First molecular typing of Mycobacterium avium subspecies paratuberculosis identified in animal and human drinking water from dairy goat farms in Brazil

brazilian journal of microbiology49(2018)358–361

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

Veterinary

Microbiology

First

molecular

typing

of

Mycobacterium

avium

subspecies

paratuberculosis

identified

in

animal

and

human

drinking

water

from

dairy

goat

farms

in

Brazil

Isis

F.

Espeschit,

Marina

C.C.

Souza,

Magna

C.

Lima,

Maria

A.S.

Moreira

UniversidadeFederaldeVic¸osa,SetordeMedicinaVeterináriaPreventivaeSaúdePública,LaboratóriodeDoenc¸asBacterianas,Vic¸osa, MG,Brazil

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t

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c

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e

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f

o

Articlehistory:

Received14November2016 Accepted5June2017

Availableonline12October2017 AssociateEditor:MilianeSouza

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Mycobacteriumaviumsubspeciesparatuberculosis,theetiologicagentofJohne’sdiseaseor paratuberculosis,wasidentifiedbycultureand/orpolymerasechainreaction(PCR)in50% and30%ofwatersamplesforanimalandhumanconsumption,respectively,fromtendairy goatfarmsinBrazil.IS1311restrictionfragmentlengthpolymorphismanalysisidentified theisolatesascattletypeC.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

ParatuberculosisorJohne’sdiseaseisachronicinfectious dis-ease,whichaffectsmainlyruminants.1,2_{Insheepandgoats}

generally,clinicalsignsappearwhentheanimalisaboutone yearold andincludeprogressiveweightloss,anddiarrhea, that may or may notbe present.3 _{The etiological agent is} Mycobacterium avium subsp. paratuberculosis (MAP), and the maineliminationpathwaysofMAPareviafecesandmilk,1,2

whichcontaminatepastures,milkingutensils,anddirectlyor indirectlycontaminatewatercourses,thusinfectinghumans andanimals.MAPisalsoassociatedfrequentlywithCrohn’s disease,giventhesimilaritiesbetweenthediseasesandthe frequentisolationofthebacteriainintestinalbiopsiesfrom thesepatients;however,theroleofMAPinthepathogenesis isunclear.4,5

∗ _{Correspondingauthor.}

E-mail:[email protected](M.A.Moreira).

Davisetal.describesindetailthehistoryofCrohn’sdisease and reportsthatDalzielwas thefirstclinician torecognize thesimilaritybetweenparatuberculosiswithchronic enteri-tis observed in humans, leading to believe that MAP was involvedinthecausationofthedisease.Butonlyyearslater, byB.B.Crohn,thediseasewasdescribedduringthe1930s, AlthoughCrohnwasconsciousofDalziel’snotes,hedidnot hadanysuccessinisolating MAPfromthepatients,raising thecontroversyabouttheinvolvementofthebacteriainthe pathogenesisofCD.Theauthorevenanalysisthatitispossibly ainaccuracytostatethatthepatientsinfectedwithMAPas haveCD,sincethisisamultifactorialdiseasewithsomany unclear features, andthe rejectionofthemedical commu-nityunderstandMAPashumanpathogen,hashadunhappy

https://doi.org/10.1016/j.bjm.2017.06.005

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazilian journal of microbiology49(2018)358–361

359

costsforpatientsinfectedwithMAPandexposedtoimproper treatment.6

InBrazil,thereagrowinginterestinandincreased produc-tionofgoat’smilk,meaningthatitisbecominganimportant alternativelivestock.6_{IntheSoutheastregionofBrazil,Minas}

Geraisstatehasthelargestpopulationofdairygoat,which isconcentrated inthe Zonada Mata region.7 _{According to}

DubeufandLeJaouen,8_{goatmilkproductshavebecomemore}

availabletoconsumerssincetheearly2000s.Goatmilkhas hypoallergenicandtherapeuticpropertiescomparedtocow milk9_{whichhasledtotheincreasingdemandforthisproduct}

anditsderivatives.

Basedon the comparisonofthe whole MAP genome, a biphasicevolutionschemehasbeenproposed,distinguishing twomainstraintypes:bovine(C–cattle)andsheep(S–sheep), which are genotypically and phenotypically different from each other.10 _{ThegeneticvariabilityofMAP hasimportant}

implicationsforthediagnosisandcontrolofparatuberculosis duetodifferencesingrowthrate,virulenceand epidemiolog-icalcharacteristics.

Waterisoneofthemostimportantnutrientsforanimals andhumans,yetitsimportanceandqualityisoftenneglected. Waterisalsoanimportantvehicleinthespreadofzoonotic bacteria,andoneofthemajorroutesofcontaminationto ani-malsandhumans.5,11–14 _{TheoccurrenceofMAPindrinking}

waterandeveninrawwaterisnotclear,and its presence inwatersamplesanditsroleintheinfectioncycleremains unclear,butsomestudiesareavailable,withinformationin othercountries.Whanetal.reportedtheoccurrenceofMAP inuntreatedwaterinNorthernIrelandinaviableform,and Pickupetal.identifiedMAPinlakecatchments,inriverwater abstractedfordomesticuse, andineffluentfrom domestic sewagetreatmentworksintheUnitedKingdom,byPCR.15–18

ThesubjectwasalreadyresearchedbyKingetal.,Chernetal., Pickup,andBeumer,butunfortunately,therewerenostudies availablereferringtheoccurrenceofMAPinwaterinBrazilor anyotherLatinAmericancountry.5,14,23

Taking into consideration that watersamples are non-invasive,easytoobtainandcanprovidethesanitarystatus of the herd, and also inform the health condition of the owners,theaimofthis studywastoverifythepresenceof MAPinwaterforhumanandanimalconsumptionand deter-minethetypeofMAPstrainspresentinthewatersamples fromdairygoatfarmsintheZonadaMataofMinasGerais state,Brazil,oneofthemostimportantgoatmilkproducing regions.

Thestudyincluded10farmswithaminimumof50animals eachinordertoincludeonlyestablishmentsforcommercial purposes. Thefarms were distributed in the seven micro-regionsthatcomprisetheZonadaMataoftheMinasGerais mesoregion.

Twentysamplesof20L ofwaterwere collected;a sam-pleofwaterforanimalconsumptionandasampleofwater forhuman consumption from each of the ten farms. The animalsampleswerederiveddirectlyfromwaterforanimal consumption,collecteddirectlyfromtheplacewherethe ani-malsdrankthewater,whilehumanconsumptionwatercame from the farm tap and the untreatedwater publicsystem supply,orfromanalternativesource(wellshallowartesian well/semi-artesianspring).Thewaterforanimalandhuman

consumption wasfrom the same sourceofsupply ineach location.

Thecollectionvialswerepreviouslydisinfectedwith2.5% sodiumhypochlorite,leftincontactforaminimumperiodof 24h,followedbywashingwithautoclaveddistilledwater.

Processingand the microbiologiccultureofthesamples were performedaccording to Pickup et al.5 _with

modifica-tions.Thewatersamplewasfilteredusingmembranefilters ofsterilecellulose(MerckMillipore,SaoPaulo,Brazil),witha 0.22␮mporesize,asmanyasnecessaryforfiltrationof20L, dependingonthecharacteristicsofthewater.Afterfiltration, themembraneswereplacedin50mLtubes,andthen30mL 0.5×PBS,pH6.9,wasadded;thiswasvortexedandscraped withsterileplasticloopsfordetachmentoftheretentateuntil themembraneswerecompletelyclean.Themembraneswere discardedandtheretrievedcontentwasaliquotedin1.5mL microtubes toperform DNA extractionand microbiological culture.

The decontamination step comprised the addition of 15mL of 1-hexadecylpyridinium chloride 0.75% (HPC) (Sigma–Aldrich,St.Louis,MO,USA)to1.5mLofthesample, makingcontactwiththesedimentovernight(12–16h).Then, the solution was centrifuged at 3000×g for 20min. After this, 2mL of antimicrobial solution containing nalidixic acid (50mg/L) (Sigma–Aldrich), vancomycin (50mg/L) (Sigma–Aldrich) and amphotericin B (150mg/L) (Critália, Itapira, Brazil) was added to the sediment, and placed in contactwiththesamplefor72hatroomtemperaturebefore inoculation.

Aliquotsof200␮Lofeachsamplewereinoculatedintofour tubes containing HEYMmedia, two containingmycobactin

Jand twowithout.Thetubeswere incubatedat37◦Cfora minimum of 16 weeks. The MAP colonieswere submitted toZiehl-Neelsencoloring(kitstaining–Laborclin),and con-firmedbyISMav2-PCRandgeneticsequencing.MAPK10and ultra-purewaterwereusedaspositiveandnegative,controls, respectively.

For DNA extraction, Wizard® Genomic DNA Purification kitwasusedfollowingthemanufacturer’sinstructions,using an initial volume of 1mL of the sample. To perform the PCR, Go Taq® Green Master Mix kit was used according to the manufacturer’s instructions, using the primer pair ISMav2/F (5-GTGAGTTGTCCGCATCAGAT-3) and ISMav2/B2 (5-GCATCAAAGAGCACCTCGAC-3) which amplifies a frag-mentof494bp.Eachreactionhadafinalvolumeof25␮L,with 12.5␮Lmix, 1␮Lofeachprimer,6.5␮Lnucleasefreewater and4␮LofextractedDNA.16_{Theampliconswerepurifiedand}

sequencedinbothdirectionsintheNationalAgricultural Lab-oratory(LANAGRO)ofMinasGerais,locatedinPedroLeopoldo, Brazil.Thesequencesobtainedwerealigned,editedand com-paredwithothersequencesdepositedinGenBank(BLAST).

The positive samples were submitted to PCR-IS1311 usingM56 (5-GCGTGAGGCTCTGTGGTGAA-3)andM119(5 -ATGACGACCGCTTGGGAGAC-3) primers and to subsequent REAaccordingtoapreviousstudy.19EnzymesHinfI(Promega) andMseI(NEB)wereusedtodifferentiateMAPfromM.avium

subsp. avium. The restriction pattern generated by

HinfI-digestion differentiates type S (sheep) from typeC (cattle) strains, whilethe restrictionpatterngeneratedbyMseI dif-ferentiatesMAPstrainsfromM.avium.Ascontrols,theK10

360

strainofMAPtypeCfromcattleandVICAP711strainoftype Sofsheepwereusedaspositivecontrols,whilenucleasefree waterwasusedasanegativecontrol.

AsamplingoffarmswasperformedusingtheOpenEpi® software(available inhttp://www.openepi.com),considering anestimatedprevalenceof5%,precisionof4%and95% con-fidenceinterval.

MAPwasidentifiedinfivefarms.Fouranimalconsumption watersampleswerepositivebycultureand PCRandafifth samplebyPCRonly.Inaddition,threesamplesofwaterfor humanconsumptionwerefoundtobepositivetoMAPbyPCR inthesamefarms,butnonewerepositivebymicrobiological culture.

Acid-FastBacilliwereobservedinslidespreparedfromthe coloniesandinthesequencingwasconfirmedtobeMAP.The degreeofidentitybetweentheDNAsamples(watersamples andcolonies)andMAPK10rangedfrom92to99%.

ISMav2haslowcopynumbersinMAPDNA,aboutthreeto five,providingmorespecificresults.19–22_{TheMAPdetectionby}

PCRandnon-observationofcoloniesinthesamesamplemay beduetothetypeofprocessing,andalsotothesmallnumber ofbacteriapresentinthesamples.Furthermore,MAPcanform “spore-like”structures,whichwouldhinderthegrowthofMAP inculture.23,24 _{Nevertheless,thereisthepossibilitythatthe}

bacteriawerenotviable.

ItwasfoundthatallMAPstrainsidentifiedinthisstudy weretypeC(cattle).Researchintothesamepropertieswith goatmilkandfecalsamplesalsofoundtypeCMAPstrains.22,25 Infiveofthevisitedfarms,theconsortiumofcattleandgoats were assessed,which suggestedthat the contaminationof goatsoccurredbytheingestionofcontaminatedwaterorfood byMapeliminatedfrom cattle.Studies onMAPtyping iso-latedfromgoatsindifferentcountriesfoundthatmoststrains were typeC.26–30 _{TypeC hasawide range ofhosts,and is}

commonlyisolatedfromdomesticandwildanimals, includ-ingnon-ruminants29_{;thisisthepredominanttypeincattle.}30

Also,inpatientsaffectedbyCrohn’sdisease,MAPisolateshave alsobeenclassifiedastypeC,31_{displayingtheimportanceof}

thisresult.

Theresultsindicatethepossibleroleofwaterinthe main-tenanceanddisseminationofMAPintheherds.Theuseofthis typeofsampleappearstobeanefficientdiagnostictoolin pre-dictingtheinfectionstatusoftheherd.Althoughwaterplays animportantroleinthedisseminationofinfectiousagents, thesearchforpathogensinthistypeofsampleisuncommon. MAPtypeC(cattle)DNAispresentinwaterforhumanand animalconsumptionindairygoatfarmsonpropertieslocated intheZonadaMatadeMinasGerais.MAPwasfoundviablein watersamplesforanimalconsumption.Thisisthefirstreport ofisolationandtypingofMAPfromwaterofdairygoatfarms.

Conflict

of

interests

Theauthorsdeclarenopotentialconflictofinterests.

Acknowledgments

The authors acknowledge the financial support from CNPq (Conselho Nacional de Desenvolvimento Científico

e Tecnológico, Brasília, Brazil), FAPEMIG (Fundac¸ão de AmparoàPesquisadeMinasGerais,BeloHorizonte,Brazil), andCAPES(Coordenac¸ãodeAperfeic¸oamentodePessoalde Nível Superior,Brasília,Brazil). M.A.S.Moreiraissupported by CNPq. We alsothank Professor Rafael Kopschitz Xavier BastosfromtheWaterQualityLaboratoryoftheUniversidade FederaldeVic¸osa(Brazil)fortheintensecolaborationduring theentirestudy,DrYung-FuChang,fromCornellUniversity (USA)andDrRamonA.Juste,fromNeiker-Tecnalia,Animal HealthDepartment(Spain)forkindlyconcedetheC-typeand S-typestrains,respectively,andDrAntonioAugustoFonseca Junior,Lanagro-MG(Brazil)forhelpinginthesequencingof MAPamplicons.

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