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
eaDepartmentofVeterinaryMedicine,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
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t
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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/).
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.
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
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.RNAextractionwasperformedusing150Lofswab samples,inwhichwereadded700LofNucliSENSeasyMAG Lysisbuffer(NucliSENSIsokit–Biomerieux).After10minof reactionatroomtemperature, 30Lofmagneticsilicawas 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
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
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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