h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Veterinary
Microbiology
Mycobacterium
paratuberculosis
detection
in
cow’s
milk
in
Argentina
by
immunomagnetic
separation-PCR
Liliana
Rosa
Gilardoni
a,
Bárbara
Fernández
a,
Claudia
Morsella
b,
Laura
Mendez
b,
Ana
María
Jar
a,
Fernando
Alberto
Paolicchi
b,
Silvia
Leonor
Mundo
a,∗aCátedradeInmunología,FacultaddeCienciasVeterinarias,UniversidaddeBuenosAires,BuenosAires,Argentina bLaboratoriodeBacteriología,EEA-INTABalcarce,Balcarce,BuenosAires,Argentina
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received7August2014 Accepted17August2015 Availableonline2March2016 AssociateEditor:JorgeLuizMello Sampaio
Keywords:
Mycobacteriumaviumsubsp.
paratuberculosis
Immunomagneticseparation PCR
Cow’smilk
a
b
s
t
r
a
c
t
TheaimofthisstudywastostandardizeadiagnosisproceduretodetectMycobacteriumavium
subsp.paratuberculosis(Map)DNAinrawcowmilksamplesunderfieldconditions.A pro-cedurethatcombinesbothimmunomagneticseparationandIS900-PCRdetection(IMS-IS1 PCR)wasemployedonmilksamplesfrom265lactatingHolsteincowsfromMapinfectedand uninfectedherdsinArgentina.IMS-IS1PCRresultswereanalyzedandcomparedwiththose obtainedfrommilkandfecalcultureandserumELISA.Theextentofagreementbetween bothtestswasdeterminedbytheKappatest.IMS-IS1PCRshowedadetectionlimitof101
CFUofMap/mLofmilk,when50:50mixofmonoclonalandpolyclonalantibodieswereused tocoatmagneticbeads.Allofthe118samplesfromtheMapuninfectedherdswerenegative forthesetofthetests.InMapinfectedherds,80outof147cowstestedpositivebymilk IMS-IS1PCR(55%),ofwhich2(1.4%)werealsopositivebymilkculture,15(10%)byfecalculture, and20(14%)byserumELISA.Kappastatistics(95%CI)showedaslightagreementbetween thedifferenttests(<0.20),andtheproportionsofagreementwere≤0.55.TheIMS-IS1PCR methoddetectedMapinmilkofthecowsthatwerenotpositiveinothertechniques.This isthefirstreportdealingwiththeapplicationofIMS-IS1PCRinthedetectionofMapinraw milksamplesunderfieldconditionsinArgentina.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Paratuberculosis(PTB)or Johne’sdisease isachronic gran-ulomatousenteritis ofruminants, causedbyMycobacterium aviumsubsp. paratuberculosis (Map). Itischaracterizedby a
∗ Correspondingauthor.
E-mail:[email protected](S.L.Mundo).
long incubation period, weight loss, diarrhea, progressive cachexia,and death.PTBhasa worldwidedistribution and causes economic loss in dairy cattle due to reduced milk production and premature culling.1 The infected animals
starttoexcreteMapinfeces,milk,andcolostrums,beforethe appearanceofclinicalsymptoms2resultingincontamination
http://dx.doi.org/10.1016/j.bjm.2016.01.013
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
oftheenvironmentandsubsequentinfectionofsusceptible animals,primarilyincalfhood.3Unweanedcalves,whichare
moresusceptible than the adults,can be infectedbecause Mapisexcretedthroughmilkorthemilkmightbe contam-inated by feces.4 The presenceof Map has been found in
commercialcow’smilkdestinedforhumanconsumptionall overtheworld5–9 includingArgentina,10 andmightimplya
potentialrisktopublichealthduetoitspossiblerelationship withCrohn’sdisease.ParatuberculosisislistedintheWorld Organizationfor Animal Health’s(OIE) Manual ofDiagnostic TestsandVaccinesforTerrestrialAnimalsandclassifiedunder RiskGroup2forhumaninfections.11
ThereareseveralstrategiestocontrolMapdissemination withinaherdthatincludevaccination,changesin manage-mentpractices,andearlydetectionandcullingofthecows withsubclinical infection,but the currently available diag-nostictestsdonotpossessenoughsensitivity(Se)todetect infectedanimalsduringtheearlystagesoftheinfection.The fecalcultureisconsideredasthereferencetestfordetecting Mapinliveanimals,butthismethodislaborintensiveand requireslongincubationperiodsandaminimumnumberof viablebacteriainthesample.TheSeofthismethodin subclin-icallyinfectedcowsislow(23–29%),whileitsspecificity(Sp) couldreachupto100%,ifthecultureisolatescouldbe con-firmedbyPCR.12TheserumELISAhascommonlybeenused
asafastandlow-costserologytest,buttheSeofthismethod isalsolow(15%)inanimalsatsubclinicalstagewithalowor moderatefecalshedding.13
Inrecentyears,PCRhasbeenthemostwidelyused tech-niquefordetectionofMap,thoughtheSeofthismethodwhen applieddirectlytomilkislow(23%)duetothepresenceofPCR inhibitorysubstancespresentinmilk.Consequently,a suit-ablesamplepreparationpriortothePCRdetectionofMapis necessaryinordertomaximizethesensitivityofthismethod. Theuseofimmunomagneticseparation(IMS)usingmagnetic nanoparticlescoupledtopolyclonalanti-Mapantibodiesisan effectiveproceduretocaptureMapfromaheterogeneousand largevolumesample,andtoreducetheinterferencesofPCR inhibitorysubstances.2,14–17
Inaccordancewiththisapproach,adiagnosticprocedure wasstandardizedtodetectMapinrawcowmilksamples.This methodcombinestheuseofimmunomagneticbeadscoupled toMap-specificpolyclonalandmonoclonalantibodiesto iso-lateMapandmodifiedIS900PCRtodetectMapDNA(IS1PCR). Theresultswerecomparedwiththoseobtainedthrough rou-tinetestssuchasmilkand fecalculturesand serumELISA fromthefieldsamples.
Materials
and
methods
Bacterialstrains
The reference Map strain ATCC 19698 and the field Map strain Malele 35 (M35; Bacteriology Laboratory Collection, EEA-INTABalcarce)wereusedaspositivecontrols.M35was isolated from cattle and typed by IS900 PCR and RFLP.18
Both the Map strains were cultured in Middlebrook 7H10 medium (Difco Laboratories Inc., Becton, Dickinson and Company,FranklinLakes,NJ,USA),supplementedwitholeic
acid, bovinealbumin, dextrose and catalase(OADC,Difco), 2mg/LmycobactinJ(AlliedMonitor,Fayette,USA),and4.1g/L sodiumpyruvate(Sigma–Aldrich,St.Louis,MO,USA). Mouseanti-Mapantibodies
Amonoclonalantibody(mAb)specifictoMap-membrane pro-teinp34(clone1A6E10)19 aswellasapolyclonalantiserum
(pAb)specifictowholeMapweredevelopedearlierinour lab-oratory.Asciticfluidandmouseserumweresemipurifiedby precipitation with ammonium sulfateand further dialyzed againstPBS.
StandardizationofIMS-IS1PCRprocedure Coatingofimmunomagneticbeads
Goatanti-mouseIgGmagneticbeads(NewEnglandBioLabs Inc.,Ipswich,MA,USA)weremixedfor1hat4◦Cunder con-stant shaking.The beads (3.65×108)in a volume of 10L
were coatedwith 10g ofeither anti-Map1A6E10 mAb or anti-MappAb.Fornegativecontrols,immunomagneticbeads werecoatedwithanidenticalquantityofeitheramonoclonal antibodyoramousepolyclonalantiserumofanon-related specificity:anti-N-6-methyladenine.17After1hat4◦Cunder
constantshaking,eachsetofantibody-coatedbeadswas sep-aratedfor10minusingamagneticrack,washedthreetimes inPBS,resuspendedin100LofPBS,andstoredat4◦Cuntil furtheruse.
CaptureofMapbycoatedbeads
Aseriesof10-folddilutionofmilksamples(1mLaliquots) ini-tiallyspikedwith1012CFU/mLofMapstrains,ATCC19698or
M35waspreparedafterbreakingthebacterialclumpsby pass-ingthrougha25-gaugeneedle.Inordertoimprovetheamount ofMapthatcouldbeobtainedinthepellet,thesampleswere heatedfor15minat50◦C,centrifugedat6000×gfor20min at4◦C,andthepelletswereresuspendedin1mLofPBS.This suspensionwasusedforimmunocapturing.Eachsetofthe coated beadina volumeof10Lwas addedtoeach sam-pleandincubated for1hat4◦Cunderconstantshakingto enableMapimmunocapture.Then,thebeadswereseparated for10minwithamagneticrack(immunomagneticseparation: IMS),andwashedthricewithPBS.Boththesupernatantsfrom eachcoatedbeadsandoriginalsampleswere10-foldserially dilutedandculturedforthreemonthsat37◦CinMiddlebrook 7H10mediumsupplementedasdescribedabove.Mapbound toeachtypeofthecoatedbeads(bacterialrecovery)was deter-minedbysubtractingtheCFUsremainingontherespective supernatantafterimmunocapturing,fromthosethatare orig-inallypresent onthe sample.Theassay wasrepeated four timesforeachtypeofcoatedbeads.
IS900PCRdetectionofMapcapturedbycoatedbeads
After IMS, the beads were boiled at 100◦C for 30min and centrifuged at7000×g for 15min, then 5Lof super-natant was used as template for the PCR mixture of 50L reaction. IS900 PCR was performed according to the protocol of Mundo et al.17 to amplify IS1 fragment
(nucleotides1129–1283;155bp) intheIS900sequence using primers IS1-F (5-ACCCGCTGCGAGAGCAATCGCTGC-3) and
IS1-R(5-ACGTCGGCGTGGTCGTCTGCTGGG-3).Amplification wasperformedin0.2mLmicrotubesusingaPTC-100TM Pro-grammableThermalController(MJResearchInc.,Waltham, MA, USA). The DNA from Map ATCC 19698 was used as thepositivecontrolandsterilewaterasnegativecontrolfor eachofthePCRassay.Theproductswereelectrophoretically separated at50Vfor2h ina2%(w/v) agarosegel,stained withethidiumbromide(0.5g/mL),andobservedunder an ultraviolettransilluminator.TheprocedurethatincludesPCR detection ofMap captured by coatedbeads, hereinafter, is referredtoasIMS-IS1PCR.
ApplicationofIMS-IS1PCRtofieldsamples
Milk, feces, and blood samples were individually collected from265Holsteindairycows:147from5Mapinfectedherds and 118 from 4Map uninfectedherds, from Buenos Aires province,Argentina.Alltheanimalswereselectedunderno formalrandomization;thecriteriaforinclusioninthestudy beingage (4–10years old)and biological andhealth status (cowsshouldbelactating andhealthy,without anyclinical signsofPTB)atthetimeofcollectionofsamples.Theherds were considered Map infected when serum ELISA positive titersorMapisolationfromfecalormilkculturewasobtained, ortherewasahistoryofclinicalPTB.Theherdswere consid-eredMapuninfected,whennoneofthesetestswerepositive duringthepreviousfiveyears.Themilksamples(50mL)were manuallycollectedinsterilecontainersfromcranialrightand caudalleftquartersaftercleaningtheudderandnipplewith an iodide solution and foremilk stripping. The feces (10g) wascollectedfromtherectumofeachcowusingdisposable examinationgloves;10mLofwholeblood wasobtainedby venipunctureofventralmediancoccygealvein.Fecalandmilk samples from infected herds were individually processed, whilethosefromuninfectedherdswerepooledingroupsof fivesampleseach.Allsamplesweretransportedtothe lab-oratoryat4◦Candstoredat−20◦Cwithouttheadditionof
chemicalpreservativesuntiltheywereprocessedwithintwo weeks.Themilksamplesweredistributedintotwoparts,one forbacterialcultureandtheotherforIMS-IS1PCR.Theresults obtainedbyIMS-IS1PCRprocedurewerecomparedwiththose obtainedbyroutinediagnostictests(milkand fecalculture andserumELISA)fromsamplescollectedsimultaneously. IMS-IS1PCR,F57PCR,andPCRamplificationchecktest IMS-IS1 PCR was performed on 10mL of milk samples as describedabove.ToensurethatthePCRamplification prod-uctsobtainedfrommilksampleswerenotduetoIS900-like sequences,amplificationofF57insertionelement20was
per-formed in all positive samples according to the protocol describedbyVansnicketal.21usingtheF57sequenceasthe
reference(GenBankaccessionno.X70277).Briefly,theF57PCR mixturewasmade-upintoafinalvolumeof25L contain-ing10mMTris–HCl(pH8.3),50mMKCl,1.6mMMgCl2,200M
ofeachofthefourdNTPs,20pMofeachprimer,0.5UofTaq polymerase(TaqDNA PolymeraseRecombinant, Invitrogen, SaoPaulo,Brasil),and5LofthebacterialDNAextract.The amplificationreactionswereperformedinaPTC-100TM ther-mocycler(MJResearch)asfollows:onecycleofdenaturation at94◦Cfor4min,40cyclesofdenaturationat94◦Cfor45s,
annealingat68◦Cfor45s,extensionat72◦Cfor45s,anda finalextensionat72◦Cfor10min.ThePCRproductswere ana-lyzedbyelectrophoresisat50Vfor2hona2%(w/v)agarose gel,stainedwithethidiumbromide,andobservedunderan ultraviolettransilluminator.Inordertoensurethatthe neg-ativeresultsofIMS-IS1PCRwerenotduetothepresenceof inhibitorysubstances,aPCRamplificationchecktestwas per-formedoneachnegativemilksampleasfollows:aduplicateof eachmilksamplewasspikedwith105CFU/mLofM35,10-fold
seriallydilutedtoobtainasetoffivemilksamplescontaining between105and101CFU/mL,andIMS-IS1PCRwasperformed.
Milkculture
The milk samples were cultured following the protocol described by Paolicchi et al.22 Briefly, 40mL of milk was
centrifuged at 2400×g for 30min; the pellet was resus-pended in 60mL of 0.75% hexadecylpyridinium chloride (HPC,Sigma–Aldrich),allowedtostandfor5h,centrifugedat 2000×g for15min,and finallyresuspendedin1mLofPBS. Four dropsofthe samplewere culturedon threeslopesof HEYMandobservedasdescribedabove.
Fecalculture
The fecal samples were cultured following the protocol describedbyPaolicchietal.22Briefly,10goffeceswere
decon-taminatedwith100mLof0.75%HPCfor30minunderstirring, and thenallowedtostandforafurther30min.The super-natant (40mL) was transferred to aclean tube, allowedto standovernightatroomtemperature,centrifugedat2000×g
for15min,and the pelletwas resuspended in1mL ofPBS and used as the inoculum. Four drops of this inoculum wereculturedonthreedifferentslopesofHerrold’seggyolk medium(HEYM)alone,HEYMsupplementedwith2.0mg/Lof mycobactin, and4.1g/Lofsodiumpyruvate (supplemented HEYM); and supplemented HEYM with an antibiotic mix-ture(100g/mLofvancomycin,2.0mg/mLofamphotericinB, 3.0mg/mLnalidixicacid,and100g/mLnystatin).Theslopes werevisuallyobservedevery15daysforatotaloffourmonths; developingcolonieswereidentifiedbyZiehlNeelsenstaining, andconfirmedbytraditionalIS900PCR.23
ELISA
Theassaywasperformedfollowingtheproceduredescribed by Paolicchi et al.22 Briefly, Paratuberculosis Protoplasmic
Antigen(PPA-3,Allied Monitor,Fayette,MO,USA)wasused to sensitize 96-well polystyrene microplates (Immulon I, Dynatech, Arlington, TX, USA). The serum samples were pre-adsorbedwithMycobacteriumphleiandtestedata1:100 dilution;anHRP-conjugatedanti-bovineIgG(Sigma–Aldrich) at a 1:4000 dilution was used as secondary antibody. The reaction was developed with 2,2 -azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS, Sigma–Aldrich) and readat405nm.TheAbsorbanceIndices(AI)werecalculated astheratiobetweenthemeanopticaldensity(O.D.)ofeach serum sample and the mean O.D. of the negative control
Table1–Mapcapture(CFU)bybeadscoatedwithdifferentantibodiesfrommilkspikedwith108CFU.
Coatedbeadtype Mapcapture(CFU±SD) p
ReferencestrainATCC19698 FieldstrainM35
10LmAb-beadsa 3×104±2×104 5×104±3×104 0.46
10LpAb-beadsb 6×103±3×103 6×103±2×103 1.00
5LmAb-beads+5LpAb-beadsc 4×107±2×107 5×107±3×107 0.66
10Lc.mAb-beadsd 5×101±3×101 6×101±1×101 0.71
10Lc.pAb-beadsd 8×101±1×101 6×101±3×101 0.34
SD,standarddeviation;mAb,anti-Mapmonoclonalantibody1A6E10;pAb,anti-Mappolyclonalserum;c.mAb,monoclonalantibodyof non-relatedspecificity;c.pAb,polyclonalserumof non-relatedspecificity.Withineach strain,valueswith differentlettersmeansignificant differencesbyWilcoxonranksumtest(avs.c:p=0.03).
serum.Bovineserawereclassifiedasnegative(AI≤1.5)or pos-itive(AI>1.5).
Statisticalanalysis
Kappa coefficient was calculated using WinEpiscope soft-wareversion2.0,2000(http://ww.clive.ed.ac.uk/winepiscope) tomeasurethe agreement betweendifferent pairsoftests (milk culture,fecal culture,and ELISA versusIMS-IS1 PCR) performedonthe147cowsthatbelongedtoinfectedherds. TheresultswererankedasdescribedbyLandisandKoch24:
<0.00,pooragreement;0.00–0.20,slightagreement;0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantialagreement;0.81–1.00,almostagreement.The dif-ferenceinthesignificanceofthetests(p)wasdeterminedby theWilcoxonsigned-ranktest.
Results
StandardizationofIMS-IS1PCR
TheresultsofthecomparisonofrecoveryofMapfromspiked rawmilksamplesobtainedusingdifferentantibodiestocoat immunomagneticbeadsareshowninTable1.Themaximum recovery ofMap (≈5×107CFU)was obtained when a
mix-tureof5LeachofmAb-beadsandpAb-beadswasappliedto milksamplesspikedwith108CFUofMap.Nofurtherincrease
inrecoverywasobtainedathigherspikinglevels; however, nearly100%recoverywasobtainedwhenmilksampleswere spikedwith≤107CFUofMap.Hence,thismixturewasused
forfurtherstudiesonfieldsamples.Theuseofmonoclonal antibodiesorpolyclonalantiserumofanunrelatedspecificity allowedarecoveryofupto9×101CFUwhenmilkwasspiked
with≥108CFUofMap,whilenobacteriumwasretrievedwhen
milksampleswerespikedwith≤108CFU.Nostatistically
sig-nificantdifferences were detectedbetween therecovery of Mapstrains,ATCC19698andM35.
TheIMS-IS1PCRprocedureenabledthedetectionofaslittle as101CFUofMapwhenthemixtureof5LeachofmAb-beads
andpAb-beadswasused(Fig.1).PCRswerealwaysnegative whenbeads coatedwithnon-specificantibodies were ana-lyzed.
250 pb
MK 1 2 3 4 5 6 7
IS1
Fig.1–DetectionofMap(M35)byIMS-IS1PCRprocedure. Sampleselectrophoresedin2%agarose.MK:50bpmarker; Lane1:positivecontrol;Lane2:101CFU/mL,Lane3: 102CFU/mL,Lane4:103CFU/mL,Lane5:104CFU/mL,Lane
6:105CFU/mL,Lane7:negativecontrol.
ApplicationofIMS-IS1PCRtofieldsamples
Atotalof265HolsteindairycowswereanalyzedbyIMS-IS1 PCRusingmilk,fecalandmilkculture,andserumELISA.When IMS-IS1 PCRwas appliedtodiagnose the 147animals that belongedtoMapinfectedherds, 80were foundtobe Map-positive; amongthem,31 werealsotested positivewithat leastoneoftheothertechniquesperformed(Table2),whereas 49werepositiveonlytoIMS-IS1PCR.Allofthe80samples, deemedpositivebyIMS-IS1PCR,wereconfirmedbytheF57 PCR, whileallthe 67 samplesthatwere foundnegativeby IMS-IS1PCRwereconfirmednegativeforMapbyperforming the PCRamplificationchecktest.Thepooledsamplesfrom the118cowsbelongingtotheuninfectedherdsandshowing anegativetestinIMS-IS1PCRwerefurtherconfirmedwiththe PCRamplificationchecktest;andtheyalsoshowednegative resultstoalltheothertechniquesperformed.
Statisticalanalysis
The results for the different pairs of diagnostic methods are presented in Table 3a. The Kappa coefficient (95% CI) showedaslightagreementaccordingtotherankingof Lan-disandKoch,24andtheproportionsofagreementwere≤0.55
Table2–IMS-IS1PCRresultsforthestudiedanimalsin thoseMaporanti-Mapantibodiesweredetected.
Cow ID
Mapculture ELISA IMS-IS1 PCR Fecalculture Milkculture
5788 + − + + 6616 + − + + 1595 + − + + 2185 + − + + 2390 + − + + 2408 + − + − 2106 − + + + 2197 − + − + 5984 + − − + 6652 + − − + 6669 + − − + 6673 + − − + 6873 + − − + 7008 + − − + 2008 + − − + 2032 + − − + 2218 + − − + 1977 + − − + 2417 + − − − 6084 − − + + 7006 − − + + 7596 − − + + 7509 − − + + 7002 − − + + 1758 − − + + 1887 − − + + 2184 − − + + 2442 − − + + 2473 − − + + 2480 − − + + 2482 − − + + 2622 − − + + 1784 − − + + 7160 − − + − 7501 − − + − 1688 − − + − 1950 − − + − 2566 − − + − Total 17 2 26 31
Discussion
Paratuberculosishasgeneratedgreatinterestinrecentyears because of its increasing worldwide prevalence leading to severe economic losses and healthconcerns caused by its zoonoticpotential.Incattle,milkherdsaremainlyaffected,as milkingcowscanremaininthefarmsformanyyearsunder an intensive production system.3 Fecal culture and serum ELISAarethetechniquesroutinelyusedforthediagnosisof PTB, buttheirresultsarereliableonlyinthelaterstagesof thedisease.25–27ThedetectionofMapinmilktodetectand
remove infectedanimals hasan economicimpact indairy industryandcouldbeofgreatuseinrelationtofoodsafety.
In this study, a diagnostic procedure known as IMS-IS1 PCR that involved the separation of Map by immunomag-neticbeadscoatedwithMap-specificantibodiesfollowedby amodifiedIS900PCRwasstandardized.Animmunomagnetic separationtechniquewasusedinordertocaptureMapfrom milksamples,toremovethePCR-inhibitorysubstances,and to avoidthe harmful effects ofchemical decontamination. Itwasdemonstratedthattheuseofamixtureof5Leach ofmAb-beadsandpAb-beadsshowedhighercaptureofMap thaneitherof10LofmAb-beadsor10LofpAb-beadsused separatelytocaptureMap,whichinturnincreasedthe quan-tityofDNAthatwasusedforPCR.Asaresult,thedetection limitoftheIMS-IS1PCRprocedurereached101CFU/mLthat
iscomparabletothatdescribedbyMundoetal.17andKhare
et al.2 byradiometric detection and real-timePCR,
respec-tively.Thislimitofdetectionishigherthanthosedescribedby Grantetal.,14Metzger-Boddienetal.,28andStratmannetal.15
butslightlylowerthanthosedescribedbyFoddaietal.29who
usedspecificantibodiesorpeptidestodetectMapby immuno-magneticseparationtechniques.
IMS-IS1 PCR procedure was assessedunder field condi-tions,andtheresultswerecomparedwiththoseobtainedby milk culture,fecalculture, and aserum in-house ELISA.A slightlevelofagreementwasobservedbyKappatestbetween this procedure and routinediagnostic techniques,probably becauseeachtechniquedetectsdifferenttargets.Inthisstudy, morepositiveresultswereobtainedthroughIMS-IS1PCRthan
Table3–Kappatestanalysisbetweenresultsobtainedindifferentsetsofdiagnosticmethodsforthe147cowsbelonging toinfectedherds.
a.Contingencytable
IMS-IS1PCR Fecalculture Milkculture ELISA
+ − + − + −
+ 15 65 80 2 78 80 20 60 80
− 2 65 67 0 67 67 6 61 67
17 130 147 2 145 147 26 121 147
b.Kappacoefficientandproportionofagreement
IMS-IS1PCR Fecalculture Milkculture ELISA
Kappa(95%CI) 0.15(0.05–0.24) 0.02(0.01–0.05) 0.15(0.03–0.27)
Concordantresults 80 69 81
throughanyothertechnique.Thiscouldbeduetotheeffects ofconcentrationofMapandremovalofmilkinhibitorsofPCR achievedbyimmunomagneticbeads,aswellasthe possibil-itythatPCRwilldetectviableaswellasnon-viableMap.On theotherhand,thebacteriacarryingIS900-likesequences30
werenotamplified;allthesamplesthatshowedIS1 amplifi-cationgavenegativeresultswithF57PCR,confirmingthefact thattheyweretruepositivesamples.InMapinfectedherds, theproportionofpositiveresultsobtainedbyfecalandmilk culturewaslow(11.6%and1.4%,respectively).Thiscouldbe explainedbythefactthatfecalaswellasmilksheddingofMap canbelowandintermittentinsubclinicallyinfectedcows,a significantfractionofMapcanbekilledduringthe decontami-nationprocedure,andalsonotalloftheMapthatispresentin thesamplescanbeculturedsuccessfully.27,31Therefore,fecal
andmilkcultureresultsarelikelytobeunderestimates. It has been described that the measurable humoral immuneresponsetoMapinsubclinicalcowscanvarywidely overtime,evenfromdaytoday,32whileitistypicallyrelated
tothelaterstagesofthedisease.Thiscouldpossiblybethe reasonbehindthelownumberofpositiveanimalsthatwere detectedbythein-houseELISA(17.7%).Thepre-absorptionof eachserumwithM.phleireducesthenumberoffalsepositives results,butmay alsoreducethe sensitivity.33 Inthis study,
some animals (12.9%) were identified as positive by ELISA but negativebyfecalor milkculture.Thisisin agreement withNielsenandToft12whofoundthatonlyapproximately
10–30% of ELISA-positive cows could be confirmed by the culture, assome cows could be Map infected, but notyet Map-shedders.
The absence of clinical signs in the animals evaluated inthis studyindicates that theanimals mustbeidentified throughlaboratorydiagnosticteststhatcurrentlysufferfrom low sensitivity. Theapparently higher performanceshown bytheIMS-IS1PCRprocedure confirmsthedisadvantageof performing the diagnosis of PTB only by means of bacte-rialculture34orserumELISA.35,36Underfieldconditions,the
methodologydescribedinthisstudypermittedidentification ofmilkingcowsinsubclinicalstagefromMapinfectedherds thatwereelusivetoothertechniques.Werecognizedthatthe confirmation oftrue infection status can onlybe obtained byhistopathologyandcultureofintestinaltissues,butpost mortemevaluationisbeyondthescopeofthisstudy.
Insummary,thisreportisthefirstoneinwhichIMS-IS1 PCRprocedurehasbeenappliedforthedetectionofMapin rawcowmilksamplesfromArgentinaunderfieldconditions. ThecombineduseofIMS-IS1PCR,bacterialculture,andELISA couldincreasetheoveralldetectionrateforthediagnosisof paratuberculosis.
Conclusions
Theaimofthis study wastostandardizea procedurethat couldbeappliedforthedetectionofMapincowmilksamples basedonimmunomagneticseparationandIS1PCRdetection. Furtherin-depthstudiesarenecessaryinordertoestablish thetrueproportionofinfectedanimalsthatcanbedetected by this method and the stages of the disease when it is present.
Conflict
of
interest
Theauthorsdeclarethattheyhavenocompetinginterests.
Acknowledgements
Thiswork wassupportedbyGrantsfrom AgenciaNacional de Promoción Científica y Técnica, Argentina (BIDPICT 2010-2672), Universidad de Buenos Aires, Argentina (UBA-CyT 20020100100912), andInstituto Nacionalde Tecnología Agropecuaria,Argentina(INTA#202831AESA).
r
e
f
e
r
e
n
c
e
s
1.McKennaSL,KeefeGP,TiwariA,VanLeeuwenJ,Barkema
HW.Johne’sdiseaseinCanada.PartII:diseaseimpacts,risk
factors,andcontrolprogramsfordairyproducers.CanVetJ.
2006;47:1089–1099.
2.KhareS,FichtTA,SantosRL,etal.Rapidandsensitive
detectionofMycobacteriumaviumsubsp.paratuberculosisin
bovinemilkandfecesbyacombinationofimmunomagnetic
beadseparation-conventionalPCRandreal-timePCR.JClin
Microbiol.2004;42:1075–1081.
3.BarkemaHW,HesselinkJW,McKennaSLB,BenedictusG,
GroenendaalH.Globalprevalenceandeconomicsof
infectionwithMycobacteriumaviumssp.paratuberculosisin
ruminants.In:BehrMA,CollinsDM,eds.Paratuberculosis.
Organism,Disease,Control.CABI;2010:10–17.ISBN: 9781845936136.
4.RadiaD,BondK,LimonG,vanWindenS,GuitianJ.
Relationshipbetweenperiparturientmanagement,
prevalenceofMAPandpreventableeconomiclossesinUK
dairyherds.VetRec.2013;14:173–343.
5.GrantIR,BallHJ,RoweMT.IncidenceofMycobacterium
paratuberculosisinbulkrawandcommerciallypasteurised
cows’milkfromapproveddairyprocessingestablishments
intheUnitedKingdom.ApplEnvironMicrobiol.
2002;68:2428–2435.
6.AyeleWY,SvastovaP,RoubalP,BartosM,PavlikI.
Mycobacteriumaviumsubspeciesparatuberculosiscultured
fromlocallyandcommerciallypasteurizedcow’smilkinthe
CzechRepublic.ApplEnvironMicrobiol.2005;71:1210–1214.
7.EllingsonJL,AndersonJL,KoziczkowskiJJ,etal.Detectionof
viableMycobacteriumaviumssp.paratuberculosisinretail
pasteurizedwholemilkbytwoculturemethodsandPCR.J
FoodProt.2005;68:966–972.
8.ShankarH,SinghS,SinghP,SinghA,SohalJ,GreensteinR.
Presence,characterizationandgenotypeprofilesof
Mycobacteriumaviumsubsp.paratuberculosisfrom
unpasteurizedindividualandpooledmilk,commercial
pasteurizedmilkandmilkproductionproductsinIndiaby
culture,PCRandPCRREAmethods.IntJInfectDis.
2009;14:121–126.
9.CarvalhoIA,PietralongaPAG,SchwarzDGG,FariaACS, MoreiraMAS.Shortcommunication:recoveryofviable
Mycobacteriumaviumsubspeciesparatuberculosisfromretail pasteurizedwholemilkinBrazil.JDairySci.
2012;95:6946–6948,http://dx.doi.org/10.3168/jds.2012-5657.
10.PaolicchiFA,CironeK,MorsellaC,GioffréA.Firstisolationof
Mycobacteriumaviumsubsp.paratuberculosisfromcommercial
pasteurizedmilkinArgentina.BrazJMicrobiol.
2012;43:1034–1037.
11.OIEManualofDiagnosticTestsandVaccinesforTerrestrial Animals.2014Chapter.2.1.11,Paratuberculosis(Enfermedad
deJohne).http://www.oie.int/fileadmin/Home/eng/
Healthstandards/tahm/2.01.11PARATB.pdf(accessed26
May2014).
12.NielsenSS,ToftN.Antemortemdiagnosisof
paratuberculosis:areviewofaccuraciesofELISA,
interferon-␥assayandfecalculturetechniques.VetMicrobiol.
2008;129:217–235.
13.ChuiLW,KingR,SimJ.Developmentofan
immunocapture-polymerasechainreactionassayusingIgY
todetectMycobacteriumaviumssp.paratuberculosis.CanJVet
Res.2010;74:102–107.
14.GrantIR,PopeCM,O’RiordanLM,BallHJ,RoweMT.Improved
detectionofMycobacteriumaviumsubsp.paratuberculosisin
milkbyimmunomagneticPCR.VetMicrobiol.2000;77:
369–378.
15.StratmannJ,DohmannK,HeinzmannJ,GerlachGF.Peptide
aMptD-mediatedcapturePCRfordetectionofMycobacterium
aviumsubsp.paratuberculosisinbulkmilksamples.Appl EnvironMicrobiol.2006;72:5150–5158.
16.LogarK,KopinR,BandeljP,Stari ˇcJ,LapanjeA,OcepekM.
Evaluationofcombinedhigh-efficiencyDNAextractionand
real-timePCRfordetectionofMycobacteriumaviumsubsp.
paratuberculosisinsubclinicallyinfecteddairycattle:
comparisonwithfaecalculture,milkreal-timePCRandmilk
ELISA.BMCVetRes.2012;8:49.
17.MundoSL,GilardoniLR,HoffmanFJ,LopezOJ.Rapidand
sensitivemethodtoidentifyMycobacteriumaviumsubsp.
paratuberculosisincow’smilkbyDNAmethylasegenotyping.
ApplEnvironMicrobiol.2013;79:1612–1618.
18.MoreiraAR,PaolicchiFA,MorsellaC,etal.Distributionof
IS900restrictionfragmentlengthpolymorphismtypes
amonganimalMycobacteriumaviumsubsp.paratuberculosis
isolatesfromArgentinaandEurope.VetMicrobiol.
1999;70:251–259.
19.StemplerA,JollyA,ColavecchiaS,SobaM,FontanalsA,
MundoSL.Developmentandevaluationofamonoclonal
antibodyagainstMycobacteriumaviumsubsp.paratuberculosis.
VetImmunolImmunopathol.2009;128:335–336.
20.PoupartP,CoeneM,VanHeuverswynH,CocitoC.
PreparationofaspecificRNAprobefordetectionof
MycobacteriumparatuberculosisanddiagnosisofJohne’s
disease.JClinMicrobiol.1993;31:1601–1605.
21.VansnickE,deRijkP,VercammenF,GeysenD,RigoutsL,
PortaelsF.Newlydevelopedprimersforthedetectionof
Mycobacteriumaviumsubsp.paratuberculosis.VetMicrobiol.
2004;100:197–204.
22.PaolicchiFA,ZumarragaMJ,GioffreA,etal.Applicationof
differentmethodsforthediagnosisofparatuberculosisina
dairycattleherdinArgentina.JVetMedB.2003;50:20–26.
23.CollinsDM,StephensDM,deLisleGW.Comparisonof
polymerasechainreactiontestandfaecalculturefor
detectingMycobacteriumparatuberculosisinbovinefaeces.Vet
Microbiol.1993;36:289–299.
24.LandisJR,KochGG.Themeasurementofobserver
agreementforcategoricaldata.Biometrics.1977;33:159–174.
25.BakkerD.ParatuberculosiscontrolmeasuresinEurope.In:
BehrMA,CollinsDM,eds.Paratuberculosis.Organism,Disease,
Control.CABI;2010:306–315.ISBN:9781845936136.
26.NielsenSS.Immune-baseddiagnosisofparatuberculosis.In:
BehrMA,CollinsDM,eds.Paratuberculosis.Organism,Disease,
Control.CABI;2010:284–291.ISBN:9781845936136.
27.WhittingtonRJ.CultivationofMycobacteriumaviumsubsp.
paratuberculosis.In:BehrMA,CollinsDM,eds.
Paratuberculosis.Organism,Disease,Control.CABI;
2010:244–260.ISBN:9781845936136.
28.Metzger-BoddienC,KhaschabiD,SchönbaurM,BoddienS,
SchedererT,KehleJ.Automatedhigh-throughput
immunomagneticseparationfordetectionofMycobacterium
aviumsubsp.paratuberculosisinbovinemilk.IntJFood Microbiol.2006;110:201–208.
29.FoddaiA,ElliottCT,GrantIR.Maximizingcaptureefficiency
andspecificityofmagneticseparationforMycobacterium
aviumsubsp.paratuberculosiscells.ApplEnvironMicrobiol.
2010;76:7550–7558.
30.MöbiusP,HotzelH,RassbachA,KöhlerH.Comparisonof13
single-roundsandnestedPCRassaystargetingIS900,
ISMav2,ISF57andlocus255fordetectionofMycobacterium
aviumsubsp.paratuberculosis.VetMicrobiol.2008;126:324–333.
31.GrantIR,RoweMT.Effectofchemicaldecontaminationand
refrigeratedstorageontheisolationofMycobacteriumavium
subsp.paratuberculosisfromheat-treatedmilk.LettAppl
Microbiol.2004;38:283–288.
32.BarringtonGM,GayJM,EriksIS,etal.Temporalpatternsof
diagnosticresultsinserialsamplesfromcattlewith
advancedparatuberculosisinfection.JVetDiagnInvest.
2003;15:195–220.
33.McKennaSL,SockettDC,KeefeGP,McClureJ,VanLeeuwen
JA,BarkemaHW.Comparisonoftwoenzyme-linked
immunosorbentassaysfordiagnosisofMycobacteriumavium
subsp.paratuberculosis.JVetDiagnInvest.2005;17:463–466.
34.AlinoviCA,WardMP,LinTL,MooreGE,WuCC.Realtime
PCRcomparedtoliquidandsolidculturemediaandELISA,
forthedetectionofMycobacteriumaviumssp.paratuberculosis.
VetMicrobiol.2009;14:177–179.
35.PinedoPJ,WilliamsJE,MonifGRG,RaeDO,BuergeltCD.
Mycobacteriumparatuberculosissheddingintomilk:
associationofELISAseroreactivitywithDNAdetectionin
milk.IntJApplResVetMed.2008;6:137–144.
36.BadiFA,AlHaroonAI,AlluwaimiAM.Thegammadeltacells
asmarkerofnon-seroconvertedcattlenaturallyinfected
withMycobacteriumaviumssp.paratuberculosis.ResVetSci.