Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Detection

of

Mycobacterium

avium

subsp.

paratuberculosis

in

bovine

milk

from

the

state

of

Pernambuco,

Brazil

Pedro

Paulo

Feitosa

de

Albuquerque

,

André

de

Souza

Santos,

Orestes

Luiz

de

Souza

Neto,

Pomy

de

Cássia

Peixoto

Kim,

Erika

Fernanda

Torres

Samico

Fernandes

Cavalcanti,

Júnior

Mário

Baltazar

de

Oliveira,

Rinaldo

Aparecido

Mota,

José

Wilton

Pinheiro

Júnior

UniversidadeRuralFederaldePernambuco,DepartamentodeMedicinaVeterinária,LaboratóriodeBacterioses,Recife,PE,Brazil

a

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o

Received14March2015 Accepted25April2016

Availableonline8November2016 AssociateEditor:JorgeLuizMello Sampaio Keywords: Molecularbiology Diagnosis Paratuberculosis Milk

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TheaimofthisstudywastodetecttheIS900regionofMycobacteriumaviumsubsp. paratuber-culosis(MAP)inbovinemilksamplesusingreal-timepolymerasechainreaction(qPCR)and conventionalPCR,andtostudytheagreementbetweenthesetests.Atotalof121bovine milksampleswerecollected fromherdsconsideredpositiveforMAP,fromtheStateof Pernambuco,Brazil.MAPDNAwasdetectedin20samples(16.5%)usingconventionalPCR andin34samples(28.1%)usingqPCR.MAPDNAwasdetectedinallofthe6animalfarms studied.ModerateagreementwasfoundbetweenqPCRandconventionalPCRresults,where thesensitivityandspecificityofconventionalPCRinrelationtoqPCRwere50%and96.6%, respectively.Thus,theIS900regionofMAPwasfoundinbovinemilksamplesfromtheState ofPernambuco.Tothebestofourknowledge,thisisthefirstreportofMAPDNAfoundin bovinemilkinNortheastBrazil.WealsodemonstratedtheqPCRtechniqueismoresensitive thanconventionalPCRwithrespecttodetectionofMAPinmilksamples.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Mycobacteriumaviumsubsp.paratuberculosis(MAP)isa gram-positive,slow-growingbacillusofthefamilyMycobacteriaceae

that possesses a cell wall rich in lipids, characteristic of thisfamily.Thismicroorganismisanintracellularpathogen, responsibleforJohne’sdiseaseorparatuberculosis.1,2

∗ _{Correspondingauthor.}

E-mail:[email protected](P.P.Albuquerque).

Paratuberculosishasbeenpreviouslyinvestigatedin sev-eralstudiesinBrazil.3_{ItsoccurrenceinPernambucohasbeen}

describedindairycattlebasedonclinicalsigns, histopatho-logy,andresultsfromenzyme-linkedimmunesorbentassay (ELISA)serology(32.3%positivesamples),isolation(50% pos-itivesamples),andpolymerasechainreaction(PCR)studies.4

Additionally, a prevalence rate of 2.7% (11/408) has been

http://dx.doi.org/10.1016/j.bjm.2016.10.010

1517-8382/©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

reportedinthemicro-regionofGaranhuns,with47.4%(9/19) outbreaks.5

ThemostcommonmethodsofMAPdiagnosisininfected animalsincludeisolationofbacteriafromfecesusing selec-tiveculturemediaandantibodydetection techniquessuch asELISA.However,thegreatestdisadvantageofusingculture mediaisthelongincubationperiod,whichcanbeaslongas 16weeksforadefinitivediagnosis.ELISAcanbeperformed withinafewhours,althoughits sensitivityisestimatedat only45%,sinceantibodiestoMAPmaynotbedetectablein theinitialstagesoftheinfection.6,7

Molecular techniques to detect MAP, such as PCR, are rapidandqualitativeinnature.Real-timePCR(qPCR)exhibits greatersensitivitythanconventionalPCR,andcandetermine theinfectiveloadinenvironmentalsamples,feces,milk,and cultures.7,8_{OneofthetargetgenesusedtodetectMAPviaPCR}

istheIS900region,firstdescribedbyGreenetal.9_and

indepen-dentlyidentifiedbyCollinsetal.10_{ThediscoveryoftheIS900}

regionofMAPenablesthediagnosisofparatuberculosiseven intheinitialstagesofinfection.Thespecificityandsensitivity ofPCRhavebeenenhanceduptothepointofdetecting1CFU ofMAPinsamples.11

MAPhas been detectedin several animal productsand byproducts.Astudy conductedinSwitzerland detectedthe presenceofthe IS900region in19.7%(273/1384) milk sam-plescollectedfrommilkstoragetanks.12 _{AstudyinCyprus}

reported63(28.6%)positiveoutofatotalof220milksamples fromtanks,usingreal-timePCRforIS900andF57.13

Theaimofthisstudy was todetectthe IS900 regionof MAPinbovinemilksamplesfromtheStateofPernambuco (Brazil)usingPCRandqPCR,andtoinvestigatetheagreement betweenthesediagnostictests.

Materials

and

methods

Sampling

In total, 121 bovine milk samples from the State of Pernambucowerecollected from6dairyherdsthatalready hadahistoryofparatuberculosis.Theanimalswereclinically healthyatthetimeofcollection.

Samplecollectionandprocessing

Samplecollection

Thecowteatswerecleanedwithwateranddisinfectedwith 70%alcoholpriortocollectionofmilksamples.Thefirst3jets ofmilkwere discarded.Subsequently,approximately 50mL milkwaspooledfromthe4mammaryglandsusingsterilized andseparatepolypropylenetubes.Thesampleswerestoredin coolboxescontainingrecyclableiceandsenttotheLaboratory ofBacteriaintheFederalRuralUniversityofPernambucofor processing.

DNAextraction

DNAextractionwas performedusing2mLofeach sample, whichwascentrifugedat12,000×gfor10min,andthepellet wasresuspendedin100␮Lbufferedsalinesolutionwithsterile

phosphate(pH7.2).Acommercialkita_{wasusedforextraction,}

followingthemanufacturer’sinstructions.

Polymerasechainreaction(PCR)

The extracted DNA was amplified in a final volume

of 15␮L, containing the following: 5␮L genomic DNA; 0.5␮L each, of primers specific for IS900 at 20pM (DF: 5-GACGACTCGACCGCTAATTG-3 and DR-1: 5 -CCGTAACCGTCATTGTCCAG-3); 2.75␮L ultrapure Milli-Q waterand6.25␮LPCRkitmixture,b _{asperthemanufacturer}

instructions.TheDNAwasamplifiedinathermocyclerc_using

the following conditions: initial denaturation at 96◦C for 5min,followedby35cyclesofdenaturationat95◦Cfor1min, annealingat58◦Cfor1minandextensionat72◦Cfor3min, with a final extensionat 72◦C for 10min.14 _{Theamplified}

product of 99bp, corresponding to MAP, was detected by electrophoresisinagarosegel(2%),followedbystainingwith bluegreen,d_{andvisualizationunderultravioletlight.Images}

ofthebandswerecaptured.

Polymerasechainreactioninrealtime

TheextractedDNAwasamplifiedinafinalvolumeof25.0␮L containing:5␮LgenomicDNA;1␮Leach,ofprimersspecific forIS900at10pM(DF:5-GACGACTCGACCGCTAATTG-3 and the DR-1:5-CCGTAACCGTCATTGTCCAG-3),5.5␮Lultrapure Milli-Qwater,and12.5␮Lreal-timePCRkitmixture,e

accord-ingtothemanufacturerinstructions.TheDNAwasamplified inathermocyclerf_{usingthefollowingconditions:initial}

dena-turationat95◦Cfor5min,followedby40cyclesat95◦Cfor 20sand60◦Cfor30s.Thethermocyclersoftwarewasusedto monitorandinterprettheresults.

DNA from a MAPstrain provided bythe National Agri-culturalLaboratoryinMinasGeraisidentifiedas“Nakajima 1991”wasusedtostandardizetheqPCR.Themeltingcurve consisted of65◦C for90s forpreparation,with aposterior gradual increase of 1◦C every 5s from 60◦C to 95◦C.The peak ofdenaturationwasat82.9◦C.Thenumber ofcopies wasdeterminedaspreviouslydescribedbyRodrígues-Lázaro etal.15_{Thecopynumberdetectedwasdividedby15,which}

correspondstotheaveragenumberofIS900elementsinthe MAPgenome.16_{TheDNAoftheMAPstrainwas10-foldserially}

dilutedandusedtoobtainthestandardcurverangingfrom 2.27×107to22.6MAPcells.Theefficiencyoftheprimerswas 95.0%andthecoefficientoflinearcorrelation(R2_)was0.99909,

whilethedetectionthreshold(Ct)wasbetween9.58and30.08. Thereactionswereperformedinduplicate.Finally,the num-berofcopiesinthepositivefieldsampleswasquantifiedusing thestandardcurve.

a_{DNAEasyBloodandTissuesKit}®_{,QiagenBiotechnologyBrazil}

Ltda.,SãoPaulo,Brazil.

b_{MasterMix,Promega}®_{Corp.,Madison,WI.}

c_{BioerXPcycler}®_{,BioerTechnology,Hangzhou,China.} d_{BlueGreenLoadingDye(LGCBiotecnologia,SãoPaulo,Brazil).} e _{QuantiFastSYBRGreenPCRKit}®_{,QiagenBiotechnologyBrazil}

Ltda.,SãoPaulo,Brazil.

f _{Rotor-GeneQthermocycler,QiagenBiotechnologyBrazilLtda.,}

Table1–Frequencyofpositivemilksamplesbyanimal farmusingconventionalPCRandqPCRwiththemean valueofMAPcells/mLperanimalfarmdeterminedby qPCR. Animal farm Quantityof samples Conventional PCR qPCR MeanofMAP cells/mL 1 22 3(13.6%) 8(36.4%) 297.78 2 19 3(15.8%) 7(36.8%) 59.88 3 21 5(23.8%) 5(23.8%) 128.4 4 19 3(15.8%) 7(36.8%) 368.15 5 20 4(20.0%) 5(25.0%) 73.33 6 20 2(10.0%) 2(10.0%) 35.97 Total 121 20(16.5%) 34(28.1%) –

Sequencingofthesamplesandvalidationofconventional PCR

In order to validate the amplifications, one of the posi-tives samples from the conventional PCR was purified using a commercial extraction kit,g _{as per the}

manu-facturer instructions, before being sent for sequencing. Sequences with a Phred quality score above 20 were ana-lyzed using the Basic Local Align Sequence Tool (BLAST;

www.blast.ncbi.nlm.nih.gov/Blast.cgi).

Statisticalanalysis

The Kappa coefficient (K) was used to assess the agree-ment between tests. The qPCR was considered as a gold standardandtheconventionalinterpretationoftheKvalues adoptedwasasfollows:0.00–0.20=weak;0.21–0.40=regular; 0.41–0.60=moderate; 0.61–0.80=good; and 0.81–1.00=very goodagreement;negativevalueswereinterpretedas0.00.17

Biostatsoftwareh_{wasusedtocalculatetheagreement,}

sen-sitivity, and specificity. The chi-squared test was used to determineassociationsbetweenthetests,withaconfidence intervalof95%.

Results

Ofthe121milksamplesanalyzed,theIS900regionofMAPwas detectedin20(16.5%)samplesusingconventionalPCRandin 35(28.1%)samplesusingqPCR.Milksamplescontaminated withMAPwerefoundinallsixfarmsstudied,rangingfrom 10.0to23.8%usingconventionalPCRandfrom10.0to36.8% usingqPCR(Table1).

ThenumberofDNAcopies,determinedbyqPCRinall pos-itivesamplesexhibitedameanvalueof160.59MAPcells/mL, with minimum and maximum values of 12.2 and 1126.66 MAPcells/mL,respectively. Themean valueofthe quantifi-cationsperanimalfarmforthepositivesamplesrangedfrom 35.97to368.15MAPcells/mL(Table1).

TheagreementbetweenconventionalPCRandqPCR, ana-lyzedbytheKappacoefficient,was0.53,whichismoderate

g _{QIAquickGelExtractionkit}®_{,QiagenBiotechnologyBrazilLtda.,}

SãoPaulo,Brazil.

h _{BioEstatversion5.0,UniversidadeFederaldoPará,Pará,Brazil.}

(2_{=38.08;p<0.000).Thevaluesforsensitivityandspecificity}

oftheconventionalPCRwere50%and96.6%,respectively. Thesequencing ofthe field sampleexhibited a similar-ity of98%withtheDNA ofM.aviumsubsp. paratuberculosis

(MAP4,completegenome)depositedattheNational Center forBiotechnologyInformation.

Discussion

ThisisthefirstrecordofthedetectionofMAPinbovinemilk inNortheast Brazil.ThepresenceofMAPinmilkhasbeen previouslyreportedinBrazilbyCarvalhoetal.,18_whofound

theIS900regionofDNAin8/222(3.6%)samplesofrawbovine milkfromVic¸osa,MinasGerais.

MAPhasbeendetectedpreviouslyinbovinemilkinother countries. In Switzerland, DNA of IS900 was found in 273 (19.7%)of1384samplesfrommilktanks.12_Slanaetal.13

col-lectedatotalof220milksamplesfromtanksinCyprusand found63(28.6%)positivesamples(IS900andF57)using real-timePCR.

ParatuberculosisisconsidereduncommoninBrazil; how-ever,outbreaksofthediseasehavebeenreportedin11states oftheFederation.3,19_{InPernambuco,Motaetal.}4_reportedthe

occurrenceofparatuberculosisinabovinedairyherdby iso-latingMAPfromfecessamples,withgeneticconfirmationof theIS900regionusingPCR.

Thehighfrequencyofpositivemilksamplesfoundinthis study couldbeexplainedbythe factthatthemilksamples werecollectedfromherdsconsideredpositivefor paratuber-culosis.Itisimportanttonotethattheactualprevalencerate couldbeevenhigher,sincenotallinfectedanimalseliminate thebacteriumviamilk.12

Based on the health risks due to MAP and considering that this microorganism is implicated in Crohn’s disease in humans, rapid and sensitive tests for detection of this bacterium in milk are urgently required.7 _{In this study,}

the number of milk samples found positive via qPCR (34 samples) was higher than that via conventional PCR (20 samples).TheqPCRtechniqueismoreefficientindetecting pathogenicbacteriaandhasseveraladvantagesover conven-tionalPCR,including thepotentialtoquantifythebacteria, theminimalriskofcross-contamination,aswell asgreater sensitivityandspecificitythatcanbeenhancedbytheuseof probes.20

SonawaneandTripathi21_{reportedthat2groupsoftissues}

(multibacillaryandpaucibacillary)wereusedtoperform con-ventionalPCRtodetecttheIS900regionofMAP.Theyrecorded sensitivitylevelsof82.6%and13.3%forthemultibacillaryand paucibacillarygroups,respectively.Inthesamestudy,qPCR recordedsensitivitylevelsof100%inthedetectionofMAPfor bothgroups.

UponanalysisoftheagreementbetweennestedPCRand qPCRintermsofdetectingMAP,Fangetal.22_{obtainedaKappa}

indexof0.85.Thisvalue,whichishigherthanthatfoundin thepresentstudy,couldbeexplainedbythefactthatnested PCRismoresensitivethanconventionalPCR,whichwasused inthepresentstudyforcomparisonwithqPCR.23

Inthepresentstudy,3samplestestedpositiveusing con-ventionalPCRandnotbyqPCR.Thereasonforthisisunclear;

however,anon-homogenousdistributionand/orsmall quan-tityofMAPbacteriainthesamplecouldberesponsiblefor theresult.22_{Anotherpossiblereasonisthepresenceofsome}

inhibitors, suchas calcium,in the extracts, whicheven in smallquantities,couldinhibitTaqand/orcompetewith mag-nesium,thusreducingtheefficiencyofqPCR.24_{This,however,}

does not refute the finding that the qPCR method reliably detectedthepresenceofMAPDNA inagreater proportion whencomparedwithconventionalPCR.

Direct detectionofviable MAPusing culturesis consid-eredthegoldstandard.However,MAPcanbedetectedmore quicklyandwithapproximately 10timeshigher sensitivity usingqPCR.13,25_{Thisdiscrepancybetweenthemethodscould}

beexplainedbythelowconcentration ofMAPinthe sam-ple,sinceqPCRisamoresensitivedetectiontechniquethan culturing,fromboth,fecesandmilk.26

Consideringthatbovineswithsub-clinicalinfectionscan eliminateverylowquantitiesofMAPviamilk,27_itis

essen-tialtouseadequateandsensitivediagnosticteststodetect this bacterium. ThesensitivityofqPCR, interms of detec-ting MAP in milk samples, has been reported to range from 1 to 100CFU/mL milk.28 _{Additionally, determination}

of MAP bacterial load in milk samples is also possible with qPCR. Conventional PCR as well as culture meth-odsare not quantitativeand culturemethods also cannot assess this factor due to the decontamination process of the samples, which decreases the quantity ofMAP in the sample.7

Rawbovinemilkhasreceived considerableattention,as it is known tobe animportant factor in the transmission ofMAPfromcowtocalf.Astudyhasshownthatmilkand thecolostrummay berisk factorsfortransmissionofMAP infection.29

MAPstillremainstobeimplicatedinCrohn’sdisease,and ifhumans canbeinfectedbyMAP,thereare severalforms of transmission, including transmission between humans. However,sinceanimalsarethemainsourceofthese bacte-ria,transmissionfrom animalstohumans ismorelikely,30

which can occur via food. Viable MAP have been recov-ered from pasteurized milk samples in Brazil31 _{and other}

countriesas well.32 _{ThepresenceofMAP inmilkproducts}

suchascheesehasbeenconfirmedinGreeceandtheCzech Republic.33

Conclusion

Inthisstudy,theIS900regionofMAPwasdetectedinbovine milksamplesobtainedfromtheStateofPernambuco,andto ourknowledge,thisisthe firstsuchreportofMAPDNA in bovinemilkinNortheastBrazil.Wealsodemonstratedthat theqPCRtechniqueismoresensitivethanconventionalPCR intermsofdetectingMAPinmilksamples.

Ethical

approval

TheEthicsCommitteeonAnimalUseoftheFederalRural Uni-versityofPernambucoapprovedtheethicalproceduresofthis studyunderlicenseNo.034/2013.

Conflicts

of

interest

The authors declare nopotential conflicts ofinterest with respecttothisstudyortheauthorshipand/orpublicationof thisarticle.

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