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Isolation of Salmonella spp. in cattle egrets (Bubulcus ibis) from Fernando de Noronha Archipelago, 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

Isolation

of

Salmonella

spp.

in

cattle

egrets

(Bubulcus

ibis)

from

Fernando

de

Noronha

Archipelago,

Brazil

Marcio

A.

Silva

a,b,c,∗

,

Érika

F.S.T.

Fernandes

a

,

Sandra

C.

Santana

d

,

Maria

Fernanda

V.

Marvulo

c,e,f

,

Mércia

R.

Barros

a

,

Sineide

M.O.

Vilela

a

,

Eliane

M.F.

Reis

g

,

Rinaldo

A.

Mota

a

,

Jean

C.R.

Silva

a,c

aUniversidadeFederalRuraldePernambuco,DepartamentodeMedicinaVeterinária,Recife,PE,Brazil bParqueEstadualDoisIrmãos,Recife,PE,Brazil

cInstitutoBrasileiroparaMedicinadaConservac¸ão,Recife,PE,Brazil

dAdministrac¸ãodoDistritoEstadualdeFernandodeNoronha,Recife,PE,Brazil eFaculdadeMaxPlanck,Indaiatuba,SP,Brazil

fUniversidadePaulista,Campinas,SP,Brazil

gInstitutoOswaldoCruz,FIOCRUZ-RJ,LaboratóriodeEnterobactérias,RiodeJaneiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4July2017

Accepted14January2018

Availableonline3March2018

AssociateEditor:CristianoGallina

Moreira Keywords: Ardeidae Oceanicislands Conservationmedicine Enterobacteriaceae Publichealth

a

b

s

t

r

a

c

t

Thegrowthofthepopulationofcattleegrets(Bubulcusibis)inthearchipelagoofFernando

deNoronhaconstitutesathreattopublichealthandbiologicaldiversitybecauseoftheir

competitionwithandpredationonnativespeciesandthepossibilityoftransmissionof

pathogenstohumanbeings,livestockandnativewildlife.Theaimherewastosearchfor,

isolateandidentifyserovarsofSalmonellainclinicallyhealthylocalcattleegrets.Cloacal

swabswereobtainedfrom456clinicallyhealthycattleegretsofbothsexesandavarietyof

ages.Theswabsweredividedinto51pools.Sixofthese(11.7%)presentedfourserovarsof

Salmonellaentericasubspeciesenterica:SalmonellaserovarTyphimurium;Salmonellaserovar

Newport;SalmonellaserovarDuisburg;andSalmonellaserovarZega.Onesamplewas

identi-fiedasS.entericasubspeciesentericaO16:y:-.Resultsinthisstudysuggestthatcattleegrets

maybereservoirsofthisagentonFernandodeNoronhaandrepresentarisktopublichealth

andbiologicaldiversity.

©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade

Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

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

Correspondingauthor.

E-mail:[email protected](M.A.Silva).

https://doi.org/10.1016/j.bjm.2018.01.004

1517-8382/©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle

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Introduction

Exoticinvasivespeciesareanimalsand/orplantsthathave

beenintroducedintoaplacewheretheydidnotpreviously

exist naturally. They are one of the biggest threatsto the

environment, causing enormous harm to biodiversity and

tonaturalecosystems.Furthermore,theymaypresentrisks

tohumanhealth,giventhattheymaytransmitdiseases to

endemicspeciesandcauseecologicalimbalances.1,2

FernandodeNoronha isanarchipelagocomposed by21

oceanicislandswith26km2oftotalareathatarepartofthe

BrazilianstateofPernambuco.Theyarelocatedinthe

equa-torialzoneoftheSouthAtlantic(3◦5028.9S,32◦2439.4W),

atadistanceof545kmnortheastwardsfromRecife,thestate

capitalofPernambuco.Themainislandhasaresident

pop-ulationofapproximately2930inhabitantsandisdesignated

asaMarineNationalParkandanEnvironmentalProtection

Area.3,4

Amongtheexoticinvasivespeciesseeninthearchipelago

of Fernando de Noronha, cattle egrets (Bubulcus ibis) have

causedemerging problems.2 Theseincludecompetition for

resources and predation of endemic species; potential for

transmissionofdiseasestothehumanpopulationand

pro-ductionanimals;andpotentialforcollisionswithaircraft.2,5,6

CattleegretsbelongtothefamilyArdeidae,order

Pelecan-iformes.Theyareinsectivorousandarecommonlyobserved

foraginginopenfield areas,closetocattle,andfeedingoff

insectsthatdisturbthecattle.7Thefirstreportsofoccurrences

ofthisspeciesonFernandodeNoronhadatefromthe1980s,

andtherehasbeenexponentialgrowthofthepopulationsince

then.2

Amongthepathogenstransmittedbywildbirds,Salmonella

spp.hastakenonanimportantroleandhasevenbeen

indi-catedasathreattowildlifeconservationingeneralterms.8,9

Salmonellabelongs to the family Enterobacteriaceae; it

col-onizes the intestines of reptiles, birds and mammals and

hasgreatimportanceinrelationtobothhumanandanimal

pathology.9,10 Some serovars like Typhimurium and

New-port arecommonlyassociatedtofood poisoningorenteric

infectionoutbreaksworldwide,beingagreatthreattopublic

health.8–10,24,28,30,32,34

Presence of this genus has been reported in several

bird species and groups, such as domestic poultry,11,12

Psittaciformes,13,14 ratites,15,16 raptors,1 Passeriformes18,19

and Ardeidae.8,10,19–23 Incattle egrets,Salmonellahas

previ-ouslybeenisolatedonlyintheUnitedStates.8,19,23,24

Considering the scarcity of studies on isolation of this

enterobacteriaincattleegrets,theaimherewastosearchfor,

isolateandidentifyserovarsofSalmonellainclinicallyhealthy

birdsinthearchipelagoofFernandodeNoronha,Pernambuco,

Brazil.

Material

and

methods

Studyarea

Thecoveredareaofthecompostingunitofwastetreatment

stationinthemainislandofthearchipelagoofFernandode

Noronhawasusedasthesiteforcatchingcattleegrets.

Capture,physicalrestraint,clinicalexaminationand collectionofbiologicalmaterial

BetweenDecember2007andJuly2008,456cattleegrets(B.ibis)

ofbothsexesandvariousageswerecaught.Thebirdswere

attractedintothecoveredareaofthecompostingplantbythe

presenceofprey(larvaeand insects).They werecontained

there by closing the doors and were physically restrained

byusingnetsandglovessuitablefortheprocedure. Allthe

birds weresubjected toclinicalexaminationatthetimeof

capture.25Aftertheyhadbeenconfirmedasclinicallyhealthy,

sterileswabs(CBProducts,Corumbataí,SãoPaulo,Brasil)were

introducedintothecloacaofeachbird.Thoseswabswere

sub-sequentlykeptunderrefrigerationandsenttothemainland

inStyrofoamboxescontainingrecyclableice,nomorethan

48hafterwards,forlaboratoryprocedurestobeperformed.

Laboratoryprocedures

Thelaboratoryanalysesrelatingtoisolationand

identifica-tion were performed inthe Infectious-Contagious Diseases

Laboratory of the Department of Veterinary Medicine of

theUniversidadeFederalRuraldePernambuco(UFRPE).The

swabs weredivided into pools ofnotmorethan 10 swabs,

inaccordancewiththeorderofcaptureofthecattleegrets,

thus totaling 51 pools. To isolate the agent, the classical

scheme for investigating Salmonella recommended by the

NationalProgramforPoultry-rearingHealth(PNSA)wasused.

This involved use of 1% buffered peptone water (Oxoid®,

Cambridge, England) as a pre-enrichment medium and

tetrathionate (Oxoid®,Cambridge,England)and

Rappaport-Vassiliadis(Acumedia®,Lansing,Miami,USA)asenrichment

broths, with subsequent seeding into brilliant green

(Bio-brás Diagnósticos®, Montes Claros, Minas Gerais, Brasil)

and xylose lysine deoxycholate (XLD) (Oxoid®, Cambridge,

England)selectiveagars.Coloniesthatwerecharacteristicfor

SalmonellawerecrushedintothescreeningmediumTSI(triple

sugarironagar)(Oxoid®,Cambridge,England),whichenabled

presumptiveidentificationofthegenus.26,27

Simplified biochemical profiling was used, consistingof

characterizationoftheisolatesaccordingtotheircapacityfor

decarboxylationoflysine,useofcitrateasthesolesourceof

carbon,mobilityandproductionofhydrogensulfide(H2S)and

indoleinthefollowingmedia:lysineironagar(LIA)(Biobrás

Diagnósticos®,MontesClaros,MinasGerais,Brasil),Simmons’

citrateagar(Oxoid®,Cambridge,England),ureabroth,methyl

redbroth(Oxoid®,Cambridge,England),VogesProskauer(VP)

(BiobrásDiagnósticos®,MontesClaros,MinasGerais,Brasil)

andsulfideindolemotility(SIM)agar(BiobrásDiagnósticos®,

MontesClaros,MinasGerais,Brasil).28

Afterbiochemicalprofilehadbeenconfirmed,thesamples

weresenttotheNationalReferenceLaboratoryfor

Enterobac-terialInfections(LRNEB)ofInstitutoOswaldoCruz,FIOCRUZ,

RiodeJaneiro.Atthislocation,antigencharacterizationwas

performedbasedonserologicalclassificationof

Kauffmann-WhiteandLeMinor,withrepresentationinaccordancewith

thecriteriaofGrimontandWeill.29Alltheantiserausedinthe

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Table1–Salmonellaserovarsisolatesfrom51poolsofcloacalswabsfromcattleegrets(Bubulcusibis)ofarchipelagoof

FernandodeNoronha,Pernambuco,Brazil,2008.

Identificationofpool Numberofcattleegrets Serovar

P10 10 SalmonellaentericasubspeciesentericaO16:y:–NDa

P13 10 SalmonellaentericasubspeciesentericaO16:y:–NDa

P20 10 SalmonellaserovarDuisburg

P42 4 SalmonellaserovarZega

P45 9 SalmonellaserovarNewport

P47 6 SalmonellaserovarTyphimurium

a ND,undetectable(flagellatestructureundetectableafterphaseinduction).

Noneofthecattleegretspresentedanyclinicalsignsofsalmonellosis,suchasruffledfeathers,fever,edemaordiarrhea.Allthebirdspresented weightswithinthenormalpatternforthisspecies,rangingfrom272to543g.

Results

Outofthe51poolsanalyzed,27 (52.9%)presentedcolonies

suggestive of Salmonella spp. Among these 27 pools, after

confirmation through biochemical tests, six (11.7% of the

total number of pools) presented test results compatible

with the genus Salmonella, and all of these were

subse-quentlyconfirmedthroughserotyping(Table1).Presenceof

fivedifferentserovarswasconfirmed:Salmonellaserovar

Duis-burg; Salmonella serovar Zega; Salmonella serovar Newport;

Salmonellaserovar Typhimurium;and onesample thatwas

identifiedasSalmonellaentericasubspeciesentericaO16:y:-.

Discussion

Theabsenceofclinicalsignsofsalmonellosisandthegood

bodyscoresofthecattleegretsthatwerecaughtdemonstrated

thatallofthemwereclinicallyhealthy.Someofthebirdshad

previouslybeenringedaspartofecologicalstudiesconducted

previouslybyprofessionalsattheBrazilianNationalCenterfor

ResearchandConservationofWildBirds(CEMAVE).However,

nopreviousstudiesoninfectiousorparasiticdiseasesinthese

birdsonFernandodeNoronhawerefound.

Most of the studies that have described isolation of

Salmonella in cattle egrets were conducted in the United

States.Intwostudies,thegenuswasidentifiedbut notthe

speciesorserovar.21,23 SerovarsofSalmonellaincattleegrets

were described inthreestudies.8,19,24 Locke atal.8 isolated

SalmonellaserovarTyphimuriumfromtheinternalorgansof

asingle individual cattle egret that had been heldin

cap-tivityanddied ataresearchcenterinMaryland.Thesame

serovarwasalsoisolatedfromotherbirdsinthefamily

Ardei-daethatdiedduringthesameoutbreakofsalmonellosis:little

blueheron(Egrettacaerulea),greategret (Ardeaalba),snowy

egret(Egrettathula),tricoloredheron(Egrettatricolor)and

black-crownednightheron(Nycticoraxnycticorax).Allofthesespecies

alsoformpartofBrazilianfauna.5

Phalenetal.24isolated30differentserovarsofSalmonella

injuvenileandadultcattleegretsthatwereculledfor

popula-tioncontrolpurposesatvariouscollectionpointsinTexas,and

observedthecharacteristics ofthesebirds’ bacteremiaand

death.Callawayetal.19isolatedSalmonellaserovarMontevideo

fromthe cecumofcattleegrets,underisolationconditions

greatlyresemblingthoseofthepresentstudy.Serovars9

iso-latedfromcattleegretsofFernandodeNoronhaseemednotto

causeillnessesinthesebirds,whichcorroboratedthefindings

ofCallawayetal.,19whosestudyincludedclinicallyhealthy

birds.

AmongtheSalmonellaserovarsfoundincattleegretsof

Fer-nando deNoronha,the serovarTyphimurium wastheonly

one similar to those found byPhalen et al.24 inthe same

speciesin TX,UnitedStates.Thus, thepresent report

pro-videsthefirstdescriptionofoccurrencesofserovarsDuisburg,

ZegaandNewportandofonesamplecharacterizedasS.

enter-icasubspeciesentericaO16:y:-,incattleegretsinBrazil,and

particularlyonoceanicislands.

S.enterica subspeciesentericaisfrequently isolatedfrom

poultry, exotic birds, pigs and chicken carcasses, and has

becomeespeciallycommonsincethelatterpartofthe20th

century. TheserovarEnteritidisistheonemostcommonly

encountered.12,30 On the other hand, S.enterica subspecies

entericaserovarO16:y:–hasnotbeen isolatedinother

stud-ies,evenfrompoultry,althoughsomeauthorshavereported

isolationofS.entericasubspeciesentericainassociationwith

otherserovarsinpoultryandlivestock.11,24

Bada-Alambedjiet al.31 isolatedSalmonellaserovar

Duis-burgfromtwoofthe75samplesthatwere positiveforthis

genus,fromchickencarcassesthatwerebeingsoldin

mar-ketsandopenfairsinDakar,Senegal,andotherserovarswere

isolatedfromtheremainingsamples.HoszowskyandWasyl30

isolatedSalmonellaspp.fromseveralpoultryspeciessuchas

chickens,turkeys,ducks,geese,pheasantsandpigeonsand

from exotic birds. However, in comparisonwith the

num-bers of isolations ofother serovars, it was foundthat the

Duisburg serovar had lowfrequency ofoccurrence.Like in

the presentstudy,theabovementionedtwostudiesdidnot

showanyindicationsofillnesscausedbythisserovarinthese

birds.

The prevalenceof Salmonellaserovar Zega inwild birds

seemstobelow,giventhescarcityofstudiesdescribingthis

agent.Evenincasesinvolvinghumans,theprevalenceseems

to below.32 Onthe other hand, the serovarsNewport and

Typhimuriumseemtobemorecommonandtohavegreater

potentialtocauseillnessinanimalsandhumans.8,10,24,28,32

From the point of view of animal health, cattle egrets

habituallycomeinto closeproximitytocattle,sheep,goats

andhorses,6,7whichsuggeststhattheymayserveassources

of infection for humans and livestock.5,6 Several authors

have suggested that contact between synanthropic birds

(4)

forSalmonellato poultryspeciesand consequently totheir

carcasses.24,31,33

Thus,cattleegrets mayperform thisrole ofasourceof

SalmonellainfectiononFernandodeNoronha,withdispersion

ofserovarsisolatedinthisstudytothehumanpopulationand

todomesticandwildanimals,bymeansoffecal

contamina-tionoffoodsourcesornaturalandartificialwaterreservoirs

suchasmillponds,watertanksandcisterns,wherethesebirds

habituallylandtocatchinsectsandsmallamphibians.This

possibility has already been described by other authors in

otherlocalities.5,10,17,25,33,34

Regardingpublichealth,Salmonellaspp.hasbeenshown

to be the main agent of foodborne diseases in Brazil

and worldwide17,24,35 and any of its serovars may be

involvedinoutbreaksoffoodpoisoninginfectionswithmild

symptoms.9,12,17,28,30 The serovarsdescribed inthe present

study have little importance as causative agents for

non-typhoid enteritis in humans, with the exception of the

serovars Typhimurium and Newport, which are commonly

associatedwiththistypeofillness.8–10,24,28,30,32,34

Inrelationtobiodiversityconservation,pathogenic

sam-plesofSalmonellaspp.may presentrisksofhigh morbidity

andmortalityamongwildanimals,9withconsequentlossof

geneticvariabilitywithinpopulations.Thismaygiveriseto

evenmorecriticalproblemsforendangeredspecies,thereby

acceleratingtheextinctionprocess.20HallandSaito10 cited

Ciconiiformesastheorderofbirdsrankedasthirdmost

sus-ceptible todeathdue to salmonellosis.However, regarding

salmonellosisorinfectionbySalmonellaspp.inwildbirds,the

majorityofauthorshavestatedthatthesebirdsaremore

com-monlyasymptomaticcarriers.20,22,25Itshouldbenotedthatin

2014,thefamilyArdeidaewasrelocatedfromtheorder

Ciconi-iformestotheorderPelecaniformes.25

Although the most native marine birds of Fernando

de Noronha are not endangered species, infected cattle

egretsmayhaveanegativeimpactthroughtransmissionof

Salmonellatothesespecies.Throughcompetitionfor

ecologi-calnicheswithnativespecies,presenceofcattleegretsmay

thusincreasethemortalityratesamongthelocalavifauna2,5,6

iftheSalmonellaserovarsthattheycarryhappentocause

dis-easeoutbreaksinthelocalpopulationsofwildbirds.

Even though there have not been any reports of

out-breaks of salmonellosis in the avifauna of Fernando de

Noronha,itwould beprudentnottodismissthis

hypothe-sis, giventhat there isno in-depthknowledge ofthe wild

epidemiological chain of this disease in this locality, and

considering the pathogenic potential of isolates found in

thiswork.8–10,24,28,30,32,34Thus,itispossiblethatcattleegrets

reallyareactingasreservoirsand/ordisseminatorsofthese

pathogensontheseislands.Thedataobtainedinthisstudy

areofrelevanceforpublichealth,sincetheycontributetoward

environmental monitoring through knowledge of the

epi-demiologyofSalmonellaserovarsencountered.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The research was supportedby the Administrationof

Fer-nando de Noronha, state district, Pernambuco state. The

authorsarealsogratefultotheInstitutoChicoMendespara

Conservac¸ão da Biodiversidade (ICMBio), and to the

Labo-ratóriodeEnterobactériasofFIOCRUZ-RJ,forserotypingthe

samples.R.A.Mota,andJ.C.R.Silvareceivedfellowshipsfrom

theNationalResearchCouncil(CNPq),Brazil.

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