brazilian journal of microbiology49S(2018)76–82
h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Veterinary
Microbiology
Survey
on
pathogenic
Escherichia
coli
and
Salmonella
spp.
in
captive
cockatiels
(Nymphicus
hollandicus)
Patricia
Silveira
de
Pontes
a,
Selene
Dall’
Acqua
Coutinho
a,∗,
Renata
de
Oliveira
Iovine
a,
Marcos
Paulo
Vieira
Cunha
b,
Terezinha
Knöbl
b,
Vania
Maria
de
Carvalho
caUniversidadePaulista,ProgramadePós-Graduac¸ãoemPatologiaAmbientaleExperimental,SãoPaulo,SP,Brazil
bUniversidadedeSãoPaulo,FaculdadedeMedicinaVeterináriaeZootecnia,DepartamentodePatologia,SãoPaulo,SP,Brazil cUniversidadedeSãoPaulo,FaculdadedeMedicinaVeterináriaeZootecnia,DepartamentodePatologia,LaboratóriodePatologia
ComparadadaVidaSelvagem,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received6December2017 Accepted10May2018
Availableonline20August2018 AssociateEditor:WaldirElias
Keywords: Escherichiacoli Salmonellasp Cockatiel Nymphicushollandicus Antimicrobialresistance
a
b
s
t
r
a
c
t
We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and
Salmonellaspp.Cloacalswabswerecollectedfrom94cockatielskeptincommercial breed-ers,privateresidenciesandpetshopsinthecitiesofSãoPaulo/SPandNiterói/RJ(Brazil). ThreestrainsofE.colifromeachindividualweretestedforthepresenceofExPEC-, APEC-andDEC-relatedgenes.WeevaluatedtheblaTEM,blaSHV,blaOXA,blaCMY,blaCTX-M,tetA,tetB,
aadA,aphA,strAB,sul1,sul2,sul3,qnrA,qnrD,qnrB,qnrS,oqxAB,aac(6)-Ib-cr,qepAresistance genesandmarkersforplasmidincompatibilitygroups.Salmonellaspp.wasnotdetected.
E.coliwasisolatedin10%oftheanimals(9/94).FourAPECgenes(ironN,ompT,iss and
hlyF)weredetectedintwostrains(2/27–7%),andiss(1/27–4%)inoneisolate.Thehighest resistancerateswereobservedwithamoxicillin(22/27–82%),ampicillin(21/27–79%), strep-tomycin(18/27–67%),tetracycline(11/27–41%).Multiresistancewasverifiedin59%(16/27)of theisolates.WedetectedstrAB,blaTEM,tetA,tetB,aadA,aphaA,sul1,sul2,sul3resistancegenes
andplasmidIncgroupsin20(74%)ofthestrains.E.coliisolatedfromthesecockatielsareof epidemiologicalimportance,sincethesepetscouldtransmitpathogenicandmultiresistant microorganismstohumansandotheranimals.
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:RuaJorgeAmericano337apto71,SãoPaulo,Brazil.
E-mail:selene@uol.com.br(S.D.Coutinho).
https://doi.org/10.1016/j.bjm.2018.05.003
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Keepingpetsisassociatedwithphysicalandemotional bene-fitsduetoitspositiveeffectonpeople’slifequality;however, itmaypresentarisktopublichealth.1,2Thisisbecausethe relationshipbetweenmenandanimalsenablestransmission ofseveraldiseasesthroughpathogens’abilitytocolonize sev-eralhosts.3Therefore,petbirdssuchascockatielsmayharbor andtransmitzoonoticagentsthroughclosecontactwiththeir owners.1,4,5
Salmonellosis, a disease caused by bacteria of the
Salmonella genus, has great relevance due to its lethality andzoonoticpotential.6Infecteddomesticchickensare con-sideredthe mostcommon sourceofhumansalmonellosis; contaminated chicken meat and eggsare one ofthe main causesoffoodpoisoningworldwide.6Furthermore,wildand exoticavianspeciesarealsoconsideredSalmonellareservoirs.7
Escherichiacoliisacommensalbacteriumoftheintestinal microbiomeofhomeothermicanimals.However,pathogenic strainsarecapableofcausingintestinalandextraintestinal diseasesinhumans,andmammalandavianspecies,leading toseriouseconomiclossesandpublichealthissues.1,5,8
Aside from the zoonotic potential of these enterobac-teriaceae, concerns regarding antimicrobial resistance are currently on the rise.8,9 Broad use of antimicrobial drugs, eithertotreatdiseasesorinlivestockproduction,resultedin selectivepressureandtheconsequentappearanceof multire-sistantbacterialstrains.10
Antimicrobialresistantenterobacteriamaybetransmitted tohumansthroughanimalcontact,contaminatedfoodorthe environment.5,10Aftercolonizingnewhosts,theymay trans-ferresistancegenestomicroorganismsofthelocalmicrobiota. Antimicrobialresistancegenesmaythenrecombineamong thesestrains,creatingnewonesresistanttoseveraldrugs.5,10 Despite the great global concern with microbial resis-tances,thereislittleinformationavailableon the epidemi-ologicalrole ofpetbirds inthe epidemiologyofE. coliand
Salmonellaspp.6 Recentstudies inBrazil showed that free-ranging wild birds may harbor potentially pathogenic and antimicrobialresistantstrains.11
The aim of this study was to survey cloacal samples of captive cockatiels (Nymphicus hollandicus) for potentially pathogenic Salmonella spp. and E. coli. We also evaluated the antimicrobial resistanceprofile ofthe isolates, as well as resistance genes belonging to the main antimicrobial classes.
Materials
and
methods
Allanimal proceduresfollowed ethicalprinciplesand were approvedby the Ethical Committee inAnimal Use (237/14 CEUA/UNIP).
Wecollectedsamplesfrom94clinicallyhealthymaleand femalecockatiels(N.hollandicus) keptincaptivity:8from a petshop,28from differentprivateresidenciesand 58from commercialbreederslocatedinthecities ofSão Pauloand Niterói,intheStatesofSãoPauloandRiodeJaneiro(Brazil), respectively.
Upon physical restraining and after pericloacal asepsis with70%alcoholicsolution,twouretralswabswererubbed againstthecloacalmucosa,keptinrefrigeratedStuartmedia andprocessedwithin48h.
CloacalswabsamplesforE.coliscreeningwereseededin MacConkeyagar(DifcoTM,Maryland,EUA)andidentified fol-lowing routinebiochemical identification,12 including EPM, MILi and citrate(ProbacTM, SãoPaulo, SP,Brazil) identifica-tionkits.ThreedifferentE.coliisolatesfromeachanimalwere storedfortheremainingtests.
Swabs for Salmonella testing followed the protocol sug-gestedbyMichaeletal.,2003.13Theswabswerecultivatedin bufferedpeptonewaterat37◦Cby24h.Aliquotsof1mlwere subculturedin9mloftetrathionateMüller–Kauffmann(TMK, DifcoTM,Maryland,EUA).After24hofincubation,aliquotsof theselectivebrothswerestreakedontoxylose-lysine-tergitol 4agar(XLT4,DifcoTM,Maryland,EUA).After24hof incuba-tion (37◦C),typicalcoloniesofSalmonellawereisolated and subjectedtobiochemicalidentification.13
Weperformedpolymerasechainreaction(PCR)ontheE.coli
isolatesinsearchofextraintestinalpathogenicstrains(ExPEC) characteristicgenesbyaccessinggenespapC,14papEF,15sfa,14
fyuA,16 cnf1,15 hlyA,15 cvaC,16 and malX,16 aside from five avianvirulencepredictinggenes:ironN,17ompT,17hlyF,17iss,17 and iutA.17 Thefollowingdiarrheagenic-relatedgeneswere alsostudied:stx1,18 stx2,18 ST,19 LT,19 astA,20 ipaH,21 aggR,22
eae23, and bfpA.24 All E. coliisolates were submittedto the Fundac¸ãoOswaldoCruz(FIOCRUZ-RJ),areferencelaboratory, tobetestedwithO157andH7antisera.
Drugsensitivitywasevaluatedbydiffusingplates, follow-inginternationalestablishedstandards.25,26 Theuseddrugs (CefarTM,SãoPaulo,SP,Brazil)belongtothefollowing antimi-crobialcategories:-lactams–penicillin(amoxicillin–AMO andampicillin–AMP),-lactams–cephalosporins(cephalexin – CFE, cefoxitin – CFO and ceftiofur – CTF) and -lactams – thienamycin(imipenem– IPM);aminoglycosides (strepto-mycin–ESTandgentamicin–GEN);tetracyclin(tetracyclin– TET);quinolones(ciprofloxacin–CIP,enrofloxacin–ENOand nalidixicacid –NAL);nitrofuran(nitrofurantoin–NIT); sul-fonamide(cotrimoxazol–SUT);anfenicol(chloramphenicol– CLO).Strainswereconsideredmultiresistantwhenresistant tothreeormoreantimicrobialcategories.27
Insearchofresistancegenes,weemployedPCRtechniques on the E. coli strains that presented antimicrobial resis-tantphenotypes.Wesearchedfor-lactamresistancegenes (blaTEM,28 blaSHV,28 blaOXA,28 blaCMY,29 and blaCTX-M28); and employedmultiplexto accesstetracycline(tetAand tetB),30 aminoglycoside (aadA,31 aphA,32 and strAB33), sulfonamide (sul1,33 sul2,31 and sul334), and quinolone resistance genes (qnrA,35qnrD,36qnrB,35qnrS,37oqxAB,35aac(6)-Ib-cr,38qnrC,35 andqepA39).
WeusedthePCR-basedreplicontyping(PBRT)techniqueon allE.coliisolatesinsearchofcharacteristicmarkersfor differ-entplasmidsoftheIncK/B,W,FIIA,FIA,FIB,Y,I1,F,X,HI1,N, H12andL/Mgroups.40
The chi-square test was employed to compare the fre-quency of bacteria from pet shop, private residences and commercial breeders. p-values ≤0.05 were considered significant.
78
brazilian journal of microbiology49S(2018)76–82Results
Salmonellaspp.wasnotisolatedfromthecloacalsamples,on theotherhand,E.coliwasisolatedin10%(9/94)oftheanalyzed animals:onefrom apetshop(1/8–13%),threefromprivate residencies (3/28–11%), and five from commercial breeders (5/58–9%),withnostatisticaldifferencesbetweenbacterial iso-lationregardingtheindividual’sorigin.
Resultsofthe virulencepredictor genes and antimicro-bialresistanceprofilesofall27analyzedstrainsareshown
inTable1.
With the exception of APEC-relatedgenes, identified in twobirds,wedidnotdetectanyothermarkersrelatedtothe remainingExPEC(papC,papEF,sfa,iucD,fyuA,cnf1,hly,cvaC, malXandiutA)anddiarrheagenicE.coli(stx1,stx2,ST,LT,ial, aggR,eaeandbfpA).
NoneoftheevaluatedstrainswerepositiveforO157and H7antiseraagglutination.
All strainswere sensitive tonitrofurantoin. Thehighest resistancepercentagewasto-lactams(93%).Amoxicillinand ampicillinweretheantimicrobialswiththehighestnumber ofresistantstrains,81%(22/27)and78%(21/27),respectively, followedbystreptomycin(74%–20/27)(Fig.1).
Thecomparisonbetweenisolates’ resistancesand cock-atieloriginshowedthatbirdsfrompetshopandcommercial breeders were predominantly resistant to penicillins, 89% and100%,respectively,whileprivateresidencesisolateswere mostlyresistanttoaminoglycosides(100%)(Fig.2).
Table1showsthat30%(8/27)oftheisolateswereresistant
to seven or more of the tested antibacterial drugs. Resis-tancetooneormoreantimicrobialcategorieswasobserved in 67% (18/27) of the strains, while 59% (16/27) of them weremultiresistant(Table1).Themostfrequentlyobserved multiresistance profile was a combination of penicillins, cephalosporinsandaminoglycosides(Table1).
Weobservedthat96%(26/27)oftheE.colistrainspresented atleast one ofthe surveyedresistance genes (Table 1). In regardsto antimicrobialcategories,resistancegenesofthe aminoglycosideweredetectedin77% (20/26)ofthestrains, 54% (14/26) of the penicillin, 35% (9/26) of the tetracyclin and sulfonamide, and 4% (1/26) of the quinoloneisolates. Cephalosporin-relatedresistancegeneswerenotobserved.
Themostfrequentlydetectedgenesinthestudiedstrains werestrAB(17/26–65%)andblaTEM(14/26–54%).Themost var-iedresistancegenotypeprofilewastheblaTEMtetBaadaphaA
sul3,presentinthreestrainsisolatedfromonesingleanimal thatbelongedtoacommercialbreeder(Table1).
ThePBRT techniqueallowed usto identifyand classify plasmidsin74%(20/27)oftheE.coliisolates.TheIncFIBwas themostrecurrentInc,presentin67%(18/27)ofthestrains, followedbyIncI1(4/27–15%),IncFIA(3/27–11%)and IncY (2/27–7%).Fivesamplespresentedmorethan oneplasmid Incgroup.
Discussion
Dataon Salmonellaspp. epidemiologyinwildand domestic animalsareveryimportantinthedetectionoftheseagents’
potentialreservoirs.41Wedidnotisolatebacteriafrom this genusinthisstudy–asuggestionthatthestudiedcockatiels didnotpresentanyrisksoftransmittingsuchpathogensto humans oranimals, aspreviouslyobservedinastudy per-formedinpsittacines.41
Salmonellaspp.canbeconsideredoneofthemost impor-tant pathogen in commercial poultry industry, and the presenceofthesemicroorganismsisassociatedwith inten-sive breeding.At the same time, previous studies show a lowprevalenceofSalmonellaspp.inwildbirds,andmostof the reports are related to the illegal wildlife traded.7,29 In thisstudy,Salmonellaspp.wasnotdetectedinbirdsfrompet shop,privateresidencesorcommercialbreeders.However,the zoonoticimportanceofthisagentjustifiesitsmonitoringin aviaryspecies.
Oneofthefirstsurveystudies,performedin125psittacines of12differentspecies,detectedE.coliin14%ofthebirds42– resultssimilartoourfindings.However,laterstudies men-tionedhigherisolationpercentages,upto48%.41
Inthisresearch,wedidnotobserveanydifferencesinthe isolationofE.coliregardstoorigin (privateresidencies,pet shopsor commercialbreeders),concluding thatthe people handlingthesebirdswereexposedtosimilarrisks,regardless ofthebirds’origin.
Herein wetested avianvirulencepredictinggenes in27 strains:three(11%)isolatesfromcockatielslivinginprivate residencies presentedthe iss gene, two ofthem with iroN, ompTandhlyFgenes,allcharacteristicoftheAPECsubgroup. Therefore,althoughthesebirdswereapparentlyhealthy,they carriedpotentiallypathogenicstrains.
Studieshaveshowntheexistenceofgenotypicand phe-notypic similarities between avian extraintestinal E. coli
strains (APEC), urinary infections (UPEC),4,43 and neonatal meningitis.3Suchresultsreinforcethehypothesisthatbirds maybereservoirsofE.colipathogenictomammals.3,4,43Thus, itispossibletosuggestthepotentialtransmissionriskofthese zoonoticdiseasestotheownersandcaretakersoftheanalyzed birds.
Eventhoughotherresearchershavedetectedtheeae44and
stx245genesinpsittacinefecalsamples,suggestingthatthese birdscouldbereservoirsofEPECandSTECtohumans,wedid notobservevirulencegenesforthediarrheagenicE.coliinthis survey.
Allstrainsevaluatedinthisstudywereresistanttoatleast oneofthetestedantimicrobialscategories,exceptfor nitrofu-rantoin,similarlytowhatwasobservedinastudyperformed inpsittacines from aconservationist breeder.41 Ourresults showed that 30% of the strainswere resistant toseven or moreantimicrobialdrugsand59%weremultiresistant.E.coli
resistancetotwoormoreantimicrobialgroupsiscurrently consideredacommonfinding,bothinhumanandveterinary medicine.27,33Thisrepresentsagreatimpactoverviable ther-apeuticoptionsandpotentialdispersionofthesepathogensin thecommunity,oneofthemostrelevantglobalpublichealth issues.27
Thehighestresistancepercentagewasrelatedto-lactams (93%),ofwhich89%ofthestrainswereresistanttopenicillin. Severalauthorshavealsoobservedahighpercentageof peni-cillinresistance,upto100%,inpsittacinesandpasseriforms.9
b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 S (2 0 1 8) 76–82
79
Phenotype Genotype Plasmids
1 Petshop 1 – EST strAB IncFIB
2 – CFE;EST;GEN strAB –
3a – AMO;CFE;CFO;EST;GEN;TET;ENO strAB IncFIB
2 Privateresidence 1 – AMP;CTF;EST;NAL strAB IncFIB;IncFIA
2a – AMP;EST;TET;CIP strAB IncFIB;IncFIA;IncY
3a – CTF;EST;CIP strAB IncFIB;IncFIA;IncY
3 Commercialbreeding 1a – AMP;AMO;EST;TET;CLO;ENO;CIP;NAL blaTEMtetBstrAB IncFIB
2a – AMP;AMO;EST;TET;CLO;ENO;CIP;NAL blaTEMtetBstrAB IncI1
3a – AMP;AMO;EST;TET;CLO;ENO;NAL blaTEMtetBstrAB IncFIB
4 Commercialbreeding 1a – AMP;AMO;EST;TET;CLO;ENO;CIP;NAL;SUT blaTEMtetBaadA;aphaAsul3 –
2a – AMP;AMO;CFE;EST;TET;CLO;ENO;CIP;NAL;SUT blaTEMtetBaadA;aphaAsul3 –
3a – AMP;AMO;CFE;EST;TET;CLO;ENO;CIP;NAL;SUT blaTEMtetBaadA;aphaAsul3 –
5 Commercialbreeding 1a – AMP;AMO;CFE;CTF;EST blaTEMstrAB IncFIB
2 – AMP;AMO;CFE blaTEMstrAB IncFIB
3 – AMP;AMO;EST blaTEM IncFIB
6 Commercialbreeding 1a – AMO;CFE;EST blaTEMstrAB IncFIB
2a – AMO;IPM;EST blaTEMstrAB IncFIB
3a – AMP;AMO;IPM;EST blaTEMstrAB IncFIB
7 Commercialbreeding 1 – AMP;AMO blaTEM IncFIB
2 – AMP;AMO;EST blaTEMstrAB IncFIB
3 – AMP;AMO - IncFIB
8 Privateresidence 1a – AMP;AMO;EST;TET;SUT tetBstrAB;aadAsul2 IncFIB;IncI1
2a ironN,ompT,hlyFandiss AMP;AMO;CFE;TET;CLO;ENO;CIP;NAL;SUT tetAsul1 IncI1
3a ironN,ompT,hlyFandiss AMP;AMO;EST;TET;SUT tetBstrAB;aadAsul2 IncFIB;IncI1
9 Privateresidence 1 iss AMP;AMO;SUT sul1 –
2 – AMP;AMO;SUT sul1 –
3 – AMP;AMO;SUT sul1 –
AMO,amoxicillin;AMP,ampicillin;CFE,cephalexin;CFO,cefoxitin;CTF,ceftiofur;IPM,imipenem;EST,streptomycin;GEN,gentamicin;TET,tetracyclin;CIP,ciprofloxacin;ENO,enrofloxacin;NAL, nalidixicacid;SUT,cotrimoxazol;CLO,chloramphenicol.
80
brazilian journal of microbiology49S(2018)76–82 SUT NIT CLO NAL CIP ENO GEN EST TET CFO CTF CFE IPM AMP AMO 0% 10% 20% 30% 40% 50% 70% 80% 90% 81% 78% 30% 41% 74% 7% 11% 4% 7% 30% 30% 30% 26% 0% 33% 100% 60%Fig.1–ResistantEscherichiacolistrainsisolatedfromcockatielsaccordingtothetestedantimicrobialdrugs.SUT, cotrimoxazol;NIT,nitrofurantoin;CLO,chloramphenicol;NAL,nalidixicacid;CIP,ciprofloxacin;ENO,enrofloxacin;GEN, gentamicin;EST,streptomycin;TET,tetracyclin;CFO,cefoxitin;CTF,ceftiofur;CFE,cephalexin;IPM,imipenem;AMP, ampicillin;AMO,amoxicillin.
00% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Private residencies Pet shop Commercial breeders
Penicillin Cephalosporins Tetracyclin Aminoglycosides Quinolones Anfenicol sulfonamides
Fig.2–Escherichiacoliresistantstrainsaccordingtocockatielorigin(petshop,privateresidency,commercialbreeder).
Weverifiedthat52%ofthestrainspresentedtheblaTEM,28 sug-geststhatupto90%ofE.coliampicillinresistanceisdueto TEM-1andTEM-2enzymescodedbytheblaTEMgene.
The isolates presented increased resistance to amino-glycosides(74%),particularlytostreptomycin(67%).Similar resistancelevelshavebeenobservedinwildbirds(63%).46We detectedthat85%ofthestreptomycin-resistantstrains pre-sented the strABgene,justifyingthe high resistancelevels noticedforthisdrug.
Similarly, the tetB gene was detected in 80% of the tetracycline-resistantstrains.Researchershaveobtainedhigh frequenciesoftetracyclineresistanceinE.colistrainsof ani-maloriginandtetBgenehasalsobeenthemostcommonly reportedgeneinhumanisolates.47
InastudyperformedinBrazil,E.colistrainsisolatedfrom wild frigates presented low ciprofloxacin and enrofloxacin
resistance indexes.11 However, the strains isolated in our studypresented41%resistancetoquinolones.Thishigh resis-tancepercentagemayberelatedtoselectivepressure,aresult ofveterinary therapiesestablishedwithno laboratory sup-portandempiricaltreatmentswithnoveterinarysupervision. Thisfactorisespeciallyrelevantwhenoneconsidersthe com-mercialformulationofthisantimicrobialcategory,whichis focusedontheavianpetmarketandcommercializedwithout anygovernmentalcontrol.
Inthisstudy,33%ofthesulfonamide-resistantE.colistrains presentedthesul1,sul2andsul3genes,frequentlyreportedin resistantisolatesofhumanorigin.48
IncreasedGram-negativeresistanceismainlyattributedto mobilegenespresentinplasmids,whichmaybedisseminated withinbacterialpopulations.40,49 Airtravels,human migra-tionsandanimaltransitallowrapidtransportationofbacterial
plasmids among countries and continents. Four plasmids weredetectedinthisstudy:IncFIB,IncFIA,IncYandIncI1.The FIBgroupwasobservedinstrainsthatpresented phenotypi-calresistanceto-lactams,aminoglycosidesandquinolones. Ourfindingsareinaccordancewiththeavailable bibliogra-phy,whichstatesthatplasmidsoftheIncFfamilyarebroadly distributed inE. coli commensals,but carry quinoloneand aminoglycosideresistancegenesandESBLencodinggenes.40 StrainspresentingplasmidIncI1werephenotypically resis-tanttopenicillins,andoneofthemtocephalexin.Plasmids IncI1and IncY are also related tothe distribution ofESBL acquisitiongenes andquinoloneresistance.40 Furthermore, IncI1ischaracterizedbyencodingthetypeIVpili,avirulence factor that contributes tobacterial adhesion and invasion. ThisvirulencecharacteristichasbeenrelatedtoShigatoxin producingE.coli(STEC)40andtothehighlypathogenicAPEC strains,50 which may contribute to the pathogenic poten-tial presentedby the APECstrains with virulence markers detectedinthisstudy.
Weobservedhighantimicrobialresistanceinthestrains isolatedfromhealthycaptivecockatiels,including multiresis-tance,aswell,wedetectedthepresenceofplasmidsandgenes relatedtoresistancephenotype.E.colistrainswithpathogenic potentialpresentedimportantAPECvirulencefactors.Froma zoonoticpointofviewitisimportanttohighlightthe rele-vanceofmaintainingpetsanddisseminatingthesebacteria tootheranimals,tohumans,andtheenvironment.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
FabianaT.Konno,SuzanaM.BezerraandCleideM.daSilva Santanaprovided technicalassistanceduringthe research. CAPES-Coordenac¸ãodeAperfeic¸oamentodePessoaldeNível SuperiorprovidedascholarshiptoPatriciaSilveiradePontes. Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectors.
Appendix
A.
Supplementary
data
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2018.05.003.
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