REVISTA COL DE CIENCIAS PECUARIAS 30-2-2017 colorbibliotecadigital.udea.edu.co/bitstream/10495/10670/1/CorreaNathali… · Revista Colombiana de Ciencias Pecuarias riial articles

Rev Colomb Cienc Pecu 2017; 30:101-115

Revista Colombiana de Ciencias Pecuarias

Original articles

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Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd¤

Cultivo fecal y dos métodos de PCR en materia fecal para el diagnóstico de Mycobacterium avium subsp. paratuberculosis en un hato seropositivo

Cultivo fecal e dois métodos de PCR em matéria fecal para o diagnóstico de Mycobacterium avium subsp. paratuberculosis em um rebanho soropositivo

Nathalia M Correa-Valencia1, MV, MSc, DSc(c); Nicolás F Ramírez1, MV, MSc, Dr An Sci; Michael Bülte2, Prof. Dr. Med. Vet.; Jorge A Fernández-Silva1*, MV, MSP, Dr Med Vet.

1Línea de Investigación en Epidemiología y Salud Pública Veterinaria, Grupo de Investigación CENTAURO, Escuela de Medicina Veterinaria, Facultad de Ciencias Agrarias, Universidad de Antioquia, Medellín, Colombia.

2Institut für Tierärztliche Nahrungsmittelkunde, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Frankfurter Straße 92, Giessen, Germany.

(Received: June 10, 2016; accepted: October 18, 2016)

doi: 10.17533/udea.rccp.v30n2a02

¤ Tocitethisarticle:CorreaNM,RamírezNF,BülteM,FernándezJA.Fecalcultureandtwofecal-PCRmethodsforthediagnosisofMycobacterium avium subsp.paratuberculosis inaseropositiveherd.RevColombCiencPecu2017;30:101-115.

* Correspondingauthor:JorgeArturoFernándezSilva.EscueladeMedicinaVeterinaria,FacultaddeCienciasAgrarias,UniversidaddeAntioquiaUdeA,Calle70No.52-21,Medellín,Colombia.E-mail:[email protected]

Abstract

Background: paratuberculosis is a slow-developing infectious disease, characterized by chronicgranulomatousenterocolitis.Thisdiseasehasavariableincubationperiodfrom6monthstoover15years,andiscausedbyMycobacterium aviumsubsp.paratuberculosis(MAP).Itsdetectionbydirectandindirectdiagnostictechniqueshasbeenofspecialinterest.Objective:toreportthediagnosisanddetectionofMAPusingseveraldiagnostictestsinaherdoftheNorthernregionofAntioquia,Colombia.Methods:serumsamplesfromthestudyherdwereanalyzed,usingacommercialELISA(enzyme-linkedimmunosorbentassay)kit.FecalsampleswereculturedbyduplicateusingHerrold´seggyolkmedium(HEYM),andanalyzedbyanend-pointIS900-specificnestedPCRprotocol,andacommercialF57-real-timePCRkit.Results:eightoutof27serumsamplesinthestudyherdresultedELISA-positive.NoneoffecalsamplesresultedpositivetoHEYMculturebyduplicateandnonewerefoundtobepositivebyF57-real-timePCR.Sevenofthe27fecalsamples

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Correa NM et al. Diagnosis of Mycobacterium avium subsp. paratuberculosis

werefoundtobepositivebyend-pointIS900-specificnestedPCR.AgreementwasfoundbetweenELISAandend-pointIS900-specificnestedPCRinoneoftheanimals.Conclusion:thepresentstudygivesinformationabouttheagreementbetweendirectandindirectMAP-detectiontechniques,usingdifferentmatrixesfromanimalsunderthesamehusbandryconditions.

Keywords: culture medium, ELISA, Johne´s disease, MAP, molecular diagnosis.

Resumen

Antecedentes: laparatuberculosisesunaenfermedadinfecciosadedesarrollolento,caracterizadaporunaenterocolitisgranulomatosacrónica.Estaenfermedadtieneunperiododeincubaciónquevaríaentrelos6meseshastapormásde15años,yescausadaporMycobacterium aviumsubsp.paratuberculosis (MAP). Sudetecciónportécnicasdiagnósticasdirectaseindirectashasidodeinterésespecial.Objetivo:reportareldiagnósticoydeteccióndeMAPutilizandovariastécnicasdiagnósticasenunhatodelaregiónnortedeAntioquia,Colombia.Métodos: seanalizaron lasmuestrasdesuerodelhatodeestudioutilizandounkitcomercialdeELISA(enzyme-linkedimmunosorbentassay).LasmuestrasdemateriafecalfueroncultivadasporduplicadoenHerrold´seggyolkmedium(HEYM),yanalizadasmedianteunprotocolodePCRanidadoespecíficodeIS900yunkitcomercialdePCRentiemporealparaF57. Resultados:ochodelas27muestrasdesueroresultaronpositivasporELISA.NingunadelasmuestrasdemateriafecalresultópositivaalcultivoenHEYMporduplicadoniporPCRentiemporealparaF57. Sietedelas27muestrasdemateriafecalresultaronpositivasaPCRanidadoespecíficodeIS900.SeencontróconcordanciaentreelresultadodeELISAydePCRanidadoespecíficodeIS900enunodelosanimales.Conclusión:elpresenteestudiobrindainformaciónacercadelaconcordanciaentretécnicasdirectaseindirectasdedeteccióndeMAP,utilizandodiferentesmatricesapartirdeanimalesbajolasmismascondicionesdemanejo.

Palabras clave: diagnóstico molecular, ELISA, enfermedad de Johne, MAP, medio de cultivo.

Resumo

Antecedentes: aparatuberculosis é umadoença infecciosa de evolução lenta, caracterizadapor umaenterocolitegranulomatosacrônica.Estadoençatemumperíododeincubaçãoquevariade6mesesa15anoseécausadapelo Mycobacterium aviumsubsp.paratuberculosis(MAP).Suadetecçãoportécnicasdediagnósticodiretoseindiretostemsidodeespecialinteresse.Objetivo:reportarodiagnósticoeadetecçãodeMAPutilizandováriastécnicasdediagnósticoemumrebanhonaregiãonortedeAntióquia,Colômbia.Métodos:foramanalisadasamostrasdesorodorebanhoutilizandoumkitcomercialdeELISA(enzyme-linkedimmunosorbentassay).AsamostrasdefezesforamcultivadasemduplicadoemHerrold´seggyolkmedium(HEYM)eanalisadasutilizandoumprotocolodePCRaninhadaespecíficodeIS900eumkitdePCRemtemporealcomercialparaF57. Resultados:oitodas27amostrasdesoroforampositivasparaELISA.NenhumadasamostrastestadasnaculturadefezesHEYMduplicadoforampositivasounaPCRemtemporealparaF57.Setedas27amostrasdefezesforampositivasnaPCRaninhadaespecíficaparaIS900. Foi encontradaconcordânciaentreoresultadodeELISAePCRaninhadaespecíficaparaIS900emumanimal.Conclusão: esteestudoforneceinformaçõessobreacorrelaçãoentretécnicasdedetecçãodiretaeindiretadoMAP,utilizandodiferentesmatrizesdeanimaissobasmesmascondiçõesdecondução.

Palavras chave: diagnóstico molecular, doença de Johne, ELISA, MAP, meio de cultura.

Introduction

Mycobacterium avium subsp.paratuberculosis (MAP) is a slow-growing,mycobactin-dependent,acid-fast bacterium that causes Johne’s disease orparatuberculosis(PTB)incattleandothersusceptiblespecies(HarrisandBarletta,2001).Thediseaseproduces

a significanteconomic impacton thecattle industry,especiallyonmilkandmeatproduction(Sweeney,1996;Chacon et al.,2004;Garcíaand Shalloo,2015;McAloonet al.,2016).Theagenthasalsobeenassociatedtothechronic human enteritis known asCrohn’s disease(Atreyaet al.,2014;Hanifian,2014;Liveraniet al., 2014;Waddellet al. 2015;2016).

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For the ante-mortemdiagnosisofPTBincattle,several types of test are available and proposed.These include tests to detect antibodies againstMAP, detection ofMAP genes, bacterial cultureof fecal samples and test to detectMAPon tissuesamples(Collinset al.,2006;NielsenandToft,2008;Stevenson,2010a;2010b).Sensitivityandspecificityof tests for the ante-mortemdiagnosisofPTBvarysignificantlydependingonMAPinfectionstageandintrinsic characteristics of each test (Nielsen and Toft,2008).

The antibody detection test known as enzyme-linkedimmunoassay(ELISA)isthemostpopulartesttodetectanimmuneresponsetoinfectionbyMAP.TheELISAisalsothemostwidelyusedtechniqueto establishPTB status of herds, but it has shownlimitations in someextend relating lowsensitivity,primarilybecauseof theslowprogressionofMAPinfection.Thisdoesnotensureanadequatedetectioncapacityofanimalsinanearlystageofinfectionwhenfecalsheddingislow(Kaliset al.,2002;McKennaet al.,2006;Nielsen,2010).Onthecontrary,ELISAis highly specific,with a lowpresentation of falsepositiveresults(HarrisandBarletta,2001).

CultivationofMAPfromtissuesandfecalsamples(individual,inpool,andenvironmental)isthemostreliablemethodofdetectinginfectedanimals(NielsenandToft,2008;2009;FecteauandWhitlock,2010).Usually,thespecificityoffecalcultureisconsideredto be almost 100% if the isolates obtained areconfirmedtobeMAPbymolecularmethodssuchaspolymerasechainreaction(PCR;NielsenandToft,2008;Schönenbrücheret al.,2008;Whittingtonet al., 2011).Fecalculturehasbeenusedasanacceptablestandardtechniquefordetectingtheinfectionstatusofanimals —related to elimination rate—, for estimating thesensitivityofotherdiagnostictests(e.g.ELISA,PCR), and as an excellent confirmatory test foranimalsthattestedpositivewithimmunologicaltests(Motiwalaet al.,2005;Alyet al.,2012).Herrold´seggyolkmedium(HEYM)isthemostfrequentlyusedtechniquefortheprimarycultivationofMAPfromclinical samples(fecesandtissue),anditssensitivityhasbeenreportedfrom39to82%,comparedtoliquidmedia (Collins et al.,1990;Eamenset al.,2000;Stichet al.,2004,Motiwalaet al.,2005;Cernicchiaroet al.,2008;Whittington,2009).

SpecialaspectsofMAPandthediseasedynamicscan affect the fecal culture accuracy, for example,MAP´seliminationthroughfecesisintermittentandoccurs inanadvanced stage (stages III and IV)ofthedisease,mainlywhentheanimalshaveclinicalsymptoms(Clarke,1997;Whittington,2010;Salemet al., 2013).Although the fecal culture hasmanylimitations,suchasalongincubationperiod(18to24weeks),highcosts,riskofcontaminationwithothermycobacteriaorfungi,andtimerequiredtoreporttheresults,itisstillconsideredtobethegoldstandardfor thedetectionofMAP(vanSchaiket al.,2007;NielsenandToft,2008;Whittington,2010).

ThedetectionofMAPgenesbyPCRhasshownadvantages (rapidity, identification of agent, andlackofcontamination)anddisadvantages(moderatesensitivity,highcost,specialequipment,andskilledpersonnelrequired;Collins,1996).However,duetorecent developments, PCRhas been suggested forherd screening (Collins et al., 2006;Anonymous,2010),andithasbeenrecentlydiscussedasapossiblenew gold standard for PTB (Stevenson, 2010a;2010b).ThePCR technique is rapid and specific,and in contrast to a culture-based diagnostic, noadditional testsarerequiredtoconfirmtheidentityoftheorganismdetected(Collins,1996).

ThemostpopulartargetgeneforthedetectionofMAPisthemulti-copyelementIS900 (BolskeandHerthnek, 2010;NationalAdvisoryCommittee onMicrobiologicalCriteriaforFoods,2010;Stevenson,2010b; Gill et al., 2011).However,mycobacteriaotherthanMAPhavebeenfoundtocarryIS900-likeelementswithnucleotidesequencesthatareupto94%identicaltothenucleotidesequenceofMAPIS900 (Cousinset al.,1999;Ellingsonet al.,2000;Englundet al.,2002;Kimet al.,2002;Taddeiet al.,2008).SomePCRsystemsthattargetIS900 also can give false-positiveresultswithDNAfrommycobacteriaotherthanMAPandwithDNAfromothertypesoforganisms (Möbiuset al., 2008a; 2008b). Due tothis, newprotocols avoiding cross-reactions havebeen reported (Bull et al., 2003; Herthnek andBölske,2006;Kawajiet al.,2007).InresponsetotheuncertaintyaboutthespecificityofPCRsystemsthattarget IS900fortheidentificationofMAP,theuseofseveralothertargetsequencesforMAPidentificationsystems have been proposed: ISMap02, ISMav2,

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hspX, locus255, andF57 (Stabel andBannantine,2005;Slanaet al.,2009;Kraliket al.,2010;Sidotiet al.,2011;Kelleret al.,2014).

ThePCRperformswell as a confirmatory teston cultures, being its sensitivity close to 100%(ManningandCollins,2001),butitsapplicationtoclinicalsampleshasbeenproblematic,mainlyduetotheproblemsassociatedwithDNAextractionfromcomplexmatrices such asmilk, feces, and blood,andthepresenceofPCRinhibitors(StevensonandSharp, 1997;Grant et al., 1998;Alyet al., 2010;Stevenson, 2010b), decreasing its sensitivity.Thelimitsofdetection, sensitivity, and specificityvarywith the targeted sequence andprimer choice, thematrixtested,andthePCRformat(conventionalgel-basedPCR,reversetranscriptasePCR,nestedPCR,real-time PCR, ormultiplex PCR;Möbius et al., 2008a;BolskeandHerthnek,2010;NationalAdvisoryCommittee onMicrobiologicalCriteria forFoods,2010;Stevenson,2010b).Ideally,samplingalladultcattleineveryherd,environmentalsampling,serialtesting,andtheuseoftwotothreediagnostictestswouldbetherecommendationforherdscreening,toincreasetheaccuracyofMAPdiagnosis(Collinset al.,2006;Clarket al.,2008;Stevenson,2010b).

TheaimofthisstudywastodiagnoseMAPusingfecal culture, F57-real-time PCR and end-pointIS900-specific nestedPCR in one herd previouslyscreenedpositiveforMAPantibodiesbyanindirectserum-ELISA.

Materials and methods

Ethical considerations

This research was approved by the EthicsCommittee forAnimal Experimentation of theUniversidad deAntioquia,Colombia (Act number88,March,2014).

Study herd

ThestudyherdwaslocatedinthemunicipalityofSanPedrode losMilagros,Antioquia (Colombia),oneofthemaindairymunicipalitiesofthecountry,locatedintheAndeanregionofColombia,withan

areaof229Km2,analtitudeof2,468m.o.s.l,ameanannualtemperatureof16°C,andacattlepopulationofapproximately71,395animals.Thestudyherdwasvisitedonlyonceaspartofapreviousstudyin2015,thataimed thedeterminationof theseroprevalenceofMAPandtheexplorationofthemainriskfactorsassociatedwithELISApositiveresultsindairycowsofthemunicipalityofinterest(Correa-Valenciaet al., 2016).Thestudyherd,reportedacattlepopulationof 39 bovines, including 27 cows over 2 years ofageatthemomentofthesampling,thepredominantbreedwasclassifiedasother in thepreviousstudy(differentfromHolsteinandJersey),withouthistoryof farmingother ruminants different frombovines(i.e.goats,sheep,buffaloes),spreadingmanureasafertilizerinthepastureswasacommonpracticeintheherd,aswellas,leavingthecalveswiththeirdamsafter parturition in direct contact, certified as freeof tuberculosis andbrucellosis, andnever reportedany compatible clinical case and/or followed anystructuredcontrolprogramforpreventionorcontrolofPTBbeforethesamplingin2015.

Blood and fecal sampleswere taken from allanimals over 2 years of age (n=27).The samplecollectionwas conducted according to standardmethods to avoid unnecessary pain or stress toanimals. Blood samples were taken from thecoccygealorjugularvein,collectedinred-topplasticVacutainer®(BectonDickinson,Sparks,NV,USA)tubesandtransportedrefrigeratedtothelaboratory,where they were centrifuged at 1,008 RCF for 5min.Fecalsamplesweretakenwithacleanglovedirectlyfromtherectumofeveryadultanimal,andthen,transportedrefrigeratedtothelaboratory.Theobtainedserumandthefecalsampleswerestoredat-20°Cuntilanalysis.

ELISA

SerumELISAwas performed using the pre-absorbed ELISA kit Parachek®2 (PrionicsAG,Schlieren,Switzerland)followingthemanufacturer’sinstructions.The sampleswere read usingEpochMicroplateSpectrophotometer® (BioTek,Winooski,VT,USA).TheELISAtestincludedapre-absorptionstepwithMycobacterium phlei to reduce cross-reactions.AnanimalwasconsideredELISA-positive

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if serumsamplewas aboveor equal to the cut-offof15percentpositivity(%P),asitisdefinedbythemanufacturerofthediagnostictestused.

Fecal culture

Feces from all animalswere thawed leavingthe samples under 4 °C for 24 h prior to thedecontaminationprocedure.FecalculturewascarriedoutaccordingtotheprotocolreportedpreviouslybyFernández-Silva et al.(2011a).Briefly,3goffeceswereaddedtoa50mLsteriletubecontaining30mLof a 0.75%HPC (hexadecyl pyridinium chloride)weight/volume(w/v)solution.Thissuspensionwasmanuallymixedbyshaking,andletinaverticalpositionfor5minatroomtemperaturetoallowprecipitationand sedimentation of big particles.Approximately20mLof theupperportionof thesupernatantwastransfertoanother50mLsteriletube,inwhichthewholesuspensionwasagitatedat200RPMfor30min.Tubeswereplaceinverticalpositioninthedarkfor24h at room temperature.Decontaminatedpooledfecalsampleswerecentrifugedat900xgduring 30 min, supernatant was discarded. DuplicatedHEYMslants,supplementedwithmycobactinJandamphotericinB, nalidixic acid, and vancomycinmix (BectonDickinson,New Jersey,USA)wereinoculatedwith300μLofthedecontaminatedpellet.Allculturemediawereincubatedat37°Cfor24weeksandwerecheckedweeklyformycobacterialgrowthorcontaminationwithundesirablegerms.MAPgrowthwas visuallymonitored for typical slow growthrate and colonymorphology according topreviousdescriptions (coloniesdevelopingafter≥3weeksofincubation, initially round, smooth andwhite, thentending toheapup slightlyandbecomingdull lightyellowwithwrinklingofthesurface;Whittington,2010).

DNA isolation from individual fecal samples

Each fecal samplewashomogenized for 5minprior to DNA extraction procedure. DNA from individualfecalsampleswasextractedaccordingtothefollowingprocedurereportedpreviouslybyLeiteet al. (2013)usingacommercialDNApreparationkit (ZRFecalDNAKit™,ZymoResearch, Irvine,CA,USA).Processingwasdoneaccordingtokit´sprotocolforisolationofnucleicacidsfrombacteriaandyeast.Amechanicalcelldisruptorstepwascarried

outinanautomatedbiologicalsamplelyzer(DisruptorGenie® 120V,ThomasScientific,Swedesboro,NJ,USA)toachieveamoreefficientcelllysis.

End-point IS900-specific nested PCR

DNA from individual fecal sampleswas testedforMAPby end-point IS900-specificnestedPCR,usingprimerstargetingIS900designatedTJ1-4[TJ1(5´-GCTGATCGCCTTGCTCAT-3´)andTJ2(5´-CGGGAGTTTGGTAGCCAGTA-3´)inthefirst-round-PCR,andprimerpairTJ3(5´-CAGCGGCTGCTTTATATTCC-3´)andTJ4(5´-GGCACGGCTCTTGTTGTAGT-3´) in the second round-PCR]accordingtoBullet al.(2003),modifiedbyFüllgrabe(2009)andBulander (2009).Thefirst andsecond-roundPCRmixturecomprisedthesamemixvolumesin afinalvolumeof50µLwith5µLofTaqDNApolymerasebuffer-MgCl2,1µLofdNTPmix,1µLofeachprimer,and0.4µLofTaqDNApolymerase(AmpliTaqGold®DNAPolymeraseLD,recombinant;5U/µL;AppliedBiosystems™,FosterCity,CA,USA),and5µLofDNA(ata1:10dilution) fromsampleorfromthefirst-round-PCR.Additionallytothesamples,apositive(Mycobacterium aviumsubsp.pararuberculosis, strainK10;ATCC®BAA-968TM) andanegativecontrol,aswellas,ablankcontrolwereincluded.Cyclingconditionsforbothroundswere:1cycleof95°Cfor10minandthen35cyclesof 94°Cfor30sec,60°Cfor30sec,and72°Cfor30sec,followedby1cycleof72°Cfor7min.Ampliconsoftheexpectedsize(355and294bp,forthefirstandsecond round, respectively)were visualizedwithethidiumbromideon1.5%agarosegels.

F57-real-time PCR

DNAfromindividualfecalsampleswastestedforMAPconfirmationbyF57usingacommercialReal-TimePCRkit,whichincludesaninternalamplificationcontrol (IAC) to avoid themisinterpretation offalse negative results (MAPsureEasy® Kit-MSE,TransMIT,Giessen,Germany).The componentsof theMAPsureEasy®Kit-MSEare the 25xMAPOligonucleotideMixincludingprimers[F57po-244F5‘–TACGAGCACGCAGGCATTC–3‘andF57po-306R5‘–CGGTCCAGTTCGCTGTCAT–3‘]andprobes[F57po-TaqMan®ProbeVIC-CCTGACCACCCTTC-MGBandIACMSETaqMan®

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ProbeFAM-AGCAATAAACCAGCCAGC-MGB];the2xqPCRMasterMix(fromqPCRMastermixplusw/oUNG*ofEurogentec,Ireland,2xPCRMMforProbeassay);theIAC(DNAIAC);andthepositivecontrol DNA of MAP strainK10 (ATCC® BAA-968TM). ThePCRmixturewaspreparedaccordingtotheprotocol,onesampleinafinalvolumeof25µL:5.5µLofmoleculargradewater,12.5 µLof2xqPCRMasterMix,1µLof25xMAPOligonucleotideMix;1µLoftheIAC-DNA,5µLofDNAprobe,and5µLof DNA.

Results

ELISA

Eightofthe27(29.6%)animalswerepositivebyserum-ELISAinthestudyherd(Table1).

Fecal culture

None of the 27 fecal samples from animals ofthestudyherdwerepositivebyfecalculturebasedon growth rate and colonymorphology (Table 1).Two duplicated cultures (four slants) presentedcontamination(7.4%).

End-point IS900-specific nested PCR and F57-real-time PCR

All samples resulted negative by F57-real-timePCR, and seven (25.9%) resultedpositive byend-point IS900-specific nested PCR (Table 1).Amplifications for end-point IS900-specific nestedPCR inagarosegel resultsareshown inFigures1and2.Acompilationofindividualinformationandtestsresultsforanimalstested(n=27)ofthestudyherdareshowninTable1.

Discussion

ThepresentstudyaimedtodiagnoseMAPusingfecal culture, F57-real-time PCR, and end-pointIS900-specific nestedPCR in one herd previouslyscreenedpositiveforMAPantibodiesbyanindirectserum-ELISA.

TheconfirmationofELISAtestresultsusingfecalcultureandPCRwasconsiderednecessarytoobtaina precise detection ofPTB infected animals in anELISApositiveherd.Nevertheless,weexpectedtofind a higher proportion ofMAP-positive animals(byELISA, aswell as, by fecal culture andPCR)in the study herd, considering inappropriate herdmanagementpracticespresentandknowntoberiskfactors for the disease (e.g. presenceofanimalsbornatotherdairies,exposureofcalves0-6weekstoadultsfeces, fecesspreadonforagefed toanyagegroup(Collins et al.,1994;Goodgeret al.,1996;Jakobsenet al.,2000;WellsandWagner,2000;Diéguezet al., 2008;Tiwariet al.,2009;Sorgeet al.,2012;Künzleret al.,2014;Fernández-SilvaandRamírez-Vásquez,2015;Vilaret al.,2015).Whenatestcombinationisconsidered,itmustbetakenintoaccountthatsomeinfectedcowsproduceantibodies for severalyearsprior to the fecal-shedding of detectable quantitiesofMAP.However,inotheranimals,antibodiesmaynotbedetectableduringtheearlystagesofinfectionwhenMAPfecal-sheddingisminimal(Kaliset al., 2002;McKennaet al.,2006;Nielsen,2010).

TheELISAresultsshouldbeanalyzedcautiously,mainlyconsideringitssensitivitybecauseofthesilentandlong-lastingbehaviorofthedisease,morethanasafailureofthetestitself(Sweeneyet al., 1996;Collinset al., 2005;Monet al., 2012;Sorge et al., 2012).AccordingtoLaverset al. (2015), thesensitivityofserumELISA is approximately25.4-45.3%and itsspecificityof97.6-98.9% inasymptomatic animals,whichcanleadtoamisclassificationofthecowsandreportinginfectedcowsasnegative.Ontheotherhand,theresultscouldberelatedtosamplehandling.Inthepresentstudy,theserumsampleswerefrozenfor30to45daysat-20°C,whichcouldhaveledtolowerscoresfortheMAPELISA(Alinoviet al.,2009).

Fecal culturedidnot report anypositive result,whichcouldbeexplained,amongotheraspects,bythestorageconditions(4°Cfor12hmax,andthenat-20°Cfor7months).AccordingtoKhareet al. (2008),tostorefecalsamplesat4°Cfor48h,andthenat-20°Cforatleastoneweekislimitingfortheculturesensitivity,contrarytoshort-termstorageat4°Candlongertermstorageat-70°C,whichappeartohavenodamagingeffectsonMAPviabilityinthefecalsample.

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Table 1. Animal-level information and MAP-diagnostic tests results in a study herd in the municipality of San Pedro de los Milagros, Antioquia (Colombia).

Animal ID

Breed* Parity Days in milk

Milk production per day (L)

Productive stage

Born in herd

Serum ELISA

Fecal culture

IS900-nested PCR

F57-real-time PCR

1 Other 2 192 23 Milking Yes − − + −

2 Other 6 163 33 Milking Yes + − − −

3 Other 2 372 n.d. Dry Yes − − − −

4 Other 5 72 34 Milking No − − − −

5 Holstein 1 4 n.d. Dry Yes − − − −


7 Other 6 182 21 Milking No + − − −



10 Other n.d. n.d. n.d. Heifer Yes + − − −

11 Other 1 37 27 Milking Yes − − − −


13 Holstein 2 88 31 Milking Yes + − − −




17 Holstein 6 324 n.d. Dry Yes − − − −


19 Holstein 7 72 51 Milking Yes − − − −


21 Other 3 192 25 Milking No − − − −

22 Holstein n.d. n.d. n.d. Heifer Yes − − + −

23 Other 5 161 22 Milking Yes + − − −

24 Holstein 5 89 37 Milking Yes − − + −

25 Other 3 409 18 Milking Yes + − + −


27 Jersey 1 40 23 Milking Yes − − + −

* Other breeds included Guernsey, Ayrshire, Swedish Red, Swiss Brown, Jersey, and several crossbreeds of Holstein with Jersey, Ayrshire, Angus, Blanco Orejinegro, Brahman, and Gir. n.d.: no data available at the moment of sampling; +: positive result; −: negative result.

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Figure 1. End-point IS900-specific nested PCR in agarose gel (final product of 294 bp), samples of cows 1-17. Molecularsizemarker(100bpDNAladder;Roche,Mannheim,Germany;lane1and20),animal1(lane2),animal12(lane13),animal15(lane16),positivecontrol(Mycobacterium avium subsp. pararuberculosis,strainK10,ATCC®BAA-968™;lane19),negativeresults(lanes3-12,14-15,and17-18).

Figure 2. End-point IS900-specific nested PCR in agarose gel (final product of 294 bp), samples of cows 18-27. Molecularsizemarker(100bpDNAladder;Roche,Mannheim,Germany;lane1and20),animal22(lane6),animal24(lane8),animal25(lane9),animal27(lane11),positivecontrol(Mycobacterium avium subsp. pararuberculosis, strainK10,ATCC® BAA-968™;lane18),blankcontrol(master mixture blank;lane19),negativeresults(lanes2-5,7,10),emptylanes(12-17).

Ontheotherhand,therewouldbefalse-negativefecalcultureforsamplesthatcontainfeworganismsduetolessofMAPduringtheculturingasadirectconsequence of the process (Whittington, 2010).Dehydration and the possible reduction of viablemicroorganism by chemical decontamination areimportantdatatointerpretnegativeresults,especiallyin low intensity fecal shedders (Reddacliff et al., 2003).

Another point that should be considered toexplain some of our results is the low-shedderstatus,considering that literaturereports thatabout75%ofpositiveanimalsareeitherloworverylowshedders (vanSchaiket al.,2003;USDA,APHIS,VS,CEAH,2008).InviewoftheminimalamountofdetectableMAP(100CFU/goffeces;Merkal,1970),only 15-25% of subclinical low and/ormoderatefecal shedderscanbedetectedbybacterial culture(Whitlock and Buergelt, 1996). The sensitivityof the fecal culture in clinical stages can be 91%(Álvarez et al.,2009),avalue thatcanbe reducedto 45-72% (Crossley et al., 2005;Alinovi et al., 2009)insubclinicalstages,whereasthespecificityisverygood(100%)inallstages(Ayeleet al.,2001).This information can explain someof our results,consideringtheseroprevalenceresultsforthewholemunicipality(3.6and2%atherd-levelandanimal-level, respectively;Correa-Valencia et al., 2016),wherenoclinicalanimalsweresampled.

TheuseofdirectPCRtofecalDNAhasseveraladvantagesasforexampleshortertimestodiagnosiscompared to culture (3 days vs. 14-22weeks). Inaddition, the procedure for the extraction of fecalDNA in preparation for PCR has become easierandlessexpensiveintherecentyears(Stabelet al., 2004).Consideringaneffectivemethodtoensureacomplete-DNAextraction, amechanicaldisruptionstep (bead-beating)was included—which breaksup bacterial cellwallmechanically by vibratingbacteriaathighspeed, in addition to the commercial kitprotocol (Odumeroet al.,2002;Zecconiet al., 2002;Herthnek,2009)improvingthesensitivityoftheprotocolapplied,alsoreportedbyLeiteet al.(2013)withthecomparableperformanceresults.

SpecialattentionshouldbegiventotheinhibitoryeffectsofcertaincomponentsofthesamplesonTaqpolymerase,whichcouldcausefalsenegativeresults,beingaprobableexplanationforsomeofournegativeoutcomes(Tiwariet al.,2006).Feces,especiallythosefromruminants,areexpectedtoincludehighlevelsofPCRinhibitors(Al-SoudandRadstrom,1998;InglisandKalischuck,2003;ThortonandPassen,2004),andoneofthemaindifficultiesistoremovethemtoimprovedPCRsensitivity(HarrisandBarletta,2001).Althoughnoclinicalcowswerefoundinourstudyherd,insomecasesishighlyprobablethatfecesfrom

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cowswithclinicalPTBmaycontainheme(acomplexofironwithprotoporphyrinIX)andepithelialcells,beingthesecomponentsreportedtobeinhibitorytoPCR(InglisandKalischuck,2003).

The sensitivity and specificity of the end-pointIS900-specificnestedPCRusedtotestoursamplesare reported tobe increased(Englundet al.,2001;Ikonomopouloset al.,2004;BölskeandHerthnek,2010).AnyPCRinhibitorsinthefirstrunwouldbedilutedwhen transferredas template to the secondPCR(BölskeandHerthnek,2010).

Our assays used twomolecular elements foundin different loci and ratios in MAP genome (IS900 and F57),leadingtonon-comparableresultsrelatedto their specificity and sensitivity.The IS900 is a repetitiveDNA sequence present in 15-18 copiesof MAP genome (Collins et al., 1989;Green et al., 1989). However, IS900-like elements havebeen described at low copy numbers in rarelyencounteredenvironmentalmycobacteria(Cousinset al.,1999;Englundet al.,2002;Tasaraet al., 2005),compromising its specificity.On the other hand,F57, asinglecopy-segment,hasdemonstratedhighspecificityforthedetectionofMAP(Coetsieret al., 2000;Ellingson et al., 2000;Harris andBarletta,2001;Strommengeret al.,2001;Vansnicket al.,2004;Rajeevet al.,2005).ThenestedIS900assaycandetect0.01pgofDNA(correspondingto10genomes)whenextractedfromapureculture,whiletheF57assaycandetect0.1pgofDNA(correspondingto100genomes;Radomskiet al.,2013). Vansnickaet al.(2004),TasaraandStephan(2005),andSchönenbrücheret al.(2008) recommendincludingtheF57-PCRassaytoconfirmthe presence ofMAPafter a positive IS900-PCR. According to this, our results (F57-PCR negative resultsandsomepositiveresultsbyIS900-PCR), can beconsideredMAP-unspecificbyIS900-PCR, and confirmedasnegativebytheF57 insertion detection.

Nevertheless, our results in thePCRprotocolsapplied could be better explained by the alreadyreported behavior of the disease than to PCRmisclassification.According toWithlock et al. (2000),thedisadvantagesofsomedetectiontestareduemainlybecauseoftheintermittentsheddingofmicroorganisms.Thismeans that the sensitivityofdirect tests to detect symptomatic animals is high,

butlowfordetectionofinfected/subclinicalanimals(NielsenandToft,2008;Schönenbrücheret al.,2008;Whittingtonet al.,2011).

On theotherhand, the thawingof fecalsamplesstoredat-20°CwasdoneindifferenttimesforfecalculturingprocessandforDNAextractionwhatcouldhaveaffectedthedetectionbyPCR, leading to false negative results because ofDNAdamage duringthawing-freezing re-processes,which can explainPCRresultsinthisstudy(BölskeandHerthnek,2010;Whittington,2010).

The low agreement between tests results hasbeen also reported before (Muskens et al., 2003;Glanemannet al.,2004;Dreieret al.,2006)andcouldbeexplainedduetothefactthatELISAnegativeorELISAfalse-positiveresultshavealowprobabilityofdeliveringapositivecultureresultifjustasinglesampling is planned as normally done in a cross-sectional study,whichwas the caseof the presentstudy(Sweeneyet al.,2006).SimilarresultsonlowagreementbetweenELISAandculture(Fernández-Silva et al., 2011b) andELISAandPCR toMAP(Fernández-Silva et al.,2011a)werefoundinpreviousstudies inasymptomaticanimalsfromherdsof thesamedairyregion.

Ourresultsforallthetestsuseddoesnotnecessarilymeanthattheanimalswerenotreallyinfected,becausethe shedding phase has probably not yet started(infected animal in a noninfectious phase) orwasabsentatthemomentoffecalsampling(intermittency).Another possibility is that in these animalsMAP-antibodiesweredetectedpriortothestartofbacterialshedding,whichcouldbeginlaterandcouldbethendetected byPCRor fecal culture (Nielsen, 2008).Considering MAP-shedding characteristics as the majorlimitationinthedetectionofinfectedanimals,itshouldbetakenintoaccountthattheeliminationofthebacteria through feceshappens at all stagesbutatdifferent levels and sporadically,whichdemandsrepeatedtestingtodetectanimalssheddingverylownumberofMAP,whichcouldanywaygoundetected(Stevenson,2010b).Nevertheless,wefoundapositiveresultbyserum-ELISAandfecalPCRinoneofthecowsinthestudyherd,revealingparalleldetectableantibodylevelsanddetectableMAPfecal-shedding,beingthisabiologicallyplausibleresult.

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Alinovi et al.(2009)reportedthattestsensitivityforculturemethodsandreal-time PCR,aswellas,testaccuracy,arecomparable.Thisclearlydemonstratesthatinfieldapplications,real-time PCRisasusefulassolidorliquidculturemethodswhileprovidingtheproducerwith test resultswithinhours,notweeks.SerumELISA,althoughnotasaccurateastheothertestsevaluated,continuestobeausefulalternativebecause of its rapid turn-around.Now,withPCR,resultsthataremoreaccuratecanbeavailableasfastasforELISA.

Ourresults inaseropositiveherddeliveredoneasymptomaticELISA-positivecowwithanegativefecalculture,andapositiveend-pointIS900-specificPCRresult.Inaddition,therewere13asymptomaticELISA-negativecows,producingnegativeresultsbyfecal culture, andnegative resultsby twodifferentPCRmethodsinaninfectedherd.Wedetectedalowagreementbetweenthediagnostictestsused(ELISA,fecalculture,andPCR).Theseresultsevidencetheperfect examples ofMAP´sdetection paradox andthemostconfoundingcomponentinPTBcontrol:thedetectionof truly infected anduninfected animals.TheinformationinthisstudyindicatestheimportanceofMAP detection and its direct impact in theimplementationofstrategicmanagementpracticestoensurethecontrolofthediseaseandthedisseminationof the agent.Thus, itwill be necessary to designrisk-basedprograms in each region in the country,adaptedtoitsspecificconditions,evenconsideringproduction systems. Follow-up studies on herdswithPTBover a long time to investigatewhetherthechangeofindividualandherd-levelmanagementpracticesleadtochangesinPTBcontrolonthisherdshouldbeperformed.

Acknowledgments

ThisresearchwasfundedbyVecolandEstrategiadeSostenibilidadCODI2013-2014,UniversidaddeAntioquia (CENTAURO).The authors thankProf.Dr.MarthaOlivera, JulianLondoño,MV (Vecol),and to farmers for allowing animal testing andinformation collecting.Authors also thank alltechnical and laboratory teamwho supported alltestinganddiagnosticprocedures,especiallyClaudiaWalteratIFTN,Giessen.

Conflicts of interest

Theauthorsdeclaretheyhavenoconflictsofinterestwithregardtotheworkpresentedinthisreport.

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