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Survey of pathogens in threatened wild red-tailed Amazon parrot (Amazona brasiliensis) nestlings in Rasa Island, Brazil

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

Veterinary

Microbiology

Survey

of

pathogens

in

threatened

wild

red-tailed

Amazon

parrot

(Amazona

brasiliensis)

nestlings

in

Rasa

Island,

Brazil

Frederico

Fontanelli

Vaz

a,∗

,

Patrícia

Pereira

Serafini

b

,

Rosangela

Locatelli-Dittrich

a

,

Rafael

Meurer

b

,

Edison

Luiz

Durigon

c

,

Jansen

de

Araújo

c

,

Luciano

Matsumiya

Thomazelli

c

,

Tatiana

Ometto

c

,

Elenise

Angelotti

Bastos

Sipinski

d

,

Rafael

Meirelles

Sezerban

d

,

Maria

Cecília

Abbud

d

,

Tânia

Freitas

Raso

e

aDepartmentofVeterinaryMedicine,FederalUniversityofParaná,Curitiba,Paraná,Brazil

bNationalCenterforBirdConservationandResearch,ChicoMendesInstituteforBiodiversityConservation,Estac¸ãoEcológicadeCarijós,

Florianópolis,SantaCatarina,Brazil

cDepartmentofMicrobiology,BiomedicalSciencesInstitute,UniversityofSãoPaulo,SãoPaulo,Brazil dSocietyforWildlifeResearchandEnvironmentalEducation,Curitiba,Paraná,Brazil

eDepartmentofPathology,SchoolofVeterinaryMedicineandAnimalScience,UniversityofSãoPaulo,SãoPaulo,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4April2016 Accepted23March2017 Availableonline3June2017 AssociateEditor:JoãoAraujoJr.

Keywords:

Avianinfluenzavirus

Chlamydiapsittaci

Microbiology Newcastledisease WestNilevirus

a

b

s

t

r

a

c

t

Thered-tailedAmazonparrot(Amazonabrasiliensis)isathreatenedspeciesofpsittacinebird thatinhabitcoastalregionsofBrazil.Inviewofthethreatofthisspecies,theaimofthis studywastoperformahealthevaluationinwildnestlingsinRasaIsland,determiningthe prevalenceofenterobacteriaandinfectiousagentsaccordingtotypeofnest.Bloodsamples werecollectedfrom64birdsandevaluatedforantibodiesofChlamydiapsittaciby commer-cialdot-blotELISA.Cloacalandoropharyngealswabssampleswerecollectedfrom23birds fromartificialwoodennests,15birdsfromPVCnestsand2birdsfromnaturalnestsfor microbiologicalanalysis.Swabsampleswerecollectedfrom58parrotsforC.psittaci detec-tionbyPCRandfrom50nestlingsforAvianInfluenza,NewcastleDiseaseandWestNile viruses’detectionanalysisbyreal-timeRT-PCR.Tenbacterialgeneraand17specieswere identified,andthemostprevalentwereEscherichiacoliandKlebsiellaoxytoca.Therewasno influenceofthetypeofnestinthenestlings’microbiota.AllsamplestestedbyELISAand PCRwerenegative.Thereiscurrentlyinsufficientinformationavailableaboutthehealthof

A.brasiliensisanddataofthisstudyprovideareferencepointforfutureevaluationsandaid inconservationplans.

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

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

Correspondingauthor.

E-mail:fredfontanelli@yahoo.com.br(F.F.Vaz).

http://dx.doi.org/10.1016/j.bjm.2017.03.004

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

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driverofpopulationdeclines.2Moreover,wildbirdsmaycarry

orbeinfectedwithmicroorganismsthatcanaffecttheirhealth andthehealthoflivestock,petanimalsandhumanbeings.3

Thered-tailedAmazonparrot(Amazonabrasiliensis)islisted asvulnerable according to the International Union forthe ConservationofNatureRedListofThreatenedSpecies.4This

speciesisendemictotheAtlanticForest,inhabiting south-erncoastalregionsofSãoPaulo,ParanáandnorthernSanta Catarinainanarrowcoastalstrip.5Breedingareasaremostly

locatedinsmallestuarineislands,andthesefactsmakethe parrotespeciallyvulnerabletoenvironmentaldisturbance.6

Since1997,theSocietyforWildlifeResearchand Environ-mentalEducation(SPVS)hasdevelopedthered-tailedAmazon ConservationProjectinanattempttominimizepopulation decline.7Environmentalmodificationbyhumanactivitiesin

RasaIslanddestroyedmanynaturalnestsandthelackoftree cavities canlimitthe populationgrowth ofparrots. Oneof theeffortsofSPVSistobuildartificialnestsmadefromwood (Fig.1A)andpolyvinylchloride(PVC)(Fig.1B),andinstallthem nexttothelostnaturalnestsinlargenativetreesthathavethe potentialtobecomefuturenaturalsheltersalongthecoastof Paraná.Since2003,theartificialnestshavebeenwidelyused bytheparrotsforbreeding.8

Inthecaseofthethreatenedred-tailedAmazon popula-tion,ahealthstudyisessentialtoaddressdiseaseriskandto establishnormalparameterspreviouslyunavailable,assisting theconservationofthisspecies.9Itwouldbealsoimportant

todifferentiatetheparrots’microbiotainthedifferenttypes ofnests,sincetheaviangastrointestinalmicrobiota canbe affectedbyenvironment.10Nomicrobiologicalstudies

com-paringwildbirdsfromdifferentnestsundersameconditions areavailableintheliteratureforparrotspecies.

Afew studies reporting health statusofwild psittacine species in southern Brazil have been described in the literature.9,11Chlamydiosisisoneofthemaininfectious

dis-easesfortheorderPsittaciformesbecausethisgrouphasthe highestpositivityrate(45%)amongbirds.12 Avianinfluenza

virus (AIV), Newcastle disease virus (NDV),and West Nile virus(WNV) arethreeofthemostimportantviraldiseases inindustrialavicultureandhaveasignificantburdento pub-lichealth.13–15Besidesholdingseveralbirdmigratoryroutes,

Brazil is considered free from high pathogenic viruses of theseviral diseases,andmonitoring wild birdsisessential toanalyzeviruscirculation,becausetheriskofintroduction is always present. Until now, wild red-tailed Amazon par-rotpopulationsinBrazilhadneverbeenevaluatedforsuch viruses.

Inviewofthelimitedinformationavailableonthespecies, theaimofthisstudywastoperformahealthevaluationin wildA.brasiliensisnestlingsinRasaIsland,Paraná,Brazil,by assessingthecloacalandoropharyngealmicrobiotaaccording totypeofnest;andbyevaluatingthepresenceofChlamydia psittaci,AIV,NDV-1and-2andWNV.

University of the State of Paraná, Southern Brazil (proto-col number 050/2013)and bythe SISBIO(number 41035-1). SamplecollectionwasperformedinRasaIsland, locatedin theEnvironmentalProtectionAreaofGuaraquec¸aba,Paraná, Brazil (Fig.2).Itisaprotected areawithanextensive area ofAtlanticforest,consistingofestuaries,islands,mangrove forests, plains, mountains and plateaus where the parrot breeds.

Sampling

SamplecollectionofA.brasiliensiswascarriedout concomi-tantlywiththemonitoringofnestlingsduringthe2013/2014 breedingseasoninfivefieldexpeditions(Decemberto Febru-ary). Thenests on the trees were accessed using climbing equipmentandladders.Thebirdsweretakenfromthenest andcarefullysampled.Theagewasestimated,thenestlings wereweighed,andimmediatelyputbackintotheirnests.

Blood samples were collected from nestlings from the superficialulnar veinusing1mLsterilesyringespretreated with1000IUsodiumheparinforantibodytest.Thesamples wereplacedintubesandrefrigeratedforupto24h.Thetubes werecentrifugedfor5mintoobtainplasma,whichwasfrozen at−40◦CuntilanalysisforC.psittaci.

Cloacaland oropharyngealswabsampleswere collected fromnestlingsformicrobiologicalanalysisandkept refriger-atedinStuarttransportmediumupto48huntilanalysisin thelaboratory.Cloacalandoropharyngealswabsampleswere collectedforC.psittacidetectionbyPCRinmicrotubes contain-ing1.0mLofPBSpH7.4,andforAIV,NDVandWNVdetection byrealtimeRT-PCR(rRT-PCR)in2mLmicrotubescontaining antifungals, antibioticsand glycerol.All sampleswerekept frozenat−40◦Cuntilanalysisinthelaboratory.

Microbiologicalanalyses

Sampleswereplatedonbrainheartinfusionandincubatedfor 24hat37◦C.Subsequently,theywerestoredin30%glycerol solution at−20◦C.Whenthawed,the sampleswere

inocu-lated bystreakingmethod onblood and MacConkey’s agar mediaandincubatedat37◦Cfor24hforthebacteriagrow. Later,theculturedbacteriawere pickedupforthe prepara-tionofsmear,stainedwithGram’sstainandexaminedunder microscope forstainingand morphologicalcharacterization oftheisolates.Catalasetestwerealsoperformedinthe cul-turedbacteria.Eachpurecolonywasinoculatedintriplesugar ironagar bystabbingthroughthe centerofthe mediumto thebottomofthetubeandthenstreakingthesurfaceofthe agarslantandincubatedat37◦Cfor24h,fordetecting carbo-hydratefermentationandproductionofH2Sandgas.Finally,

thefermentingcolonieswereinoculatedinanenterobacteria kit(Newprov–Pinhais,Paraná,Brazil)forbiochemicaltestsat 37◦C,examinedat24and48hofincubation.

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Fig.1–Red-tailedAmazonparrotinawooden(A)andpolyvinylchloride(B)nestinRasaIsland,Paraná,Brazil.

The kit provides the following biochemical tests: tryp-tophan deaminase reaction, hydrogen sulfide production, glucose fermentation, gas production from glucose, l-Lysine decarboxylation, indole production, decarboxylation

of ornithine, motility,citrate as the sole source ofcarbon, and rhamnosefermentation. Identificationof bacteria was performedaccordingtomanufacturer’sinstructionsandthe literature.16

Fig.2–MapoftheEnvironmentalProtectionAreaofGuaraquec¸aba(whiteborder)inthestateofParaná,Brazil.RasaIsland inthereddot.Figureadapted.37

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ConventionalPCR

C.psittaciDNAanalysisbyconventionalPCRwasperformed. Swab samples were vortexed for 2min and centrifuged at 20,000gfor30minat8◦C.Thesupernatantwasdiscardedand DNAextractionwasperformedusingaDNApurificationkit (NucleoSpinTissue–Macherey-Nagel,Germany)accordingto manufacturer’sinstruction.

Thecyclingwasperformedwithaprogramof5minat94◦C; 40cyclesat94◦Cfor1min,50◦Cfor1minand72◦Cfor2min; andafinalextensionat72◦Cfor10min,producinga300-bp fragment.17Positiveandnegativecontrolswereincludedand

thePCRproductswereanalyzedbyelectrophoresison1.5% agarosegelsstainedwithGelRed(Biotium).

Real-timeRT-PCR

OnesteprRT-PCRwascarriedout fordetectionofAIV,NDV andWNV.RNAextractionwasperformedusing150␮Lofswab samples,inwhichwereadded700␮LofNucliSENSeasyMAG Lysisbuffer(NucliSENSIsokit–Biomerieux).After10minof reactionatroomtemperature, 30␮Lofmagneticsilicawas addedinthesolution,andthewholeextractionprocesswas carried out in automated equipment (NucliSENS Iso kit – Biomerieux)accordingtomanufacturer’sinstructions. Ampli-ficationreactionsforAIVdetectionanddetectionofclassIand IINDVusingamultiplexrRT-PCRassaywereperformed.13,15

SpecificprimersandprobeswereusedforWNVdetection.14

Thecycling was performed inan ABI 7300 PCRSystem (Applied Biosystems) with a program of 45◦C for 20min (reversetranscription);95◦Cfor10min;40cyclesof95◦Cfor 15sand60◦Cfor45s.

Statisticalanalysis

AnANOVAanalysis(atP<0.05)wascarriedouttodetermine theinfluenceofthetypeofnestonprevalenceofbacteriain thenestlingsbyusingPortalActionsoftware.Alldatawere verifiedfornormaldistributionbytheShapiro–Wilktest.

Results

Atotalof74nestlingsweresampledfrom38artificialnests (21woodenand17PVC)andtwonaturalnests(representing 5%ofthetotal).The74birdshadestimatedagebetween25 and56days(averaging42days)andweighedbetween275and 540g(averaging420g).

Cloacalandoropharyngeal swabsampleswere collected from40nestlingsformicrobiologicalanalysis:23individuals from12artificialwoodennests,15parrotsfrom10PVCnests andtwoparrotsfromtwonaturalnests.Anaverageoftwo nestlingspernestwasobserved.Inthesamenest,threebirds

One hundred eighteen bacterial colonies were isolated from73samplesof40nestlings,totaling91.25%(73/80)of pos-itivesamplesforenterobacteria.Onespeciesofgram-positive bacteria, Staphylococcus sp., was isolated from two parrots in artificial woodennests.Colonies were notisolated from four cloacalsamplesofparrots inPVCnestsandfrom two oropharyngealsamplesandonecloacalsampleofparrotsin artificialwoodennests.

Theprevalenceofbacterialspeciesobtainedfromcloacal and oropharyngeal samples according to the nest can be observedinTable1.Therewasnoinfluenceofthetypeof artifi-cialnestinthebirds’microbiota(P<0.05).Thenestlingsinthe naturalnestswerenotcomparedbecauseofthesmallnumber ofsamples.

AllsamplestestedforantibodydetectionandbyPCRforC. psittaciandrRT-PCRforAIV,NDVandWNVwerenegative.

Discussion

Thisstudyprovideshealthbaselineparametersforthe red-tailed Amazon parrot in the state of Paraná, Brazil. The results indicate that the microbiota of wild A. brasiliensis

nestlings typically havegram-negative bacteria inits com-position.Someliteraturestatesthatnormalbacterialfloraof parrotsiscomposedpredominantlybygram-positivebacilli,18

butthenormalityandsignificanceofthepresenceof gram-negativebacteriaisdivergent,sincesomestudiesindicatethat theirpresencedoesnotimplydisease.10,11

Theaviangastrointestinalmicrobiotacanbeaffectedby manyfactorssuchasdiet,age,environment,antibiotic admin-istration and infection with pathogenic organisms.10 Many

microbiologicalstudiesincludecaptiveadultpsittacinebirds inthesample,18andhighpercentageofgram-positiveandlow

percentageofgram-negativebacteriacanbefound.10Inthe

presentstudy,onlywildparrotnestlingsupto56dayswere sampled,showinghigh prevalenceofgram-negative bacte-ria(91.25%).Youngbirdsarestillestablishingtheirbacterial floraandthediversityofbacterialcommunityincreaseswhen someavianspeciesgrowolder.19Immediatelyafterhatching,

the digestivesystemofbirdsispresumedtobesterileand subsequentlyeitherpassivelyoractivelywillacquire micro-biota from the environment, suchas nesting environment and food.19,20 In the case of parrots, the parents

regurgi-tatefoodtotheiryoung,thuspermittingamodeofvertical microbial transmission.20 The influence of age (adult and

young)andenvironment(captiveandwild)couldexplainthe difference inthe diversityofbacterialcommunitybetween thewild nestlingssampledinthepresentstudyand others researches.

InBrazil,thedetectionofgram-negativebacteriahasbeen reported in asymptomatic wild A. brasiliensis nestlings in Paraná and São Paulo coast.11,21 In São Paulo coast, swab

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Table1–PrevalenceofEnterobacteriaceaeisolatedfromcloacalandoropharyngealswabsamplesofwildred-tailed Amazonparrot(Amazonabrasiliensis)nestlingsinRasaIsland,Paraná,Brazil,accordingtothenest.

Bacterialspecies Prevalenceinnestlings fromartificialwoodennest

Prevalenceinnestlings fromPVCnest

Prevalenceinnestlings fromnaturalnest Cloacal samples Oropharyngeal samples Cloacal samples Oropharyngeal samples Cloacal samples Oropharyngeal samples Escherichiacoli 72.7%(16/22) 19.0%(4/21) 72.7%(8/11) 46.7%(7/15) 100.0%(2/2) 100.0%(2/2) Escherichia fergusonii 0 14.3%(3/21) 0 20.0%(3/15) 0 0 Klebsiellaoxytoca 22.7%(5/22) 23.8%(5/21) 18.2%(2/11) 20.0%(3/15) 50.0%(1/2) 100.0%(2/2) Klebsiella pneumoniae 9.1%(2/22) 23.8%(5/21) 0 20.0%(3/15) 50.0%(1/2) 0 Proteusmirabilis 13.6%(3/22) 14.3%(3/21) 0 13.3%(2/15) 0 0 Proteusvulgaris 0 0 9.1%(1/11) 0 0 0 Citrobacter diversus(koseri) 0 0 9.1%(1/11) 0 0 0 Citrobacter werkmanii 0 4.8%(1/21) 0 0 0 0 Citrobacter freundii 0 0 0 6.7%(1/15) 0 0 Enterobacter aerogenes 22.7%(5/22) 19.0%(4/21) 9.1%(1/11) 13.3%(2/15) 0 0 Enterobacter cloacae 4.5%(1/22) 0 18.2%(2/11) 0 0 50.0%(1/2) Enterobacter gergoviae 4.5%(1/22) 0 0 0 0 0 Enterobacter sakazakii 4.5%(1/22) 4.8%(1/21) 0 0 0 0 Serratia liquefaciens (group) 13.6%(3/22) 0 0 13.3%(2/15) 50.0%(1/2) 0 Serratiarubidaea 0 4.8%(1/21) 0 0 0 0 Morganella morgani 0 4.8%(1/21) 9.1%(1/11) 0 0 0 Hafniaalvei 0 4.8%(1/21) 0 6.7%(1/15) 0 0 Staphylococcus sp.* 0 9.5%(2/21) 0 0 0 0 Kluyveraspp. 0 0 0 6.7%(1/15) 0 0 ∗ FamilyStaphylococcaceae.

samplesof14A.brasiliensisshowedpredominanceofE.coliand

Proteusmirabilisinbothcloacalandoropharyngealsamples.21

Microbiologicalanalysesofcloacalswabsampleswithhigh positivity(74.46%) for Enterobacteriaceaewere observed in a previousstudywith19A.brasiliensisfromfourislandsofthe Environmental Protection Area of Guaraquec¸aba, including RasaIsland.11HighprevalenceofE.coli(84.2%)wasobtained

inthisstudy,11andPseudomonasspp.(31.5%),Enterobacterspp.

(26.3%), Proteus vulgaris (26.3%), Citrobacter spp (21.0%) and

Staphylococcusspp.(5.2%)werealsofound.Similarspeciesof bacterialcoloniesobservedinthepresentstudyareconsistent withthefindingsoftheseinvestigations,showingthe normal-ityofgram-negativebacteriainthemicrobiotaofpsittacine species.

All bacteriagenera showedin Table1were observed in previousstudieswithpsittacinespecies.11,21–24These

microor-ganisms probably compose the normal microflora of this avianfamily,andthemicrobiologicalprofileofthenestlings sampled was within normality. Concerning the different bacteriaspecies,toourknowledge,thisisthefirstisolationof

EscherichiafergusoniiandCitrobacterwerkmaniiinapsittacine bird.

Generally,theEnterobacteriaceaeareconsideredsecondary pathogens,butcanfunctionastheprimarypathogenin cer-taincircumstances,dependingonthevirulenceofthebacteria andthehostresponsetoinfections.AmongCitrobacterspecies,

C.freunddiappearstobethemostpathogenicofthegroup,and theotherspeciesarelesscommonlyencounteredinbirdsand donotappeartohavegreathealthrelevance,aswellasE. fergu-sonii,alreadyisolatedfromasymptomaticBrazilianbirds.25,26

ThegeneraEnterobacter,Hafnia,SerratiaandProteusareoflow pathogenicity,andMorganellamorganiiandKluyveraspp.are infrequentopportunisticpathogensinbirds.25Differently,the

speciesE.coli,Klebsiellaoxytoca,K.pneumoniaandStaphylococcus

spp.cancauseprimaryorsecondarydiseaseinavianspecies andhavebeenassociatedwithdiseaseinAmazonparrots.25

Pathogenicityandantimicrobialresistancetestscouldbeused toassesstherealrisksofthesebacteriatothered-tailed Ama-zonparrots’healthinfutureevaluations.

Concerningthenests,theexcretionsofthebirdscan accu-mulateovertime,increasingexposurelevelsofnestlingsto fecalbacteria.Differentmaterialsusedtobuildnestsprobably provide different environments for wild nestlings, increas-ing or decreasing the presenceof microorganisms, but no

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artificialnestsarestillneededintheRasaIsland,sinceitisa smallbreedingarea(10.5km2)fortheparrots,and

deforesta-tionleadstoalackofsufficientnaturalcavities.

Analyses for antibody detection by ELISA and for DNA detectionbyPCRforC.psittaciwerenegative,aswellasthe analysisforRNAdetectionbyrRT-PCRforAIV,NDVandWNV. Theabsence of pathogens such asC. psittaci can likely be justified bythe isolation ofthe population studied, which mostlyuseislandsforbreeding,feedingandnightrest.6On

theotherhand,thisisolationandlimiteddistributionofthe parrotscouldbeconcerning,sinceislandendemicbirds prob-ablyhavebeenexposedtofewpathogens,27andemergence

andintroductionofdiseasescanaffectthepopulation dynam-icsandregulateitsabundance.1Pathogenicorganismshave

beeninvolvedinnumerousdeclinesofendemicspecieson islands.27Thesefactsreinforcetheimportanceof

establish-ingmonitoringprogramstoallowearlydetectionandprevent furtherspreadofaviandiseases.

Opportunely, the Environmental Protection Area of Guaraquec¸aba is located within the most preserved area of continuous Atlantic forest in Brazil and is the largest conservationunit oftheregion. Muchofthe populationof parrotsisfoundinprotectedareasinParaná,butthelackof supervisionintheregionenablesthecuttingofimportanttree speciesforreproduction,shelterandfeedingoftheparrot.28

Besides this,these birds probably have less anthropogenic interference,less contactwithhumanbeingsand domestic animals, andsuffer lessstress byenvironmentaldisorders, whencomparedtobirdsfromotherareassuchasPantanal, where modifications in the habitat are largely due to the plantingofpasture forcattle.Inthis region, C.psittaciwas detectedin6.3%(2/32)ofwildAmazonaaestivabysemi-nested PCR.29 Environmental conditions are often a basic cause

associatedwithdiseaseemergence,persistence,andspread, andmustbeaddressedtoavoiddisease.1

UnlikeconditionsintheGuaraquec¸abaarea,thered-tailed AmazonparrotstillfacesgreatadversityinthestateofSão Paulo becauseof urbangrowth, deforestation and removal ofnestlingsbyhumansforillegaltrade,andbecauseofthe lackofprotectedareasandlackofsupervision.5Thesefactors

endangerthefutureofthespeciesinthisregionandcanaffect their health. Sera examined from this speciesin the state ofSãoPaulousingdotblotELISAdemonstrated42.85%of14 birdsolderthan10dayspositiveforantibodiestoC.psittaci.21

Butthishighpercentageofpositiveanimalscanalsobedue tomaternalimmunity,whichcanlastupto2–4weeksafter hatch.30These resultsarenotsimilar tothepresent study,

inwhichall64nestlings serologicallyevaluatedwere nega-tiveforC.psittaci.ThisagentisconsideredendemicinBrazil andhasbeenreported incaptive parrotsonoutbreaksand wildprevalencestudies.29,31InthestateofParaná,C.psittaci’s

DNAwasdetectedin1.2%ofthesamplesfrom117free-living

A.brasiliensisnestlingsanalyzedbysemi-nestedPCR,9alow

prevalence,whichisinaccordancewiththepresentstudy.

donotappeartoplayamajorroleintheepidemiologyofthe disease.33Negativeresultsofthepresentstudymayreinforce

thelittleimportanceoftheseagentsforpsittacinespecies. Concerningother countries,similarresultstothis study wereobservedinwildpsittacinebirds.AntibodiestoAIV,NDV andC.psittacihasnotbeenfoundinblue-frontedAmazon par-rots(A.aestiva)inBolivia.34Serologictestinginwildparakeets

(AratingaweddelliiandBrotogerissanctithomae)inPeruwere neg-ativetoantibodiesforC.psittaciandNDV,35andfourspeciesof

wildMexicanparrotsshowednegativeserologictestsforAIV andNDV.36

Theresultsofthisstudyindicatethatthered-tailed Ama-zon populationlocated inRasaIsland isnotinfectedbyC. psittaci,AIV,WNVorNDV,andthedataobtainedissupported bypreviousstudiesinvolvingwildpsittacinespecies. Concern-ingthebacteriaobservedmoreinformationandstudiesare necessary forestablishingtheirpotentialas diseaseagents fortheparrots.ThemicrobiotaofwildA.brasiliensisnestlings is predominantly composed of gram-negative bacteria and was not influenced by the type of artificial nest. Unfortu-nately, few naturalnests were found in the study area to compare the microbiota ofnestlings between artificial and naturalnests.Datanotpreviouslydescribedforthespecies was reported here, providing a reference point for future evaluationsandinterpretationsoflaboratoryfindingsin con-servationplans.Healthmonitoringinthreatenedfree-living speciesisanimportanttoolwhenitcomestoavian conserva-tionandshouldbeincreasinglyencouraged.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WegratefullyacknowledgetheSocietyforWildlifeResearch and EnvironmentalEducation(SPVS)forlogisticsand tech-nical support in Guaraquec¸aba area and forthe efforts to conserve the red-tailed Amazon parrot. We would like to thank J.V.B. Agottani for the ELISA test provided. Fund-ing forthis project was inpart provided bythe São Paulo ResearchFoundation–FAPESP(2011/13821-7),(2013/05485-2) and(2009/05994-9).

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