Clinical and epidemiological use of nested PCR targeting the repetitive element IS1111 associated with the transposase gene from Coxiella burnetii

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

Clinical

Microbiology

Clinical

and

epidemiological

use

of

nested

PCR

targeting

the

repetitive

element

IS1111

associated

with

the

transposase

gene

from

Coxiella

burnetii

Maria

Angélica

M.M.

Mares-Guia,

Alexandro

Guterres,

Tatiana

Rozental,

Michelle

dos

Santos

Ferreira,

Elba

R.S.

Lemos

OswaldoCruzInstitute,LaboratoryofHantavirusandRickettsioses,Fiocruz,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Received18May2016 Accepted21April2017 Availableonline24August2017 AssociateEditor:RoxanePiazza

Keywords: Qfever Coxiellaburnetii Moleculardiagnosis NestedPCR IS1111

a

b

s

t

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a

c

t

QfeverisaworldwidezoonosiscausedbyCoxiellaburnetii—asmallobligateintracellular Gram-negativebacteriumfoundinavarietyofanimals. Itistransmittedtohumansby inhalationofcontaminatedaerosolsfromurine,feces,milk,amnioticfluid,placenta, abor-tionproducts,wool,andrarelybyingestionofrawmilkfrominfectedanimals.NestedPCR canimprovethesensitivityandspecificityoftestingwhileofferingasuitableampliconsize forsequencing.Serialdilutionswereperformedtenfoldtotestthelimitofdetection,and theresultwas10×detectionofC.burnettiDNAwithinternalnestedPCRprimersrelative totrans-PCR.DifferentbiologicalsamplesweretestedandidentifiedonlyinnestedPCR. Thisdemonstratestheefficiencyandeffectivenessoftheprimers.Ofthe19samples,which amplifythepartialsequenceofC.burnetii,12werepositivebyconventionalPCRandnested PCR.Sevensamples—fivespleentissuesamplesfromrodentsandtwoticksamples—were onlypositiveinnestedPCR.Withthesenewinternalprimersfortrans-PCR,wedemonstrate thatournestedPCRassayforC.burnetiicanachievebetterresultsthanconventionalPCR.

PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons. org/licenses/by-nc-nd/4.0/).

Introduction

Qfever isaworldwidezoonosis caused byCoxiellaburnetii, a small obligate intracellular bacteria and Gram-negative pleomorphicmemberoftheorderLegionellales.1_Itremains

infectiousintheenvironmentforalongtimeandisresistant tochemicalandphysicalfactors.2_{Theinfection existsina}

varietyofanimalsincludingvariousruminants,domesticand wildanimals,birds,andarthropods(especiallyticks).2_Cattle,

∗ _{Correspondingauthorat:PavilhãoHélioePeggyPereira,salaB115,LaboratoryofHantavirusandRickettsiosesoftheOswaldoCruz}

Institute,Fiocruz,AvenidaBrasil4365,Manguinhos,21040-900RiodeJaneiro,RJ,Brazil. E-mails:elemos@ioc.fiocruz.br,elba.lemos@ioc.fiocruz.br(E.R.Lemos).

sheep,andgoatsaretheprimaryreservoirsofC.burnetii.2–5

Transmissiontohumansusuallyoccursthroughinhalationof contaminatedaerosolsfromurine,feces,milk,amnioticfluid, placenta,abortionproducts,wool,orlesscommonlythrough ingestionofrawmilkfrominfectedanimals.2,5

TherearevariouswaysofdetectingC.burnetiiinfection.3

Several PCR-basedDNAdetection methodshavebeenused to diagnosis Q fever including conventional PCR, nested PCR,real-timePCR(qPCR),andtheloop-mediatedisothermal amplification(LAMP)assay.3,6,7_Berrietal.8_{showedthatthe}

https://doi.org/10.1016/j.bjm.2017.04.009

1517-8382/PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

sensitivityoftrans-PCRis100-foldgreaterthanthatobtained byPCRusingprimerCB1–CB2.CompletesequencingoftheC. burnetiigenomeshowed20copiesoftheIS1111transposase. ThereweremanyelementnumbercopiesinthegenomeofC. burnetii,andthiswasausefultoolforthedetectionand dif-ferentiationofC.burnetiiisolates.9_{Theobjectiveofthisstudy}

wastoincreasethesensitivityofthetrans-PCRprimerusing anestedPCRassayand todemonstrateitseffectivenessin differentspeciesofanimalsanddifferenttypesofsamples.

Materials

and

methods

ClinicalsamplesandDNAextraction

Patientsamples(n=24)withsuspectedQfever weretested alongwith68domesticanimals,74rodents,and180ticksfrom RiodeJaneiro,Brazil.Domesticanimalsamplesandtickswere obtainedfromareaswhereC.burnetiiinfectionwasdetectedin humanandanimals,10–12_{androdentsampleswerecollected}

fromvariousregionsofRiodeJaneirostateaspartofan eco-logicalresearchprojectconductedonwildanimalsfrom2008 and2015.ConventionalandnestedPCRwereusedtoassay bio-logicalspecimensfromthesuspectedQfevercasesaswellas clot,tissue,milk,andanalandvaginalswabsamplesextracted fromanimals.Inaddition,tissuesamplesfromrodentsand tickswerealsotestedwithconventionalandnestedPCR.

DNAwasextractedfromgoats,sheep,rodents,dogs,and cats. Serum and clot samples were used for human DNA extraction.ThesamplesweretestedusingaQIAampDNAMini Kit(QiagenTM_{,Valencia,CA,USA)accordingtotheinstruction}

manual.AfterextensivewashinginPBS,theDNAfrommilk aswellasanalandvaginalswabswasextractedusingATL (QIAampDNAMiniKit,QiagenTM_{)andproteinaseK.Each}

sam-ple(200␮L)wasmixeddirectlywiththeATLandproteinase Kandincubatedat56◦Covernight.Thiswasfollowedbyan incubationstepwithALbuffer for10minat70◦C.10,13 _The

extractionprocesscontinuedwithaQIAmpDNAkit(QIAamp DNAMiniKit,QiagenTM)followingthemanufacturer’s instruc-tions.Negativecontrolswereincludedineachextractionto checkforpossibleDNAcontamination.

ObtainingandpurifyingC.burnetiiDNA

C.burnetiiNineMilephaseIIstrainisolatefromtheUNIFESP São Paulo Culture Collection was used as a the positive control.14_{AccordingtoZamboniandcollaborators,thestock}

suspensionscontainingthebacteriaweremildlysonicatedat 35kHzfor15minatroomtemperaturetodisruptaggregates.C. burnetiiweregrowninVerocells(about3×105_cellsper2cm2_).

In2–3days,theVerocellsdevelopedlargevacuoles contain-ingbacteria.There were3–10␮Lofbacterialstockper well. After24h,cultureswerevigorouslywashedandfreshmedium added(minimalessentialmedium,MEM,containing15mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonicacid–HEPES, 2g/Lsodiumbicarbonate, 1mMl-glutamineand5%(v/v)of fetalbovineserum).14_{TheDNAwasextractedfrom200␮Lof}

thissolutionusingaQIAampDNAMiniKit(QiagenTM_,

Valen-cia,CA,USA)followingthemanufacturer’sinstructions.The DNAwasthenquantifiedandusedasapositivecontrolfor

thefirstPCR.Serialtenfolddilutionswere usedtoexamine thedetectionlimit.

PCRassay

ThePCR assayuseda pairofprimers thattarget the gene IS1111transposaseelementsinthegenomeofC.burnetii.15,16

Theexpectedamplificationproductusingtheseprimerswas 687bp.ThepairofprimerswereTrans1(5TATGTATCCACC GTA GCCAGT C-3)and Trans2 (5-CCC AAC AACACC TCC TTATTC-3).These primerswere usedtodoa PCRreaction using4␮LofeachDNAsamplein25␮Lfinalreactionvolume. Thefinalreactionmixturecontained1×PCRbuffer10×,0.2␮M ofeachprimer(RTD/ProdimolTM_{,BeloHorizonte,MG,Brazil),}

1.5mMofMgCl2,200␮MofdNTPmix(20mMofeach

deoxynu-cleotidetriphosphate),0.5UofPlatinumTaqDNApolymerase (InvitrogenTM, Carlsbad,CA,USA),4␮LofDNAsample,and nuclease-freewater(PromegaTM_{,Madison,WI,USA).}

Ampli-fication used a GeneAmp PCR System 9700 thermalcycler (Applied BiosystemsTM_{, FosterCity, CA, USA): initial}

dena-turationat95◦Cfor5min,followedby40successivecycles ofdenaturationat95◦Cfor30s,annealing at60◦Cfor30s, andextensionat72◦Cfor1min.Thiswasfollowedbyafinal extensionof7minat72◦C.Allstepsusednegativecontrolsto controlcontamination.

NestedPCRassay Primersdesign

Oligonucleotide primersto detectC. burnetiiwere designed from conservedregionsofdifferentIS1111genesequences. The MUSCLE tool was used to perform multiple sequence alignment17; MUSCLE is part of the MEGA 6 software package.18 _{The nucleotide sequences of the IS1111 gene}

were analyzed(GenBankaccessionno.AE016828,CP001020, CP001019, CP000890, CP000733, CP007555, HG825990, LK937696, and M80806). The highly conserved regions wereidentified,andthedegenerateoligonucleotideprimers weredesignedtocorrespondtonucleotidesintheseregions. Standardizationandgradient

Weusedprimersfrom thetrans-PCR forthe firstPCR; N3+ (5-AAG CGTGTG GAG GAG CGAACC-3)and N4− (5-CTC GTAATCACCAATCGCTTCGTC-3)wereusedforthe sec-ondPCRdesignedfromthegeneIS1111transposaseelements inthegenomeofC.burnetii.15,16_{Theexpectedamplification}

productofthetargetsequencewiththeseprimerswas440bp long.The25␮Lmixturecontained2␮LofDNAsamples(2␮L offirstPCRreactionmixtureinsecondPCR).Thefinal reac-tion mixturecontained 1× PCRbuffer 10×, 0.2␮M ofeach primer(RTD/ProdimolTM_{,BeloHorizonte,MG,Brazil),1.5mM}

ofMgCl2,200␮MofdNTPmix(20mMofeachdeoxynucleotide

triphosphate), and 0.5U of Platinum Taq DNA polymerase (InvitrogenTM_{,Carlsbad,CA,USA).}

AgradientPCRamplificationwasperformedinaVeriti 96-well thermalcycler (Applied BiosystemsTM_{, FosterCity,CA,}

USA).Thisconsistedofinitialdenaturationfor5minat95◦C, followedby30successivecyclesofdenaturationat95◦Cfor 30s,annealinginagradientof65/66/67/68/69/70◦Cfor30s, and extension at 72◦C for 1min. This was followed by a

687bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fig.1–PCRassaywithC.burnetiiperformedwithprimersoffirstPCRasdescribedinthematerialsandmethods.Lanes:1, negativecontrol;2,100bpDNAladder;3,C.burnetiisuspension1×[21.4␮g/mL];4,C.burnetiisuspension10×;5,C.burnetii suspension102_{×;6,C.burnetiisuspension10}3_{×;7,C.burnetiisuspension10}4_{×;8,C.burnetiisuspension10}5_{×;9,C.burnetii} suspension106_{×;10,C.burnetiisuspension10}7_{×;11,C.burnetiisuspension10}8_{×;12,C.burnetiisuspension10}9_×;13,C. burnetiisuspension1010_{×;14,C.burnetiisuspension10}11_{×;15,C.burnetiisuspension10}12_{×;and16,C.burnetiisuspension} 1013_×.

finalextensionof5minat72◦C.Toconfirmamplification,the productswereanalyzedbyelectrophoresisin1%agarosegel stainedwithGelRedTM_{solution(Biotium,Hayward,CA,USA).}

A100-bpDNAladder(InvitrogenTM_{,Carlsbad,CA,USA)served}

asamolecularweightmarker.Theamplifiedproductswere visualizedunderaUVlighttrans-illuminatorandstoredina digitalsystemforgel documentation(CarestreamGelLogic 212Pro,Rochester,NY,USA)toselecttheannealing tempera-ture.Thus,theselectedreactionusedinitialdenaturationfor 5minat95◦Cfollowedby30successivecyclesofdenaturation at95◦Cfor30s,annealingat66◦Cfor30s,extensionat72◦C for30s,andafinalextensionto72◦Cfor5min.

SensitivityofPCRandnestedPCRassays

VerocellswithC.burnetiiwereextractedusingtheQIAamp DNA Mini Kit (QiagenTM_{, Valencia, CA, USA) following the}

manufacturer’sinstructions.Afterextraction,thetotalDNA wasquantifiedusingaQubit©_{2.0fluorometer(Invitrogen}TM_,

Carlsbad,CA,USA)indicatingaconcentrationof21.4␮g/mL. SolutionsofpurifiedC.burnetiiwereusedaspositivecontrol, anddilutionswerepreparedrangingfrom1to10.13Dilutions used2␮Lofthesolutionextractedwith18␮Lofnuclease-free wateruntilthe1013_{dilution.Four␮LoftheDNAsolutionwas}

usedforthePCR,and2␮LwasusedforthenestedPCRassays.

SpecificityofPCRandnestedPCRassays

DNAsamplesofC.burnetiiand15otherbacteriawere used in the PCR and the nested PCR assays to evaluate speci-ficity. The bacteria were Rickettsia rickettsii, Rickettsia typhi, Ehrlichiachaffeensis,Ehrlichiacanis,Anaplasmaphagocytophilum, Bartonellahenselae,Borreliaburgdorferi(Hantavirosesand Rick-ettsiosesLaboratory,FIOCRUZ),PseudomonasaeruginosaINCQS 00025(ATCC15442),BrucellaabortusINCQS00242(ATCC7705), Legionellapneumophilasubsp.pneumophilaINCQS00451(NCTC

11232 ATCC 33155), Staphylococcus aureus CCBH 3853(ATCC 25923), Streptococcus pneumonia,Haemophilus influenzae, Neis-seria meningitidis (Epidemiology and Molecular Systematics Laboratory, FIOCRUZ), and Listeria monocytogenes (Bacterial ZoonosesLaboratory,FIOCRUZ).

Results

SensitivityofthePCRandnestedPCRassays

SensitivitywascomparedbetweenthePCRandthenestedPCR assaysinthedetectionofC.burnetiiDNA.Theamplification productbyPCRassaywiththetrans-PCRprimersforthefirst PCRwasdetecteduntilthe108_{dilution(Fig.1).ThefirstPCR}

datashowedthattheannealingtemperaturegradientswere negativeforsolutionsof109_to1013_{.Thedetectionbynested}

PCRassaywithprimersN3+andN4−waseffectivetothe109

dilutionindicatingthatitis10-foldmoresensitivethanthe first PCRassay(Fig.2).Thus,the selectedreactionwas the initialdenaturationfor5minat95◦Cfollowedby30successive cyclesofdenaturationat95◦Cfor30s,annealingat66◦Cfor 30s,extensionat72◦Cfor30s,andafinalextensionto72◦C for5min.

SpecificityofPCRandnestedPCRassays

DNAsamplesfromthe15otherbacteriausedinthefirstPCR and nested PCRassays toassessthe specificityshowed no amplification(datanotshown).

DetectionofC.burnetiiDNAfromclinicalspecimens

ThesamplesweretestedforC.burnetiiDNAbythefirstPCR andthenestedPCRassays.Of24patients,2werepositive(clot sample).Onesheep(tissue)andtwodogs(bloodsamples)were alsopositive.Of10goats,sixwerepositive(milksample),and

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

440bp

Fig.2–PCRassaywithC.burnetiiperformedwithprimersofnestedPCRasdescribedinthematerialsandmethods.Lanes: 1,negativecontroloffirstPCR;2,negativecontrolofnestedPCR;3,100bpDNAladder;4,C.burnetiisuspension1×;5,C. burnetiisuspension10×;6,C.burnetiisuspension102_{×;7,C.burnetiisuspension10}3_{×;8,C.burnetiisuspension10}4_×;9,C. burnetiisuspension105_{×;10,C.burnetiisuspension10}6_{×;11,C.burnetiisuspension10}7_{×;12,C.burnetiisuspension10}8_×; 13,C.burnetiisuspension109_{×;14,C.burnetiisuspension10}10_{×;15,C.burnetiisuspension10}11_{×;16,C.burnetiisuspension} 1012_{×;and17,C.burnetiisuspension10}13_×.

of13cats,onewaspositive(analswab)(Table1).Of74rodents, thespleentissuesamplefromfiverodentsandtwotick sam-plesofthe 180tested(Table2)wereonlypositivewiththe nestedPCR.Therefore,theyweresequenced,andthepartial sequencesweredepositedinGenBank.

Other samplesincludingmilk, vaginalswab,tissue,and serumsampleshadinitialPCRsthatwerepositive.Theywere thensequencedusingthenestedPCR.Thetissuesamplefrom sheep(GenBankaccessionno.KC854154–373bp),EDTAblood samplefromdog(GenBankaccessionno.KT867377–439bp), andfecalswabfromcat(GenBankaccessionno.KC854155– 394bp)werealsodepositedinGenBank.

Discussion

PCRhasbeenusedwidelyforthediagnosisofinfectious dis-easesincludingthosecausedbyC.burnetti.Hemi-nestedand nestedPCRincreasethesensitivityofPCRandleadtoatleast a10,000-foldenhancementinPCRproductovernonspecific productsthatcouldbeco-amplifiedwhenusingonlytheouter primers.TheamountofPCRproductusedinthenestedPCR shouldbeadjustedaccordingtotheresultsofthefirstPCR. Infuturediagnosticwork,allsamplesshouldbetestedwith nestedprimersregardlessofthefirstPCRresult.False nega-tiveresultsafterthefirstPCRcouldoccurduetoaverysmall numberofcopies.19,20

Theobjectiveofthisstudywastodesignandusenested PCRforatargetgenethathasalreadybeen showntohave sufficientspecificityandsensitivity.8,15,16_{Thesenewinternal}

primersfortrans-PCRprovedthattherewasa10×increasein sensitivitywithanoptimalsequencesizeof440bp.

Although Boden and colleagues developed Nested PCR usingthe IS1111elementasthetarget,21 _{ourstudyverified}

notonlythepresenceofthepartialsequenceoftheC.burnetii gene in serum samples but also used it to test for other samples used for epidemiological studies such as swabs, milk, and ticks. Samples forepidemiological research may have small amounts of microorganisms.22 _{In contrast to}

Duron,23 _{smallsizeamplificationscanactuallycharacterize}

the detection offragmentsand not C.burnetii inits whole form.Thus,wedevelopedthisprimer,whichislocatedata differentsiteandwithareasonablesizeforsequencing.We determinedthatourampliconsweredifferentfromtheqPCR DNAsequencesstudiedpreviously.24_{Theprimersdeveloped}

inthepresentstudyarenotlikelytoresultinamplification ofendosymbiontsduetothedegradation inthisparticular regionoftheirIS1111gene.24

Pearson and colleagues24 _{detected Coxiella-like bacteria}

throughtheIS1111target.Thesearerareintickpopulations, whichlimitstheimpactoffalsepositivesonprevalence esti-matesofC.burnetii.WedistinguishedC.burnetiifrom Coxiella-likebacteriausingC.burnetiiSNPgenotypingassaysduetothe extremesensitivityoftheIS1111multiplecopyassay.

Although several qPCR techniques have been shown to be sensitive for diagnosis,3,7 _{this study aimed to find an}

alternative to conventional PCR in laboratories with few applicablefinancialresourcesforthe realizationofaqPCR. Chenetal.alsoproposedanoveldetectionmethodforserum samples.6_{However,nestedPCRcanbeusedindifferenttypes}

ofsamplessuchasmilk,swabs,andtissuesamples.25

Recently,thepresenceofIS1111inbothC.burnetiiandin theCoxiella-likeendosymbiontsledtoquestioningthe speci-ficityofthediagnostictestsinQfeverepidemiology.However,

Table1–DetectionofCoxiellainfectionbyPCRofprimers(trans1/2)andnestedPCR(+N3/N4)amonghuman,domestic animals,rodents,andticksamplescollectedinRiodeJaneirobetween2008and2015.

Samples PCRpositivea_{(No.tested) NestedPCRpositive}b_{(No.tested) Sourcelocation}

Patients Serum 0(24) 0(24) Clot 2(24) 2(24) Itaboraí,RJ Sheep Serum 0(18) 0(18) Clot 0(18) 0(18) Tissue 1(1) 1(1) Itaboraí,RJ Dogs Serum 1(19) 1(19) Clot 0(19) 0(19)

EDTAblood 1(8) 1(8) Itaboraí,RJ

Vaginalswabs 0(5) 0(5) Fecalswabs 0(8) 0(8) Goats Serum 0(10) 0(10) Clot 0(10) 0(10) Itaboraí,RJ Milk 6(7) 6(7) Vaginalswabs 0(8) 0(8) Fecalswabs 0(3) 0(3) Cats Serum 0(13) 0(13) Clot 0(13) 0(13)

Fecalswabs 1(7) 1(7) Itaboraí,RJ

Rodents

Spleentissue 0(74) 5(74) Piraí/Valenc¸a,RJ

Ticks 0(180) 2(180) Itaboraí,RJ

a _{PrimerstargetthegeneIS1111transposaseelementstrans1/trans2.} b _{PrimerstargetthegeneIS1111transposaseelementsofnestedN3+/N4−.}

Table2–PartialsequencesofthegeneIS1111transposaseelementsinthegenomeofC.burnetiiatGenBankfrom samplesobtainedfromnestedPCR.

Register Rodent/tick Sourcematerial Trans1–2 N3+/N4− GenBankaccessionnumber a(%);b(%)

Size(bp)

LBCE11866 Akodoncursor1 Spleen N P KT965026

a(100%);b(100%)

438

LBCE11914 Oligoryzomysnigripes Spleen N P KT965028

a(99%);b(100%)

326

LBCE11924 Musmusculus Spleen N P KT965029

a(100%);b(100%)

440

LBCE13257 Akodoncursor2 Spleen N P KT965030

a(100%);b(100%)

432

LBCE13265 Oxymycterusdasythricus Spleen N P KT965031

a(100%);b(100%)

440

C59 Rhipicephalussanguineus Tick N P KT867378

a(100%);b(100%)

370

C154 Amblyommasculptum Tick N P KT970067

a(99%);b(99%)

439

a(%)maximumsimilaritywiththesequenceoftheIS1111geneofC.burnetiiRSA331. b(%)querycoverwiththesequenceoftheIS1111geneofC.burnetiiRSA331. N,negative;P,positive.

IS1111amino-acidsequencesinCoxiella-likeendosymbionts

revealedthepresenceofdegradedsequencescontainingan

internal stop codon(s). These are likely non-functional.23

Thus,PCRorqPCRtechniquesforQfeverdiagnosisusingsmall fragmentsoftheIS1111-DNAmayleadtomisidentification.21

Conversely, our new internal primers (N4−) for IS were developedconsideringthedegradedregionsforCoxiella-like endosymbionts.Therefore,weprovideanestedPCRtechnique withbettersensitivityforC.burnetiiandasuitableDNA frag-mentsizeforgeneticcharacterization.

Usingtheseprimers,weamplifiedandsequenced440bp partialsequencesthatdonotalignwiththe15other bacte-riatestedhere.Thismakestheprimersagreatalternativefor qPCR.Wealsousedsamplesfromdifferenthostsand differ-enttypes,andweimprovedthespecificityoftheamplified material.Inanimals,weidentifiedpositivecasesinsample typesotherthanblood.Thismakesthistechniquevaluablefor surveillance.WedemonstratedthatournestedPCRassayfor C.burnetiiachievedbetterresultsthanconventionalPCRand thatthismethodmaybeagoodalternativeforthediagnosis ofC.burnetiiinfection.

Financial

support

This study was supported by FIOCRUZ and grants from ConselhoNacionalparaoDesenvolvimentoCientificoe Tec-nológico(CNPq)Project407664/2012andFundac¸ãodeAmparo àPesquisa doEstado doRiode Janeiro (FAPERJ),Project E-26/010.001567/2014.

Conflicts

of

interest

Theauthorshavenoconflictofinterestordisclosures con-cerningthispaper.

Acknowledgments

PhaseIINineMilestrainofC.burnetiiwasprovidedbyDr. Fer-nandoRealandDr.AlexisMelofromEPM,UNIFESP,SãoPaulo, SP,Brazil. Bacterial DNA was kindly providedby Dr. Ivano de Filippis, Collection Reference Microorganisms in Health Surveillance, CMRVS Fundac¸ão Oswaldo Cruz – FIOCRUZ, Brazil;theEpidemiologyandMolecularSystematics Labora-tory–Fundac¸ão Oswaldo Cruz –FIOCRUZ,Brazil;and Dra. Martha Pereira, Bacterial Zoonoses Laboratory – Fundac¸ão OswaldoCruz–FIOCRUZ,Brazil.

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