Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC)

brazilian journal of microbiology48(2017)760–763

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

Veterinary

Microbiology

Captive

wild

birds

as

reservoirs

of

enteropathogenic

E.

coli

(EPEC)

and

Shiga-toxin

producing

E.

coli

(STEC)

Lilian

Aparecida

Sanches

_,

_Marcelo

_da

_Silva

_Gomes

_,

_Rodrigo

_Hidalgo

_Friciello

_Teixeira

_,

Marcos

Paulo

Vieira

Cunha

_,

_Maria

_Gabriela

_Xavier

_de

_Oliveira

_,

Mônica

Aparecida

Midolli

Vieira

_,

_Tânia

_Aparecida

_Tardelli

_Gomes

_,

_Terezinha

_Knobl

a_{DepartamentodePatologia.FaculdadedeMedicinaVeterináriaeZootecnia,UniversidadedeSãoPaulo(USP),SãoPaulo,Brazil} b_{ParqueEstoril,ZoológicoMunicipaldeSãoBernardodoCampo,SP,Brazil}

c_{QuinzinhodeBarrosZoologicalPark(PZMQB),Sorocaba,SP,Brazil}

d_{DepartamentodeMicrobiologia,ImunologiaeParasitologia,EscolaPaulistadeMedicina,UniversidadeFederaldeSãoPaulo,SãoPaulo,}

Brazil

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o

Articlehistory:

Received8September2016 Accepted8March2017 Availableonline2June2017 AssociateEditor:CristianoGallina Moreira Keywords: DiarrheagenicE.coli AtypicalEPEC STEC Aviandiseases

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b

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PsittacinebirdshavebeenidentifiedasreservoirsofdiarrheagenicEscherichiacoli,asubset ofpathogensassociatedwithmortalityofchildrenintropicalcountries.Theroleofother ordersofbirdsassourceofinfectionisunclear.Theaimofthisstudywastoperformthe moleculardiagnosisofinfectionwithdiarrheagenicE.coliin10differentordersofcaptive wildbirdsinthestateofSãoPaulo,Brazil.Fecalsampleswereanalyzedfrom516birds belongingto10orders:Accipitriformes,Anseriformes,Columbiformes,Falconiformes, Gal-liformes,Passeriformes,Pelecaniformes,Piciformes,PsittaciformesandStrigiformes.After isolation,401E.colistrainsweresubjectedtomultiplexPCRsystemwithamplificationof geneseaeandbfp(EPEC),stx1andstx2forSTEC.Theresultsofthesetestsrevealed23/401 (5.74%)positivestrainsforeaegene,16/401positivestrainsforthebfpgene(3.99%)and3/401 positiveforstx2gene(0.75%)distributedamongtheordersofPsittaciformes,Strigiformes andColumbiformes.Noneofstrainswerepositiveforstx1gene.Thesedatarevealthe infec-tionbySTEC,typicalandatypicalEPECincaptivebirds.Thefrequencyofthesepathotypes islowandrestrictedtofeworders,butthedatasuggestthepotentialpublichealthriskthat thesebirdsrepresentasreservoirsofdiarrheagenicE.coli.

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

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

∗ _{Correspondingauthor.}

E-mail:tknobl@usp.br(T.Knobl).

http://dx.doi.org/10.1016/j.bjm.2017.03.003

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

brazilian journal of microbiology48(2017)760–763

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Introduction

Many zoonoses are associated with ecological imbalances as a result of deforestation, human population expan-sion,changesinagriculturalpracticesandencroachmenton wildlifehabitats.1_{Urbanareas representimpacted} environ-mentandwildanimalsaremaintainedincaptivity,livingin zoos.Captivewildlifeanimalsareverysusceptibleto oppor-tunisticdiseasesandtheymayactasreservoirofpathogenic bacteria.2,3

Escherichiacolicanbeconsideredthemostprevalent oppor-tunisticenterobacteriaincaptiveanimalsandwereassociated withsystemicdisease inbirds.4 _{Airsaculitis and sepsisare} oftencausedbyavianpathogenicEscherichiacoli(APEC),which are considered asExtraintestinal pathogenic E. coli(ExPEC) pathotype.5 _{ThepathogenesisofenteritisbyE.coliinbirds} isstillunclear,butthepresenceofdiarrheagenicstrainsmay representapublichealthrisk. Shigatoxin-producingE. coli (STEC)andenteropathogenicE.coli(EPEC)representtwoofat leastsixpathotypesofhumandiarrheagenicE.colithataffect birdsandmaybeconsideredzoonoticpathogens.6,7

TheEPEC pathotypeleadstoahighchildmortalityrate indeveloping countries. Diarrhea isa consequenceof loss ofintestinalmicrovillus,afterbacterialadherenceon entero-cytes,withensuingattachingandeffacing(A/E)lesions.8_The AElesion dependsonintimin,anoutermembraneprotein, codifiedbytheeaegene,whichispresentinthepathogenicity islandtermedthelocusofenterocyteeffacement(LEE).9_The identificationoftypicalEPECstrains(tEPEC)isperformedby moleculardetectionoftheeaeandbfpgenes(“bundle-forming pili”encodedbytheEAFplasmid).TheEPECareregardedas atypical(aEPEC)incaseofabsenceoftheEAFplasmid,making iteae+andbfp−.10

TheattachingandeffacinggenesontheLEEmaybealso present in STEC, but Shiga toxin productionis considered themostrelevantvirulencefactorofthisdiarrheagenicE.coli patothype.8,11 _{Shiga toxinscanbeclassifiedintotwotypes,} Stx1(subtypesa,candd)andStx2(subtypesatog).Bothtoxins areencodedonprophagesthatareintegratedintothe chro-mosomeandleadtoinhibitionofproteinsynthesis,causing celldeath.8

In humans, STEC infection causes hemorrhagic colitis followinginjuryoftheintestinalepithelium,inducedby Shiga-toxinproduction.Stx-2 toxin ismoretoxicthan Stx-1,and isoftenassociated withhemolyticuremic syndrome.8 _The transmissionroutes ofSTEC includeingestion of contami-natedfood or waterand contact withinfected companion animals(dogs,catsandbirds).7,8,11

Theaimofthisstudy wastosearchforthepresenceof EPECandSTECisolatesincaptivebirdsfromdifferentorders locatedatzoosfromSãoPaulo,state,Brazil.

Material

and

methods

Birds

Thisprojectwasapprovedbythe Ethics CommitteeofSão Paulo University (2984230514) and authorized for scientific

purposes(SISBIO43541-1).Weexaminedatotalof516fecal samplesisolated from captive birds belongingto10 orders (including 70 species): Accipitriformes (hawk, n=14), Galli-formes(guanandcurassow,n=50),Anseriformes(duckand goose, n=80), Psittaciformes (macaw, parrot and parakeet, n=99), Passeriformes (canary and thrush, n=88), Falconi-formes (falcon, n=46), Strigiformes (owl, n=48), Columbi-formes(pigeons,n=72);Piciformes(toucanandaracari,n=10) Pelecaniformes(pelicanandegret,n=9).

ThesampleswerecollectedfromSeptember2013toJune 2015,intwomunicipalzooslocatedinSãoPauloState,Brazil. FecalswabswereseededinAmiestransportmediaandsent tothelaboratory,underrefrigeratedconditions.

CultureandIdentificationofE.coli

The fecal samples were enriched in brain heart infusion broth,seededonMacConkeyagar,andincubatedat37◦Cfor 24h.Bacteriawereidentifiedbybiochemicaltests,usingan Enterokit(Probac® –SãoPaulo,Brazil).

PCRAmplificationforvirulencegenes

SearchforvirulencegenesinthediarrheagenicE.coliisolates performedbyPCRforamplificationofeae(454bp),bfp(550bp), stx1(349bp)andstx2(110bp)genes,accordingtothemethod describedbyCostaetal.(2010).12_{Thefollowingstrainswere} used ascontrol ofthe PCR: E. coliDH5␣ (negative control); O157:H7(STECpositivecontrol)andO55:H7(EPECpositive con-trol).

The DNA extraction was performed as described by Boom et al. (1990).13 _{The amplification mixture consisted} of Tris-HCl buffer (pH 8.3) 10mM, MgCl2, deoxynucleotide

triphosphates 200mM, pairs of primers, Taq DNA poly-merase 0.5U, and ultrapure water autoclaved in a final volumeof25␮l.Amplifiedproductswereseparated in1.5% agarosegelandexaminedafterstainedwithBlueGreen® (LGC Biotecnologia, SãoPaulo, Brazil).A 100bpDNA ladder (LGC Biotecnologia,SãoPaulo,Brazil)wasusedasamolecularsize marker.

Results

Atotalof401isolateswereidentifiedasE.coli.AfterPCR inves-tigation,23/401isolateswerepositiveforeae,16/401positive forbfp and 3/401 positiveforstx2 genes(Table1). Noneof strains(0/401)werepositiveforstx1gene.

The pathotype classification of the isolates is pre-sented inTable2.These resultsshowedthatatypical EPEC were detectedin3/10 orders,including Psittaciformes(2/99 birds), Columbiformes (2/72 birds) and Strigiformes (1/48 birds). TypicalEPECwere detectedin2/10 orders, including Psittaciformes(11/99birds)andColumbiformes(4/72birds). STEC wasdetectedonlyinColumbiformes, presentin 3/72 birds.

Thetotal prevalenceofdiarrheagenicE. colistrains was 23/401(5.74%),whichincluded13/76(17.03%) from Psittaci-formes, 9/72 (12.48%) from Columbiformes and 1/37 (2.7%) fromStrigiformes.

762

Table1–IsolationofE.coliandvirulencegenesdistributedaccordingtotheordersofcaptivewildbirds.SãoPaulo, 2013–2015.

Orders Birds(n) E.coliisolates Genes

eae bfp stx2 Accipitriformes 14 12/14 Anseriformes 80 28/80 Columbiformes 72 72/72 9/72(12.5%) 5/72(6.94%) 3/72(4.17%) Falconiformes 46 44/46 Galliformes 50 50/50 Passeriformes 88 56/88 Pelecaniformes 9 16/9 Piciformes 10 10/10 Psittaciformes 99 76/99 13/76(17.11%) 11/76(14.47%) Strigiformes 48 37/48 1/37(2.7%) Total 516 401/516 23/401(5.74%) 16/401(3.99%) 3/401(0.75%)

Table2–PathotypesofdiarrheagenicE.colidistributedaccordingtotheordersofcaptivewildbirds.SãoPaulo, 2013–2015.

Orders E.coli(n) PathotypesofdiarrheagenicE.coli

TypicalEPEC AtypicalEPEC STEC

Columbiformes 72 1/72(1.38%) 5/72(6.94%) 3/72(4.16%)

Psittaciformes 76 11/76(14.4%) 2/76(2.63%)

Strigiformes 37 1/37(2.7%)

Total 401 12/401(2.99%) 8/401(1.99%) 3/401(0.74%)

Discussion

Brazilhas greatwildlifebiodiversity. Awide rangeofbirds speciesare alsokeptinzoosforentertainment,education, searchandconservation.Ourstudyanalyzed10orderswith 70speciesofbirdsforthepresenceofdiarrheagenicE.coli.

Unfortunately, due to the small number of birds in some

orders, a fair sampling was somewhat compromised. Our

resultsdemonstratethatmoleculartechniqueswereusefulfor diagnosisofthediarrheagenicE.colipathotypes,identifying captivebirdsinfectedbytypicaloratypicalEPECandSTEC.

TheresultsshowedthatEPECwerefoundin3/10orders,

including Psittaciformes (2/99 birds), Columbiformes (2/72 birds)andStrigiformes(1/48birds).Likewise,Kobayashietal.

(2009) evaluated the prevalence of eae- and stx-positive

E. coli strains in 447 wild birds belonging to 62 species

inTokyo.14 _{Eae-positive strains were found in 7/10 orders:} Columbiformes,Passeriformes,Anseriformes,Ciconiformes, Procellariformes,Pelecaniformesand Galliformes. However, the prevalence of eae-positive strains in Tokyo was 25% (11/447), higher than the prevalence of this study (5.74%). PsittaciformeswerenotincludedinaTokyosurvey,but Passer-iformes,ColumbiformesandPelecaniformeswereimplicated asareservoirofEPEC.14_{InBrazil,noneofthePasseriformes,} PelecaniformesorAnseriformesinvestigatedwereinfectedby EPEC.However,wefoundoneatypical EPECinStrigiformes (1/37owl).Toourknowledge,thisisthefirstreportofinfection byEPECinowls.

RelativeanalysesshowedthatPsittaciformesisthemost prevalentorderofbirdspositivelyinfectedbyEPEC(Table2), with14.4%oftypicalEPECand2.63% ofatypical EPEC.The

EPECinfectionofpsittacinebirdsinBrazilwasreported pre-viouslyinparrots(Amazonaaestiva,Amazonaamazonica)and macaws(Anodorhynchusleari,Guaroubaguarouba)with preva-lencerangingfrom2.27%infree-rangingbirds15 _to6.5%for captivepsittacinebirds.16

Marietto-Gonc¸alveset al.(2011)investigatedswabsfrom 86psittacidaesrecoveredfromillegalwildlifetradeinBrazil and foundonlyonestrain(1/86–1.1%) classifiedastypical EPEC,isolatedfromablue-frontedparrot.17 _{Webelievethat} thehighprevalencereportedinourstudy(11.11%)isrelated tozooenclosuresthatallowintensecontactbetweenbirds, mammalsandparkvisitors.Bacterialdiversitywasreported previouslytobesignificantlylower inwild parrotsand the compositionofcloacalbacterialmicrobiotamightundergo sig-nificantchangesincaptivebirdsif theyare overexposedto contactwithmammals.18

Farooq et al. (2009)found a high prevalenceof atypical EPEC(15.56%)fromavianspeciesinIndia.6_{Thefrequencywas} greaterinfarmedanimals(chickenandduck–27/112)than inpigeons(6/100),buttheauthorsstillbelievethatpigeons actasaninfectioussourceforcommercialpoultry.Our sur-veyalsohighlightstheroleofColumbifomesasareservoirof EPECandSTEC.Wedetected6/72EPECand3/72STECstrains inferalpigeons(Columbalivia).Thisdataaresimilartothose reportedbyKobayashietal.(2009),with5/67EPECand2/67 STECstrainsinTokyo.14

Feralpigeonsaresynanthropicbirds.Inzoos,thesebirds invadetheenclosureslookingforwaterandfood,andtransmit diseases or even acquire pathogens from animals belong-ing to other classes, such as mammals and reptiles. The zoonoticriskassociatedwithEPECinfectioninpigeonswas firstdocumentedbySilvaetal.(2009)inBrazil,reporting3.3%

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prevalenceofinfectedpigeonsinurbanareas.19_Population controlmeasuresinurbanenvironmentsareverydifficultdue totheabsence ofnaturalpredators.Thepresenceofthese birdsinsomehabitatshasbeenassociatedwithtransmission ofmany zoonosessuchaschlamydiosis,salmonellosisand campylobacteriosis.20

Sacristanetal.(2014)reportedthatferalpigeonsfromSpain wereinfectedwithatypicalEPEC(8%).21_{Thefrequencyofeae} geneinurbanpigeonswas6%and4%inruralspecies.The authorshighlighted thepublic healthrisksassociatedwith antibioticresistance,becausesomestrainspresentedclassI integronscontaininggenes cassetesencodingforantibiotic resistance.

Webelievethatthecolonizationofbirdsmayvary accord-ingtothesusceptibilityofthespecies,withinfluenceofdiet, microfloraandmanagement.Apparently,theordersof Galli-formesandAnseriformesarelesssusceptible,andthereports ofdiarrheagenicE.coliinthesebirdsarerare,evenintheface ofmany managementrisk factors,suchasaccess tolakes andwatercollection, contactwithother animalsinshared enclosures and difficulty in maintaining hygiene on dirt floors.

Conclusion

This study highlights the presence ofdiarrheagenic E. coli (EPECandSTEC)incaptivePsittaciformes,Columbiformesand Strigiformes.Thezoonoticpotentialofthesestrainsmaybe investigatedbecauseofthesanitaryimpactonzoobird col-lections,whichareimportantforthe“insitu”conservationof thespecies.

Conflicts

of

interests

Theauthorswishtostatethattheyhavenocompeting inter-estswithregardtothepublicationofthismanuscript.

Acknowledgements

TheauthorsarethankfultoQuinzinhodeBarrosZoo (Soro-caba,SP–Brazil)andParqueEstoril.ZoológicoMunicipalde SãoBernardodoCampoforfecalsamplesdonation.

This study was conducted with financial support from CNPqandFAPESP(2014/07837-6).

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