Bartonella species pathogenic for humans infect pets, free-ranging wild mammals and their ectoparasites in the Caatinga biome, Northeastern Brazil: a serological and molecular study

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w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Bartonella

species

pathogenic

for

humans

infect

pets,

free-ranging

wild

mammals

and

their

ectoparasites

in

the

Caatinga

biome,

Northeastern

Brazil:

a

serological

and

molecular

study

Mariana

Campos

Fontalvo

a

_,

_Alexsandra

_Rodrigues

_de

_Mendonc¸a

_Favacho

b

_,

Andreina

de

Carvalho

Araujo

a

_,

_Naylla

_Mayana

_dos

_Santos

a

_,

Glauber

Meneses

Barboza

de

Oliveira

a

_,

_Daniel

_Moura

_Aguiar

c

_,

Elba

Regina

Sampaio

de

Lemos

b

_,

_Mauricio

_Claudio

_Horta

a,∗

a_Universidade_Federal_do_Vale_do_São_Francisco_(Univasf),_Laboratório_de_Doenc¸as_{Parasitárias,}_Petrolina,_PE,_Brazil b_Instituto_Oswaldo_Cruz_(FIOCRUZ),_Laboratório_de_Hantaviroses_e_{Rickettsioses,}_Rio_de_Janeiro,_RJ,_Brazil

c_Universidade_Federal_de_Mato_Grosso,_Laboratório_de_Virologia_e_{Rickettsioses,}_Cuiabá,_MT,_Brazil

a

r

t

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c

l

e

i

n

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o

Articlehistory:

Received4October2016

Accepted3February2017

Availableonline27February2017

Keywords: Bartonella Flea Tick Lice Brazil

a

b

s

t

r

a

c

t

ThisstudyveriﬁedtheoccurrenceofBartonellaspp.indogs,cats,wildmammalsandtheir

ectoparasitesinPetrolina andLagoaGrande Counties,Pernambuco,located ina

semi-aridregioninNortheasternBrazil.Anti-Bartonellaspp.antibodiesweredetectedbyindirect

immunoﬂuorescenceassay(IFA)in24.8%ofdogs(27/109)andin15%ofcats(6/40).Bartonella

sp.DNAwasidentiﬁedbyPCRperformed onDNAextractedfromblood and

ectopara-sitesusingprimerstargetingBartonellasp.gltAandribCgenesin100%(9/9)ofPulexirritans

fromCerdocyonthous,57.4%(35/61)ofP.irritansfromdogs,2.3%(1/43)ofCtenocephalidesfelis felisfromdogs,53.3%(24/45)ofC.felisfelisfromcats,and10%(1/10)ofPolyplaxspp.from

Thrichomysapereoides.DNAsequencingidentiﬁedBartonellaclarridgeiaeandBartonella hense-laeinC.felisfelisfromcats,BartonellarochalimaeinP.irritansfromdogandC.thous,and

BartonellavinsoniberkhoﬁiinP.irritansfromdog.

openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/

by-nc-nd/4.0/).

∗ _{Corresponding}_author.

E-mailaddress:horta.mc@hotmail.com(M.C.Horta).

http://dx.doi.org/10.1016/j.bjid.2017.02.002

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Introduction

Bartonellaspp.comprisesagroupofemergingpathogensthat

areprevalentinalargevarietyofvertebratesandcause

diver-siﬁedsymptomsandclinicalmanifestations.1_Transmission

predominantlyoccursviaavector,andﬂeas,hematophagous

lice,andtickshaveconﬁrmedvectorcompetence.2_Bartonella

DNAhasalsobeenisolatedfrommites.3

Bartonellahenselaeisthemaincausalagentofbartonellosis

inhumans.Althoughcatsareamajorreservoirforhuman

infectionswiththeseBartonellaspecies,thisagentcaninfect

manymammalianhosts.Thus,rodentsandotherwild

mam-malscan actasnaturalreservoirsfornumerousspeciesof

thesebacteria and the presenceof the bacteriahave been

describedintheseanimalsonﬁvecontinents.3–7

InBrazil, the circulationofthe agent hasbeen

demon-strated in humans, cats, dogs, and wild animals using

serologicaland moleculartechniques. However,little

infor-mationisavailableabouttheoccurrenceofBartonellainthis

country, and to date no study has veriﬁed the occurrence

ofBartonella spp. in the Caatinga biome. Therefore, in the

presentstudyweinvestigatedthecirculationofBartonellaspp.

indomesticmammals(dogsandcats),free-rangingsmallwild

mammals(rodents,marsupials,andcanids)andtheir

ectopar-asites(ﬂeas, ticksandhematophagouslice)intheCaatinga

biome.

Materials

and

methods

Studysite

This study was conducted in the Petrolina (9◦1941S,

40◦3330W)andLagoaGrande(8◦401S,40◦842W)

munic-ipalities,StateofPernambuco,Brazil(Fig.1).Thisregionhas

semi-aridtropicalweatherandispartoftheCaatingabiome

inNortheasternBrazil.8_The_{municipalities}_are_located_in_the

regionoftheSãoFranciscoValleyandrepresenttwodistinct

areaswithinadegradedenvironment.

Samplecollection

Atotalof77smallmammalsweretrappedineighttrialsfrom

August2014toMay 2015.Eachtrialwasinadifferentarea,

withfour in each municipality and two ineach season of

theyear(spring,summer,autumn,andwinter).Animalswere

chemically immobilized as recommended by Mares-Guia.9

Bloodsampleswerecollectedfromtheseanimalsviathe

cau-dalveinorintra-cardiacpuncture.Additionally,ectoparasites

werecollectedfromtheanimalsandstoredin1.5mL

micro-tubescontainingabsoluteethylalcohol(C2H5OH)at−20◦C

priortolaboratoryanalysis.Aftercollectionofbiological

mate-rials,theanimalsweremarkedbycuttingthehairfromthe

sacralregionandweresetfreeinthesamepointofcapture

afterfullrecoveryofconsciousness.

Domiciledogsandcatswereanalyzedinrural dwellings

aroundeachcollectionarea.Informationincludingage,

gen-der,countyoforigin,tick,ﬂeaand/orlousepresence,history

of ectoparasitism, and access to the forest was obtained.

Bloodsampleswerecollectedfromthecephalicveinintubes

containing EDTA, properly identiﬁed, and centrifuged at

3000rpmfor15min.Plasmaandwholebloodwerestoredin

1.5mLmicrotubesat−20◦_C_prior_to_analysis.

Ticks, ﬂeas, and lice were collected and conserved in

absolute ethanol and stored at room temperature until

identiﬁcation,accordingtoLinardiandGuimaraes,10

Barros-Battesti, Arzua and Bechara,11 _and _Pereira _et _al.,12 _and

moleculartesting.

All procedures followed the ethicalstandards ofanimal

experimentationestablishedbytheCommitteeonEthicsand

StudiesandResearchattheFederalUniversityofSãoFrancisco

Valley – CEDEP/Univasf (protocol number 9/021014) and by

theBrazilianInstituteofEnvironmentandRenewable

Natu-ralResources-IBAMA(protocolnumber45764-1)accordingto

therecommendationsandlawsregardingthemaintenanceof

animalwelfare.

Indirectimmunoﬂuorescenceassay(IFA)

Plasmasamplesfromdogsandcatsweresubjectedto

indi-rectimmunoﬂuorescentantibodyassay(IFA)(Bion,IL,USA)to

detectanti-Bartonellasp.antibodiesasrecommendedbythe

manufacturer.Acutoffvalueof1:64wasused.13

Polymerasechainreaction(PCR)

DNAwasextractedfromwholebloodofdogs,cats,andwild

animals and from individual ticks using a commercial kit

(Promega,Madison,WI,USA)asrecommendedbythe

man-ufacturer. A negative DNA extractioncontrol consisting of

100␮Lofsteriledistilledwaterwasincludedineachbatchof

samples.Wholeﬂeasandlicewereindividuallysubjectedto

DNAextractionbyboilingat100◦Cfor20min.14

AllsampleswereindividuallyprocessedbyPCRusingthe

primersBhcs.781p(5-GGGGACCAGCTCATGGTGG-3)and

Bhcs.1137n(5-AATGCAAAAAGAACAGTAAACA-3),which

ampliﬁeda380-bpfragmentofthecitratesynthasegene(gltA)

ofBartonellaspp.,andBARTON-1(5-TAACCGATATTGGTT

GTGTTGAAG-3)andBARTON-2(5-TAAAGCTAGAAAGTC

TGGCAACATAACG-3),whichampliﬁeda580-bpfragment

ofthe riboﬂavin synthaseC gene(ribC). ThePCR reactions

were performedasdescribedpreviously.15,16 _The_PCR

prod-uctswerestainedwithethidiumbromideandvisualizedby

electrophoresisina1.5%agarosegel.

PCR products ofthe expected amplicon size were

puri-ﬁed,andtheirforwardandreversenucleotidesequenceswere

subjectedtodirectsequencingusingtheABIPrismBigDye

Ter-minator v.3.1cyclesequencingkit (AppliedBiosystems,CA,

USA).Partialsequencesobtainedfromthis studywere

sub-mittedtoBLAST(BasicLocalAlignmentSearchTool)analysis

todeterminesimilaritiestootherBartonellaspeciessequences

availableintheGenBankdatabase.17

Statisticalanalysis

Categorical variables are described as proportions with

the respective 95% conﬁdence interval, and compared

by the Chi-square test (X2₎ _using _the _software _Epi

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Legend

Brazil

State of Pernambuco

Municipality of Petrolina Municipality of Lagoa Grande

0 10 20

40

60 N

W

E

S

80 km

Fig.1–LocationofthemunicipalitiesofLagoaGrandeandPetrolina,PernambucoState,Brazil.

Results

Seventy-sevensmallwildmammalsweretrappedover2944

trapnights,showingasuccessrateof2.6%.Themammals

wereidentiﬁedasRodentia [Thrichomysapereoides(30

speci-mens),Wiedomyspyrrhorhinus(3),Galeaspixii(3),andCalomys

expulsus(2)],Marsupialia[Didelphisalbiventris(27),Monodelphis domestica (10), and Gracilinanusagilis (1)], and Canid

[Cerdo-cyonthous(1)].Byseason,15mammalsweretrappedinwinter

(September2014),17inspring(November2014),20insummer

(January2015),and25inautumn(May2015).

Hematophagousectoparasiteswerefoundin51.94%(40/77)

ofthe wildanimals.AmongRodentia,52.63%(20/38)ofthe

animalswere infested: T. apereoidesbyAmblyomma sp.

(lar-vae), Amblyomma auricularium(nymphs), Argasidae (larvae),

andPolyplaxsp.(adult)andW.pyrrhorhinusbyAmblyommasp

(larvae).AmongMarsupialia,50%(19/38)oftheanimalswere

infested:D.albiventrisbyAmblyommasp.(larvae),A.

auricu-larium(nymphs),Amblyommadubitatum(nymphs),Argasidae

(larvae), Ctenocephalides felis felis (adults), M. domestica by

Amblyommasp.(larvae),A.auricularium(nymphs),and

Argasi-dae(larvae).AmongCanidae,100%(1/1)oftheanimalswere

infested:C.thousbyPulexirritans(adults).Therewasno

infes-tationinC.expulsus,G.spixii,andG.agilis.

Approximately56%ofthedogs(61/109)wereinfestedwith

Riphicephalussanguineusadultticksand19.26%(21/109)with

C.felisfelis(11/109)and/orP.irritans(16/109)ﬂeas.Fifteendogs

(13.76%)wereparasitizedbybothticksandﬂeas.Around22%

ofthecats(9/40)wereinfestedwithC.felisfelisﬂeas.

Anti-Bartonella sp. antibodies were detected in 24.77%

(27/109)ofthedogsand15%(6/40)ofthecats.Nosigniﬁcant

differenceswerefoundamonganyofthevariablesanalyzed

(p>0.05).

Bartonella sp. DNA was identiﬁed in nine (100%) P. irri-tansﬂeasfrom C.thous,one(10%)Polyplaxsp.lousefromT. apereoides, one(2.32%)C.felisfelisand 35(57.37%) P.irritans

fromdogs,and24(53.33%)C.felisfelisfromcats.Alltickstested

negativebyPCR(Table1).

OnedogwasinfestedsimultaneouslywithpositiveC.felis

felisandP.irritans.Twodogswhoseﬂeaswerepositivewere

reactivetotheIFA.Fleasfromeight(88.88%)catsshowed

Bar-tonellasp.DNA,ofwhichtwoanimalshadtitersofantibodies

detectablebytheIFA.

Partial sequences of the gltA gene were shown by

the BLAST analysis to be 100% identical to the

corre-sponding sequences of Bartonella clarridgeiae (KJ170236),

Bartonella henselae (HG965802), Bartonella rochalimae

(DQ676484.1) and Bartonella vinsoni berkhoﬁi (AF143445.1)

(Table1).

Discussion

Thestudyofwildlifeallowsforanimprovedunderstanding

of aspects related to the maintenance of various

etiologi-calagentsintheepidemiologicalchainofdifferentdiseases

as well as their sustainability and viability in nature. In

thisrespect,itisimportanttoemphasizestudiesrelatedto

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Table1–Bartonellaspp.infectioninhematophagousectoparasitesinwildanddomesticmammalsinasemi-aridregion ofPernambuco,Brazil. Host %infestation (presenceof ectoparasite/total) Ectoparasites Infectionof Bartonellaspp.by PCR DNAsequencing

Thrichomysapereoides 63.3(19/30) Amblyommasp. 0/3 –

A.auricularium 0/23 –

Argasidae 0/8 –

Polyplaxsp. 1/20 no

Wiedomyspyrrhorhinus 33.3(1/3) Amblyommasp. 0/1 –

Galeaspixii 0(0/3) – 0/0 –

Calomysexpulsus 0(0/2) – 0/0 –

Didelphisalbiventris (15/27) Amblyommasp. 0/13 –

A.dubitatum 0/4 –

Argasidae 0/5 –

C.felisfelis 0/3 –

Monodelphisdomestica 40.0(4/10) Amblyommasp. 0/2 –

Argasidae 0/1 –

Gracilinanusagilis 0(0/1) – 0/0 –

Cerdocyonthous 100(1/1) P.irritans 9/9 B.rochalimae Canisfamiliaris 61.5(67/109) R.sanguineus 0/230 –

C.felisfelis 1/43 no

P.irritans 35/61 B.rochalimae,B.vinsoniberkhoﬁi Feliscatus 22.5(9/40) C.felisfelis 24/45 B.clarridgeae,B.henselae

byoriginallysylvanareas.18 _In_this _study,_a_data _survey_of

faunaectoparasiteswasofgreatrelevanceduetothevector

transmissionofBartonellaspp.19

Thecapturesuccessrateinthisstudy(2.6%)wassimilar

tothatobtainedbyDantas-Torresetal.20_(1.7%)_in_the_Atlantic

ForestfragmentsintheStateofPernambuco,whichisabiome

thathassufferedseveredegradationsimilartotheCaatinga.

However,the successratewaslowerthan thatachievedby

Guimarães21_(6.1%)_in_Serra_das_Confusões_National_Park,_State

ofPiauí,whichisanenvironmentalpreservationareawithlow

humaninterventionrates.Inlinewiththeseﬁgures,Fonseca22

comparedanon-degradedareatoanotherdegradedareain

theStateofMinasGeraisandobtainedsuccessratesof6.2%

and2.9%,respectively.

Eightdifferentmammalianspeciesweredetected,which

was equivalentto approximately 5.4%ofthe total number

ofnaturally occurring mammalianspecies inthe Caatinga

(n=148).23 _The _most _frequently _caught _species _was _T.

apereoides,representing39%(30/77)ofthewildanimalsinthe

presentstudy.Thisspecieswasequivalentto79%(30/38)of

therodents,whichwassimilartothe80%reportedinSerra

dasConfusõesNationalPark.21

Weobtainedasmallnumberofanimalsinfestedwithﬂeas

in the present study compared to similar studies in both

domesticand wild animals.24,25 _This_result_may _be_related

to climatic factors of the study site, which is located in

the Drought Polygonregion that presents featuressuch as

anegativewaterbalance (resultingfrom anannualrainfall

lessthan500mm),averageinsolationof2800h/year,annual

averagetemperatureofapproximately28◦C(reachingupto

44.1◦C insummer and 39.1◦C inwinter) and average

rela-tivehumidityof50%.26,27_These_factors_indicate_that_the_site

ofthepresent studyisa relativelyhostileenvironment for

orderSiphonaptera,whichgenerallyprefersatemperatureof

28±1◦Candarelativehumidityofapproximately75%forits

development.28

Althoughnopositivediagnosiswasobtainedinwild

ani-malsbloodsamples,itwaspossibletodemonstratethewild

cycleofBartonellaspp.intheCaatingabythepresenceof

bac-terialDNAintheﬂeasandblood-suckinglicethatparasitized

C.thousandT.apereoides,respectively.

ThepresenceofBartonellaspp.inPolyplaxspp.hasbeen

pre-viouslydescribedinTaiwan.24_The_same_study_also_detected

DNAoftheagentinﬂeasfromwildanimalsbutnotintheir

ticks, in line with the results in our study. More recently,

Bartonellavinsoniisubsp.arupensiswasidentiﬁedindifferent

speciesofwildrodentscollectedinMatoGrossodoSul,which

istheBrazilianstatemostcoveredbythePantanalbiome.29

None ofthe dogsparticipatinginthepresent study had

any apparent sign ofdisease. Amongthe seropositive

ani-mals(titers≥64),twohadapositivediagnosisofBartonellasp.

DNAintheirﬂeasbutnotintheirbloodsamples.Dogsactas

accidentalreservoirsofsomespeciesofBartonella.However,

asymptomaticdogsrarelyshowpersistentbacteremia.30,31

ThepresentstudyconﬁrmedtheoccurrenceofBartonella

spp.bydetectingantibodiesindogsandcatsandthe

pres-enceofbacterialDNAinectoparasitescollectedfromdomestic

andwildanimals.Theseresultssuggestedthatthehostsmay

harborthemicroorganism,althoughBartonellaDNAwasnot

foundinanybloodsamples.Thus,thepresenceorabsence

of Bartonella spp. in the host does not necessarily reﬂect

the conditionoftheirﬂeas.32,33 _Negative_PCR_results_in_the

blood are often indicativeof the bacterialDNA

concentra-tion inthe sample,which maybeinsufﬁcientfordetection

by the technique.34,35 _{Additionally,} _PCR _of _blood _samples

is commonly not successful due to the inhibitory factor

hemin.36_Furthermore,_Bartonella_causes_a_{characteristic,}_cyclic

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AllpositivesamplestestedbyPCRtargetingtheribCgene

werealsopositivebyPCRtargetingthegltAgene,butnotvice

versa.Thisdiscrepancymaybeduetovariabilityinthe

sensi-tivityoftheassaydependingontheprimerusedortheprimer

targetinggltA may havebeen morecomprehensive forthe

detection ofa muchwider range of Bartonella speciesthat

theprimertargetingribC.15,16,38

The ﬁnding that an animal could be PCR-positive and

IFA-negativemaysuggestthattheinfectionisrecent,the

indi-vidualhasnotproducedanIgGantibodyleveldetectablebythe

test,theanimalisimmunosuppressed,ortheinfectionisbya

Bartonellaspeciesthatdoesnotpresentcross-reactivitywithB. henselae.39_This_possibility_may_also_explain_why_ﬂeas_from_an

animalarepositiveforBartonellaDNAwhentheanimaldoes

nothavedetectableantibodiesbyIFAaswasobservedinsome

animalsinthisstudy.

Atotalof24.77%(34/109)ofthedogshadasuggestive

diag-nosisforinfection byBartonella spp.,and PCR-positiveﬂeas

were present on 42.3% (9/21) ofthe infesteddogs. Among

these,twodogswerebothseropositiveandhadpositiveﬂeas.

AlowerprevalencewasdescribedbyBrenneretal.40_in_stray

dogsinSãoPaulo(9.3%,11/118animals).InBotucatu,stateof

SãoPaulo,only3.5%of198dogswereseropositiveforBartonella

spp.30_In_a_study_conducted_in_Peru,_a_63%_(68/108)

seroposi-tivityrateforBartonella spp.wasdetectedinasymptomatic

dogs.Theagegroupwasshown tobeariskfactorfor

ani-malsagedlessthanoneyear,41_which_was_in_contrast_to_the

present study in which there was no such correlation. Of

the40cats,six(15%)werepositiveintheIFAandseven(17.5%)

inthePCRtestofﬂeas.Onecatwasconcurrentlypositiveinthe

IFAandPCRtestofﬂeas.Crissiumaetal.25_reported_the

occur-renceofBartonella DNAinﬂeasfrom 20%(4/20)ofinfested

cats,ofwhichonlyonehaddetectablebacterialDNAinthe

blood.

Theserologicalresultsincatsinthisstudyaresimilarto

thoseofLoureiroandHagiwara,42_who_detected_16%_positivity

foranti-B.henselaeantibodiesin200domicilecatsinSãoPaulo.

Amongstudies conductedwithcatsinBrazil,the

seroreac-tivityrangedfrom1.6%indomesticcatsandcatsfromthe

ZoonosisControlCenterofSãoLuís,Maranhão,andCuiabá

and Várzea Grande,Mato Grosso43,44 _to _up _to _97% _in _cats

fromsheltersinVassouras,RiodeJaneiro,45_using_molecular

and/orserologicalmethods.Thestudieswere performedin

theStatesofSãoPaulo[4.3%,46_30%47_],_Rio_de_Janeiro_[42.5%,25

35.7%,13_and_56.1%48_],_Mato_Grosso_[2.2%49_]_and_Rio_Grande_do

Sul[25.5%50_].

Conclusions

Theresultsof the present study conﬁrmfor the ﬁrsttime

theoccurrenceofBartonellaintheCaatingabiomeandinthe

semi-aridregionandtheidentiﬁcationofatleastfourdifferent

species(B.clarridgeiae,B.henselae,B.rochalimaeandB.vinsoni

berkhoﬁi)inthesamegeographicregionofNortheasternBrazil.

ThisistheﬁrstreportofBartonellaspp.inPolyplaxsp.andPulex

sp.inBrazil.Furtherinvestigationsareneededtoidentifythe

prevalentBartonellaspecies,toverifythevectorcompetence

oftheseﬂeaspecies,andtoelucidatetheirepidemiologyat

thestudysite.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

WethankAnaIsabelA.Santos,Lais F.Santos,JoseniltonR.

Santos,IvoW.G.daSilva,andDáliaM.R.Machadofortheir

valuablehelpduringtheﬁeldworkandlaboratorytestsand

theCentrodeConservac¸ãoeManejodeFauna(Cema-Fauna)

for conﬁrmation of the identiﬁcation of wild mammalian

species.Thiswork wassupportedbytheBrazilianResearch

FundingAgency(CNPq).

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