First report of a naturally patent infection of Angiostrongylus costaricensis in a dog

VeterinaryParasitology212(2015)431–434

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Short

communication

First

report

of

a

naturally

patent

infection

of

Angiostrongylus

costaricensis

in

a

dog

Alejandro

Alfaro-Alarcón

_,

_Vincenzo

_Veneziano

_,

_Giorgio

_Galiero

_,

_Anna

_Cerrone

_,

Natalia

Gutierrez

_,

_Adriana

_Chinchilla

_,

_Giada

_Annoscia

_,

_Vito

_Colella

_,

Filipe

Dantas-Torres

_,

_Domenico

_Otranto

_,

_Mario

_Santoro

e_{DipartimentodiMedicinaVeterinaria,UniversitàdegliStudidiBari,Valenzano,Bari,Italy}

f_{DepartmentofImmunology,AggeuMagalhãesResearchCentre,OswaldoCruzFoundation,Recife,Pernambuco50670-420,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received15June2015

Receivedinrevisedform4August2015 Accepted14August2015 Keywords: Abdominalangiostrongyliasis Angiostrongylidae Pet Zoonotichelminth Reservoir Molecularanalyses

a

b

s

t

r

a

c

t

Angiostrongyluscostaricensisisthezoonoticagentofabdominalangiostrongyliasisinseveralcountries inNorthandSouthAmerica.RodentsarerecognizedasthemaindefinitivehostsofA.costaricensis,but otherwildlifespeciescandeveloppatentinfections.Although,severalhumancaseshavebeendescribed intheliterature,theroleofdomesticanimalsintheepidemiologyoftheinfectionisnotclear.Herewe reviewtheliteratureavailableonA.costaricensisinmammalsanddescribethefirstconfirmedfatalcase ofabdominalangiostrongyliasisina4-month-olddog,presentedwithintestinalperforation,peritonitis andfaecalsheddingoffirst-stagelarvae.Parasiteidentitywasconfirmedbymorphology,histologyand molecularcharacterizationoftargetgenes.Thisisthefirstrecordofanaturallyinfecteddogactingasa definitivehostforA.costaricensis.Thesedatasuggestthatdogsmaypotentiallyspreadthisparasitein urbanizedareas.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction

The genus Angiostrongylus (Strongylida, Angiostrongylidae) includes at least 21 species of nematodes infecting the lungs and blood vessels of a variety of mammals including insecti-vores,rodents,felidsandcanids(Anderson,2000;Spratt,2015). Angiostrongyluscostaricensis is thezoonotic agentof abdominal angiostrongyliasis,which wasoriginally described in1971 in a humanpatientfromCostaRica(MoreraandCéspedes,1971).Later, A.costaricensiswasreportedinseveralcountriesoftheAmericas, fromtheUnitedStatestoArgentina,withthedescriptionofnearly 200humancasesoccurringwithinthisgeographicalrange (Graeff-Teixeiraetal.,1991;Penaetal.,1995;Romero-Alegríaetal.,2014). HumaninfectionshavealsobeenreportedfromAfrica(Zaire)and Europe(FranceandSpain)(Romero-Alegríaetal.,2014).

Adult nematodes of A. costaricensis residein the mesenteric arteriesofrodents,wherefemaleslaytheireggs,whichhatchinthe

∗ Correspondingauthor.

E-mailaddress:[email protected](M.Santoro).

intestine,andfirst-stagelarvae(L1)arepassedintheenvironment withthefaeces(Anderson,2000).Tocontinuethelife-cycle,the L1needtobeingestedoractivelypenetratethetissueofan inter-mediatehostwheretheymoulttwicetothethird-stagelarvae(L3), whichistheinfectivestageforthedefinitivehosts(Thiengo,1996).

Inthedefinitivehost,L3followtwoprincipalmigratoryroutesafter infection:(i)throughthelymphatic/venous–arterialsystem,or(ii) throughthevenousportalsystem(MotaandLenzi,1995,2005; Spratt,2015).

InCostaRicathemostimportantintermediatehostofA. costari-censisistheveronicellidslugVaginulusplebeius(MoreraandAsh, 1970;Morera,1973;Moreraetal.,1988),while,atleast12rodent specieshave beenimplicatedasdefinitivehosts (Morera,1973; Teshetal.,1973),withthecottonrat(Sigmodonhispidus)beingthe mostwidespread(MoreraandCepsedes,1971).Humansacquire theinfection viatheconsumption of infected molluscs(mainly slugsofthefamilyVeronicellidae),orvegetablescontaminatedby L3releasedintheirslimetrails(Ubelakeretal.,1980).However, theriskofhumanstobeinfectedbyL3releasedinthe environ-ment has been considered of minor importance (Cowie, 2013) althoughthesnail-to-snailtransmissionofL3hasbeenreported http://dx.doi.org/10.1016/j.vetpar.2015.08.016

432 A.Alfaro-Alarcónetal./VeterinaryParasitology212(2015)431–434 asasurvival strategyfor somenematodes (i.e.,Aelurostrongylus

abstrusus)inthesamefamilyofA.costaricensis(Colellaetal.,2015). InspiteofthezoonoticrelevanceofA.costaricensis,humans rep-resenta dead-end host for this parasite,which does notreach adulthood.In addition, A. costaricensis hasbeen reported para-sitizingzoo-bornsiamangs(Hylobatessyndacttylus),nightmonkeys (Aotusnancymaae),free-rangingraccoons(Procyonlotor)and opos-sums(Didelphisvirginiana)intheUnitedStates(Milleretal.,2006), although,faecalsheddingofL1wasnotdemonstratedforanyof thesespecies.However,viableL1areshedbynaturallyinfected white-nosedcoati(Nasuanarica)(Mongeetal.,1978)andby cap-tivenon-humanprimates(Slyetal.,1982;BrackandSchöpel,1995; Berrocaletal.,1997).Arroyoetal.(1998)describedafatalcaseof abdominalangiostrongyliasisinadog,presumptivelyinfectedby A.costaricensis,butwithoutanyparasitologicaldescriptions.Later on,anexperimentalinfectionstudysuggestedthatadultdogsmay besuitablereservoirhostsinthedomesticenvironment(Rodriguez etal.,2002),representingapotentialriskofinfectionforhumans.

Here we describe a fatal case of canine abdominal angiostrongyliasis caused by A. costaricensis and report, for the first time, the faecal shedding of A. costaricensis L1 in a naturallyinfecteddog.Clinical,haematologicalandpathological data are also reported along with the molecular characteriza-tionof A.costaricensis specimens collectedduring post-mortem examinationofthesameanimal.

2. Clinicalpresentationanddiagnosticprocedures

OnNovember2014a4-month-oldFrenchpoodlemale weigh-ing1.1kgandboughtinapetshopinSanJosé(CostaRica)was hospitalizedinaprivateveterinaryclinicwithahistoryof7days ofhemorrhagicdiarrhoeaandoccasional vomitussincethefirst dayofitspurchase.Thedogwascachectic,dehydrated,anaemic andintermittentlylethargic. Ithad alargeabdomenwithacute abdominalpainandnofever.Atthefaecalexaminationthe ani-malscorednegativeforhelmintheggs(bydirectfaecalsmearand flotationmethods).Haematology confirmedanaemia(redblood cells:2.24M/␮L;haematocrit:15.5%;haemoglobin:5.1gr/dL),and moderate neutropenia (2.5K/␮L). Blood biochemistry revealed hypoglycaemia(glucose:70mg/dL)andhypoproteinaemia(total protein:4gr/dL) andhighureanitrogen(60mg/dL)andalanine aminotransferase(85U/L)levels.Radiographoftheabdomenand abdominal ultrasound showed prominent intestinal distension with abundant pneumo-faecal material in the large intestine. A radiopaqueimage resembling intussusception withintestinal obstructionwasalsoobservedinthecaudaldextralquadrantofthe abdomen.Thedogreceivedsymptomatictreatmentandfluid ther-apy.Attheexploratorylaparotomy,necroticileocaecallesionswere observedalongwithintestinalperforationofthedistalileum.The livershowedayellowishpalediscoloration.Aresection (approx-imately 6cm inlength) around theileal lesionand subsequent anastomosiswasperformed.Thefollowingday,thegeneral con-ditiondegeneratedandthedogdiedwithin48hafterthesurgery. 2.1. Anatomopathologicalfindings

Post-mortemexaminationperformedattheVeterinarySchool oftheNationalUniversityofCostaRicarevealedthepresenceof suppurativeexudate(about 25mL)intheabdominalcavity.The abdominalwallhadsmallaggregatesoffibrinwithassociated peri-tonitis.Thelungs,liver,kidneysandspleenwerecongested.The intestinalserosa (mainly atthe ileocaecaljunction) wasfocally thickened with presence of greyish tissue areas and multifo-calfibrinaggregation.Mesentericlymphnodeswereenlargedto twicethenormalsize and haemorrhagic(Fig.1).Theintestinal

Fig.1.Enlargedmesentericlymphnodesandiliocaecalmucosawithmultifocal areasofhaemorrhage.

Fig.2.Crosssectionsofadultwormsinthemesentericarteryoftheiliocaecal segment;pyogranulomatousinflammationattheintestinalserosaisevidentHE.

mucosawaserodedwithmultifocalpetechialandaslightly pres-enceoffibrin.Mesentericarteriesweredilatedbyadultparasites. CoprologicalexaminationperformedbyBaermanntestonfaeces collectedatnecroscopyrevealedthepresence ofnematode lar-vae.Histopathologically,theintestinalmucosashowedsegments ofmassivecoagulativenecrosisin associationwithdegenerated nematodelarvaeandeggs.Thelaminapropriawasdiffusely thick-enedbyprofuseinflammatoryinfiltratecomposedoflargeamounts ofmacrophageswithpresenceofepithelioidmacrophages, neu-trophils,lymphocytes,plasmacellsandeosinophils(Figs.2and3). Inflammatoryinfiltratesextendedthroughthemucosa,the muscu-larisexterna,serosaandtheassociatedadiposetissue.Moderate fibrosis of the submucosa was present at some parts of the ileum.Transmuralnecrotizingpyogranulomatousandeosinophilic inflammationwasmoreprominentattheterminalpartoftheileum (Fig.2).Sectionsofnematodesindifferentstagesofdevelopment wereobservedinthesuperficialmucosa,laminapropria, submu-cosa,muscularisexterna,andserosaoftheintestine(Figs.2and3) Adiposetissueandmesentericlymphnodeswereassociatedwith prominenteosinophilicinfiltratesandmultifocalmultinucleated foreignbodytypemultinucleatedgiantcellwithupto12nuclei.The livershowedseveralfociofgranulomatousinflammation associ-atedwithnematodesections.Adultnematodeswerealsodetected in thelumen of dilatedmesenteric arteries,occludingpartially orcompletelythearteriallumen,featuredbygranulomatousand eosinophilicperivasculitiswithmoderatethickeningof endothe-liumandtunicamedia.Adultnematodesfrommesentericarteries were stored in 70% ethanol and clarified in lactophenolto be

A.Alfaro-Alarcónetal./VeterinaryParasitology212(2015)431–434 433

Fig.3. MigrationoflarvaeofA.costaricensis(arrow)withintheilealwallin associ-ationwithgranulomatousinflammation(asterisk)HE.

identified.AdultwormsandL1obtainedbyBaermanntechnique weremorphologicallyidentifiedasA. costaricensisbased onthe species-specificfeaturesdescribedinMoreraandCéspedes(1971), and Rebello et al. (2013). Histopathologically,characteristics of nematodesectionsobtainedfromtheileumatlaparoscopywere compatiblewiththoseofA.costaricensisextractedfromvesselsas describedinGraeff-Teixeiraetal.(1991).

2.2. Molecularanalyses

The nematode identity was confirmed molecularly by spe-cific amplificationof 12S and 18Sgene fragments accordingto previouslydescribedmethodologies(Patterson-Kaneetal.,2009; Otrantoetal.,2011).Briefly, followinggenomicextractionfrom asinglespecimenusinga commercialkit(DNeasyBlood& Tis-sue Kit, Qiagen, GmbH, Hilden, Germany), 12S (∼300bp) and 18S (∼1700bp) gene fragments were amplified and amplicons wereresolvedin(2%)agarosegel,purifiedandsequenceddirectly in an automated sequencer (ABI-PRISM 377). Sequences were determinedfrom both strands and compared withthose avail-ableinGenBankTM_{datasetbyBasicLocalAlignmentSearchTool}

(BLAST—http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1997).The BLAST analysis of the12S and 18S sequences pro-ducedhereshowed96%and100%homology,respectively,tothose available in GenBankTM _{(Accessionnumber: LK939409 for 12S,}

LK942974for18S).

3. Discussion

Theresultsofthisstudyindicate,forthefirsttime,thatdogs nat-urallyinfectedbyA.costaricensismayshedlarvaeintheirfaeces, therefore,actingasadefinitivehostforthisnematode.Although, A.costaricensisL1werealreadyfoundinthefaecesofan experi-mentallyinfecteddog(Rodriguezetal.,2002),dataontheroleof thisanimalasaspreaderoftheinfectionhasneverbeen demon-stratedundernaturalconditions.Todate,avarietyofmammalian species(e.g.,non-humanprimates,raccoons,opossumand procy-onids)havebeeninvolvedasdead-endhostsinthelife-cycleofA. costaricensisandtherefore,thisnematodemayrepresentathreat forwildlifeanimals(Milleretal.,2006).

The severity of the clinical presentation in the dog herein observedwasnotconsistentwiththeclinicalsignsdescribedin apreviouscase(Rodriguezetal.,2002).Thisapparentdifference inclinicalpresentationmightbeduetothefactthatthedoghere examinedwasmuchyoungerthanthatreportedbyRodriguezetal. (2002)andconsequentlyajuvenileanimalmaybemoresusceptible

totheinfectionbyA.costaricensis.Thishypothesisisalsosupported bythecasereportofa9-month-oldpuppypresumptivelyinfected byA.costaricensisthatdisplayedasevereclinicalpresentation char-acterizedbyabdominalpainandanorexia(Arroyoetal.,1988).

Fromanepidemiologicalstandpoint,theidentificationofdog asdefinitivehostforA.costaricensisandtheoccurrenceofL1 shed-dinginurbanizedareamaybeofpublichealthconcern.Indeed, thepresenceofrodents,slugsandsnailsintheperidomesticarea representsariskfordogtoacquireinfections,and,eventually,to humanbeings.

Inaddition,thisisthefirstmolecularlyconfirmedcaseofadog naturallyinfectedbyA.costaricensisandindicatingthatthisspecies ofanimalsmayactasareservoir hostinthedomestic environ-ment.Additionalstudiesarerequiredtoascertaintheroleofdogs intheepidemiologyofA.costaricensisinareaswherethiszoonotic nematodeisendemic.

Acknowledgment

AuthorsthankMikeKinsella(HelmWestLaboratory,USA)for Englishrevisionofthemanuscript.

References

Altschul,S.F.,Madden,T.L.,Schäffer,A.A.,Zhang,J.,Zhang,Z.,Miller,W.,Lipman, D.J.,1997.GappedBLASTandPSI-BLAST:anewgenerationofproteindatabase searchprograms.NucleicAcidsRes.25,3389–3402.

Anderson,R.C.,2000.NematodeParasitesofVertebrates:TheirDevelopmentand Transmission.CABIInternational,Wallingford(UK),pp.650.

Arroyo,R.,Rodriguez,F.,Berrocal,A.,1988.AngiostrongilosisabdominalenCanis familiaris.Parasitol.Dia12,181–185.

Berrocal,A.,Arroyo,R.,Bulgarelli,G.,Valverde,A.,1997.Abdominal

angiostrongyliasisinaspidermonkey(Atelesgeoffroyi).Eur.J.Vet.Pathol.3, 35–37.

Brack,M.,Schöpel,M.,1995.Angiostrongyluscostaricensisinablack-eared marmoset.Trop.Geogr.Med.47,136–138.

Colella,V.,Giannelli,A.,Brianti,E.,Ramos,R.A.,Cantacessi,C.,Dantas-Torres,F., Otranto,D.,2015.Felinelungwormsunlockanovelmodeofparasite transmission.Sci.Rep.5,13105,http://dx.doi.org/10.1038/srep13105. Cowie,R.H.,2013.Pathwaysfortransmissionofangiostrongyliasisandtheriskof

diseaseassociatedwiththem.HawaiiJ.Med.PublicHealth72,70–74. Graeff-Teixeira,C.,Camillo-Coura,L.,Lenzi,H.L.,1991.Histopathologicalcriteria

forthediagnosisofabdominalangiostrongyliasis.Parasitol.Res.77,606–611. Miller,C.L.,Kinsella,J.M.,Garner,M.M.,Evans,S.,Gullett,P.A.,Schmidt,R.E.,2006. EndemicinfectionsofParastrongylus(=Angiostrongylus)costaricensisintwo speciesofnonhumanprimates,raccoons,andanopossumfromMiami,Florida. J.Parasitol.92(2),406–408.

Monge,E.,Arroyo,R.,Solano,E.,1978.Anewdefinitivenaturalhostof Angiostrongyluscostaricensis(MoreraandCespedes1971).J.Parasitol.64,34. Morera,P.,1973.LifehistoryandredescriptionofAngiostrongyluscostaricensis

MoreraandCéspedes,1971.Am.J.Trop.Med.Hyg.22,613–621. Morera,P.,Andrews,K.L.,Rueda,A.D.,1988.Theintermediatehostof

AngiostrongyluscostaricensisinHonduras.Rev.Biol.Trop.36,575–576. Morera,P.,Ash,L.R.,1970.Investigacióndelhuéspedintermediariode

Angiostrongyluscostaricensis(MorerayCéspedes,1971).Bol.Chil.Parasitol.25, 135.

Morera,P.,Céspedes,R.,1971.Angiostrongyluscostaricensisn.sp.(Nematoda Metastrongyloidea),anewlungwormoccurringinmaninCostaRica.Rev.Biol. Trop.18,173–185.

Mota,E.M.,Lenzi,H.L.,1995.Angiostrongyluscostaricensislifecycle:anew proposal.Mem.Inst.OwaldoCruz90,707–709.

Mota,E.M.,Lenzi,H.L.,2005.Angiostrongyluscostaricensis:completeredescription ofthemigratorypathwaysbasedonexperimentalSigmodonhispidusinfection. Mem.Inst.OswaldoCruz100,407–420.

Otranto,D.,Brianti,E.,Dantas-Torres,F.,Weigl,S.,Latrofa,M.S.,Gaglio,G.,Cauquil, L.,Giannetto,S.,Bain,O.,2011.Morphologicalandmoleculardataonthe dermalmicrofilariaeofaspeciesofCercopithifilariafromadoginSicily.Vet. Parasitol.182,221–229.

Patterson-Kane,J.C.,Gibbons,L.M.,Jefferies,R.,Morgan,E.R.,Wenzlow,N., Redrobe,S.P.,2009.PneumoniafromAngiostrongylusvasoruminfectioninared panda(Ailurusfulgensfulgens).J.Vet.Diagn.Invest.21,270–273.

Pena,G.P.,Andrade-Filho,J.,deAssis,S.C.,1995.Angiostrongyluscostaricensis:first recordofitsoccurrenceinthestateofEspiritoSanto,Brazil,andareviewofits geographicdistribution.Rev.Inst.Med.Trop.SaoPaulo37,369–374. Rebello,K.M.,Menna-Barreto,R.F.S.,Chagas-Moutinho,V.A.,Mota,E.M.,Perales,J.,

Neves-Ferreira,A.G.C.,Oliveira-Menezes,A.,Lenzi,H.,2013.Morphological aspectsofAngiostrongyluscostaricensisbylightandscanningelectron microscopy.ActaTrop.127,191–198.

434 A.Alfaro-Alarcónetal./VeterinaryParasitology212(2015)431–434 Rodriguez,R.,Agostini,A.A.,Porto,S.M.,Olivaes,A.J.O.,Branco,S.L.,Genro,J.P.,

Laitano,A.C.,Maurer,R.L.,Graeff-Teixeira,C.,2002.Dogsmaybeareservoir hostforAngiostrongyluscostaricensis.Rev.Inst.Med.Trop.SaoPaulo44,55–56. Romero-Alegría,A.,Belhassen-García,M.,Velasco-Tirado,V.,Garcia-Mingo,A.,

Alvela-Suárez,L.,Pardo-Lledias,J.,CorderoSánchez,M.,2014.Angiostrongylus costaricensis:systematicreviewofcasereports.Adv.Infect.Dis.4,36–41. Sly,D.L.,ToftII,J.D.,Gardiner,C.H.,London,W.T.,1982.Spontaneousoccurrenceof

Angiostrongyluscostaricensisinmarmosets(Saguinusmystax).Lab.Anim.Sci. 32,286–288.

Spratt,D.M.,2015.SpeciesofAngiostrongylus(Nematoda:Metastrongyloidea)in wildlife:areview.Int.J.Parasitol.ParasitesWildl.4,178–189.

Tesh,R.B.,Ackerman,L.J.,Dietz,W.H.,Williams,J.A.,1973.Angiostrongylus costaricensisinPanama,reportoftheprevalenceandpathologicfindingsin wildrodentsinfectedwiththeparasite.Am.J.Trop.Med.Hyg.22,348–356. Thiengo,S.,1996.ModeofinfectionofmolluscswithAngiostrongyluscostaricensis

larvae(Nematoda).Mem.Inst.OswaldoCruz91,277–288.

Ubelaker,J.E.,Bullick,G.R.,Caruso,J.,1980.Emergenceofthird-stagelarvaeof AngiostrongyluscostaricensisMoreraandCespedes1971fromBiomphalaria glabrata(Say).J.Parasitol.66,856–857.