotdirectvirusinducedendothelialcytolysisthatisresponsibleforfulminantBT.

CRWAD2017 ABSTRACTS


219-Foot-and-mouthdiseasevirusnon-structuralprotein2BfunctionsasaviroporinM.V.Borca1,E.Largo2,E.Ramirez-Medina3,L.Holinka1, I.de laArafa4, J.L.R.Arrondo5,V.M.Aguillella6,A.Alcaraz2,K.Berggren7,E.Brocchi8,J.Nieva9,D.P.Gladue1.1PlumIslandAnimalDiseaseCenter,USDA,Greenport,NY,USA,2BiophysicsUnitandDepartmentofBiochemistry andMolecular Biology, University of the Basque Country, Bilbao, Spain, 3University of Connecticut, storrs, CT, USA,4BiophysicsUnitandDepartmentofBiochemistryandMolecularBiology,UniversityoftheBasqueCountry,Bilbao,Spain,5:BiophysicsUnitandDepartmentofBiochemistryandMolecularBiology,UniversityoftheBasqueCountry,Bilbao,Spain,6UnitandDepartmentofBiochemistryandMolecularBiology,UniversityoftheBasqueCountry,Bilbao,Spain,7OakRidgeInstituteforScienceandEducation,OakRidge,TN,USA,8NationalReferenceCentreforVesicularDiseases–Dpt.Biotechnology,IstitutoZooprofilatticoSperimentaledellaLombardiaedell’EmiliaRomagnaandUnitedStatesDepartmentofAgriculture,Brescia, Italy, 9BiophysicsUnit (CSIC-UPV/EHU)andDepartmentofBiochemistryandMolecularBiology,UniversityoftheBasqueCountry,Greenport,[email protected]:VirologyKeynote/ViralPathogenesis,Room9,12/4/201711:30AMPurpose:Foot-and-mouthdiseasevirus(FMDV),asingle-strandedpositive-senseRNAvirus,isthecausativeagentoffoot-and-mouthdisease (FMD), a highly contagious and economically important viral disease of domestic and wild cloven-hoofed animals.Understandingviralproteinfunctioniscriticaltounderstandpathogenesisofthiseconomicallyimportantdisease.Herewedescribethe viroporin activitiy of non-structural protein 2B.Methods: The structure of FMDV 2B was determined using ATR-polarizedtechniques,andoverlappingpeptidesPoreformationof2Bwasdeterminedinaminimalmodelsystem.Results:The2BproteinofFMDVislargerthanentrovirusorrhinovirus2Bproteins,andispredictedtobeaviroporinthatformsthreetransmembranedomainswiththesecondandthirdhelicaldomainseparatedbyabetafragment.Thethreedomainswereconfirmedbycirculardichroismtobehelical. Using artificialmembranes that resemble both the plasmamembrane and the endoplasmic reticulum2B peptide analysisshowedthatpermeabilizationactivityoccurredforboththesecondandthirdtransmembranedomains.Permeabilizationwasinhibitedby viroporin inhibitors amantadine and verapamil. By using ATR-polarized techniques we were able to determine the angle ofmembraneinsertionforthesetransmembranedomains.TheviroporinfunctionisessentialforFMDVassubstitutionsofresiduesin2Bthatarecriticalforthestructuralintegrityoftheviroporin,impededvirusviability.Thus,FMDV-2Bcontainstwoindependentpore-forming domains,with one pore formedwith the second transmembrane domain and the second pore is formed from the thirdtransmembranedomain.Conclusions:FMDV2B isaviroporin that shares little sequence identity fromotherpicornavirusesand isuniquefromotherpicornavirus2Bproteins,whichformonlyasinglepore.FMDV2Bformstwodistinctpores.Disruptionofeitherporewasfoundtobelethalforvirusreplication,suggestingthatbothporeshaveessentialfunctions.ThisisthefirsttimeFMDV2Bproteinhasbeendescribedwiththecapabilitytoformtwodistinctpores.220-ComparisonofhistoricandcontemporarystrainsofSenecavirusAA.Buckley1,B.Guo2,V.Kulshreshtha1,A.vanGeelen1,K.-J.Yoon2,K.Lager3.1ORISE,NADC,USDA,Ames,IA,USA,2CVM,ISU,Ames,IA,USA,3NADC,USDA,Ames,IA,[email protected]:VirologyKeynote/ViralPathogenesis,Room9,12/4/201711:45AMOnehypothesisforthesuddenincreaseinSVAcasesintheUnitedStateswasthatcontemporarystrainsweremorepathogenicthanhistorical strains. Our objective was to study disease progression of historical and contemporary SVA isolates in growing pigs.Commercialswineaged16-20weeksold(n=54)weresplitinto6challengegroups(n=8)and1controlgroup(n=6).Threehistoricalisolates(2001,2011,2012)andthreecontemporary isolates(2015)with inoculumtitersrangingfrom105.1–106.8TCID50/mLweregivenintranasally.Animalswereregularlybled,rectalswabbed,andoralswabbed.Animalswerealsoobserveddailyforanyclinicalsignsofvesiculardisease.Thesamplingperiodrangedfrom0dayspostinoculation(dpi)to14dpi.Serumandswabsweretestedbyreal-timePCRforSVAnucleicaciddetection.Serumwasalsotestedforneutralizingantibodyresponsetothechallengevirusbyvirusneutralizationassayandcrossneutralizingantibodiestootherchallengeisolates.All isolatesusedinthestudywereableto induceclinicaldiseasewiththedevelopmentofvesicleseitheronthecoronarybandsorthesnout.Thenumberofpigspresentingwithclinicalsignsineachchallengegrouprangedfrom5/8-8/8.Allanimalsineachchallengegroupreplicatedvirus.Therewereslightdifferencesinonsetanddurationofsheddingamongthesixdifferentisolates,butoverallmostpigswerePCRpositiveforSVAinoraland/orrectalswabsby4dpiandwerestillsheddingvirusat14dpi.Neutralizingantibodytiterstobothhomologousandheterologousstrainsusedinthisstudyarepending.Sequencingresultsandcomparisonbetweenthe6isolatesisalsopending.ThisstudydemonstratedthatvesiculardiseasecanbeexperimentallyreproducedingrowingpigswithbothhistoricandcontemporaryisolatesofSVA.Inaddition,theresultssuggestedtherewerenotlargedifferencesinclinicalpresentationbetweenstrains.FurtherresearchwillbeneededtohelpdeterminethecauseofthesuddenincreaseinvesiculardiseaseduetoSVAinfectionintheUnitedStatesswinepopulation.

CRWAD2017 ABSTRACTS


221-SusceptibilitytoandsheddingofSenecavirusAinboarsinoculatedwithanhistoricalandacontemporarystrainM.Sturos1,D.Reicks2,J.Cano3,S.Rossow1,F.Vannucci1.1UniversityofMinnesota,St.Paul,MN,USA,2ReicksVeterinaryResearchandConsulting,St.Peter,MN,USA,3PigImprovementCompany,Hendersonville,TN,[email protected]:ViralDiseasesinSwine–1,Room9,12/4/20172:00PMIntroductionSenecavirusA(SVA)isapicornaviruswhichcausesvesiculardermatitisinpigsandhasbeenassociatedwithincreasedneonatalmortality.Theobjectiveofthisstudyistoevaluatethesusceptibilityandshedding(fecal,oral,semen)ofSVAinmatureboarsinoculated with an historical and a contemporary strain.Methods Twelve boars were intranasally inoculated with SVA (6.0x108TCID50/boar),sixwithanSVAisolateobtainedin2017fromthesemenofanaturallyinfectedboarinMinnesota(contemporarystrain)andsixwithanSVAisolateobtainedin1999fromanabortedporcinefetusinMinnesota(historicalstrain).Fecalswabs,oralswabs,serum,andsemenwerecollectedonDay1post-inoculation(D1),D2,D3,D5,D7,D14,D22,D28,D35,andD42.ResultsBoarswerepositiveforSVAinserumbyreal-timeRT-PCRfromD1-D14(D1-D3:12boars,D7:8,D14:1;Ctvaluesbetween19and35).OralfluidswerepositivefromD1-D42(D1:11,D2-D7:12,D14:8,D22:5,D28:3,D35:1,D42:2;Ctbetween21and35).FeceswerepositivefromD1-D42(D1:3,D2:4,D3-D5:11,D7-D14:12,D22:9,D28:10,D35:6,D42:3;Ctbetween24and35).SemenwaspositivefromD1-D22(D1:1,D2:5,D3:1,D5:6,D7:2,D14-D22:1;Ctbetween30-35).SVA-specificIgGantibodiesweredetectedinserabyIFAfromD5onward(D5:2,D7:6,D14-D42:12).Onecontemporary-strainboardevelopedanulceratedareaonthedorsalnoseonD1andulceratedareasatthemarginofthesolesoftherearfeetonD5,withmoderatelameness.Asecondcontemporary-strainboardevelopedanulceratedareaonthenasalplanumonD2.ConclusionsThisstudyshowedthatmatureboarsaresusceptibletoinfectionwithhistoricalandcontemporarySVAstrains,withdifferencesinsheddingandclinicalsignsbetweenstrains.Contemporary-strainboarshadhigherlevelsofviremiawithprolongedvirussheddinginoralfluidsandfeceswhencomparedwiththehistoricalisolate.Allcontemporary-strainboarswerePCRpositiveinthesemen(upto22dayspost-inoculation)butonlyonehistorical-strainboarwaspositiveinsemenatonetimepoint.Onlyboarsinfectedwiththecontemporarystrainexhibitedclinicalsigns.222-SenecavirusAinfectionandtransmissioninmiceunderexperimentalconditionsE.Houston1,G. Temeeyasen2, L.Gimenez-Lirola1, R.Magtoto1, P. Piñeyro1. 1DepartmentofVeterinaryDiagnostics andProductionAnimalMedicine, Iowa State University College of VeterinaryMedicine, Ames, IA, USA, 2Department of VeterinaryMicrobiology,ChulalongkornUniversityFacultyofVeterinaryScience,Bangkok,[email protected]:ViralDiseasesinSwine–1,Room9,12/4/20172:15PMSenecavirusA(SVA)isaRNAvirusinthefamilyPicornaviridaethathasbeenidentifiedasthecausativeagentofvesicularlesionsandincreasedneonatemortalityinswine.Neutralizingantibodyandnucleicacidhavebeenreportedinmousesamplescollectedatswinefacilities. The objective of this study was to evaluate SVA transmission and clinical outcomes in experimentally infected mice.Micestrainsusceptibility:8-week-old(BALB/C,SWISS,SJL/J,C57BL/6,n=5perstrain)and4-week-oldmice(n=5,SCID)wereinoculatedsubcutaneously(SC)with100µLofSVAat109TCID50/mL.Onesentinelmousewasaddedpercageasnegativecontrol.Clinicalsigns,body weight (BW) and feces were collected daily for 14 days. Blood and full set of tissues were collected at euthanasia.Infectious route: 10BALB/C7-week-oldmicewere inoculated SC, intraperitoneal (IP), and intranasal (IN)with the samedoseandvolume.Feces,clinicalsignsandBWwerecollecteddailyfor28days.Bloodwascollectedatdaypostinoculation(dpi)0,3,5,7,10,14,21and28.Afullsetoftissueswerecollectedateuthanasia.BALB/CmicedevelopedmildclinicalsignsincludingaroughcoatandslightdecreaseinBW,butnoclinicaldifferencewasobservedwithothermicestrainsused.Inall infectedanimalsexceptSCIDandnegativecontrol,SVAIgGantibodyresponsewasdetectedat14dpibySVArVP1andwholevirusELISA,andIFA.SVAnucleicacidwasdetectedinalltissuesat14dpi.Viralsheddinginfeceswasdetected2dpi,lastinguntil10-12dpi.Sentinelanimalsshedvirusfrom5-10dpi.AnimalsinfectedviaSCandIProutesshowedviremiaat3dpi.Viralsheddingwasobservedasearlyas3dpiinallthreeinfectedgroups. SVA IgG antibodies were detected in both SC and IP groups from 5 and 7 dpi, respectively, to the end of the study.Inconclusion,micearesusceptibletoSVAinfectionunderexperimentalconditions.Theviruscanbedetectedinmultipletissuesandisshedthroughfeces.SeroconversionoccursafterSCorIPinoculationwithoutclinicalsigns.Viralsheddingandseroconversioninsentinelmicesupportpreviousfieldobservationssuggestingthatmicecouldbepotentialreservoirand/orvectorforSVAinswineherds.

CRWAD2017 ABSTRACTS


223-AnaturallyoccurringcrossorderrecombinantofenterovirusandtorovirusP.Shang1,S.Misra2,B.Hause1,Y.Fang1.DepartmentofDiagnosticMedicineandPathobiology,Kansasstateuniversity,Manhattan,KS,USA,2DepartmentofBiochemistry&MolecularBiophysics,Kansasstateuniversity,Manhattan,KS,[email protected]:ViralDiseasesinSwine–1,Room9,12/4/20172:30PMEnteroviruseshavebeenimplicatedinawiderangeofdiseasesinhumanandanimals.Inthisstudy,anovelenterovirus(speciesG;EVGKS16-08)wasidentifiedfromadiagnosticsampleusingmetagenomicsandcompletegenomesequencing.Theviralgenomeshared87.5%aminoacididentityand73.5%nucleotideidentitywithprototypeEVGstrain(PEV9UKG/410/73).Remarkably,a582nucleotideinsertionwasdeterminedinthe2C/3Ajunctionregionoftheviralgenome,whichwasflankedby3Cprocleavagesitesat5’-and3’-ends.Sequenceanalysisrevealedthatthisinsertionregionencodesapredictedproteasethatismostlyclosetotorovirus(ToV)papain-likeprotease(PLP)with54-68%aminoacididentity.StructurehomologymodelingpredictsthatthisproteaseadoptsafoldandcatalyticsitecharacteristicofaminimalPLPcatalyticdomain.Thestructureissimilartothatoffoot-and-mouthdiseaseleaderproteaseandtothecorecatalyticdomainsofcoronavirusPLPs,allofwhicharede-ubiquitinatinganddeISGylatingenzymestowardhostcellsubstrates.More importantly, recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deuibiquitination anddeISGylationactivity,anddemonstratedthecapabilitytosuppressIFN-betaexpression.Subsequently,ToV-PLPknockoutrecombinantviruswasgeneratedusingreversegenetics.Incomparisontothatofwildtypeclonedvirus,themutantvirusinfectedcellsshowedimpairedgrowthpropertyandincreasedexpressionlevelofinnateimmunegenes.TheseresultssuggestthatToV-PLPfunctionsasaninnateimmuneantagonist,whiletheenterovirusmaygainfitnesswithacquisitionofToV-PLPthroughtherecombinationevent.224-MultiplexeddigitalmRNAprofilingofimmuneresponsesinpigspersistentlyinfectedwithporcinereproductiveandrespiratory

syndromevirusP.Shang1,R.Guo1,C.A.Carrillo2,C.J.Jaing2,Y.Fang1.1DepartmentofDiagnosticMedicineandPathobiology,Kansasstateuniversity,Manhattan,KS,USA,2LawrenceLivermoreNationalLaboratory,Livermore,CA,[email protected]:ViralDiseasesinSwine–1,Room9,12/4/20173:00PMThecapacitytoestablishandmaintainanasymptomaticpersistentinfectionisoneofthehallmarksofPRRSVinfection.Trackingoftheglobalchangesinthehostgeneexpressionprofileduringviralinfectioncontributestothebetterunderstandingofviralpathogenesisand identification of potential diagnostic / therapeutic targets. To better understand the host-pathogen interaction during PRRSVpersistentstage,wedevelopedaswinegenespecificmultiplexedimmunegenemRNAprofilingassaybasedonthenanostringnCounterRNA-arraytechnology,ahigh-throughputdigitalgeneexpressionsystem.ThenCounterswineimmunegenepanelincludes189widelystudiedinnate/adaptiveimmune-relatedgenesplus3internalcontrols.Theassaywasusedtoanalyzeswineimmunegeneexpressionin tracheobronchial lymph nodes from PRRSV-infected pigs during acute and persistent infection stages. Results showed that 55immunegeneswere significantlyupregulated inacutely infectedpigs compared tomockorpersistently infectedpigs.Persistentlyinfectedpigshadgeneexpressionpatternmoreclosetothatofmockinfectedpigs.Furthergeneontologyanalysisshowedthatmostof genes in acutely infected pigs were enriched in innate immune pathways and cytokine responses. Taken together, this studyestablishedaswineimmunegenemRNAprofilingassayanddemonstratedthefeasibilityofutilizingnCounterRNA-arraytechnologyinrapidanalysisofhostimmunegeneexpressionsininfectedanimals.

CRWAD2017 ABSTRACTS


225-AdualribosomalframeshiftingmechanismtransactivatedbyanarterivirusproteinandhostcellularfactorsY.Li1,A.E.Firth2,I.Brierley2,E.Snijder3,J.Kuhn4,Y.Fang1.1DiagnosticMedicine/Pathobiology,KansasStateuniversity,Manhattan,KS, USA, 2University of Cambridge, Cambridge, UK, 3Molecular Virology Laboratory, Leiden University Medical Center, Leiden,Netherlands,4IntegratedResearchFacilityatFortDetrick,NationalInstituteofAllergyandInfectiousDiseases,NationalInstitutesofHealth,FortDetrick,MD,[email protected]:ViralDiseasesinSwine–1,Room9,12/4/20172:45PMArterivirusesareagroupofenveloped,positive-strandedRNAvirusesthatinfectanimals.Theycancausepersistentorasymptomaticinfections,andalsoacutediseaseassociatedwitharespiratorysyndrome,abortionorlethalhaemorrhagicfever.Thefamilyincludesporcinereproductiveandrespiratorysyndromevirus(PRRSV),equinearteritisvirus(EAV),mouselactatedehydrogenase-elevatingvirus(LDV),simianhemorrhagicfevervirus(SHFV),andanumberofmorerecentlyidentifiedmembers,manyofwhichareofsimianorigin.Recently,anovelcaseof-2/-1programmedribosomalframeshifting(-2/-1PRF)wasidentifiedinPRRSV.The-2/-1PRFleadstothetranslationoftwoadditionalviralproteins,nsp2TF(from-2PRF)andnsp2N(from-1PRF).Remarkably,thisdualribosomalframeshiftingmechanismistransactivatedbyaviralproteinnsp1βandhostfactorsPCBPs.Criticalelementsfor-2/-1PRFinPRRSV,includingtheslippery sequence (RGGUUUUUorRGGUCUCU)and3' C-richmotif,werealso identified in all nine speciesof simianarteriviruses.Interestingly, in four simian arteriviruses (MYBV-1, KRCV-1, KRCV-2, and SHEV), the slippery sequence (XXXUCUCU instead ofXXXUUUUU)cannotfacilitate-1PRFtogeneratensp2N.Thensp1βofsimianhemorrhagicfeverviruswasidentifiedasakeyfactorthattransactivatesboth-2and-1ribosomalframeshifting,andauniversallyconservedArg114inarterivirusesisessentialtoitsfunction.TheinvolvementofPCBPs in -2/-1PRF inPRRSVand simianarteriviruseswasalsodemonstratedusing the in vitro translation system.Furthermore,PRRSVnsp1βcouldstimulate-2/-1PRFwiththeSHFV-2/-1frameshiftingsequences,whileSHFVnsp1βcouldstimulate-2/-1 PRF with the PRRSV -2/-1 frameshifting sequences. Taken together, these data suggest that -2/-1 PRF is an evolutionarilyconservedmechanismemployedinarterivirusesfortheexpressionofadditionalviralproteins.226 - RNA stem-loop structures and conserved regions in ORF6 are important for the replication of porcine reproductive and

respiratorysyndromevirusP. Shang1,Y. Li1, I. Brierley2, A.E. Firth2, Y. Fang1. 1Department of DiagnosticMedicine and Pathobiology, Kansas state university,Manhattan,KS,USA,2DepartmentofPathology,UniversityofCambridge,Cambridge,[email protected]:ViralDiseasesinSwine–1,Room9,12/4/20173:15PMIn Nidoviruses, the high-order RNA structures in untranslated regions and transcription regulating sequences are crucial for virusreplication.Bioinformaticanalysisonallavailable full-lengthgenomesequencesofporcine reproductiveandrespiratorysyndromevirus (PRRSV) inGenBank suggested theexistenceof two stem-loop (SL1 and SL2)RNA structures, anextended stem-loop (extSL)formedbyaconservedregion(CR)anddownstreamcomplementarysequencewithinORF6regionofbothgenotypes.Apaneloffull-lengthcDNAclonesthatcontainssynonymousmutationsattheseregionswasconstructedtoinvestigatetherolesofSL1,SL2andCRinvolvedinviralreplication.Twopanelsoffull-lengthcDNAclonescontainingsynonymousmutationsattheseregionsweregeneratedtoinvestigatetherolesofSL1,SL2andextSLplayedinRNAtranscription.IntypeIPRRSV,SL1andextSLmutantsshowed2-loglowervirus titer than that ofwild-type (WT) virus, indicating that SL1 andextSL are important for virus replication. Similar resultswereobtained in type 2 PRRSV; especially,mutations disrupting extSL significantly impaired the virus replication, generated nonviableviruses.ThestemregionofSL2iscrucialforvirusreplicationinbothgenotypes,whilevirusreplicationwasseverelyattenuatedonlyintype2PRRSVwhentheloopregionofSL2(B-TRS7.1)wasmutated.QuantitativeRT-PCR(qRT-PCR)analysisofminusstrandRNAsontype2PRRSVfurthershowedthatcomparedwiththoseofWTvirus,lowerlevelsofminusgenomicRNAswereproducedbySL1andextSLmutants,whiletherelativeratiosofminussgRNA7.1weredecreasedinloop2andextSLmutants.ThesedatasuggestthatSL1,SL2andCRarecrucialforvirusreplication.ThedetailedmechanismoftheseRNAstructuresinvolvedinvirusreplicationisunderactiveinvestigation.

CRWAD2017 ABSTRACTS


227-PorcinereproductiveandrespiratorysyndromevirusnucleocapsidproteinactivatesNF-kBthroughbindingtoPIAS1H.Ke,S.Lee,D.Yoo.DepartmentofPathobiology,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,[email protected]:ViralDiseasesinSwine–2,Room9,12/4/20174:00PMPorcinereproductiveandrespiratorysyndromevirus(PRRSV)triggerstheonsetofinflammationduringinfection,andpro-inflammatorycytokines including interleukin (IL)-1β, IL-6, IL-8, and TNFα have been shown to be upregulated in virus-infected porcine alveolarmacrophages(PAMs),suggestingtheactivationofNF-κBbyPRRSV.WeshowinthepresentstudythatincellsinfectedwithPRRSVorcellsexpressingPRRSVnucleocapsid(N)protein,theRelA(p65)subunitofNF-κBwasincreasinglyphosphorylatedandtranslocatedtothenucleustoresultintheactivationofNF-κB.ByyeasttwohybridscreeningusingNasabait,theproteininhibitorofactivatedSTAT1(PIAS1)wasidentifiedfromPAMsasamolecularpartnerofN.PIAS1bindstoRelAandpreventsNF-κBactivationbyinterferingRelA-DNAbindinginthenucleusandthusisanegativeregulatorofNF-kB.TheNbindingtoPIAS1wasconfirmedbyco-immunoprecipitationandcolocalizationstudies.TomapthebindingdomainsofthePIAS1andPRRSVNproteins,deletionsandtruncationsweremade,andthebindingdomainofPIAS1wasmappedtotheN-terminalfragment.ThisdomainwasshowntobethesoledomainthatbindstoRelAtopreventitfrombindingtoκBsites,demonstratingthecorrelationbetweentheN-PIAS1interactionandtheNF-κBactivation.ForN,theregionbetween37and72aminoacidswasfoundtointeractwithPIAS1,andthisregionoverlappedwiththenuclearlocalizationsignalofN.AndthisregionwasalsoshowntoactivatetheNF-κBsignalingintheNF-κBpromoter-basedreporterassay,confirmingthecorrelationbetweenN-PIAS1bindingandNF-κBactivation.Bycompetitionassay,weshowthat,comparedtoRelA,thebindingofPIAS1toNispreferred,andthisinteractionwasvalidatedinPRRSV-infectedcells.Takentogether,PRRSVNbindsPIAS1toreleaseNF-kBfromPIAS1,andasaconsequence,NF-kBbecomesactivated.ThisisanovelstrategyofPRRSVforNF-κBsignalingactivation.228-ImportanceofcellularcholesterolfortheentryprocessofporcinenidovirusesJ.Jeon,C.Lee.AnimalVirologyLaboratory,SchoolofLifeSciences,BK21PlusKNUCreativeBioResearchGroup,KyungpookNationalUniversity,Daegu,Korea,[email protected]:ViralDiseasesinSwine–2,Room9,12/4/20174:15PMPorcinereproductiveandrespiratorysyndromevirus(PRRSV)andporcineepidemicdiarrheavirus(PEDV)representemergingandre-emergingporcinenidovirusesthatcontinuetothreatenpork-producingcountriesaroundworld,leadingtohugefinanciallossestotheglobalswineindustry.Althoughcholesterolisknowntoaffectthereplicationofabroadrangeofvirusesinvitro,itssignificanceandroleinporcinenidovirusinfectionremainstobeelucidated.Inthepresentstudy,therefore,weinvestigatedtherequirementforcellularor/andviral cholesteroland itsmechanismofaction inporcinenidovirus infection. Independentdepletionof cholesterol fromtheplasma membrane of target cells by methyl-β-cyclodextrin (MβCD) significantly impaired PRRSV and PEDV infection in a dose-dependentmanner.Theseinhibitoryeffectsonviralreplicationwerepartiallyreversiblebyreplenishmentwithexogenouscholesterol.Incontrast,porcinenidoviruseswereshowntoberesistanttopharmacologicalreductionofcholesterolcontentintheviralenvelope.Thesedataindicatedthatcholesterol-enrichedmicrodomainsareessentialforPRRSVandPEDVinthecellularmembrane,butnotintheviralmembrane.TheantiviralactivityofMβCDonporcinenidovirusinfectionwasfoundtobepredominantlyexertedwhenusedasatreatmentpre-infectionorpriortotheviralentryprocess.Furtherexperimentsrevealedthatpharmacologicaldepletionofcellularcholesterolprimarilyinterfereswithvirusbindingandpenetrationandsubsequentlyinfluencespost-entrystepsofthePRRSVandPEDVreplicationcycle,includingviralRNAandproteinbiosynthesisandprogenyvirusproduction.Inaddition,pharmacologicalsequestrationof cellular cholesterol suppressed the replication of a newly emerged porcine deltacoronavirus (PDCoV), suggesting its essentialfunctioncommontonidovirusinfection.Altogether,ourresultssuggestthatcholesterolinthecellularmembraneiscriticalforporcinenidovirusentry,andthatdisruptionofthecholesterol-dependententryprocessmaybeanexcellenttherapeuticoptionfornidovirusinfectioninhumanorveterinarysubjects.

CRWAD2017 ABSTRACTS


229-AssessingofthezoonoticpotentialofswineinfluenzavirusesinaprimarycellculturemodelC.S.Kyriakis,M.Krunkosky,J.M.Luczo,S.M.Tompkins.CenterforVaccinesandImmunology,CollegeofVeterinaryMedicine,UniversityofGeorgia,Athens,GA,[email protected]:ViralDiseasesinSwine–2,Room9,12/4/20174:30PMAviruses(IAVs),membersoftheOrthomyxoviridaefamily,aresinglestranded,negativesenseRNAviruseswitheightgenesegmentsthat encode between 10 and 18 proteins. IAVs are characterized by two important genetic traits: (a) genetic drift, resulting frommutationsusuallyingenesegmentsexpressingthesurfaceglycoproteinshemagglutinin(HA)andneuraminidase(NA),whichaltertheirantigenicphenotypeand(b)geneticshiftorreassortment,whichoccurswhentwovirusesco-infectthesamehostandexchangegenesegments.Geneticshiftisthemainmechanismfortheemergenceofnovelepizooticandpandemicviruses.Pigshavebeenproposedas“mixingvessels”linkedtothegenerationofnovelIAVsthatcaninfecthumans,sincetheyaresusceptibletobothavianandhumanIAVs.SwineIAVscausewidespreadrespiratorydiseaseinpigswithhighmorbidity,lowmortalityandasubstantialeconomicimpacttotheindustry.ThreesubtypesofIAVscirculateinswineinNorthAmerica:H1N1,H3N2andH1N2.Theyareofdiversegeneticorigin,frequentlycombininggenesegmentsfromavianorhumanviruses.SomeIAVsthatemergeinpigsoccasionallyinfecthumanseitherinisolatedzoonotictransmissioneventsorviathegenerationofviruseswithpandemicpotential.Toinvestigatethezoonoticcapacityofswine IAVs we compared the replication properties of human and swine IAVs in normal human bronchoepithelial (NHBE) cells.Specifically,weusedtheprototype2009H1N1pandemic(pdmH1N1)virusandarecenthumanH3N2strainaspositivecontrolsandcomparedtheirreplicationkineticstohistoricalandrecentswineIAVsofdifferentsubtypes.Wefoundthatcontemporaryswineviruseswithspecificinternalproteingenesegment-origincombinationsreplicatebetterinNHBEcellsindicatingzoonoticpotential.Togetherwithdetailedantigenicandgeneticcharacterization,ourresultsprovideinsightsintothezoonoticthreatcurrentlycirculatingswineIAVspose.230-NLRC5,acriticalplayerininfluenzaviruspathogenesisinchickensS.K.Chothe,R.Nissly,M.Sill,L.Lim,G.Bhushan,P.Ibrahim,M.Keller,I.Bird,B.Jayarao,S.Kuchipudi.AnimalDiagnosticLaboratory,ThePennsylvaniaStateUniversity,UniversityPark,PA,[email protected]:ViralDiseasesinSwine–2,Room9,12/4/20174:45PMInfluenzaAviruses(IAVs)continuetobemajorthreattothepoultry industryandpublichealthglobally.Avianinfluenzavirus(AIV)infection in chicken ranges frommild to severe fatal disease depending on the virus type involved and the underlyingmolecularmechanismsofAIVpathogenesisareyettobefullyunderstood.ItiswidelyknownthathostimmuneresponsesplayakeyroleinIAVpathogenesis.MembersofNodlikereceptor(NLR)family,inparticular,NLRC5,thelargestNLRmember,regulatesimmuneresponsesagainstinvadingpathogens.However,theroleofNLRC5inmediatingantiviralresponseappearstobecomplex,asNLRC5wasshowntobothpositively andnegatively regulateNFκB and type I interferon (IFN) signalingpathways. The role ofNLRs in avian immuneresponseispoorlyunderstood.Hence,toestablishtheroleofNLRC5inAIVpathogenesisinchicken,weinvestigatedtheregulationofNLRC5inchickenmacrophages(MQ-NCSU)infectedwithalowpathogenicAIV(LPAIV)H5N2virus.WefoundthatNLRC5expressionwasupregulatedinMQ-NCSUcellsduringH5N2virusinfection,from4hourspostinfection(hpi)to48hpi.Further,siRNAmediatedNLRC5knockdown(KD)inMQ-NCSUcellsresultedinsignificant(P<0.01)reductioninLPAIH5N2virusreplication,whichwasassociatedwithupregulationof type I IFN (IFNβ)andLGP2expression.Notably,expressionofMDA5,and IRF7wasnot significantlydifferentbetweenNLRC5KDcellsandwildtypecells.Cytokinesimportantininflammasomeformation,andmediatinginflammatorydisordersnamelyInterleukin1β(IL1β)andIL18weresignificantlydownregulatedinNLRC5KDMQ-NCSUcells.Insummary,NLRC5couldbeanegative regulatorof innateantiviral responseandpromotes inflammatory response inAIV infectedchickencells.OurpreliminaryresultsraiseastrongpossibilitythatNLRC5couldbeacriticalplayerinAIVinfectioninchickenandassuchwarrantsfurtherindepthstudies.

CRWAD2017 ABSTRACTS


231-Developmentofanewprotocolforquantitativereal-timePCR(qPCR)forthedetectionofAfricanswinefevervirusinfromformalin-fixed,paraffin-embeddedtissues

K.Urbaniak,D.A.Meekins,J.A.Richt,A.S.Davis,J.D.Trujillo.DepartmentofDiagnosticMedicine/Pathobiology,CollegeofVeterinaryMedicine,KansasStateUniversity,Manhattan,KS,[email protected]:ViralDiseasesinSwine–2,Room9,12/4/20175:00PMAfricanswinefevervirus(ASFV)isendemicinAfricaandSardiniaandcausesahighlycontagious,fataldiseaseinpigs.RecentspreadtoTranscaucasia,NorthernAsiaandEasternEuropecausesconcernforcontinuedoutbreaks.Formalin-fixed,paraffin-embeddedtissues(FFPET)arenon-infectiousspecimenswithapplicationinsurveillanceanddiagnostics.However,recoverymethodsforASFVDNAfromFFPET are poorly developed. Recently,wedeveloped a successful protocol for rapid recovery ofASFVDNA fromFFPET.Here,weevaluate the FFPET protocol for quantitate qPCR detection of ASFV DNA and compare it to fresh, frozen tissue (FFT).Tissuesfrompigs(n=15)experimentallyinfectedwithASFVArm07,E70andKen05weretested,specificallyheart,liver,spleen,tonsil,andsuperficial cervical lymphnodes (LN)aswellaskidney, lung,and renalandgastrohepatic LN.DeparaffinizationofFFPET,ATL-proteinaseKdigestionandDNAde-crosslinkingwereperformedinasingletube.FFTlysatesderivedfrom20%tissuehomogenatewereprocessedusingATL-proteinaseKdigestion.LysateswereprocessedusingautomatedmagneticbeadextractionsoptimizedforDNArecovery.QuantitativeqPCRforthedetectionoftheASFVp72andactinDNAwereperformedusingQuantaFastMixII.Thegenecopynumber (CN) was calculated via plasmid standard curves. The results were presented as ASFV CN per 1000 CN of actin.OverallsensitivityfordetectionofASFVinFFPETwas100%ascomparedtoFFT.TwopigshadASFVCNoutsidethequantitativerangeofqPCRandwerenot included in inter-protocolequivalencyanalysis.Eighty-sevenpercentofthe100tissuesamples from13pigsresulted in equivalentdetectionASFVCNbetween FFPET and FFT. The remaining13%processedas FFPEThad1-2 logs lessASFVdetectedascomparedtoFFT.ForthetwopigswiththelowASFVCN,FFPETwasmoresensitiveforonepig(Arm07),whileforthesecond(Ken05)FFTwasmoresensitive.Toourknowledge,thisisthefirststudytoquantifyASFVCNinFFPET.ItshighsensitivityandcomparabilitytoFFTconfirmsFFPETasanalternativespecimenforASFVqPCRdiagnostics,vitaltoadvancingASFeradication,control,andsurveillanceeffortsglobally.232-PointofneedmolecularbaseddetectionofAfricanswinefevervirusJ.Trujillo1,K.Urbaniak1,T.Wang2,I.Morozov1,J.Richt1.1CollegeofVeterinaryMediine,,CenterofExcellenceforEmergingandZoonoticAnimalDiseases,KansasStateUniversity,Manhattan,KS,USA,2GeneReach,USA,Boston,MA,[email protected]:ViralDiseasesinSwine–2,Room9,12/4/20175:15PMAfricanswinefever(ASF)isahighlycontagious,commonlyfataldiseaseofswinecausedbyalarge,macrophage-tropic,doublestrandedDNAvirus,ASFvirus(ASFv).NaturalhostsforASFVincludewildsuidsandarthropodvectors(Ornithodorosgenus).ASFVisendemicinAfricaandSardinia.TherecentspreadtoTranscaucasia,theRussianFederationandEasternEuropewarrantseriousconcernoffurtherspreadintoEurope.RapiddetectionandresponsetoASFoutbreaksisparamounttomitigateeconomicandanimallosses.Forrapidon-sitedetection,theqPCRassayforASFvp72genewasadaptedforinsulatedisothermalPCR(iiPCR)ontheportabledevice,POCKIT(GeneReachUSA).LOD100forthisassaywasdeterminedontheportabledeviceandcomparedtotheNAHLN(USDA)referenceassayonthelaboratorythermocyclerutilizingnon-infectiousASFvcontrolsandpurifiedASFvDNAfromthefollowingASFviruses:Arm07,E70,Ken05andOURT88/3.LOD100forthereferenceassayperformedonthelaboratorythermocycler(BioRadCFX)usingQuantaqPCRFastmixIIwasbetween1.2and12ASFvDNAcopies.TheiiPCRassaysonPOCKITyieldedsimilarsensitivitieswithLOD100between1-50ASFvDNAcopies.ThedeployabledevicedemonstratedexcellentagreementandreproducibilitywhencomparedtotheUSDAreferenceassayperformedonthelaboratorythermocycler.Testingofnegativeserumsamples(n=25)yieldednofalsepositivesandaclinicalspecificityof100%.Studiesusingformalininactivatedtissues(n=36testedinduplicate)attainedfromexperimentallyinfectedpigsresultedinequivalentsensitivityforthedeployabledeviceandthereferenceassay(100%sensitivity).Samplestestedcontainedfrom1to106copiesofASFvDNAandcomprise6diagnosticallyrelevanttissuetypes.Testingofwholeblood,freshtissuesandoralfluidsareunderway.ThesedatademonstratethefutureutilityofiiPCRassaysonthedeployabledevice,POCKITdeviceforaccurateandsensitivedetectionofhighimpactpathogensunderfieldconditions.

CRWAD2017 ABSTRACTS


233-Oralfluidspecimenscanbeclarifiedwithoutaffectingporcineepidemicdiarrheavirus(PEDV)isotype-specific(IgG,IgA)ELISAresponses

K.Poonsuk,L.Giménez-Lirola,R.Magtoto,D.Baum,C.Rademacher,J.Brown,J.Ji,J.Zhang,C.Wang,R.Main,J.Zimmerman.VeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversity,Ames,IA,[email protected]:SwineImmunology,Room9,12/5/20179:00AMINTRODUCTIONTheoralfluid-basedPEDVIgAELISAisanefficientapproachforevaluatingimmunityinpopulations.However,oralfluidsamplesmaycontainparticlesoffeed,feces,andothermaterialsfromtheenvironmentthatpotentiallyaffectpipettingaccuracyandtestperformance.Therefore,theobjectiveofthisstudywastoimprovethequalityandreliabilityoforalfluidspecimenbyremovingtheparticulatessuspendedinthematrixusingchemical"clarification",butwithoutadverselyaffectingthedetectionofPEDVantibody.METHODSOralfluidswerecollectedondayspost inoculation(DPIs) -3,0,5,10,15,20,25,30,35,and42frompensofpigs inanexperimentalstudyandonDPIs-4,0,3,7,10,14,17,21,24,28,31,35,38,and42fromafieldstudy.Ineachstudy,aliquotsoforalfluidsamplesweretreatedwithoneof3lyophilizedchemicaltreatments(A,B,C,plusnegativecontrol)andwerethentestedbyIgGandIgAwholevirusPEDVELISAs.Thereafter,treatedandcontroloralfluidsampleswerestoredat4°Candtestedagainondayspost-treatment(DPTs)2,4,and6toevaluatetheresidualeffectofthetreatment.Likewise,serumsamplesweretestedbyPEDVIgGandIgAELISAsonday0,thenheldat4°Candtestedagainon2,4,and6tobecomparedwithoralfluidantibodyresponses.Datawereanalyzed for the effect of treatment overDPT (0, 2, 4, 6) on thediagnostic performanceof PEDVELISA S/P ratios.RESULTSANDCONCLUSIONSTreatmentswereeasilyadministered,i.e.,thelyophilizedchemicalswereeasilyresuspendedinoralfluidsamplesandthe clarifying effect was immediate. Statistical analysis (nonparametric ANOVA) of oral fluid IgA and IgG S/Ps found that neithertreatmentnortimeaffectedtheELISAS/Presults(p>0.05).Thatis,pairwisecomparisonsofDPTs2,4,and6resultstoDPT0IgAandIgGS/Psdetectednosignificantdifferences(p>0.05).Thus,thechemicaltreatmentsremovedparticulatesfromoralfluidswithoutaffecting testperformance.Thisapproachcouldbeusedeither in the fieldor inveterinarydiagnostic laboratories to improve thecharacteristicsoforalfluidstestedforPEDVantibody.234-Developmentofafluorescent imagingcytometry-basedimmunoassayforthedetectionofporcineepidemicdiarrheavirus

(PEDV)antibodiesJ.MoraDíaz,L.Sarmento,K.Poonsuk,D.Baum,R.Main,J.Zimmerman,L.Gimenez-Lirola.VDPAM,IowaStateUniversity,Ames,IA,[email protected]:SwineImmunology,Room9,12/5/20179:15AMPorcineepidemicdiarrheavirus(PEDV)isresponsibleofsignificanteconomiclossesintheswineindustrydueanelevatedmorbidityandmortality inneonatalpigs.PEDV infection is clinicallyandpathologically indistinguishable fromthosecausedbyotherporcineentericcoronaviruses.Therefore,PEDVdiagnosisreliesonlaboratorydiagnosticmethods.Nowadays,thereareseveralPEDVdirect(e.g.,virusisolation,insituhybridization,electronmicroscopy,PCRmethods)andindirectdetectionmethods(e.g.,ELISA,IFA,VN,FFN).TheIFAwasthefirsttechniqueavailableforPEDVantibodydetectionafterthefirstPEDVoutbreakintheUSin2013.However,thistechnique is time consuming, labor intensive, and inherently variable due to the subjectivenature. In this study,wedescribe theadaptationofstandardIFAtoahigh-throughputformatusingfluorescent imagingcytometry.Forthispurpose,96well flatbottomblack tissue culture treated microplates were seeded with Vero 81 cells, and subsequently infected with PEDV. The fluorescentcytometryassaywasdesignedandoptimizedforaSpectraMaxMiniMax300imagingcytometer.Thisinstrumentpresentsimaging,cellanalysis and fluorescent reading capabilities all in one platform. The time of detection, duration of the detection and diagnosticsensitivityofthetestwasevaluatedon160serumsamplescollectedfromPEDVinfectedanimalsatdaypost-inoculation(DPI)-4,0,7,14,21,28,35,and42.Thediagnosticsensitivitywasevaluatedon132serumsamplescollectedfromnegativeanimalsatDPI-7,0,3,7,10,14,17,21,28,35,and42.ThePEDVIgGantibodydetectionratevariedwiththetimepostinoculation.ThefirstantibodyresponsewasdetectedbyDPI7,peakingatDPI28,anddecliningthereafter.Theoveralldiagnosticspecificitywas96%.Resultsdemonstratedclear advantages over IFA standard method including, reduction of time for plate reading (< 3 min), improvement of testrepeatability/reproducibility,andimprovementoftheprecisionofantibodyresponseestimates.Thishighthroughputtestingapproachcanbebroadlyapplicabletoothercell-basedimmunoassaysandfordifferentpathogens.

CRWAD2017 ABSTRACTS


235-ProductionandunderlyingmechanismsoftheStreptococcussuisserotype2-inducedinterleukin-1beta(IL-1B)A.Lavagna,J.-P.Auger,D.Roy,M.Segura,M.Gottschalk.DepartmentofMicrobiologyandPathology,FacultyofVeterinaryMedicine,UniversityofMontreal,Saint-Hyacinthe,QC,[email protected]:SwineImmunology,Room9,12/5/20179:30AMStreptococcussuisserotype2(SS2)isanimportantswinepathogenresponsibleformeningitisandsuddendeath.Itisalsoanimportantzoonoticagentcausingmeningitisandsepticshock.Infact,exacerbatedinflammationisahallmarkoftheSS2systemicinfection.IL-1βisacytokinecentraltotheinflammatorycascadethatrequirespreciseregulationsinceexcessiveanddefectiveresponsescanhavedetrimentalconsequences.However,theroleofIL-1βintheSS2infectionandtheunderlyingmechanismshavebeenlittlestudied.Herein,systemiclevelsofIL-1βwereevaluatedusingaSS2mousemodelofsystemicinfection(sepsiswithsepticshock).Whileplasmalevelswerebarelydetectable throughout the infection, includinguponpresentationof severeclinical signs, liverandspleen levelsradically increased following infection, suggesting the participation of resident immune cells. Consequently, IL-1β production bydendriticcells(DCs)andmacrophages(Mθ)wasevaluated.DCs,andtoalesserextentMθ,wereshowntobeasourceofIL-1β.Tobetter understand the underlying mechanisms involved in this production, different cellular pathways were studied. Resultsdemonstrated thatMyD88 and Toll-like receptor (TLR) 2were required for production of IL-1β, unlike TRIF and TLR4.Moreover,maturationofSS2-inducedIL-1βwascaspase-1-dependent.Consequently,theroleofdifferentinflammasomeswasevaluated:S.suis-induced IL-1β production required NLRP3 but not NLRP1. The participation of other inflammasomes is being evaluated. SincestreptococcalhemolysinsmayberesponsibleforactivationoftheNLRP3inflammasome,theroleofthesuilysin,asecretedhemolysinofSS2,wasevaluatedusinganisogenicmutant.Resultssuggestthat,possiblyduetoitspore-formingcapacity,thesuilysinisimplicatedinNLRP3 inflammasomeactivation involved in IL-1βmaturation. Inconclusion,SS2 induceshigh levelsof IL-1βduringthesystemicinfection,restrictedtoorgans,inwhichDCsandMθmightparticipate.Consequently,theprotectiveordetrimentalroleofthismediatorduringtheSS2infectioniscurrentlybeingdeterminedusingIL-1receptorknock-outmice.236-DynamicsofadaptiveimmuneresponsesfollowingSenecavirusAinfectioninpigsM.F.Maggioli1,S.Lawson1,M.DeLima2,L.R.Joshi1,T.C.Faccin3,F.V.Bauermann1,D.G.Diel1.1AnimalDiseaseResearch&DiagnosticLaboratory,SouthDakotaStateUniversity,Brookings,SD,USA,2UniversidadeFederaldePelotas,Pelotas,Brazil,3UniversidadeFederaldeSantaMaria,SantaMaria,[email protected]:SwineImmunology,Room9,12/5/20179:45AMSenecavirusA(SVA)isanemergingpicornavirusthatcausesvesiculardisease(VD),clinically indistinguishablefromfoot-and-mouthdisease(FMD) inpigs.ManyaspectsofSVA infectionbiologyandthehost immuneresponsesto infectionremainunknown. Inthepresentstudy,finishingpigswereinfectedwithacontemporarySVAstrain(SD15-26),andtheensuinghumoralandTcellresponseswereevaluatedduringacuteviralinfection(14days)orafterdiseaseresolution(atday35pi).Weevaluatedtheneutralizingantibodiestiters,andthelevelsofIgMandIgGdirectedagainstexternalcapsidproteinsVP1,VP2andVP3.CellularimmuneresponsestoSVAwereassessedbyflowcytometricanalysisofIFN-γexpressionandproliferativeresponsesbyTcellsuponrecallstimulationwithuv-inactivatedvirus(uvSVA)orwithcapsidproteins(VP1,VP2andVP3).Infectionelicitedneutralizingantibodyasearlyas5dayspi,whichcoincidedandwas strongly correlatedwithVP2- andVP3-specific IgM responses. Levels of anti-VP1, -VP2and -VP3 IgG increasedstartingatday10(forVP-3)andatday14(forVP1andVP2).BothIgMandIgGlevelswanedbyday35pi(exceptforantiIgM-VP1).SpecificTcellresponsestoSVAwerefirstobservedatday3pi,andwerehighlysignificantstartingatday7pi(asmeasuredbybothIFN-γexpressionandproliferation).Tcellresponsestoallthreeexternalcapsidproteinsweredetected,butresponsestoVP1tendedtobedelayed.Ingeneral,CD4TcellscontributedgreatlyfortheTcellresponses,butrespondingCD8Tcellswerealsodetected.Cellularresponses were still present at day 35 pi. In summary, SVA elicits robust B and T cell activation early upon infection, with highneutralizationtitersassociatedwithIgMactivity.TandBcellresponsestargetedallthreeexternalcapsidproteins.

CRWAD2017 ABSTRACTS


237-ComparativeanalysisofsignaturegenesinPRRSV-infectedporcinemonocyte-derivedcellstodifferentstimuliL.C.Miller1,D.Fleming1,X.Li2,D.Bayles3,F.Blecha4,Y.Sang5.1VPDRU,USDA-ARS-NADC,Ames,IA,USA,2PulikeBiologicalEngineering,HenanLuoyang,China,3InfectiousBacterialDiseasesResearchUnit,USDA-ARS-NADC,Ames,IA,USA,4DepartmentsofAnatomyandPhysiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA, 5Department of Agricultural andEnvironmental Sciences, College of Agriculture, Human and Natural Sciences, Tennessee State University, Nashville, TN, [email protected]:SwineImmunology,Room9,12/5/201710:00AMMonocyte-derivedDCs(mDCs)aremajortargetcells inporcinereproductiveandrespiratorysyndromevirus(PRRSV)pathogenesis;however,theplasticityofmDCsinresponsetoactivationstimuliandPRRSVinfectionremainsunstudied.Inthisstudy,wepolarizedmDCs, andappliedgenome-wide transcriptomicanalysis andpredictedprotein-protein interactionnetworks to compare signaturegenesinvolvedinmDCsactivationandresponsetoPRRSVinfection.PorcinemDCswerepolarizedwithmediatorsfor30hours,thenmock-infected,infectedwithPRRSVstrainVR2332,orahighlypathogenicPRRSVstrain(rJXwn06),for5h.TotalRNAwasextractedandused to construct sequencing libraries for RNA-Seq. Comparisonsweremade between each polarized and unpolarized group (i.e.mediatorvs.PBS),andbetweenPRRSV-infectedanduninfectedcellsstimulatedwiththesamemediator.Differentiallyexpressedgenes(DEG)fromthecomparisonswereusedforpredictionofinteractionnetworksaffectedbythevirusesandmediators.TheresultsshowedthatPRRSV infection inhibitedM1-prone immuneactivity,downregulatedgenes,predictednetwork interactions relatedtocellularintegrity, and inflammatory signaling in favor ofM2 activity. Additionally, the number ofDEG andpredictednetwork interactionsstimulatedinHP-PRRSVinfectedmDCswassuperiortotheVR-2332infectedmDCsandconformedwithHP-PRRSVpathogenicity.238-DeletionoftheORF2geneoftheneurogenicequineherpesvirustype1strainAb4reducesvirulencebymaintainingstrong

immunogenicityC.L.Schnabel1,C.Wimer1,G.Perkins1,S.Babasyan1,H.Freer1,C.Watts1,A.Rollins1,N.Osterrieder2,B.Wagner1.1CollegeofVeterinaryMedicine,CornellUniversity,Ithaca,NY,USA,2FreieUniversitaetBerlin,Berlin,[email protected]:EquineImmunology,Room9,12/5/201710:45AMEquineherpesvirustype1(EHV-1)inducesrespiratoryinfections,abortion,andneurologicdiseasewithsignificantimpactonhorsesworldwide.Virulencefactorscontributingto infectionandimmuneevasionareofparticular interesttoelucidatethe interactionofEHV-1andthehorse’simmuneresponse.ApotentialvirulencefactoroftheneurogenicEHV-1strainAb4istheORF2geneproduct.Inthisstudyweaimedtocharacterizeitseffectsinvivo.Icelandichorses(2-4yearsold)wereinfectedwithEHV-1Ab4,oritsORF2genedeletionmutant(Ab4ΔORF2).Thehorses’clinicalpresentation,virusshedding,viremia,antibodyandcellularimmuneresponsesweremonitoreduntil260daysafterexperimentalinfectionandcomparedtonon-infectedcontrols(n=8pergroup).TheAb4ΔORF2virusreducedfeverandminimizedvirussheddingafterinfectioncomparedtotheparentAb4strain,whileAb4ΔORF2establishedviremiasimilartoAb4.TheserumantibodyresponsetoEHV-1,evaluatedbyabead-basedmultiplexassay,wassimilarinhorsesinfectedwithAb4andAb4ΔORF2.EHV-1specificIgG1andIgG4/7dominatedthehumoralresponseafterinfection.EHV-1specificantibodiesweredetectableasearlyaseightdaysafterinfectionandEHV-1specificIgG4/7remainedelevatedinserumofbothinfectedgroupsuntiltheendofthestudy.Incontrasttothelong-lastingantibodyresponse,EHV-1specificcellularresponseswereonlyidentifiedduringviremia,ondays5-10afterinfectionwitheithervirus.Thehorses’peripheralbloodmononuclearcells(PBMC)secretedincreasedinterferon-γand interleukin-10 after ex vivo re-stimulation with EHV-1. However, EHV-1 specific T-cells were not detected in PBMC by flowcytometricanalysis.Insummary,ORF2isavirulencefactorofEHV-1Ab4.ThedeletionoftheORF2genereducesfeverandnasalvirusshedding. Incontrast,ORF2deletiondoesnot influenceviremia.The immunogenicityoftheAb4ΔORF2andparentAb4virusesareidentical.

CRWAD2017 ABSTRACTS


239-QuantificationofequineimmunoglobulinAinserumandmucosalsecretionsbyafluorescentbead-basedassayC.L.Schnabel,S.Babasyan,H.Freer,B.Wagner.CollegeofVeterinaryMedicine,CornellUniversity,Ithaca,NY,[email protected]:EquineImmunology,Room9,12/5/201711:00AMOnlyfewquantitativereportsexistabouttheconcentrationsandinductionofimmunoglobulinA(IgA)inmucosalsecretionsofhorses.Despitethis,itiswidelyassumedthatIgAisthemajorimmunoglobulinonmucosalsurfacesinthehorse.Here,twonewmonoclonalantibodies(mAbs)againstequineIgA,clones84-1and161-1,weredevelopedandcharacterizedindetail.BothIgAmAbsspecificallyboundmonomericanddimericequineIgAindifferentapplications,suchasWesternblotsandfluorescentbead-basedassays.Cross-reactivitywithotherequine immunoglobulin isotypeswasnotobserved.ThenewIgAmAb84-1wasused incombinationwiththepreviouslycharacterizedanti-equineIgAmAbBVS2forthedevelopmentofafluorescentbead-basedassaytoquantifytotalIgA.Forthe quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA),respectively.ThedifferentstandardswereneededforaccurateIgAquantificationintherespectivesamplestakingthedifferentsignalintensitiesofmonomericanddimericIgAontheflorescentbead-basedassayintoaccount.IgAwasquantifiedbythebead-basedassayestablished here in different equine samples of healthy adult individuals. In serum, the median total IgA was 0.45 mg/ml forThoroughbredhorses(TB,n=10)and1.16mg/mlinIcelandichorses(ICH,n=12).InnasopharyngealsecretionsofTB(n=7)0.13mg/mlmediantotalIgAwasmeasured,and0.25mg/mlforICH(n=12).SalivaofICH(n=6)containedamedianof0.15mg/ml,colostrumofWarmbloods(n=8)amedianof1.89mg/mlIgA.ComparedtoIgG1andIgG4/7quantifiedinthesamesamples,IgAappearedasthemajorimmunoglobulinisotypeinnasopharyngealsecretionsandsalivawhileitisaminorisotypeinserumandcolostrum.Thenewlydevelopedmonoclonalantibodiesagainstequine IgAandtheresultingbead-basedassayforquantificationoftotal IgAcannotablyimprovetheevaluationofmucosalimmunityinhorses.240-IntracellularsurvivalandreplicationofRhodococcusequiinequinemacrophagesstimulatedwithcytokinesL.J.Berghaus.LargeAnimalMedicine,UniversityofGeorgia,ATHENS,GA,[email protected]:EquineImmunology,Room9,12/5/201711:15AMRhodococcusequiisacommoncauseofpneumoniainfoals.TheabilityofR.equitosurviveandreplicateinmacrophagesisthebasisofitspathogenicity.MostofwhatweknowregardingtheroleofcytokinesinhostdefenseagainstR.equicomesfromstudiesinmiceandthemechanismsleadingtomacrophageactivationandkillingofR.equiinhorsesareunknown.Theobjectivesofthisstudyweretodeterminetheeffectofprimingwithinterferon(IFN)-γ,interleukin(IL)-1β,IL-4,IL-6,IL-10,ortumornecrosisfactor(TNF)-αatvariousconcentrationson intracellularsurvivalofvirulentR.equi inequinemonocyte-derivedmacrophages (MDM),andtodeterminetheeffectsofvariouscombinationsofthesamecytokinesonintracellularsurvivalofR.equi.MDMfrom10adulthorseswerepre-incubatedwithrecombinantequinecytokinesatdoublingconcentrationsrangingfrom25to200ng/mlpriortoinfectionvirulentR.equi.PrimingwithIFN-γ,TNF-α,andIL-6significantlydecreasedintracellularreplicationofR.equicomparedtounprimedmonolayers.Incontrast,primingwithIL-10andIL-1βsignificantly increasedintracellularreplicationofR.equi.Pairwisecombinationsofthecytokines listedabovedidnot results insynergismorantagonism.However,combinationwith IFN-γprevented thedetrimentaleffectsof IL-10onintracellular replicationofR.equiwhereascombinationwithTNF-αor IL-6didnotprevent thenegativeeffectof IL-10.This studydemonstratethatIFN-γ,TNF-α,andIL-6improveequineMDMfunctionagainstR.equiwhereasIL-1βandIL-10aredetrimental.

CRWAD2017 ABSTRACTS


241-ContactnetworkanalysisbetweenswineherdsandfeedsuppliersduringtheearlyphaseoftheporcineepidemicdiarrheaoutbreakinCanada

A.M.Perri1,Z.Poljak1,C.Dewey1,J.C.S.Harding2,T.L.O'Sullivan1.1PopulationMedicine,UniversityofGuelph,London,ON,Canada,2LargeAnimalClinicalSciences,UniversityofSaskatchewan,Saskatoon,SK,[email protected]:HealthinSwinePopulations,Room4,12/4/20172:15PMNetworkanalysiscanbeusedtodescribelivestockcontactandmovementpatternstovisualizeandestimatethepotentialfordiseasetransmission.Theaimofthisstudywastodescribethecontactstructureofatwo-modedirectednetworkoffeedsuppliersandporcineepidemicdiarrhea(PED)caseandcontrolherdsduringearlyphaseoftheCanadianoutbreak.Separatecaseandcontroltwo-modedirectednetworkswerecreatedtorepresentcontactpatternswithfeedsuppliersandherdsbasedonfarmquestionnairedatabetweenDec2013-Feb2014.Acasewasdefinedasaherdwithconfirmeddiagnostic test (RT-PCR)result forPEDVpluspresenceof typicalclinicalsignsfromJan22-Mar1,2014.Controlherdswererandomlyselectedandmatchedonprovince,herdsize,type,andtimeofPEDonsetinthematchedcaseherd.Thecaseherdnetworkhadatotalof21nodes(n=9caseherds;n=12feedsuppliers)with161edges.Anedgerepresentedonefeeddeliveryfromasinglefeedsuppliertoaherd.Thisnetworkwasmadeupof2weakcomponentsandhadadensityof0.05.Atthenode-level,feedsuppliershadameanout-degreeof1.8(range:1-8),ameank-reachoutof3.0(range:2-9.5)andamaximumoutgoingcontactchain(OCC)of9.Thecontrolherdnetworkhad27nodes(n=13controlherds;n=14feedcompanies)with105edges.Thenetworkcontained5weakcomponentsandhadadensityof0.02.Atthenode-level,feedsuppliersinthecontrolherdnetworkhadameanout-degreeof1.4(range:1-3),ameank-reachoutof3.0(range:2-4.5)andamaximumOCCof4.Compared to thecontrolnetwork, thecasenetworkhadahigherdensityof connectionsbutwitha smallernumberofgroups.Similarly,forthecasenetworktheindividualfeedcompanieshadahigheraveragereach,regardingthenumberofherdsthattheyweresupplying.Funding:OMAFRA,OntarioPork,andNSERC-CRD.242-EvaluationoftheimpactofdentalprophylaxisontheoralmicrobiotaofdogsR.Flancman1,A.Singh2,J.S.Weese1.1Pathobiology,UniversityofGuelph,Guelph,ON,Canada,2ClinicalStudies,UniversityofGuelph,Guelph,ON,[email protected]:EcologyofInfectiousAgents–2,Room4,12/5/201711:30AMPurpose:Dentalprophylaxisisaroutinelypracticedveterinaryprocedure.Insightintohowtheplaqueandcompositeoralecologicalnicheswithinthecanineoralcavitychangeovertimeiscrucialtounderstandingtheeffectsofdentalcleaning.Theobjectivesofthisstudyweretoevaluatetheimpactofdentalprophylaxisontheoralmicrobiotaofdogsandtoassessiforalsamplingcouldbeusedasaproxy fordentalplaquecollection.Methods: Thirtydogs receivedadentalprophylaxis.Supragingivalplaqueandcompositeoralswabswerecollectedjustpriorto,andoneweekafterdentalprophylaxis.Asubsampleof10dogswasfollowed,andadditionalsampleswerecollectedtwoandfiveweekspost-prophylaxis.TheV4regionofthe16SrRNAgeneanda2x250chemistrywasusedforIlluminaMiSeqnext-generationsequencing.Results:ResultsdemonstratethatdecreasesinTreponemaandPasteurellaaswellasincreasesinMoraxella and Neisseria distinguished the plaque pre- and one week post-prophylaxis time points. Within the composite oralmicrobiota,Psychrobacterhadarelativeabundanceof20%priortoprophylaxisandPseudomonashadan80%relativeabundanceoneweekafterwards.Arapidtransitionbacktothepre-dentalprophylaxismicrobiotabyfiveweekspost-treatmentwasseenforbothnichesforalphadiversityaswellascommunitymembershipandstructureindices.Directcomparisonofthetwoenvironmentsyieldedstrikingdifferences,withcompleteseparationofgroups.Firmicutes(40%)andSpirochaetes(22%)predominatedintheplaquewhileProteobacteria (58%)waspredominant inthecompositeoralmicrobiota.Greaterrichnesswasalsoseen intheplaquemicrobiota.Conclusions:Thisstudyrevealsthatprophylaxishadaprofoundimpactonboththeplaqueandcompositeoralmicrobiota.Resultssuggestthecanineoralmicrobiotaisresilientandthatoralswabsareapoorproxyforplaquesamples.Further,thegreaterrichnessinplaque samples suggests that studies targeting the presence of specific microorganisms (e.g. periodontal pathogens, zoonoticpathogens)thatonlyinvolveoralsamplingmightunderestimatethetrueprevalenceinthemouthenvironment.

CRWAD2017 ABSTRACTS


243-EvaluationofCampylobacterjejuniisolatestoexperimentallycolonizeturkeypoultsM.J.Sylte1,T.Looft1,M.H.Inbody1,E.L.Meyer1,T.A.Johnson1,L.Susta2,Z.Wu3,Q.Zhang3,J.E.Line4.1FoodSafetyandEntericPathogensResearchGroup,USDAARSNationalAnimalDiseaseCenter,Ames,IA,USA,2DepartmentofPathobiology,UniversityofGuelph,Guelph,ON, Canada, 3Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA, 4PoultryMicrobiological Safety and Processing Research Unit, USDA ARS US National Poultry Research Center, Athens, GA, [email protected]:EcologyofInfectiousAgents–2,Room4,12/5/201711:45AMPurpose: Consumption of contaminated poultry products is the main source of human campylobacteriosis, caused mainly byCampylobacter jejuni.Little isknownaboutthehostresponseof turkeystoC. jejunicolonization,andenumerationfromintestinalsamplescanbechallengingbecauseCampylobacterselectivemediasupportthegrowthofnon-Campylobacterorganisms.Wesoughtto identifya)C. jejuni isolates thatpersistentlycolonizepoults,andb) selectivemedia toenumerate theirabundance in intestinalsamples.Methods:ThreeweekoldpoultswereorallycolonizedwithdifferentC.jejuniisolatesormock-colonized,andwereeuthanizedup to 14 days post-colonization. For ease of isolation, mutants of C. jejuni strain NCTC 11168 were constructed resistant tochloramphenicol (CjCm) or kanamycin (CjK). CjCm and CjK were enumerated on Campy-Line agar with sulfamethoxazole (CLA-S)supplementedwithchloramphenicolorkanamycin,respectively.WildtypeisolatesNCTC11168andNADC20827wereenumeratedusingCampylobacterselectivemedia(Campycefex,CLA-SandChromeAgarCampylobacter(CAC)).PCRwasusedwasusedforpost-culturevalidationofrecoveredcolonies.HostresponsewasevaluatedbyhistologicalscoringoftissuesandqPCRofhostgenesfromcecaltissue.Results:CecalcolonizationbyCjCmandCjKsignificantlydroppedby14days.Wild-typeisolatesNCTC11168andNADC20287persistentlycolonizedthececumforupto21daysusingCLA-SandCAC.Enumerationfromilealandcolonsamplesdiminishedthroughoutthestudy,indicatingthatthececumwastheprimarysiteofC.jejunicolonizationinturkeys.SignificantdifferencesinIL-1β,IL-10, IL-13, IL-17Aand IL-22mRNAexpressionweredetected2daysafter colonization,whichcorrelatedwithhistological lesions.Conclusions:Datafromthisstudydemonstratedthatwild-typeisolatesNCTC11168andNADC20827persistentlycolonizedthececum,andCLA-SorCACwerethebestselectivemediatoenumeratewild-typeCampylobacterfrompoults.Thesefindingswillbeusefultoevaluatethehost-responsebyC.jejunicolonizationinturkeysandevaluatestrategiestoreduceitscolonizationtopromotefoodsafety.244-IntroductionandOverview:TheVaccineNetworkYoungAJ1,ChaseCCL1.DepartmentofVeterinaryandBiomedicalSciences,SouthDakotaStateUniversity,Brookings,SD,[email protected]:VaccinesandVaccinology–1,Room5,12/4/20179:00AMThereareuniqueopportunitiesfortheResearchandDevelopmentofvaccinestoaddresstheneedsofanimalsincomparisontohumans,giventheabilitytodirectlytestformulationsintheintendedspeciesduringdevelopment.Thisisparticularlyadvantageousgiventheneedtoaddressthespreadofemergingorre-emerginginfectiousdiseasesthatmaythreatenAnimalAgriculture.Tothatend,researchershaveaddressedchallengesintheproductionofnewvaccineformulations,andtheregulatoryenvironmenthasrespondedtopermitnewapproachestorapidlydevelopvaccinesforemergingorrapidlymutatingdiseases.Currentchallengestovaccinedevelopmentandimplementationincludetheneedtoexaminenewadjuvantapproachestopromoteearlyandlong-lastingimmunity,newtechnologiesforthedevelopmentofmodifiedlive,subunit,andkilledvaccinesthataddressrapidlychangingorganisms,andimplementationofnewtechnologiesintolicensedproducttoaddressindustryneed.Successfuldevelopmentofnewvaccineapproachesbenefitsmostfromaclearunderstandingofdiseasepathogenesisandepidemiology,immunology,andregulatoryorcommercialrelevanceforthefinalproduct.Currentapproachestoaddresstheseissues,aswellasinitiativestopromotefurtheractivitytoforwardthedevelopmentanduseofnewapproaches,willfurtherenhancedevelopmentofnewapproachestoreducechallengestoanimalagriculture.

CRWAD2017 ABSTRACTS


245-Assessmentofabivalentprobioticlivevaccinecandidateforporcinepost-weaningdiarrhea(PWD)B.Ou1,D.Jin1,M.-X.Xu1,T.Lu2,C.Garcia2,G.Zhu1,W.Zhang2.1CollegeofVeterinaryMedicine,YangzhouUniversity,Yangzhoucity,China, 2Diagnostic Medicine/Pathobiology, Kansas State University College of Veterinary Medicine, Manhattan City, KS, [email protected]:VaccinesandVaccinology–5,Room5,12/5/201710:45AMEnterotoxigenicEscherichia coli (ETEC) strainsexpressingK88 (F4)or F18 fimbriaeandenterotoxins are thepredominant causeofporcinepost-weaningdiarrhea(PWD).PWDcontinuescausingsignificanteconomiclossestoswineproducersworldwide.VaccinesareconsideredthemosteffectivepreventiveapproachagainstPWD.Inthisstudy,weappliedprobioticE.coliNissle1917(EcN)asavectortoexpressF4andF18fimbriaeandassessedtheimmunogenicityofvaccinecandidatesinamurinemodel.ByintegratingF4orF4andF18geneclusters(underPtetpromotor)intothechromosomeDNAofamutantEcNstrain(EcNc,EcNwithdeletionofcrypticplasmidspMut1andpMut2)usingtheCRISPR-cas9technology,weconstructedtworecombinantprobioticstrains,EcNcΔnth/tppB::ptetK88(nthstrain) to express F4 fimbriae and cNcΔyjcS::ptetF18ΔcadAp::ptetF18ΔlacZ::ptetF18ΔyieN/trkD::ptetF18ΔmaeB::ptetK88Δnth/tppB::ptetK88(multicopyintegrationstrain)toexpressbothF4andF18fimbriae.ExpressionofF4orF4andF18fimbriaewasverifiedwithWesternblotusinganti-F4andanti-F18antibodies.TheexpressedF4andF18fimbriaewereconfirmedtoadheretoporcine cell lines IPEC-1 and IPEC-J2.Mice gavage immunizedwith thenth strain developed anti-K88 IgG response, and themiceimmunized with themulticopy integration strain developed anti-K88 and anti-F18 IgG antibody responses.Moreover, the serumantibodiesfromthemiceimmunizedwiththemulticopyintegrationstrainsignificantlyinhibitedtheadherenceofF4+ETECwildtypestrain(3030-2)toIPEC-J2cells.Keyword:bivalentPWDvaccinestrains;E.coliNissle1917;CRISPR/cas9;K88(F4);F18.246 -Method fordetectingLawsonia intracellularisoutermembraneproteins that interactwith intestinal cells to reveal strong

candidatesforsubunitvaccineformulation.M.R.O.Obradovic1,J.Pasternak2,S.Ng1,H.L.Wilson1.1labA133,VIDO-Intervac,Saskatoon,SK,Canada,2Largeanimalclinicalsciences,WesternCollegeofVeterinarymedicine,Saskatoon,SK,[email protected]:VaccinesandVaccinology–5,Room5,12/5/201711:00AMPurpose:Lawsoniaintracellularisisanintracellularmicroorganismandthecausativeagentofporcineproliferativeenteropathy.Itisanobligateintracellular,Gramnegative,bacteriumthatinfectsenterocytesindistalileum.Attachmentandadherenceofbacteriatoenterocytesareimportantstepsinbacterialinfectionbutthemechanismofbacterialcontactwiththehostcellshasnotyetbeenwellelucidated.Bacterialproteinsthatfacilitatecontactwithenterocytesarepossiblygoodimmunogensastheyaresurfaceexpressedandaccessibletothehostimmunesystem.Thusweperformedexperimentstoidentifyantigenicproteinsimportantforattachmentandinvasionthatcouldinducehostimmuneresponseandhavepotentialtobeusedtoformulateasubunitvaccine.Methods:Todetectimmunogenicproteinsthatplayaroleinadherenceorinvasion,welysedthebacterialcells,extractedproteinsandincubatedthemwith intact, live intestinal pig epithelial cells (IPEC-1). Next, we centrifuged andwashed IPEC-1 to remove any unbound bacterialproteins.TheIPEC-1cellsandadherentbacterialproteinsweresubjectedto2DSDSpagegelelectrophoresisfollowedbyWesternblotwith L. intracellularis specific hyperimmune serum. Bacterial proteins recognized by serum antibodieswere isolated from a silverstainedgelandsentforMassspectroscopyanalysis(MS).Results:MSresultsrevealed11uniqueL.intracellularisproteins,fromwhich4arepredictedtobeoutermembraneproteinsusingbioinformaticstools.Proteinswerecloned,expressedandpurifiedfromE.coli.Thetruncatedanddenaturedrecombinantproteinsretainepitopesrecognizedbyhyperimmuneserumthusmakingthempotentiallygoodvaccinecandidates.Conclusions:Usingthistechniquewewereabletoidentifyimportantproteinsonthesurfaceofbacteriumthat interactwithhostcellsandshow immunogenicproperties. Inour futureworkwewillperformexperiments todetermine theimportanceofeachoftheseproteinsinattachmentandinvasionandtheirpotentialtobeusedasimmunogenstoformulatesubunitvaccine.Theserecombinantproteinswillbeformulatedassubunitvaccinesandevaluatedinvivo.

CRWAD2017 ABSTRACTS


247-Characterizingtheinnateimmuneresponsetovaccineadjuvantsdeliveredbyintrauterinevaccinationinsows.G.Hamonic1,J.Pasternak2,N.Forsberg1,H.L.Wilson1.1VIDO-InterVac,Saskatoon,SK,Canada,2LargeAnimalClinicalSciences,WesternCollegeofVeterinaryMedicine,Saskatoon,SK,[email protected]:VaccinesandVaccinology–5,Room5,12/5/201711:15AMPurpose:SignificantinvestmentsinstringentbiosecurityandvaccinationsareemployedtoprotectthehealthandproductivityofsowsandgiltsinNorthAmericanfarrowingbarns.Mucosalvaccinestargetingtheporcineuterusdeliveredalongwithsemenduringartificialinsemination(AI)wouldtakeadvantageofthenaturallordosisresponseduringswinebreeding,simultaneouslyeliminatingtheneedforneedlesandanimalrestraint.However,inordertodevelopaneffectiveintrauterinevaccine,anappropriateadjuvantformulationshould be identified that will induce an effective response by recruiting the necessary immune cells to the uterine lumen andendometrium.Methods: Sowswere synchronized following standard fixed timeAI protocols and subsequently bredwith either astandard semen dose or a standard semen dose containing polyI:C, a host defense peptide and a polyphosphazene (tri-adjuvantcombination).Twentyfourhourspostinsemination,animalswereeuthanizedandbloodanduterinetissueswerecollected.Cellularrecruitmenttouterinelumenwasdeterminedbyflowcytometry.CytokineandchemokineexpressionwasmeasuredbyqPCR.Uterineepithelialcellswere isolatedandstimulatedexvivowithpolyI:C,ahostdefensepeptideandapolyphosphazenetodeterminetheimmune response to individual adjuvant components.Results: Significant reductions in the numbers of gamma delta T cells andmonocytes inbloodwereobservedaftertheadjuvantsandsemenwereadministeredtotheuterus.Geneexpressionanalysiswascarried out on the uterine endometrium and showed significantly increased expression of chemokines associatedwithmonocyterecruitment.Whenuterineepithelial cellswere stimulatedby individual adjuvants,poly I:Cwas found tobe theprimarydriverofchemokineexpression.Conclusions:Theseresults indicate that thetri-adjuvantcombination inducedan immuneresponsedistinctfromtheinflammatoryresponsetosemen.Furtherstudiesandanalysiswilldeterminetheirmechanismofactionfortheseandotheradjuvantcomponentstotailortheformulationforuseaspartofaneffectiveintrauterinevaccine.248-CytokineandinflammatoryresponseprofilesinducedbyadjuvantsPCEPandEmulsigenattheinjectionsiteandthedraining

lymphnodesinpigsR.B.Magiri1,K.Lai2,Y.Huang3,H.Wilson1,G.Mutwiri1.1VIDO-InterVac,SchoolofPublicHealth,UniversityofSaskatchewan,Saskatoon,SK,Canada,2VIDO-Intervac,Saskatoon,SK,Canada,3PrairieDiagnosticServicesInc,Saskatoon,SK,[email protected]:VaccinesandVaccinology–5,Room5,12/5/201711:30AMPurpose:Adjuvantsareformulatedwithantigeninvaccinestoenhancethetypeandmagnitudeoftheimmuneresponse,buttheirmechanismofactionremainpoorlyunderstoodespeciallyinlargeanimals.InductionofaninflammatoryresponseandincreasedcellrecruitmenttothesiteofinjectionwasobservedinmicevaccinatedwithvaccinescontainingtheadjuvantPCEP.Methods:Toascertainwhetherasimilarresponsewasobservedinresponseinpigs,weinjectedPCEP,CpGandEmulsigenintradermallyinpigsandevaluatedinflammatoryresponsesatsiteofinjectionthroughhistopathology.Cytokineproductionattheinjectionsiteandthedraininglymphnodes, and systemic cytokine secretion in peripheral bloodwas alsomonitoredover 2weeks.Results:PCEP induced a significantinflammatoryresponseatthesiteofinjectionandthedraininglymphnodewithouttissuedamage,whileemulsigenonlyinducedtheinflammatoryresponsesatthesiteofinjection.CpGdidnottriggeranyinflammatoryresponsesateithersite.IntradermallyinjectedPCEPledtosignificantproductionofIL-1β,andIL-13atthesiteofinjectionafter4daysandIL-1β,andIL-6atthedraininglymphnodes24hourspostinjection.IntradermallyinjectedEmulsigenpromotedproductionofIL-1βandIL-12onlyatthesiteofinjectionafter4daysbutnotdraininglymphnodes.Inperipheralblood,nosignificantchangeincytokineprofileinresponsetotheadjuvantswereobservedovertime.Conclusions:Together,thesedatademonstratethatadjuvantsPCEPandEmulsigenstimulatetheearlyeventsofinnate immune responseat the injection site, suggesting their ability to increase the immunogenicity to co-administeredantigensthroughcreationofanimmune-competentenvironmentatthesiteof injection.Moreover,thisworkprovidesrelevantinformationaboutelementsofinnateandacquiredimmuneresponseinducedbyvaccineadjuvantsadministeredintheabsenceofantigen.

CRWAD2017 ABSTRACTS


249-PEDvaccinedevelopmentusingnewgeneticvariantPEDVstrainisolatedinfieldH.Jang.WOOGENECo.,GyeonginRo775,Yedeungpogu,Seoul,Korea,[email protected]:VaccinesandVaccinology–5,Room5,12/5/201711:45AMPurpose:PEDVinfectspigsandcausesanentericdisease.Specifically,in2013newvariantstrainofPEDVraisingconcernsregardingtheirprotectiveefficienciesandtheneedfornewvaccinedevelopment.InthisstudywehavebeennewgeneticvariantfieldisolatedPEDVwasadaptedVerocell,geneticanalysis,andvirulencetest,inactivatedvaccinedevelopmentandsafetyandefficacytestinthelaboratoryandinthefiledMethod:Fecalandsmall intestinewerecollectedfrompigletswithseverewaterydiarrhea.TheRT-PCRpositivesamlesusedverocellculture.Inordertotestthepathogenicityofpassagedviruses,thevirusconcentrationwasadjustedto105TCID50permlforthree-dayoldpigletswereorallyinoculatedvirusesgrowninthe15,40,and60passagesofthePED-CUP-B2014strain.Theseverityofdiarrheawasscored,vaccinesafetyandefficacytestwasdoinginthevivariumandfarmvaccineandELISA,SNtest was doing follow the basicmethod, clinical symptomsweremonitored. Result: PEDV isolated from RT-PCV positive samplesinfectedcellswereshowedCPEsuchascelldetachmentandrounding.PhylogeneticanalysisofSsequencesindicatedthattheisolatedPEDVisclusteredwithotherstrainsofPEDVcurrentlycirculatinginUnitedStates.PED-CUP-B2014cultivatedinthepassagenumber65,wasshowgoodimmunogenicityandnovirulence.TodetermineimmunogenicityandprotectiveeffectsofPED-CUP-B2014,SNtiterof serumof sowand colostrumandpigletwerehigher thanother vaccine. These results suggest thatPED-CUP-B2014 inactivatedvaccineestablishstronghumoralimmunity,whichissuperiortotheefficacyofconventionalcommercialinactivatedvaccine.AtthechallengetesttothepigletshowPED-CUP-B2014vaccinegoodprotectandvaccinatedsowsexhibitedagradualincreaseinbodyweightgain and consistently. Conclusion:the present work demonstrated that immunization with the new inactivated vaccine providesprotective immunity topiglets. It canbeexpectedthat large-scaleclinical trialson the field farmfor thevaccinecandidatesnewlyproducedbyisolatingtherecentlyintroducedUS-typemutantstraincanbeexpected.250-BVDVandBHV-1antibodylevelsincolostrumofbeefheifersvaccinatedorunvaccinatedduringgestationwithamultivalent

killedviralrespiratoryvaccineE.J.Reppert1,M.F.Chamorro1,L.A.Robinson1,J.Wick1,N.Cernicchiaro2,S.Lacoste3,R.Sargent4,D.M.Haines4.1ClinicalSciences,KansasStateUniversityCollegeofVeterinaryMedicine,Manhattan,KS,USA,2DiagnosticMedicineandPathobiology,KansasStateUniversityCollegeofVeterinaryMedicine,Manhattan,KS,USA,3DepartmentofVeterinaryMicrobiology,UniversityofSaskatchewanCollegeofVeterinaryMedicine,Saskatoon,SK,Canada,4TheSaskatoonColostrumCompany,Saskatoon,SK,[email protected]:BovineImmunology–1,Room6,12/4/201711:15AMRespiratorydiseaseinnursingbeefcalvesoccursbetween30and150daysoflife.Failureofpassivetransferofimmunityorrapiddecayofmaternally-derivedantibodiesagainstrespiratorypathogenssuchasBVDVandIBRhavebeenassociatedwiththiscondition.Theobjectiveofthisstudywastodetermineeffectofvaccinationofpregnantbeefheiferswithtwodosesofakilledvirus(KV)multivalentvaccinecontainingBVDV1,BVDV2,andBHV-1duringlategestationontotalIgGandantibodiesagainstBVDV1,BVDV2,andBHV-1incolostrum.54pregnantbeefheiferswereallocatedintotwogroups.GroupA(n=24)receivedtwodosesofKVvaccine21daysapartat6. 5 to8monthsof gestation.GroupB (n=30) received5mL salineasunvaccinated controls.Heifersweremonitored for vaccinereaction,abortion, andparturitionbypersonnelblinded to treatment.Newborncalveswereallowed tonurse colostrumnaturallywithoutassistance.ColostrumsampleswerecollectedatcalvingformeasurementoftotalIgGandspecificantibodiestoBVDV1,BVDV2,andBHV-1.Serumsamplescollectedfromcalvesat24hrs.ofagefordeterminationoftotalIgGandneutralizingantibodiestoBVDV1,BVDV2,andBHV-1.ThemeanlevelsofIgGincolostrumatcalvingwerehigheringroupAcomparedwithgroupB(140.17g/Lvs.130.03 g/L; P < 0.05). Themean level of colostrum specific antibodies toBHV-1 andBVDV1werehigher in groupA vs. groupB.ColostrumantibodiestoBVDV2weresimilaramonggroups.ThemeanserumIgGlevelsincalvesat24hrs.weresimilarincalvesborntoheifersingroupAandgroupB(A=30.2g/L;B=32.3g/L;P>0.05).ThemeanLog2serumantibodytiterstoBVDV1,BVDV2,andBHV-1at24hrs.werehigherincalvesborntoheifersingroupA.VaccinationofpregnantbeefheifersduringlategestationwithtwodosesofKVmultivalentvaccinedemonstratedincreasesincolostraltotalIgGandspecificantibodiestoBVDVandBHV-1.Higherserumantibody titers toBVDV1,BVDV2,andBHV-1 incalvesborn tovaccinatedheifersmayextendthedurationofmaternally-derivedimmunityandbetterprotectagainstrespiratorydiseaseduringthepre-weaningperiod.

CRWAD2017 ABSTRACTS


251-NorthwardrangeexpansionofIxodesscapularisevidentovershorttimescaleinOntario,CanadaK.M.Clow1,P.A.Leighton2,N.H.Ogden3,L.R.Lindsay4,P.Michel5,D.L.Pearl6,C.M.Jardine1.1DepartmentofPathobiology,UniversityofGuelph,Guelph,ON,Canada,2DepartmentofPathologyandMicrobiology,UniversityofMontreal,Saint-Hyacinthe,QC,Canada,3NationalMicrobiologyLaboratory,PublicHealthAgencyofCanada,Saint-Hyacinthe,QC,Canada,4NationalMicrobiologyLaboratory,PublicHealthAgencyofCanada,Winnipeg,MB,Canada,5OfficeofChiefScienceOfficer,PublicHealthAgencyofCanada,Ottawa,ON,Canada,6DepartmentofPopulationMedicine,UniversityofGuelph,Guelph,ON,[email protected]:PathobiologyofEntericPathogensKeynote/VectorborneDisease,Room7,12/4/20172:45PMTheinvasionoftheblackleggedtick,IxodesscapularisintoOntario,Canadaposesasignificantrisktopublichealthbecauseitisavectorfornumerouspathogens,includingBorreliaburgdorferisensustricto,thecausativeagentofLymedisease.Baselinefieldsamplingin2014and2015detectedI.scapularisandB.burgdorferiatsitesacrosssouthern,easternandcentralOntario,includingahotspotineasternOntario.A“speedofspread”modelfor I.scapularisdevelopedbyLeightonandcolleagues(2012)estimatedthatthetick’srangewasexpandingnorthwardat46km/year.In2016,werevisitedasubsetofsitessampledin2014and2015tounderstandthechangingnatureofrisk,andassesswhethertherateoftickinvasionisconsistentwiththespeedofspreadestimate.Tickswerecollectedviatickdraggingat17outof36sites,5ofwhichwerenewsitesforI.scapularis.SampleswerepositiveforB.burgdorferiat8sites.Noother I. scapularis-borne pathogenswere detected. Centrographic statistics revealed an increase in the dispersion of I. scapularispositivesitesineasternOntario.Fielddataforeachsitewerethencomparedtothemodel’spredictedyearofestablishmentforeachcensussubdivision.OurfindingsillustratethattherangeexpansionofI.scapularisandtheemergenceofB.burgdorferiisongoing,andprovideshorttime-scaleevidenceoftheprocessesassociatedwithI.scapularisspread.Therangefrontappearstobemovingatarateof~46km/year,withcolonizationofthetickbehindthisrangefrontoccurringataslowerandheterogeneousrate.Assessmentofsite-levelecologicalfactorsdidnotprovideanyinsightintotheunderlyingprocessesthatmaybeinfluencingthecolonizationofI.scapularisinspecificareas.Ongoingfieldsamplingisneededtomonitorthisdynamicprocess.ThisstudyhighlightsthecurrentgeographicriskassociatedwithLymedisease,whichcanbeusedtotargetpublichealthinterventionstotheareasofgreatestrisk.252-AnalysisofBVDV-1a,1band2areplicationandco-infectiondynamicsinepithelialandlymphoidcelllinesbyPrimeFlowRNA

assayS.Silveira1,S.M.Falkenberg1,R.P.Dassanayake1,P.H.Walz2,J.D.Neill1,C.W.Canal3,J.F.Ridpath1.1NADC-ARS,USDA,Ames,IA,USA,2Department of Pathobiology, Auburn University, College of VeterinaryMedicine, Auburn, AL, USA, 3Virology Laboratory, FederalUniversityofRioGrandedoSul-UFRGS,PortoAlegre,[email protected]:EquineandBovineImmunology,Room9,12/5/201711:45AMBovineViralDiarrheaViruses(BVDV)areglobally-distributedpathogensofcattleresponsiblefornumerousclinicalsyndromesthatleadtomajoreconomiclosses.Twospecies,BVDV1andBVDV2,belongingtothegenusPestivirus,Flaviviridaefamily,canbesegregatedintoseveralsubgenotypes.BVDV-1band1aarethepredominantsubgenotypesworldwide,followedby2ainUSA.Thesubgenotypes1aand2aarecommonlyusedinmodifiedlivedvaccines(MLV).Variationinviralreplicationandclinicalpresentationareobservedinsingleandco-infectionsandaretheresultofgeneticandantigenicdifferencesbetweenthespecies,subgenotypesandevenstrains,presenceofpersistentinfections,immunomodulationanduseofmultivalentMLV.TheaimofthisstudywastoexaminethedynamicsofreplicationofBVDV-1a,1band2ainsingle,dual,andtripleinfectiondynamicsusingtwocelllines,onederivedfromepithelialcells(bovineturbinate;Btu)andonederivedfromlymphoidcells(bovineBlymphoma;BL3)usingthePrimeFlowRNAassay.EvaluationbyPrimeFlowRNAassay,aflowcytometry-basedtechniquethatallowstheamplificationofasingleRNAtranscript,wascarriedoutondays2,9and30postinfection.AllstrainsreplicatedmoreeffectivelyintheBtucelllinethantheBL3cellline.Therelativeprevalenceofviralstrainsinco-infectionsoftheBtucelllinevariedovertime,withnostrainexcluding/outcompetingotherstrains.Incontrast,exclusion/competition was observed in co-infections of the BL3 cell line, leading to one strain predominating over time. DNAsequencing, confocal and electronmicroscopywere also performed and similar results were observed. The interaction of strainsobservedinthesestudiesmayhelpexplainvariationsobservedinthefrequencyofsubgenotypesdetectedinthefiled,pathogenesisand immune response tomultivalent vaccines. These studies demonstrate that PrimeFlowRNAassay is an important tool for thedetectionandquantificationofviralinfectionatthesinglecelllevel.

CRWAD2017 ABSTRACTS


253-ComparativeproteomicanalysisoffourstrainsofMycoplasmabovis,strainsG45,M23,428EandDSA16bydifferentialproteinextractionusingTX-114

L.B.Tabatabai1,M.K.Zimmerli2,R.E.Briggs3,F.M.Tatum3.1RoyJ.CarverDepartmentofBiochemistry,BiophysicsandMolecularBiology,Iowa State University, Ames, IA, USA, 2Biochemistry,Western Governors University, Salt Lake City, UT, USA, 3Bovine RespiratoryDiseasesandImmunology,NationalCentersforAnimalHealth,Ames,IA,[email protected]:EquineandBovineImmunology,Room9,12/5/201711:30AMMycoplasmabovisplaysanimportantroleinbovinerespiratorydiseaseandbovinemastitis.SomestrainsofM.boviswerethoughttoplayaroleinlungabscessformation.Toourknowledge,thereisnopublishedinformationonstudiescomparingtheproteomesofabscess-formingandnon-abscessformingstrainsofM.bovis.Theproteomesofanabscess-formingfieldisolate(strainDSA16)andonenon-abscess-forming isogenic field isolate (428E) ofM. bovis were compared to the type strain, PG45 and an additional well-characterizedfieldisolate,M23.SDS-PAGEgelplugsofTX-114solubleproteins,TX-114aqueous-phaseproteins,andTX-114-insolubleproteinswereanalyzedbymatrix-assistedtime-of-flight(MALDI-TOF)massspectrometryandtandemmassspectrometry.Theresultsrevealedthatdifferenceswereobservedinthe1-DgelproteinprofilesoftheTX-114-solubleproteinsofallfourstrains.Specifically,theabscess-formingstrainDSA16showedoneproteinwithapparentmolecularweights79.7kDathatwasnotpresent inthenon-abscess-formingstrain428E,thetypestrainPG45orinthefieldstrainM23.Massspectrometryanalysis,usingScaffoldandMascotsoftwareprograms identifiedmostproteinswitha score>50andusing> than2matchedpeptides.Of the109M.bovisproteinsanalyzed,nohomologsofthe79.7kDaDSA16proteinwerefoundintheNCBIorSwissProtdatabases.Asthedatabasesarecontinuallyupdated,wesuspectahomologofthisproteinwillbeidentifiedsoon.TheseresultssuggestthatthismajorexpressedproteinofDSA16couldbeatargetedfordevelopmentofadiagnostictestforabscess-formingM.bovisincattle.Thesignificanceoftheproteomeanalysisfindingssuggeststhreeorfouridenticalproteinsthatareexpressedbyallfourstrainsmaybetargetedfordevelopmentofinterventionanddiagnosticstrategies.P001-WholegenomesequencingandcomparativegenomeanalysisofBrucellaisolatesfromhumanandanimalsamplesfromthe

countryofGeorgiaK.Sidamonidze1,G.Dzavashvili1,T.Tevdoradze1,E.Zhgenti1,G.Gogoladze1,P.Chain2,T.Erkkila2,G.Chanturia1,P.Imnadze1.1NationalCenterforDiseaseControlandPublicHealth/R.LugarCenter,Tbilisi,Georgia,2LosAlamosNationalLaboratory,LosAlamos,NM,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose:BrucellosisisaglobalzoonoticinfectionthatcausessubstantialeconomiclossesinlivestockandisendemictoGeorgia.Thecausativeagents,BrucellaabortusandB.melitensis,areclassifiedasCategoryBbiologicalthreatagents.Lackofgeneticresolutionwithavailablemethodshasmade itchallengingtounderstand itsevolutionaryhistoryanddeterminetheglobalspread.Wholegenomesequencing(WGS)allowsforadeeperunderstandingofphylogeneticrelationshipsamongbacterialstrains.Methods:Inthisstudy,weassessthevariantsofBrucellaspp.thatarecirculatinginGeorgia.StrainsofB.melitensis(n=5)andB.abortus(n=5)wereselectedfromtheNationalCenterforDiseaseControlandPublicHealth’s(NCDC)LiveCultureRepository,andweresequenced,andanalyzed.Thestrainswerechosenasrepresentativesofmajorgeneticclusters,previouslydeterminedbymultiple-locusvariable-numbertandemrepeat (VNTR) loci (MLVA). All ten strainswere cultured, and genomicDNAwas extractedusing a chloroformextractionmethod.Brucella DNA fragment library preparation and sequencing was performed on aMiSeq platform using the Illumina v2 500-CycleSequencingKit.The rawdatawereused toperformreferencebasedanalysisusingEmpoweringDevelopmentGenomicsExpertise(EDGE) bioinformatics. Results: Phylogenetic and molecular evolutionary (PhaME) analysis was used for whole genome singlenucleotidepolymorphisms(SNP)-basedphylogeneticanalysis.Asaresult,GeorgianstainsB.abortus-2308andB.melitensis-16Marefromuniquecladesandseparatefromthereferencegenomes.Also,B.abortusstrainsfromGeorgiawerefoundtobemorediversefromthereferencegenomeofB.melitensis.Conclusions:ThisstudywasafirstwholegenomesequencingandphylogeneticcomparisoneffortforBrucellastrainsinGeorgia.Thisapproachwillbeincorporatedinfuturesurveillanceefforts.

CRWAD2017 ABSTRACTS


P002-InvasionefficiencyandcytokineproductionsofBrucellaabortuswildtypeandmutantstrainsinRAW264.7cellsS.Shim1,Y.Im1,S.Soh1,S.Kim2,H.Yoo1.1DepartmentofInfectiousDiseases,CollegeofVeterinaryMedicine,SeoulNationalUniversity,Seoul,Korea,Republicof,2CollegeofVeterinaryMedicine,GyeongsangNationalUniversity,Seoul,Korea,[email protected]:BacterialPathogenesis,12/3/20176:30PMBrucellosisisazoonoticdiseasethatcanbeeasilyinfectedinhumanandotheranimals.Althoughseveralresearcheshavebeendoneto reveal the immune responses, the mechanism of Brucella infection still remains for further investigation. Recognition of theinteractionbetweenthebacteriumandhostcellsiscrucialtoelucidatetheinfectiousprocess.B.abortusmutantsweregeneratedusingtransposonmutagenesis.TodemonstratetherolesoftheB.abortusgenes,RAW264.7cells(includingHeLacellsandTHP-1cells)wereinfectedwithB.abortuswildtypeandmutantstrains.Growthrate,internalization,andcytokineproductionofB.abortusmutantstrainsweredeterminedexperimentally.AllofB.abortuswildtypeand28mutantstrainswereinvadedintoRAW264.7cellsshowingdifferentinternalizationefficiency.Wildtypeshowedthehighestinvasionabilityamongthe28strainsinRAW264.7cells,butnotinHeLaandTHP-1cells.Inaddition,theproductionofthecytokines(TNF-α,IL-6andIL-1β)wereanalyzedinRAW264.7cellsstimulatedwiththemutants.Especially,fourB.abortusmutantstrains,C1,C10,C27andC32,showeddifferentproductionlevelsofcytokinescomparedtothewildtype.MutantC1showedlowerproductionlevelsofTNF-αthanthewildtypeat12hrsafterstimulation.Incontrast,mutantsC27andC32showedhigherproductionlevelsofTNF-αandIL-6thanthewildtypeat24hrsafterstimulation.Plus,IL-1βproductionwas increasedat24hrsafterstimulationwithmutantsC1andC10comparedtothewildtype. Inconclusion,theseresultsmaybesuggestingthatinvasionabilitiesandcytokineproductionsdependonthegeneticcharacteristicsoftheB.abortusmutantstrains.ThisworkwassupportedbyKHIDI(No.HI16C2130),theBK21PLUSprogramandtheRIVS,SeoulNat’lUniversity,RepublicofKorea.P003-CoincidencecloningrecoveryofBrucellamelitensisRNAfromgoattissues:advancingtheinsituanalysisofpathogengene

expressioninbrucellosisP.Boggiatto,D.Fitzsimmons,D.O.Bayles,D.Alt,C.E.Vrentas,S.C.Olsen.USDA/NADC,Ames,IA,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose:Brucellamelitensiscausepersistent, intracellular infections insmallruminantsaswellas inhumans, leadingtosignificantmorbidityworldwide.TheintracellularnatureofBrucellacomplicatesthestudyofhost-pathogeninteractionsinvivo.MoststudiesofgeneexpressionprofilesofBrucellaareperformedinvitrooncellculturesorviashort-termmacrophageinfections.Whileseveralgenesassociatedwithpathogenicityhavebeenidentified,littleisknownregardingthegeneexpressionprofilesofBrucellawithintheinfectedhost.OurgoalwastoutilizecoincidencecloningtorecoverB.melitensisRNAandcharacterizepathogengeneexpressionprofilesfrominfectedgoat tissues.Characterizationof suchgenescould identify targets for therapeutic intervention.Methods:Weutilizedandvalidatedthepreviously-developedcoincidencecloningtechniquetoisolateB.melitensisRNAfrominfectedhosttissuesandperformedRNA-sequencing to characterizepathogengeneexpressionpatterns.Results:Wedemonstrate that coincidence cloning is a viabletechniqueforthestudyofhost-pathogeninteractions.Wereportadistincttranscriptionalprofilepresentinsamplesrecoveredfromlong-termB.melitensisgoatinfectionsandwealsopresentthechallengesofvalidatingRNA-SeqexpressiondifferencesintissueswithlowrelativeratiosofpathogenRNA.Conclusions:WeprovidethefirstexampleofrecoverypluscharacterizationofB.melitensisRNAfrominfectedhosttissuesviacoincidencecloning.

CRWAD2017 ABSTRACTS


P004 - Characterizationof theNOD-scid IL2rγnullmousemodel to study the safetyofB. abortus S19ΔvjbR vaccine candidate inBrucella-inducedosteoarticulardisease

O.Khalaf,D.G.GarciaGonzalez,S.P.Chaki,T.A.Ficht,A.M.Arenas-Gamboa.VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,[email protected]:BacterialPathogenesis,12/3/20176:30PMOsteoarticularbrucellosis isthemostcommoncomplication(46.5%)inBrucella-infectedhumansregardlessofage,sex,or immunestatus.ThemechanismofbonedestructioncausedbyBrucellaspeciesremainspartiallyunknownduetothelackofananimalmodelforthestudyofBrucella-inducedosteoarticulardisease.However,duringthepastyears,theuseofImmunocompromisedmicehasproven to be a valuable tool to understandbasic host-agent interactions duringBrucella infection. In this study,we explored thesuitabilityoftheuseofNOD-scidIL2rγnullmouseasamodeltostudyosteoarticularbrucellosisandexaminedthepotentialuseofthismodel to study the safetyof liveattenuatedvaccinecandidates, and specifically for this case theB.abortusS19ΔvjbR.Micewereinoculated intraperitoneallywitha singledoseofeither1x104,1x105,or1x106CFUofB.abortusS19orB.abortusS19ΔvjbRandmonitoredforthedevelopmentofsideeffects,includingosteoarticulardisease,for13weeks.Hypothermia,decreasedbodyweight,splenomegaly,anddeformationoftailswasobservedinmiceinoculatedwithB.abortusS19butnotwithS19ΔvjbR.Histologically,allS19vaccinatedmicehadaseveredose-dependentinflammatoryresponseinmultipleorgansincludingthetail.Specifically,atthetail,the inflammatoryresponsewascharacterizedbytherecruitmentof largenumbersof inflammatorycells includingneutrophilsandmacrophages with marked bone destruction surrounded by large numbers of osteoclasts which histologically resembles what istypically observed inpatientswithosteoarticular brucellosis. In contrast,mice inoculatedwithB. abortusS19ΔvjbRdidnot showsignificantbonechangeswiththeexceptionofscatteredareasofneutrophilinfiltration.Immunofluorescence,insituhybridizationandconfocalimagingdemonstratedthatBrucellawaspresentattheareaofinflammationbothintraandextracellularlywithtropismtoosteoclasts.TheseresultsdemonstratethepotentialuseoftheNOD-scidIL2rγnullmousemodelasaviabletooltostudyosteoarticularbrucellosisandprovidesupportforthepotentialuseofthismodelinevaluatingvaccinesafety.P005-AneffectiveimmunofluorescencestainingassayfordiagnosisortreatmentevaluationofhumanbrucellosispatientsW.Wang,H.Yang,J.Li,X.Yang,C.Li.DepartmentofTransfusionmedicine,SchoolofLaboratoryMedicineandBiotechnology,SouthernMedicalUniversity,Guangzhou,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose: Brucellosis is oneof themost severewidespread zoonoses. It's important todevelopaneffective assay for diagnosis ortreatmentevaluationofhumanbrucellosisinChina.Methods:Threeserologicassays(RBPT,SATandELISA)andthreepathogenictestmethods(nestedPCR,bacteriumculturingandimmunofluorescencestaining, IFS)wereusedfordetectingBrucella infection.Bloodsampleswerecollectedfrom154brucellosispatientsinahospital,Harbin,northofChina.TheIFSwasdevelopedfordetectingbrucellaeinPBMCsofpatientsbyusingthemonoclonalantibodiesagainstmajoroutermembraneproteinsofBrucellamelitensisprovidedinourlaboratory.Results:TheBrucellosispatientswerecategorizedasacutephase(withsymptomslessthan6months)andchronicphase(morethan6months),respectively.Among69bloodsamplescollectedfromacutebrucellosispatients,49(71.0%)werereactivebyIFS,39(56.52%)detectedbyPCRand4(5.8%)positivebybacteriumculturing.However, fortheBrucellaantibodies,68(98.55%)werepositivebySAT,61(88.4%)byRBPTand65(94.2%)byELISA.Among85bloodsamplesfromchronicbrucellosispatients,57(67.06%)weredetectedbyIFS,43(50.59%)byPCR,andnonebybacteriumculturing,while55(64.71%)weretestedpositivebySAT,28(32.94%)byRBPTand82(96.47%)byELISA.Conclusions:TheIFSisaneffectiveandusefulimmunoassayfordiagnosisortreatmentevaluationofhumanbrucellosisinthehospital.

CRWAD2017 ABSTRACTS


P006-TowardsthedevelopmentofaliveattenuatedvaccineforcaninebrucellosisL.W.Stranahan,D.G.GarciaGonzalez,S.P.Chaki,M.E.Hensel,A.M.Arenas-Gamboa.VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,[email protected]:BacterialPathogenesis,12/3/20176:30PMCaninebrucellosis,acontagiousdiseasecausedbyBrucellaspp infectionindogs,constitutesaseriousproblemforpetownersandbreedersintermsofreproductivelossandveterinarycare,andmostimportantly,isconsideredapublichealthconcernasitcanbetransmittedtohumans.Caninebrucellosis isontherise intheUnitedStatesandthere iscurrentlynovaccineforuse indogs.TheobjectiveofthisstudywastoevaluatealiveattenuatedvaccineforcaninebrucellosisbythedeletionofthevjbRgene(BAB2_0118),whichencodesaquorumsensingtranscriptionalregulatorimportantforvirulenceandsurvivalinmultipleBrucellaspp.TheattenuationandimmunepotentialoftheBrucellacanisΔvjbRmutantwasinvestigatedbyinfectionofthecaninemacrophage-likecelllineDH82withwild-typeB. canisRM-666,B. canisΔvjbR, andotherBrucella spp that commonly infectdogs, suchasB. suis.Monolayersofmacrophageswereinfectedatamultiplicityofinfectionof1:100.Interestingly,B.canissuccessfullyenteredcellsatalevelsimilartothatobservedforotherBrucellastrains.However,by24and48hourspost-infection,bacterialnumbershadsignificantlydecreased,suggesting that replicationwithinmacrophages is restrictedcompared tootherBrucellastrains that can infectdogs.Aspredicted,deletionof theΔvjbRgene fromB.canisresulted inattenuation,demonstratedby increasedabilityof themacrophagestokill themutantcomparedtotheparentalstrain.ThesedataconfirmthatthevjbRgeneinB.canisisinvolvedinvirulenceasobservedinotherBrucellastrainssuchasB.abortus(cattle)andB.melitensis(sheepandgoats),andthatthesafetyandefficacystudiesofthisvaccinecandidateobservedinotherspeciesmaytranslateintodogs.P007-RiskofhumanbrucellosisamongpastoralistcommunityinKajiadoCounty,Kenya:acase-controlstudyM.M.Kung'u1,A.B.Orinde2,A.J.Mwatondo3,E.Osoro4,S.Thumbi5,K.Njenga6.1KenyaMinistryofAgriculture,LivestockandFisheries,KenyaZoonoticDiseaseUnit,Nairobi,Kenya,2FoodandAgricultureOrganisationoftheUnitedNations,Nairobi,Kenya,3KenyaMinistryof Health, Kenya Zoonotic DiseaseUnit, Nairobi, Kenya, 4KenyaMinistry of Agriculture, Livestock and Fisheries,Washington StateUniversity,Nairobi,Kenya,5PaulG.AllenSchoolforGlobalAnimalHealth-WashingtonStateUniversity,Nairobi,Kenya,6KenyaMedicalResearchInstitute,CenterforGlobalHealthResearch-KenyaMedicalResearchInstitute,Nairobi,[email protected]:BacterialPathogenesis,12/3/20176:30PMBrucellosis,anendemicdiseaseinKenya,isrankedasoneofthetopfourpriorityzoonoticdiseasesinthecountry.Acase-controlstudywas conducted in 2015 to determine risk factors for human infection among residents of Kajiado-West sub-county, Kenya.Ahospitalbased,unmatchedcase-controlstudywasconductedinthreehealthfacilitiesservingthestudyarea.Casesandcontrolswererecruitedfromindividualsparticipatinginanongoingcommunitybasedbrucellosisinlivestockcohortstudytominimiseselectionbias.ThecasedefinitionwasadaptedfromtheWorldHealthOrganisationwithenzymelinkedimmunosorbentassaytestusedtoclassifycases.Riskfactordatawascollectedusingastructuredquestionnaireandassociationbetweenexposurevariablesanddiseaseoutcomedeterminedusingunconditionallogisticregression.Weenrolled43casesand86controlsintothestudy.Themeanageforthecaseswas48.7(SD±20)range(10-85)whilethatofthecontrolswas37.6(SD±18.8)range(8-72).Thedominantgenderforbothcases(62.7%)and controls (58.1%) groups was female. There was no significant difference in socio-demographic characteristics (sex, religion,occupation,maritalstatusandeducation)betweencasesandcontrolsbesidesage.Consumptionofun-boiledrawmilkregularly(OR7.7,95%CI1.5–40.1)andassistinglivestockindelivery(OR3.7,95%CI1.1-13.5)weresignificantlyassociatedwithbrucellosis.Publichealtheducationonriskbehaviorandinterventionsthatminimizeconsumptionofrawmilkwillreduceincidenceofdisease.

CRWAD2017 ABSTRACTS


P008-Detectionofcirculatingimmune-complexesincalvesinfectedwithMycobacteriumbovisS.A.Hadi1,R.Waters2,K.Lyashchenko3,S.Sreevatsan1.1PathobiologyandDiagnostic Investigation,CollegeofVeterinaryMedicine,MichiganStateUniversity,EastLansing,MI,USA,22InfectiousBacterialDiseasesResearch,VeterinaryMedicalScience,UnitedStatesDepartmentofAgriculture,Ames,IA,USA,3ChembioDiagnosticSystemsInc.,Medford,NY,[email protected]:BacterialPathogenesis,12/3/20176:30PMWehypothesized thatMycobacteriumbovis-host interactionswould lead to accumulationof pathogen specific smallmolecules inserum that lead to accurate TB diagnosis in animal populations.We appliedmultidimensional proteomics (LC-MS-MS) to analyzecirculatingimmunecomplexesincalvesexperimentallyinfectedwithMycobacteriumbovis(MBO)usingdualpathplatform(DPP)testtechnologywithrabbitpolyclonalantibodiesspecifictoMycobacteriumtuberculosiscomplex(MTBC).Calves(n=4)wereprospectivelysampledatbaselineandweeks9,14,15,31and36post-infection.AllsamplesweretestedwithDPPandtest-zonesexcised.Thesewere subjected to proteomics anddata analyzed against a databaseofMTBCpeptides. Thirty-twoand twenty-oneMTBC specificproteins (minimum2peptidehits per proteinwith95%protein threshold)were identified atweek14 andweek36, respectively.FindingsfromourstudysuggestthattheMTBCpeptidesidentifiedarehighlyspecifictoMTBCinfectionandshowpromiseofahighlyaccuratediagnosticpanelforbovinetuberculosis.P009-Seroprevalence&associatedriskfactorsofparatuberculosisamongsmallholderdairyfarmsinwesternChitwan,NepalR.Acharya1,S.Shrestha2.1AgricultureandForestryUniversity(AFU),Rampur,Nepal,2AnimalHealthResearchDivision(AHRD),Khumaltar,Lalitpur,[email protected]:BacterialPathogenesis,12/3/20176:30PMCross-sectional study was carried out to determine the animal level and herd level prevalence and associated risk factors ofparatuberculosisinsmallholderdairyfarmsinWesternChitwan,Nepal.Atotalof115serumsampleswerecollectedfromcattleof62farms.Herdlevelandanimallevelprevalencewereestimatedbytwostagesamplingsurvey.Inthefirststage,numberofherds(primarysamplingunits)wererandomlyselected;inthesecondstage,numberofcows(>1yearage)wererandomlyselected.Enzyme-linkedimmunosorbantassay(ELISA)testkitswereusedforMycobacteriumaviumsubsp.Paratuberculosis (MAP)antibodydetection.TheherdwasdeemedpositiveforthepresenceofMAPifit includedatleastonepositiveanimalinherds.Animallevelprevalencewas12.17%(95%CI)andherdlevelprevalencewas19.36%(95%CI).Theriskfactorswereidentifiedasfollows:Cownotcalvingincleancalvingarea(OR=6.38(1.52-26.78))andmanurestoragewithcattleaccess(OR=4.89(1.17-20.37)).Despitethelimitednumberofsampleincluded,thisstudyisthefirsttoreportandestimatetheseroprevalenceofparatuberculosisinsmallholderfarmsinWesternChitwan,Nepal. Study supports the idea that cow should be calves in clean calving area andmanure storagewith no cattle access for thepreventionoftransmissionofMAPintheherds.

CRWAD2017 ABSTRACTS


P010-MoleculargeneticanalysisofalargeMycobacteriumaviumsubsp.paratuberculosismutantbankG.Rathnaiah1,D.O.Bayles2,J.R.Stabel2,D.K.Zinniel1,J.P.Bannantine2,Y.T.Grohn3,R.G.Barletta1.1SchoolofVeterinaryMedicineandBiomedicalSciences,UniversityofNebraska,Lincoln,NE,USA,2NationalAnimalDiseaseCenter,AgricultureResearchService,UnitedStatesDepartmentofAgriculture,Ames,IA,USA,3CollegeofVeterinaryMedicine,PopulationMedicineandDiagnosticSciences,CornellUniversity,Ithaca,NY,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose:Mycobacteriumaviumsubsp.paratuberculosis(MAP)istheetiologicagentofJohne’sdiseaseinruminantsandapotentialcauseofCrohn’sdiseaseinhumans.OurgoalwastoidentifyMAPgenesencodingessentialandnon-essentialfunctionsunderinvitroandinvivoconditions.Methods:MycoMarT7transposonmutagenesiswascarriedoutandaboutonemillionindependentMAPK-10mutantswerecollected.ChromosomalDNAwasisolatedandfragmentsweresubjectedtoIlluminasequencing.TheMAPmutantpoolwasorallyinoculatedintofivecalvesbyfeedingmilkreplacerthreetimescontaining5.0x105CFUlivebacilli.Fecesandbloodwerecollectedonthedaypriortoinoculationandduringthecourseofthe12-monthinfection.Animalswerenecropsiedtoobtainsectionsof jejunum, ileumand their associated lymphnodes, ileoceccal valveand ileac lymphnodes. Sampleswereprocessed for culture,mutantpoolanalysis,RT-PCR,andhistopathologicandgeneexpressionanalyses.Results:Toclassifygenes,weapplieda4-stateHiddenMarkovmodelanalysisthatassignseachTAsitetoa“statecall”basedontheincreasingnumberofsequencereads:ES(essential),GD(growthdefect),NE(non-essential)andGA(growthadvantage).Intheinvitrostudy,outof4,350genesweidentified328ES,1,103GD,2,603NE,258GAand58unassigned.SomeessentialgenesmatchfunctionsforDNAreplication(dnaE),transcription(rpoB),translation(rpmC,rpsL)andlipidbiosynthesis(fabD2,fadE9).Fortheinvivostudy,analysisofanimalsamplesindicatedthattheIFN-gtestwaspositiveafter90daysupuntilday360forthemutantpool.ForwildtypeK-10,thetestwaspositiveat180days.ThisindicatesthattheanimalsbecameMAPpositive.Serumantibodiesremainednegativethroughouttheentirestudy.Analysisofthetransposonmutantpoolinvivoisstillinprogress.Conclusions:ThisstudydefinesforthefirsttimetheinvitrogeneessentialityinMAPonawholegenomebasis.IncreaseofIFN-gsecretionandthelackoffecalsheddingareconsistentwithalowinfectionmodelthatoccursintheearlystagesofdiseaseprogression.P011-GeneticdiversityandenterotoxinproductionprofilesofStaphylococcusaureusstrainsfromcasesofbovinemastitisJ.Vaughn,R.DugumaAbdi,B.Gillespie,C.Merrill,O.KerroDego.AnimalScience,TheUniversityofTennessee,Knoxville,TN,[email protected]:BacterialPathogenesis,12/3/20176:30PMStaphylococcus aureus is an important zoonotic pathogen that causes mild to life threatening infections in animals and human.Staphylococcusaureus isalsothemost frequentandmajorcausativeagentofmastitis indairycows.S.aureus isoneof themanyfoodborne pathogens that causes food poisoning through its diverse enterotoxins. It is believed thatS. aureus strains that infectdifferenthostspeciesaregeneticallydistinctalthoughsomestrainsarebelievedtobeinfectivetowiderangeofhostspecies.SomereportsalsoshowedthepresenceofdistinctstrainsofS.aureuswithspecifictissue-tropismsuchasthemammarygland.ThereisnoclearlydefinedgeneticmakeupofS.aureus that is responsible for specificdisease inanygiven speciesof animalsorhuman.Theobjectivesofthisstudyare:1)evaluategeneticdiversityofS.aureusisolatesfromcasesofbovinemastitis,2)determineenterotoxinproductionprofilesofS.aureusisolatesfromcasesofbovinemastitis.Atotalof120S.aureusisolatesfromcasesofbovinemastitiscollectedfromdifferentdairycattlefarmsintheEasternTennesseewereevaluatedfortoxingenessuchastoxicshocksyndrometoxin1(tsst-1),Panton-valentineleucocidin(luK)andenterotoxingenes(seA,seB,seC,seD,seE,seG,seH,seI,seJ,seK,seL,seM,seN,seO,seP,andseQ)byPCRusingeachgenespecificprimerpairs.Similarly,geneticdiversityofall120isolateswerealsoanalyzedbypulsedfieldgelelectrophoresis(PFGE).Ourresultsshowedthatthemajorityofthetestedisolateswerenegativeforenterotoxins.TheseBisthemostprevalentgeneamongthe120isolates.ThePFGEresultsshowed35clonalpatternsamongtestedstrainswhichweregroupedinto7majorPFGEtypes.Resultsofthisstudyshowedpresenceofdominantlineagethatareresponsibleformastitisinthisstudyarea.Therefore, developing effective vaccine using conserved immunogenic surface proteins from these dominant strains is the bestapproachtocontrolagainstmastitiscausedbydifferentstrains.

CRWAD2017 ABSTRACTS


P012-AminoacidinhibitionofbiofilmformationbyStaphylococcusaureus,Pseudomonasaeruginosa,andListeriamonocytogenesA.L.Brown1,K.S.Brandenburg1,J.F.McAnulty2,C.J.Czuprynski1.1PathobiologicalSciences,UniversityofWisconsin-Madison,SchoolofVeterinaryMedicine,Madison,WI,USA,2SurgicalSciences,UniversityofWisconsin-Madison,SchoolofVeterinaryMedicine,Madison,WI,[email protected]:BacterialPathogenesis,12/3/20176:30PMObjective:Biofilmbacterialcellsgenerallyaremoreresistanttohostdefensemechanismsandantimicrobialtherapy.TheopportunisticpathogensStaphylococcusaureusandPseudomonasaeruginosaarecommonlyisolatedfromchronicwoundsinanimalsandhumanbeings.Theirimportanceinnosocomialinfectionsismediatedinpartbytheircapacitytoformbiofilms.ListeriamonocytogenesandS.aureus,areimportantcausesoffoodbornedisease.Biofilmformationisrecognizedasanimportantreasonforbacterialcontaminationandpersistenceinfoodprocessingenvironments.Severalpreviousreportssuggestedthataminoacidshavepotentialasnaturalanti-biofilmagents.Methods:InthisstudywesystematicallyevaluatedtheefficacyofD-andL-isomersofall26essentialaminoacidsatinhibitingbiofilmformationbyS.aureus,P.aeruginosaandL.monocytogenes.AcrystalvioletstainassaywasusedtoquantifybiomassandIC50valueswerecalculatedbynon-linearregression.Results:Atotalof21isomersinhibitedbiofilmformationbyP.aeruginosa,withD-Serexhibitingthemostpotencyat0.3mM.ForS.aureus24isomersinhibitedbiofilmformation,with1.2mML-Leubeingmostpotent.BiofilmformationbyL.monocytogeneswasinhibitedby9isomers,withD-TyrandL-Ileshowingsignificantinhibitionat0.5mM.InterestinglyD-Tyrinhibitedbiofilmformationbyallthreeorganisms,whileconverselyL-AspandL-Proenhancedtheirbiofilmformation.Inadditionweobservedadditiveactivityforseveralaminoacidcombinations.AnequimolarmixtureofL-ArgandD-His(4mM)inhibitedS.aureusbiofilmby99.95%at24h.Similarly,anequimolarmixtureofD-SerandD-Tyr(2mM)inhibitedP.aeruginosaby85%at24h.Conclusions:Theseresultshighlightseveralaminoacidsthatpreventbiofilmformationbydiversebacterialspecies,providingpromisingavenuesforthepreventionandtreatmentofbiofilm-mediatedcontaminationanddisease.P013-ThepathogenicroleofMoraxellabovoculiininfectiouskeratoconjunctivitisinsemi-domesticatedreindeerJ.SanchezRomano1,I.H.Nymo2,K.K.Sørensen3,T.Mørk2,J.A.Angelos4,M.Tryland1.1ArcticandMarineBiology-ResearchGroupofArctic InfectionBiology,TheArcticUniversityofNorway (UiT),Tromsø,Norway, 2NorwegianVeterinary Institute,Tromsø,Norway,3Department of Medical Biology - Vascular Biology Research Group, The Arctic University of Norway (UiT), Tromsø, Norway,4Department of Medicine and Epidemiology, University of California, Davis - School of Veterinary Medicine, Davis, CA, [email protected]:BacterialPathogenesis,12/3/20176:30PMTheaimofthisstudywastoinvestigateapossibleroleforMoraxellabovoculi inthepathogenesisofinfectiouskeratoconjunctivitis(IKC)insemi-domesticatedEurasiantundrareindeer(Rangifertarandustarandus).Fivesemi-domesticatedreindeer,culturenegativeforthepresenceofM.bovoculiintheireyes,wereinoculatedwithM.bovoculiisolatedfromtheIKC-affectedeyeofareindeeraspartofachallengeexperimentin2014(Group3).AlthoughthisisolatefailedtocauseIKC,animalsinoculatedwithCervidHerpesvirus2(CvHV2;Group1)oracombinationofCvHV2andM.bovoculi(Group2)developedclinicalsignsofIKCwithin2daysafterinoculation.Tissues (upper and lower eyelids, cornea and lacrimal gland) collected from each eye after fixationwere processed routinely forhistologicalexamination.Paraffin-embedded sectionsof5μmwere stainedwithhaematoxylinandeosinand subjected toablindhistological examination. Histopathological analysis of the tissues collected from the experimental animals in group 3 showed noapparentdamageof themucosalorcornealepithelium,andtheeyesremainedhealthy. Incontrast,exudates,edema,hyperemia,haemorrhage,necrosis,vascular thrombosis,vascularnecrosis, infiltrationofmononuclearcellsandneutrophils,pusand lymphoidfolliclereactionwerepresentinthetissuesfromtheanimalsinoculatedwithCvHV2(group1and2).InordertoinvestigatepotentiallypathogeniccharacteristicsofM.bovoculi inreindeer,38isolateswerecollectedfromreindeerwithandwithoutclinicalsignsofIKCfrom11geographicallocationsinthereindeerherdingareasofNorway,SwedenandFinland.Theseisolates,togetherwiththeisolateusedinthechallengeexperiment,willbeinvestigatedbywholegenomesequencingandcomparativegenomicanalysistocompareM.bovoculi isolatedfromreindeerandcattle.ThisstudywillhelptofurthercharacterizepotentiallypathogenicfactorsofM.bovoculiisolatedfromreindeeranditspossibleroleinIKC.

CRWAD2017 ABSTRACTS


P014-AnalysisofEscherichiacolistrainsassociatedwithpersistentandtransientbovinemastitisandtheroleofcolanicacidonmotilityandcomplementresistance

J.D.Lippolis1,D.B.Holman2,B.W.Brunelle2,T.C.Thacker3,B.L.Bearson4,T.A.Reinhardt1,R.E.Sacco1,T.A.Casey2.1RuminantDiseaseandImmunology,NationalAnimalDiseaseCenter/ARS/USDA,Ames,IA,USA,2FoodSafetyandEntericPathogens,NationalAnimalDiseaseCenter/ARS/USDA,Ames,IA,USA,3BacterialDiseasesofLivestock,NationalAnimalDiseaseCenter/ARS/USDA,Ames,IA,USA, 4Agroecosystems Management, National Laboratory for Agriculture and the Environment / ARS / USDA, Ames, IA, [email protected]:BacterialPathogenesis,12/3/20176:30PMEscherichiacoliisaleadingcauseofbacterialmastitisindairycattle.Infectionismostoftentransient,causingamammaryinfectionthatlasts2-3days.However,E.colihasbeenshowntocauseapersistentinfectioninaminorityofcases.MechanismsthatallowforapersistentE.coliinfectionarenotfullyunderstood.ThegoalofthisworkwastodeterminedifferencesbetweenE.colistrainsoriginallyisolatedfromdairycattlewithtransientandpersistentmastitis.WesequencedthegenomesoftheE.colistrainsandreportgenesuniquetothetwophenotypes.Inaddition,weusedRNAsequencingandshowgeneexpressiondifferencesinnearly200geneswhencomparing bacteria from the two clinical phenotypes. Our previous work demonstrated that E. coli strains that cause persistentinfectionsweremoremotilethanthosethatcausetransientinfections.Inadditiontogenesinvolvedinmotilityweobserveddifferencesinthewcaoperon,whichencodesforcolanicacidacapsularpolysaccharide.DNAaswellasRNAsequencingofthewcaoperonshoweddifferencesforthetwophenotypes.Deletionofgenesinthewcaoperonfromapersistentstrainresultedinareductionofmotilityasmeasuredinswimmingandswarmingassays.WeshowthattransientE.colistrainsaremoresensitivetocomplement-mediatedkilling.ThedeletionofgenesfromthewcaoperoncausedapersistentE.colistraintobecomesensitivetocomplement-mediatedkilling.ThisworkidentifiesimportantdifferencesbetweenE.colistrainsthatcausepersistentversusatransientmammaryinfectionindairycattle.P015-Currentresearchondigitaldermatitis:lessonsfromthemodelJ.Wilson-Welder,J.Nally,D.Alt.InfectiousBacterialDiseaseofLivestock,NationalAnimalDiseaseCenter,ARS-USDA,Ames,IA,[email protected]:BacterialPathogenesis,12/3/20176:30PMIntroduction:Digitaldermatitisisaninfectiouscauseoflamenessaffectingdairycattle,beefcattle,sheep,goats,andasmallpopulationofNorthAmericanwildelk.ItisassociatedwithseveralTreponemaspecies,andotheraerobicandanaerobicbacterialspecies.Whilethe exact etiology has not been demonstrated, a number of bacterial, host and environmental factors contribute to diseasedevelopment.Inordertostudyhost-bacterialinteractions,areproduciblelaboratorymodelofinfectionisrequired.Theobjectiveofthisexperimentwastofurtherdefineanddelineateapreviouslysuccessfulmodelofexperimentaldigitaldermatitisinsheep.Method:Crossbredsheepwereobtainedfromaflockfreeofhoofdisease.Anareaofskinaboveeachheelbulbandbelowthedewclawswasabraded;feetwerethenwrappedtocreateamoist,anaerobicenvironment,believedtopredisposeanimalstoinfection.After3days,theabradedareaswereinoculatedbyexposuretomaceratedlesionmaterialfrompriorexperimentallyinduceddigitaldermatitisinsheep. Animals weremonitored for lameness and lesion development for 4 weeks. Results: Experimentally inoculated hind feetdevelopedlesionsby2weekspost-inoculation.Histologicchangesinthedermisandepidermiswereevident9-10dayspost-inoculationand were consistent with those described for bovine digital dermatitis, including erosion, ulceration, ballooning degeneration ofkeratinocytes andneutrophilic inflammatory infiltrates. Silver stainingof lesionbiopsies at 5 dayspost-inoculation confirmed thatspirochetespenetratedhosttissue.Conclusion:Digitaldermatitis isaninfectiousdiseasethatcanbereproducedinexperimentallyinoculatedsheep.Lesionmaterialcanbepreparedinmassandstoredallowingforanimalsatdifferenttimestobeinoculatedwiththesamelot.Serialpassageoflesionmaterialwithinthesheepmodelappearstohaveshortenedthetimerequiredfordevelopmentoflesions.Thecontinueddevelopmentandrefinementofthisovinemodelofdigitaldermatitiswillallowfor insights intopathogenicmechanismsofinfection,andthedevelopmentofimproveddiagnosticandtherapeutics.

CRWAD2017 ABSTRACTS


P016-MolecularanalysisoftheinteractionofLigBandtropoelastinC.-L.Hsieh1,C. Ptak1,R.Oswald2,Y.-F.Chang1. 1PopulationMedicineandDiagnostic Sciences,Cornell.University, Ithaca,NY,USA,2PopulationMolecularMedicine,Cornell.University,Ithaca,NY,[email protected]:BacterialPathogenesis,12/3/20176:30PMLeptospiraimmunoglobulin-likeproteinB(LigB)iscapableofmediatingtheattachmentofpathogenicleptospiratothehostthroughinteractionwithvariousextracellularmatrices(ECM)suchaselastin.Humantropoelastin(HTE),thebuildingblockofelastin,confersresilienceandelasticitytolungandothertissues.PreviouslyidentifiedIg-likedomainsofLigB,includingLigB4andLigB12,bindtoHTE,which is likely to promote Leptospira adhesion to lung tissue. However, the molecular mechanism that mediates the LigB-HTEinteractionisunclear.Inthisstudy,theLigB-bindingsiteonHTEwasfurtherpinpointedtoaN-terminalregionofthe20thexonofHTE(HTE20N).Alaninemutantsofbasic andaromatic residuesonHTE20Nsignificantly reducedbinding to the LigB.Additionally,HTE-bindingsitewasnarroweddowntofirstβ-sheetofLigB12.Onthisbindingsurface,residuesF1054,D1061,A1065andD1066werecriticalfortheassociationwithHTE.Mostimportantly,therecombinantHTEtruncatescoulddiminishthebindingofLigBtohumanlungfibroblastsby68%,andcouldblocktheassociationofLig-expressingspirochetestolungcellsby61%.Thesefindingsshouldexpandourunderstandingofleptospiralpathogenesis,particularlyinpulmonarymanifestationsofleptospirosisP017-Qlp19,anovelinfection-associatedleptospiralproteinB.Beigel,A.Verma.LincolnMemorialUniversityCollegeofVeterinaryMedicine,Harrogate,TN,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose:ThepathogenicmechanismsinleptospirosisarenotwellunderstoodthoughproteinsproducedbyLeptospirainterrogansappeartobeimportant.Thisstudydescribesanovel,infection-associatedleptospiralprotein,Qlp19.Methods:ThroughscreeningofalambdaphageexpressionlibraryofL.interrogans,weidentifiedanovelprotein,Qlp19.Triton-XextractionofLeptospirainterroganswasutilizedforcellular fractionationand localizationstudies.RecombinantQlp19wasusedtodevelopanELISAtoassessthehostresponsetotheproteinbyscreeningforthepresenceofQlp19-specificantibodiesineyefluidsandseraofinfectedhorses.Results:ThegeneencodingQlp19wasdetectedinallpathogenicL.interrogansbutnotinnonpathogenicL.biflexa.CellularlocalizationstudiesindicatethatQlp19islocatedintheleptospiralperiplasmoroutermembrane.Qlp19-specificantibodieswerepresentintheeyefluidsandseraofsignificantnumberofinfectedhorses,butabsentintheseraofvaccinatedanimals.Conclusions:Inthisstudy,wedescribeanovel leptospiralprotein,Qlp19.Qlp19isanimmunogenicproteinconservedamongstpathogenicLeptospiraspp.ThesequalitiesmakeQlp19anattractivecandidateforuseindiagnosisofleptospirosis.Thepresenceofanti-Qlp19antibodiesinnaturallyinfectedequinessuggestthatQlp19couldbeusefulinthediagnosisofleptospirosis.FurtheranalysisisnecessarytoevaluatethecorrelationbetweenaQlp19-basedELISAandstandardserologicalassays.

CRWAD2017 ABSTRACTS


P018-LipidbiomarkersofimmuneactivationinequineleptospirosisandLeptospira-vaccinatedhorsesP.Wood1,M.Steinman2,E.Erol2,C.Carter2,U.Christmann1,A.Verma3.1CollegeofVeterinaryMedicine,LincolnMemorialUniversity,Harrogate,TN,USA,2UniversityofKentucky,Lexington,KY,USA,3CenterofInfectious,ZoonoticandVector-bornediseases,CollegeofVeterinaryMedicine,LincolnMemorialUniversity,Harrogate,TN,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose:Currentlyavailablediagnosticassaysfor leptospirosiscannotdifferentiatevaccinefrominfectionserumantibody.Severalleptospiralproteinsthatareupregulatedduringinfectionhavebeendescribed,buttheirutilityasadiagnosticmarkerisstillunclear.Inthisstudy,weundertookalipidomicsapproachtodetermineifthereareanyreliablelipidbiomarkersofimmuneactivationinhorsesnaturallyinfectedwithpathogenicLeptospiraspp.andhorsesvaccinatedagainstacommerciallyavailablebacterin.Methods:Lipidswereextractedfrom100μLofEDTAserumwithmethyl-tert-butyletherandmethanolcontainingstabledisotopestandards.Constantinfusionhigh-resolutionESImassspectrometryoftheselipidextractswereperformedasdescribedpreviously.Results:Utilizingahigh-resolutionmassspectrometryserumlipidomicsanalyticalplatform,wedemonstratethatcyclicphosphatidicacids,diacylglycerols,andhydroperoxideoxidationproductsofcholineplasmalogensareelevatedinboththeserumofhorsesinfectedwithleptospirosisandhorsesvaccinatedagainstthis infection.Otherbiomarkersof interestweretriacylglycerolsthatwereonlyelevated intheserumofinfectedhorsesandsphingomyelinsthatwereincreasedonlyintheserumofvaccinatedhorses.Conclusions:OurdatasuggestthataugmentedserumlevelsofcyclicphosphatidicacidsmaybeusefulasglobalbiomarkersofimmuneactivationofphospholipaseD,asuggestionsupportedbyparallelincreasesinthelevelsofdiacylglycerols.Measurementsoftheselipidsalongwithsphingomyelins,hydroperoxidesofglycerophospholipids,andtriacylglycerolsalsoallowsfordifferentiationofimmuneactivationbyvaccinationfromthatinducedbyanactiveleptospiralinfection.P019-UsingActinobacillussuistoidentifyputativeinvasiondeterminantsinothermembersofthegenusActinobacillusA.R. Bujold, R. Liu, A. Shure, A.M. Kropinski, J.I. MacInnes. Pathobiology, University of Guelph, Guelph, ON, [email protected]:BacterialPathogenesis,12/3/20176:30PMMembersofthegenusActinobacillusareGram-negativebacteriatypicallyassociatedwithmucosalmembranesofmammalianhosts.Whilesomespeciesarebenigncommensals,otherscancauseimportantdiseasesindomesticanimalsandhumans.Althoughabletocause severe fibrinous hemorrhagic pneumonia in swine, A. pleuropneumoniae does not cause systemic disease whereas othermembersofthegenussuchasA.suiscaninvaderesultinginsepticemiaandsequelaesuchasmeningitis,arthritis,anddeath.TobegintounderstandtheinvasivephenotypeofActinobacillusspp.,wecomparedActinobacillusgenomes.Completegenomesof8isolateswere obtained from ncbi.nlm.nih.gov. Pseudogenomes of 5 additional isolates were assembled using DNASTAR SeqMan andprogressiveMauve and annotated with BASys. Whole genome phylogenetic trees of A. capsulatus, A. equuli subsp. equuli, A.pleuropneumoniae (4 serovars),A. suis (5 strains, 3 serovars),A. ureae, andA. succinogeneswere constructed using CVtree3. A.suisisolatesclusteredbysurfaceantigentype(O1:K1,O2:K2,O2:K3).Inaddition,A.suiswasmorecloselyrelatedtoA.ureae,A.equuliequuli,andA.capsulatus (causativeagentsofsystemicdisease inhumans,horses,andrabbits respectively) thantoanotherswinepathogen,A.pleuropneumoniae.GiventhesimilarphenotypeandhostrangeofA.suisandA.pleuropneumoniaeandtheirsharedcomplementofvirulencefactors(e.g.,cytotoxinsApxIAandApxII),thisfindingwasnotanticipated.Further,usingthelarge-scaleblastscore ratio (LS-BSR) pipeline, similarities and differences between A. pleuropneumoniae and invasive Actinobacillus species weredetectedin251putativevirulencegenesassociatedwithserumresistanceandinvasion(e.g.,autotransporters,CPS,LPS,OMPs,sialicacidbiosynthesis,IgA1proteases,opacityassociatedproteinA)andotherimportantvirulencefactors(e.g.,RTXtoxins,biofilm,ironacquisition).Toourknowledge,this isthefirstgenome-widestudyofmembersofthegenusActinobacillusanditshouldhopefullycontributetoabetterunderstandingofhosttropismandmechanismsofinvasionofpathogenicActinobacillusandrelatedgenera.

CRWAD2017 ABSTRACTS


P020-VaccinedevelopmentforprotectionagainstsystemicinfectionswithStreptococcussuisandHaemophilusparasuisinswineS.L.Brockmeier,K.C.Eberle,K.T.Mou,S.J.Hau,C.L.Loving,T.L.Nicholson,U.Waack.VirusandPrionResearchUnit,NationalAnimalDiseaseCenter,Ames,IA,[email protected]:BacterialPathogenesis,12/3/20176:30PMBothStreptococcussuisandHaemophilusparasuisareimportantinvasivebacterialpathogensofswine,commonlycausingmeningitis,arthritis,polyserositis,andsepticemia.Duetothepresenceofmanyserotypesandhighgenotypicvariability,efficaciousvaccinesarenot readily available.Weareusing various strategies such as functional genomic screens, immunoproteomics, and attenuation todevelop potential vaccine candidates, followed by testing against both homologous and heterologous protection against thesepathogens.Theselectionofproteincandidatesforinclusioninvaccinesisaccomplishedbyidentifyingfitnessgenesthroughafunctionalgenomicsscreen,selectingpredictedsurface-associatedproteins,andidentifyingproteinsconservedacrossisolatestoenhancetheprospectofcross-protection.Immunoproteomicmethodsarebeingusedtodetectproteinsthatarereactivewithantiserafrompigsthataremorebroadlyprotectedcomparedtoantisera frompigsonlyprotectedagainsthomologouschallenge inorderto identifypotentialtargetsresponsibleforheterologousprotection.Mutantsinpurportedvirulencefactorsarealsobeingtestedforattenuationandpossibleuseasvaccines.VaccinetrialsareexaminingtheefficacyofthesevaccinecandidatesagainstsystemicdiseasecausedbyS.suisorH.parasuis.Throughthisrationalapproachwehaveidentifiedseveralpromisingvaccinecandidatesagainsttheseimportantswinediseases.P021-GenerationoftheenterotoxigenicEscherichiacolivaccinecandidatebylysozyme-PMAP36fusionprotein&itsprotection

efficacyagainstpigcolibacillosisJ.Moon1,W.Kim1,J.Cho1,D.Tark2,J.HUR1.1CollegeofVetinaryMedicine,ChonbukNationalUniversity,Iksan,Korea,Republicof,2KoreaZoonosisResearchInstitute,ChonbukNationalUniversity,Iksan,Korea,[email protected]:BacterialPathogenesis,12/3/20176:30PMPurpose:F4+,F5+,F6+andF41+ETECstrainswerelysedbylysozyme-PMAP36fusionprotein.Subsequently,theefficacyofthelysedcellsasavaccinecandidateagainstcolibacillosiswasevaluatedinpiglet.Methods:Thelysozyme-PMAP36fusionproteinwasexpressedtolysetheETECstrains.Allgroupsows(n=6)wereintramuscularlyimmunizedat11weeksofpregnancyandat14weeksofpregnancy.GroupAwereimmunizedwithsterilePBS.GroupBwereimmunizedwiththemixtureofthelysedETECcells.Bloodsamplesweretakenat0,3and6weekspostprimeimmunization.Allpigletswereorallychallengedwiththemixtureofeachchallengestrainat5-day-old.Allpigletsweremonitoreddailyfordiarrheaandmortalityfor14daysafterchallenge.Results:SerumIgGtitersagainstallETECfimbrialantigensweresignificantlyincreasedinallimmunizedsowscomparedtothoseingroupA.Inimmunizedgroupsows,colostrumIgAandIgGtitersagainstallfimbrialantigensweresignificantlyincreased.TheserumIgGandIgAtitersagainstallETECfimbrialantigensingroupApigletsweresignificantlyhigherthanthoseofcontrolpiglets.GroupBpigletsdidnotexhibitclinicalsignssuchasdiarrhea.Incontrast,diarrheawasobservedin17of26groupApiglets(65.4%)and8diedduetoseverediarrhea.Conclusion:Theseresultsindicatethatintramuscularimmunizationofsowswiththemixtureofthelysedcellscaneffectivelyprotecttheiroffspringfrompigletscolibacillosis.

CRWAD2017 ABSTRACTS


P022-RoleofantigenI/IIintheStreptococcussuisserotype9virulence:Implicationinthesystemicinfectionanddevelopmentofclinicaldisease

J.-P. Auger, A. Boa, M. Gottschalk. Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, QC, [email protected]:BacterialPathogenesis,12/3/20176:30PMStreptococcussuisisanimportantporcinebacterialpathogenandzoonoticagentresponsibleforsuddendeathandsepticshock,ofwhichserotype9(SS9)isamongstthemostimportant.Ofthefewputativevirulencefactorsdescribedsofar,werecentlydemonstratedthatantigenI/II(AgI/II)isimplicatedinavarietyoffunctionsinvolvedincolonizationandpersistenceinpigs;however,itsroleinthedevelopment of the systemic infection responsible for clinical disease remains unknown. Herein, the role of the SS9 AgI/II in thesystemic infectionwas evaluatedusing an isogenicAgI/II-deficientmutant (AgI/II-) and awell-characterized intraperitonealmousemodelofinfection.While100%ofmiceinfectedwiththewild-typestrain(WT)succumbedtoinfectionwithin3days,only10%ofmiceinoculatedwith theAgI/II- died.WT-infectedmicedevelopedanelevatedbloodbacterialburden,whichalongsideanexacerbatedsystemic inflammatory reaction,was responsible for their rapiddeath.Bycontrast, little tonobacteremiawasobserved inAgI/II--infectedmice. These results suggest a role of the SS9AgI/II in the peritoneumand/or, following dissemination, the bloodstream.Consequently,theroleofAgI/II inresistancetophagocytosisand intracellularkillingbyperitonealmacrophagesanddendriticcells(DCs),importantphagocytesoftheperitonealcavityandsystemicorganssuchasthespleen,respectively,wasevaluated.WhileAgI/IIconferred resistance to phagocytosis and promoted intracellular survival, it also participated in complement-dependent pro-inflammatorymediator induction fromDCs.Additionally,AgI/IIwaspartially implicated in resisting thebactericidaleffectofwholeblood. Virulence studies following intravenous injection are currently underway to ascertain whether the role of AgI/II is in theperitonealcavityonlyoralsointhebloodstream.Takentogether,theseresultssuggestthattheSS9AgI/IIcouldplayanimportantroleinthesystemicinfectionbypromotingprotectionagainstphagocytes,bloodstreampersistence,andinductionofinflammation,thuscontributingtothedevelopmentofclinicaldisease.P023-InvestigationoftheassociationbetweentheStreptococcussuisgenomeandexpressionofclinicaldiseaseinnurserypigsL. Denich, Z. Poljak, K. Kuruppu, R. Friendship, A. Farzan, E. Arndt. PopulationMedicine,Ontario Veterinary College/University ofGuelph,Guelph,ON,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMStreptococcussuisisabacteriumthatusuallycolonizesthenasalandtonsillarareasofmanypigs,withouthavinganyknownnegativeeffectsonhealth.Insomepigs,systemicinfectioncouldresultleadingtoawiderangeofdiseaseconditions.Suchconditionscommonlyaffectpiglets4-8weeksofage.Theexactpathogenesisinvolvedinclinicaldiseaseisnotcompletelyknown,butitislikelythatmanyfactorsplayarole.Somecouldbedirectlyrelatedtothepathogenitself.Forexample,S.suishas35establishedserotypeseachhavingmanystrains;someofwhichcouldpresentasmorevirulentandtransmissiblecomparedtoothers.Recentdevelopmentsinsequencingapproachesprovideopportunitiestoexplorethesedetailsthatpreviouslywerenotpossible.TheobjectiveofthisstudyistodetermineifthereisanassociationbetweentheS.suisgenomeandclinicalstatusinnurserypigs.Acasecontrolstudyinvolving4-8weekoldnurserypigs fromOntario farms isbeingconducted.Casesweredefinedaspigsshowingclinicalsignssuchastremors,stagnation,paddling,jointswelling,inabilitytostandandconvulsions.Nasal,tonsil,rectalandmeningealswabs,spleen,wholebloodandserumarecollected.ClinicalcaseshadtobeconfirmedasS.suispositivebybacteriologicalculturefromthemeningealswab,spleenorblood.Ahealthycontrolpigwasmatchedtoeachcasepigincludedinthestudybasedonherd,timeofvisitandpen.Nasal,tonsilandrectalswabs,wholebloodand serumwere collected fromeachcontrolpig.Relevant sampleswereplatedonColumbiabloodagar, andsuspectcoloniesweresub-cultured.DNAhasbeenextractedfrompresumablypositivecoloniesandidentifiedasS.suiswithaPCRtestbasedonthegdhgene.Atwostep-multiplexPCRisfurtherusedtoserotypeisolates.Preliminaryresultsindicatethatofthe30pigssampledthusfar,10/15casesand12/15controlstestedpositiveforS.suis.Serotypes3,7-9,29anduntypablewerethemostfrequentlydetected in cases and serotypes 7,17,29 and untypable most frequently in controls. Description of clinical cases are presentedelsewhereandtheprotocolfornextgenerationsequencingiscurrentlybeingdeveloped.

CRWAD2017 ABSTRACTS


P024-AcasecontrolstudytoexploretheinteractionbetweenStreptococcussuisinfectionandrespiratoryviralpathogensofnurserypigs

K.Kuruppu,L.Denich,E.Arndt,Z.Poljak,V.Farzan,R.Friendship.UniversityofGuelph,Guelph,ON,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMStreptococcus suis is a globally reported pathogen known to causemeningitis as well as arthritis, polyserositis, endocarditis andpneumoniainnurserypigs.S.suisisabundantinallswineherdsandlong-termcontrolismadedifficultbytheemergenceofsporadicoutbreaksofdifferentserotypes.S.suisisoneofthemanypathogensthatinteractwithPRRSVasasecondaryinfectiousagent.ItishypothesizedthatPRRSV-inducedsuppressionofpulmonaryintravascularmacrophagesmayincreasetheincidenceofS.suisduetodiminishedbacterialclearance.NurserypigscoinfectedwithPRRSVandS.suisexperiencesevereCNSdisease,bacteremiaandhighermortalitythanthoseinfectedwithS.suisalone.OutbreaksofS.suisinnurserypigletsmaybesuggestiveofanunderlyingviralpathogen.TheobjectiveofthisstudyistocomparetherespiratoryviralgenomeofnurserypigsclinicallyaffectedwithS.suiswithhealthypigsfromthesamegrouptofurtherexplorethishypothesisofviral-bacterialinteractionfollowedbyclinicaldisease.Thisstudyinvolvescollectingnasal,tonsillar,rectal,meningealswabsandwholebloodfromuptoninetycasesandmatchedcontrolsfromfarmswithoutbreaksofS.suisinthenurseryinsouthwesternOntario,Canada.Sofar,wehavecollect24potentialcasesandanequalnumberofhealthy controls. Cases are selected based on clinical signs including ataxia, incoordination, convulsions, paralysis, nystagmus andcontrolsbasedonhealth,appropriatebodyconditionandexhibitionofnormalbehavior.Completeserotypingresultsofthesecaseswillbereportedelsewhere.S.suiswasisolatedfrom79%ofthepotentialcasesandeightanimalswereconfirmedascasesviameningealand/orbloodculture.Sevenpigswereconfirmedpositiveinthemeninges,oneinbloodandonewaspositiveforS.suisonboth.Nextsteps in our study design include next generation sequencing of tonsillar swabs to determine the viral genomic profiles of thoseconfirmedS.suispositivebymeningeal,splenicand/orbloodcultureandPCR,andtheirhealthycounterpartstodeterminewhethertheviralgenomicprofileofanindividualpigcanpredicttheirsubmissiontoclinicalS.suis.P025-Streptococcussuisinfections:seroprevalenceincommunityinNamDinh,VietnamO.T.Kim1,T.Q.Pham2,H.T.Ngo3,M.Imanishi4.1USAID,Hanoi,VietNam,2NationalInstituteofHygieneandEpidemiology,Hanoi,VietNam,3OxfordUniversityClinicalResearchUnit,Hanoi,VietNam,4WHO,Hanoi,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Todeterminethedetailedepidemiological,clinical,andmicrobiologicalcharacteristicsofS.suisinadultswithoccupationalexposurestopigs.Methods:Weconductedacross-sectionalsero-surveyusingclustersamplingtodeterminetheseroprevalenceofS.suis,andaKnowledgeAttitudePractice(KAP)surveyamong450participantswithoccupationalexposurestopigincludingpigfarmers,slaughterhouseworkers,andanimalhealthworkers.Agroupof200participantswhowerenotexposedtopigwasalsorecruitedasacontrol group. S. suis antibody in blood was quantified using quantitative real-time PCR. All participants were interviewed withstructuredquestionnaires includingquestionson risk factorsandcurrentKAPonpreventionandcontrolofS. suis.Results: S. suisantibodyappearedin37.1%(32.6%-41.6%)ofparticipants.Bloodpuddingor“tietcanh”(51.1%)hasbeenfoundasthemainfactorfortheS.suisinfectionwhileoccupationalfactorshaveturnedoutnosignificanteffect.Useofprotectivepersonalequipment(PPE)whileworking(77.3%)partiallyaffectedtheinfection.Household/farmsettingsalsostatisticallysignificantmatchedwithdistributionof S. suis in the geographical model. Conclusion:Moderately high seroprevalence was determined in pig-exposed populations.ConsumptionofbloodpuddingwasreconfirmedasariskfactorofS.suisinfection.UsingofPPEsmaybehelpfultoreducetheriskofS.suisinfection.Itisneededtohavefurtherstudiesusinga‘OneHealth’approach,toimproveunderstandingofdiseasestransmissionbetweenpigsandhumans,andproposerecommendationsforpolicymakerstoreducetheinfectionamongpopulation.

CRWAD2017 ABSTRACTS


P026-SurvivalanalysisofprotocolsforeradicationofMycoplasmahyopneumoniaeinswinefarmsP.Yeske1,R.ValerisChacin2,R.Singer2,M.Pieters3.1SwineVetCenter,SaintPeter,MN,USA,2VeterinaryandBiomedicalSciences,University of Minnesota, Saint Paul, MN, USA, 3Veterinary Population Medicine, University of Minnesota, Saint Paul, MN,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Mycoplasmahyopneumoniae isanimportantrespiratorypathogencausingeconomiclossesinfinisherpigs.Eradicationofthisbacteriumfromtheherdsresultsinincreasedproductivityandanimalwelfare.TheobjectiveofthisresearchwastocomparetimewithoutM.hyopneumoniaedetectionafterfarmsappliedeitherherdclosureorwholeherdmedicationasstrategiesforeradication.Methods:Fifty-sixsowfarms located intheUSMidwestconstitutedthecohort.Herdclosurewasundertaken in45of farmswhilewholeherdmedicationwasundertakenintheother11farms.Allfarmswerefollowedupforamaximumof155monthswhiletheyremainedM.hyopneumoniae-free.Twopossibleeventswererecorded:detectionofM.hyopneumoniae(theevent)orendoffollow-up (censored observation). Time-to-event data were analyzed with non-parametric (Kaplan-Meier curves and Wilcoxon test),semiparametric (Cox proportional hazardsmodel), and parametricmethods.Moreover, the proportional hazards assumptionwasassessedusingasimulationprocedure.Asensitivityanalysistoevaluatetheassumptionofindependentcensoringwasundertakenaswell.Results:TimetodetectionofM.hyopneumoniaein25%offarmswas8.2monthswhenwholeherdmedicationwasappliedand>155monthswhenherdclosurewasapplied.ThehazardofdetectingM.hyopneumoniaeinthosefarmswithwholeherdmedicationwas 4.5 times the hazard in those with herd closure (95% CI: 1.1-18.1; P<0.05). The most parsimonious parametric model wasexponential.Theestimateforthesurvival time(negativetoM.hyopneumoniae) inpig farmswithherdclosurewas4.45timesthesurvival time in thosewithwhole herdmedication (95% confidence interval: 1.11 - 17.78; P value<0.05).Conclusions: Under theconditionsofthisinvestigation,bacterialeradicationusingherdclosuresignificantlyreducedthelikelihoodofdetectingnewcasesofM.hyopneumoniaeinswinefarms,whichmakesitamoresuitablestrategytotacklethisswinehealthproblem.P027-Assessmentofreproductiveperformanceincommercialfarmswithdifferentporcinereproductiveandrespiratorysyndrome

statusinJapanA. Furutani1, S. Nakatake2, T. Kawabata3, Y. Sasaki1. 1University of Miyazaki, Miyazaki, Japan, 2Miyazaki Prefectural EconomicsFederationofAgriculturalCooperatives,Sectionofswine,Miyazaki,Japan,3KagoshimaPrefecturalEconomicsFederationofAgriculturalCooperatives,Sectionofswine,Kagoshima,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Today,porcinereproductiveandrespiratorysyndrome(PRRS)isconsideredtobeoneofthemostchallengingdiseasesinpigscausingsignificanteconomic impactontheswine industryworldwideduetoreproductive failure inbreedinganimalsandreducedgrowthperformanceinnurseryandgrow-finishingpigs.However,fewstudieshavequantifiedreproductiveperformanceinfarmswithdifferentPRRSvirusstatus.Therefore,theobjectiveofthepresentstudywastoassessreproductiveperformanceinfarmswithdifferentPRRSvirusstatus.Methods:Thepresentstudywascarriedoutfor25commercialfarmsinMiyazakiandKagoshimaPrefecture,southernpartofJapan.Datausedinthepresentstudycontained22,689servicerecordsof17,905giltsand119,743farrowingrecordsof20,708sowsatfirstservicebetween2011and2014.Amixedeffect linearmodelwasusedforstatisticalanalysis.ThesefarmsusedinthepresentstudyarecategorizedasPositiveUnstable(StatusI),PositiveStable(StatusII),ProvisionalNegative(StatusIII),orNegative(StatusIV)onthebasisofherdsheddingandexposurestatus.Results:ComparedtosowsinStatusIV,noreductionsonnumberofpigsbornalive(PBA)werefoundinsowsinStatusI,IIandIIIatallparity(LSmeans±SEM:10.5±0.2pigsvs.10.6±0.1,10.4±0.1and10.7±0.1pigs,respectively).However,atparity≥6,sowsinStatusIIIhadhigherPBAthansowsinStatusI(10.8±0.1pigsvs.10.5±0.1pigs;P<0.05).Therewasnodifferenceofweaning-to-first-matinginterval(WMI)betweensowsinStatusIVandsowsinStatusI,IIandIII,butsowsinStatusIhadlongerWMIthansowsinStatusIIIatparity1(8.4±0.5daysvs.7.5±0.5days;P<0.05).Conclusions:TherewasasignificantdifferenceinreproductiveperformanceamongfarmswithdifferentPRRSstatus.

CRWAD2017 ABSTRACTS


P028-Quantifyingporcineepidemicdiarrheavirus-specificneutralizingantibodieswitharapidcolorimetricassayG.Singh1,P.Singh1,J.Karsky1,E.Nelson2,S.Ramamoorthy1.1MicrobiologicalSciences,NorthDakotaStateUniversity,Fargo,ND,USA,2AnimalDiseaseResearchandDiagnosticLaboratory,SouthDakotaStateUniversity,Brookings,SD,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMTheporcineepidemicdiarrheavirus(PEDV)isacauseofseverediarrhea,dehydrationanddeathinnewbornpiglets.Hence,clinicaldiseaseleadstosevereeconomiclossestotheswineindustry.Thevirusneutralizationassayhaspracticalutilityinthemeasurementofprotectiveantibodiesasaresultofvaccination,orduringinfection.However,inlaboratorieswherealargenumberofsamplesareprocessed, the scoringof numerouswells of tissue cultureplates todistinguish thosewith andwithout cytopathic effects, is verylaborious.Toaddressthisproblem,acolorimetricvirusneutralizationassaywasdevelopedandvalidatedinthisstudy,using(3-(4,5-dimethylthiazol-2-yl) Tr-2,5-diphenyltetrazolium- bromide) or MTT, a chemical which is commonly used to measure cell viability.Previously,astockvirusculturewasquantifiedbytheconventionalTCID50andplaqueassaymethods.TheconventionalmethodswerecomparedagainstthecolorimetricmethodbyROCanalysis,toarriveatacut-offvaluetoenablethedifferentiationofwellswithandwithoutCPEfromanELISAread-out.Inthisstudy,thecolorimetricmethodwasadaptedtothevirusneutralizationassayformatusinga panel of sera with known ELISA values. Assay performance in terms of sensitivity and repeatability were comparable to theconventional virus neutralization assay, as only 3 of the 30 replicates tested varied by two-fold between the conventional andcolorimetricassays.Thedescribedcolorimetricmethodcansignificantlyreducetestingtime,andcanbeadaptedtovirusneutralizationassaysforotherswinecoronaviruses.P029-Acase-controlstudyinvestigatingtheearlyoutbreakofporcineepidemicdiarrhea(PED)inCanada2014A.M.Perri1,Z.Poljak1,C.Dewey1,J.C.S.Harding2,T.L.O'Sullivan1.1PopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2LargeAnimalClinicalSciences,UniversityofSaskatchewan,Saskatoon,SK,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMThefirstreportofporcineepidemicdiarrhea(PED)inCanadawasfromafarminsouthwesternOntarioinJanuary2014.1Asinglefeedcompany(FC)wasidentifiedasthelikelyoriginofthevirusfortheearlyOntarioPEDcases.Thespray-driedporcineplasma(SDPP)fromFC reproduced the infectionunderexperimental conditions,however thecomplete feeddidnot.2InFebruary2014, FCvoluntarilyrecallednurseryfeedproductscontainingSDPP.ThestudyobjectivewastoevaluatetheroleoffeedintheearlyphaseoftheCanadianPEDoutbreakin2014,aftercontrollingforpotentialconfounders.Twenty-twoherds(n=9caseherds;n=13controlherds)wereincludedinthecase-controlstudy.Acasewasdefinedasanyswineherdwithconfirmeddiagnostictest(RT-PCR)resultsforPEDvalongwiththetypicalclinicalsignsattheherdlevelfromJanuary22-March1st2014.Controlherdswererandomlyselectedandmatchedonprovince,herdsize,herdtype,andtimeofPEDonsetincaseherds.Theassociationbetweenthenumberofpig/peoplemovementson/offsites,thenumberoffeeddeliveriesreceived,whetheraherdreceivedanyfeedfromFC,thequantityofpotentiallycontaminated(PC)feedreceivedfromFC,herdbiosecuritymeasuresandtheherd-levelPEDstatuswereevaluated.MorecaseherdsreceivedfeedfromFC(n=8/9)thancontrolherds(n=3/13).TheoddsofaPEDoutbreakwas38timesgreaterforherdsthatreceivedPCfeedfromFC(P=0.007)comparedtoherdsthatdidnot.ThisstudyconfirmstheroleofPCfeedfromFCasasignificantriskfactorforPEDduringtheearlyphaseoftheCanadianoutbreak.Incontrast,thefrequencyofanimalmovementsandcontactsthroughpeopleandotherfomiteson/offsitesattheherd levelwasnotassociatedwiththePEDherdstatusduringthestudyperiod. Funding:OMAFRA,OntarioPork,andNSERC-CRD.

CRWAD2017 ABSTRACTS


P030-Prospectiverandomizedclinicaltrialcomparingtherapeuticinterventionsforcompletehealingofadvanceddigitaldermatitislesionsindairycows

J.Coatney,J.Shearer,A.Krull,P.Gorden,P.Plummer.VeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversityCollegeofVeterinaryMedicine,Ames,IA,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMDigitaldermatitisisacommoncauseoflamenessindairycattle,andofgreatsignificancetotheindustry.Whileasingletreatmentwithtopicaloxytetracyclineresultsinsomelevelofimprovementoflesionscoreinmostanimals,ourpreviousworkhasdemonstratedthatonly4/43mature lesionscompletelyhealed tonormal skin followinga single treatment.This studyseeks todeterminewhether3applicationsof topical therapy result inahigherclinical resolution rate thanasingle treatment.CowswithactiveDD lesionswereenrolledandrandomizedtooneoftwotreatmentgroups.Onegroupreceivedasingletopicalapplicationofoxytetracyclinepowderappliedtothelesion.Theotherreceived3sequentialtopicalapplicationsat1-weekintervals.Digitalphotographs,locomotionscore,andalgometerscore(measuringresponsetopressureatthelesionsite)wererecordedatdays0,7,14,30,60,and120.Photoswererandomizedandscoredbyablindedobserver.Biopsieswerecollectedformetagenomicsanalysisatdays0,14,30,and120.Atday120,5/31 lesionshadhealedtonormalskinfollowingtreatment(3singletreatmentand2triple).26/31 lesionswerestillpresent,mostlyasearlystagelesions(18/26).Theaveragelesionscoredecreasesinbothgroupsoverthefirst60days,butbeginstoriseagainbyday120.Thereisnosignificantdifferencebetweenlesionscores(orpercentreduction)inthetwotreatmentgroupsoneitherday60orday120.BothsingleandtripletreatmentseliminatesignificantlamenessattributedtoDD.Whilebothasingletreatmentwithtopicaloxytetracyclineandthreeconsecutiveweeklytreatmentseffectivelyresolveclinicallamenessforthedurationofthestudy(120days),neithertreatmentappearstoofferconsistent,long-termresolutionofdigitaldermatitislesions.Itisapparentinnearlyallcasesthata“scab”formsshortlyaftertreatment,suggestingthatthelesionisintheprocessofresolving;however,in26/31casesthelesionremains present (and in 9/31 cases, progresses) long after the “scab” is gone. Further work to identifymore effectivemeans ofcompletelyclearingDDlesionsiswarranted.P031-Prospectiverandomizedclinicaltrialexaminingtheuseofformalinfootbathstohaltdiseaseprogressioninearlystagedigital

dermatitislesionsJ.Coatney,J.Shearer,A.Krull,P.Gorden,P.Plummer.VeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversityCollegeofVeterinaryMedicine,Ames,IA,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMDigitaldermatitisisacommoncauseoflamenessindairycattle,andofgreatsignificancetotheindustry.Footbathsareacommonmeansofcontrollinginfectioushoofdisease.Formalinhasbeenshowntobeeffective;however,nopreviousstudieshaveutilizedascoringsystemthatidentifiestheearlieststageofDDlesions.Theselesionsrepresenttheinitiationofthediseaseprocess.Thisstudyseekstodeterminewhethertheapplicationofafootbathcontaining3%formalinhaltsthedevelopmentandencouragestheresolutionofearlystageDDlesionsbetterthannotherapy.48cowswithearlystageDDlesionswereenrolledandrandomizedto1of2treatmentgroups.Onegroupwasdirected througha footbathcontaining3% formalin,3 timesaweek, for4weeks.Theotherwasdirectedthroughafootbathcontainingtapwater.Animalswerehousedtogetherthroughoutthedurationofthestudy.Digitalphotographswererecordedatdays0,14,28,and42.Photographswerescoredbyablindedobserver.20/54earlylesionsreturnedtonormalskinat6weekspost-enrollment.15/29lesionsthatwentthroughthefootbathand5/25ofthecontrolsreturnedtonormalskin.Averagelesionscoredroppedfrom1.38(scaleof0to4)to0.72forthetreatmentgroup,anddroppedfrom1.48to1.24forthecontrolgroup.Thereisasignificantdifferencebetweenthedecreaseinseverityinlesionsinthetreatmentgroupoverthecourseofthestudyvs.controls(p=0.009).Inaddition,ofthe15advancedlesionsenrolled,13remainedadvancedatthesix-weekmark(5/6inthetreatmentgroup,8/9inthecontrolgroup).Resultsindicatethatthefootbathusedheresignificantlyreducestheaveragelesionscoreforearlylesionsbut,thereisnoappreciablechangeintheaveragelesionscoreforadvancedlesions,suggestingthatfootbathsaremosteffectiveatcontrolofearlystagelesions.Interventionattheearlieststagesoflesiondevelopmentresultsinahaltingofthediseaseprocesspriortothedevelopmentoflameness,andleadstothecompleteregressionofamajority(52%)ofearlylesions.Earlylesionsseemtoresponddifferentlythanadvancedlesions;thus,evaluatingthemseparatelyiscriticalinfootbathstudies.

CRWAD2017 ABSTRACTS


P032-TheroleofenvironmentaltransmissionofMycobacteriumaviumssp.paratuberculosis:anindividualbasedmodelK.Ceres,M.A.Al-Mamun,Y.T.Gröhn.PopulationMedicineandDiagnosticSciences,CornellUniversityCollegeofVeterinaryMedicine,Ithaca,NY,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:UnderstandinghowanimalsbecomeinfectedwithMycobacteriumaviumsubsp.paratuberculosis(MAP),thecausativeagentofJohne’sdiseaseisessentialinevaluatingcontrolstrategiestoreduceitsprevalenceondairyfarms.ItiswelldocumentedthatanimalsbecomeinfectedwithMAPfromingestingcontaminatedmaterialintheirenvironment,butthereareveryfewmodelsthatdescribetheroleoftheenvironment intransmission.Ourmodel isthefirst individualbasedmodel(IBM)thatdescribesthecontributionofenvironmentaltransmissionofMAPinadairyherd.Methods:Wedevelopedanindividualbasedmodelofacloseddairyherdwithtypicaldairyherddynamics.WethenconvertedanexistingMAPtransmissionmodeltotheIBMtoincludeanewinfectionstructurewhereanimalscouldbecomeinfectedfromanenvironmentcontaminatedwithMAPinadditiontobecominginfectedinuteroandfrom colostrum andmilk. Using thismodel, we explored fourmanagement strategies to simulate the effect of different hygienestrategiesonMAPprevalence.Results:Anindividualbasedmodelwithtypicaldairyherddynamicswasdevelopedsuchthatindividualanimalscouldbothcontaminateandbecomeinfectedfromtheirenvironment.WealsoshowedthatbetterhygieneleadstodecreasedMAP prevalence. Conclusions: Our model more accurately described transmission pathways than previous models because itconsideredtheenvironmentexplicitlyasamajorsourceofinfectionandincludesherdandinfectiondynamicsattheindividuallevel.OurmodelcanbeausefultoolforfarmerstocontrolMAPprevalence,andcanbeusedasaframeworkforfutureresearch.P033-TheeffectofCelmanaxTMSCPonfecalpathogenshedding,healthandperformanceofpre-weanedHolsteindairycalvesS.M.Raabis1,T.Ollivett1,J.Ding2.1DepartmentofMedicalSciences,UniversityofWisconsin-MadisonSchoolofVeterinaryMedicine,Madison,WI,USA,2DepartmentofIntegrativeBiology,UniversityofWisconsin-Madison,Madison,WI,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMDiarrheainpre-weaneddairycalvesisasignificantcauseofmorbidityandmortality.Optimizinghealthinthiscrucialperiodwithoutantibioticsisofprimaryinterest.CelmanaxTMSCP(CSCP)containsderivativesofSaccharomycescerevisiaecellwall,whichhavebeenshown to reduce bacterial adherence to gastrointestinal mucosa and increase enteric pathogen clearance. Our objective was toinvestigatetheeffectofCSCPsupplementationinHolsteindairycalvesonfecalpathogenshedding,overallhealthandaveragedailygain (ADG)during thepre-weaningperiod. This randomized, placebo-controlled studywas conductedat two commercial farms inWisconsin.ThestudypopulationincludedHolsteincalvesbornbetweenAugust-November2016(n=321).Calveswererandomizedatbirthinto4treatmentgroups:placebo,1g,2g,and4gofdailyCSCPsupplementationfor56days.Investigatorsperformedtwice-weeklyhealthscoresusingtheUniversityofWisconsinHealthScoringApp.QualitativeEnterichek®(BiovetInc.)andquantitativeRT-PCRwereusedtoanalyzefecalsamplesforC.parvum,coronavirus,rotavirus,andSalmonellaspp.Atotalof304calvessurvivedtotheendofthestudy.Therewerenosignificantdifferencesinentryweights,mortalityorpassivetransferstatusamonggroups.Incalveswithfailureofpassivetransfer,theonsetofmilddiarrheaoccurredlaterincalvesdosedwith1gcomparedtoallothergroups(p=0.01).TherewerenodifferencesintheprobabilityofsheddingC.parvum,rotavirusorcoronavirusbetweengroups.Theprobabilityofsheddingrotavirustrendedlowerinthecalvesdosedwith2gwhencomparedtoplacebo(p=0.06).CalveswithfailureofpassivetransferhadreducedsheddingofSalmonellaspp.ifdosedwith2gcomparedtotheothergroups(p=0.04).Calveswithfailureofpassivetransferdosedwith4ghadareducedoddsoflobarpneumoniacomparedtotheplacebogroup(p=0.02).TherewasnosignificantdifferenceinADGamonggroups.Inconclusion,CSCPsupplementationduringpre-weaningmayhelpdelaytheonsetofdiarrhea,reducepneumonia,andreduceSalmonellaspp.sheddingincalveswithfailureofpassivetransfer.

CRWAD2017 ABSTRACTS


P034-EpidemiologicalinvestigationoftheoccurrenceofdiseaseonacommercialdairyfarmlocatedintemperateclimatezoneinJapan

E. Kino1, T.Minamino2, Y. Horii2, K. Honkawa2, Y. Sasaki1. 1University ofMiyazaki,Miyazaki, Japan, 2Honkawa Ranch, Oita, [email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Climate isoneof the important factors indairyproduction.Heatstresshasseveral seriousandeconomicallydeleteriouseffectsoncattle,somostdairyfarmsinJapanwerelocatedinnorthernpartofJapanthatarecoldclimate.However,severalfarmsarelocatedintemperateclimatezonetosupplyrawmilk.Therearefewreportstoinvestigatetheoccurrenceofdiseaseinthisclimatezone.Therefore,theobjectivesofthepresentstudyweretodeclarethediseaseoccurrenceofdairycowsonacommercialdairyfarmlocatedintemperateclimatezoneinJapan,andtocompareincidencerateofeachdiseasebyparity,seasonanddaysinmilk(DIM).Methods:Thepresentstudywasconductedonalargedairyfarmhavingapproximately2,000HolsteincowslocatedinOita,Kyusyu,Japan.DatawerecollectedfromJanuary1st,2014toJune30th,2017,including6,500calvingrecords.Theoccurrenceofdiseasewasidentifiedbytherecordofmedicalexaminationthatperformedtocowswithclinicalsymptomsuchasoff-feed,cough,andextremelylowmilkyield.Results:Theoccurrenceratesofdiseaseswere31.1%formastitis,2.5%formilkfever,0.9%forhoofdisease,0.7%forketosis,0.5%forleftdisplacedabomasum,0.4%forbirthcanallacerationand0.3%formilkfever.Highincidencerateofmastitiswasfoundinsecondparity,winterandDIM18to60days(33.3%,34.7%and9.2%,respectively).Conclusions:Conclusions:Thepresentstudydeclaredthatthehighestoccurrenceofdiseaseonacommercialdairy farm located intemperateclimatezone in Japanwasmastitis,especiallyinthesecondparity,winter,andDIM18to60days.P035 - Evaluationof diagnostic tools for BovineRespiratoryDisease in calves challengedwithBovineRhinotracheitis Virus and

MannheimiahaemolyticaJ.Baruch1,C.A.Cull2,N.Cernicchiaro1,J.Nickell3,K.F.Lechtenberg2,D.G.Renter1.1DepartmentofDiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,USA,2VeterinaryandBiomedicalResearchCenter,Manhattan,KS,USA,3AnimalHealth,Merck,DeSoto,KS,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30:00PMPurpose:Bovinerespiratorydisease(BRD)isoneofthemostcostlydiseasestothebeefindustry;itischaracterizedbyhighmorbidity,mortality,andproductionloses.Ante-mortemdiagnosticsarechallengingasindicatedbythepoordiagnosticperformanceofcommonclinicalapproaches (approximately60%sensitivityand specificity). Theobjectiveof this studywas toevaluate theperformanceofchute-sidediagnostic tools for thedetectionofBRDusingachallengemodelwith InfectiousBovineRhinotracheitisVirus (IBR)andMannheimia haemolytica (Mh). Methods: Thirty Holstein steers were inoculated intranasally with IBR on study day 0, andintrabronchiallywithMhonstudyday6.Duringa13-dayperiod,whisperstethoscope(WS),chute-sidebloodleukocytedifferential(CBLD)andpulseoximetry(PO)wereusedondays0,1,2,4,6,6.5,7,7.5,9,11,and13,clinicalillnessscoreswererecordeddailyandthoracicultrasoundwasmeasuredondays0,6,7,9,11and13.Cattlewereeuthanizedandlungconsolidationdatawererecordedondays6,7,9,11and13.Datawereanalyzedusinggeneralizedlinearmixedmodels.Results:Thechallengemodelrepresentedclinicalsignsandlesionstypicalof IBRandMhinfection.Statisticallysignificantdifferencesbystudydaywereobservedforalldiagnostics.BeforeMhinoculation,mostcattlewereassignedclinicalillnessscoresfromnormal(0)tomoderate(2);however,afterMhinoculation,moderate,severe(3)andmoribund(4)scoreswererecorded.Oxygensaturationsignificantlydecreased12to24hafterMhinoculationcomparedtolevelsobservedondays0,1,2and4.Whisperstethoscopescoresweresignificantlylowerondaysbefore(days0,1,2and4)versusthedaysafterMhinoculation(7,7.5,9and11).Averagelungconsolidationwas1.9%(0.9%)onday6and55%(7.7%)onday10.Conclusions:Resultsfromthesediagnostictoolswereconsistentwithpathologicalandclinicaldiseaseprogressionfindings.Subjectivemeasures such as clinical illness scores could be combinedwith objective tools, such as,WS, POor CBLD, contributingtowardsanimprovedapproachtoBRDdiagnosis.

CRWAD2017 ABSTRACTS


P036-IdentifyingpathogenscausingsubclinicalmastitisinSoutheastorganicdairiesV.L.Couture1,L.J.Siebert1,P.D.Krawczel1,A.G.Rius1,J.M.Bewley2,G.M.Pighetti1.1AnimalScience,UniversityofTennessee,Knoxville,TN,USA,2AnimalandFoodScience,UniversityofKentucky,Lexington,KY,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMOneofthegreatesthealthchallengestheorganicdairyindustryfacesismastitis.Understandingthepathogeniccauseofmastitiscanaidinimprovingareasofmanagementanddecreasingincidenceofmastitis.TheobjectiveofthisstudywastoidentifytheorganismscausingsubclinicalmastitisinfectionsonorganicdairyfarmsintheSoutheast.Lactatingcows(n=83)weresampledduringvisitstofiveorganicdairies inTennesseeandKentuckyfromApril toJune2017.Eachfarmwasvisitedtwice,28daysapart.Milksampleswereasepticallycollectedfromeachindividualmammaryglandincowswithasomaticcellcountgreaterthan200,000cells/mL.Atotalof29.1%ofcows(n=83)weresampled(eachfarmrangedbetween13.9%-37.8%).Ofthese,32cowsmetthesamplingrequirementsforbothvisits.MilksampleswereculturedfollowingNationalMastitisCouncilguidelinestoidentifypathogens.Contaminatedsamples(>3colonytypes)wereexcluded(n=11)resultinginanalysisof460quartermilksamples.Datawereanalyzedusingthefrequencyprocedure in SAS (v9.4) to identify pathogen prevalence and chronic infections. Of the 460 samples evaluated,microbial growthoccurredon47.7%.ThemostprevalentpathogensacrossallfarmswerecoagulasenegativeStaphylococci(CNS;14.6%),Staphylococcusaureus(6.5%),Streptococcusuberis(5.3%),andStaphylococcushyicus(5.1%).OftheCNSspecies,Staphylococcuschromogeneswasdominant,makingup6.3%oftotalsamples,or42.5%ofCNS.Withinfarms,thedominantpathogenvariedon4outof5farms,betweenS.aureus,S.uberis,S.hyicus,andS.chromogenes. Isolationof thesamepathogenfromthesamemammaryglandonconsecutivesamplingdaysoccurredin10.5%ofthequartersthatwereresampled.Ofthesesamples,37.9%wereidentifiedasS.aureus,20.7%asS. uberis, and 17.2% as S. chromogenes. These microorganisms and their prevalence have been identified in conventional dairyoperations. In summary, pathogen prevalence between organic and conventional dairies is similar, indicating that organic andconventionaldairyproducersfacethesamechallengesofcontagiousandenvironmentalpathogens.P037-WholegenomesequenceanalysisoffourfecalgenericblaCMY-2producingEscherichiacoliisolatesfromHolsteindairycalvesB.B. AWOSILE, J. Reyes-Velez, M.E. Saab, J. Rodriguez-Lecompte, J. Sanchez, L.C. Heider, J.T. McClure. HEALTH MANAGEMENT,ATLANTICVETERINARYCOLLEGE,UNIVERSITYOFPRINCEEDWARDISLAND,CHARLOTTETOWN,PE,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:ThisstudywascarriedouttodeterminetheantimicrobialresistomeandmobilegeneticelementsoffourfecalgenericblaCMY-2 producing Escherichia coli isolated from Holstein dairy calves using whole genome sequencing.Methods: The whole-genomesequencing was carried out using the Nextera XT DNA library preparation and Illumina MiSeq platform. Genome assembly andsubsequent annotationwere performed using SPADES and RAST tool kits found in the Pathosystems Resource Integration Centerrespectively.WholegenomesequenceswereanalyzedusingthebioinformaticstoolsfromtheCenterofGenomicEpidemiologyandPathosystemsResourceIntegrationCenter.AfurthergenomicanalysiswascarriedoutusingISFinderandPHASTERtodeterminetheinsertionsequencetypeandprophageregionrespectively.Results:Genomicanalysisrevealed3isolatesfromthesamefarmsharedsimilargeneticfeaturesincludingsequencetype;ST88,serotype;O8:H17,plasmidiccharacteristics;IncFIA,IncFIB,pO111andIncN2,plasmidicrepliconsequencetypes;IncF[F67:A6:B5]andIncI1[ST-12],insertionsequencetype;IS10group(IS10L),1intactprophageregion, and similar virulence genes.While the other isolate belongs to the serotypeO9: H12, unknown sequence type, insertionsequence type; IS110 group (IS621), plasmidic replicon sequence types; IncF[F67:A6:B1], IncN[ST-6], and IncI1[ST-12], and alsopossessed2 intactprophageregions.Besides thepresenceofgenesencoding forcomplexmulti-drugresistanceeffluxsystemandproteins in the isolates, the isolates were carriers of genes conferring resistance to β-lactams, tetracycline, aminoglycosides,sulphonamides,andtrimethoprimincludingblaCMY-2,blaTEM-1B,tetA,tetB,tetD,aadA1,aph(3”)-lb,aph(6)-ld,sul2,andDfrA1resistancegenes.Conclusions:Thestudyprovidesinformationforcomparativegenomicanalysisofresistancegenesandmobilegeneticelementsthatcouldprovidesomeexplanationtothemultidrugresistancecharacteristicsofbacteriacolonizingtheintestinaltractofdairycalves.

CRWAD2017 ABSTRACTS


P038 - Extended-spectrum cephalosporin, carbapenem, and fluoroquinolone resistant coliform bacteria present on companionanimal,equine,andlivestockenvironmentalsurfaces

R.J.Adams1,D.Mollenkopf1,D.Mathys1,S.Kim1, J.Daniels2,T.Wittum1.1Dept.ofVeterinaryPreventiveMedicine,TheOhioStateUniversity, Columbus, OH, USA, 2Dept. of Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, [email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose: Antimicrobial resistant bacteria are a rapidly growing concern to both veterinarymedicine and public health. The risingprevalenceofextendedspectrumbeta-lactamase(ESBL),AmpCbeta-lactamase,carbapenemase(CRE),andfluoroquinolone-resistantEnterobacteriaceae continually decreases the efficiency of vital antibiotics.Moreover, antibiotic resistant enteric bacteria can betransmittedbetweenanimalsandhumans,thusposinganimportantzoonotichealthrisk.Ourobjectivewastoevaluatetheprevalenceofantibiotic resistantbacteriaonhumancontact surfaces invariousanimalenvironments.Methods: Environmental surfaces fromcompanion animal shelters, private equine facilities, dairy farms, livestock auctionmarkets, and county fairswere sampled usingelectro-static-cloths (Swiffer®.) Samples were screened for coliform bacteria expressing AmpC, ESBL, CRE, and fluoroquinolonephenotypic resistance using selective media. Resistance genotypes were confirmed using standard PCR techniques.Results:Livestocksalesandlocalcountyfairsrevealedhigherlevelsofbothcephalosporinandfluoroquinoloneresistancethanequine,dairy,andcompanionanimalenvironments.Equinefacilitiesharboredmorecephalosporinresistancethancompanionanimalshelters,butlessfluoroquinoloneresistance.Theregularuseofcephalosporins(ceftiofur)inlivestockspeciesmayaccountfortheheightenedlevelsofresistanceinlivestockspeciesenvironmentscomparedtocompanionanimalandequinefacilities.Humansurfacesaswellashuman and animal surfaces were contaminated with various resistant bacteria regardless of species environment.Conclusions:Detectingthesebacteriaoncommonhumancontactsurfacessuggeststhattheenvironmentcanserveasareservoirforantibioticresistancegenes.Identifyinginterventionstolowertheprevalenceofantibioticresistantbacteriainanimalenvironmentswillprotectbothanimalandpublichealth.P039-PrevalenceandantimicrobialsusceptibilityofCampylobacterinsheepJ.Pang,Y.Tang,J.Xia,Z.Wu,L.Dai,K.Singh,C.Xu,L.Xia,X.Ma,K.StillBrooks,O.Sahin,P.Plummer,Q.Zhang.Collegeofveterinarymedicine,IowaStateUniversity,Ames,IA,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMCampylobacterisamajorcauseofovineabortionsandfoodborneillnessesinhumansintheU.S.Forthepurposeofcontrolofabortionand other diseases, tetracycline is commonly used for commercial sheep production in the U.S. However, whether tetracyclinetreatmentinfluencesCampylobacterprevalenceanditssusceptibilitytoantimicrobialsremainsunknown.ToclosethisknowledgegapandfacilitatethecontrolofCampylobacterinsheep,weperformedacontrolledtreatmentstudyusinglambsthatwerederivedfromcommercialoperationsandwerenaturallyinfectedbyCampylobacter.Intotal,160feederlambswereusedinthisstudy,halfofwhichreceivednoantibioticsandtheotherhalfweremedicatedwithtetracyclineinfeedata350mg/hd/ddose.FecalsampleswerecollectedweeklyandprocessedforisolationofCampylobacterusingselectiveculturemedia.ThebacterialisolatesweresubjectedtospeciesidentificationbyMALDI-TOFandantimicrobialsusceptibilitytestsusingSensititreplates.Theresultsrevealedthat1)morethan80%ofthe fecal sampleswerepositivewithCampylobacter; 2) themajority of theCampylobacter isolateswereC. jejuni, butC. colialsoaccounted for a substantial portion of the isolates; 3) all tested C. jejuni isolates were resistant to tetracycline regardless of thetreatmentgroups;and4)asignificantnumberofciprofloxacin-resistantCampylobacteremergedinsheep.However,theCampylobacterisolates were still susceptible to many other tested antibiotics such as florfenicol and macrolide. These findings reveal the highprevalenceofresistancetotetracyclineandciprofloxacininsheepCampylobacterandprovideusefulinformationforthepreventionandcontrolofCampylobacterinsheep.

CRWAD2017 ABSTRACTS


P040-IdentificationofapredominantCampylobactercolicloneinfeedlotcattleintheUnitedStatesY.Tang,S.Muirhead,Z.Wu,N.Pavlovic,Q.Zhang.DepartmentofVeterinaryMicrobiologyandPreventiveMedicine,Ames,IA,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMCampylobacter is a major foodborne pathogen and a leading cause of human gastroenteritis in the U.S. and other countries.Campylobacter is commonly present in food-producing animals, and ruminants are important reservoirs. To determine theepidemiologyofC.coliincattle,wecollected3,184fecalsamplesfrom35feedlotsinfivedifferentstatesandisolatedCampylobacterfromthesamples.Intotal,356C.coliisolateswereobtained.Todeterminethegeneticrelationshipoftheisolates,116C.coliisolateswererandomlychosenforgenotypingusingpulsed-fieldgelelectrophoresis (PFGE),multi-locussequencetyping(MLST)andwholegenomesequencing.Basedonthe88%similaritylevel,theisolatesweregroupedinto13clustersbyPFGE.Notably,themajority(75%)ofthePFGE-typedC.coliisolatesweregroupedintothreeclustersofhighgeneticsimilarity,andMLSTrevealedtheybelongtoasinglesequencetype(ST-1068).Ofthe116isolatestested,85(73.3%)werefoundtoberesistanttotetracycline,97(83.6%)wereresistanttociprofloxacin,96(82.8%)wereresistanttonalidixicacid,and15(12.9%)wereresistanttoclindamycinandflorfenicol.Noneoftheisolateswere resistant to azithromycin, erythromycin, gentamicin or telithromycin. These results revealed the dissemination of apredominantC.colicloneondifferentcattlefarmsintheU.S.,andtheC.coli isolateswerehighlyresistanttofluoroquinoloneandtetracycline.P041-OrganictracemineralsupplementationandfecalmicrobiomewithreducedprevalenceofDDandsheddingofEscherichiacoli

0157inbeefcattleM.Montanye,K.Schultz,K.Anklam,J.-M.Ariza,M.Kulow,G.Suen,K.McFarland,M.Cox,D.Dopfer.UniversityofWisconsin-Madison,SchoolofVeterinaryMedicine,Madison,WI,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMAbstract: Lameness in beef cattle is often caused by digital dermatitis (DD). Cattle are also known for shedding Escherichia coli0157(EHEC0157).Both,DDandEHEC0157continuetobeaconcernforconsumersintheUS.TheresultsofthecurrentstudywillbeusedtohighlightandimprovefoodsafetyregardingbeefcattlebyunderstandingthefecalmicrobiomesoftheanimalsrelatedtothesheddingofEHEC0157. Analysisof fecalmicrobiomedatawillestablishanassociationbetween thesupplementationofOTMandsignificantreductionofDDprevalenceandthesheddingofEHECO157inbeefcattle.Atotalof414cowsfromfeedlotsinIowaandIllinoiswere enrolled in the study and fecal samples fromall animalswere gathered for analysis. The 16SmicrobiomedataweregeneratedfromDNAextractionsandanalyzedusingmothur(version1.37.5).Thefinaldatasethad396observationsof63variables.TheOTU,metadata,andtaxonomytableswerederivedfromdataoutputsfrommothur.Regressionmodelsforalphadiversityweremade and the output tables for each analysis were analyzed for significant association with alpha and beta diversity. SignificantrelationshipswerefoundbetweentheOTMdietswiththesheddingofEHEC0157throughevaluationofthealphadiversityofthefecalmicrobiome. Betadiversitywithin thedata setdidnothave significant relationshipsbetween the sheddingof EHEC0157and thetreatmentversuscontroldiet.Thisstudyreflectsimplicationsforimprovedfoodsafetyandcattlewelfare.EffectivepreventionandcontrolofDDbreaks thetransmissioncycleofEHEC0157resulting in reducedshedding. Thisbreak leads to improved foodsafetythroughriskfactorcontrolincattlepre-harvest.

CRWAD2017 ABSTRACTS


P042-AOneHealthapproachtobrucellosissurveillanceatthehumananimalinterfaceinruralTanzaniaC.Kilonzo1,B.H.Bird1,A.Ekiri1,D.J.Wolking1,E.VanWormer2,G.Paul3,G.Makingi3,R.D.Sumaye4,A.Abdalla5,R.R.Kazwala3,J.Mazet1,W.Smith1.1OneHealthInstitute,UniversityofCalifornia-Davis,Davis,CA,USA,2SchoolofVeterinaryMedicineandBiomedicalSciences,University of Nebraska, Lincoln, NE, USA, 3Veterinary Medicine and Public Health, Sokoine University of Agriculture, Morogoro,Tanzania, United Republic of, 4Ifakara Health Institute, Ifakara, Tanzania, United Republic of, 5Ifakara Health Institute, Bagamoyo,Tanzania,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMAcrosssectionalsurveywascarriedoutintheIringaandMorogororegionsofcentralTanzaniatodeterminetheprevalenceofandriskfactorsforhumanandlivestockbrucellosis.Livestockherdsandhumansweresampledfromareasnearbyninehealthfacilitiesfromwhichconsentinghumanfebrilepatientswereenrolledintothestudy.Thiscomprehensiveonehealthsamplingstrategyresultedinserumspecimens from1,054people, 1,621 cattle, 761goats and718 sheepduringongoing the studyperiod.All specimenswerescreenedforBrucellaantibodiesusingtheRoseBengalplatetest(RBPT).ConfirmationofRBPTpositivehumanandanimalspecimenswasdoneusingeitherRivanoltesting,oracELISA,respectively.Humanseroprevalencewas0.5%comparedwithanindividualanimalseroprevalenceof1.7%.Overall,16.7%of livestockherdswereseropositive.Studyparticipantswhoreportedlivestockabortions intheirherdswere10timesmorelikelytobeseropositivecomparedtolivestockownerswithnoreportedabortions,andtheburyingofaborted animal fetuses was positively associated with human exposure and seroconversion (OR=11.2). In livestock, there was asignificantlyhighernumberofseropositiveanimalsinIringaruraldistrictcomparedtoKilomberodistrict(OR=3.6).Goatswere3.5timesmorelikelytobeseropositivecomparedtosheepandfemalelivestockwere3.7timesmorelikelytobeseropositivecomparedtomales.Eachadditionalbreedingcycleincreasedtheoddsofseropositivityinfemalesby1.2.Femaleanimalswithpreviousabortionwere8.7timesmore likelytobeseropositivecomparedtothosethathadnotaborted.ThisstudyshowsthatcomparableregionalseroprevalenceinbothhumansandanimalsexistsinruralTanzaniaunderscoringtheimportanceofapplyingOneHealthapproachestoevaluaterisksandcontrolmeasuresforbrucellosis.P043-Biotypingandgenotyping(MLVA)ofBrucellaisolatesfromdifferentanimalsinThailandJ.J.Lee.AnimalandPlantHealthResearch,AnimalandPlantQuarantineAgency,Gimcheon,Korea,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose: Brucellosis has been constantly arising in livestock in Thailand over the last decades, but there is less reports on thebacteriological and epidemiological data. The aim of this study is to investigate the epidemiological characteristics togetherwithbacteriologicalfindingsofbrucellosisinThailand.Methods:Atotalof65Brucella(B.)isolatesfromanimalsin14regionsofThailandconfirmedtobesero-positivewereidentifiedbyBrucella-specificmultiplexPCR,real-timePCRandclassicalbiotypingassay.MolecularepidemiologywasanalyzedbyMLVA-16.Results:Brucella isolatesweredifferentiatedby4B.abortusS19,15B.abortusand46B.melitensis,andbiotypingofB.abortusisolatesfrombeefanddairycattlewereidentifiedasbiovar(bv.)1and3,andallB.melitensisfromgoats,sheepandbeefcattlewereconfirmedasbv.3.MLVAdatarevealed19B.abortusand46B.melitensisweredividedinto9and 28 genotypes, respectively, and they had the adjacent genetic profiles according to provinces and animal species; differentprovinces,atleast2ones,haveaclosegeneticrelationshipinthesamecluster.Moreover,B.melitensisisolatestendtobegroupedintothesameclusterbetweensheep,goatsandcattle.Comparedwithotherforeignisolates,2ThaiB.abortuswerecloselysimilarwithBrazilianstrain(83.34%similarity).ManykindsofThaiB.melitensisisolatesfromgoatsandsheepshowedhighgeneticsimilarity(88.75~100%)with Chinese strains.Conclusions:Molecular epidemiological findings indicate thatBrucella strains circulate amongvariousanimalspeciesandprovincesinThailandandtheyarealsopossibletobetransboundaryspreads.Takentogether,thisstudyunderscoretoarrangetheappropriatecontrolstrategiesforbrucellosisinThailand.

CRWAD2017 ABSTRACTS


P044-BrucellosisinArmenianlivestock2016H.Mkrtchyan.StateFoodSafetyServiceoftheRAMoA,Yerevan,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMBrucellosisisananthropozoonoticdiseaserecordedamongcattle,sheepandgoatsinallMarzesoftheRepublicofArmeniain2016.Annual diagnostic activities are implemented in cattle, goats and sheep via theRoseBengal test according to state anti-epidemicmeasures.Forconfirmation,asecondtestconsistingofeitheranagglutinationreactionoranELISAisrequired.AsTable1shows,therewereanaverageof111confirmedcasesofbrucellosisperMarz.However,thereisasignificantdifferencebetweenthenumberofoutbreaksbetweenMarzes.For instance, inTavush,YerevanandVayotsDzorthetotalnumberdoesnotexceed50,while inmanyMarzes there over 100 cases recorded, including 392 in Kotayk). The proportion of animals at risk to those infected was 3.3:1.Table 1. Cases of brucellosis by Marz (2016) Aragatsotn: 20 outbreaks, 510 animals at risk, 162 cases, 162 slaughtered; Ararat:15/138/55/55; Armavir: 7/395/132/132; Gegharkunik: 14/246/88/88; Kotayk: 12/981/392/392; Lori: 9/363/49/49; Shirak:14/656/222/222;Syunik:12/247/79/79;Tavush:7/32/8/8;VayotsDzor:9/413/23/23;Yerevan:11/149/11/11;AsTable2shows,anaverageof73.8casesconfirmedcasesofbrucellosisinsheepandgoatsoccurredinallMarzesexceptingYerevan.Morethan100caseswererecordedinAragatsotnandShirak.Theproportionofanimalsatrisktothose infectedis4.3:1.Table2.Casesofbrucellosis insheepandgoatsbyMarz(2016)Aragatsotn:5outbreaks,364animalsatrisk,132cases,132slaughtered;Ararat:7/303/55/55;Armavir:11/231/66/66; Gegharkunik: 10/309/56/56; Kotayk: 8/306/75/75; Lori: 1/49/12/12; Shirak: 14/846/175/175; Syunik: 7/328/53/63;Tavush:1/22/4/4;VayotsDzor:3/431/98/98;So,despitethediagnosticeffort,brucellosiscontinuestobeamajorissueforveterinaryservices inArmeniaresultingfromarangeofunsolvedproblems. It isnecessarytoundertakeamoredetailedanalysisofcurrentlyavailable epidemiological data and improve the current disease management strategy, which should be based on the givenepidemiologicalsituationandspecificsofeachMarz.P045-Socio-culturalriskfactorsforhumanbrucellosisinLolgoriandivision,TransMaradistrict,KenyaP.M. Alusi1, K. Wanjala2, M. Maichomo3, S. Bukachi4. 1Socio-economics, KALRO-Biotechnology Research Institute, Kikuyu, Kenya,2Anthropology, SouthEasternKenyaUniversity,Kitui,Kenya, 3Epidemiology,KALRO-VeterinarySciencesResearch Institute,Kikuyu,Kenya,4InstituteofAnthropologyGenderandAfricanStudies,UniversityofNairobi,Nairobi,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Todeterminesocio-culturalandeconomicriskfactorsforhumanbrucellosis.Methods:Questionnaireswereadministered,Focus group discussions, Key informant interviews and observations were made. Results: Results showed that brucellosis wasassociatedwithparturitionduringabortionandlivingincloseproximitywithlivestock.Peoplewhoconsumedrawmilkrawmeatandrawbloodwerefoundtohaveahigherriskofbrucellosis.Womenandchildrenwereseentobeatriskduetotherolesofmilkingtheanimalsandtakingcareofthesickones.Conclusions:Thestudyconcludesthatprotectinghumansfromcontactwithbirthingfluidsandtissues, livingincloseproximitywithlivestockandconsumingrawlivestockproducts,maybeimportantinreducingtheriskofcontactingbrucellosisinhumans.Thesecanbeachievedthroughhealtheducationinthecommunity.

CRWAD2017 ABSTRACTS


P046-TheroleofawarenessandrapidresponseduringoutbreaksofAfricanswinefeverL.Sargsyan.StateFoodSafetyServiceoftheRAMoA,Yerevan,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMThe firstoutbreakofAfrican swine fever (ASF) in theRepublicofArmeniawas recorded in2007 inTavushandLoriMarzes. Stateepidemiologicalsurveillancedatashowsthat,in2007-2011,24,958infectedpigsdiedorwereslaughtered.Thankstothetimelyandappropriateresponseofspecialists,furtherspreadwasprevented.Diseasepreventioninswine-breedingfarmsrequiresexecutionofbiosafetypracticeswhichdependonwell-informedfarmersandveterinarians.Forestersandhuntersneedtobeawareoftherisksposedbywildboarsandferalpigs.TheDTRA-fundedTAP-A1project“ThepublicoutreachonecologyandepidemiologyoftheAfricanswinefeverinEasternEurope,provisionoftrainingforthepurposesofsurveillanceandprevention,implementationofmethodsandstrategies” was implemented in Armenia in 2015. For this, working discussions and awareness seminars were conducted withveterinarians, swine-breeders, foresters, andhunters. Information leaflets (3,000pcs) for veterinarians and farmerswereprinted;posters (150 pcs) were designed for foresters and hunters. Questionnaires were designed tomeasure the level of knowledge ofveterinariansandfarmers,aswellastoincreaseawareness(2,000farmers,301veterinarians,100forestersandhunters).Pre-testingandpost-testingquestionnaireswereusedtoassesstargetaudienceknowledge.Theseactivitiesincreasedawarenessofproperanimalcare, ranging, and biosafety requirements to prevent ASF and contain the spread of disease by as much as 60% indicating theimportanceofinformationdisseminationinthemulti-componentchainofpreventingthedisease.P047-Spatio-temporalkriginganalysistoevaluatetheinterfacelivestock-wildlifeinthespreadofAfricanswinefeverintheRussian

FederationI.Iglesias1,F.Montes2,A.Perez3,A.Gogin4,D.Kolvasov4,A.delaTorre1.1EpidemiologyandEnvironmentalhealth,INIA-CISA,Madrid,Spain,2EpidemiologyandEnvironmentalhealth,INIA-CIFOR,Madrid,Spain,3UniversityofMinnesotaCollegeofVeterinaryMedicine,SaintPaul,MN,USA,4StateResearchInstitutionNationalResearchInstituteforVeterinaryVirologyandMicrobiologyofRussia,Pokrov,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMAfricanswinefever(ASF)isaninfectiousandnotifiableswinediseasewhichhasdevastatingconsequencesforswinesector.Inthelastyears(2007-2016)thehighspreadofthediseaseinTheRussianFederation(RF)hascausedsignificanteconomiclossesintheswineindustryandcurrentstatusisendemiccountry(OIE).Eventhoughitisknownthatbothdomesticpigsandwildboarwereinvolvedinthespreadofthedisease,theexactnatureofthedomestic-wildanimalinterfaceremainsunclear.ThereforeabetterknowledgeabouttheinteractionofASFbetweenwildboaranddomesticpigsisrequiredtogainabetterunderstandinginquantifiabletermsthepotentialmechanisms of virus spread in the Russian Federation. A spatio-temporal kriging analysiswas carried out to estimate the source(domesticorwild)of849outbreaksofASF(377inwildboarand472indomesticpigs)reportedbetweenNovember2007andDecember2015intheRF.Thekriginganalysisidentifiedareasinwhichdomesticpigsweretheprincipalsourceofinfection.OnlyinfourspecificzoneswerewildboaridentifiedasthesourceofASFoutbreaks.Resultshowsthatdomesticpigswerethesourceofinfectionin611(72%)andwildboarin237(28%)outbreaks.Inoutbreaksindomesticpigherds,thesourceofinfectioncouldbeattributedtodomesticpigsin68.29%(320)andtowildboarin31.71%(151)ofcases,whilstinwildboaroutbreaks,thesourceofinfectioncouldbeattributedtodomesticpigsin77.24%(290)andtowildboarin22.26%(86)ofcases.TheroleofwildboarintheepidemicspreadofASFintheRF(2007-2016)wasmuchlessthanthatofdomesticpigs,whichweretheoriginofapproximatelytwo-thirdsofallnotificationsindomesticpig andwildboarpopulations. These resultsprovideuseful information for evaluatingandunderstanding the transmissionofASFbetweenwildlife and livestock, and the analyses used here can be easily applied to other infectious animal diseaseswith similarcharacteristicsandtootherregionsoftheworld.

CRWAD2017 ABSTRACTS


P048-LeptospirosisinabattoirworkersinUganda:ariskassessmentpilotP.Pithua1,L.Mugisha2,M.L.Khaitsa3,S.Odemuyiwa4.1VeterinaryMedicine&Surgery,UniversityofMissouri,Columbia,MO,USA,2Wildlife&AquaticAnimalResources,MakerereUniversity,Kampala,Uganda,3Pathobiology&PopulationMedicine,MississippiStateUniversity, Mississippi State, MS, USA, 4Veterinary Pathobiology, University of Missouri, Columbia, MO, [email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMBackground.InUganda,theconditionsconducivefortheemergenceandpersistenceofLeptospiraarecommon.Eventhougharecentstudyrevealedthat≥35%ofpatientsseekinghealthcarewereseropositiveforLeptospira,howthepathogenistransmittedtopeoplehasnotbeenstudied.RiskfactorsforhumaninfectionwithLeptospiraarelargelyunknown.TheobjectiveofthisstudywastoidentifypathogenicLeptospiraserovarscirculatinginhumansandlivestockinUgandaandtodetermineriskfactorsforexposureinabattoirworkers.Methods. Cross-sectional datawere collected to (a)measure the seroprevalence, (b) identify risk factors for Leptospiraexposure inabattoirworkers,and (c)quantify theseroprevalenceofLeptospira incattle, sheepandgoatsslaughtered inUgandanabattoirs.Serumsamplescollectedfromcattle,sheep,goats,andpigsslaughteredintwoabattoirs inKampalaarebeingevaluatedusingMicroscopicAgglutinationTesting(MAT).Abattoirworkersfromthesefacilitiesarealsobeingtestedserologicallyandinterviewedforbothoccupationalandnon-work-relatedexposurestohumanpathogenicLeptospira.Results.Wehavecollectedbloodsamplesfromcattle(n=300),sheep(n=15),goats(n=160),pigs(n=250)andabattoirworkers(n=200).SerologicaltestingofthesesamplesusingMATisongoing.Weareprocessingdataonexposureriskfactorsamongabattoirworkerspendingfinalanalysis.Conclusions.Uponcompletion,weexpecttogainabetterunderstandingofLeptospiraspeciesdiversityinUganda,majorlivestockspeciescarryinghumaninfectingLeptospiraserovars,andclonalrelationshipsbetweenLeptospiraisolatedfromlivestockandhumansforinsightsintocommonsourcesoftransmission.P049-WildliferabiessurveillancestudyinGeorgiaL.Ninidze,N.Kartskhia,L.Avaliani,I.Menteshashvili.NationalFoodAgency,Tbilisi,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:RabiesvirusisapublichealththreatthatistransmittedbythebiteofaninfectedanimalandisendemictothecountryofGeorgia. In 2013, Georgia re-established its prophylactic vaccination program against rabies and increased coverage in domesticanimals.In2016,vaccinationcoveragewasupto70%indomesticanimals,andsince2013,rabiescasesdecreasedby51%.However,wildanimalsarestillreservoirsandposeariskofexposureandinfectiontodomesticanimalsthathavedirectcontactwithhumans.From2014-2017,48%ofthesuspectrabiescaseswereconfirmedpositiveforrabiesvirusamongagriculturalanimalsinGeorgia.In2017, theNational FoodAgency conducted a rabieswildlife surveillance study.Methods: A total of 31brain tissue sampleswerecollectedfromwildanimals(jackals,foxes,andwolfs)fromsixdifferentregions(Tbilisi, Imereti,Kakheti,Samegrelo,Samtskhe,andKvemo Kartli). The National Food Agencywas granted authorization from theMinistry of Environment and Protection of NaturalResourcesofGeorgiatohuntwildanimalsthatweresuspecttobeinfectedwiththevirus.ThebioassayforrabiesvirusconfirmationwasconductedattheLaboratoryoftheMinistryofAgriculture(Tbilisi,Georgia).Results:Asaresult,32%oftheanimalswerepositive(threejackals,sixfoxes,andonewolf).Testingresultsofwildanimalswillhelpsupportthedevelopmentofalong-termanti-rabiesprogram.Conclusions:Thesurveillancestudyisongoing,andourgoalistotest200wildlifeanimalstestedbytheendof2018.Ourstudyshowsthatrabiesispresentinwildanimals,butfurtherinvestigationsareneededforidentifyingtheremainingfoci,prevention,andcontrol.

CRWAD2017 ABSTRACTS


P050-Usingafactorialsurveytocharacterizeriskprofilesofcattleherdsandownerstodevelopamoreeffectivepolicyforbovinetuberculosissurveillance

Y.wang1,S.Wells2,M.J.Oakes3.1GraduatePrograminBioinformaticsandComputationalBiology,UniversityofMinnesota,SaintPaul,MN,USA,2DepartmentofVeterinaryPopulationMedicine,CollegeofVeterinaryMedicine,UniversityofMinnesota,SaintPaul,MN,USA, 3Division of Epidemiology and Public Health, School of Public Health, University of Minnesota, Minneapolis, MN, [email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMAnimalmovementisthetopcontributortobetween-herdtransmissionofinfectiousdiseases(e.g.,bovineTB)incattle.Amajorsourceofanimalmovementiscattleintroduction.Identifyingmotivatingfactorsbehindcattleintroductioniscrucialfordiseasecontrolandsurveillance.Wehypothesizedthat5factorsareimportantinthedecisionofpurchasingananimal:governmentpolicyonbovineTBcontrol,placeofpurchase,andanimal'sofficialID,originandnumberofpreviousowners.WeusedafactorialsurveyapproachthatrandomlyexposedherdownerstoscenariosofbovineTBinfectionandgovernmentinterventionpolicyandcapturedimpactsonriskbehavior.Eachherdownerwaspresentedwith2short storiesof randomlyassignedscenariosconstructedwithdifferent levelsofhypothesized factors and were asked to rate their willingness to purchase cattle. Combined with factual demographic/businesscharacteristicsalsocollectedinthesurvey,weestimatedtheirriskprofilesforcattleintroduction.Inaddition,weuseddata-miningtechniques to explore associations between actual purchase behavior and responses from the factual survey.Wemailed3994surveystwicetoastratifiedrandomsampleofMNcattleproducers,andreceived904responses(responserate22.6%).Threefactorsrelatedtotheanimalofinterest(officialID,origin,numberofpreviousowners)werehighlyinfluentialtowillingnesstopurchase.Governmentaidpolicy,however,wasnot.Additionalvariables(numberofworkingyears,age,education,mandatorydiseasetestingbeforeimport,farmdistrict)werealsoassociatedwithwillingnesstopurchase.Thisworkisanovelapproachthatcombinedanobservationalsurveywithstrengthsofexperimentaldesign,factoranalysisandpredictivelearning.Hereweidentifiedinfluentialfactorsfor producer behavior in response to government policy on disease control. The outcome provides useful insights for targetedsurveillanceintheUSwherecompletecattletraceabilitysystemisunavailable.P051-Understandingcurrentissuesonthefoot-and-mouthdiseasesusingmathematicalsimulationmodelsH. Yoon1, K.-N. Lee1, K.-H. Cho1, C.-H. Lee2, P.-W. Kim2. 1Veterinary Epidemiology Division, Animal and Plant Quarantine Agency,Gimcheon,Korea,Republicof,2DepartmentofMathematicalScience,UlsanNationalScienceandTechnologyInstitute,Ulsan,Korea,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Mathematicalsimulationmodelsonspreadoffoot-and-mouthdisease(FMD)weredevelopedattwo-stagesetting, intra-herdandbetween-farms,incooperationwithAPQAandUNISToftheRepublicofKorea.Methods:Modelsweredevelopedbothwithordinarydifferentialmethodandstochasticapproach.Results:Thesemodelsemployedthreespecificfeatures:i)informationonrealdemographyoffarms(i.e.species,herdsize,distanceamongfarmsbasedongeo-coordinates,anddensityofanimalsandfarms);ii)recordsoflivestock-relatedfarmsonlivestockfacilitiesbasedongeographicalpositioningtrackingsystem;andiii)realoutbreakdataofFMDinKorea.Thefirsttwofeatureswereprovidedbythe‘KoreaAnimalHealthIntegratedSystem(KAHIS)’operatedbyAPQA.Dataonregularvaccinationoncattle,pigs,goatsanddeerinKorea,andserologicalsurveillancewereusedasinputparameters.Conclusions:Simulationswereusefulatestimating infectionperiod (intra-herdmodel)andpre-emptivelydescribing farmsandareaathighrisk(between-farmmodel).

CRWAD2017 ABSTRACTS


P052 - A comparison of infectious agents between hatchery-enhanced and wild out-migrating juvenile Chinook salmon fromCowichanRiver,BritishColumbia

K.K. Thakur1, R. Vanderstichel1, E. Laurin1, S. Tucker2, K.M. Miller2. 1Health Management, University of Prince Edward Island,Charlottetown,PE,Canada,2PacificBiologicalStation,FisheriesandOceansCanada,Nanaimo,BC,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:Todescribetheprevalenceandspatio-temporaldistributionofinfectiousagentsinjuvenileChinooksalmonsamplescollectedfromCowichanRiversystemandtocomparedifferencesinprevalenceanddiversityofinfectiousagentsbetweenhatcheryandwildfish.Methods:Weappliedahigh-throughputmicrofluidicsplatformtoscreenfornucleicacidsof45infectiousagentsinmixedtissuesamplesfrom566out-migratingjuvenileChinooksalmoncollectedfromfreshwater(FW)andsaltwater(SW)locationsintheCowichanRiversystemduring2014.Thedatawereanalyzedusinganumberofstatisticalmethodsincludinglogisticregression,clusteranalysis,log-linearmodelsetc.Results:Wedetected19infectiousagents(5bacterial,2viral,and12parasitic)inthesesamples,withprevalencesrangingfrom0.2to57.5%.Prevalenceofspecificagentsvariedbyfishtype(hatcheryandwild),samplinglocation(FWandSW),andmonth. Individual fish harboured up to nine infectious agents. Co-infections between Candidatus Branchiomonascysticola,Paranucleosporatheridion,andGillchlamydia,allassociatedwithgilldisease,werestatisticallysignificantandweremostnotable in SW. Some of the infectious agents detected in our study are known to cause large-scalemortalities in Pacific salmon(Ceratomyxa shasta), or mortalities during specific life-history stages (Parvicapsula minibicornis), while others (C. B. cysticola, P.theridion, Facilispora margolisi and Parvicapsula pseudobranchiola) have only recently been reported in Pacific salmon in BC.Conclusions:Ourfindingsalsoindicatedthatwildandhatcheryfishweremostdivergentinagentprofilesintheirnatalsites;differencesinprevalencedissipatedoncetheyconvergedinthemarineenvironment,suggestingthathatcheryandwildfishareequallysusceptibletoinfectiousagentsinacommonenvironment.Thisstudyprovidesthefirstinsightintothedistributionandprevalenceofinfectiousagentsinhatchery-rearedandwild-caughtjuvenileChinooksalmoninsouthernVancouverIsland.P053-DetectionofDeltacoronavirus(δ-CoVs)inaviancloacalswabsfromwildbirdsintheMississippiflywayF.C.Paim1,L.J.Saif1,A.Bowman2,H.Hu3,A.N.Vlasova1.1DepartamentofVeterinaryPreventiveMedicine,FoodAnimalHealthResearchProgram,TheOhioAgriculturalResearchandDevelopmentCenter,TheOhioStateUniversity,Wooster,OH,USA,2DepartamentofVeterinaryPreventiveMedicine,CollegeofVeterinaryMedicine,TheOhioStateUniversity,Columbus,OH,USA,3CollegeofAnimalScienceandVeterinaryMedicine,HenanAgricultureUniversity,Zhegzhou,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMCoronaviruses(CoVs)arepositive-senseRNAvirusesthatarewidespreadinhumans,andvariousmammalianandavianspecies.Theycauseenteric,respiratoryorsystemicdiseasesofvariableseverity.CoVsbelongstotheCoronaviridaefamilythatissubdividedintofourgenera:Alphacoronavirus(α-CoV),Betacoronavirus(ß-CoV),Gammacoronavirus(γ-CoV),andDeltacoronavirus(δ-CoVs).Anewporcineδ-CoVwasdiscoveredthatcausesentericdiseasewithseverediarrhea,vomitinganddehydrationinneonatalpiglets,resultingineconomiclossestotheswineindustry.Furthermore,studiessuggestthatwildbirdsarepotentialreservoirsforδ-CoVs.Duetheirabilitytoflylongdistances,birdscouldplayaroleincross-speciestransmissionofδ-CoVs.Ouraimwastoinvestigatethepresenceofδ-CoVs indifferentpopulationsofwildwaterfowl inOhio,Mississippi, IndianaandArizona.Fivehundredaviancloacalswabswerecollectedduring2015-2016tosurveyforavianinfluenzavirus.AviancloacalswabRNAsamplesweretestedforδ-CoVusingapreviouslydesignedone-stepuniversalCoVRT-PCRassaywithδ-CoVnucleocapsid-specificuniversalprimers.Sevenof500aviansampleswerepositiveforδ-CoVs,correspondingwithaprevalenceof1.4%.Thisishighercomparedwiththatobservedinourpreviousstudy(0.5%)that analysed Ohio samples from 2013-2014. Our studies show that δ-CoVs circulate in wild birds in the US, demonstrating theimportanceofwaterfowlasareservoirofavianδ-CoVs.Wewillnextevaluatethephylogeneticrelationshipbetweenthecurrentavianandporcineδ-CoVstoinvestigatetheirpotentialfortransmissionbetweenavianandlivestockspecies.

CRWAD2017 ABSTRACTS


P054-SerologicalinvestigationofexposuretoinfluenzaAvirusH3N2indogsandcatsR.M.Pogranichniy1,S.Gutman2,L.Guptill3,G.Moore4.1ComperativePathobiology,PurdueUniversity,W.Lafayette,IN,USA,2PurdueUniversity,W.Lafayette, IN,USA,3VeterinaryClinicalScience,PurdueUniversity,W.Lafayette, IN,USA,4VeterinaryAdministration,PurdueUniversity,W.Lafayette,IN,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMCanineinfluenzavirus(CIV)H3N2isasubtypeofinfluenzaAvirus(IAV)ofavianlineagethatoriginatedinAsia.Recently,CIVH3N2hasbeenofinterestduetoawidespread,suddenoutbreakintheChicago,ILregioninAprilof2015.TheaimofthisstudywastoinvestigatetheassociatedriskfactorsandprevalenceofantibodiesagainstIAVandCIVH3N2inserumsamplescollectedfromrandomlyselecteddogsandcatsnativetovariousU.S.states.InordertomeasuretheseroprevalenceofantibodiesagainstCIVH3N2inthe458canineand67felinesamples,acommercialenzyme-linkedimmunosorbentassay(ELISA)andin-househemagglutinationinhibition(HI)testwereutilized.UsingROCanalysisoftheresults,itwasdeterminedata100.00%sensitivityand98.01%specificitythattheHIcutofftiterwas1:32.FromtheHIresults,itwasfoundthatdogshada2.21%seropositivityforCIVH3N2while8.96%ofcatswereseropositive.Therewerenoapparenttrendsfoundbetweenseroprevalenceandassociatedriskfactorsoftheanimals.AllpositiveanimalswerefromIndianaorIllinois.ItwasconcludedthatCIVH3N2virusshouldstillbekeptundersurveillancebecauseofitsversatileabilitytore-assortandspreadasamorevirulentstrain.P055-Inter-observeragreementincategorizationofracehorsenecropsyreportsA.E.Hill1,S.M.Stover2,M.Rhea3,F.A.Uzal3.1CaliforniaAnimalHlth&FoodSafetyLab,UniversityofCalifornia-Davis,Davis,CA,USA,2Anatomy, Physiology, and Cell Biology, University of California-Davis, Davis, CA, USA, 3California Animal Hlth & Food Safety Lab,UniversityofCalifornia-Davis,SanBernardino,CA,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMSince1990,racehorsesthatdieatsanctionedracetracksinCaliforniamustbenecropsiedtodeterminethecauseofdeath.Duringthisperiod, knowledge of racehorse musculoskeletal injuries has advanced, and necropsies have been performed at four differentlaboratorylocationsbymanypathologists.Asaconsequence,theterminologyandlevelofdetailusedinnecropsyreportsarehighlyvariable.Thisvariabilitymakescomparisonofcasesovertimeorbydifferentpathologistschallenging.Classifyingracehorsenecropsyreportsusingdefinedguidelinesandaseriesofcategoricalvariablescouldprovidestandardizeddata.Thepurposeofthisprojectwastoassessagreementamongobserverswhowerereviewingandcodingnecropsyreports fromracehorses thatdied.434racehorsenecropsyreportsfromJanuary1995toDecember1996werereviewedandclassifiedbyfourobserverswithexpertiseinracehorseanatomy,pathology,andinjuryassessment.Codingguidelinesweredevelopedbytheobserversandupdatedasneeded.Eachnecropsyrecordwasclassifiedbytwoobservers,withdescriptorsforcircumstances,injurysite(e.g.limb),syndrome(e.g.fracture,jointluxation,etc.), and anatomic structure (e.g. carpus). Additional descriptorswere included for “fetlock failure” injuries,which are themostcommon group of fatal injuries in California racehorses. Inter-observer agreement was assessed using Cohen’s kappa statistic.Agreementbetweenobserversonallaspectsofanecropsyreportrangedfrom73.4%to82.0%,withsomedescriptorshavingahigherproportionofobserverdisagreementthanothers.Recordingstandardizedvariablesfromfree-textfieldsremainssubjective,evenforknowledgeableobserversusingcodingguidelines.

CRWAD2017 ABSTRACTS


P056-IdentifyingimmunodominantandneutralizingepitopesfromK88fimbriaeofenterotoxigenicEscherichiacoliT.Lu1,W.Zhang1,R.A.Moxley2.1DiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,USA,2SchoolofVeterinaryMedicineandBiomedicalSciences,UniversityofNebraska-Lincoln,Lincoln,NE,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMPurpose:F4+(K88)enterotoxigenicEscherichiacoli(ETEC)istheprimarycauseofporcinepost-weaningdiarrhea(PWD),yetaneffectivePWDvaccineforthispathogenisnotavailable.WeidentifiedsuitableantigensforaneffectivePWDETECvaccine.Methods:Wefirstinsilico identifiedepitopesfromtheK88fimbrialmajorsubunitFaeG.Then,wegeneticallyfusedeachepitopetonon-homologoushumanETECCFA/Iadhesinsubunit(CfaB),andexaminedimmunodominantepitopesbyreactingeachfusionwithanti-K88antiserum.Subsequently, each epitope fusionwas used to immunizemice.Mouse serum sampleswere examined for anti-K88 IgG antibodyresponsesandneutralizationofK88fimbriaandporcineETECwildtypestrain3030-2toporcineintestinalIPEC-J2cells.Inaddition,weexpressedtheFaeGsubunitproteinasthecoatingantigeninELISAsandWesternBlottoexaminereactionwithantibodiesderivedfromeachepitopefusion,inordertoverifywhetherepitopeconformationalterationcouldleadtothelossinreactingwithanti-K88serumorininducinganti-K88antibodyresponses.Results:Atotalof9epitopeswereidentifiedfromFaeGmajorsubunit.Theseepitopesdifferedinimmunogenicity,indicatedbydifferentreactivitywithanti-K88antiserum.Antibodiesderivedfromtheseepitopefusionsvariedinantibodyneutralizingactivity.EpitopesMTGDFNGSVD,LNDLTNGGTK,GRTKEAFATP,PMKNAGGTKVGAVKVNandFNQAVTTSTQshowedstrongerreactivitywithanti-K88antisera,suggestingtheseepitopesimmunodominant.Moreover,mouseimmunizationdatashowed thatepitope fusions inducedvarious levelsofanti-K88antibody responses.Epitopes LGRGGVTSADGEL,PRGSELSAGSAandRENMEYTDGT were neither immunogenic nor neutralizing, while epitope ELRKPDGGTN was less immunodominant but stronglyneutralizing, epitope FNQAVTTSTQ was immunodominant but less neutralizing. However, epitopes MTGDFNGSVD, LNDLTNGGTK,GRTKEAFATPandPMKNAGGTKVGAVKVNwereimmunodominantandalsoneutralizing.Conclusions:TheseresultssuggestepitopesMTGDFNGSVD,LNDLTNGGTK,GRTKEAFATPandPMKNAGGTKVGAVKVNaresuitableK88antigensfordevelopinganeffectivevaccineagainstporcinePWD.P057-MicrobiomechangesinthesmallintestineofnewbornpigletsrelatedtoenterotoxigenicEcherichiacolichallengeandgilt

vaccinationF.L.L.Leite1,R.M.Nandre2,Q.Duan2,W.Zhang2,R.Isaacson1.1UniversityofMinnesota,StPaul,MN,USA,2KansasStateUniversity,Manhattan,KS,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMEnterotoxigenicEscherichiacoli(ETEC)areasignificantcauseofneonataldiarrheaandpost-weaningdiarrheainpiglets.ETECarealsoaleadingcauseofdiarrheatochildrenunder5yearsindevelopingcountriesandinternationaltravelers.ProtectionagainstETECcanbemediatedbyantibodiesagainstfimbrialadhesinsandenterotoxinsthatcanbegeneratedthroughvaccination.TheimpactofETECinfectiononthemicrobiomeofneonatesandtheeffectofvaccinationmediatedprotectionontheswineneonatalmicrobiomehavenotbeenfullystudied.HereweinvestigatedthecompositionofthemicrobiomeofthesmallintestineofpigletschallengedwithanETECstrainthatproducestheheat-labiletoxin(LT).Vaccinationwasperformedintwogiltswhichreceivedeitheratoxoidfusionsubunitvaccine(3xSTaN12S-dmLTwithdoublemutantLTadjuvant)oratoxoid-adhesinsubunitvaccine.Thelittersoffourgiltswereassignedtofourtreatmentsusedinthisstudy, littersfromthetwovaccinatedgiltsandonenon-vaccinatedgiltwerechallengedwiththeETECstrainafter24hrsofsuckling,anon-challenged litterservedasnegativecontrol.Bothvaccines ledtoasignificant reduction in thenumberofpigletswithdiarrheaatonedayafterchallenge(p<0.05).Thecontentsofthesmallintestineswerecollectedwhenpigletswere2daysofageandmicrobiomeanalysiswasperformedusingtheV3regionofthe16srRNAgene.Pigletsfromthechallengednon-vaccinatedgroup,whichhadmorediarrhea,hadagreaterabundanceofLactobacillusandbacteriainthephylumFirmicutescomparedtothevaccinatedgroupsandthenon-challengedcontrolgroup.InfectionwithETECledtoadecreaseofProteobacteriaregardlessofvaccination.ThisstudydemonstratesthatETECinfectionhasalargeeffectontheneonatalmicrobiomeandtheseuniquevaccinescanbeaneffectivestrategytominimizeclinicalsignsduetoinfection.

CRWAD2017 ABSTRACTS


P058-Anovelbioinformaticspipelineforefficientmetagenomicsanalysis;a16sstudyofthemicrobiomeofthepreputialskinofbulls

H.Jung1,J.Koziol2,S.L.Yingst1.1ComparativePathobiology,PurdueUniversity,WestLafayette,IN,USA,2VeterinaryClinicalSciences,PurdueUniversity,WestLafayette,IN,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMItisessentialtoestablishabaselineofthenormalbiotaofthepreputialskinofthebullinordertoassessandunderstandtheeffectofantibiotictherapyespeciallyinpredispositiontoinfectionwithTrichomonasfoetus.Thisinitselfisanoveleffort,butthebioinformaticsprocessestoaccomplishthisinanefficientandcost-effectivemannerarenotwellestablished.Weestablishedananalysispipelineforthe bullmicrobial communitywith R/Bioconductor packageswithout pickingOTUs.Differentiation of real biological variants fromsequencingerrorsisstillchallenging.ConstructionofOTUscanbeusedtoreducesequencingerrorsinIllumina-sequencedamplicons.However,currentapproachessuchasmothurandQIIMErelyonreferenceclusteringalgorithmsorhavelimitationstofullyutilizethequality informationof Illuminaamplicon sequencing.Toovercome theseobstacleswhile still producing robust relativeabundanceresults,weusedDADA2Rpackagetoinfermoreaccuratesequencesbyintroducinganewquality-awaremodelasopposedtoothercurrentmethods.Themodel-basedapproachforcorrectingampliconerrorswithoutconstructingOTUsisreferencefree,applicabletoanygeneticlocusandcanbeimportedintotheRpackagesincludingphyloseq.Wehaveanalyzed32samplesfromostensiblynormalanimalsandhavefoundroughly66diversespeciesineachsample,representingawiderangeofbacterialspecies,noneofwhichareobligatepathogens.Asaresult,themethodologyisviableforefficientandcost-effectiveanalysisofthemicrobiometosupportbothresearchandindividualanimaltesting,forexample,forpurposesofbreedingsoundnessexams.P059-RNAseqrevealsacomplexsurvivalresponsebyCampylobacterjejuniincloseassociationwiththeovinegallbladdermucosa

inanexperimentalmodelofinfectionB.Ruddell,P.Plummer,J.Schleining,M.Yaeger,A.Kreuder.IowaStateUniversity,Ames,IA,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMPurpose:Certainentericbacterialpathogensareknowntopossesstheabilitytocolonizethegallbladderandsurvivewithinthisuniqueenvironment.However, little isknownaboutthebacterialmechanismsthatallow long-termsurvival in thisotherwise inhospitablelocation.Overthepastdecade,C.jejunisheepabortion(SA)clonehasemergedasthepredominantcauseofsheepcampylobacteriosisin theUnitedStates leading toabortionstorms in flocks.Previous studieshave indicated thatC. jejuni cloneSAcan frequentlybeisolatedfromthegallbladdersofhealthysheep,suggestingthatthegallbladdermayserveasanimportantlocusforC.jejunipersistenceandtransmission.Inaddition,ourgrouphasrecentlydescribedthecompletetranscriptomeofC.jejunifreewithinthebileoftheovinegallbladder.TheobjectiveofthisstudywastogaininsightintothemolecularmechanismsofsurvivalofC.jejunibacteriafoundindirectassociationwiththemucosalsurfaceoftheovinegallbladder.Methods:AclinicalisolateoftheSAclone,C.jejuniIA3902,wasexposedforupto24htothenaturalovinehostgallbladderenvironment.Next,totalRNAwasisolatedfrombacteriadirectlyassociatedwiththe gallbladdermucosa. Subsequently, high throughput deep sequencing of strand specific rRNA-depleted total RNAwas used tocharacterize the transcriptome of IA3902 under this condition. Results: Comparison of the transcriptome of C. jejuni in directassociationwiththegallbladdermucosaascomparedtowhenfreewithinbiledemonstratedsignificantdifferencesingeneexpression,includingbothproteincodingandnon-codingRNAgenes.Anadditionalsubsetofgeneswerealsofoundtobeconsistentlydifferentiallyexpressed inboth locationswhencompared topre-inoculation.Conclusions:This studyenables further insight into themolecularmechanismsrequiredforsurvivalofC.jejuniwithinovinegallbladder.

CRWAD2017 ABSTRACTS


P060-Inter-andintra-serotypediversityinbiofilmformationbythemostprevalentpoultry-associatedSalmonellaserotypesN.C. Paul, D.H. Shah. Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, [email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMSalmonellacanattachtoabioticsurfacesandproducebiofilm.Biofilmaidssurvivalinharshconditionsandalsoenhancesresistancetoantimicrobialsandsanitizers.Theobjectivesofthisstudywastodeterminethequantityandtypeofbiofilmformationbythemostprevalentpoultry-associatedSalmonellaserotypes(MPPSTs).Atotal226fieldstrainsofSalmonellabelongingto12serotypesisolatedfrompoultryandpoultryproductsweretestedfortheirabilitytoformbiofilmsbyagold-standardcolorimetricassay(polypropylenemicrotiterplatetest).All isolateswerealsotestedfortheirabilitytoformpellicleattheair-brothinterface(borosilicatetubetest),productionofthinaggregativefimbriaeand/orcellulose(congored-brilliantblueandcalcofluortests).Atotalof145(64%)Salmonellastrainswereidentifiedasbiofilmproducersbycolorimetricassayalone.Thenumberofbiofilmpositivestrainsincreasedto160(71%)and162 (72%)whencolorimetricassaywascombinedwithcongored-brilliantblueandtubetest, respectively.Theserotype levelprevalenceofbiofilmwasasfollows:S.Infantis(6/6,100%),S.Thompson(6/6,100%),S.I4,[5],12:i:-(4/4,100%),S.Schwarzengrund(1/1,100%),S.Mbandaka(24/26,92%)andS.Senftenberg(16/19,84%),S.Typhimurium(10/12,83%),S.Montevideo(13/16,81%),S.Enteritidis (29/38,76%),S. Kentucky (35/56,62%), S.Heidelberg (7/12,58%)andS.Hadar (11/31,36%). Thesedata suggests thatdifferentserotypesmayvaryintheirabilitytoproducebiofilms.Inaddition,thereweredifferencesinthetypeofbiofilmproducedatbothinterandintra-serotypelevel,suggestingthatacombinationoftwoormoretestsshouldbeusedtoaccuratelyidentifythetypeandamountofbiofilmproductionbyfieldisolatesofSalmonella.P061-Co-circulationofhepatitisEvirusgenotypes3&4indomesticpigsinKoreaY.Kim,B.Park,H.Ahn,S.Han,H.Go,D.Kim, J. Lee,S.Park,C.Song,S. Lee, I.Choi.Konkukuniversity,Seoul,Korea,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMCo-circulationofhepatitisEvirusgenotypes3&4indomesticpigsinKorea.Yong-HyunKim,Byung-JooPark,Hee-SeopAhn,Sang-HoonHan,Hyeon-JeongGo,Dong-HwiKim,Joong-BokLee,Seung-YongPark,Chang-SeonSong,Sang-WonLee,In-SooChoi*.DepartmentofInfectiousDiseases,CollegeofVeterinaryMedicine,KonkukUniversity,120Neungdong-ro,Gwangjin-gu,Seoul05029,Korea.Purpose:HepatitisEhasbeenaseriouspublichealthconcerninAsia,theMiddleEast,andAfrica,aswellasinSouthAmerica.ThehepatitisEvirus(HEV)infectioninducesanacutehepatitiswithhighmortalityupto30%inpregnantwomen.TheHEVhasbeenisolatedinmanymammalianspeciesincludingpigsandplaysasanimportantzoonoticagent.SeveralcasesofhepatitisEwerereportedinKoreaandmostofthemwereknowntobeinfectedwithHEV-3andHEV-4.However,itwashardtoexplaintheinfectioussourceofthepatientsinKoreasufferedfromHEVinfections.Inthisstudy,wedetectedapartialHEVgenomefromfecalsamplescollectedfrom14farmslocatedat5provincesinKorea.Methods:Swinefecalsamplesweredilutedto10times(w/v)inphosphatebufferedsaline(PBS).Thefecalsupernatantswereobtainedaftercentrifugationfor30minat3,000rpm.RT-PCRwasperformedtodetectapartialHEVgenomelocatedattheORF1-2junction.PhylogenetictreeanalysiswasconductedtoanalyzetheHEVgenomicsequence.Results:HEVRNAwasidentifiedin30/148(20.3%)offecalsamplescollectedfrom14pigfarms.HEV-3a-likeandHEV-4c-likesubtypesweredetectedfrom5/14(36%)and2/14(14%)pigfarms,respectively.PhylogeneticanalysisindicatedthatthoseHEVisolateswerecloselyrelatedwithpreviouslyidentifiedzoonoticstrainsinKorea.Conclusions:WefoundforthefirsttimeinourknowledgethatbothHEV-3andHEV-4aresimultaneouslycirculatinginpigsinKorea.ThoseswineHEVisolatesseemtoberesponsibleforcross-speciestransmissiontohumans.

CRWAD2017 ABSTRACTS


P062-InhibitoryactivityofreducedpHonSalmonellasurvivalincalfmilkreplacerH.E.Pilch1,T.Earleywine2,R.Musser2,C.Czuprynksi1.1UniversityofWisconsin-Madison,Madison,WI,USA,2LandO'Lakes,Shoreview,MN,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMObjective:Calfmilkreplacer(CMR)iscommonlyusedintherearingofyoungdairycalves.ReducingpHcanpresentahurdletobacterialgrowth.InthisstudyweevaluatedthefateofSalmonellaentericaserovarsDublin,Cerro,MontevideoandHeidelberginCMRadjustedoverarangeofpH.Methods:CMRwasreconstitutedusingsteriledeionizedwaterandputintoa15mlroundbottomtube.SalmonellaDublin,SalmonellaCerro,andSalmonellaMontevideoweregrownovernightintrypticasesoybrothonarotatingrackat35°Cuntiltheculturereachedacelldensityofapproximately109CFU/ml.Thebacterialcellswereharvestedbycentrifugation,washed,dilutedinPBS and added as either a cocktail of strains (S. Dublin, S. Cerro and S.Montevideo) or a single strain (S. Heidelberg) to CMR atapproximately106CFU/ml.TheCMRwas incubatedat38.50Candsamples removedat2,4,and8hours todetermineCFU/mlofSalmonella.Results:WheninoculatedintocontrolCMRthecocktailofSalmonellastrainsincreased3log10CFUduringan8hincubation,reachingafinalconcentrationofapproximately109CFU/ml.Salmonellagrewtoalesserextent(2log10)inCMRwithapHof5.80.Incontrast,reducingthepHofCMRto5.2resultedinanapproximately2log10decreaseinCFUduringan8hincubationat38.5

0C.SimilarresultswereobservedwhentheexperimentswereperformedusingasinglestrainofS.Heidelberg.Conclusion:ThesefindingssuggestthatreducingthepHofCMRto5.2substantiallyimpairsbacterialsurvivalduringan8hincubationat38.50C.P063-Apilotseroprevalencestudyoftick-borneencephalitisvirusineasternGeorgiaT.Jashiashvili1,R.Sukhiashvili1,S.Borg2,J.Pollakova2,R.Wölfel2,P.Imnadze1.1NationalCenterforDiseaseControlandPublicHealth(NCDC),Tbilisi,Georgia,2BundeswehrInstituteofMicrobiology,Munich,[email protected]:VectorborneandParasiticDisease,12/3/20176:30PMPurpose:Tick-borneencephalitisvirus (TBEV), isa fatalneurological infectionthat isendemictotheCaucasus.Geographicdiseasespreadmay occur because of climate-change and social, political, ecological and demographic factors. TBE risk areas have beencharacterizedbysurveillancestudiesand illustratedbygeographicprevalencemapping;however, thecountryofGeorgiahasbeenincludedduetothelackofsurveillancedata.In2016,theNationalCenterforDiseaseControlandPublicHealth(NCDC)ofGeorgiacollaboratedwiththeBundeswehrInstituteofMicrobiology(BwIM)andperformedapilotstudyofTBEVseroprevalenceinEasternGeorgia.ThisprojectwaspartoftheGermanPartnershipProgramforExcellenceinBiologicalandHealthSecurity(GPEBHS).Methods:TheaimofthisstudywastoestimatetheseroprevalenceofTBEVinhealthyadultpopulationsfromEasternGeorgiaandidentifyhigh-riskareas.WedesignedthisstudybasedonthedistributionofmostcommonTBEVvectorsinEasternGeorgia:Ixodesricinus,andI.persulcatus.Atotalof1,821humanserumsampleswererandomlyselectedfromtheHepatitisCeliminationprogramandtestedforthe presenceof TBEV IgG antibodies by anti-TBEV ELISA (IgG). For confirmation,weused immunofluorescence assay (EuroimmunFlavivirusBiochip).Results:Asaresult,eightsamples(0.44%)wereTBEV-positive(fivefemalesandthreemales,withagesbetween28to74years).GPEBHS’sgoalistoimplementlong-termbiosecurityprojectsinvariouscountriesundertheGlobalPartnershipAgainsttheSpreadofWeaponsandMaterialsofMassDestruction.Conclusions:DespitethelowseroprevalenceofTBEV,furtherinvestigationsshouldbeconductedwithalargersamplepopulation,whichwillincludehuman,animal,andticksamples.

CRWAD2017 ABSTRACTS


P064-MolecularsurveryofAnaplasmabovisinHolsteincattleintheRepublicofKoreaK.-S.Choi1,J.Park2,J.-S.Chae3,B.-K.Park4,H.-C.Kim5,D.-H.Yu6.1KyungpookNationalUniversity,Sangju,Korea,Republicof,2ChonbukNationalUniversity, Iksan,Korea,Republicof,3SeoulNationalUniversity,Seoul,Korea,Republicof,4ChungnamNationalUniversity,Daejeon,Korea,Republicof, 5KangwonNationalUniversity,Chuncheon,Korea,Republicof, 6GyeongsangNationalUniversity, Jinju,Korea,[email protected]:VectorborneandParasiticDisease,12/3/20176:30PMPurpose:Anaplasmosis isatick-borneinfectiousdiseasethat impactsbothhumanandanimalhealth.ThisstudywasperformedtocharacterizeandinvestigatetheprevalenceofAnaplasmabovisinHolsteincattlefromtwodifferentregionsintheRepublicofKorea(ROK).Methods:Onehundredfifty-onebloodsamples(80fromNamwonand71fromJejuIsland)wereanalyzed,andtheprevalenceofA.boviswascomparedbeforeandaftergrazing.Results:InNamwon,A.bovisinfectionwasnotdetected,whileinJejuIsland,A.boviswasdetected in3animals (23.1%,3/13)aftergrazing.Theoverall infectionrateofA.boviswas1.9%(3/151)basedonPCR.PhylogeneticanalysisrevealedthatourA.bovisisolatesshowed99%homologywithaKoreanspotteddeerisolateandHaemaphysalislongicornis tick isolates identifiedon Jeju Island. This is the first report to identifyA.bovis infection inHolstein cattle in theROK.Conclusions:ThisstudyshowsthatA.bovis infection iscloselyrelatedtograzingandtheseasonalactivityof ticks.FurtherstudiesshouldfocusonbloodsamplesobtainedfromvariousclimaticregionstoidentifythedistributionofA.bovis,aswellastheassociationbetweenthisdiseaseandpathogenicity,andtoclarifythevectorsandreservoiranimalsofA.bovis.P065-Plant-basedvaccinetopreventnecroticenteritisinchickensK.L.Roland1,A.M.Gonzales1,S.Wilde1,A.Diamos2,J.Hunter2,H.Mason2.1BiodesignInstitute,ArizonaStateUniversity,Tempe,AZ,USA,2SchoolofLifeSciences,ArizonaStateUniversity,Tempe,AZ,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMNecroticenteritis(NE)iscausedbytypeAstrainsofthebacteriumClostridiumperfringens.TotalglobaleconomiclossestothepoultryindustryduetoNEisestimatedtobeover2billiondollarsannually.Traditionally,NEhasbeeneffectivelycontrolledbyinclusionofantibiotics inthediet.However, recentconcernsregardingthe impactof thispracticeon increasingantibioticresistance inhumanpathogenshaveledustoconsideralternativeapproaches,suchasvaccination,forcontrollingthisdisease.NEstrainsofC.perfringensproducetwomajortoxins,α-toxinandNetB.Immuneresponsesagainstα-toxinarepartiallyprotective,althoughthetoxindoesnotplayadirect role inNE.TheNetB toxin is responsible for the symptomsassociatedwithNEandanti-NetBantibodiesarepartiallyprotective.Wehavedeveloped a fusionprotein combining a non-toxic carboxy-terminal domainofα-toxin (PlcC) and a non-toxicmutantformofNetB(NetB-W262A)foruseasavaccineantigentoimmunizepoultryagainstNE.WeutilizedaDNAsequencethatwascodon-optimizedforplantstoenablehighexpressioninatobaccorelative,Nicotianabenthamiana.The6-HistaggedPlcC-NetBfusionproteinwassynthesizedinN.benthamianausingageminiviralreplicontransientexpressionsystem,purifiedbymetalaffinity,andusedtoimmunizebroilerbirds.ImmunizedbirdsproducedastrongserumIgYresponseagainsttheplantproducedPlcC-NetBproteinandalsoagainstbacteriallyproducedHis-PlcCandHis-NetB. Immunizedbirdsweresignificantlyprotectedagainstasubsequent in-feedchallengewithvirulentC.perfringens.Theseresultsindicatethataplant-producedPlcC-NetBtoxoidisapromisingvaccinecandidateforcontrollingNEinpoultry.

CRWAD2017 ABSTRACTS


P066-MolecularandgenomicsbasedapproachestoassesspublichealthrisksassociatedwithbushmeatconsumptioninTanzaniaR.Katani1,B.Lyimo2,A.Martin2,E.Eblate2,F.Stomeo3,P.Gwakisa4,T.Buza1,I.Albert1,A.Estes1,F.Mosha5,B.Jayarao1,W.R.McVey1,I.Cattadori1,P.Hudson1,G.Shirima2,M.Schilling1,L.Li1,T.Tonui3,A.Joseph6,D.Rentsch7,E.Gill1,L.Samson2,L.Munuo2,J.Buza2,V.Kapur1.1PennState,UniversityPark,PA,USA,2NelsonMandelaAfricanInstituteofScienceandTechnology,Arusha,Tanzania,UnitedRepublicof,3BioscienceseasternandcentralAfrica-InternationalLivestockResearchInstitute(BecA-ILRI)Hub,Nairobi,Kenya,4SokoineUniversityofAgriculture,Morongoro,Tanzania,UnitedRepublicof,5MinistryofHealthandSocialWelfare,NationalHealthLaboratoryandQualityAssuranceandTrainingCentre,DaresSalaam,Tanzania,UnitedRepublicof,6SokoineUniversityofAgriculture,Morogoro,Tanzania,UnitedRepublicof,7LincolnParkZoo,Chicago,IL,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMBushmeat,themeatderivedfromwildlifespecies,isthecommonsourceofanimalproteinforhumanconsumptioninmanypartsofAfrica, including Tanzania. Given the documented evidence of the presence of dangerous zoonotic pathogens amongst wildlifeharvestedforbushmeatinTanzania,ourstudywasdesignedtoassessthebiologicalriskandpotentialforimpactonhumanhealthfromfreshandprocessedbushmeat.Acomprehensivestratifiedrandomsamplingapproachwasusedtomaptheprevalenceandthedistribution of anthrax, Brucella and Coxiella during rainy and dry seasons in villages and surroundingmarkets in three targetedecosystems (Serengeti,Ruaha-Rungwa,andSelous) inTanzania.Preliminary resultsof real timePCRanalysisofover1800samplescollectedfrommorethan150regionsacrossthethreeecosystemsidentifiedsignaturesofBacillusanthracis(1.2%),Brucella(0.80%),andCoxiella(0.57%)inbushmeatharvestedandsoldinthisregion.Thedataalsorevealahigherabundanceofanthraxinfreshversusprocessedsamples.MicrobiomesequencinganalysesoftheV3-V4regionofthe16SrRNAgenewasperformedonasubsetof30freshandprocessedbushmeatsamplesrecoveredfromtheSerengetiecosystem,andprovidefurtherevidenceforthepresenceofnucleicacidsignaturesofgenerarepresentingthesethreeselectpathogensaswellasotherdangerouspathogens.AdditionalstudieswereperformedtodeterminethespeciesoforiginofthebushmeatsampleswithPCR-basedamplificationandmolecularcharacterizationofthecytochromeBandcytochromeCoxidaseIgenessequences,andourpreliminaryresultssuggestthatthespeciesoforiginofafractionof~40%ofbushmeatsamplesismisrepresentedwhensoldinthemarkets.Takentogether,theresultsofourinvestigationsprovideevidenceof thepresenceofDNAsignaturesofespeciallydangerouszoonoticpathogens inbushmeatsoldorpreparedforconsumptioninTanzania.Inthelong-term,ourresearchwillprovidearationalbasisfordefiningandmitigatingthepublichealthrisksassociatedwiththeharvesting,trade,andconsumptionofbushmeat.P067-DetectionandcontrolofnodulardermatitisintheRepublicofArmenia,2015-2016N.Hakobyan,H.Mkrtchyan,A.Arzumanyan.StateFoodSafetyServiceoftheRAMoA,Yerevan,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMLumpyskindisease(LSD)isaviralinfectiousdiseaseofcattlecharacterizedbyfever,necroticskinnodes,enlargedlymphnodes,swellingofextremities,andanorexia.Itcausesadecreaseinmilkproductionandtemporaryorpermanentinfertilityinbulls.InDecember2015cattleinSyunikwerefirsttopresentsymptoms.PathologicalmaterialwassenttothereferencelabwhichconfirmedthediagnosisviaPCRinJanuary2016.Quarantinewasimposedwherethediseasewasdetected.Infectedanimalswerequicklyisolatedandvaccinatedandthesurroundingareadisinfected.Movementofanimalsfromtheinfectionfocuswasprohibitedwithallroadsclosedandround-the-clock quarantine checkpoints established. To determine the source of LSD and identify new disease cases an epidemiologicalresearchandanalysiswascarriedoutinthesurroundingareas.TopreventfurtherspreadofLSDVwithinArmeniaandtopreventitsre-emergenceinthecountry,vaccinationswereperformedinthespringof2016alongborderregionswithIran,Turkey,GeorgiaandAzerbaijan.A total of 137, 440 cattlewere vaccinated. Thenumber and locales are shownbelow: Syunik: 9570 vaccinated cattle;Aragatsotn: 1800 vaccinated cattle; Ararat: 24275 vaccinated cattle; Vayots Dzor: 1370 vaccinated cattle; Gegharkunik: 41330vaccinated cattle; Tavush: 1000 vaccinated cattle; Shirak: 28330 vaccinated cattle; Lori: 995 vaccinated cattle; Armavir: 28300vaccinatedcattle.Becauseofthesemeasures,thefocaldermatitisofcattlewaseliminated.SincethiswasthefirstreportofLSDintheRepublicofArmeniaanepidemiologicalinvestigationofcattleisnowbeingregularlycarriedout.Toavoidnewcasesofthedisease,itwasproposedandapprovedtovaccinatethecattleagainstfocaldermatitisaccordingtothe2017-2019ProgramonVaccinationofFarmAnimals.

CRWAD2017 ABSTRACTS


P068-InactivationofvirulentBrucellaspeciesincultureandanimalsamplesS.Olsen,P.Boggiatto,C.Vrentas.NationalAnimalDiseaseCenter,Ames,IA,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMPurpose:Severalpublicized failuresof inactivationprocedures relatedtobiological selectagentsandtoxins (BSAT)coveredby theSelectAgentprogramhaverecentlyoccurred.Althoughsomedataon inactivationofBrucella spp. isavailablewithinthehistoricalliterature,reportsareprimarilylimitedtoheatinactivationofmilkandtherearediscrepanciesbetweenstudies.Inaseriesofstudies,validationdataonsomecommoninactivationproceduresweregeneratedasrelatedtoB.abortus,B.suis,andB.melitensis.Methods:Inactivationmethods tested included:heat treatment,methanol andmethanol:acetone solutions, 0.22um filtration, and formalintreatment.Results:Bufferedneutralformalin(10%concentration)washighlyeffectiveininactivatingBrucellabacteriaby4hoursfromtissuesectionsthathadhighlevelsofcolonization.Solutionscontainingmethanolormethanol:acetoneat50to70%concentrationsinactivatedviableBrucellafromliquidsampleswithin3to5days.Afterpassagethrougha0.22umfilter,noBrucellawererecoveredfromspikedserumsamplesorserumsamplesfromexperimentallyinfectedanimals.CompleteeliminationofviableBrucellabacteriawithin30to60minutesrequiredtemperaturesapproachingboiling,whereaslowertemperaturesrequiredmuchlongerheatingtimes(hours).Conclusions:OurdatasuggeststhatcommoninactivationmethodscanbeeffectivelyusedtoeliminateviableBrucellabacteriawithinsamples.Becauseofdifferencesbetweenlaboratories, it ishighlyrecommendedthatothersvalidateanysimilarproceduresused under their own in vitro conditions. Culturemethods should still be used, as possible, on inactivated samples to verify theeffectivenessofthevalidatedprocedureandprotectagainstinadvertentlaboratoryerrors.P069-“Ondemand”vaccinedesign:applicationtoAfricanswinefevervirusM.Parrillo.InstituteforImmunologyandInformaticsattheUniversityofRhodeIsland,Providence,RI,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMPurpose:AfricanSwineFeverVirus(ASFV)isahighlycontagiousviraldiseaseofswine,withthepotentialforgreateconomiclossestoporkproducersintheUSasthereisnoeffectivevaccineavailable.TheiVAXsoftwaretoolkitwasrecentlyadaptedtoswinevaccinedesign.Inthisstudy,weanalyzedfiveASFVproteinstoidentifyputativeTcellepitopes.WedesignedavaccineforanAfricanstrainofASFV,Benin97/1andfortheGeorgia2007/1ASFVstrainMethods:ThePigMatrixandVaxCADwereappliedtoidentifyputativeTcellepitopes in five proteins from Georgia 2007/1 and Benin 97/1 ASFV. Clusters of T cell epitopes were found and analyzed usingJanusMatrixtoevaluateconservationacrosstwelveASFVstrainsandconservationwiththeswineproteome.TheclusterswererankedbasedonclassIIepitopecontent,conservationwith12ASFVstrainsandconservationwiththeswinegenome.Theselectedpeptideswereassembledin“stringsofbeads”whileeliminatingpotentialjunctionalimmunogenicityusingVaxCAD.Results:Inunder48hours,ASFVclass IIepitopeswere identifiedandvaccinesweredesigned.49potentially immunogenicclustersderived fromfiveproteinsfoundintheGeorgia2007/1strainandBenin97/1strainwereidentified;theseclusterscontainhitsforfourormoreclassIISLAalleles.ThesepeptidesboundtomultipleSLAalleles,werenotconservedwiththeswinegenomeandhighlyconservedwithotherASFVstrains.Two vaccine designswere generatedusingVaxCAD, one for class I andone for class II peptides.Conclusions:This computationalvaccinesondemandapproachtovaccinedevelopmentleveragesanexistingimmunoinformaticstoolkitforthebenefitofbiosecurityintheUnitedStatesandporkproducers.VaccineproductionandchallengestudieswillbeconductedincollaborationwithUniversityofScience,TechniquesandTechnologiesofBamako,MaliandevaluatedattheLaboratoireCentralVeterinaire.

CRWAD2017 ABSTRACTS


P070-UsingmodifiedradiofrequencyidentificationtagstoquantifycontactpatternswithinanOntarioequinefacility:avalidationstudy

R.Milwid1,T.L.O'Sullivan1,Z.Poljak1,M.Laskowski2,A.L.Greer1. 1PopulationMedicine,UniversityofGuelph,Guelph,ON,Canada,2MathematicsandStatistics,YorkUniversity,Toronto,ON,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMThisstudydocumentstheutilityof InternetofThings(IoT)devicestostudycontactpatternsthatoccurwithinagriculturalsettingsrelevanttocomplexphenomenasuchasthespreadofinfectiousdisease.Theuseofradiofrequencyidentification(RFID)tagsisonemethodofquantifying suchcontactsand typically comprises:RFID tagsandRFID readers.RFID readers requireanexternalpowersource,limitingtheutilityofthesysteminoutdooragriculturalsettings.Thisworkpilottestedamodificationtoanopen-source,open-hardwaresystem(OpenBeacon)eliminatingtheneedforon-siteRFIDreaders.ThismodificationenabledtheincreasedutilityoftheRFIDsystemsoitcouldbeusedinanoutdooragriculturalsetting.TheOpenBeaconRFIDtagfirmwarewasmodifiedtomakeuseofthe8MBof onboard storage to store collected contact data on the tags, thusmaking the readers redundant. Themethodology andvalidationofcollectingnetworkdatawithinanequinefacilityusingthesemodifiedRFIDtags ispresented.Thestudytookplace inNovember2016overa7-dayperiodonanequinefacilityinSouthwesternOntario.ThemodifiedRFIDtagswereplacedonparticipatinghorses’(N=9)halters,aroundthefacility(i.e.onthepasturefence, inthearena)(N=17),andwerewornbystaffduringshifthours(N=6).Whentwotagscamewithinapre-defineddistanceofeachother(2meters),thetagscommunicatedinapeer-to-peerfashion.Thetagsrecordedthespecifictagidentificationnumbersoftheindividualswhocamewithin2metersofeachother,aswellasthetimeofthecontactevent.Horseswererecordedashavingmostcontactwithotherhorsesintheirpastureorbarnaisle,whichcorrespondedto the turnout and training schedules. Furthermore, themost contact occurred between horses, followed by horses and specificlocationsatthefacility(i.e.crossties,pastures).Althoughthefacilitycontained6pastures,onlythreewereusedduringthestudy,withan extra pasture being used on theweekend. This changewas reflected the network graphs, confirming the validity of the data.Approximately94%ofthebatterieslostchargeonthe6thdayofthestudy,withonly~12%losingchargebeforethe6thday.P071-Phylogeneticanalysesofthewild-typestrainsofcaninedistemperviruscirculatingintheUnitedStatesE.A.Anis,R.Wilkes.UniversityofGeorgia,Tifton,GA,[email protected]:CompanionAnimalEpidemiology,12/4/20175:45PMCaninedistemperisahighlycontagious,systemic,viraldiseaseofdogsseenworldwide.Despiteintensivevaccinationindevelopedcountries,recentreportssuggestboththere-emergenceandincreasedactivityofCaninedistempervirus(CDV)worldwide,includingtheUnitedStates.CDVisanRNAvirusofthegenusMorbilliviruswithinthefamilyParamyxoviridae.TheviralgenomicRNAencodessixstructuralproteins.Ofthesixstructuralproteins,ofwhich,theHemagglutinin(H)genehasthegreatestgeneticvariationandisthereforeasuitabletargetformolecularepidemiologicalstudies.ThemajorityofneutralizingepitopesarealsofoundontheHprotein,makingthisgenealsoimportantforevaluationofchangesovertimethatmayresultinantigenicdifferencesamongstrains.TheaimofthisstudywastodeterminethephylogeneticrelationshipofCDVstrainscirculatingintheU.S.Forty-twopositivecaninedistemperviruscasescollectedfromdogsfromdifferentregionsandstatesfrom2014to2017weretested.SequenceanalysisoftheHgenerevealedthatthereareatleast3differentcladesofCDVcurrentlycirculatingintheUS.ThesecladesincludeAmerica-3(Edomex),America-4andacladethatwaspreviouslyreportedinassociationwithanoutbreakinWyoming,whichwaslinkedtoadomesticdog-breedingfacilityinKansasin2010.ThesecladesdifferfromthehistoricallyidentifiedcladesintheUS,includingAmerica-1,whichcontainsthevaccinestrains.Geneticdifferencescanresultinsignificantchangestotheneutralizingepitopesthatconsequentlymayleadtovaccinefailure.Therefore,furtherstudyisrequiredtodeterminewhetherthesegeneticvariationsrepresentsignificantdifferencesinantigenicity,particularlybetweenvaccine-strainsandthesewild-typestrainscirculatingintheU.S.

CRWAD2017 ABSTRACTS


P072-DiagnosticefficiencyofsomediagnosticproceduresofbovinebrucellosisH.Abdalaal1,A.Mahrous2,L.Bakkar1. 1VeterinaryMedicine,BeniSuefuniversity,BeniSuef,Egypt, 2Zoonoses,GOVS,Egypt,Cairo,[email protected]:CompanionAnimalEpidemiology,12/4/20175:45PMThepresentstudywascarriedoutforinvestigationofthesensitivityandspecificityofsomediagnosticproceduresusedfordiagnosisof bovine brucellosis on serological, bacteriological andmolecular basis. A total of 141 cows from brucella infected farms underquarantineoftheveterinaryauthoritieswereemployed.Relativesensitivity,relativespecificity,positivepredictivevalueandnegativepredictivevalueofBPAT,RBTandCFTwereestimatedas,(98.04%,76.92%,91.74%and93.75%);(94.33%,85.71%,95.24%and83.33%)and (93.46%,88.23%,96.15%and88. 24%) respectively.Different tissues specimensof 104 confirmed seropositive cowsunderinvestigationincluding,retropharyngeal,prescapular,prefemoral,internaliliac,supramammarylymphnodes,udderandspleenaswellasmilkof46lactatingcowsweresubjectedforbacteriologicalstudiesforisolationandidentificationofBrucellaorganisms.Brucellamelitensisbiovar3couldberecoveredfrom64(45.39%)tissuespecimensand28(60.87%)milksamples.BrucellacultureswerefurtheridentifiedonmolecularbasisusinguniversalPCR throughamplificationof targetgene (Immunodominantantigen,genebp26)andBruceladderemployingfiveprimerspairswithamplificationofthreefragmentsof587bp,1071bpand1682bpsizes.P073-AerotoleranceofCampylobacterjejuniandCampylobactercoliisolatedfromvariousretailmeatandliverproductsA.B. Karki, C.K. Oakey, K. Mar, M.K. Fakhr. Dept of Biological Science, The University of Tulsa, Tulsa, OK, [email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMCampylobacterisagramnegative,microaerophilicbacteriaandacommonfoodbornepathogencausingapproximately1.3millioncasesofCampylobacteriosis in theUSeachyear.AhighprevalenceofCampylobacter specieshasbeenreported in retailmeatand liverproducts.Despitebeingmicroaerophilic,aerotolerancecapabilityofsomeCampylobacterisolateswasrecentlyreportedwhichmighthavearoleinincreasingthetransmissionandsurvivalofthosestrainsinfoodsduringstressfulprocessingandstorageconditions.Inthisstudy,screeningforaerotolerancewasperformedfor176Campylobacterisolates(75C.jejuniand91C.coli)whichwerepreviouslyisolatedfromchickenmeat,chickenlivers,chickengizzards,turkey,porkandbeefliver.BacterialcultureswereincubatedaerobicallyinMuellerHintonBrothwithshakingat200rpm,andviablecellcountwasdeterminedat0,6,12and24hours.Approximately41%ofthe totalCampylobacter isolateswere found tobeaerotolerant (viable after 12hrsunder aerobic incubation)whereas22%werehyperaerotolerant(viableafter24hrsunderaerobicincubation).Ahigherprevalenceofaerotolerantstrains(70%)wasfoundamongC.coliisolateswhencomparedtotheC.jejuniones(6%).AllC.colistrainsfromchickengizzardsandpork,and79%ofchickenliverisolateswereaerotolerant,whereas47%ofchickenliverisolateswerehyperaerotolerant.Interestingly,comparativegenomicsoffewWholeGenomesequencedisolatesalongwithPCRshowedthepresenceofagenecodingforacatalaselikeproteinin75%(68/91)ofallscreenedC.colistrains,butwascompletelyabsentinalltestedC.jejunistrains.Thecatalaselikegenewasfoundin83%(29/35)ofthehyperaerotolerantC.colistrains.AlmostallC.colistrainsfromchickengizzards(3/3),chickenlivers(54/57)andchickenmeat(8/9)harboredthecatalaselikegene.Inconclusion,aerotolerantCampylobacterstrainsareprevalentamongthoseisolatedfromvariousretailmeats.CampylobactercoliappeartobemoreaerotolerantthanCampylobacterjejuni.ApotentialroleforacatalaselikeproteinfoundinmostofC.coliisolatesinaerotolerancyneedsfurtherinvestigation.

CRWAD2017 ABSTRACTS


P074-Prevalenceofextended-spectrumß-lactamasesproducingGramnegativebacteriainbackyardpoultryflockenvironmentinWashingtonState

D.H.Shah,N.C.Paul,A.M.Clarridge,M.Cook-Webb,R.Crespo.DepartmentofVeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMBackground:TheobjectiveofthisstudywastodetermineprevalenceofESBLproducingGramnegativebacteriainthebackyardpoultryflockenvironmentintheWashingtonState.Methods:Dragswabsampleswerecollectedfromnests,waterer-feeder,coopfloorandarandomsitewithincoopsof34smallscalebackyardpoultryflocksfromNorthWestWA.SampleswereprocessedforisolationofGramnegativebacteriabyplatingonMacConkey’sagarcontainingceftiofur(8µg/ml).Arepresentativecolony(lactosefermentingand/ornon-lactosefermenting)wasselectedfromeachpositivesampleforfurtheridentificationusingMALDI-TOF.Antimicrobialsusceptibilityagainst17antibioticswastestedforallrecoveredisolates.Results:Atotalof125ceftiofurresistantgramnegativebacteriabelongingtoninegenerawereisolatedfrom34flocks.Majorityofisolateswereresistantto3ormoreantibioticclasses(MDR)includingE.coli(44/49,90%),Acinetobacterspp.(34/42,81%),Pseudomonasspp.(21/22,95%),Achromobacterspp.(5/8,62%),Bordetellaspp.(1/1,100%),Enterobacterspp.(1/1,100%),Hafniaalvei(1/1,100%),Ochrobactrumintermedium(1/1,100%),Stenotrophomonasmaltophilia(1/1,100%).SeveralisolateswereESBLpositiveincludingE.coli(15),Acinetobactercalcoaceticus(1)andbaumannii(1),Achromobacterspanius(3)andpiechaudii(1)andStenotrophomonasmaltophilia(1).Inaddition,atotalof34isolatesincludingAcinetobacterspp.(20),Pseudomonasspp.(11),E.coli(2)andS.maltophilia(1)werealsoresistanttocarbapenemantibioticsertapenemand/orimipenem.Conclusions:SeveralGramnegativebacteriaidentifiedinthisstudyhavebeenreportedascontaminantsofpoultrymeatandareoftenassociatedwith spoilageof refrigerated rawpoultrymeat.Fewbacterialgenerahavebeen reported tobeassociatedwithhumaninfection.ThisisthefirststudytodemonstratetheprevalenceofESBLGramnegativebacteriainthebackyardpoultryflockenvironmentintheUSandwillprovideimportantbaselineinformationforfurtherinvestigations.P075-MetagenomicanalysisontheeffectsofchlortetracyclineandceftiofurongrowerswineintestinalmicrobiotaJ.Y.Morales,F.Lopez,J.Vinasco-Torres,H.M.Scott,K.N.Norman.VeterinaryInegrativeBiosciences,TexasA&MUniversity,CollegeStation,TX,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMInthefoodanimalindustry,theadministrationofantibioticstoswineisimportantfortheprevention,control,andtreatmentofdisease.Previousstudiessuggestthattheuseofantibioticsinthefoodanimalindustryhascontributedtotheemergenceofantibioticresistantbacteria.Inaddition,antibioticsmayinduceselectivepressuresonthegutmicrobiotaofswine,potentiallyaffectingtheiroverallhealth.Theobjectiveofthisstudywastoobservealterationsintheintestinalmicrobiomeofgrowerswine,challengedwithmulti-drugresistant(MDR)andpan-susceptibleSalmonellaspp.,inresponsetotreatmentwithchlortetracyclineand/orceftiofur.CommunityDNAfromswine fecal samples was extracted using the QIAamp DNA stool mini kit and QIAcube automated platform. 16S MetagenomicsSequencingLibrarieswerepreparedusingtherecommendedIlluminaprotocol.SequencingwasperformedontheIlluminaMiSeq,andoutput datawas analyzed using the 16sMetagenomic application on Illumina Basespace, Explicet software, and Stata. Themostprevalent microbiota found from bacterial libraries were from the families Prevotellaceae, Ruminococcaceae, Veillonellaceae,Lachnospiraceae,andClostridiaceae,respectively.Significantdifferencesinmicrobialcommunitieswerefoundbetweendayfouranddayeighteenlibraries,aswellaslibrariesbetweenthecontrolandcombinedchlortetracycline-ceftiofurtreatmentgroup.Knowledgeontheimpactofhowantibioticsaffectthebacterialpopulationsinswinecontributestotheunderstandingofthepotentialimpactstoswinehealth.Furthermore,comprehendingthelengthoftimeatwhichantibioticsaffectgutmicrobiotacanaidindecision-makingaboutantibiotictreatmentoptions.

CRWAD2017 ABSTRACTS


P076-GenotypicallydistinctlineagesofSalmonellaTyphimuriumST313andST19arecirculatinginBrazilP.N.Panzenhagen1,N.C.Paul1,C.A.Conte,Junior2,R.G.Costa3,D.P.Rodrigues4,D.H.Shah1.1DepartmentofVeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA,USA,2FoodScienceProgram,UniversityFederalofRiodeJaneiro,RiodeJaneiro,Brazil,3NationalReferenceLaboratoryforDiagnosisofEntericBacteria,OswaldoCruzInstitute,RiodeJaneiro,Brazil,4NationalInstituteofQualityControlinHealth,OswaldoCruzFoundation,RiodeJaneiro,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMTwo genetically distinct lineages ofmulti-drug resistant non-typhoidal Salmonella (NTS) serovar Typhimurium sequence type 313(ST313)areknowntocauseinvasivediseaseamongpeopleinsub-SaharanAfrica.S.TyphimuriumST313hasevolvedtobecomemorehumanadaptedandiscommonlyisolatedfromsystemicsites(eg.,blood)fromfebrilepatientsinsub-SaharanAfrica.Interestingly,S.TyphimuriumisfrequentlyisolatedfromsystemicsitesfromhumanpatientsinBrazil,however,itiscurrentlyunknownifthispathogenhas also evolved to becomemore invasive and human adapted in this country. Here, we determined genotypic and phenotypicdivergenceamongclinicalS.Typhimuriumstrains isolatedfromsystemicandnon-systemicsites fromhumanpatients inBrazilandcomparedthesewiththereferenceS.TyphimuriumST19lineagethatisassociatedwithhumangastroenteritisworldwide.Wereportthat a sub-population (8/38, 20%)of epidemiologicallydiversehuman clinical strainsof S. Typhimurium isolated fromBrazil showsignificantlyhigher intra-macrophage survival, indicating that theseare likelymore invasive.Usingwhole genome sequencingandphylogenetic approaches, we report the discovery of a unique S. Typhimurium ST313-lineage in Brazil that is genetically andphenotypicallydistinctfromtheAfricanST313-lineages.WealsoreportthediscoveryofauniqueS.TyphimuriumST19-lineageinBrazilthatisevolvingsimilartoST313lineagesfromAfricahoweverisgeneticallyandphenotypicallydistinctfromST19-lineagecommonlyassociatedwiththegastrointestinaldiseaseworldwide.ThediscoveryofnewS.TyphimuriumST313andST19lineagesresponsibleforhumanillnessesinBrazilwarrantsfurtherepidemiologicalinvestigationstomonitorthespreadofthisgeneticallydivergentpopulationofanimportanthumanpathogen.P077-MultiplexmolecularserotypingofSalmonellausingLuminexxMAPassayandCheck&TraceSalmonellakitcomparingwith

serologicalmethodX.Liu1,Y.Wang1,X.Shi2,R.Ransburgh1,H.Zhang1,L.Noll1,E.Poulsen1,V.Pasupuleti3,A.Karczmarek4,T.Nagaraja2,D.Renter2,Y.Fang2,G. Anderson1, J. Bai1. 1KSVDL, Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA,2DepartmentofDiagnosticMedicine/Pathobiology, Kansas StateUniversity,Manhattan, KS,USA, 3InnovativeMolecularDiagnosticSystemsInc,Geneva,IL,USA,4Check-PointsBV,Binnenhaven,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMSalmonellaaremajorfoodbornepathogens,andSalmonellaserotypingisanessentialandintegralpartofSalmonellasurveillanceandoutbreakinvestigations.Inthisstudy,atotalof188Salmonellaisolates(strains)wereblind-serotypedusingthebead(microsphere)-basedxMAPSalmonellaserotypingassay(Luminex,Austin,TX)andthemicroarray-basedCheck&TraceSalmonellaKit(Check-Points,Wageningen,TheNetherlands),andcomparedwiththeresultsobtainedfromtheclassicalagglutinationtest.Ingeneral,thetraditionalSalmonella serotyping is time-consuming, labor-intensive,and itsun-typablerate is relativelyhigh.BothLuminexandCheck-Pointsassaysarenucleicacid-based,multiplexed,andhighthroughputdetectionmethods.Atotalof25serotypeswereidentifiedfromthe188Salmonellastrains(rangingfrom1~30 isolatesperserotype).Check-Pointsdetected184 isolatesthatmatchedwithtraditionaland/orLuminexresults,andtheadditional4strainsresulted inauniquemicroarraypatternthat istranslatedbythesoftware into“Genova14959”.ThisuniquepatternispotentiallyanewAnatumvariantforCheck-Pointsdatabase.Assumingtheserotypingresultiscorrectifitisdetectedandconfirmedbyatleast2ofthe3methods,thentheCheck-Pointsassay’scorrectrateis97.8%or100%afterthedatabaseisupdated.Luminexdetected180samples(95.7%)thatmatchedtothetraditionaland/orCheck-Pointsresults,andtheother8strainsresultedininconclusivedualserotypes.Thetraditionalserologicalmethoddetected169samples(89.9%)matchedwiththeLuminexand/orCheck-Pointsresults,andtheserotypesof15strains(mismatchrate8.0%)didnotmatcheitherLuminexorCheck-Points results. The other 4 strains (2.1%) were not viable for agglutination testing. In conclusion, our results demonstrated thatmolecularserotypingmethodswithCheck-PointsorLuminexassaysareaccurateandrapidalternativestothetraditionalantigen-basedmethodforSalmonellaserotyping.

CRWAD2017 ABSTRACTS


P078-AntimicrobialresistancetrendsinnorthernCaliforniadairycattleSalmonellaisolates,2002-2016K.E. Davidson1, R.V. Pereira2, B.A. Byrne3. 1University of California, Davis, Davis, CA, USA, 2Population Health and Reproduction,UniversityofCalifornia,Davis,Davis,CA,USA,3Pathology,Microbiology,andImmunology,UniversityofCalifornia,Davis,Davis,CA,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMPurpose:NontyphoidalSalmonellainfectionscontributetoapproximately1.2millionannualillnessesintheUnitedStates.Historicalandrecentoutbreakshavebeenassociatedwithdairyproducts,groundbeef,anddirectcontactwithcattle.Salmonellaantimicrobialresistance(AMR)isaseriousconcernthatcanreducesuccessfultreatmentofinfections,increasingrecoverytime,medicalcosts,andmortalityratesinhumansandanimals.ThishighlightstheneedtotrackAMRinSalmonellaisolatedfromcattletoimprovetreatmentplans,managetrendsinAMR,andpreventfutureAMRdevelopment.Methods:Atotalof242Salmonellaisolateswereretrievedfromatotalof9,162cattlefecalsamplessubmittedtotheUniversityofCalifornia,DavisVeterinaryMedicalTeachingHospitalfrom2002to2016.Theseisolatesweretestedforantimicrobialsusceptibilityusingastandardizedbrothdilutionpanel.Results:Multidrugresistance(MDR)tothreeormoreclassesofantimicrobialswasobservedin50.8%ofisolates,andthemostcommonMDRpatternwasamoxicillin-ampicillin-cefoxitin-ceftiofur-ceftriaxone-chloramphenicol-streptomycin-sulfisoxazole-tetracycline (23.2%). There were significantlygreateroddsfornonsusceptibilitytoaminoglycosides(OR:2.03,95%CI:1.1-3.7),beta-lactam/beta-lactamaseinhibitorcombinations(OR: 1.79,95% CI: 1.0 - 3.4), folate pathway inhibitors (OR: 2.45,95% CI: 1.3 - 4.5), penicillins (OR: 1.87,95% CI: 1.0 - 3.5), andtetracyclines(OR:1.87,95%CI:1.0-3.4) forthe2002-2009periodwhencomparedtothe2010-2016period.Conclusions:DespitereducedoddsofAMRtothesedrugclassesintherecentyearperiod,lackofasignificantreductioninAMRforimportantdrugclassessuchascephalosporinsandquinoloneshighlighttherelevanceofAMRsurveillanceincattlewithSalmonellainfectionswiththeaimoftargetingfutureinterventions.P079-Frequencyandgenusdistributionofchromosomally-mediatedcarbapenem-resistantEnterobacteriaceaeinsurfacewaterE.Sechrist1,D.Mollenkopf1,S.Lee2,J.Lee2,T.Wittum1.1VeterinaryPreventativeMedicine,TheOhioStateUniversity,Columbus,OH,USA,2EnvironmentalHealthScience,TheOhioStateUniversity,Columbus,OH,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMMultiplegenuswithintheEnterobacteriaceaefamilyareintrinsicallyresistanttocarbapenemantimicrobialsthroughchromosomallyencodedcarbapenemasegenes.Thesecarbapenem-resistantEnterobacteriaceae(CRE)lacktheepidemiologicsignificanceofCREwithplasmid-mediatedcarbapenemasegenes,buttheycanbehighlyrelevantclinicallyforindividualpatients.Whenpatientsaretreatedwith carbapenems, the resulting selection pressure strongly favors resistant bacteria.We believe that these resistant pathogenseventuallyspreadintothecommunityduetoapatient’sfecalshedding,eventuallyenteringwastewater,wheretheyaremaintainedandreleasedintothedownstreamsurfacewater.CREinpublicwaterwaysposebothadirectand,moreproblematically,anindirectthreattopublichealth.Overthecourseof12months,wastewaterwascollectedattheWWTPservicingalargemetropolitantertiarycare hospital in order to assess the degree of hospital-associated CRE environmental contamination. Influent and effluent watersamplesfromtheWWTPwerecollected,aswellaswatersamplesupstream,downstream,andfurtherdownstreamoftheWWTP.Collectionsiteswereanalyzedtodeterminethefrequencyandgenusdistributionofchromosomally-mediatedCRE.Wefoundthatthefardownstreamsiteproducedthehighestnumberofchromosomally-mediatedCREcomparedtoallothersamplingsites.Weidentifiedatotalof129CREwithchromosomalcarbapenemasegenes,withtheAeromonasgenusthemostfrequent.ThisoutcomesuggeststhathospitalsmaybecontributingtothespreadofclinicallyrelevantCREwithchromosomalcarbapenemasegenesintotheenvironmentinsurfacewater.

CRWAD2017 ABSTRACTS


P080-ImpactsoffeedingpreweanedcalvesmilkcontainingdrugresiduesonthefunctionalprofileofthefecalmicrobiotaR.Pereira1,L.Carroll2,S.Lima,148503,C.Foditsh3, J.Siler3,R.Bicalho3,L.Warnick3. 1UniversityofCaliforniaDavis,Davis,CA,USA,2CornellUniversity,Ithaca,NY,USA,3CornellUniversityCollegeofVeterinaryMedicine,Ithaca,NY,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMFeedingdrugresidue-containingmilk(wastemilk)tocalvesiscommonworldwide,howevernoinformationisavailableontheimpactoffeedingdrugresidue-containingmilk(wastemilk)onthefunctionalprofileofthefecalmicrobiota.Theobjectiveofthisstudywastocharacterize the functional profile of the fecalmicrobiota of preweaneddairy calves fed rawmilkwith residual concentrations ofantimicrobialscommonlyfoundinwastemilkfrombirthtoweaning.Atbirth,thirtycalveswererandomlyassignedtoacontrolledfeedingtrialwhere:14calveswerefedmilkwithnodrugresidues(NR),and15calveswerefedmilkwithdrugresidues(DR)byaddingceftiofur,penicillin,ampicillin,andoxytetracycline.Fecalsamplescollectedfromeachcalfonceaweekstartingatbirth,priortothefirstfeedinginthetrial(pre-treatment),until6weeksofage.ShotgunsequencingofthemicrobiotawasconductedusingtheIlluminaplatform.Milkdrugresiduesresultedinasignificantdifferenceinrelativeabundanceofmicrobialcellfunctions,especiallywithgeneslinkedwithstressresponse,regulationandcellsignaling,andnitrogenmetabolism.Thesechangescoulddirectlyimpactselectionanddisseminationofvirulenceandantimicrobialresistanceinthemicrobiota.DrugresiduesalsoimpactedselectionanddistributionofgeneticfunctionsinResistancetoAntibioticsandToxicCompounds(RATC),resultinginatransitiontoamicrobialRATCfunctionmoresimilaroverdifferentsamplingtimepointsinDRwhencomparedtoNRcalves.OurdataalsoidentifiedastrongassociationbetweenageinweeksandabundanceofRATC, independentoftreatmentgroup.Regardlessoftreatmentgroup,theageofcalves inweeksrevealedasignificantshift inabundanceofRATCfunction, includingincreases infunctionsrelatedtoresistancetovancomycinandfluoroquinolones as calves became older. Findings from this study support the hypothesis that drug residues, even at very lowconcentrations,impactthegutmicrobiotaofcalvesandresultinchangesinthefunctionalprofileofmicrobialpopulations.P081-FecalmicrobiomeofperiparturientdairycattleandassociationswiththeonsetofSalmonellasheddingL.M.Muñoz-Vargas1,S.O.Opiyo2,R.Digianantonio1,M.Williams3,A.Wijeratne2,G.Habing1.1Dpt.ofVeterinaryPreventiveMedicine,The Ohio State University, Columbus, OH, USA, 2Ohio Agricultural Research and Development Center, The Ohio State University,Wooster,OH,USA,3BiologyandMarineScience,JacksonvilleUniversity,Jacksonville,FL,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMSalmonellaentericaisazoonoticpathogenwithcriticalimportanceinanimalandpublichealth.ThepersistenceofSalmonellaonfarmsaffectsanimalproductivityandhealth,andrepresentsariskforfoodsafety.Theintestinalmicrobiotaplaysafundamentalroleinthecolonizationandinvasionofthisubiquitousmicroorganism.Toovercomethecolonizationresistanceimpartedbythegutmicrobiome,Salmonellausesinvasionstrategiesandthehostinflammatoryresponsetosurvive,proliferate,andestablishinfectionswithdiverseclinicalmanifestations.CattleserveasreservoirsofSalmonella,andperiparturientcowshavehighprevalenceofSalmonellashedding;however, little is known about the association between the gut microbiome and the onset of Salmonella shedding during theperiparturientperiod.Thus,theobjectiveofthisstudywastoassesstheassociationbetweenchangesinbacterialcommunitiesandtheonsetofSalmonellasheddingincattleapproachingparturition.Inaprospectivecohortstudy,fecalsamplesfrom98dairycowsoriginatingfromfourdifferentfarmswerecollectedatfourtimepointsrelativetocalving.All392sampleswereculturedforSalmonella.SequencingoftheV4regionofthe16SrRNAgeneusingtheIlluminaplatformwascompletedtoevaluatethefecalmicrobiomeinaselectedsamplesubset.Analysesofmicrobialcomposition,diversity,andstructurewereperformedaccordingtotimepoints,farm,and Salmonella onset status. Individual cow fecal microbiomes, predominated by Bacteroidetes, Firmicutes, Spirochaetes, andProteobacteria phyla, significantly changed before and after parturition. Microbial communities from different farms weredistinguishablebasedonmultivariateanalysis.AlthoughthereweresignificantdifferencesinsomebacterialtaxabetweenSalmonellapositiveandnegativesamples,ourresultsdidnotidentifydifferencesinthefecalmicrobialdiversityorstructureforcowswithandwithout the onset of Salmonella shedding. These data suggest that determinants other than the significant changes in the fecalmicrobiomeinfluencetheperiparturientonsetofSalmonellasheddingindairycattle.

CRWAD2017 ABSTRACTS


P082 - Modulation of bovine mammary gland epithelial cell permeability and actin distribution by extracellular adenosinetriphosphate

D.Huisman,M.Shatek,H.Salzbrenner,D.McClenahan.DepartmentofBiology,UniversityofNorthern Iowa,CedarFalls, IA,[email protected]:Immunology,12/4/20175:30PMEpithelial cells lining the secretoryunits and theductsof thebovinemammaryglandperforman important role in regulating themovement of various macromolecules and whole cells during normal lactation and mastitis. Many host and bacterial producedsubstancescangreatlyaffectthebarrierfunctionofthisepithelialmonolayerduringmastitis.Onepotentialplayerinthisprocess,thatcanbe releasedbyboth thehostand throughbacterialdegradation isadenosine triphosphate (ATP).ATP likely interactswith thepurinergicreceptorP2X7inmediatingsomeoftheeffectsassociatedwithmastitis.Abovinemammaryglandepithelialcellline,Mac-Tcells,weretestedwithvariousconcentrationsofATPandinhibitorsofP2X7todeterminetheireffectsonepithelialcellmonolayerpermeability bymeasuring the transwell epithelial electrical resistance (TEER) of themonolayer. In addition, the ability of ATP toactivate thepore forming functionof P2X7was testedbymeasuring the change in intracellular Ca+2 andmovementof the largemoleculeYo-Prointothecell.Finally,changesinthecytoskeletonofthecellsasaresultofP2X7interactionswasmeasuredbystainingthe actin cytoskeleton with phalloidin and examination with a flourescent microscope. ATP did increase Mac-T cell monolayerpermeabilityalmost immediatelyafter itsaddition,andthiswasreversiblebytheuseofP2X7inhibitors.WhileCa+2levelsdidnotincrease incellsexposedtoATP,Yo-Pro levelsdidsignificantly increase in theMac-Tcells treatedwithATP.Finally,anobservableincreaseinactinfibrilsizewasnotedinATPtreatedcellswhichwasnotseenincellsinitiallytreatedwithP2X7inhibitorspriortoATPexposure.TheseresultswouldindicatethatATP,throughitsinteractionswiththeP2X7receptor,modulateepithelialcellfunctioninthebovinemammarygland,especiallyinregardstothebarrierfunctionthatepithelialcellsnormallyprovide.P083 - Identification of C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells by newly

generatedmonoclonalantibodiesC.L.Schnabel, S.Babasyan,H.Freer,M.Wemette,B.Wagner.CollegeofVeterinaryMedicine,CornellUniversity, Ithaca,NY,[email protected]:Immunology,12/4/20175:30PMChemokinesaresolublemoleculeskeytotheinnateimmuneresponse.Theydirectimmunecelltrafficking,homingandresponsestoinfectionandinflammation.Inhorses,theanalysisofchemokineshasbeenhamperedbythelackofspecificantibodies.Wegeneratedmonoclonalantibodies(mAbs)forequineC-Cmotifchemokineligands(CCL)CCL2(MCP-1),CCL3(MIP-1α),CCL5(RANTES)andCCL11(eotaxin)usinghybridomatechnology.AntibodyspecificitywasconfirmedbystainingofChineseHamsterOvarycellstransfectedwithexpressionvectorsforCCL2,CCL3,CCL5,orCCL11.TransfectantswerestainedwiththedifferentmAbsandevaluatedbyflowcytometry.Inaddition,equineperipheralbloodmononuclearcells(PBMC)wereculturedwithorwithoutstimulationwithlipopolysaccharide(LPS)orphorbol12-myristate13-acetate(PMA)andionomycin.PBMCwerethenstainedintracellularlywithindividualCCLmAbstoidentifythecellularsourcesofeachchemokine.CCL2andCCL3wereproducedbyCD14+monocyteswithoutstimulation.CCL3wasadditionallyproducedinlymphocytes,mainlyCD4+T-cellsafterstimulationwithPMA/ionomycin.CCL5wasproducedtosimilarextendswithandwithoutstimulationinCD4+andCD8+T-cells.CCL11wasproducedbyCD4+T-cellsafterstimulationwithPMA/ionomycin.ChemokineproductionafterLPSstimulationwascomparabletoPBMCincubationinmediumalone.Pairsofanti-equineCCLmAbsweretestedtogenerateenzyme-linkedimmonosorbentassays(ELISAs)foreachchemokine.CCLmAbpairswereidentifiedforallfourchemokinesandusedtoquantifythem.SupernatantsofPMA/ionomycinstimulatedPBMCcontaineddetectableCCL2,CCL3andCCL5,whileCCL11secretion couldbe stimulated fromequine tracheal epithelial cells in response to IL-4. Thenewly generatedmAbs for equineCCLchemokinesenablethequantitativeanalysisofintracellularchemokineproductionbyflowcytometryandsolublechemokinesbyELISA.Theyarevaluabletoolstoimprovetheevaluationofinnateimmuneresponsesinhorses.

CRWAD2017 ABSTRACTS


P084-ImproveddeliveryofaHAandM2ebasedpeptidevaccineagainstswineinfluenzaviruses.G.Singh1,O.Zholobko2,A.Pillatzki3,B.Webb4,E.Nelson3,A.Voronov2,S.Ramamoorthy1.1MicrobiologicalSciences,NorthDakotaStateUniversity,Fargo,ND,USA,2CoatingsandPolymericMaterials,NorthDakotaStateUniversity,Fargo,ND,USA,3AnimalDiseaseResearchandDiagnosticLaboratory,SouthDakotaStateUniversity,Brookings,SD,USA,4VeterinaryDiagnosticLaboratory,NorthDakotaStateUniversity,Fargo,ND,[email protected]:Immunology,12/4/20175:30PMSwineinfluenzaviruses(SIVs),areagroupofgeneticallydiverseandeconomicallyimportantpathogensofswineandhumans.Despitedecadesofresearch,thedevelopmentofvaccinesthatcaninducerobustandbroadprotectionagainstthemanystrainsofinfluenzavirusesremainsachallenge.Recentbreakthroughsonepitope-basedimmunizationforinfluenzavirusesidentifyconservedregionsoftheHA2andM2eproteinsascapableofinducingbroadprotectionagainstmultipleinfluenzastrains.TheM2epeptidehasnotbeenevaluatedasanSIVvaccinecandidate inswine.Whileconferringtheadvantageofbroadprotection,peptidesare inherentlyweakimmunogens.Toenhancethedeliveryandimmunogenicityofpeptidebasedvaccines,threeSIVepitopes(conservedM2eandHA2,strain-specificHA1)wereexpressedasachainofepitopesinabacterialexpressionsystem,andentrappedinanovelbiodegradablepolymer,PEG600PTHF650.Pigletsvaccinatedwithpolymericpeptidevaccinemountedsignificantlystrongerantibodyagainstthetrimericpeptideconstruct,whencomparedtopigletsvaccinatedwiththepeptideconstructalone.Viralsheddinginnasalsecretionswashigherinpigsvaccinatedwiththepolymericpeptidevaccineorpeptidealoneat3dayspost-challengewiththevirulentA/CA/2009/H1N1virus.However,thetrendreversedbyday6post-challenge,whenasharpdropinviralsheddingwasevidentinthevaccinatedpigs,butnot the controls, indicating a delayed but effective clearing of the challenge virus in vaccinated pigs. Hence the combination ofPEG600PTHF650 polymer and trimeric peptide construct appeared to enhance delivery of the peptides and act as an adjuvant instimulatingantibodyresponses,whileinducingprotectiveimmunityinvaccinatedpigs.P085-Exploringtheeffectsofpegbovigrastim(Imrestor)therapyonexperimentalmastitisinHolsteinsduringlactationE.J.Powell, J.D. Lippolis, T.A.Reinhardt, E.Casas.RuminantDiseasesand ImmunologyResearch,USDAARSNADC,Ames, IA,[email protected]:Immunology,12/4/20175:30PMPurpose:Mastitiscontinuestoleadhealthandeconomicconcernsforthedairyindustry.Betterunderstandingofdairycattle’sresponsetopathogensisimportantfordevelopmentofnext-genantibioticalternatives.Neutrophilsarethefirst-acting,mostprominent,cellulardefense against mastitis causing pathogens. This makes neutrophil activation and expansion obvious candidates for targetedtherapeutics.Onesuchtreatmentincludespegylatedgranulocytecolony-stimulatingfactor(gCSF)knownaspegbovigrastim(Imrestor,ElancoAnimalHealth).ThegCSFcytokinetargetsneutrophilregulation,andhasbeenwellassociatedwithneutrophilia.Pegbovigrastimhas been shown to significantly decrease naturally-occurring cases of mastitis compared to un-treated control cows. However,previously,pegbovigrastimhadnotbeenevaluatedinresponsetoanexperimentalmastitischallenge.Methods:Wechallenged11lactating Holsteins with ~400 cfu Escherichia coli P4 by intra-mammary infusion. Five cows received subcutaneous injections ofpegbovigrastim14daysand7days,priortodiseasechallenge.Toevaluatetheresponsetopegbovigrastim,wemeasuredcompleteblood counts (CBCs), somatic cell counts (SCCs), bacterial counts, milk yield, and feed intake data from 20 days prior to diseasechallenge,throughday11postchallengeincludingrelevant6and12hourintervalsimmediatelypostinfection.Results:Pegbovigrastimtreatedcattlehadsignificantlyincreasedcirculatinglevelsofneutrophilsandlymphocytesaftereachpegbovigrastiminjection,aswellasfollowingmastitischallenge(P<0.01).Treatedcowshadsignificantlylowerbacterialcountsinthe48hourspostinfection(P<0.05),butdidnotdifferinSCCspreorpostinfectioncomparedtocontrolcows.Inaddition,pegbovigrastimtreatedcattlehadlessmilkyieldreductionday1postchallenge(P=0.02),andtrendedtohavelessreducedfeedintake.Conclusions:Collectively,thisdatasuggeststhe utilization of gCSF as a potential antibiotic alternative tomastitis treatment and prevention and supports neutrophil focusedtherapiesasanimportantfacetofmastitisdiseaseprotection.

CRWAD2017 ABSTRACTS


P086-TheuseofIgGtasadiagnostictoolinfoalswithnaturallyacquiredRhodococcusequipneumoniaF.Cesar,S.Paudel,D.W.Horohov.VeterinaryScience,UniversityofKentucky,Lexington,KY,[email protected]:Immunology,12/4/20175:30PMFordecades,rhodococcalpneumoniahasbeenamongtheleadingcausesoffoalmortalityworldwide.However,itsdiagnosisremainsachallenge.TheroleoftheVapA-specific(virulence-associatedproteinA)immunoglobulinG(IgG)anditssubclasseswaspreviouslyevaluatedasadiagnostictoolamongfoalsthathadbeenchallengedwithRhodoccocusequi.Withinthispopulation,VapA-specificIgGsubclasses,withtheexceptionofIgGt,werepoorpredictorsofdisease.TheobjectiveofthisstudywastofurtherinvestigatetheuseofIgGtasadiagnostictoolafternaturalinfection.SerialserumsampleswerecollectedfromaThoroughbredbreedingfarmlocatedincentralKentucky,duringthe2016foalingseason.Allhealthyfoalsnaturallydeliveredatthefarm,andthathadachievedoptimumpassivetransferby24-48hoursoflife(IgGgreaterthan800mg/dL),wereenrolledinthestudy(n=98).Aserumsamplefromeachfoalwascollectedwithintheirfirst7daysoflife,atultrasoundscreeningtimes(1,2,and3monthsofage),andatdiseasediagnosistime.ThesampleswerestoredforbatchanalysisutilizingapreviouslyvalidatedELISAforVapA-specifictotalIgGanditssubisotypes(IgGa,IgGb,andIgGt).Basedonthefoal’ssubsequentphysicalandultrasonographicexaminationrecords,theywereclassifiedintooneofthethreegroups:norespiratoryabnormalities(NRA),subclinicaldisease(regressors),orrhodococcalpneumonia(progressors).RepeatedmeasuresANOVAwasusedtocomparetheconcentrationsofIgGsamongthethreedifferentgroupsateachtimepoint,andovertimeusingcumulativescores.LinearregressionwasusedtoassesshowreliabletheIgGsubisotypeswereinpredictingclinicaldisease.Serumconcentrationsof IgGtwerenot significantly higher inprogressor foals in comparison to regressor foals, and itwasnot a reliablepredictor of clinical disease at any timepoint, or as a cumulative score.While IgGa and IgGboutperformed total IgG and IgGt inpredicting clinical diseasewhenmeasuredeither at onemonthof ageor as cumulative scores, however theuseof these IgGs asdiagnostictestsisnotadvisedduetotheirwideconfidenceintervals.P087-BovineneutrophilsformextracellulartrapsinresponsetothegastrointestinalparasiteOstertagiaostertagi.J.Mendez1,W.Tuo2,Z.Xiao1.1Dept.ofAvianandAnimalSciences,UniversityofMaryland,CollegePark,MD,USA,2BARC.NEA,AnimalParasiticDiseasesLaboratory,Beltsville,MD,[email protected]:Immunology,12/4/20175:30PMPurpose:Ostertagiaostertagiisawidespreadparasitethatcausesbillionsofdollarsinproductionlossesinthecattleindustryannually.Itisunknownwhythereisnoeffectiveprotectiveimmunityincattledespitealargeinfluxofimmunecells.Neutrophils,theimmunesystems first-responders, are crucial in early immune response formation, and neutrophil extracellular traps (NETs), an importanteffectorresponseagainstpathogens,arealsosuggestedtobeinvolvedinregulationoftheoverallimmuneresponse.Inthepresentstudy,theabilityandmechanismsbywhichO.ostertagiinfluencesbovineneutrophilsandNETformationwereinvestigatedinvitro.Methods:Bovineneutrophilswereincubatedwithlipopolysaccharide(LPS),O.ostertagilarvalextract,liveorheat-killedL4larvaeforupto3hours.UnstimulatedandPMA-activatedneutrophilswereusedasnegativeandpositivecontrols,respectively.BlockageofToll-likereceptor4(TLR4)wasconductedbypreincubationwithaTLR4-specificinhibitor.QuantificationofNETformationwasdeterminedbyreleaseofextracellularDNAusingafluorescentdye.Fluorescenceimagingwasusedto imageandconfirmNETstructurebyco-localizationwithhistone.Results:ExposureofneutrophilstoLPSandO.ostertagilarvalextractresultedinareleaseoflargeamountsofextracellularDNA.Fluorescenceimagingwithco-localizationofhistoneproteinconfirmedtheclassicalstructureofNETs.Inresponsetobothliveandheat-killedlarvae,therewasasimilarlystrongreleaseofNETs,andimagingdemonstratedtheNETsabilitytoentraptheparasite.BlockageofTLR4signalinghadnoeffectonNETreleasebyO.ostertagi.Conclusions:ThisisthefirstreportindicatingO.ostertagi-inducedNETformation.Interestingly,thereleaseofNETsinresponsetoO.ostertagidoesnotseemtobemediatedbyTLR4,eventhoughTLR4ligandLPScaninduceNETsefficiently.NETsmaybeanearlyresponseagainstO.ostertagiinfection,howeverfurtherresearch isneeded todetermine roleofNETs in theoverall immune responseagainstO.ostertagi andwhetherNETspossess thecapabilitytocompletelyimmobilizeorkilltheparasite.

CRWAD2017 ABSTRACTS


P088-ImmunogenicityofamicroencapsulatedBacillusanthracisSternestrain34F2vaccinebysubcutaneousandoraladministrationJ.Benn1,S.P.Chaki1,A.Rice-Ficht2,T.A.Ficht1,W.E.Cook1.1VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,USA,2MolecularandCellularMedicine,TexasA&MHealthScienceCenter,CollegeStation,TX,[email protected]:Immunology,12/4/20175:30PMAnthrax(Bacillusanthracis)isazoonoticdisease,endemictoenvironmentsworldwide.Spores,thedormantformofthebacteria,cansurvivefordecadesinsomeofnature’sharshestenvironments.Anthraxoutbreaksarecommoninfree-ranginglivestockandwildlife,thusmakinganthraxaneconomicallyandecologically importantdisease.TheAnthraxSporeVaccine(ASV), thecurrentvaccineforlivestock andwildlife, is a suspensionofBacillus anthracisSterne Strain 34F2 spores in saponin; however, it is only available as asubcutaneousinjectionwhichisanimpracticalmethodofpreventionforwildlife.Therefore,thegoalofthisstudyistocreateanoralformulationoftheASVthatwillgenerateaprotectiveimmuneresponse.PriorresearchhasshownthatamicroencapsulatedvaccineagainstBrucellaabortusresultedinanantibodytitertwo-foldhigherthananun-encapsulatedcounterpart.Thesecontrastingimmuneresponsessuggestthatacontrolledreleasevehicle,suchasmicroencapsulation,mayhavesimilarbenefitsforananthraxvaccine.Toevaluatethepotentialforthecurrentvaccinetobeusedorally,varyingdosesoftheASVwereadministeredsubcutaneously(SC)andorallyinBALB/cJmice.AntibodytitersagainstanthraxprotectiveantigenweremeasuredbyELISAinweeklyserumsamplesovertwomonths.TheantibodylevelsoftheSCadministeredvaccines(Abs450nm=2.0)farexceededthoseoftheorallyadministeredvaccines(Abs450nm=0.03,p<0.01),indicatingthattheASValoneisnotsufficientasanoralvaccine.AlginatemicrospherescontainingSternestrain34F2sporescoatedwithaproteolysisresistantproteinaretheninoculatedSCandorallyinBALB/cJmice.Biweeklyserumsamplesarecollectedover4monthstomeasureantibodytitersbyELISA.Themicroencapsulatedanthraxvaccineshouldsignificantlyincreasetheantibody titeragainstanthraxprotectiveantigenwhenadministeredSCandorally (datawillbeavailablebyconference time).ApplyingthisinnovativeformulationtotheASVprovidesessentialbaselinedataneededtoestablishanoralanthraxvaccinethatwillconvenientlyandeffectivelypreventanthraxinwildlifepopulations.P089-AtypicalgranulomaformationbyMycobacteriumbovisinyoungcalvesJ. Carrisoza Urbina1, J.Á. Gutierrez Pabello2, M.A. Bedolla Alva3, E. Morales Salinas3, R. Hernández Pando4. 1Microbiology andimmunology, Universidad Nacional Autónoma deMéxico,,Mexico,Mexico, 2microbiology and immunology, Universidad NacionalAutónomadeMéxico,,Mexico,Mexico,3pathology,UniversidadNacionalAutónomadeMéxico,,Mexico,Mexico,4pathology,NationalInstituteofMedicalSciencesandNutrition,Mexico,[email protected]:Immunology,12/4/20175:30PMMycobacteriumbovisistheetiologicalagentofbovinetuberculosiswhichisachronicinflammatorydiseasecharacterizedbyproductionofgranulomatouslesions.Severalstudieshavebeenfocusedoncharacterizinggranulomasinexperimentallyinfectedcattle,whichhasbeen useful to understand the pathogenesis of the infection and to allow assessment of the protection of prototype vaccines.Nevertheless,fewstudieshavereportedmacroscopicandmicroscopiccharacteristicsoftheselesionsincattlenaturallyinfectedbyM.bovis.Therefore,inthepresentresearchgranulomasfrom32HolsteinFriesiancattleofadairyareaincentralMexicowerestudied.Inthis opportunity sampling 46.8% (15/32) cattlewere less than4monthsof age and53.2% (17/32)weremore thanone year old.Macroscopically,100%(32/32)oftheanimalsincluded,developedlesionssuggestiveoftuberculosisinmediastinallymphnodes,fromthesetissues700granulomaswereanalysedmicroscopicallyandwereclassifiedaccordingtothemethodologyofWangooetal.2005.Lesionsincattle1-year-oldorolderweremainlystageI35.8%(124/346)andstageIV32.6%(113/346).Surprisingly,lesionsincattleunder4monthsofageshowedanunusualpatternthatinterferedwithclassification.Granulomafeaturesincludelargeareasofnecrosisextendinginmostoftheaffectedorgan,centralcalcification,absenceofconnectivetissuecapsule,abundantpresenceofneutrophils,anaverageof1.6giantcellsperlesionandalargenumberofpositiveBAARs.Ourobservationssuggestthatcattleunder4monthsofageareunabletocontrolM.bovisinfection.Thisknowledgecanbeusefulinunderstandingnaturalhostresistancetomycobacterialinfections.Thisworkwassupportedbygrants:PAPIITIN-220415fromtheUniversidadNacionalAutónomadeMéxico.

CRWAD2017 ABSTRACTS


P090-Thermography,serumamyloidA,andprostaglandinE2toassessinflammationinareversibleequinefootlamenessmodelT.Brunner1,S.Grady2,T.Lescun3,A.Davern3,G.Moore4,S.Taylor3.1PurdueUniversity,WestLafayette,IN,USA,2VeterinaryTeachingHospital, Purdue University, West Lafayette, IN, USA, 3Veterinary Clinical Sciences, Purdue University, West Lafayette, IN, USA,4VeterinaryAdministrationDepartment,PurdueUniversity,WestLafayette,IN,[email protected]:Immunology,12/4/20175:30PMNon-steroidalanti-inflammatorydrugs(NSAIDs)areusedtoprovidepainreliefinhorses.AreversiblefootlamenessmodelhasbeenusedtodemonstrateanalgesicefficacyofNSAIDsinhorses,butthemechanismbywhichNSAIDsmitigatepaininducedbythismodelhasnotbeendetermined.Aspartofalargerstudytocomparetheanalgesicefficacyofketorolac(KT),phenylbutazone(PBZ),flunixinmeglumine (FM) and saline (control) using this lamenessmodel,we aimed to determinewhetherNSAIDs decrease pain primarilythroughanti-inflammatoryoranalgesiceffects.Indicatorsofinflammationincludedfoottemperature(thermography),serumamyloidA(SAA),andcephalicveinPGE2.Wehypothesizedthatfoottemperature,SAAandPGE2wouldincreasefollowinginductionoffootpain,indicatingthepresenceofinflammation.WeexpectedthattreatmentwithanyNSAIDwoulddecreasefoottemperature,SAAandPGE2twohoursafterNSAIDadministration(timeofpeakdrugeffect)comparedtotheplacebogroup.Arandomizedcrossoverwasdoneusing5healthyhorses.Allhorsesreceivedeachof3NSAIDsandsaline.Treatmentwasgiven1hafterlamenesswasinduced,andlamenesswasassessedhourlyfor12h.Foreachofthetrials,thermographyandcephalicbloodcollectionforSAAandPGE2weredonepriortolamenessinduction(baseline),immediatelyfollowinglamenessinduction(0h),immediatelypriortotreatment(1h),timeofpeakdrugeffect(3h)andpriortothenextdose(13h).Meandifferencesbetweentreatmentgroupsandacrosstimewereassessedinlinearmixedmodelswithhorseconsideredarandomeffect.Significancewassetatp<0.05.Foralltreatmentgroupscombined,therewasanincreaseinfoottemperaturefrom0hto1h(p<0.001).TherewasnodifferenceinmeanSAA(p=0.282)orPGE2(p=0.416)amongtreatmentgroups.TimedidnotaffectmeanSAA(p=0.272)orPGE2(p=0.460)inanytreatmentgroup.Thereisnoevidencethatthismodel induces inflammation.Althoughall 3NSAID treatments improved lameness, the inflammatorymarkersdidnotdecrease inresponsetoNSAIDs.Theincreaseinfoottemperaturefrom0hto1hmighthavebeenduetoincreasedpoolingofbloodinthenon-weightbearingfoot.P091-Developmentandcharacterizationofimmunereagentsforswinehealth,vaccine,anddiseasestudiesJ.Lunney1,M.Bailey2,J.Manirarora1,G.Renukaradhya3,S.Kenney3,J.LaBresh4,Y.Sang5,O.Francis2,L.Wooldridge2.1USDAARSBARCAPDL,Beltsville,MD,USA,2BristolVeterinarySchool,UniversityofBristol,Bristol,UK,3FoodAnimalHealthResearchProgram,OARDC,OhioStateUniv.,Wooster,OH,USA,4KingfisherBiotech,Inc.,St.Paul,MN,USA,5FoodAnimalHealthResearchProgram,TennesseeStateUniversity,Nashville,TN,[email protected]:Immunology,12/4/20175:30PMTo perform complex immune studies panels of immune reagents are required; those available for pigs are limited. The US-UKCollaborativeSwineImmuneToolkitInitiativehasasitsgoaltogeneratepriorityimmunereagents,basedoninternationalinput,andpipelinethemformarketing.UKresearchershavefocusedonmucosaltargets,includingproductionofmonoclonalantibodies(mAbs)tochemokinereceptorsandIgE.Targetpeptides,fromthreadedproteinstructuresofchemokinereceptorextracellularloop2,wereusedtoprobephagedisplaylibrariesandhaveidentifiedmAbsforCCR3andCCR9;verificationisunderway.USeffortsareaimedatexpressionofsolubleproteinsandswineCDmolecules,andproductionofpanelsofmAbs.TheteamhassetupcollaborationswithcommercialpartnersforproteinexpressionandmAbproduction,andupdatedprotocolstoevaluatereagentspecificity.NewpanelsofmAbsreactivewithCXCL10,CX3CL1(fractalkine),IL-6,IL-13,IL-17A,IFNβ,andIFNγ,havebeenproducedaswellasafewmAbstoIFNω1andIFNω5.EachofthesepanelsofmAbshasbeentestedforreactivityonyeastexpressedproteinsfromotherspeciesaswellasforepitopereactivityusinginhibitionELISAs.Severalhavebeenscreenedforintracellularstaining.ThesemAbreagentsarebeingscreenedforbestpairstodevelopnewmultiplexassaysforeachmarkerwithourgoaltoprovidetheveterinarycommunitywithnewcommercialreagentsandtechniquesfortheirresearchefforts.PlansforproducingmAbstoporcineCD1dusingsyntheticpeptidesareinprogressusingCD1dknockout(KO)miceandscreeningonCD1dKOpigcells.Toolsandreagentsgeneratedbythisprojectwillundoubtedlyadvanceswineimmune,diseaseandbiomedicalresearchefforts.SupportedbyUSDAARS,NIFAAFRIgrant#2015-67015-23216andBBSRCgrantBB/M028232/1.

CRWAD2017 ABSTRACTS


P092 - Effect of cell immortalization on TLRs expression and transcriptomeprofiling of TLRs and cytokine genes in bovine ilealepithelialcellsstimulatedwithbacterialligands

T.Uprety1,P.Katwal1,L.Antony1,M.Thomas2,R.S.Kaushik2.1BiologyandMicrobiology,SouthDakotaStateUniversity,Brookings,SD,USA, 2Biology and Microbiology; Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD, [email protected]:Immunology,12/4/20175:30PMTheintestinalepithelialliningisthinandextendsoveralargeareamakingitvulnerabletoattackbypathogensandcommensals.Toovercometheattackbypathogens,intestinalmucosalsurfaceinadditiontoepitheliallayerisequippedwithlargenumbersofimmunecells.PatternRecognitionReceptors(PRRs)playakeyroleininnateimmunitybyrecognizingPathogenAssociatedMolecularPatterns(PAMPs).Toll-Like-Receptor(TLR)family isoneamongdifferentfamiliesofPRRs.TLRsrecognizebothextracellularandintracellularpathogens.TLR-3,7,8,and9arepresentinendosomalcompartmentwhereasTLR-1,2,4,5,6and10arepresentonthecellsurface.Aprimarybovineilealepithelialcellline(BIEC)wasestablishedearlierfroma2-dayoldcalfinourlaboratory.Thiscelllinewasalsoimmortalizedbyhumantelomeraseencodedreversetranscriptase(hTERT),simian-virus40largeTantigen(SV40)andhumanpapillomavirusE6/E7protein(HPVE6/E7).TheeffectofimmortalizationonexpressionofTLRs1-10werestudiedbyreal-timeqPCR.TLR-1wasdownregulatedincellsimmortalizedbyhTERTandE6/E7whiletherewerenochangesinexpressionofTLRs2-10.Further,BIECcelllinewas used for transcriptome profiling of TLRs after 3 and 24h stimulation with: lipopolysaccharide (LPS) from E. Coli O55:B5,peptidoglycan(PGN)fromStaphylococcusaureus,andflagellin(FL)fromSalmonellatyphimurium.Upon3hstimulation,FLsignificantlydownregulatedTLR-1,LPSdownregulatedTLR-4,5,6,7,9,and10,andPGNdownregulatedTLR-5andTLR-9.Upon24hstimulation,TLR-3wasupregulatedbybothLPSandPGNandTLR-9byPGN.Alterationsintheexpressionofvariouscytokinessuchasinterleukin1alphaandbeta,TNF-alpha,interleukin6(IL-6),interleukin8(IL-8)andinterleukin10(IL-10)geneswerealsoinvestigatedforstudyingchangesindownstreamsignaling.Upon3hstimulationwithLPS,IL-8andIL-10wereupregulatedwhileIL-1betawasdownregulatedbyLPSandFL.Upon24hstimulation,IL-6andIL-8wereupregulatedbyLPSandTNF-alphawasdownregulatedbyFL.Overall,thisstudysuggeststhatBIECcellsmayserveasagoodmodelforstudyingtheintestinalinnateimmuneresponses.P093-Caninedistempervirusandparvovirus:howlongdoesimmunememorylastandareweover-vaccinating?D.Leistikow,L.Larson,R.Schultz.PathobiologicalSciences,UniversityofWisconsin-MadisonSchoolofVeterinaryMedicine,Madison,WI,[email protected]:Immunology,12/4/20175:30PMThecurrentstandardofveterinarypracticeistoadministercaninecorevaccinesincludingCanineDistemperVirus(CDV)andCanineParvovirus(CPV-2)everythreeyears.Adverseeffectsduetoovervaccinationareanincreasingconcerninbothhumanandanimalhealth.Agrowingnumberofveterinariansutilizeantibodytestingtodeterminetheneedforrevaccination.Therefore,thedurationofimmunityengenderedbycaninecorevaccineshasbeensubjecttoinvestigation.Thecurrentstudyaimstodetermineifrevaccinationintervalsgreaterthanthreeyearscanbesuccessful.31beagleswereheldinavirus-freeenvironment;16werelastvaccinatedthreeyearspriorand15nineyearsprior.18dogsreceivedacommercialmodifiedlivevaccinecontainingCDVandCPV-2,9dogsreceivedrecombinantCDVandkilledCPV-2vaccine,and4dogsreceivedkilledCPV-2vaccineonly.Serawerecollectedatpredeterminedtimepoints.SerumVirusNeutralization(SVN)forCDVandHemagglutinationInhibition(HI)forCPVwereperformed.Immuneresponseswerecomparedbetweenthegroupsbasedontimeandvaccineadministered.Ofthedogsthatwerevaccinatednineyearsprior,93%retainedprotectivetitersforCDVand42%forCPV-2.Intheabsenceofprotectivetiter,immunememorywasmaintainedforuptonineyears.Immuneresponsetorevaccinationiscorrelatedwithantibodytiteratday0;hightitersneutralizemodifiedlivevaccinevirus.Inconclusion,dogsmaintainimmunememorytoCDVandCPVforuptonineyears.Therefore,titertestingcontinuestobeanexcellenttooltodeterminetheneedforrevaccination.Lastly,geriatricdogsmaybenefitfromboostingiftheirtitersarebelowprotectivelevels.

CRWAD2017 ABSTRACTS


P094 - Investigation of circulating immunoglobulin-gamma (IgG) in swinewith Severe Combined Immunodeficiency (SCID) anddevelopmentofanIgGsupplementationregimen

L.M.Varley1,E.Powell1,A.N.Boettcher1,S.Charley1,J.Cunnick2,C.K.Tuggle1.1AnimalScience,IowaStateUniversity,Ames,IA,USA,2Microbiology,IowaStateUniversity,Ames,IA,[email protected]:Immunology,12/4/20175:30PMSevereCombinedImmunodeficiency(SCID)pigsareavaluableorthologousbiomedicalmodelwithsimilarimmunology,genetics,andsizetohumans.Theyareusefulinmechanisticstudiesofporcineandhumaninfectiousdisease.RecentlydiscoveredSCIDpigsatIowaStateUniversityhavemutationswithintheArtemisgeneandhaveaT-B-NK+cellularphenotype.Wehavedevelopedprotocolsforraisingthesepigsinsidebubblefacilitiestoreducetheirpathogenexposure.Newbornpigletsreceiveimmunoglobulin-gamma(IgG)richcolostrumduringthefirst24hoursoflifethroughtraditionalsuckingortubefeedingmethods.Weareinterestedifadifferenceexists between colostral IgGhalf-life of SCID andnon-SCID piglets. Additionally, it is also of interest tomonitor the decay rate ofcirculating IgGwithin theSCIDs todeterminewhen theyaremostvulnerable,as this timing is important forpotential therapeuticadministrationofIgG.Tocharacterizethedecayofcolostrum-derivedantibodiesintheseSCIDs,circulatingtotalIgGconcentrationsofSCIDandnon-SCIDpigswereanalyzedbyELISAfromserumofcordbloodandweeklycollectionsovereachpigs’lifetime.Forsucklingpigs,SCIDs(n=7)absorbedonaveragemorecolostrum-derivedIgGthannon-SCIDlittermates(n=7).TheIgGhalf-lifevariedfrom9-18days,butwasnotsignificantlydifferent(p=0.56)betweenSCID(12.7days,n=12)andnon-SCID(13.3days,n=12)pigs.Thus,theseSCIDpigsappeartoprocessIgGnormally,andcouldbecandidatesforIgGsupplementation.SwineIgGwaspartiallypurifiedfromabattoirbloodusingammoniumsulfatefractionations.TheabsenceofspecificviralorbacterialpathogensintheIgGsupplementwereverifiedbyTSAcultureand‘SpecificPathogenFree’PCRs.Inapreliminarytest,~80mg/kgIgG(n=2)orequivalentvolumePBSvehicle(n=1)wasadministeredsubcutaneouslyto18-dayoldcolostrum-deprivednon-SCIDpiglets.SuccessfulIgGabsorptionwasconfirmedbyELISA;circulatingIgGconcentrationincreasedanaverageof2.78foldcomparedtothevehiclecontrol.Thus,IgGtherapymaybeavaluabletoolinSCIDpighealthandisunderfurtherdevelopment.P095-Bovineviraldiarreavirusinducedifferentinterleukin-1betareleasepatternsonbovinemacrophagesY.F. López-Reyes, Jr.1, A. Morales-Aguilar1, M.-H. Regalado1, R.E. Sarmiento-Silva1, L. Arriaga-Pizano2, A. Benítez-Guzmán1.1DepartamentodeMicrobiologíaeInmunología,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico,Mexico,2UnidaddeInmunobioquímica,CentroMédicoSigloXXI,CiudaddeMéxico,[email protected]:Immunology,12/4/20175:30PMBovineviraldiarrheavirus(BVDV)isoneofthemostwidelydistributedviraldiseasesintheworldandcausesmillionsofeconomiclossestodairyproductionsystems.BVDVinfectsdifferentcelltypesincludingantigen-presentingcells,likemacrophages.Theinfectionwiththeviruscouldaffecttheirabilitytoproducemicrobicidalmoleculeslikecytokines,necessaryfortriggerasuccessinflammatoryresponseagainstinfections.Severalauthorshavereporteddifferencesoninterleukin1beta(IL-1β)productionininfectedmacrophages,thesepatternscouldberelatedwithinflammasomeassembling,becauseofthese,ouraimwastoassessBVDVcapabilitytoinduceIL-1betaanditsreleasebyinflammasomeinbovinemacrophages.Toaddressthisissueweinfectedperipheralbloodmonocyte-derivedmacrophagesusingNADLstrainat0.001,0.1&10multiplicitiesofinfectionandwemeasuredIL-1βproduction(ELISA)andnitricoxide(Griess)atdifferenttimes.Theresultsshowedanincreaseof700pgforaMOIof10:124hafterinfection.Incontrast,wedidnotfindanychangeinGriess’reactionfornitricoxideproduction.Inordertolinkthesefindingstoinflammasomeassembling,wealsoshowedcaspase-1cleavagebywesternblot.TheseresultssuggestIL-1βreleasebyinflammasomedependentpathway.

CRWAD2017 ABSTRACTS


P096-EvaluationoftheimmuneresponseandprotectionindairycowsvaccinatedwithimmunodominantStaphylococcalsurfaceproteins

C.Merrill1,D.B.Ensermu1,R.D.Abdi1,B.E.Gillespie1,J.Vaughn1,R.A.Almeida1,S.P.Oliver1,S.I.Headrick1,K.Hash2,T.B.Walker2,O.K.Dego1.1DeptofAnimalScience,TheUniversityofTennessee,Knoxville,TN,USA,2EastTennesseeResearchandEducationCenter-LittleRiverAnimalandEnvironmentalUnit,TheUniversityofTennessee,Walland,TN,[email protected]:Immunology,12/4/20175:30PMStaphylococcusaureusmastitisisthemajorcauseofeconomiclossesindairyproductionworldwide.Staphylococcusaureusalsoposesasignificantpublichealthconcernsinceitcancausemildtolifethreateninginfectionsthatareresistanttoantimicrobialtreatment.Dairycowsareverysusceptibletomastitisduringperiparturientandearlydryperiodsoflactationcycle,withS.aureusbeingreportedasamajorpathogen.TherearenoeffectivevaccinesforS.aureusmastitis.CurrentS.aureusmastitiscontrolmeasuresarebasedongoodmilkingtimehygiene,drycowantibiotictherapyandpromptdetectionandtreatmentofinfectedquarters.Despitetheadoptionofthesecontrolmeasures,S.aureusmastitiscontinuestobethemostcommondiseaseofdairycattlethroughouttheworld.Duetothetendencyofthisorganismtocausechronicmastitis,treatmentwithantibioticsisoflimitedsuccess.TheobjectiveofthisstudywastoevaluatetheimmuneresponsesandprotectionindairycowsvaccinatedwithStaphylococcusaureussurfaceproteins(SASP)andStaphylococcuschromogenessurfaceproteins(SCSP).Atotalof17Holsteindairycowsat2ndlactationwererandomlydividedintothreegroups.Cows ingroup1(n=5)andgroup2(n=6)werevaccinatedwith1.2mgofSASPandSCSP,respectivelywithEmulsigen-Dasadjuvant in a total volume of 3ml. Similarly, cows in a group 3was injectedwith PBS and used as a control. Three consecutivevaccinationsweregivensubcutaneouslyintheneckareaat28daysbeforedryingoff(D-28),14daysbeforedryingoff(D-14)andatdryingoff (D0).Milkand serumantibody titerswereevaluatedbyenzyme linked immunosorbentassay (ELISA)andall cowswerechallengedwithS.aureusstrainUT1byteatdippinginabacterialsuspensionatthecelldensityof107-108CFU/mlofgrowthmedia.Results showed that vaccinated cowshad increasedmilk and serumantibody titers and reducedbacterial shedding throughmilk.Interestingly, SCSP vaccine induced immune response that cross-protect against S. aureus clinical mastitis and reduced bacterialsheddingthroughmilkconfirmingthatSCSPiseffectivevaccinetocontrolbovineS.aureusmastitis.P097 - Novel multispectral cell sorting protocol and immunophenotyping panel for lymphocyte subpopulations from equine

peripheralbloodmononuclearcells(PBMC)J.E. Tomlinson1, B.Wagner2, J. Felippe3,G. Van deWalle1. 1Baker Institute for AnimalHealth, CornellUniversity, Ithaca,NY,USA,2DepartmentofPopulationMedicineandDiagnosticSciences,CornellUniversity, Ithaca,NY,USA,3DepartmentofClinicalSciences,CornellUniversity,Ithaca,NY,[email protected]:Immunology,12/4/20175:30PMPurpose:Ourobjectivewastodevelopanovelmultispectralpaneltoidentifyandsortsubpopulationsofequinelymphocytesusingcommercially available, conjugated monoclonal antibodies (mAbs) to surface markers, to allow for downstream gene expressionanalysis.Methods: Equine PBMCwere prepared by Ficoll Paque Plus density gradient centrifugation. Antibody specificities wereconfirmedbymulticolorflowcytometry.CD4,CD8,andBcellsweresortedonFACSFusiondirectly intocell lysisbuffer. IdentityofsortedpopulationswasconfirmedbyqRT-PCRforcell lineagemarkersCD4,CD8b,CD21,IgG1,CD19,andCD79a.Results:Anti-CD8cloneCVS21boundCD3-,non-TcellsinadditiontoCD3+Tcells.Anti-equineCD8(CVS8)andCD4(CVS4)onlylabeledCD3+cells.Anti-equine IgM (I-22) produced indistinct populations. Pan B-cell (CVS36) performed as expected butwas not available in conjugatescompatiblewiththepanel.CD21(B-ly4)andIgG1(WS45)labeledincompletelyoverlappingsubsetsofCD3-PanB-cell+cells.Afinalpanelofanti-CD4(CVS4),CD8(CVS8),IgG1(WS45),andCD21(B-ly4)antibodieswasusedtosortCD4+,CD8+,andCD21/IgG1+Bcells.CD3wasnotincludedduetolackofavailableconjugates,howeverthiswasconsideredacceptableafterthevalidationstepsdemonstratedhere.PCRresultsconfirmedeffectiveenrichmentoftheappropriatecellpopulations.Conclusions:Ourfindingofnon-Tcell labelingwithCD8(CVS21)emphasizestheneedforincreasedavailabilityofreagentsanduseofmultispectralflowforequineimmunophenotypicanalysis.Thismethodwillfacilitatestudyofequineimmuneresponsesandwillhavebroadapplicationacrossstudiesofinfectiousandimmune-mediateddisease.

CRWAD2017 ABSTRACTS


P098-Isolationandcharacterizationofaporcineparainfluenzavirus-1associatedwithrespiratorydiseaseinweanedpigsJ.Park,M.Welch,K.M.Harmon,J.Zhang,P.E.Piñeyro,G.Li,P.C.Gauger.VDPAM,CollegeofVeterinaryMedicine,IowaStateUniversity,Ames,IA,[email protected]:RespiratoryDiseases,12/4/20175:30PMPorcine parainfluena virus type 1 (PPIV-1) is a member of the Paramyxoviridae family and the genus Respirovirus. PPIV-1 is anenveloped,single-stranded,negativesenseRNAvirusthatwasfirstdetectedfromdeceasedpigsatslaughterhouseHongKong,Chinain2013.Currently,PPIV-1isconsideredwidespreadinUnitedStates(US).TheIowaStateUniversityVeterinaryDiagnosticLaboratory(ISUVDL)hasdetectedPPIV-1byreal-timereversetranscriptionPCR(RT-qPCR) in lung,nasalswabs,andoral fluidsfrompigswithclinicalrespiratorydisease.PositivePPIV-1samplesbyRTqPCRattheISUVDLwithcyclethresholdvalueslessthan25wereselectedforvirusisolation.ThePPIV-1isolate,USA/MN25890NS/2016(GenBank#:MF681710),wassuccessfullyisolatedinLLC-MK2(ATCC®CCL-7™),aRhesusmonkeykidneyepithelialcellline,inthepresenceoftrypsin.ThePPIV-1cytopathiceffectincludingsyncytiaformationwasdemonstratedandenvelopedviralparticleswithadiameterofapproximately200nmwasobservedbyelectronmicroscopy.Thewhole genome sequence of USA/MN25890NS/2016 PPIV-1 is closely related to the PPIV-1 strains detected in the US in 2016,KT749882.1 and KT749883.1, with 98.1% and 98.2% nucleotide homology, respectively. Amino acids of the hemagglutinin-neuraminidase (HN) share 95.7% -98.2% identitywith recently reported HN in theUS. PPIV-1 polyclonal and specificmonoclonalantibodiesweregeneratedbywholevirusimmunizationofthree,5-week-oldBALB/cmicewith100µLof106TCDI50/mL.ThemonoclonalAbsspecificallybindtoPPIV-1infectedLLC-MK2cells.ThevirusisolatecanbeusedforpathogenesisstudiestodeterminetheroleofPPIV-1 in respiratorydisease in swine, andevaluationof vaccinesefficacyusing appropriate challengemodels. In addition, PPIV-1specificmonoclonalantibodiescanbeusedtodevelopvariousserologicalassaysfordetectingviralexposureinfieldsamples.P099-UpdateofrealtimeRT-PCRforthedetectionandtitrationofswineinfluenzavirusesM.Elaish1,E.Cho2,J.M.Ngunjiri1,A.S.Bowman3,C.W.Lee1.1FoodAnimalHealthResearchProgram,OhioAgriculturalResearchandDevelopment Center., The Ohio State University, Wooster, OH, USA, 2Williams College,Williamstown, MA, USA, 3Department ofVeterinaryPreventiveMedicine,CollegeofVeterinaryMedicine,TheOhioStateUniversity,Columbus,OH,[email protected]:RespiratoryDiseases,12/4/20175:30PMSwineinfluenzaisacontagiousrespiratorydiseaseofpigscausedbyinfluenzaAvirus.Pigscansupportthereplicationof influenzavirusesfromdifferenthostsandthusplayanimportantrole in interspeciestransmission.Moreover,frequentswineinfluenzavirus(SIV)infectioninhumaninrecentyearshasraisedpublichealthconcerns.Forinfluenzadiagnosisandsurveillanceinswine,areal-timeRT-PCR(RRT-PCR)protocolthatwasvalidatedbyNationalVeterinaryServiceLaboratories(NVSL)hasbeenwidelyusedintheUnitedStates.Inthisstudy,wedesignedanewRRT-PCRprimerbasedontheSIVMatrixgenesequencesavailableintheGenBankdatabase.RRT-PCRwasconductedusingdifferentprimercombinationsandRNAfromSIVsthatwereselectedbasedonprimer-targetsequencecomparison.TheresultsindicatethatRRT-PCRusingtheNVSLprotocolcaneffectivelydetectdiverseSIVsthatarecurrentlycirculatinginthefield.However,thedetectionlimitwasratherhigh(approximatelyrangingfrom102.0to103.0TCID50)

formanySIVsthatbelongtopandemicH1N1lineage.ComparedwithNVSLmethod,ournewlydesignedprimerwasatleast100timesmoresensitivefordetectionofaspecificgroupofSIVsthatbelongtoNorthAmericantriplereassortantlineagewhilemaintainingsimilarorbettersensitivityforotherSIVswhenusedincombinationwithexistingprimers.Withfurtheroptimization,weexpecttodevelopaRRT-PCRassaythatismoresensitivethanthecurrentNVSLprotocolforthedetectionofdiverseSIVscirculatinginthefield.

CRWAD2017 ABSTRACTS


P100-Amultiplexreal-timeRT-PCRassayforsimultaneousdetectionanddifferentiationofInfluenzaD,C,BandAvirusesinswineandcattle

H.Zhang1,X.Liu1,Y.Wang1,Y.Li2,Y.Lang2,E.Poulsen1,L.Noll1,N.Lu1,R.Ransburgh1,W.Zheng1,W.Ma2,L.Peddireddi1,Y.Fang2,G.Anderson1, J. Bai1. 1KSVDL, Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA,2DepartmentofDiagnosticMedicine/Pathobiology,KansasStateUniversity,Manhattan,KS,[email protected]:RespiratoryDiseases,12/4/20175:30PMInfluenzaisahighlycontagiousviralrespiratorydiseasecausedbydifferentinfluenzavirusesincludinginfluenzaA,B,C,andDviruses(IAV,IBV,ICV,andIDV).Timelydetectionanddifferentiationofinfluenzavirusesisimportantforpreventionandintervention.IAVinhumanandanimalshasbeenwellstudiedandvariousmaturediagnosticassaysareavailable.Inthisstudy,TaqManreal-timeRT-PCRassaysweredevelopedandvalidatedfordetectionofIBV,ICV,andIDVinsingularandtriplexformats.ThecurrentUSDAandCDCIAVreal-timeRT-PCRassayswere incorporated, andamultiplex real-timePCRpanel assay (IDV, ICV, IBV, IAV, andan internal controltargetingthehost18SrRNAgene)hasbeendevelopedforrapiddetectionanddifferentiationofthefourinfluenzaviruses.Specificreal-timePCRprimersandprobesweredesignedtotargetthemostconservedgeneregionsupontheanalysesofallavailableandfull-ornear-fullsegmentsequencesof IBV, ICV,and IDV.Cloningprimers flankingthereal-timePCRtargetregionsweredesigned,andpositivecontrolplasmidsharboringthereal-timePCRtargetregionswereconstructed.BoththeplasmidDNAsamplesandtheinvitrotranscribedRNAsamplesfromtherespectiveplasmidswereusedtodeterminetheanalyticalsensitivity/limitofdetection(LOD).Theassaycoveragerate(perfectmatchesofallreal-timePCRprimersandprobe)ofIBVqPCRis95.8%over5,261Matrixsequences,ICVqPCRis99.4%over157Matrixsequences,andIDVqPCRis100%over23PB1sequences.ThesingularandmultiplexRT-qPCRprotocolswere optimized and validated for the identification of IBV, ICV, IDV, and IAV,with PCR efficiencies (E) 90%~110%and correlationcoefficients(R2)>0.99.Themultiplexassaywasalsohighlyspecificindetectingthetargetinfluenzaviruswithoutcross-reactivityamonginfluenza viruses and other common pathogens in swine and cattle. In conclusion, the high-coverage, high-throughput, low-cost,multiplex real-timeRT-PCRassayestablished in thisstudywillbeefficientlyandconvenientlyused forsimultaneousdetectionanddifferentiationofIAV,IBV,ICV,andIDV.P101-PorcinereproductiveandrespiratorysyndromevirusJA142inducesaprotectiveTcellmediatedimmuneresponsesinpigsS.-M. Lee1, B. Kim2,A. Khatun2, S.Nazki2, S. Salam1, S.Gu1,C.-G. Jeong2,W.-I. Kim2. 1DivisionofBiotechnology,ChonbukNationalUniversity, Iksan, Korea, Republic of, 2College of Veterinary Medicine, Chonbuk National University, Iksan, Korea, Republic [email protected]:RespiratoryDiseases,12/4/20175:30PMImmuneresponseagainstPRRSVinfectionisoftendelayedandinsufficienttoclearthisvirusfrompigsandmechanismsinvolvedarestillremainedunclear.Itiswellknownthatcell-mediatedimmuneresponse(CMI)drivenbyCD4+helperTcellsandcytotoxicCD8+Tcellsisacrucialfactortoremovevirusinfectedcellsandmodulateshumoralimmuneresponse.Therefore,thepresentstudywasaimedtostudytheresponsesofhelperCD4+T(Th)cellsandcytotoxicTlymphocytes(CTL)inpigsinfectedwithPRRSVJA142andpossibleroleofthesecellsinvirusclearanceinpigs.Four-weeksoldpigswereinfectedintramuscularlywithJA142orkeptasuninfected.Weightgainwasmonitoreddailyandserumwascollectedat0,3,10,14,21and28dpc.Pigsweresacrificedat3,10and28dpcandPBMCs,BAL,lungs,lymphnodesandtonsilswerecollectedforTcellanalysusbyflowcytometry.Viralloadsweremeasuredinserumandlungs;anti-PRRSVIgG,virusneutralizingantibody(VNA)andmRNAforvariouscytokineswereevaluatedaswell.JA142infectedpigsexhibitedhighestviralloadsinserumandlungsbetween3and10dpc,whileviruswasalmostclearedby28dpc.PRRSVspecificantibodiesweredetectedat10dpcandincreasedupto28dpc.VNAresponsewasdelayedandobservedatlowlevelswhenmostvirushasalmostcleared. Interestingly, T cell responses including Th1, Th17 andCTLwere significantly higher in JA142 infectedpigs at 10dpc andremainedoreven increasedby28dpc.On theotherhands, immunosuppressiveTregswerenot significantly changedupon JA142infectioninallsamplesexcepttemporalincreaseinlymphnodeat10dpc.CytokinessuchasTNF-α,IFN-α/β/γ,IL-1β,IL-6,IL-8andIL-10wereinducedathighlevelsinPBMCofJA142infectedpigsreflectingon-goinginnateandadaptiveimmuneresponsesagainstJA142infection.Takentogether,thepresentstudydemonstratedthatpigsinfectedwithPRRSVJA142developedsignificantlevelsofTcellresponses,possiblyleadingtoclearanceofthisvirusat28dpc,whileanweakneutralizingantibodyresponsewasdetectedonlyat28dpc.ThesefindingsimplythatcellmediatedimmunitycouldbeakeyforsuccessfulremovalofPRRSVJA142.

CRWAD2017 ABSTRACTS


P102-EpiCC:anewtoolforselectingtheoptimalvaccineduringanemergingoutbreakM.Parrillo.InstituteforImmunologyandInformaticsattheUniversityofRhodeIsland,Providence,RI,[email protected]:RespiratoryDiseases,12/4/20175:30PMPurpose:WehavedevelopedanovelimmunoinformaticstoolforcomparingpotentialvaccinestrainstooutbreakstrainsusingHLA-orSLA-epitopeforhumansandpigs.HereweappliedthistooltoexistingPorcinereproductiveandrespiratorysyndromevirus(PRRSV)vaccinesandcirculatingstrainsofPRRSVinUSswineherds.EpiCCcanbeusedtocompareTcellepitopecontentbetweenvaccinestrainsandoutbreakstrains. ItevaluatesTcellepitopecross-conservationtodevelopanepitope-basedrelatednessorEpiCCscore.Methods:WeusedPigMatrixtoidentifyTcellepitopesinthecompleteproteomesof70non-redundantPRRSvstrainsandexistingPRRSVvaccines(GP5InglevacMLV,GP5InglevacATP,GP5Fostera).EpitopespredictedtobindtocommonclassIandclassIISLAalleleswereidentified.TheepitopesofvaccineandoutbreakstrainswerecomparedandanEpiCCscorewascalculated.Results:WeobservedthatepitopecontentandconservationvariedbetweenUS-basedpigherdoutbreakstrains.AthresholdofprotectionwasdefinedusingpreviousEpiCCstudies;usingthisthresholdthevaccineefficacyagainsttheselectedstrainswasestimated.Wefoundthatnoneofthevaccines ispredicted toprotectagainstall PRRSV strains,however,GP5 InglevacMLVwouldprotectagainst7PRRSV strains,GP5InglevacATPwouldprotectagainst4PRRSVstrainsandGP5Fosterawouldprotectagainst15PRRSVstrains.Conclusions:EpiCCgivesvaccineresearchersanadditionaltoolfordevelopingvaccinestrainsandcouldalsohelpporkproducerspicktheappropriatevaccineinaPRRSvoutbreak.P103-Genetic&antigeniccharacterizationofH5N6virusesinVietnam2015-2016T.D.Nguyen,T.L.To.DepartmentofAnimalHealth,NationalCentreforVeterinaryDiagnostics,Hanoi,[email protected]:RespiratoryDiseases,12/4/20175:30PMPurposes:ThehighlypathogenicH5N6avianinfluenza(HPAI)viruseswerefirstreportedfrompoultryoutbreaksinVietnamin2014.AlthoughnoremarkablewaveofH5N6epidemicsappeared,H5N6virusesspreadedtomanyprovinces intheNorthandCentreofVietnamfrom2015-2016.TocontrolAI,vaccineisstillavaluabletoolinVietnamhusbandarypractices,thereforegeneticandantigeniccharacterization of H5N6 viruses is needed for vaccine updates. Methods: H5N6 viruses were HA sequenced and analyzedphylogeneticallyusingMEGA6withthethegeneraltimereversibleplusgammadistributionplusinvariantsites(GTR+G+I)model.H5N6viruseswere antigenically characterizedusing ahemagglutination inhibition test. The antigenic relationshipbetween thedifferentviruseswas expressed as the r value,whichwas calculated using the Archetti and Horsfall formula (Archetti and Horsfall, 1950).AntigenicassociatedsitesinHAwereanalyzedaccordingtothedesignbyCaietal.,2012;Duvvurietal.,2009.Results:Intheperiodof2015-2016,VietnamH5N6viruresarephylogeneticallyclassifiedintoclade2.3.4.4(Smithetal.,2015).Howeverthesevirusesformed2separatelineagesco-circulatinginVietnam.ThefirstlineagewassimilartoSichuan-H5N6virusesandthesecondlineagewassimilartoJiangxi-H5N6viruses.ThesetwolineagesmettheWHO/OIE/FAO’scriteriatobenewsubclades,2.3.4.4aforSichuanlineageand2.3.4.4bforJiangxilineage.Regardingtoantigenicrelationship,H5N6viruseshadstillcloseantigenicrelationshipbutH5N6virusesofsubclade2.3.4.4bseemedtobemorevariableinHIreactionindicatingantigenicdriftoccurred.AnalysisofantigenicsiteinHAalsoshowthatH5N6viruses,subclade2.3.4.4bhadvariablemutationsatantigenicsites.Conclusion:TheHPAIH5N6virusesinVietnamintheperiodof2015-2016belongedtoclade2.3.4.4andweresubdividedinto2subclades,2.3.4.4aand2.3.4.4b.H5N6virusessubclade2.3.4.4bhadundergonecertainantigenicdrift thereforevaccineevaluation isneededtoensurethesufficientefficacyagainstnewH5N6variants.

CRWAD2017 ABSTRACTS


P104-SuccessionalchangesinrespiratorymicrobiomeinclinicallyhealthychickenlayersM.C.Abundo1,J.M.Ngunjiri1,H.Jang1,M.Elaish1,M.KC1,A.Ghorbani1,B.P.Youmans2,T.J.Johnson2,C.-W.Lee1.1FoodAnimalHealth,TheOhioStateUniversity,Wooster,OH,USA,2DepartmentofVeterinaryandBiomedicalSciences,UniversityofMinnesota,St.Paul,MN,[email protected]:RespiratoryDiseases,12/4/20175:30PMComplexmicrobialcommunities(microbiome)thatinhabittherespiratorysystemsarethoughttohaveasignificantinfluenceontheimmune status and animal health. However, these communities remain largely unknown. To define the baseline respiratorymicrobiomeofchickenlayers,wefollowedacommercialflockforoneyearandsampled12-32birdsatdifferentagestocollectsinusandtracheawashesforbacterialcommunityprofiling,throughNGSsequencingoftheV4regionofthe16SribosomalRNAgeneanddownstreamanalysis. The beta-diversity of bacteriawas highly influencedby their habitatwithin the respiratory system (sinus vstrachea)andage.Therespiratorymicrobiomefromindividualbirdsclusteredaccordingtothebrooding,grow-out,andtheegglaying(adult)stagesofthechicken.Inthesinus,speciesrichnesswashighestin51weekoldchickensandlowestat1week,whileallotherageshad subtledifferences in species richness. In trachea, therewasnoapparent relationshipbetweenageand species richness,suggestingthattracheacolonizationwastransient.Attheclassleveloftaxonomicresolution,therespiratorybacterialcommunitieswerecharacterizedbyhighlevelsofActinobacteriaandDeinococciinthesinus,Betaprotebacteriaintrachea,andBacilliinbothsinusandtrachea.Eventhoughthisflockwasclinicallyhealthy,serologicalandsequenceanalysesshowedevidenceofmycoplasmasynoviaeinfection in trachea during the middle and late stages of egg production. Serological evidence also indicated that the flock waspersistentlyinfectedwithreovirusthroughoutthesamplingperiod.Thisstudyhasprovidedbaselinedataforrespiratorymicrobiomeincommercialchickenlayersendemicallyinfectedwithreovirusandisimmunizedagainstinfectiousbronchitisvirus,infectiousbursaldisease virus, Newcastle disease virus, and avian encephalomyelitis virus. Controlled experiments are planned to investigate theimpactsofubiquitousrespiratorymicrobiomeinpathogenesisofrespiratorypathogensandefficacyofvaccines.P105-Theeffectoflungconsolidationonaveragedailygainuntil50daysofageinpreweanedgroup-houseddairycalvesM.C. Cramer1, T.L.Ollivett2. 1Dairy Science,University ofWisconsin-Madison,Madison,WI,USA, 2School of VeterinaryMedicine,UniversityofWisconsin-Madison,Madison,WI,[email protected]:RespiratoryDiseases,12/4/20175:30PMThoracicultrasonographywasvalidatedfortheidentificationoflungconsolidationinpreweaneddairycalves,butthereislittleresearchthataddresseshowlungconsolidationaffectscalfperformance.Thestudyobjectivewastodeterminetheeffectoflungconsolidationonaveragedailygain(ADG;kg/day)inpreweaneddairycalves.Calves(n=200)onacommercialherdinOhio,USAwereenrolledatentrytoanautomatedmilkfeederbarnandhousedingroupsof13±3(mean±stdev).Calveswere21±6daysoldatenrollmentandwerefolloweduntil50daysofage.Twiceweeklyhealthexamsincludedaclinicalrespiratoryscore,thoracicultrasoundscore(USS),fecalscore,andbodyweight.TheUSSrangedfrom0to5(0=normal;1=diffusecomettails;2=lobularlesions≥1cm;3=1entirelunglobeconsolidated;4=2lunglobesconsolidated;5=≥3lunglobesconsolidated).Calveswithlungconsolidation(CON+;atleastoneUSS≥3)werecompared tocalveswithout lungconsolidation (CON-;USSalways<3).Amultivariable linear regressionmodelwas fit todetermineifADG(outcome)wasassociatedwithCON,aftercontrollingforbreed,cohort,anddiarrheapriortostudyenrollment.CalvesidentifiedasCON-hadsignificantlygreaterADGcomparedtoCON+calves (0.88vs.0.73, respectively;P=0.0004).CalveswithoutdiarrheapriortostudyenrollmenthadsignificantlygreaterADGcomparedtocalveswithdiarrheapriortostudyenrollment(0.87vs.0.74,respectively;P<0.01).Breed(P<0.0001)andcohort(P=0.0003)werealsoassociatedwithADG.Resultsfromthisstudyshowthatcalveswithlungconsolidationhavea-0.15kg/daydisadvantageinweightgainduringthepreweanedperiod.Calvesidentifiedwithdiarrheaatayoungagehada-0.13kg/dayweightgaindisadvantagethatlasteduntillateintothepreweanedperiod.Managementpracticesshouldfocusonpreventionoflunglesionsanddiarrheainyoungdairycalves.Futureresearchshouldaddresstheeffectsoflunglesionsonpost-weaningperformance.

CRWAD2017 ABSTRACTS


P106-PhylogeneticanalysisofemergingzoonoticcoronavirusinGeorgianbatsL.Urushadze1,G.Babuadze1,I.Natradze2,A.Velasco-Villa3,P.Imnadze1.1NationalCenterforDiseaseControlandPublicHealth/R.LugarCenter, Tbilisi, Georgia, 2Ilia State University, Tbilisi, Georgia, 3Centers for Disease Control and Prevention, Atlanta, GA, [email protected]:ViralPathogenesis,12/4/20175:30PMPurpose:Someoftheworld’smostdeadlyvirusesoriginateinbats,includingNipah,Hendra,severeacuterespiratorysyndrome(SARS),and Middle East respiratory syndrome coronavirus (MERS CoV). In the past decade, numerous novel coronaviruses have beendiscovered in a great variety of bat species throughout Asia, Europe, Africa, and America. In this investigation, we conducted acomprehensivesamplinginGeorgiatoinvestigatethepresenceofAlphacoronavirusandBetacoronavirusinbatpopulations.Methods:A total of 189batswere collected: eight species captured in 2014 fromeight locations inwestern andeasternGeorgia. RNAwasextractedfrombatfecesandpan-coronavirusRT-PCRscreeningassaywasperformedusinghighlyconservedRbRpgeneprimers.RT-PCRpositive coronavirus ampliconswere gel purifiedusingQIAGENGel ExtractionKit and sequenced inbothdirections to assureaccuracy. Sequences were edited using Sequencher 5.0. NCBI BLAST. Results: AS a result, 45 (24%) bats were positive forAlphacoronavirus(n=26)andBetacoronavirus(n=19).Interestingly,32ofthosebats(71%)werecollectedinImereti(westernGeorgia),and4(9%)inSamegrelo(westernGeorgia).ThreebatsthatwerecollectedinTskaltubo’sGlianaCave(west-centralGeorgia)presentedBetacoronavirussequencesthatseemcloselyrelatedtoMERSbeta-coronavirusessequencesobtainedfromafatalhumaninfectioninSaudiArabiaandCamelusdromedarius fromDubai(phylogenetictree),aswellaswithbatcoronavirusessequencesobtainedfromEptesicus isabellinus fromSpain,Nyctalusnoctula,Eptesicusserotinus fromItaly,andVespertiliosuperans fromChina.Conclusions:Futurestudiesshouldbeconductedtoprovidingalargersamplesizeofbats,otherwildlifespeciesforCoVinfection,andtheirroleinhumaninfection.FurtheranalysisandinterpretationofresultswillbenefitGeorgia’ssurveillancesystems.P107-InhibitionoftypeIIIinterferonsintheintestinalepithelialcellsbyporcineepidemicdiarrheavirusandinnateimmuneevasionQ.Zhang1,H.Ke1,T.Fujita2,D.Yoo1.1DepartmentofPathobiology,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,USA,2InstituteforVirusResearch,KyotoUniversity,Kyoto,[email protected]:ViralPathogenesis,12/4/20175:30PMPorcineepidemicdiarrhea(PED)isahighlycontagiousacuteentericdiseasecharacterizedbyvomiting,waterydiarrhea,andseveredehydrationaccompaniedbyhighmortalityinsucklingpiglets.PEDVemergedintheUSin2013andbecameendemic,posingsignificanteconomicconcerns.PEDVinfectsepithelialcellsofthesmallintestine.AccumulatingevidencesuggeststhattypeIII interferon(IFN-lambda)playsakeyroletomaintaintheinnateantiviralstateinthemucosalepithelialcellsurfaceinthegut,andinturn,entericvirusesmayhaveevolvedtoevadetheIFN-lambdaresponsesduringinfection.TostudytheinnateimmuneevasionofPEDVfromthetypeIIIIFNresponse,wefirstdevelopedapigintestinalepithelialcelllinesusceptibleforPEDVinfectionandnameditIPEC-DQ.IPEC-DQcellsefficientlysupportedPEDVreplication,andpotentlyandselectivelyinducedtheproductionoftypeIIIIFNs.OffourisotypesoftypeIIIIFNs,IFN-lambda-1,-lambda-3,and-labmda-4wereexpresseduponstimulationwithdouble-strandedRNAanalog.Interestingly,IFN-lambda-2wasdeficientinvariousporcinecells, indicatingthatpigsmaybedeficientforIFN-lambda-2expression.InPEDV-infectedcells, the production of type III IFNswas inhibited, suggesting the PEDV-mediated suppression of type III IFNs in these cells. Therecombinant IFN-lambda-1 and IFN-lambda-3 restricted the PEDV replication over time in a dose-dependentmanner, indicating apotentantiviralactivityofthetypeIIIIFNs.IFNregulatoryfactor1(IRF1)isakeyregulatorfortypeIIIIFNs,andwefurthershowthatPEDVblockedtheIFN-lambdapromoteractivationbyinterferingbothIRF1andNF-κB.PEDVdidnotinterferetheexpressionofIRF1,butratherinhibiteditsnucleartranslocation.ThedecreaseinthenumberofperoxisomeswasevidentinPEDV-infectedcells.OurstudyforthefirsttimedemonstratesthatPEDVevadesthetypeIIIIFNresponseintheintestinalepithelialcells.OurfindingmayfacilitatetodesignanovelapproachtocontrolnotonlyPEDVbutalsootherentericviralinfections.

CRWAD2017 ABSTRACTS


P108-MolecularcharacterizationofporcinedeltacoronavirusesinSouthKorea,2014-2016C.Lee1,G.Jang1,S.-H.Kim2,K.-K.Lee2.1AnimalVirologyLaboratory,SchoolofLifeSciences,BK21PlusKNUCreativeBioResearchGroup,KyungpookNationalUniversity,Daegu,Korea,Republicof,2AnimalDiseaseDiagnosticDivision,AnimalandPlantQuarantineAgency,Gimcheon,Korea,[email protected]:ViralPathogenesis,12/4/20175:30PMPorcinedeltavoronavirus(PDCoV),amemberofthegenusDeltacoronavirusinthefamilyCoronaviridaeoftheorderNidovirales,isanewlyemergedswineentericcoronaviruscausingsevereclinicaldiarrheaandintestinalpathologicaldamageinpiglets.Inthepresentstudy,weaimedtofurtherinvestigatetheprevalenceandfull-lengthgenomesequenceanalysisofPDCoVfromclinicalcasesassociatedwith diarrhea from Korean swine farms. Here, a nested reverse transcription (RT)-PCR approach for the detection of PDCoVwasdeveloped to identifyandcharacterizeetiologicagent(s)associatedwithdiarrhealdiseases inpiglets inSouthKorea.APCR-basedmethodwasappliedtoinvestigatethepresenceofPDCoVin683diarrheicsamplescollectedfrom449commercialpigfarmsinSouthKoreafromJanuary2014toDecember2016.Themolecular-basedsurveyindicatedarelativelyhighprevalenceofPDCoV(19.03%)inSouthKorea.Amongthose,themonoinfectionofPDCoV(9.66%)andco-infectionofPDCoV(6.30%)withporcineepidemicdiarrhea(PEDV)werepredominantindiarrhealsamples.Thefull-lengthgenomesorthecompletespike(S)genesofthemostrecentstrainsidentifiedin2016(KNU16-07,KNU16-08,andKNU16-11)weresequencedandanalyzedtocharacterizePDCoVcurrentlyprevalentinSouthKorea.We founda single insertion-deletion signature anddozensof genetic changes in the S genesof theKNU16 isolates.PhylogeneticanalysisbasedontheentiregenomeandspikeproteinsequencesofthesestrainsindicatedthattheyaremostcloselyrelatedtootherKoreanisolatesandallpreviousandrecentKoreanstrainsaregroupedwithintheUSPDCoVclade.However,KoreanPDCoVstrainsformeddifferentbrancheswithinthesamecluster,implyingthattheviruscontinuestoevolveandadapttoitsnaturalhostinthefield.OurdatawilladvancetheunderstandingofthemolecularepidemiologyandevolutionarycharacteristicsofPDCoVcirculatinginSouthKorea.P109-EfficacytestnovelPRRSVlivevaccinecandidateD.Sung,H.Jang,J.Jang.VaccineBusinessDivision,WoogeneB&G,Yesan-gun,Korea,[email protected]:ViralPathogenesis,12/4/20175:30PMPurpose: WGV-0917 (KCTC 12783BP), WGV-1014 (KCTC 12784BP), and WGV-1107 (KCTC 12785BP) were originally isolatedfromneonatalpigletsthatwereshowntypicalPRRSVinfectedsymptoms.ThesestrainshadgeneticallycharacterizedtheNA(WGV-0917andWGV-1014)andEU(WGV-1107)strainsandculturallypassagedonPAMcellsforadaptationandMARC-145cellsforattenuation.This studywas tocompare theirimmunogenicityofattenuatedvirulentPRRSVNAandEUstrainswithMLVvaccine inguineapigs.MaterialsandMethods:PhylogeneticanalysiswasperformedusingORF5genesequencesofPRRSV-WGV-0917,PRRSV-WGV-1014andPRRSV-WGV-1107virusesfromthisstudyaswellasotherPRRSVstrains.Animalandexperimentaldesign.Atotalof12CrlOri:HAVFAguineapigs,4weeksofage.Allgroupsofguineapigswereinoculated½dosebyIMroute.Group1(n=3),2(n=3)and3(n=3)wereinoculatedwith104.5TCID50/mlofstrainWGV-0917,WGV-1014andWGV-1107,respectively.Group4(n=3)wasinoculatedwith104.9TCID50/mlofMLVvaccine.Group5(n=3)was injectedwithPBSascontrols.Bloodwastakenon0,7,14,21daysaftervaccination.Serumsampleswerecollectedandstoredat-70℃beforetestingwithVN.PRRSVVNtiterassay.ResultsandConclusions:OurresultsshowedthatWGV-1014begantoinduceNAtitersatWPI2andthetitersweresignificantlyhigherin10^6.5TCID50/mlvaccinegroupatWPI6(p<0.001).Inconclusion,thisstudyindicatedthatnovelPRRSVvaccinecandidatesof10^6.5TCID50/mlraisedVNtiter(<32)enoughtoprotectPRRS,butfurtherstudieslikechallengeareneededinordertoconfirmthisPRRSVvaccinecandidatescouldfullyprotectPRRSVinpigs.

CRWAD2017 ABSTRACTS


P110 - Evaluationof theefficacyof anattenuated live vaccinebasedona virulent type2porcine reproductiveand respiratorysyndromevirusstraininyoungpigs

S.Lee1,S.-C.Lee2,H.-W.Choi2,Y.-H.Noh2, I.-J.Yoon2,C.Lee1. 1AnimalVirologyLaboratory,SchoolofLifeSciences,BK21PlusKNUCreativeBioResearchGroup, KyungpookNationalUniversity,Daegu, Korea,Republic of, 2ChoongangVaccine Laboratory,Daejeon,Korea,[email protected]:ViralPathogenesis,12/4/20175:30PMPRRSVisagloballyubiquitousswineviralpathogenthatcausessignificantfinanciallossesworldwide.Thetype2PRRSVlineage1thatincludesvirulentMN184strainanditsrelativestrainshasseverelyaffectedtheporkindustryinSouthKoreasincetheearly2010s.Anattenuatedstrain,CA-2-MP120,wasobtainedbysequentiallypassagingthevirulentMN184-relatedstrainCA-2onMARC-145cellsfor100passagesandonculturedporcinealveolarmacrophagecells foradditional20passages. In thepresentstudy,weassessed theefficacy of the cell-attenuated CA-2-MP120 strain as a modified live vaccine. Three-week-old PRRSV-free pigs were inoculatedintramuscularlywithCA-2-MP120(106.0TCID50)andthenchallengedintramuscularlywiththe20thpassagevirusofCA-2strain(CA-2-P20,106.3TCID50/2ml)at57dayspost-immunization(dpi).Allanimalswereeuthanizedat14dayspost-challenge(dpc)forpost-mortemexamination.Noneofpigletsshowedanyobviouschangesindailyweightgain,bodytemperature,orclinicalsignsofdiseaseduringtheexperiment.Theresultsshowedthatseroconversionoccurredinallvaccinatedpigsby11dpi,reachingthemaximumS/Pratiovalueat21dpi.Althoughallpigsinthevirus-challengedunvaccinatedcontrolgroupseroconvertedby7dpc,theirmeanS/Pratiosweresignificantlylowerthanthevirus-challengedvaccinatedgroup.Allvaccinatedanimalsdevelopedviremiaby3dpithatpersisteduntil54dpi,whileunvaccinatedcontrolgroupsremainedviremia-negativeduringthe54-daypost-immunizationperiodbutwereviremicat4dpc(61dpi).Interestingly,noviremiawasdetectedinthevaccinatedpigsduringthe14-daypost-challengeperiod.Furthermore,nopigsinthevirus-challengedvaccinatedgroupshedvirusnasally,orallyorrectallythroughoutthestudy.Comparedtothechallengecontrol,vaccinatedpigsshowedmuchmilderpathologicallungandlymphnodelesions.TheseresultsindicatedthatCA-2-MP120canprovideeffectiveprotectionagainstthevirulentwild-typestrain.Altogether,ourdatasuggestthattheattenuatedCA-2-MP120strainisapromisingcandidatefordevelopingasafeandefficaciouslivePRRSVvaccine.P111-Double-strandedviralRNApersistsinvitroandinvivoduringprolongedinfectionofPRRSVR.Guo1,P.Shang1,C.A.Carrillo2,X.Yan1,T.Wang1,C.J.Jaing2,M.Niederwerder1,R.R.R.Rowland1,Y.Fang1.1DiagnosticMedicine&Pathobiology, Kansas state University, Manhattan, KS, USA, 2Lawrence Livermore National Laboratory, Livermore, CA, [email protected]:ViralPathogenesis,12/4/20175:30PMPorcinereproductiveandrespiratorysyndromevirus(PRRSV)infectioncanbedividedintoatleasttwodistinctstages:acuteinfectionandpersistence.Initialacuteinfectionleadstotherapidcytopathicreplicationofthevirusinhostcells,resultinginthereleaseoflargeamountofPRRSVintothebloodcirculation.Theinfectionthenprogressesintoasymptomaticpersistentstage,inwhichthevirusisnolongerdetectedinbloodandviralreplicationisprimarilylocalizedinlymphoidorgans, includingtonsilandlymphnodes.Currently,littleisknownaboutthemechanismofPRRSVpersistence.Inthisstudy,acellularmodelofpersistentinfectionwasestablishedusingPRRSV-infectedMARC-145cellspassaging109timesinvitro.Strand-specificquantitativeRT-PCRanalysisrevealedthatplus-andminus-strand viral RNAswerepresent at nearly equivalent levels; and immunofluorescencemicroscopyandRNAase I treatment analysisshowedthatdouble-strandedRNA(dsRNA)conformationexistedinpersistentlyinfectedcells.Incontrast,plus-andminus-strandviralRNAswerepresentatabout46:1ratioinacuteinfectedcells.Consistentwiththedatageneratedfrominvitrocellculturesystem,therewasabout3.3-foldreducedratioofplusversusminus-strandviralRNAsinlymphoidtissuesfromPRRSV-infectedpigsat52dayspostinfection (dpi), in comparison to thatofPRRSV-infectedpigs at10dpi. The resultswere further confirmedusing lymphoid tissuescollected from pigs at 70 dpi, in which reduced ratios of plus versus minus-strand viral RNAs were consistently detected.ImmunohistochemistryanalysisshowedthatviraldsRNAsweredetectedaggregatinginsidethegerminalcentersoftonsilandlymphnodesfromPRRSVpersistencepigs,whilemostofthedsRNAsweredetectedinmarginalzonesoflymphoidtissuesinacutePRRSV-infectedpigs.OurresultssuggestthatthePRRSVdsRNAcouldfunctionasamediatorforviralpersistence.TheviraldsRNApersistenceingerminalcentersoflymphoidtissuesmayrevealanovelmechanismforPRRSVtoescapeantiviralimmuneresponses.

CRWAD2017 ABSTRACTS


P112-Theeffectsofbovineherpesvirus1onmonocyte-deriveddendriticcellcytokineproductionandsurfacemarkerexpressionJ.Sobraske,K.Abdelsalam,C.Chase.SouthDakotaStateUniversity,Brookings,SD,[email protected]:ViralPathogenesis,12/4/20175:30PMPurpose:Cowsinfectedwithbovineherpesvirus1(BHV-1)areknowntohaveincreasedratesofabortions,respiratoryproblems,andotherdiseases.IthasbeenhypothesizedthatBHV-1negativelyaffectsbovinedendriticcells.Thisspecificstudywastodetermineifmonocyte-deriveddedriticcell(MDDC)surfacemarkersandcytokineproductionwasaffectedafterinfectionwithBHV-1.Methods:Bloodwas collected from5-6monthHolstein Friesians andmonocyteswere isolated from the collectedblood.Monocyte-deriveddendriticcellswereproducedfromthemonocytes.TheMDDCswereinfectedafter7dayswithstrainsofCooperandLABHV-1andtimepointsweretaken.Aftersamplecollection,theMDDCswereanalyzedusingflowcytometryandqPCR.FortheqPCR,cytokinesproducedbyMDDCwereanalyzed.ResultsfortheqPCRwererunintheprogramREST,indicatingifthecytokineswereupordown-regulated.Results:Coopersignificantlydown-regulatesMHCI,MHCII,andCD86comparedto thecontrol.LAdown-regulatesCD86comparedtothecontrol,howeverCooperdown-regulatesmuchfaster.Coopershowssignificantdown-regulationofIL6,IL10,IL12,and IFN-gamma. LA shows significant up-regulation of IFN-alpha and IFN-beta as well as late down-regulation of IFN-gamma.Conclusions:Cooper,amuchmorevirulentviralstrain,hasamuchgreaternegativeeffectonMDDCsthanLAinaffectingbothsurfacemarkerexpression.P113-DevelopingnovelmoleculardiagnosticassaysforbovineleukemiavirusC.J.Droscha1,M.C.Frie2,O.J.Benitez3,P.C.Bartlett1,V.Ruggiero4.1AntelBioSystems,NorthStartCooperative,EastLansing,MI,USA,2CellandMolecularBiologyProgram,MichiganStateUniveristy,EastLansing,MI,USA,3ComparativeMedicineandIntegrativeBiologyProgram,MichiganStateUniveristy,EastLansing,MI,USA,4LargeAnimalClinicalSciences,MichiganStateUniveristy,EastLansing,MI,[email protected]:ViralPathogenesis,12/4/20175:30PMRecentresearchhasshownthatcattleinfectedwithbovineleukemiavirus(BLV)haveseriouslyalteredimmunesystemswhichprobablyaccountfortheirreducedmilkproduction,shortenedcowlongevityandlymphoma.Whileover21nationshaveeradicatedBLV,theU.S.hasseenprevalencegrowtoalmost50%ofalldairycows.Recentfindingsshowthataminorityofcattlehaveahighconcentrationofprovirus(proviralloadorPVL)andmayberesponsibleformosttransmissionwithintheherd.However,dairyproducersonlyhaveaccesstoanantibody-captureELISAthatcannotdistinguishthemostinfectiouscattleandcullingorsegregatingtheapproximately2/3ofELISA-positivecattlewhoselowinfectivityposeslittlethreattotheirherdmatesisnotfeasible.Developmentoffollow-updiagnostictestsareneededtostratifyanimalsbythreatoftransmission.NorthStarCooperativeincollaborationwiththeMichiganStateUniversityBLV research teamaredevelopingnovel PCR-basedmethods that can categoricallydefinediseaseprogressionwithinBLV-positivecattle.WepresenttheinitialdesigngoalsforaBLVPVLassaywhichshouldbespecificandsensitiveforallknownBLVgenotypes.InadditiontoPVL,quantificationofBLV-derivedmicroRNAsasapotentialbiomarker fordiseaseprogression isalsobeingpresented.ThesenewdiagnostictoolsforBLVareessentialcomponentsofaBLVcontrolprogramthatcanpreservethesustainabilityoftheU.S.dairyindustryinaglobalmarketwheremanyothernationshavemadeBLVcontrolapriority.

CRWAD2017 ABSTRACTS


P114-Regionalseroprevalence&identificationoflocaldominantepitopesforthedevelopmentofimmunodiagnosticassaysT.Osorio-Gallardo1,F.Espinosa-Castillo2,L.Martínez-Chavarría3,A.Benítez-Guzmán4,J.Hernández-Medrano2.1FacultaddeMedicinaVeterinariayZootecnia,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico,Mexico,2Reproduction,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico,Mexico,3Pathology,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico,Mexico,4Microbiology and Inmunology, Universidad Nacional Autónoma de México, Ciudad de México, [email protected]:ViralPathogenesis,12/4/20175:30PMPurpose:Tropicalcattleproductionsystems(TCPS)holdnearly55%ofMexico’snationalherd,yethealth&epidemiologicalinformationisscarceandinnacurate.InfectiousdiseasesinTCPS,suchasBVDareamongthemaincausesoflowproductivity&infertility.BovineViralDiarrhea(BVD)isaworldwidespreaddiseasecausedbyaPestivirus,withprevalencerangingfrom25to96%inTCPS.Thankstoacollaborativeworkbetweengovernment(SAGARPA)&theVetSchoolatUNAM,anationalserumbank(NSB)wascreated.Theaimof thepresent studywas toassessBVDherdseroprevalence in themainTCPSstates inMexicousing theNSB.Also,we identifiedinmunodominantepitopesbywesternblot(WB)todeveloprecombinantproteinsforaimmunodiagnosticmultiplexsystem(Luminex).Methods:Pooledserumsamplesbyherd(n=12animals)wereassayedusingIDEXXBVDVELISAkitsfollowingmanufacturer’sadvice.WBstoidentifyimmunodominantepitopesusedBVDVNADLstrainincubatedwithcommercial&wild-typepositiveantisera.Results:Inthe5mostimportantTCPSstates,seroprevalenceforBVDatherd-levelrangedfrom67.2to92.6%(mean=81%).UsingWB,threebandsof~78,47&40kDawereidentified,correspondingtoviralproteinsNS3&E2.Thesewillbeusedforamplification&subsequentrecombinantimmunodominantproteinproductionusingE.coli.Thesynthesisoftherecombinantproteinisanticipatedtoserveinthedesignofanewmultiplextestthatisintendedtosimultaneouslydiagnoseseveralabortivediseasesinthenearfuture.Conclusions:Usingpositiveserawewereabletodetect2mainepitopesthatarerecognisedbyantibodiesofnaturallyinfectedanimalswhichwillallowthedevelopmentofspecificimmunodiagnosticassay.Thisstudy,whichispartofaconsortiumtoidentify&produceproteinswithimmunodiagnosticvaluetodevelopamultiplexdiagnosticsystem(Luminex)forthemostimportantcattleabortivediseasesinMexico,showstheveryhighprevalenceofBVDinthenationalherdthatcouldbethecauseoflowproductivity.Thus,developmentofacountry-specificdiagnostictooltoidentifyBVDinfectedcattleisparamount.P115-Surveillanceforandcharacterizationofnon-primatehepacivirusineastAlabamaM.M.Mitchell,N.Pastrana-Negron,A.Mohamed,P.G.Reddy,S.O.Odemuyiwa.Dept.ofPathobiology,CollegeofVeterinaryMedicine,TuskegeeUniversity,Tuskegee,AL,[email protected]:ViralPathogenesis,12/4/20175:30PMPurpose:InfectionwithhumanHepatitisCvirus(HCV)isstronglyassociatedwithhepaticcirrhosisandhepatocellularcarcinoma.HCVinfection is the leading cause of liver transplants in the world. The virus is not readily cultured in vitro and there are noimmunocompetentsmallanimalmodelsofHCVinfection.ItisthereforeverydifficulttoconductinvitroandinvivostudieswithHCV.In2012,non-primatehepacivirus(NPHV),avirushomologoustoHCVwasfoundinhorses.TheaimofthisstudyistodeterminethegenotypiccharacteristicsofNPHVinEasternAlabama.Methods:UsingnestedRT-PCRwithdegenerateprimerstargetinga300-base-pairfragmentintheNS5Bregionofthevirus,totalRNAextractedfromseraofhorsesvisitingtheTUEquineHealthFairbetween2015and2016wereprobed forNPHV.Gel-purified ampliconswere sequenced.Nucleotide sequenceswere alignedusing theClustalWprogram.PhylogenetictreeswereconstructedandvisualizedusingMEGA7.Results:Of283serumsamplesanalyzed,15(5.3%)yieldedNPHV isolates. Alignment and phylogenetic analysis of nine isolates showed clustering of NPHV and HCV. Isolates of NPHV fromTuskegee are closely related to isolates from other parts of theworld.Conclusions: These results suggest that NPHV infection iswidespread inhealthyhorses inAlabamaas reported inotherpartsof theworld. Inaddition, the rapidglobal spreadofdisparategeographicalvariantsofNPHVprovidesauniqueepidemiologicalmodelofemergenceandspreadofnewblood-bornepathogensinthetwenty-firstcentury.

CRWAD2017 ABSTRACTS


P116-InvitroinvestigationofthepotentialofZikavirustoinfectlivestockandthedevelopmentofanalternativeanimalmodelC.S.Kyriakis1,R.A.Palomares2,S.M.Tompkins1.1CenterforVaccinesandImmunology,CollegeofVeterinaryMedicine,UniversityofGeorgia,Athens,GA,USA,2FoodAnimalHealthandManagement,DepartmentofPopulationHealth,CollegeofVeterinaryMedicine,UniversityofGeorgia,Athens,GA,[email protected]:ViralPathogenesis,12/4/20175:30PMZikavirus(ZIKV)isamosquito-borneflavivirus,firstidentifiedinmonkeysin1947inUganda.Humaninfectionsweredetectedin1952inUgandaandTanzania.ZIKVwasneglectedfordecadesbecause(a)itwasnotassociatedwithdiseaseand(b)ZIKVinfectionsweresignificantlyunderestimatedinserologicalstudies,sinceantibodiesagainstZIKVcross-reactedwithotherflaviviruses.In2007,amorevirulentZIKVstrainemergedinMicronesia,accompaniedwithsymptomsthatincludedfever,skinrashes,conjunctivitis,muscleandjointpain,andheadache.OtheroutbreaksfollowedinthePacificandin2015thevirusreachedtheAmericaswithoutbreaksoccurringfromBraziltoPuertoRico.MicrocephalyassociatedwithZIKVwasreportedinBrazilandwaslaterconfirmedwithinvitroandinvivostudies.WhilethecurrentZIKVoutbreakisongoing,aquestionthatneedstobeanswerediswhetherfarmanimalsmightserveasreservoirsforthevirus.Furthermore,althoughmousemodelsareavailable,amorereliableexperimentalanimalforthestudyofZIKVpathogenesis, transmissionandvaccineefficacy isdesirable.To investigate thepotentialofZIKV to infect livestock,bonemarrow-derivedmesenchymalstemcells(MSCs)ofswine,ovineandequineoriginwereinfectedwithtwoZIKVstains:Nigeria/1968andPuertoRico/2015at amultiplicity of infection (MOI) of 0.05 and0.5. Cell supernatantswereharvested at different timepoints and ZIKVreplicationwasquantifiedbyplaqueassay.Nigeria/1968atlowMOIwasnotrecoveredfromequineMCSandinlowtitersfromswineandovinecells,whilePuertoRico/2015replicatedmoreconsistently inovineMCScomparedtoswineandequineMSCs.ThesametrendwasnoticedinMSCsinfectedwiththehighMOI.OvinecellsweremorepermissibletoZIKVcomparedtoswineandequineMSCs.Overall,PuertoRico/2015replicatedathigherlevelscomparedtoNigeria/1968.TheseresultsindicatepossiblesusceptibilityofsheeptoZIKVcomparedtopigsorhorses,stressingouttheimportanceofanimalsurveillance.Additionally,anovinemodelforthestudyofZIKVcouldbedevelopedasanalternativetothelessreliablemousemodels.P117-TheeffectofmacrophagedepletionagainstH4N6lowpathogenicavianinfluenzaviralinfectioninchickenM.S. Abdul-Cader1, G. Ehiremen2, E. Nagy3,M. Abdul-Careem4. 1University of Calgary, University of Calgary, Calgary, AB, Canada,2UniversityofCalgary,Calgary,AB,Canada,3DepartmentofPathobiology,UniversityofGuelph,Guelph,ON,Canada,4DepartmentofEcosystemandPublicHealth,UniversityofCalgary,Calgary,AB,[email protected]:ViralPathogenesis,12/4/20175:30PMClodronateliposomeshavebeenusedforthedepletionofmacrophagesinmouse-viralinfectionmodels,butithasnotbeenextensivelyused to study the roleofmacrophagesduring viral infections in chickens.Wehypothesized that thedepletionofmacrophages inchickens using clodronate liposomes will alter the replication of H4N6 low pathogenic avian influenza virus (LPAIV) infection inrespiratoryandintestinaltractsofchickens.Ourobjectivesaretoseewhether1)H4N6LPAIVinfectionalterthemacrophagenumbersinrespiratoryandgastrointestinaltractsand2)clodronateliposomesdepletemacropahgesintherespiratoryandgastrointestinaltractsof chickens changing the H4N6 LPAIV replication in these tissues.We used clodronate liposomes intra-abdominally in 5 days-oldchickensandfoundsignificantdepletionsofmacrophages inrespiratory(trachea)andgastrointestinal (duodenum)tractsat4dayspost-treatment.WhenweinfectedthechickensthataredepletedofmacrophagesalongwithmacrophageintactchickenswithH4N6LPAIVatday6,wefoundincreasedH4N6LPAIVreplicationintrachea,butnotinduodenumofclodronateliposometreatedchickens.Furthermore,weobservedthatH4N6LPAIVinfectionitselfinducedexpansionofmacrophagenumbersintracheaandduodenuminchickensat3dayspost-infection.TheresultsofthisstudyindicatetheimportanceofmacrophagesininnateantiviralresponseagainstH4N6LPAIVinfectionatleastintracheaofchicken.

CRWAD2017 ABSTRACTS


P118-Developmentofareal-timeRT-PCRassayfordetectingporcineparainfluenzavirus1infectioninpigsY.Li1,Y.Fang1,C.Stahl2, J.Bai3,X.Liu3,L.Peddireddi3,G.Anderson1. 1DiagnosticMedicine/Pathobiology,KansasStateuniversity,Manhattan,KS,USA, 2FairmontVeterinaryClinic,Fairmont,MN,USA, 3KansasStateVeterinaryDiagnosticLaboratory,KansasStateuniversity,Manhattan,KS,[email protected]:ViralPathogenesis,12/4/20175:30PMPorcineparainfluenzavirus1(PPIV1)wasfirstreportedinthepignasopharyngealsamplesinHongKongin2013.Recently,PPIV1wasdeterminedtobewidespreadinUScommercialswineherds.However,novalidateddiagnosticassayisavailableforPPIV1detection.Inthisreport,aone-stepreal-timequantitativeRT-PCRassay(qRT-PCR)targetingtheviralhemagglutinin-neuraminidase(HN)geneofPPIV1wasdevelopedandvalidated.No cross-reactivitywasobservedwithnucleic acidprepared fromcommon swinepathogens,includingPRRSV,PCV2,IAV.Thelimitofdetectionwasdeterminedtobe10copiesper20μlreactionusinginvitrotranscribedHNRNA.Theperformanceofthisassaywasfurthervalidatedwith120pignasalswabs(60knownpositiveand60knownnegativeforPPIV1)and20oral fluid (8knownpositiveand12knownnegative forPPIV1).TheqRT-PCRresultswereconsistentwithRT-PCRandDNAsequencingofHNgene.Thisassaywasfurtherusedtoscreenthediagnosticsamplescollectedfrom10differentfarms.Among310nasalswabsamplesthatwehavetested,202samplesfrom8farmswerePPIV1positive.Overall,thisqRT-PCRassaydemonstratessufficientsensitivityandaccuracyfordetectingPPIV1RNA.ItwillbeausefultoolfortherapiddiagnosisofPPIV1infectionandinaidofPPIV1epidemiologicalsurveillance.P119-AdversefetaloutcomesinpregnantrabbitsexperimentallyinfectedwithrabbithepatitisEvirusH.Ahn,B.Park,S.Han,Y.Kim,H.Go,D.Kim,J.Lee,S.Park,C.Song,S.Lee,I.Choi.CollegeofVeterinaryMedicine,KonkukUniversity,Seoul,Korea,[email protected]:ViralPathogenesis,12/4/20175:30PMPurpose:HepatitisEvirus(HEV)causesseverehepatitisinpregnantwomen,withassociatedpoorfetaloutcomes.TostudyHEVviralpathogenesis,pregnantrabbitswereinfectedwithlow-andhigh-doserabbitHEVat2weeksgestation.Methods:Atotalof18pregnantSPFrabbitsweredividedintothreegroups:6rabbitseachofnegativecontrol,low-dose(103GEofrabbitHEV),andhigh-doseHEV(106GE of rabbit HEV) infection groups. Serum and fecal samples were collected before virus inoculation and at 7 and 14 days postinoculation(dpi).ThelevelsofALT,AST,TNF-α,IL-2,IL-4,IL-10,IL-12,andIFN-γweredeterminedwithserumsamplescollectedfrompregnantrabbitsofthethreeexperimentalgroups.Results:HEVwasidentifiedintheserum,feces,andlivertissueofinfectedrabbits,anddose-dependentfetalmortalityratesrangingfrom67-80%wereobserved.Theaspartatetransaminase(AST)/alaninetransaminaseratiowassignificantlyhigher(P<0.01)inhigh-doseinfectedrabbitsthanlow-doseinfectedandnegativecontrolrabbits14dayspostinfection(dpi).Tumornecrosisfactor-α(TNF-α)wassignificantlyhigherinlow-dose(P<0.01)andhigh-doseinfectedrabbits(P<0.001)thaninnegativecontrols7dpi.High-doseHEV-infectedrabbitsproducedsignificantlymoreinterferon-γ(IFN-γ;P<0.05)thannegativecontrolrabbitsat7and14dpi.HighlevelsofAST,TNF-α,andIFN-γmaysubstantiallyinfluenceadversefetaloutcomesinpregnantrabbits infectedwith high-dose HEV.Conclusions: Collectively, high production of AST, TNF-α, and IFN-γ seemed to substantiallyinfluenceadversefetaloutcomesinpregnantrabbitsinfectedwithrabbitHEV.AbstractNumber:

CRWAD2017 ABSTRACTS


P120-EfficacyofacommercialPCV2vaccineagainstthecontemporaryPCV2dstrainO.Kolyvushko1,G.Singh1,B.Webb2,A.Pillatzki3,D.Diel3,S.Dillberger-Lawson3,E.Nelson3,S.Ramamoorthy1.1MicrobiologicalSciences,NorthDakota StateUniversity, Fargo,ND,USA, 2VeterinaryDiagnostic Laboratory,NorthDakota StateUniversity, Fargo,ND,USA,3AnimalDiseaseResearchandDiagnosticLaboratory,SouthDakotaStateUniversity,Brookings,SD,[email protected]:ViralPathogenesis,12/4/20175:30PMSince its identificationasthecausativeagentofpost-weaningmulti-systemicwastingdiseasesyndrome,porcinecircovirusstrain2(PCV2)hasperiodicallyevolvedtoemergeasnewstrains.ThecurrentlycirculatingPCV2strainsincludePCV2a,b,c,dande,withPCV2dbeingthelatesttoemergein2012.Fieldevidencesuggeststhatthenewlyemergingstrainsdifferinvirulencefromcirculatingstrains,whileexperimentalevidenceisinconsistent,andallcurrentcommercialvaccinesagainstPCV2containthePCV2avirusoritscapsidprotein.Inthisstudy,wetestedtheefficacyofacommercialPCV2a-basedvaccineagainstthenewlyemergedPCV2dvirus.PureculturegeneratedfromaPCV2dinfectiousclonewasusedtochallengepigsimmunizedwiththeselectedcommercialvaccine,atadoseof105TCID50/ml. Strong PCV2-specific antibody responses and virus neutralizing responseswere detected in immunized pigs. Viremia inserum,asdetectedbyqPCR,showedthatthevaccinatedpigshadameanvalueoflessthan0.5logTCID50/ml,whilecontrolpigshadavalueof 2.5 log TCID50/ml.As assessmentof antigen load in lymphoid tissuesby immuno-histochemistry, showed that vaccinatedanimals had no antigen accumulation in themesenteric, tracheal lymphnodes or tonsils,while the unvaccinated controls had anaveragescoreof2.14.Hence, thecommercialvaccinetestedwashighlyeffectiveagainst thePCV2dvirus,under theexperimentalconditionsofthestudy.P121-Developmentofareal-timepolymerasechainreaction(qPCR)methodforrapidandaccuratedetectionoftoxicMicrocystis

spp.basedonmicrocystinsynthetaseC(mcyC)geneJ.Yuan1,H.-J.Kim2,S.Ensley1,B.Guo1,K.-J.Yoon1.1DepartmentofVeterinaryDiagnosticandProductionAnimalMedicine,IowaStateUniversity,Ames,IA,USA,2FoodScienceandHumanNutrition,IowaStateUniversity,Ames,IA,[email protected]:BiosafetyandBiosecurity,12/4/20175:45PMPurpose:Microcystis areubiquitously living in freshwater and some species are known toproducemicrocystin - a hepatotoxin toanimalsandhuman.MostMicrocystisspp.areabletoformalgalbloomsincludingtoxicspeciesundercertainconditions,andthetoxicblooms can be harmful to animals and human. Therefore, it is crucial to detect the presence of toxigenicMicrocystis spp. in theenvironmentespeciallyattheearlystageofbloomformation.Nowadays,moleculartechniquespresentafastandconvenientwayformicroorganismdetection.ThefactisalsoadvantageousformoleculardetectionthatalltoxigenicMicrocystisspp.sharetheidenticalgeneticfeatureinmicrocystinsynthesis,i.e.allpertainingproteinsaretranslatedfromthesamemicrocystinsynthetase(mcy)genecluster.Methods:Thisstudywastodeveloparapidandaccuratenucleicacidbasedmethodinaquantitativemanner(i.e.,quantitativereal-timePCR)fordetectingthetoxigenicMicrocystisspp.inwatersamplesbasedonmcyCgene.Inaddition,anotherqPCRmethodtodetect allMicrocystis spp.was establishedbasedon16S rRNAgene.Results:Bothmethodshad the same limit of detection as 5copies/reaction.Whenapplyingon100watersamplesfrom5pondsaroundswineoperationsintheUSMidwestcollectedweeklyfromAugusttoOctoberin2016,97sampleswerefoundcontainingvariousdensitiesoftoxigenicMicrocystisspp.,rangingfrom0.1to1.7×103cells/mL.Bycontrast,Microcystisspp.existedinall100sampleswithadensityof17.4to7.6×104cells/mL,suggestingthattoxigenicMicrocystisspp.werenotdominantinthedetectedMicrocystispopulation.Twenty-oneoutofthe97toxigenicMicrocystis-positivesampleswerefoundbyacommercialELISAkittocontainmicrocystin(0.01-0.49ppb);however,toxinconcentrationwasnotcorrelatedwiththeamountoftoxigenicMicrocystisspp.Conclusions:Inconclusion,thePCRmethodcanproviderapidandaccuratedetectionoftoxigenicMicrocystisspp. inpondwaterservinganimalproduction.It,however,remainstobefurtherstudiedifanimalhealthandproductioncanbeimpairedbyuseofpondwatercontainingalowlevelofmicrocystin.

CRWAD2017 ABSTRACTS


P122-IdentificationandcharacterizationofnovelBrucellamelitensisvirB-T4SSeffectorproteinsA.L.C. Cabello1, K. Patrick2, J.A. Wojcechowskyj3, D.F. Meyer4, C. Noroy4, R. Watson2, T. Ficht1, P. DeFigueiredo2. 1VeterinaryPathobiology,TexasA&MUniversity,CollegeStation,TX,USA,2DepartmentofMicrobialPathogenesisandImmunology,TexasA&MUniversityHealthScienceCenter,Bryan,TX,USA,3DepartmentofCellularandMolecularPharmacology,UniversityofCalifornia,SanFrancisco,CA,USA,4CIRAD,UMRCMAEE,Petit-Bourg,Guadeloupe,[email protected]:BacterialPathogenesis,12/3/20176:30PMAnimportantmechanismthatBrucellausestosurviveandreplicateinsidethehostisamacromolecularmachinecalled“virB/type4secretionsystem(virB-T4SS)”.VirB/T4SStranslocatesproteinsthatareessentialforBrucellatoestablishareplicativenicheandcausediseasebymodulatinghostfunctions.AmajorobstacletoourunderstandingoftheintracellularlifecycleofBrucellaisthepoorrateofidentificationoftheseeffectorsproteins,coupledwiththefailuretodefinetheirmoleculareffectsonthehostcell.Currentscreeningmethods for identification of putative effectors proteins fail to consider all known effector protein features and are incapable ofscreening the complete bacterial genome. To address these concerns, theBrucella genomewas analyzed by an enhanced searchalgorithmforT4SSeffectors(T4Es)(Meyeretal.2013).Weanalyzedthetwomostprevalentstrains,B.melitensisstrain16MandB.abortusstrain2308,aswellasB.abortusstrain19,avaccinestrainthathasbeenusedinbrucellosiseradicationprograms.Theanalysisrevealed45T4EscommontoallthreeBrucellastrains,whichwereselectedforfurtheranalyses.Aproteincomparisonanalysis(PATRIC)revealedthat34outof45T4Eswerefoundinother intracellularbacteriawitha low%of identity,whereas11werefoundonly inBrucella.TogaininsightintothebiologicalfunctionofBrucella’sT4Es,wedeterminedtheirsubcellularlocalizationinHeLacells.TheT4EsdisplayedhighlyvariedsubcellularlocalizationssuchasthenucleusandER.TounderstandthemolecularmechanismsofBrucella’sT4Es,weperformedepistaticminiarrayprofile(E-MAP)experimentsinyeastSaccharomycescerevisiae.GeneticinteractionsbetweenthehostandBrucellaeffectorswererecognizedandclassifiedaccordingtogeneontologyterms.ThisanalysisclarifiedthespecificandconservedeukaryoticpathwaystargetedbyBrucella’sT4EsforfuturestudiesinthecontextofBrucellainfection.FutureworkincludesvalidatingtheVirB/T4SSdependenttranslocationofidentifiedT4EsinvitroandabiologicalanalysistodemonstrateT4Esfunctionandvirulenceinvitroandinvivo.P123-InitialinvestigationofBurkholderiapseudomalleiinpigsinNgheAnprovince,VietnamH.T.T.Nguyen1,H.T.T.Tran1,T.T.Trinh2.1GeneralLaboratoryandConservationGene,NationalInstituteofVeterinaryResearch,Hanoi,VietNam,2InstituteofMicrobiologyandBiotechnology,VietnamNationalUniversity,Hanoi,[email protected]:BacterialPathogenesis,12/3/20176:30PMMelioidosisisabacterialinfectionofanimalsandpeople.InVietnam,casesofmelioidosisinhumanhavebeendiscoveredrecently.However,since1972therewasnofurtherstudiesofmelioidosisinanimals.WeconductedasurveytodeterminethepresenceofB.pseudomalleiinfectioninpigsinNgheAnprovincewheretherewerepatientswithmelioidosis.Thefirstsamplingincluded119samples(43blood,40swabthroat,11urine,9lungsofasymptomaticpigsofdifferentages;8soiland8water),takenatthebeginningofrainyseason(30/07-01/8/2016)inhouseholdfarmers,farmsandslaughterhouses,culturedonTSBmediumthentransferredtobloodagar,MacConkey,TSA,thenAshdownagar.Thesecondsamplingcontained100samplesofpigswabthroatcollectedlate-rainyseason(15-20/10/2016) in household farmers, farms and slaughterhouses, cultured on Ashdown broth and then if positive Realtime-PCR,transferredtoAshdownagar.Isolation,identificationofbacteriaandantimicrobialsusceptibilitytestweredoneasusual.119samplescollectedduringthefirstrounddidnotisolateB.pseudomallei.Ofthe100samplestakeninthesecondround,25samples/5householdfarmersdidnotisolatethebacterium.From35swabthroatsamples(amongthem9sickpigs)/02pigfarmsisolatedonestrainofB.pseudomalleiinsickpigwhichskippedmealfordays(2.86%).B.pseudomalleiisolatewashighlysusceptibletoamoxicillin/A.clavulanic,ciprofloxacin,chloramphenicol,tetracylin.Itwasresistanttogentamicin,colistinandceftazidim.From35swabthroatsamplesofpigs(9sickpigs)/02farmscollectedattheendofrainyseason,culturedinAshdownbroththenifRealtimePCRpositivetransferedonAshdown agar, we isolated 01 specimen of B. pseudomallei (2.86%). This B. pseudomallei was highly susceptible toamoxicillin/A.clavulanic, ciprofloxacin, chloramfenicol, tetracylin and resistant to gentamicin, colistin antibiotics andat the limit ofresistancetoceftazidim.WeneedmoreresearchinparticularonsickpigswithhypothesisthatsamplesbeingculturedonAshdownbrothandthenrealtimePCR,ifpositive,culturedonAshdownagarcouldfindthebacteria.

CRWAD2017 ABSTRACTS


P124-CharacterizationofCampylobacterjejunitlp2anditsroleinpathogenesisandcolonizationofchickengutK.Chandrashekhar1,V.Srivastava1,S.Hwang2,B.Jeon3,S.Ryu2,G.Rajashekara1.1FoodAnimalHealthResearchProgram,DepartmentofVeterinaryPreventiveMedicine,TheOhioStateUniversity,Wooster,OH,USA, 2DepartmentofFoodandAnimalBiotechnology,Seoul National University, Seoul, Korea, Republic of, 3School of Public Health, University of Alberta, Edmonton, AB, [email protected]:BacterialPathogenesis,12/3/20176:30PMCampylobacterjejuniisacommoncauseofbacterialfoodpoisoninginhumansworld-wide.Chickensarethemajorsourceofhumaninfections.Despiteoftherecognizedsignificanceofthispathogen,noeffectiveandpracticalinterventionsavailabletoreducehumaninfectionsortolimitC.jejunicolonizationinchickens.Thus,thereisanimmediateneedforidentifyingnovelpathogenicityandvirulencedeterminantsinordertodevelopeffectivecontrolandpreventivestrategiesagainstC.jejuni.ItiswellestablishedthatC.jejuniemploysmotilityandchemotaxistocolonizeintheavianandmammaliangastrointestinaltract.DirectionalmotilityinC.jejuniismediatedbythechemotaxissystem,composedofchemoreceptorsandothercoresignaltransductionproteins.Transducerlikeproteins(Tlps)arethekeycomponentsinvolvedinsensingenvironmentalsignalsthroughchemotaxisinC.jejuni.Inthisstudy,wecharacterizedtheroleoftlp2inchemotaxis,stresssurvival,in-vitrovirulenceandcolonizationinchickengut.OurstudyshowsthatΔtlp2mutantwasdefectivein chemotaxis towards aspartate, pyruvate, Pi and iron (FeSO4)with RCR values < 2 (P≤0.05). Tlp2 expressionwas induced aftertreatmentwithPiandFeinWTaswellasinΔtlp2mutantbypromoterreportergeneassays.Themutantphenotypeswererestoredaftercomplementationwithtlp2gene.Further,overlappingRT-PCRindictedthatthephoXgene,locatedimmediatelydownstreamoftlp2,isco-transcribedwithtlp2.Althoughdefectiveininvasion,theΔtlp2mutantexhibitedanincreasedintracellularsurvivalinINT407cells,comparedtotheWTstrain.Thetlp2geneisalsorequiredforachievingoptimalcolonizationofC.jejuniintheproximalanddistalsegmentsofthegastrointestinaltractofchicken,asΔtlp2mutantshowed4to5loglessCFUcomparedtotheWT.Thisstudyemphasizestheimportanceoftlp2inchemotaxis,infectionofhostcellsandcolonizationinchickengastrointestinaltract.P125-RecenttrendsofcanineleptospirosisintheUnitedStates:spatial,temporal,environmentalandanimal-levelriskfactorsA.M. Smith, A.G. Arruda, T.E.Wittum, J.W. Stull. Veterinary PreventiveMedicine, College of VeterinaryMedicine, TheOhio StateUniversity,Columbus,OH,[email protected]:CompanionAnimalEpidemiology,12/4/20175:45PMPurpose:Canineleptospirosisisareemergingzoonoticdiseaseofconcern,yetinformationonitsepidemiologyislacking.Theobjectivesof this study were to describe the recent temporal and spatial distribution of canine leptospirosis in the U.S., and to identifyenvironmental, seasonal, dog- andhuman-level factors associatedwith canine leptospirosis infection.Methods:Data from18,727canineleptospirosisPCRtestsrunintheU.S.betweenJanuary2015andDecember2016wereacquiredfromanationalcommerciallaboratory.Data on temperature, precipitation,water cover, human income and educationwere obtained frompublicly availabledatabases. Maps were created to identify states of high risk for leptospirosis, and generalized mixed logistic regression modelsaccountingforcountyandstatewereusedtoidentifysignificantpredictors.Results:Overalltest-positiveprevalenceacrosstheU.S.was5.5%;Texas(10%prevalence),Illinois(8.5%),Nebraska(8.2%),IowaandWestVirginia(each8.1%)hadthehighestprevalence.Thegreatestpercent increase inprevalenceover the2-yearperiodoccurred inArizona (197%).Prevalencevariedtemporally,with thegreatesttest-positiveprevalenceinthesummerandfall.RegionaldifferenceswerenotedwithinseasonaltrendsforthewesternandsouthernU.S. regions: prevalence in thewestern regionpeaked in fall andwinter, andprevalence in the southernU.S. remainedrelativelyconstantthroughouttheyear.Femaledogswereatloweroddsoftestingpositiveascomparedtomales(OddsRatio=0.80,p-value=0.001).Dogs≤4yearsofagewereatgreateroddsoftestingpositivethanallotheragegroups(p-value<0.001).Dogstestedinstateswithgreatermonthlyprecipitationhadagreateroddsoftestingpositive(OR=1.2,p-value=0.023).Conclusions:Theseresultshighlightimportantrecentchangesintheepidemiologyofcanineleptospirosis,allowingforabetterunderstandingofthiscomplexdiseaseandtargetededucationandpreventioneffortsatclients/dogswithgreatestdiseaserisk.

CRWAD2017 ABSTRACTS


P126-LeptospirosisinruralAppalachiaB.Beigel,C.Smola,K.Murphey,R.Goss,B.Kitts-Morgan,U.Christmann,P.Nader,J.Roberson,A.Verma.LincolnMemorialUniversityCollegeofVeterinaryMedicine,Harrogate,TN,[email protected]:CompanionAnimalEpidemiology,12/4/20175:45PMPurpose: In this study, we investigated leptospiral contamination of surface water, presence of leptospires in wild rodents andseroprevalenceofleptospirosisincattleandhorsesinthetri-stateareaofTennessee,VirginiaandKentucky.Methods:Wescreenedsurfacewatersamples,andkidneysoftrappedrodentsbyarealtimequantitativePCRthattargetsLeptospira-specificlipl32gene.Serafromcattleandhorseswerescreenedfor leptospiralantibodiesusingmicroscopicagglutinationtest,which is thegoldstandard inserodiagnosisofleptospirosis.Results:Ourresultsshowleptospiralcontaminationofopenwatersourcesonanimalfarms(5%)andpresenceofleptospiralDNAinkidneysof60.3%oftrappedrodents(n=101).Moreover,MATanalysesoffarmanimalsshowpresenceofleptospiralantibodiesinapproximately40%ofhorses(n=31)and33.3%ofcattle(n=21).Conclusions:Inconclusion,resultsfromourstudyshowthepresenceofthepathogenintheenvironmentwater,carriagebywildrodentsandexposureinfarmanimals.Thepublichealthimplicationsofthesefindingsremaintobeassessed.P127–Evaluationofantimicrobialresistancegenesduringavianmanurecompostingprocess.F.Esperón1,M.Delgado2, I. Iglesias,Sr.3,M.Carballo1,M.Ugarte-Ruiz4,M.Moreno4,J.Tadeo2,A.de laTorre1.1INIA-CISA,Madrid,Spain,2INIA,Madrid,Spain,3UniversityofMinnesotaCollegeofVeterinaryMedicine,Madrid,Spain,4UCM-VISAVET,Madrid,[email protected]:EcologyandManagementofFoodborneAgents,12/4/20175:45PMPurpose:Antimicrobialresistanceisanemergingandglobalproblem.Itisestimatedthatby2050mortalityfrommultidrug-resistantbacteriawilloutlastcancermortality.Therefore,thereiscurrentlyaremarkableefforttounderstandthemechanismsofresistance,topromotetheresponsibleuseofantimicrobialsandtoseekeffectivetherapeuticalternatives.Whilemostlivestockstudiesarefocusedalongthefoodchain(“fromfarmtofork”),therearefewavailablepapersontheroleoflivestockmanureinthespreadandpersistenceofantimicrobial resistance.Animalwaste representsanenvironmentalproblemof livestock industry.Onepossible solution to thisenvironmentalproblemisthedirectapplicationofslurrytocrops;however,itmayfavorthetransmissionofantimicrobialresistancefromcattletovegetables.Analternativeontheenvironmentalmanagementofmanureistherealizationofacompostingpriortotheapplication of the slurry. The objective of this work is to evaluate the impact of the composting process on the persistence ofantimicrobialresistancegenes.Methods:Acompostingof10weeksofdurationhasbeencarriedoutfromstrawandavianmanure,fromalayinghenproduction.Compostingsamplesweretakenintriplicateattheendofeachweek,andtotalDNAwasextractedfromeach.21genescodingforresistancetotetracyclines,sulfonamides,phenicols,aminoglycosides,quinolones,betalactams,vancomycinandcolistinweredetectedandquantifiedbyreal-timePCR.Results:16ofthe21genesweredetectedinatleastonesample.Analysisofthetemporalevolutionofthesamplesshowsthatthereisamarkedreduction(>97%)inthegenescodingfortetracycline,quinoloneandmacrolideresistance,whileanincreaseinaminoglycosideandsulfonamideresistancegenesisobserved.Conclusions:Althoughthecompostingprocessdoesnotendupeliminatingtheantimicrobialresistancegenes,itcanbeconsideredagoodalternativetotheenvironmentalmanagementoftheavianmanure.

CRWAD2017 ABSTRACTS


P128-HusbandrypracticesandfactorsassociatedwithriskofbrucellosisinwinterpastureflocksinAzerbaijan.A. Azimov1, M. Khatibi1, D. Agalarov1, M. Shikhiyev1, T. Seyidov1, C. Suleymanova2, R. Jackson3. 11Agricultural CompetitivenessImprovementProject,MinistryofAgriculture,Baku,Azerbaijan,2AzerbaijanStateAgriculturalUniversity,Ganga,Azerbaijan,3MasseyUniversity,PalmerstonNorth,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMSmallruminantwinterpastureflocksarelargeflockscomprisedmainlyofsheepwithrelativelyfewgoats.Somemigrateannuallyfromtheirlowlandwinterbasestoalpinepasturesinthesummerwhileothersstayatorclosetohome.Aspartofanationwidebrucellosisserosurvey,100randomlyselectedmaturefemaleanimalsinuptofiverandomlyselectedwinterpastureflocksineachof43districtswere tested for presence of antibodies with the Rose Bengal Plate Agglutination Test and a confirmatory cELISA. The overallseroprevalencesinwinterpastureandvillagebasedflockswere2.47%and1.15%.Noseropositiveswerefoundin88ofthe197winterpasture flockswhichweretested.Aretrospectivecasecontrol study involving88negative flocks (controls)and108positive flocks(cases)wasconductedusingpersonal interviewswithflockmanagersto identifymanagementfactorsassociatedwithflockdiseasestatus.ResultsfrombivariateanalysesshowingRiskRatios(RR)with95%confidenceintervals inbracketsindicatedalowerriskforflockswhichmigratedthanforflockswhichstayedathome,RR0.4(0.3,0.7)andhigherrisksforchangingthemakeupofbreedsintheirflocks,RR1.6(1.3,2.1),buyingbreedinganimalsfrommarkets,RR1.6(1.2,2.2),havingcontactwithotherflocksinthesummerpastures,RR1.8(1.4,2.2)anddisposalofplacentasbyfeedingtodogs,RR2.5(1.7,3.5)asopposedtodisposalbyburying,RR0.5(0.4,0.7).Casefarmswerelesslikelythancontrolstoaskaveterinarianforhisrecommendationwhenbuyinganimals,RR0.6(0.5,0.8),quarantinenewlypurchasedanimals,RR0.5(0.4-0.7),considerbrucellosistobeaproblem,RR0.6(0.4,0.7)andonlybuyvaccinated,RR0.7(0.6,0.9).AmultivariablelogisticregressionanalysisinwhichallsignificantvariableswereenteredproducedamodelwithOddsRatiosof0.22(0.09,0.54)forstayingathomeandnotmigrating,2.32(1.12,4.81)forchangingbreeds,0.08(0.03,0.25)forconsideringbrucellosistobeaproblemand4.38(2.18,8.78)forfeedingplacentastodogs.Thestudyproducedimportantfindingsforincorporationintotheawarenesscampaignwhichisacomponentofthenationalbrucellosiscontrolprogram.P129-Theidentificationofriskfactorsassociatedwithfoot-and-mouthdiseaseinSriLankaU. Gunaskekera1, M.P.O. Baumann2, N. Wedasinghe3, V. Punyapornwithaya4. 1Veterinary Population Medicine, University ofMinnesota,VeterinaryMedicine,STPAUL,MN,USA,2FAOReferenceCentreforVeterinaryPublicHealth,DepartmentofVeterinaryMedicine,FreieUniversitätBerlin,Berlin,Germany,3VeterinaryMedicine,DepartmentofAnimalProductionsandHealth,NorthCentralProvince,SriLanka,4VeterinaryMedicine,VeterinaryPublicHealthCentreForAsiaPacificandDepartmentofFoodAnimalClinic,FacultyofVeterinaryMedicine,Chiangmai,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMFootandMouthDisease(FMD)isahighlycontagiousdiseasethataffectsalltheclovenhoofanimalsandcausesconsiderableeconomiclossestothecattleandbuffalofarmersworldwide.TheinitialcountryleveleradicationofFMDis importantatthetaskofthefinalglobaleradicationofFMD.FMDisendemictoSriLanka.Theobjectivesofthisstudyweretoidentifyrelevantriskfactorsassociatedwiththe2014outbreakandtoobtainfarmersperceptionofFMDbymeansofparticipatoryapproach.TheNorthCentralProvinceofthecountrywhereFMDoutbreaksarefrequentlyreportedwasselectedasthestudyarea.AquestionnairewasusedtocollecttheinformationonpotentialriskfactorsforFMDfortheyear2014outbreakfromcasefarm(n=83)andcontrolfarm(n=161).Sevenfocusgroup(FG)discussionswithfarmersandfivein-depthinterviewswithveterinariansandlivestockofficerswereconducted.TheVenndiagrams,proportionalpiling,seasonalcalendarandtheparticipatorymappingwerethetechniquesusedintheFGdiscussion.Alogisticregressionmodelwasusedtodeterminetheassociationbetweenpotentialriskfactorsanddiseasestatusofthefarm.Basedonthecases-controlstudytherewerefivevariablessignificantlyassociatedwiththeFMDspread:cattle/buffalocontactwithnearbyvillages(OddRatio=2.88:95%CI1.23-6.72),cattle/buffalograzingnearwatertankareas(OR=3.11:1.21-7.97),animalsboughtorsoldduringtheoutbreak(OR=3.3:1.39-7.83),beingneartoaroadwhereanimaltraderstravel(OR=3.44:1.10-10.79),andbeingfedonthefloorinstead of feed troughs (OR=2.61,1.08-6.31). The major risk factor identified here was cattle/buffalo movement by means ofgrazing/trading.TheresultsfromtheFGandin-depthinterviewswerequalitativelyanalyzed.ThishelpedtoidentifytheimpactandseasonaldistributionofFMDwhilesupportingtheriskfactorfindings.Bothfocusgroupdiscussionsandthequestionnaireidentifiedthatthevaccinationhadnoeffectinthemostrecentoutbreak.ResultsfromthisstudysupportveterinaryservicesinSriLankainregardtodevelopingeffectivecontrolmeasuresduringfutureoutbreaks.

CRWAD2017 ABSTRACTS


P130-TraditionalandlocalknowledgeformuskoxhealthandfoodsecurityintheCanadianArcticM.Tomaselli1,S.C.Gerlach2,S.J.Kutz1,S.L.Checkley1,&thecommunityofIqaluktutiaq.1FacultyofVeterinaryMedicine,Departmentof Ecosystem and Public Health, University of Calgary, Calgary, AB, Canada, 2Faculty of Arts, Department of Anthropology andArchaeology,UniversityofCalgary,Calgary,AB,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose:InthecommunityofIqaluktutiaq(CambridgeBay),Nunavut,Canada,muskoxhealthandpopulationsustainabilityareofgreatimportanceforthelocalcommunityfortheInuitcultureaswellasasourceofhealthyfoodandincomethroughemploymentinsporthunting and commercial harvest. Over two-thirds of households in Nunavut are considered food insecure, which underlines theimportanceoffoodandincomethroughwildliferelatedemployment.Theobjectiveofthisstudywastogatherandexploretraditionalandlocalknowledgeconcerningmuskoxhealthtoinformdevelopmentofcommunitybasedmuskoxsurveillanceforthecommunity.Methods:Qualitativeresearchandparticipatoryepidemiologymethodswereused.Semi-structuredindividualinterviewswerecarriedinitiallywithparticipantsidentifiedthroughlocalagencies,thencontinuedthroughsnowballsamplinguntilthematicsaturationat30participants.Afterinitialsummaryoffindings,groupinterviewswereimplementedwith19participantsin7groupstofurtherexplorecertain themes, giving a total of 38 participants overall. Summarized findingswere later reviewedwith 31 participants to ensureaccuracyofinterpretation.Results:Keyfindingsincludedasignificantmuskoxpopulationdeclineinthestudyareawhichconcurredwithsubsequentmuskoxpopulationestimates.Participantsalsomadeotherimportantobservationsrelatedtodisease,bodycondition,populationstructureandmortality.Theareaundersurveillancewasmappedalongwithmuskoxmortalities.Conclusions:Overall,localandtraditionalknowledgecontributedtoknowledgeofmuskoxpopulationsize,distributionandhealth.ThesemethodscanbeusedinwildlifesurveillanceframeworksintheCanadianArctic.Inclusionoflocalandtraditionalknowledgeinsurveillancewillimprovelocalengagementandenrichdiscussionsrelatedtoco-management.P131-Foot-and-mouthdiseasevirusantibodyscreeningusingLPB-ChromatographicstripstestJ.Choi,Y.Kang,J.Jeong,H.Choi,S.Lee,B.Lee,C.Kim,S.Lee,I.Choi,C.Song,S.Park,J.Lee.KonkukUniversity,Seoul,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMPurpose: There are three methods in OIE Terrestrial Manual for Foot-and-mouth disease(FMD) serological test, which are virusneutralizingtest(VNT),solid-phasecompetitionELISA(SPCE),liquid-phaseblockingELISA(LPBE).TheVNTrequirescellculturetechnique,liveFMDvirusandtakes2-3daystogetresults.ELISAmethodsarehighthroughputandproviderelativelyaccurateresults.AmongtwoELISAs, the outcomes of LPB-ELISA present strong correlation with those of VNT. In previous studies, we appliedimmunochromatographicstriptestinsteadofELISAtomodifyLPBEsuitableforfieldsurveillance.Inthisstudy,wescreenedvaccinatedpigsandcattleserumcollectedincommercialfarmwiththeLPB-chromatographicstripstestandcomparedtheresultsofthestriptestswiththatofSPCEandLPBE.Methods:Theantigen-serummixturewasincubatedinatubelikeLPBEandtheeffectofblockingbyFMDV-specificantibodyintheserumwasconfirmedbyappearanceofthebandontheteststrip.Results:Inconsequence,therewascloseconnectionamongtheresultsofimmunologicaltests,butmismatchinsomecases.Conclusions:Tosolvethisproblem,weneedextrastudyonthecauseofnon-specificreactionoftheantigen-antibodycomplexandserumsfortests. , Ifwesolvesometroubles,PB-chromatographicstriptestwouldbeausefultoolforserologicaltest.

CRWAD2017 ABSTRACTS


P132-DetectionandgeneticcharacterizationofPorcinecircovirustype3inRussiaA.G.Yuzhakov1,S.A.Raev1,K.P.Alekseev1,A.M.Mishin1,T.V.Grebennikova1,O.A.Verkhovsky1,A.D.Zaberezhny2,T.I.Aliper2.1Virology,DiagnosticsandPreventionResearch Institute forHumanandAnimalDiseases,Moscow,RussianFederation, 2Virology,All-RussianResearchInstituteofExperimentalVeterinaryMedicinenamedYa.R.Kovalenko,Moscow,[email protected]:ViralPathogenesis,12/4/20175:30PMPurpose:InthepresentstudywesumuptheresultsofthedetectionandcompletegenomesequencingoftwoPorcinecircovirustype3(PCV3)strainsinRussia,performedforthefirsttime.Theviruseswereisolatedfromtwogeographicallydistantcommercialfarmswithrecordsofreproductivefailure,porcinedermatitisandnephropathysyndrome(PDNS),oneofthefarmswaslocatedintheregionofSmolensk(WestpartofRussia,isolate"SM"),whilsttheotheronewasintheregionofTyumen(WestSiberia,isolate"TU").Methods:AmplificationandsequencingprimersweredesignedonthebasisofexistingGenBankPCV3sequences.TheresultingPCRfragmentscoveredtheentiregenomeofthevirus.TotalDNAwasextractedfrom10%suspensionoftissueinphosphate-bufferedsalineandusedasatemplateforPCR.Sangersequencingwasperformed,withacoverageofatleasttworeadsforeveryDNAfragment.ThegenomesequencesoftheRussianPCV3isolateswerecomparedwitheachotherandwithcurrentlyavailablecompletegenomicPCV3sequencesfromGenBank.Results:Thefullgenomesequencesof thePCV3 isolateswere2000nucleotides in length.Fullsequencealignmentrevealeda99,2%identitybetweentheSMandTUstrains.TheSMandTUisolateswerefoundtoformamonophyleticgroupinPCV3phylogenetictree.Whencomparedwithavailablefull-lengthgenomePCV-3sequencesfromGenBank,theSMandTUisolateswerefound to be the closest related to Chinese (KY778776/CN/Shandong-1/20703) and South Korean (KY996337/KU/1602) strains,demonstratinga98,9%identityforSMisolateanda99,5%identityforTUisolate.Conclusions:TheresultsofthisstudyconfirmthatPCV3ispresentwithinpigpopulationinRussia.WehavesequencedandanalyzedthecompletegenomesoftheSMandTUisolates.WehavereasonstobelievethatPCV3couldplayaroleinreproductivedisorders,porcinedermatitisandnephropathysyndrome(PDNS)observedatthesurveyedfarms.FurtherstudiesarecurrentlyinprogresstobetterunderstandtheepidemiologyandpathogenicityofPCV3.P133-TheEffectsofsalinewaterconsumptionontheultrasonographicandhistopathologicalappearanceof liverandkidney in

sheepM.M.Ghanem1,M.M.Zeineldin2,A.Eissa3,E.ElEbissy3,R.Mohammed3. 1AnimalMedicineDepartment,BenhaUniversity,Benha,Egypt,2DepartmentofVeterinaryClinicalMedicine,IllinoisUniversityatUrbana-Champaign,Urbana,IL,USA,3AnimalHealthDesertResearchCenter,Materia,Cairo,[email protected]:EpidemiologyandAnimalHealthEconomics,12/3/20176:30PMThisstudywascarriedoutatSouthSinaiResearchStationtoevaluatetheultrasonographical,andhistopathologicaleffectsofsalinewaterconsumptiononliverandkidneyinbarkisheep.Atotalof30Barkisheepwereallottedintotwogroups(1stgroupcontains15males,and2ndgroupcontains15females).Eachgroupsubdividedinto3sub-groupsA,BandC(n=5pergroup).GroupAmaleandgroupAfemalewereallowedtodrinktapwaterwith350ppmtotaldissolvedsolids(TDS)andusedasacontrol.GroupBmaleandgroupBfemalewereallowedtodrinkmoderatesalinewaterwith4557ppmTDSandgroupCmaleandgroupCfemalewereallowedtodrinkhighsalinewaterwith8934ppmTDSfor9months.Attheendofthestudy,ultrasonographicexaminationrevealedincreasethelengthofrightandleftkidneywithcrystalsformationinbothkidneyandurinarybladder.Theprevalenceofthecrystalformationshowed a significant increase in case of males than females. Ultrasonographic examination of liver revealed formation of hyperechogenicdotswhichincreaseinnumberandsizeinfemalesmorethanmales.Histopathologicalexaminationofthekidneyrevealedhyalineformation,widenessofbowmanspace,atrophyofglomeruliwithinnecrosisandmassivefibrosisandhemorrhageespeciallyinmalegroup.Histopathological examinationof the liver revealed that therewas fatty liver change, signet ringappearance,hepaticsinusoiddilatationandmassivefibrosiswithmassiveinfiltrationofchroniccellsmainlyinfemalesgroup.Inconclusion,werecommendthatthebreedersofsheepinEgyptespeciallyinsouthSinaishouldusegroundsalinewatermixedwithfreshwatertoavoidtheharmfuleffectofsalinewateronthekidneyandliverfunction.

CRWAD2017 ABSTRACTS


P134-OralVaccinationofgoatswithheat-inactivatedMycobacteriumbovisA.Menshawy.CollegeofVeterinaryMedicine,BeniSuefUniversity,Egypt,BeniSuef,[email protected]:Immunology,12/4/20175:30PMVaccinationagainsttuberculosis(TB)isprohibitedincattleorotherspeciessubjectedtospecificTBeradicationcampaigns,duetotheinterferencethatitmaycausewiththeofficialdiagnostictests.However,immunizationwithaheat-inactivated(HI)Mycobacteriumbovisvaccineviatheoralroutehasbeensuggestedtoovercomethisissue.Inthisstudy,themaingoalwastoassesstheinterferenceoftheHIvaccinebydifferentroutesofadministrationusingapreviousvaccinationandre-vaccination(boosting)protocol.TB-freekidgoatsweredividedintothreegroups:oral(n=16),intramuscular(IM;n=16),andcontrol(n=16).Resultsshowedthattherewasasignificantdifference in thepercentageof animalspositive to the single intradermal test (SIT) andbloodbased interferon-gammareleaseassay(IGRA)causedbyvaccinationwhenperformedintheIMgroupcomparedtotheoralgroup(p<0.001).Nevertheless,nopositivitytotheSITorIGRAtestwasobservedinorallyvaccinatedgoatsregardlessofthedifferentinterpretationcriteriaapplied.Noneofthegroupspresentedpositiveantibodytitersusinganin-houseELISAandsamplescollected2monthsaftertheboost.P135-PCV2serologyELISAcomparisonJ.Toepfer,M.Bandrick,H.Fan,A.Kenworthy.Zoetis,Kalamazoo,MI,[email protected]:Immunology,12/4/20175:30PMSerologicalELISAassaysmeasuringAnti-PCV2antibodiesinporcineserumareusedtosupportclinicalstudiesandunderstandPCV2antibodylevelsinherds;however,thereisnogold-standardPCV2serologicassay.Further,themanyassayoptionsformeasuringPCV2antibodiesincreasesthecomplexityofcommunicatingaboutPCV2serology.AninternalstudywasperformedtocomparetheZoetisinternalPCV2serologicalELISAwiththeZoetis(Synbiotics)SERELISAPCV2AbMonoBlockingkit(inboththeSERELISAEndpointandSERELISASingleDilutionformats),theIngenasaIngezimCircoIgGassaykit,andtheKansasStateUniversityIFA.Ninetyporcineserumsamples fromhigh,mid-low, andnegative antibody level groups, as determined fromprevious testing in the Zoetis internal PCV2serologicalELISA,wereevaluated inall assays. Study sampleswere testedatKansasStateUniversityvia IFA for comparison toanexternalassaycurrentlybeingusedwithhighregardinthefield.Assayswouldbeconsideredequivalentifmeantitersineachantibodylevelgroupwerewithina20%differenceof theZoetis internalPCV2serologicalELISA results.The IngezimCirco IgGhighpositivesampleshadidenticalresultstotheZoetisinternalPCV2serologicalELISA;however,themeanpositivesfortheIngezimassaydidnotmeet acceptance criteria in themid-low and negative antibody level groups. The Zoetis internal PCV2 serological ELISA, SERELISAEndpoint,SERELISASingleDilutionandtheKSUIFAassaysperformedsimilarlyinalltitergroups.TheSERELISAEndpointassayandtheSERELISASingleDilutionassaywereequivalentandsatisfactoryforusebasedontheacceptancecriteria.TheabilitytousetheZoetis(Synbiotics)SERELISAPCV2AbMonoBlockingkithasmanypositive impacts includingbutnot limitedto:providingacommerciallyavailablekitforPCV2serumsampletesting.Thiswillfacilitatecommunicationandunderstandingofclinicalresultsamonglaboratoryandfieldstudiesacrosstheglobe.

CRWAD2017 ABSTRACTS


P136-ComparativegenomicstudyofSalmonellaheidelbergfromchickenandturkeypoultryproductionsystemsL. Deblais1, B. Lorentz1, J. Scaria2, K.V. Nagaraja3, M. Nisar3, G. Rajashekara1. 1Veterinarian Preventive Medicine, The Ohio StateUniversity,Wooster,OH,USA,2VeterinarianPreventiveMedicine,SouthDakotaStateUniversity,Brookings,SD,USA, 3VeterinarianPreventiveMedicine,UniversityofMinnesota,Minneapolis,MN,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMPurpose:Non-typhoidalSalmonellaareoneoftheleadingcausesofhumanfoodbornegastroenteritisintheUnitedStatesandcostupto$3.6billionannually.Salmonellaareincreasinglyproblematicissuesinlivestockproductionduetolackofeffectivecontrolmethodsand constant adaptationsof thepathogen towardsnewmanagementpractices. Theseadaptationsareoften related tohorizontalacquisitions of virulence or antibiotic resistance genes. We hypothesize that poultry production systems significantly influenceSalmonella genomic content, and by consequence its survival and virulence abilities.Methods: This study aimed to compare thegenomecompositionofS.Heidelbergisolatedfromenvironmentalsamplesoftwodifferentproductionsystems(turkeys,n=19;andchicken,n=12)breederfarmsintheMidwestoftheUS).FollowingHiSeqsequencing,the31genomeswereassembled,annotated,andanalyzedusingSPADES,RAST,andSEED.Results:Wholegenomecomparisondatashowedthat63%ofchickenand58%ofturkeyfarmsisolateswereclusteredbyfarmproductionsystem,indicatingthatatsomeextentthefarmsystemseemedtohaveanimpactonthegenome content of S. Heidelberg. Further, gene composition and diversity studies revealed that specific pathways linked toantimicrobialresistanceandsurvivalinhostileenvironmentweredifferentbetweenfarmingsystems.GenesassociatedtothetypeIVsecretionsystem(n=15)weremorerepresentedinchickenisolates(P>0.01);while,turkeyisolateswereenrichedwithDNAencodingphage proteins (n= 50) and resistance genes to several aminoglycoside antibacterials (n= 2; P>0.01). Conclusions: This studycorroboratesthatdefinefarmproductionsystemscouldinduceenvironment-drivenadaptationsinSalmonellaviapotentialhorizontaltransfersbetweenbacteriaorviralinfections.ComplementarymicrobialandviralpopulationstudiesofthesesampleswouldprovidecriticalinsightsthataidinthedevelopmentoffuturemanagementpracticestocontrolSalmonella.P137-Co-occurrenceofmcr-1andextendedspectrumβ-lactamaseinEscherichiacoliisolatedfromcommercialchickenmeatinBrazilD.F. Monte1, A. Mem1, M.R. Fernandes2, L. Cerdeira2, P. Fedorka-Cray3, N. Lincopan4, M. Landgraf1. 1Department of Food andExperimentalNutrition, School of Pharmacy,Universityof SaoPaulo, SaoPaulo,Brazil, 2DepartmentofClinicalAnalysis, School ofPharmacy, University of Sao Paulo, Sao Paulo, Brazil, 3Department of Population Health and Pathobiology, College of VeterinaryMedicine,NorthCarolinaStateUniversity,Raleigh,NC,USA,4DepartmentofMicrobiology,InstituteofBiomedicalSciences,UniversityofSaoPaulo,SaoPaulo,[email protected]:PathobiologyofEntericandFoodbornePathogens,12/3/20176:30PMPurpose:Thedetectionandrapidspreadofcolistin-resistantEscherichiacoliisolatescarryingthemcr-1genehascreatedanurgentneed to strengthen surveillance mainly because these isolates have been identified in the one health interface: foods, animals,environments,andhumans.Inthisstudy,wedescribedthepresenceofeightclonallyunrelatedcolistin-resistantE.coliisolatescarryingmcr-1,blaCTX-M-2,blaCTX-M-8,orblaCMY-2genesisolatedfromcommercialchickenmeatinBrazil.Methods:Duringalocalsurveillancestudyconducted tomonitoring the presence of colistin and extended spectrum β-lactamase (ESBL) resistant bacteria, 41 chickenmeatsampleswerecollectedfrom12marketsindifferentregionsofSaoPaulocity(north,south,eastandwest).Afterwards,antimicrobialsusceptibilityprofilesandMICvaluesweredeterminedbydiscdiffusionandmicrodilutionmethod,respectively,andmcr-1,ESBL,andpAmpCgeneswerescreenedbyPCRandwholegenomesequencing(WGS).Results:Co-existenceofmcr-1andblaCTX-M-2(4/8;50%),blaCTX-M-8(2/8;25%),orblaCMY-2 (1/8;12.5%)geneswereconfirmedforsevencolistin-resistantE.coli (17%)collected frommarketslocatedinthenorth,south,andwestregionofSaoPaulocity.TheseisolateswerefoundtobegeneticallyunrelatedbyPFGE.Ontheotherhand,amongcolistin-susceptibleE.colistrainsrecoveredfrom29(70%)chickenmeatsamplesfrommarketsdistributedinallregionsstudiedwerepositiveforblaCTX-Mgenes.Mostmcr-1-E.colistrainscarriedIncX4plasmidswhichhasbeengloballyreported.Additionally, in silico assays revealed thepresenceofplasmid-mediatedquinolone resistance (PMQR)genesamongmcr-1-positivestrains,suchasqnrB19(2/8;25%)orqnrS1(1/8;12.5%)inthreeisolates(37.5%).Conclusions:TheseresultshighlightthatcommercialchickenmeatcanbeanimportantreservoirofE.colicarryingcolistin,ESBLandPMQRgeneswhichisacauseforpublichealthconcern,sincethiscouldcontributetotheaccelerationofthespreadofthesegenes.

CRWAD2017 ABSTRACTS


P140-Prevalenceoffourvector-bornepathogensinCanadiandogs,2007-2016M.D.Evason.Pathobiology,UniversityofGuelph,Guelph,ON,[email protected]:VectorborneandParasiticDisease,12/3/20176:30PMVector-bornepathogensareemerginginmultipleregionsofCanada.Studyobjectiveswereto:1)investigatenationalandprovincialvector-bornepathogencaninetestpositivestatus,and2)assesschangeintestpositiveprevalenceovertime(2008-2015),nationallyandwithin individualCanadianprovinces.Adatasetof937,004 test results forDirofilaria immitisantigenandBorreliaburgdorferi,Anaplasmaspp.andEhrlichiaspp.serologyperformedondogsresidinginCanadawasidentified.DatawereavailablefromMarch2007until July 2016, along with canine geographic distribution. Descriptive statistics and X2 and X2 for trendwere used for analyses.There were significant geographic differences in dog pathogen prevalence between provinces (p<0.001). Seroprevalence for B.burgdorferiwashighestinCentral(Manitoba),Eastern(Ontario,Quebec)andAtlanticCanada(NewBrunswick,NovaScotia).Overthe7years(2008comparedto2015),asignificanttemporaldifferenceinpathogenprevalencewasobserved.ThiswasmostnotableforCanadiandogprovincialseroprevalenceforB.burgdorferi(p<0.001).TheincreaseinprevalencedetectedforB.burgdorferiisconsistentwithanecdotal informationonheightenedseropositivityanddisease insomeregionsofCanada;specifically,a rise incanineLymedisease.P141-SeroprevalenceofBorreliaburgdorferiandAnaplasmaphagocytophiluminOntariohorsesM.Neely1,S.Weese1,L.Arroyo2,H.Archer1,A.Moore3,M.Hazlett4,K.Clow1.1Pathobiology,UniversityofGuelph,Guelph,ON,Canada,2Clinical Sciences,University ofGuelph,Guelph,ON, Canada, 3OntarioMinistry ofAgriculture Food andRuralAffairs,Guelph,ON,Canada,4UniversityofGuelph,Guelph,ON,[email protected]:VectorborneandParasiticDisease,12/3/20176:30PMLymediseaseisamulti-systemictick-bornedisease,causedbythebacteriumBorreliaburgdorferi,andisofgrowingconcerninOntario.Recently, reports of Lyme disease in horses in the United States have been increasing; however, very little is known about theprevalenceofLymedisease inhorses inOntario.Anaplasmaphagocytophilum - thecauseofequineGranulocyticAnaplasmosis, isanothertickbornebacteriaofconcern.InnortheasternNorthAmerica,thevectorofthesetwopathogensisIxodesscapularis.Thistickspecies isexpanding isrange inOntario, therefore, it is imperativetodevelopanunderstandingoftheprevalenceoftheseequinediseases.Theobjectivesofthisstudywereto1)toidentifytheprevalenceofB.burgdorferiandA.phagocytophilumseropositivityinOntariohorses;2)identifyriskfactorsforinfection;and3)compareanin-clinicSNAPtesttoaLymemultiplexassay.Veterinaryclinics(n=80)fromacrossOntarioenrolledtoparticipateinthestudy.Bloodserumsamplesfrom551horsesweresubmittedalongwithaquestionnairewhichevaluateddemographics,clinicalhistoryandfarmmanagementofeachhorseinthestudy.SerawereexaminedwithanIDEXXSNAP®4Dx®test,whichdetectsserumantibodiestoC6antigenofB.burgdorferiandanequineLymemultiplexassaythatquantifiesB.burgdorferiantibodiestoOspA,OspCandOspFantigens.Inparalleltesting,wefoundtheoverallprevalenceofLymedisease to be 17%. On the SNAP® 4Dx® test, 5% of the samples were positive for B. burgdorferi, and 1% were positive for A.phagocytophilum.3horseswerecoinfectedwithB.burgdorferiandA.phagocytophilum.Onthemultiplexassay,theprevalencewas15%.SeropositivesforOspFweremorethan2-foldgreaterthanOspAandOspC.ThehighestprevalencerateswerefoundinEasternOntario,whichisaknownhotspotforthetickinOntario.Theseresultsprovidepreliminarydataforunderstandinghowseropositivesrelatetothediseaseandtherelevanceofdiagnostictesting.Furthermore,asthereisongoingexposurerisktohorsesforequineLymediseaseinOntario,knowingthedistributionandriskfactorswillaidinthecontinuedmonitoringandpreventionofthedisease.

CRWAD2017 ABSTRACTS


P142-Productionoffree,infectiousT.parvasporozoitesfromticksusingacontinuousflowinvitrofeedingsystemK.Dinkel1, L. Fry2,D. Knowles2,W. Johnson2, R.Vimonish1, R. Bastos1, S.Madsen-Bouterse1, R. Bishop1,M.Ueti2. 1DepartmentofVeterinaryMicrobiologyandPathology,WashingtonStateUniversity,Pullman,WA,USA,2UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,AnimalDiseaseResearchUnit,Pullman,WA,[email protected]:VectorborneandParasiticDisease,12/3/20176:30PMTick-bornediseasesofcattleseverelyimpedelivestockproductionglobally.Ofthesediseases,themosteconomicallyimportantinsub-SaharanAfricaisEastCoastFever(ECF),causedbytheintra-lymphocyticprotozoanparasite,Theileriaparva.T.parva,transmittedbythetickRhipicephalusappendiculatus,causespulmonaryedemaandrespiratoryfailureinsusceptiblecattle.Currently,ECFpreventionreliesalmostentirelyontheinfectionandtreatmentmethod(ITM)vaccine,inwhichcattleareinfectedwithliveT.parvasporozoitesandco-treatedwithoxytetracycline.ITMelicitsimmuneprotectionagainstmultipleparasitestrains;however,productionofthelivesporozoitestabilate incattle,ticks,andrabbits iscumbersome, inconsistent,andexpensive.Currently,thehighcostof ITMgreatlylimitswidespreadadoptionbysmallholderfarmers.OurobjectivewastosimplifyandstandardizetheITMproductionprocessusingacontinuousflow, invitrotickfeedingsystemtoproduceT.parvasporozoites.Inthis,T.parva-infectedR.appendiculatustickswereplacedonasiliconemembraneovercirculatingbloodandallowedto feed formultipledays.Eachday, for threehours,bloodwasreplaced with cell-free medium and secreted T. parva sporozoites collected and enumerated by quantitative PCR. Incubation oflymphocytes with as few as 1000 sporozoites led to infection, schizogony, and cell transformation, verified by microscopy, flowcytometry,andimmunocytochemistry.Threecalveswereinoculatedsubcutaneouslywith3x105sporozoites.Withtheexceptionofamild,transientfeverinonecalf,noclinicalsignsofECFwereobserved.Atfourweekspost-infection,eachcalfwasPCR-positiveforT.parva.Ourinvitrotickfeedingsystemprovidesasimplifiedmethodtoproducelive,infectiousT.parvasporozoitesforuseinvaccinedevelopment.Sincefewanimalsarerequired,andfreesporozoitesareeasilyquantified,oursystemwillgreatlyreducethecostperdoseofITM,allowingwidespreadadoptionofthevaccineamongsmallholderfarmersinsub-SaharanAfrica.

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Author Index Presentation Abstracts · Conference of Research Workers in Animal Diseases Page 2 CRWAD 2017 Dedicatee: Katherine M. Kocan, PhD Regents Professor, Veterinary Pathobiology