VeterinaryParasitology228(2016)93–95
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
The
white-nosed
coati
(Nasua
narica)
is
a
naturally
susceptible
definitive
host
for
the
zoonotic
nematode
Angiostrongylus
costaricensis
in
Costa
Rica
Mario
Santoro
a,∗,
Alejandro
Alfaro-Alarcón
b,
Vincenzo
Veneziano
c,
Anna
Cerrone
a,
Maria
Stefania
Latrofa
d,
Domenico
Otranto
d,
Isabel
Hagnauer
b,
Mauricio
Jiménez
b,
Giorgio
Galiero
aaIstitutoZooprofilatticoSperimentaledelMezzogiorno,Portici,Naples,Italy bEscueladeMedicinaVeterinaria,UniversidadNacional,Heredia,CostaRica
cDepartmentofVeterinaryMedicineandAnimalProduction,UniversityofNaplesFedericoII,Naples,Italy dDipartimentodiMedicinaVeterinaria,UniversitàdegliStudidiBari,Valenzano,Bari,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received17June2016
Receivedinrevisedform23August2016 Accepted24August2016 Keywords: Abdominalangiostrongyliasis Procyonlotor Zoonotichelminth Parasiticdisease Publichealth Reservoir
a
b
s
t
r
a
c
t
Angiostrongyluscostaricensis(Strongylida,Angiostrongylidae)isaroundwormofrodents,whichmay causeasevereorfatalzoonosisinseveralcountriesoftheAmericas.Asinglereportindicatedthatthe white-nosedcoati(Nasuanarica),actsasapotentialfree-rangingwildlifereservoir.Hereweinvestigated theprevalenceandfeaturesofA.costaricensisinfectionintwoprocyonidspecies,thewhite-nosedcoati andtheraccoon(Procyonlotor)fromCostaRicatobetterunderstandtheirpossibleroleinthe epidemi-ologyofthiszoonoticinfection.Eighteenof32(56.2%)white-nosedcoatiscollectedbetweenJuly2010 andMarch2016wereinfectedwithA.costaricensisbutnoneof97raccoonsfromthesamelocalitieswere diagnosedwiththisinfection.First-stagelarvaeofA.costaricensiswereobtainedfromfecesof17fresh white-nosedcoaticarcassesbyBaermanntechnique.Parasiteidentitywasconfirmedbymorphology, histologyandmolecularcharacterizationoftargetgenes.Thesedatademonstratethatthewhite-nosed coatiisanaturallysusceptibledefinitivehostforA.costaricensisinCostaRicacontrarytofindingsinthe raccoon.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
Angiostrongylus costaricensis (Strongylida, Angiostrongyli-dae), a zoonotic nematode causing severe or fatal abdominal angiostrongyliasisinhumans,developsinawiderangeofrodents (i.e.,cottonratSigmodon hispidus)whichact asdefinitivehosts (MoreraandCéspedes,1971;Spratt,2015).Inaddition,naturally infected white-nosed coati (Nasua narica) and dogs have been suggestedtoactasreservoirforthisparasite(Mongeetal.,1978; Alfaro-Alarcónetal.,2016)andtheymayshedfirst-stagelarvae (L1)intheirfeces.Angiostrongyluscostaricensiswasalsoreported frommesentericarteriesoffree-rangingraccoons(Procyonlotor) andanopossum(Didelphisvirginiana)inFlorida(UnitedStates)
∗ Correspondingauthor.
E-mailaddresses:marisant@libero.it,mario.santoro@izsmportici.it (M.Santoro).
although,fecalsheddingofL1wasnotdemonstrated(Milleretal., 2006).
Adult wormslivein themesentericarteriesofthedefinitive hosts,theyshedL1whichdevelopintheintermediatehosts(snails and slugs)totheinfectivethird stage(L3).Thedefinitivehosts andhumansmaybecomeinfectedingestingraworundercooked intermediateorparatenichostsandvegetablescontaminatedwith infectedslugandsnailmucus(Spratt,2015).
Angiostrongyluscostaricensisisreportedinseveralcountriesof theAmericas, fromtheUnitedStates toArgentina, withnearly 200humancasesdescribed,mostofwhichoccurredinchildren (Graeff-Teixeiraetal.,1991;Romero-Alegríaetal.,2014). How-ever,MoreraandAmador(1998),onthebasisofserologicalstudies suggested thatthenumber ofhumancases inCosta Ricacould beupto600annually,alsobeingthecausativeagentfor20%of acuteabdominalconditionsreferredtothelocalNationalChildren’s Hospital(Abrahams-Sandietal.,2011).HumaninfectionswithA. costaricensishavealsobeenreportedfromAfrica(Zaire)andEurope
http://dx.doi.org/10.1016/j.vetpar.2016.08.017 0304-4017/©2016ElsevierB.V.Allrightsreserved.
94 M.Santoroetal./VeterinaryParasitology228(2016)93–95
(Franceand Spain)(Graeff-Teixeiraetal.,1991;Romero-Alegría etal.,2014).
White-nosedcoatiand raccooninCostaRicahave expanded theirrange intoboth peri-urbanand urbanareas,and are con-sideredaspotentialreservoirsofseveralzoonoticpathogens,such asTrypanosomacruzi,Mycoplasmaspp.,Babesiaspp.,and Baylisas-carisprocyonis(Mehrkensetal.,2013;Baldietal.,2016).Herewe investigatedtheprevalenceandfeaturesofA.costaricensis infec-tioninwhite-nosedcoatisandraccoonsfromCostaRicatobetter understandtheirpossibleroleintheepidemiologyofthiszoonotic infection.
2. Materialsandmethods
Freshcarcassesof17adultwhite-nosedcoatis(tenfemalesand sevenmales)and44raccoons(27femalesand17males)were pre-sentedfornecropsytothePathologyDepartmentoftheVeterinary MedicineSchooloftheUniversidadNacional(PSVMUN)inHeredia (CostaRica).CarcasseswereobtainedfromtheNorthPacific, Cen-tralValley,andSouthPacificareasofCostaRicafromFebruary2014 toMarch2016.FecalsampleswereprocessedbytheBaermann techniqueforthedetectionofL1.Adultnematodesfrom mesen-tericarteriesandL1fromfecalsampleswerestoredin70%and 100%ethanol formorphologicalandmolecularanalysis, respec-tively.Retrospectivecasesfromfree-rangingwhite-nosed coatis andraccoonssubmittedasbiopsyornecropsymaterialtothe PSV-MUNwerealsoreviewedforabetterappreciationofA.costaricensis orassociatedlesions.
2.1. Molecularanalyses
Thenematodeidentitywasconfirmedmolecularlybyspecific amplificationofcytochromecoxidasesubunit1(cox1)and18S rRNA gene fragments of adult worms according to previously describedmethodologies(Folmeretal.,1994;Patterson-Kaneetal., 2009).Briefly,followinggenomicextractionusingacommercial kit(DNeasyBlood&TissueKit,Qiagen,GmbH,Hilden,Germany), cox1(∼710bp) and 18SrRNA (∼1700bp)gene fragments were amplifiedandampliconswereresolvedin(2%)agarosegel, puri-fiedandsequenceddirectlyinanautomatedsequencer(ABI-PRISM 377).Sequencesweredeterminedfrombothstrandsandcompared withthoseavailableinGenBankTMdatasetbyBasicLocal
Align-mentSearchTool(BLAST—http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschuletal.,1997).
3. Resultsanddiscussion
Fourteen of 17 (82%) fresh carcasses of white-nosed coatis werepositiveforA.costaricensisbutnoindividualofraccoonwas foundinfected.Infectedcarcasseswereinmoderatetopoorbody conditionwithvariableamountsofabdominaltransudate(from 10 to35ml),enlargement of mesentericlymph nodes, marked dilationofmesentericarteriesassociatedwithlargegranulomas rangingfrom2to5cmindiameterinassociationwithadult nema-todes(Figs.1and2).Amoderatetoseverecatarrhalenteritiswas observedinallinfectedwhite-nosedcoatis,mainlyaffectingthe jejunumandileum.Theintestinalserosawashyperemicwith pres-enceoffibrin.Theliver,spleenandkidneyswerecongestedinall cases.
Atthehistopathologicalexam,allinfectedcasesshoweda mod-eratetoseveretransmuralenteritiswithareasofepithelialerosion, bluntingand atrophy of villi.The lamina propria wasdiffusely thickenedbyinflammatory infiltratecomposedoflymphocytes, macrophages, and plasma cells. Several nematode larvae were observedinthemucosa,sub-mucosaandmusculariswithpresence
Fig.1. Multiplelargegranulomasrelatedtomesentericarteriesofthesegmentsof thejejunum,ileum,caecumandproximalcolon.
Fig.2.Sectionofalargepyogranulomaassociatedwithmesentericarteries,note theadultindividualsofA.costaricensisinthevascularlumen.
Fig.3.PyogranulomatousandeosinophilicileitiswithpresenceofA.costaricensis larvaeinthemucosa.HE.
of granulomatousand eosinophilic inflammation(Fig.3).Adult nematodes were alsodetected in thelumen of dilated mesen-tericarteries,occludingpartiallyorcompletelythearteriallumen
M.Santoroetal./VeterinaryParasitology228(2016)93–95 95
Fig.4.SectionsofadultA.costaricensisinthevascularlumenofthemesenteric artery;thearteryissurroundbyamassiveproliferationofgranulationtissueand pyogranulomatousandeosinophilicinflammation.HE.
(Fig.4).Thesewormswereassociatedwithsevere pyogranuloma-tousandeosinophilicperivasculitiswiththepresenceofepithelioid macrophages and multinucleated foreign body type giant cells withupto28nuclei.Mesentericlymphnodeswerehyperplasic inassociationwithaseverepyogranulomatousand eosinophilic inflammationandthepresenceofseveralparasiticlarvae.Ina sin-glecase,extensivepyogranulomatousandeosinophilicconfluent andbridgingnecrotizinghepatitiswasobservedassociatedwith nematodelarvae.
Adult wormsand L1 obtainedbyBaermann techniquewere morphologicallyidentifiedasA.costaricensisbasedonthespecies specificfeatures described inMoreraand Céspedes (1971),and
Rebello et al. (2013). Histopathologically, the characteristics of nematodesectionswerecompatiblewiththoseofA. costaricen-sisextracted from vessels as described in Graeff-Teixeira et al. (1991).TheBLASTanalysisofthecox1and18SrRNAsequences producedhereshowed99%and100%homology,respectively,to thoseofA.costaricensisavailableinGenBankTM(Accession
num-ber:GQ398122forcox1,LK942974for18SrRNA).Sequenceswere depositedinGenBankTM underaccessionnumberKX378964for
18SandKX378965forcox1.
RetrospectivecasesobtainedbetweenJuly2010andDecember 2013included15white-nosedcoatis(eightmalesandsixfemales) fromSouthandNorthPacific,and53raccoons(21malesand32 females)fromNorthPacific,CentralValley,andSouthPacificareas. Ofallretrospectivecases,only4(27%)white-nosedcoatiswere foundhistopathologically infected with A. costaricensis (3from SouthandonefromNorthPacificrespectively).
ThehighprevalenceofA.costaricensissuggestedthatthis infec-tionisenzooticamongwhite-nosedcoatisinCostaRica,itbeing anaturallysusceptible definitivehostfor thenematodein con-trasttoraccoons.L1ofanAngiostongylusspeciescollectedfrom fecesofawhite-nosedcoatiwerefoundtoexperimentallydevelop in Biomphalaria glabrata snails (Monge et al., 1978). L3 larvae obtained30days later fromthose snailswereorally inoculated intothreecottonratsand,22dayslater,L1werefoundinfeces andadultnematodesofA.costaricensiswereretrievedin
mesen-tericarteriesofthesameanimals.Asidefromthisexperimental trial(Mongeetal.,1978),nodatahave beenpublishedon free-rangingwildlifereservoirsofA. costaricensisotherthan rodents (Spratt,2015).Indeed,thereportofA.costaricensisfrommesenteric arteriesoffree-rangingraccoonsandanopossuminFloridawas neverconfirmedbyfecalsheddingofL1(Milleretal.,2006).Our findingstogetherwiththeconsistentoccurrenceofpopulationsof infectedmolluskintermediatehostsinurbanandperi-urbanareas of Costa Ricawithprevalence ofinfection>50% for veronicellid slugs (Conejo and Morera, 1988), highlighttherisk of abdomi-nalangiostrongyliasistohumansand theirpets,which,in turn, mayactasreservoirhostsinthedomesticenvironment( Alfaro-Alarcón etal., 2016).Localresidents shouldbeinformed about theriskoftransmissionandpublichealthauthoritiesshould con-sider surveillanceand controlstrategies toreduceintermediate hostpopulations.
References
Abrahams-Sandi,E.,Mesén-Ramírez,P.,Suarez-Chacón,D.,Fernández-Quezada,K., 2011.Anindirectimmunofluorescenceantibodytestemployingwholeeggsas theantigenforthediagnosisofabdominalangiostrongyliasis.Mem.Inst. OswaldoCruz.106(4),390–393.
Alfaro-Alarcón,A.,Veneziano,V.,Galiero,G.,Cerrone,A.,Gutierrez,N.,Chinchilla, A.,Annoscia,G.,Colella,V.,Dantas-Torres,F.,Otranto,D.,Santoro,M.,2016. FirstreportofanaturallypatentinfectionofAngiostrongyluscostaricensisina dog.Vet.Parasitol.212,431–434.
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.
Baldi,M.,Alvarado,G.,Smith,S.,Santoro,M.,Bola ˜nos,N.,Jiménez,C.,Hutter,S.E., Walzer,C.,2016.Baylisascarisprocyonisparasitesinraccoons,CostaRica,2014. Emerg.Infect.Dis.22(8),1502–1503.
Conejo,M.E.,Morera,P.,1988.Influenceoftheageofveronicellidslugsin Angiostrongyluscostaricensisinfection.Rev.Biol.Trop.36(2B),519–526. Folmer,O.,Black,M.,Hoeh,W.,Lutz,R.,Vrijenhoek,R.,1994.DNAprimersfor
amplificationofmitochondrialcytochromecoxidasesubunitIfromdiverse metazoaninvertebrates.Mol.Mar.Biol.Biotechnol.3,294–299.
Graeff-Teixeira,C.,Camillo-Coura,L.,Lenzi,H.L.,1991.Histopathologicalcriteria forthediagnosisofabdominalangiostrongyliasis.Parasitol.Res.77,606–611. Mehrkens,L.R.,Shender,L.A.,Yabsley,M.J.,Shock,B.C.,Chinchilla,F.A.,Suarez,J.,
Gilardi,K.V.K.,2013.Whitenosedcoatis(Nasuanarica)areapotential reservoirofTrypanosomacruziandotherpotentiallyzoonoticpathogensin Monteverde,CostaRica.J.Wildl.Dis.49(4),1014–1018.
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.,Amador,J.,1998.Prevalenciadelaangiostrongilosisabdominalyla
distribuciónestacionaldelaprecipitación.Rev.Costarric.SaludPública.7(13), 1–14.
Morera,P.,Céspedes,R.,1971.Angiostrongyluscostaricensisn.sp.(Nematoda Metastrongyloidea):anewlungwormoccurringinmaninCostaRica.Rev.Biol. Trop.18,173–185.
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.
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.
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. Spratt,D.M.,2015.SpeciesofAngiostrongylus(Nematoda:Metastrongyloidea)in
