Seroprevalence and risk factors of Neospora spp. in donkeys from Southern Italy.

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VeterinaryParasitology198 (2013) 201–204

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j ourn a l h o m e pa 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

Seroprevalence

and

risk

factors

of

Neospora

spp.

in

donkeys

from

Southern

Italy

T. Machaˇcová

a

_,

_E.

_Bártová

a,∗

_,

_A.

_Di

_Loria

b

_,

_K.

_Sedlák

c

_,

_J.

_Guccione

d

_,

D. Fulgione

e

_,

_V.

_Veneziano

d

a_Department_of_Biology_and_Wildlife_Diseases,_Faculty_of_Veterinary_Hygiene_and_Ecology,_University_of_Veterinary_and_{Pharmaceutical}

Sciences,Palackéhotˇr.1/3,61242Brno,CzechRepublic

b_Department_of_Health_Science,_University_of_Magna_Græcia,_Catanzaro_88100,_Italy

c_Department_of_Virology_and_Serology,_State_Veterinary_Institute_Prague,_{Sídliˇstní}_136/24,₁₆₅₀₃_Prague_6,_Czech_Republic d_Department_of_Veterinary_Medicine_and_Animal_Production,_University_of_Naples,_Federico_II,_Naples_80137,_Italy e_Department_of_Biology,_University_of_Naples,_Federico_II,_Naples,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received3May2013

Receivedinrevisedform9August2013 Accepted11August2013 Keywords: Equusasinus Neosporosis Seroprevalence cELISA Italy

a

b

s

t

r

a

c

t

InsomeEuropeancountriesthereisanincreasinginterestondonkey.Despitetherearefew dataregardingthedonkey’sparasiticdiseasesespeciallythosewithaprotozoaletiologyas neosporosis.Samplesusedinthestudywerecollectedfrom238domesticdonkeysduring year2010inSouthernItalyfrom207femalesand31malesoffivebreeds(Martina-Franca, Amiata,Sicilian-Grey,Ragusano,Sardinian)andcrossbreedswiththeaverageage9years(1 month–24year).Seraweretestedbyacompetitive-inhibitionenzyme-linked immunosor-bentassayforantibodiesagainstNeosporacaninum;theseraweremarkedpositive,ifmore than30%inhibitionwasfound.Outofatotal238donkeys,28(11.8%)werefound posi-tiveforNeosporaantibodieswith12%infemalesand6%inmales.Differentseroprevalence 15.4%,16%,12%and8.8%werefoundinagecategories<1year,1–4years,5–9yearsand≥10 years,respectively.Theseroprevalencerangedindifferentbreedsfrom36%(Sicilian-Grey) to0%(Sardinian)andindifferentusefrom17%(forbreeding)to0%(formeatproduction). Logisticregressionanalysisdemonstratedevidenceofasignificant(P<0.05) associa-tionbetweencrossbreedoriginofsamplesandriskofprotozoaninfection;ageofdonkeys wasalsosignificantriskfactorforprotozoaninfection.Nostatisticalsignificantdifference (P>0.05)wasfoundamonggendersanduseofdonkeysandriskofN.caninuminfection. ThisisthefirstserologicalsurveyforNeosporaspp.performedindonkeys.

1. Introduction

Thedonkeys(Equusasinus)weretraditionallyusedas workinganimalfortransportandridingandforfarm activ-ities.Intherecentyears,anincreasinginteresthavebeen shownfordonkeysandinparticularlyfortheiruseasapet animal,inleisureactivities,foronotherapyandespecially

∗ Correspondingauthor.Tel.:+420541562633;fax:+420541562633. E-mailaddress:bartovae@vfu.cz(E.Bártová).

fortherediscoveryofdonkeymilkasafoodsourcefor chil-drenaffectedwithcowmilkallergy.Equineneosporosisis aprotozoaldiseasecausedbyacyst-formingcoccidianof thephylumApicomplexa.Twospecies,Neosporacaninum and Neospora hughesi, have been identified as infecting thehorseandwereassociatedwithneurologicaldisease and fetal loss(Dubeyand Porterfield, 1990; Pitelet al., 2003).Basedonexperimentalstudies,domesticdogs(Canis lupus),Australiandingoes(Canisdomesticus)andcoyotes (Canislatrans)wereconfirmedasdefinitivehostsofN. can-inumwhilethedefinitivehostofN.hughesiremainstobe 0304-4017/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved.

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202 T.Machaˇcováetal./VeterinaryParasitology198 (2013) 201–204

elucidated. Seroprevalence of Neospora spp. in asymp-tomatic horses were reported from North America (Dubey, 2003), South America (Villalobos et al., 2006; Dangoudoubiyametal.,2011)andEurope(Pronostetal., 1999;Piteletal.,2001;Ciaramellaet al.,2004;Jakubek et al., 2006; Kligler et al., 2007; Kilbas et al., 2008; Piantedosietal.,2009;Bártováetal.,2010).

InSouthernItaly,werethepopulationofdonkeyisgoing toincrease,Neosporainfectionhasbeenreportedinhorses (Ciaramellaetal.,2004),cattle(Guarinoetal.,1998),water buffaloes(Guarinoetal.,2000)andindogs(Cringolietal., 1996).Since,thepresenceofdonkeysisinpromiscuitywith thesespeciesinmanyfarms,thisconditionletustoassume apossiblespreadingofNeosporainfectionalsoindonkeys. Untilnow,noreportsareavailableabouttheprevalenceof thisprotozoaldiseaseindonkeys.

Theaimofthepresentstudyistoimprovethe informa-tionabouttheseroprevalenceofNeosporasppinanimals fromsouthernItaly.

2. Materialsandmethods

2.1. Samplesizeanddonkeyssampled

Thesurvey wasconducted on238 clinically healthy donkeysbred and raisedin Southern Italy. Thissample size was calculated by using the formula proposed by Thrusﬁeld (1995) inserting the following values: study population(9991donkeys,datasuppliedbyItalian Associa-tionofBreeders)expectedprevalenceofneosporosis(20%, datareportedin horses in Southern Italy byCiaramella etal.,2004),conﬁdenceinterval(95%),anddesiredabsolute precision(5%).Duringautumn2010bloodsampleswere collectedin20donkeyfarms(5largefarms≥10donkeys and15smallfarms<10donkeys)of16municipalitiesof SouthernItaly.Generaldataincludinggender,age,breed, useandperiodofgrazingduringyear,presenceofdogsand ruminantsinthefarmswereobtainedthrougha question-nairescompletedduringsamplecollection.Thedonkeys weredividedintofouragecategories:<1year,1–4years, 5–9years,≥10years;theaverageageofdonkeyswas8 yearsand11month(1month–24year).Furthermore,a completeclinicalexaminationwasdoneoneachdonkey.

2.2. Serapreparationandserologicaltest(cELISA)

Allbloodsampleswereobtainedfromthejugularvein byusingavacuumtubewithoutanticoagulant.Serawere obtainedfromclottedbloodsamplesbycentrifugationand storedat−20◦_C _until_analysis._Sera_were_tested_for _the presenceofantibodiestoN.caninumusinga competitive-inhibitionenzyme-linked immunosorbentassay(cELISA, VMRD,Inc.,Pullman,WA,USA).Serologicalexamination andevaluationwasdoneaccordingtothemanufacturer’s instructions;samplesassociatedwithpercentinhibition value≥30%were consideredpositives. Theoptical den-sityvalueswereobtainedusinganautomaticplatereader (DynexTechnologyMRXII,Prague,CzechRepublic).

Table1

RiskfactorsforNeosporaspp.infectionindonkeysasaresultofthelogistic regressionmultivariateanalysis.

Coefﬁcient Stderror z P Intercept −1.98 0 0.9998 Gender(male) −17.0 −0.002 0.9980 Age(year) −0.709 −2.011 0.0443 0.01 Breed Crossbreed −1.37 −2.277 0.0228 0.01 Sicilian-Grey 1.15 1.569 0.1167 Martina-Franca −381 −0.667 0.5047 Amiata −18.0 −0.004 0.9964 Ragusano 0.176 0.192 0.8479 Sardinian −18.5 −0.005 0.9963 Use Milk 1.19 −0.004 0.9999 Pet 1.58 −0.002 0.9998 Breeding 17.9 −0.007 0.9944 Presenceofdog −0.28 1.05 0.1750 Presenceofruminants −0.31 1.21 0.0980 2.3. Statisticalmethods

Statisticalanalyseswereperformedonthebasisofthe

individualanimalastheunit.Amultivariateanalysiswas

usedtoevaluatethecontributionofeachvariablesinvolved

ininfectionrisk.Alogisticregression(generallinear

mod-els,GLM)wasusedtopredictseropositivityaccordingto

additiveandlinearrelationshipbetweenvariables.

Statis-ticalanalysiswasperformedusingGraphPadPrismversion

6.00forMacOSX,GraphPadSoftware,LaJolla,California,

USA.P<0.05wasconsideredstatisticallysigniﬁcant.

3. Results

Out of a total 238 donkeys, 28 (11.8%) were found

positiveforNeosporaspp.antibodieswithinhibition

ran-gingfrom30.07%to44.34%.Neosporaspp.antibodieswere

foundin12%(26/207)femalesand6%(2/31)males.The

following seroprevalences 15.4%(4/26), 16%(8/50), 12%

(7/60) and 8.8% (9/102) were found in age categories

<1 year, 1–4 years, 5–9 years and ≥10 years,

respec-tively.We alsofounddifferentseroprevalencein breeds

with 36% (5/14), 25% (2/8), 20% (9/46), 13% (7/53), 5%

(5/110) and0%(0/7)in Sicilian-Grey,Ragusano,Amiata,

Martina-Franca, crossbreedsand Sardinian, respectively.

Thefollowingseroprevalences 17%(2/12),13%(23/183),

11%(3/27)and0%(0/16)weredetectedindonkeysused

forbreeding,milkproduction,asapetanimalandformeat

production,respectively.Incaseof28donkeyspositivefor

Neosporaspp.,82%camefromlargefarms(≥10donkeys),

64%donkeysgrazedduringwholeyear,in29%cases,the

dogshadaccesstofoodandwaterusedforfeedingdonkeys,

in100%and21%caseshorsesandruminantswerebredon

thesamefarms.

Logistic regression analysis involving all considered

variablesdemonstratedevidenceofasigniﬁcant(P=0.01)

associationbetweencrossbreedoriginofsamplesandrisk

ofprotozoaninfection(Table1).Ageofdonkeyswasalso

signiﬁcant(P=0.01)riskfactorforprotozoaninfection.It isevidentthatthedonkeysolderthantwoyearscan high-lightN.caninumpositivity(Fig.1).Nostatisticalsigniﬁcant

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T.Machaˇcováetal./VeterinaryParasitology198 (2013) 201–204 203

Fig.1. AssociationbetweenageofdonkeysandtheirpositivitytoNeosporaantibody.Itisevidentthatthedonkeysolderthantwoyearscanhighlight positivity(brokenlineontheleft).Thisarrangementseemstoendattheageof20years(brokenlineontheright),althoughthenumberofsamplesolder thanthisageislow.

differencewasfoundbetweengendersanddifferentuseof donkeysandriskofN.caninuminfection.

Theclinicalexaminationofalltheseropositivedonkeys didnotshowanyneurologicalsigns,reproductive disor-dersandabortion.

4. Discussion

ThisistheﬁrstserologicsurveyforNeosporaspp. anti-bodies performedondonkeys.Asregardstotheclinical investigations,theabsenceoffetallossandneurological signs,itindicatesthatasubstantialdegreeofNeosporaspp. infectionoccurssub-clinicallyintheseanimals.Wefound Neospora spp. antibodies in 11.8% donkeys.Since there arenosimilarstudiesondonkeys,wecancompareour resultswithseroprevalenceinhorses.ConsideringELISA asdiagnostictest usedinEurope,similarprevalence9% wasfoundinhorsesfromTurkey(Kilbasetal.,2008)and Sweden(Jakubeketal.,2006),whileahigherprevalence 24%wasfoundinhorsesfromtheCzechRepublic(Bártová etal.,2010).Indirectﬂuorescentantibodytest(IFAT)was usedforsurveysinItalyandIsrael.Theprevalenceof28% (Ciaramellaetal.,2004)and10%(Piantedosietal.,2009) wasfoundinhorsescomingfromdifferentItalianregions, while12%wasfoundinhorsesfromIsrael(Kligleretal., 2007).InFrance,23%prevalencewasobtainedinhorses testedbydirectagglutinationtest(Piteletal.,2001).

In ourstudy,no statisticalsignificantdifferencewas foundbetweengendersanduseofdonkeysandtheir pos-itivity,whileageofdonkeyswassignificantriskfactorfor protozoaninfection.Sincethemajorityofdonkeysbecame infecteduntiltheageof2years,itletustoassumethat donkeyswereexposedtotheparasiteinfectionattheearly stagesoftheirlife.Wefoundtwoyoungdonkeys(2and 1 monthsold) positive for N.caninum antibodies;their motherswerealsopositive.Recently,Pusterlaetal.(2011) confirmedthatN.hughesicanpersistinhorsepopulation viaendogenoustransplacentalinfection.Consideringthis factandpositivityofshe-assofyoungdonkeysleadsusto considerpossibleverticaltransmissionofNeospora infec-tionindonkeysinfurtherstudies.Thecrossbreedseems tobemoreresistanttoNeosporaspp.infectionsincefour timesdifferencewasfoundbetweenpositivityof cross-breedsandbreeds.

Itisdifficulttoexplain whatisthecriticalpointsfor theNeosporainfection,consideringthatalimitationofthe presentstudyistheimpossibilitytodiscriminateN.hughesi versusN.caninumwecanonlypostulatesomehypothesis: thepossiblesourceofNeosporaspp.infectionfordonkeysin farmscouldbewater,foodorpasturescontaminatedwith Neosporaspp.oocysts.In29%donkeyspositiveforNeospora spp.,thedogshadaccesstofoodandwaterusedforfeeding donkeys.Althoughthepresenceofdogsinfarmshasnot beenconfirmedasariskfactorforNeosporaspp.infection, ThedonkeysaswellashorsesinSouthernItalycouldbe exposedtoNeosporaspp.infection.Furtherstudiesshould focusonconcretefarmstofindthepotentialriskfactors androutesforinfectiontransmission.

Conﬂictofintereststatement

Noneoftheauthorsofthispaperhasaﬁnancialor per-sonalrelationshipwithotherpeopleororganizationsthat couldinappropriatelyinﬂuenceorbiasthecontentofthe paper.

Acknowledgements

ThisstudywasfundedfromtheMinistryofEducation, YouthandSportsoftheCzech(MSM6215712402)andfrom IGAVFUBrno,CzechRepublic(6/2012/FVHE).

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