Toxoplasma gondii in stranded marine mammals from the North Sea and Eastern Atlantic Ocean: Findings and diagnostic difficulties

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Contents lists available atScienceDirect

Veterinary

Parasitology

j o u r n a l h o m e 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

Research

paper

Toxoplasma

gondii

in

stranded

marine

mammals

from

the

North

Sea

and

Eastern

Atlantic

Ocean:

Findings

and

diagnostic

difﬁculties

Norbert

van

de

Velde

a

_,

_Brecht

_{Devleesschauwer}

b,c,∗

_,

_Mardik

_Leopold

d

_,

Lineke

Begeman

e,1

_,

_Lonneke

_IJsseldijk

f

_,

_Sjoukje

_Hiemstra

f

_,

_Jooske

_IJzer

f

_,

Andrew

Brownlow

g

_,

_Nicholas

_Davison

g

_,

_Jan

_Haelters

h

_,

_Thierry

_Jauniaux

i

_,

_Ursula

_Siebert

j

_,

Pierre

Dorny

b,k

_,

_Stéphane

_De

_Craeye

l

a_Department_of_Pathology,_Bacteriology_and_Avian_Diseases,_Faculty_of_Veterinary_Medicine,_Ghent_University,_Merelbeke,_Belgium b_Department_of_Virology,_Parasitology_and_Immunology,_Faculty_of_Veterinary_Medicine,_Ghent_University,_Merelbeke,_Belgium c_Department_of_Public_Health_and_{Surveillance,}_Scientiﬁc_Institute_of_Public_Health_(WIV-ISP),_Brussels,_Belgium

d_Wageningen_IMARES_–_Institute_for_Marine_Resources_and_Ecosystem_Studies,_Den_Helder,_The_Netherlands e_Viroscience,_Erasmus_Medical_Centre,_Rotterdam,_The_Netherlands

f_Utrecht_University,_Faculty_of_Veterinary_Medicine,_Dept._{Pathobiology,}_The_Netherlands

g_Scottish_Marine_Animal_Stranding_Scheme,_SAC_Consulting._Veterinary_Services,_{Drummondhill,}_Inverness,_IV2_4JZ_Scotland,_UK h_Royal_Belgian_Institute_of_Natural_Sciences_(RBINS),_Ostend,_Belgium

i_Department_of_Morphology_and_Pathology,_Faculty_of_Veterinary_Medicine,_University_of_Lie`_ge,_Liège,_Belgium j_Institute_for_Terrestrial_and_Aquatic_Wildlife_Research,_University_of_Veterinary_Medicine_Hannover,_Büsum,_Germany k_Department_of_Biomedical_Sciences,_Institute_of_Tropical_Medicine,_Antwerp,_Belgium

l_National_Reference_Laboratory_for_{Toxoplasmosis,}_Department_of_Communicable_and_Infectious_Diseases,_Scientiﬁc_Institute_of_Public_Health_(WIV-ISP),

Brussels,Belgium

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received16June2016

Receivedinrevisedform19October2016 Accepted22October2016 Keywords: Toxoplasmagondii Marinemammals Seroprevalence PCR MAT IFA ELISA

a

b

s

t

r

a

c

t

Theoccurrenceofthezoonoticprotozoanparasite Toxoplasmagondiiin marinemammals remains apoorlyunderstoodphenomenon.Inthisstudy,samplesfrom589marinemammalspeciesand34 Europeanotters(Lutralutra),strandedonthecoastsofScotland,Belgium,France,TheNetherlands andGermany,weretestedforthepresenceofT.gondii.Brainsampleswereanalysedbypolymerase chainreaction(PCR)fordetectionofparasiteDNA.Bloodandmusclefluidsamplesweretestedfor spe-cificantibodiesusingamodifiedagglutinationtest(MAT),acommercialmulti-speciesenzyme-linked immunosorbentassay(ELISA)andanimmunofluorescenceassay(IFA).Outof193animalstestedbyPCR, onlytwoharbourporpoise(Phocoenaphocoena)cerebrumsamples,obtainedfromanimalsstrandedon theDutchcoast,testedpositive.Theserologicalresultsshowedawidevariationdependingonthetest used.Usingacut-offvalueof1/40dilutioninMAT,141outof292animals(41%)werepositive.Using IFA,30outof244testedsamples(12%)werepositiveata1/50dilution.ThecommercialELISAyielded 7%positiveswithacut-offofthesample-to-positive(S/P)ratio≥50;and12%whenthecut-offwasset atS/Pratio≥20.ThehighnumberofpositivesinMATmaybeanoverestimationduetothehighdegree ofhaemolysisofthesamplesand/orthepresenceoflipids.TheELISAresultscouldbean underestima-tionduetotheuseofamultispeciesconjugate.OurresultsconfirmthepresenceofT.gondiiinmarine mammalsinTheNetherlandsandshowexposuretotheparasiteinboththeNorthSeaandtheEastern AtlanticOcean.WealsohighlightthelimitationsofthetestsusedtodiagnoseT.gondiiinstrandedmarine mammals.

∗ Correspondingauthorat:DepartmentofPublicHealthandSurveillance, Sci-entiﬁcInstituteofPublicHealth(WIV-ISP),RueJulietteWytsmanstraat14,1050 Brussels,Belgium.

E-mailaddress:brechtdv@gmail.com(B.Devleesschauwer).

1 _During_this_research,_LB_was_working_at_Utrecht_University,_Faculty_of_Veterinary

Medicine,TheNetherlands.

1. Introduction

Toxoplasma gondiiis a globallydistributed zoonotic parasite. Itisassumedthatallwarm-bloodedanimals,includinghumans, canact asintermediate hosts, whileonly felidsactas theﬁnal hosts(Tenteretal.,2000).AprimaryinfectioninfelidsallowsT. gondiitocompleteitssexualcycle,resultinginthefaecalshedding ofmillionsofoocystsintotheenvironment(Dubey,2010).These http://dx.doi.org/10.1016/j.vetpar.2016.10.021

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oocystsareabletoreachcoastalseawatersandseveralstudieshave demonstratedthepresenceofT.gondiiinmarinemammalspecies includingpinnipeds,cetaceans,sirenians,seaotters(Enhydralutris) andpolarbears(Ursusmaritimus)(Coleetal.,2000;Dubey,2010; Mathewsetal.,2012;Oksanenetal.,2009).Different transmis-sionroutessuchascoastalrun-offandmechanicalvectorshave beenhypothesized,buttheexactmodeoftransmissionremains unknown(Arkushetal.,2003;Lindsayetal.,2001;Massieetal., 2010).Immunosuppressivefactors,suchaspolychlorbiphenylsand Morbillivirusinfection couldmakemarine mammalsmore sus-ceptibletodiseaseinducedbyT.gondiiinfection(Mazzarioletal., 2012).Besidesbeingapathogenicagentcapableofaffectingmarine mammalhealth,T.gondiiactsasanindicatorofmarinepollution (Jessupetal.,2004),withmarinemammalsbeingexcellentsentinel species(Conradetal.,2005;deMouraetal.,2014;Jessupetal., 2004;Moore,2008;Stewartetal.,2008).

VariousserologicaltestsareavailabletodetectthepresenceofT. gondiiantibodiesinhumansandanimals.TheSabin-Feldmandye testisconsideredthegoldstandard test,butistimeconsuming andmakesuseofliveparasites.Althoughnotvalidatedformarine mammals,themodifiedagglutinationtest(MAT)maybean appeal-ingalternativemethod,becauseofitsspeedandeaseofuse,butalso becauseitdoesnotrequirespecies-specificsecondaryantibodies (DesmontsandRemington,1980).Thisisparticularlyusefulwhen testingwildlife,suchasmarinemammals(Cabezónetal.,2011; Santosetal.,2011;Jensenetal.,2012).Recently,however,Blanchet etal.(2014)questionedthereliabilityofMATintheserological diagnosisofT.gondiiinfectioninharbourporpoises(Phocoena pho-coena).ThiswasbasedonacomparisonofMATonuntreatedserum sampleswithenzyme-linkedimmunosorbent assay(ELISA) and polymerasechainreaction(PCR),whichsuggestedthatMATmay givefalsepositiveresults.Adherenceofcomponentstotachyzoites otherthanspecificantibodies,suchassamplesrichinparticulate matterandlipids,mayberesponsibleforthesefalsepositive reac-tions.Achloroform‘clean-up’ofserumsamplesmaypreventthis problem(Blanchetetal.,2014).Withtheexceptionofharbour por-poises(Phocoenaphocoena),however,thismethodhasnotbeen evaluatedforothermarinemammalspecies.

TheaimofthisstudywastoassesstheprevalenceofT.gondii invariousmarinemammalspeciesstrandedonScottish,Belgian, French,Dutchand Germancoastlinesof theNorth Seaand the AtlanticOceanaroundScotland.AsthereliabilityofMATinmarine mammalsampleshasbeenquestioned(Blanchetetal.,2014), it wasdecidedtoperformadditionaltestingtoevaluateandcompare variousserologicaldetectionmethods.

2. Materialsandmethods 2.1. Samples

From589free-rangingstrandedmarinemammalsnecropsiedat differentinstitutesinTheNetherlands,Belgium,France,Germany and Scotland (Table 1), a selection of samples including cere-brumandcerebellum, serumand/ormusclefluid wasobtained. Additionally,samplesfrom34free-rangingEuropeanotters(Lutra lutra)livingalongtheNorthSeacoastinScotlandwerealso ana-lysed.Convenience-basedsamplingfromthesedifferentinstitutes resultedinacollectionofsamplesfromatimeperiodof1992–2013 thatcovered17differentmarinemammalspecies.Anoverviewof thenumbersandspeciesofcollectedandtestedanimalsisgivenin Table2.Brainsamples(n=193)weretestedbyPCR.PositivePCR sampleswerefurtheranalysedbyhistopathologyand immunohis-tochemistry.Allserum(n=292)andmusclefluid(n=193)samples wereanalysedwithMATandalargepartofthecollectedserum sampleswasfurtheranalysedwithanimmunofluorescenceassay

(IFA)(n=244)andamultispeciesELISA(n=241)(Table3).Dueto therelativelylargevolumesrequiredbyMATandIFA,itwasnot possibletotestallsampleswithallthreetests.

2.2. PCR

2.2.1. DNAextraction

DNAfromthebrainsampleswasisolatedwiththeQIAmpDNA MiniKit(Qiagen,Venlo,TheNetherlands)aspreviouslydescribed byDeCraeyeetal.(2011).Brieﬂy,400mgofeachbrainsample wasmixedwith20␮lofproteinaseKand400␮l ofaﬁrstlysis bufferand vortexedthoroughlypriortoovernightincubationat 56◦C.Thenextday,400_␮lofasecondlysisbufferwasaddedand themixturewashomogenizedandfurtherincubatedat70◦Cfor 20min.Aftercoolingonice,600␮lofachloroform/isoamylalcohol (24/1)mixturewasaddedandeachsamplewasvortexedandthen centrifugedat22,000×g(4◦C)for30min.Thesupernatant was transferredintoanewtubeandtheDNAwasprecipitatedbyadding 400␮lof95%ethanolcooledat−20◦_C._From_this_point_onwards,_the

manufacturer’sprotocolwasfollowed.Themixturewastransferred toaspin-columnandwashedoncewith500␮lofaﬁrstwashing bufferandoncewith500␮lofasecondwashingbuffer.Thepuriﬁed DNAwasthenelutedfromthecolumnusing200␮lofanelution buffer.Thesampleswerestoredat−20◦_C_until_further_testing.

2.2.2. Real-timePCR,histopathologyandimmunohistochemistry TheisolatedDNAsamplesweretestedbyduplexreal-timePCR (qPCR)asdescribedbyDeCraeyeetal.(2011).The529-basepair repeatelementwasusedasatarget(Homanetal.,2000).Table4 givesanoverviewoftheprimersandprobesusedandtheirfinal concentrationperreaction.DNAqualityandPCRinhibitionwere assessedusingcellularr18Sasa target(Table4).Sampleswere runandanalysedonaBioRadCFX96thermocycler/detection sys-tem(BioRad,Hercules,CA,USA).Ineachrun,apositiveextraction control sample consisting of DNA extracted froma brain sam-plefromamouseexperimentallyinfectedwiththe1984-IPB-GAN T. gondii strainwas includedas wellas onesample containing onlyphosphatebufferedsaline(PBS)asanegativecontrol. Pos-itiveresultsfromPCRwereevaluatedusinghistopathologyand immunochemicalstainsfordetectingT.gondiitachyzoites. Neutral-buffered 10% formalin fixed samples were routinely processed in paraffin for light microscopy using haematoxylin-eosin and immunohistochemicalstaining.For immunohistochemical stain-ing,anavidin-biotincomplexmethodusingpolyclonalantibodies againstT.gondii(1:100)wasapplied(Key,2009).

2.3. Serologicalexamination

2.3.1. Modiﬁedagglutinationtest(MAT)

TheseraweretestedwithacommercialMATkit(Toxo-Screen DA® _kit,_Biomérieux,_{Marcy-l’Etoile,}_France)_according_to

manu-facturer’sinstructions.Brieﬂy, samplesweredilutedin PBSand 25␮lofeachdilutionwasappliedinaroundbottommicrotiter platewith25␮l2-mercaptoethanolat0.2mol/l.Tothis,50␮lof a1/5dilutionofwholeformolizedtachyzoitesinbovinealbumin boratesaline(suppliedinthekit)bufferwasadded.Theplatewas mixedfor1minonaplatemixerat300revolutionsperminute andincubatedovernightatroomtemperature.Thenegativeand positivecontrolswerebothsuppliedbythemanufacturerand con-sistedofgoatserum.Thepositivecontrolwascalibratedagainstthe WorldHealthOrganizationstandard,apositivestandardserum cal-ibratedtointernationalunits.Agglutinationina1/40dilutionwas consideredtobeapositiveresultaccordingtothemanualofthe diagnostickitandearlierstudiesonmarinemammalsera(Jensen etal.,2010,2012;Oksanenetal.,1998).Accordingtothe

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manufac-Table1

Institutesfromwhichsampleswerecollected.

Name Location Locationofthestrandedanimals

UtrechtUniversity FacultyofVeterinaryMedicine,Dept. Pathobiology,Utrecht,TheNetherlands

Dutchcoastline–NorthSeaand WaddenSea

InstituteforMarineResourcesand EcosystemStudies(IMARES)

Texel,TheNetherlands Dutchcoastline–NorthSeaand WaddenSea

InstituteforTerrestrialandAquatic WildlifeResearch(ITAW)

UniversityofVeterinaryMedicine Hannover,Büsum,Germany

Germancoastline–NorthSeaand WaddenSea

RoyalBelgianInstituteofNatural Sciences

Ostend,Belgium Belgiancoastline–NorthSea

UniversityofLiège FacultyofVeterinaryMedicine,

UniversityofLiège,Belgium

Belgian,DutchandnorthernFrench coastline–NorthSea

ScottishMarinemammalStranding Scheme(SMASS)

Scotland’sRuralCollege,Inverness, Scotland

Scottishcoastline–AtlanticOceanand NorthSea

Table2

NumberandspeciesofanimalstestedforToxoplasmagondiifromthedifferentlocations.

Serum Serum+Brain Brain Brain+Muscle Muscle Total

Belgiancoastline–NorthSea

Finwhale(Balaenopteraphysalus) 1 1

Greyseal(Halichoerusgrypus) 2 2

Harbourporpoise(Phocoenaphocoena) 20 1 48 69

Harbourseal(Phocavitulina) 8 1 9

Hoodedseal(Cystophoracristata) 1 1

White-beakeddolphin(Lagenorhynchusalbirostris) 1 1

NorthernFrenchcoastline–NorthSea

Finwhale(Balaenopteraphysalus) 1 1 2

Harbourporpoise(Phocoenaphocoena) 3 12 15

Stripeddolphin(Stenellacoeruleoalba) 1 1

Dutchcoastline–NorthSeaandWaddenSea

Harbourporpoise(Phocoenaphocoena) 16 15 68 5 81 185

Harbourseal(Phocavitulina) 28 28

Germancoastline–NorthSeaandWaddenSea

Harbourporpoise(Phocoenaphocoena) 2 19 21

Scottishcoastline–AtlanticOcean

Atlanticwhite-sideddolphin(Lagenorhynchusacutus) 7 7

Bottlenosedolphin(Tursiopstruncatus) 4 4

Finwhale(Balaenopteraphysalus) 1 1

Harbourporpoise(Phocoenaphocoena) 47 47

Killerwhale(Orcinusorca) 2 2

Long-ﬁnnedpilotwhale(Globicephalamelas) 6 6

Minkewhale(Balaenopteraacutorostrata) 2 2

Risso’sdolphin(Grampusgriseus) 4 4

Short-beakedcommondolphin(Delphinusdelphis) 9 9

Sowerby’sbeakedwhale(Mesoplodonbidens) 2 2

Spermwhale(Physetermacrocephalus) 1 1

Scottishcoastline–NorthSea

Atlanticwhite-sideddolphin(Lagenorhynchusacutus) 11 11

Bottlenosedolphin(Tursiopstruncatus) 2 2

Harbourporpoise(Phocoenaphocoena) 56 56

Killerwhale(Orcinusorca) 1 1

Long-ﬁnnedpilotwhale(Globicephalamelas) 5 5

Minkewhale(Balaenopteraacutorostrata) 3 3

Risso’sdolphin(Grampusgriseus) 4 4

Seiwhale(Balaenopteraborealis) 1 1

Short-beakedcommondolphin(Delphinusdelphis) 4 4

Sowerby’sbeakedwhale(Mesoplodonbidens) 2 2

Spermwhale(Physetermacrocephalus) 1 1

Europeanotter(Lutralutra) 34 34

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Table3

ResultsbyspeciesofserumsamplestestedforToxoplasmagondiiwithmodiﬁedagglutinationtest(MAT),enzyme-linkedimmunosorbentassay(ELISA)witha sample-to-positiveratio(S/P)cut-offat20and50,andimmunoﬂuorescentassay(IFA).

MAT ELISA(S/P≥20) ELISA(S/P≥50) IFA

No.tested No.positive(%) No.tested No.positive(%) No.tested No.positive(%) No.tested No.positive(%)

Dutchcoastline–NorthandWaddenSea 31 4(13)

Harbourporpoise(Phocoenaphocoena) 31 4(13)

Scottishcoastline–AtlanticOcean 105 54(51) 98 7(7.1) 98 3(3.1) 100 7(7.0)

Atlanticwhite-sideddolphin(Lagenorhynchusacutus) 7 7(100) 7 0(0) 7 0(0) 7 0(0)

Bottlenosedolphin(Tursiopstruncatus) 4 3(75) 4 0(0) 4 0(0) 4 0(0)

Finwhale(Balaenopteraphysalus) 1 0(0) 1 0(0)

Greyseal(Halichoerusgrypus) 5 2(40) 4 0(0) 4 0(0) 4 0(0)

Harbourporpoise(Phocoenaphocoena) 47 22(47) 43 0(0) 43 0(0) 44 1(2)

Harbourseal(Phocavitulina) 6 0(0) 6 0(0) 6 0(0) 6 0(0)

Killerwhale(Orcinusorca) 2 1(50) 2 0(0) 2 0(0) 2 0(0)

Long-ﬁnnedpilotwhale (Globicephalamelas) 6 5(83) 5 3(60) 5 2(40) 5 3(60)

Minkewhale (Balaenopteraacutorostrata) 2 0(0) 2 0(0) 2 0(0) 2 0(0)

Risso’sdolphin(Grampusgriseus) 4 2(50) 4 1(25) 4 0(0) 4 1(25)

Short-beakedcommondolphin(Delphinusdelphis) 9 5(56) 9 1(11) 9 0(0) 9 1(11)

Sowerby’sbeakedwhale (Mesoplodonbidens) 2 1(50) 2 0(0) 2 0(0) 2 0(0)

Spermwhale(Physetermacrocephalus) 1 1(100) 1 0(0) 1 0(0) 1 0(0)

Stripeddolphin(Stenellacoeruleoalba) 5 2(40) 5 0(0) 5 0(0) 5 0(0)

White-beakeddolphin(Lagenorhynchusalbirostris) 4 3(75) 4 2(50) 4 1(25) 4 1(25)

Scottishcoastline–NorthSea 156 63(40) 143 21(15) 143 13(9.1) 144 23(16)

Atlanticwhite-sideddolphin(Lagenorhynchusacutus) 11 4(36) 11 0(0) 11 0(0) 11 0(0)

Bottlenosedolphin(Tursiopstruncatus) 2 0(0) 1 0(0) 1 0(0) 1 0(0)

Greyseal(Halichoerusgrypus) 12 3(25) 9 1(11) 9 0(0) 9 0(0)

Harbourporpoise(Phocoenaphocoena) 56 21(38) 53 0(0) 53 0(0) 54 1(2)

Harbourseal(Phocavitulina) 14 4(29) 11 2(18) 11 1(9) 11 2(18)

Killerwhale(Orcinusorca) 1 1(100) 1 0(0) 1 0(0) 1 0(0)

Long-ﬁnnedpilotwhale (Globicephalamelas) 5 3(60) 5 1(20) 5 1(20) 5 1(20)

Minkewhale(Balaenopteraacutorostrata) 3 1(33) 3 0(0) 3 0(0) 3 0(0)

Risso’sdolphin(Grampusgriseus) 4 2(50) 3 1(33) 3 0(0) 3 1(33)

Seiwhale(Balaenopteraborealis) 1 1(100) 1 0(0) 1 0(0) 1 0(0)

Short-beakedcommondolphin(Delphinusdelphis) 4 3(75) 4 1(25) 4 0(0) 4 1(25)

Sowerby’sbeakedwhale (Mesoplodonbidens) 2 1(50) 2 0(0) 2 0(0) 2 0(0)

Spermwhale(Physetermacrocephalus) 1 0(0) 1 0(0) 1 0(0) 1 0(0)

Stripeddolphin(Stenellacoeruleoalba) 4 2(50) 4 0(0) 4 0(0) 4 0(0)

White-beakeddolphin(Lagenorhynchusalbirostris) 2 1(50) 2 1(50) 2 0(0) 2 0(0)

Europeanotter(Lutralutra) 34 16(47) 32 14(44) 32 11(34) 32 17(53)

Total 292 121(41) 241 28(12) 241 16(6.6) 244 30(12)

Table4

Primersandprobesusedinthereal-timePCR.

Target Name Sequence5_→₃ ₅_{Modiﬁcation}a ₃_{Modiﬁcation}b _{Concentration}c

Primers T.gondii T2 CGGAGAGGGAGAAGATGTT 400nM

T3 GCCATCACCACGAGGAAA 400nM

Cellularr18S VF1 GATTAAGTCCCTGCCCTTT 200nM VR1 CACACCGCCCGTCGCTACTACC 200nM Probes T.gondii TP CTTGGCTGCTTTTCCTGGAGGG FAM488 BHQ1 66nM

Cellularr18S VP CACACCGCCCGTCGCTACTACC TexasRed547 BHQ2 66nM

a_Fluorophore_used_as_reporter. b _Quencher_molecule.

c _Final_{concentration}_per_reaction.

turer’sinstructions,sampleswerealsotestedata1/4000dilution tomonitorforapotentialprozoneeffectata1/40dilution.

2.3.2. Enzyme-linkedimmunosorbentassay(ELISA)

Two hundred and forty-one sera of the 292 collected sera were tested for the presence of anti-T. gondii IgG withthe ID Screen Toxoplasmosis Indirect Multi-species ELISA kit (ID.vet, Montpellier,France).Samplesweretestedand analysed accord-ingtothemanufacturer’sinstructions.Brieﬂy, eachsamplewas diluted1/10inthesupplieddilutionbufferand100␮lwasapplied in the antigen-coated 96 well plate. After 45min of incuba-tionat room temperature,the platewas washed 3 timeswith the supplied washing buffer. 100_␮l of the multispecies con-jugate diluted 1/10 in the supplied buffer was then applied in each well.The plate was incubated for another 30min and

washed3times.Thecolorimetricreactionwasdonewiththe sup-plied3,3,5,5-tetramethylbenzidineand stoppedafter15min withthesuppliedstopsolution.Foreachsamplethe sample-to-positive(S/P)ratiowascalculatedusingtheopticaldensity(OD) as (ODsample−ODnegativecontrol)/(ODpositivecontrol−ODnegativecontrol),

with the positive and negative controlssupplied with the kit. Accordingtothemanufacturer’sinstructions,asamplewas con-sideredpositiveiftheS/Pratiowas≥50.

2.3.3. Immunoﬂuorescentassay(IFA)

Twohundredandforty-fourofthe292collectedserumsamples weretested forthepresenceofanti-T.gondiiIgGusingIFA.The IFAwasperformedusingcommerciallyavailableantigencoated slides(Toxo-SpotIF,Biomerieux,Capronne,France).Theserawere diluted1/50inPBSand50␮lofeachserumdilutionwasappliedon

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Table5

Conjugatesusedintheimmunoﬂuorescenceassay.

Fluoresceinisothiocyanatelabelledconjugatea _Manufacturer _Used_for

goatanti-bovineIgG(H+L) KPL(Gaithersburg,Maryland,USA) Dolphins,porpoises,whalesb

goatanti-dogIgG(H+L) KPL(Gaithersburg,Maryland,USA) Sealsc

goatanti-ferretIgG(H+L) KPL(Gaithersburg,Maryland,USA) Otterc

a_(H₊_L)_conjugate_against_IgG_heavy_and_light_chains. b_Nollens_et_al.₍₂₀₀₈₎_.

c _Miller_et_al.₍₂₀₀₂₎_.

theantigencoatedslides.Theslideswereincubatedat37◦Cina sat-uratedmoistatmospherefor30min.Theslideswerethenwashed oncewithPBSandoncewithdistilledwater.Afterdrying,30␮lof theﬂuoresceinisothiocyanatelabelledconjugatewasappliedina 1/25dilutioninPBSwithEvansBlueascounterdye.Thefollowing conjugateswereused:anti-bovineIgGtotestcetaceansera(Jaber etal.,2003),anti-ferretIgGtotestottersera(Milleretal.,2002)and anti-dogIgGtotestsealsera(Carlson-Bremeretal.,2015).Table5 liststheconjugatesusedpertestedanimalspecies.Afterwashing anddrying,theslideswerereadwithaﬂuorescencemicroscope (NikoneclipseNi-U).Samplesthathadtestedpositiveand nega-tiveinELISAwereusedaspositiveandnegativecontrolsforeach typeofconjugate.

2.3.4. Muscleﬂuid

MusclefluidsamplesweretestedusingMAT.Musclefluidwas collectedbysqueezingthedefrostedmusclesamplesabovea15ml centrifugetube.Firstly,47samplesfromharbourporpoises (Pho-coenaphocoena)fromTheNetherlandsweretestedatthefollowing dilutions:1/4,1/200and1/400.Sincenopositivitywasfoundat the1/200and1/400dilution,theremaining146musclefluid sam-plesweretestedata1/2,1/10and1/64dilutiontobetterestimate theantibodytitres.Doubtfulresultswereretestedfor confirma-tion.Forbesetal.(2012)showedthata1/10dilutionofmuscle fluidsamplesfrombothexperimentallyinfectedandnegativepigs identifiedallpositivecases.Therefore,samplesinthissurveythat showedagglutinationat1/10dilutionwereconsideredpositive. 3. Results

3.1. PCR

Thepositiveandnegativecontrolsandthenegativeresultsofthe non-templatecontrolsconﬁrmedpropertestingconditions with-outcontaminationduringextraction.

Twooutof193brainsampleswerepositiveforT.gondiiDNA:a cerebrumsamplefromayoungmaleharbourporpoise(Phocoena phocoena)strandedin2010nearKatwijk,TheNetherlands(Ctvalue of 34.4)and a cerebrum samplefrom anadult femaleharbour porpoise(Phocoenaphocoena)strandedin 2010nearNoordwijk, TheNetherlands(Ctvalueofcycle40.4).Thecerebellumsamples frombothporpoisestestednegative.Furtherhistopathologicaland immunohistochemicalexaminationsofbraintissuesofboth ani-malscouldnotconﬁrmthepresenceofT.gondii.Othertissuesof theseanimalswerenotavailableforfurtherserologicaltesting. 3.2. Serology—serumsamples

Table3summarizestheresultsoftheserologicalassaysper ani-malspecies.MarinemammalscollectedfromtheDutchcoastline, theScottishcoastline alongtheAtlanticOceanand theScottish coastlinealongtheNorthSeashowedaseroprevalenceof13%,51% and39%,respectivelyinMAT.Sixteenofthe34(47%)Europeanotter (Lutralutra)samplesfromtheScottishcoastlinealongtheNorthSea

testedpositive.Fromthe261seracollectedfromScotland,244and 241serawereretestedusingIFAandELISA,respectively.InIFA,13 outof212marinemammalsamples(6.1%)testedpositiveata1/50 dilutionand17ofthe32(53%)Europeanotter(Lutralutra)samples testedpositive.ThecommercialELISAyieldedaseroprevalenceof 2%inmarinemammalsampleswhenusingaS/Pratiocut-offof50, and7%whenloweringthecut-offto20.ELISAidentiﬁed11(34%) and14(44%) ofthe32Europeanotter(Lutralutra)samplesper cut-offrespectively.

Theoneharbourporpoise(Phocoenaphocoena)fromtheAtlantic Ocean,whichtestedpositiveinIFA,alsotestedpositiveinMAT. Theoneharbourporpoise(Phocoenaphocoena)fromtheNorthSea, whichtestedpositiveinIFA,testednegativeinMAT.Theonegrey seal(Halichoerusgrypus)fromtheNorthSea,which tested posi-tiveinELISAwitha cut-offvalueof20, testedpositivein MAT. Table 6 shows the agreement betweenresults in testing long-finnedpilotwhale(Globicephalamelas),Risso’sdolphin(Grampus griseus), short-beaked common dolphin (Delphinus delphis) and white-beakeddolphin(Lagenorhynchusalbirostris)samples. Agree-mentwasdefinedasasimilarresultinallthreediagnosticmethods. Ofthe209marinemammalstestedwithallthreeserological tech-niques,4(1.9%)marinemammals(1harbourseal(Phocavitulina),2 long-finnedpilotwhales(Globicephalamelas)and1white-beaked dolphin(Lagenorhynchusalbirostris))testedpositiveonalltestsand 104marinemammals(50%)testednegativeonalltests,leaving101 marinemammals(48%)withinconsistentresultsacrosstests.Nine outof32(28%)Europeanotter(Lutralutra)samplestestedpositive onallthreetests.

3.3. Serology—muscleﬂuidsamples

Out of the ﬁrst batch of 47 muscle samples from The Netherlands,21(43%)testedpositiveina1/4dilutioninMAT.No positivitywasfoundat1/200and1/400dilutions.Furthertesting wasdoneusinglowereddilutions,i.e.,1/2,1/10,1/64.Outofall146 animalstestedwiththeselatterdilutions,only12samples(8.2%) showedagglutinationina1/2dilution(datanotshown).Allthese sampleswerenegativeusingthecut-offvalueof1/10.

4. Discussion

Inthisstudy,589marinemammalsof17differentspeciesand 34Europeanotters(Lutralutra)wereexaminedforthepresence ofT.gondii,usingvariousdetectionmethods.PreviousstudiesonT. gondiiinmarinemammalsshowedthatthisparasiteisabletoreach andinfectmarinemammals(Cabezónetal.,2011;Rengifo-Herrera etal.,2012).InfectionwithT.gondiiinmarinemammalsmayresult inclinicaldisease(Carlson-Bremer etal.,2015;Mikaelianetal., 2000; Van Bressemet al., 2009). Thishasbeenclearly demon-stratedinCaliforniaseaotters(Euhydralutrisnereis)(Milleretal., 2004),Hector’sdolphins(Cephalorhynchushectori)(Roeetal.,2013) andharbourporpoises(Phocoenaphocoena)(Herderetal.,2015).T. gondiiclinicaldiseasehasalsobeenrecordedwithco-infectionsof Brucellaceti,andListeriamonocytogenesinstripeddolphins(Stenella

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Table6

Agreementbetweenresultsinserologicaltestingoflong-ﬁnnedpilotwhale,Risso’sdolphin,short-beakedcommondolphinandwhite-beakeddolphinsamples.

Animalno. MAT ELISA(S/P≥20) ELISA(S/P≥50) IFA Agreement Long-ﬁnnedpilot

whale

(Globicephalamelas)

AtlanticOcean 1 N N N N Yes

2 P P P P Yes

3 P P N P No

4 P − − − −

5 P P P P Yes

6 P N N N No

NorthSea 1 N N N N Yes

2 P N N N No 3 N N N N Yes 4 P N N N No 5 P P P P Yes Risso’sdolphin (Grampusgriseus)

AtlanticOcean 1 N N N N Yes

2 N N N N Yes 3 P P N P No 4 P N N N No NorthSea 1 N − − − − 2 N N N N Yes 3 P P N P No 4 P N N N No Short-beakedcommon dolphin (Delphinusdelphis) AtlanticOcean 1 P N N N No 2 P N N N No 3 P N N N No 4 P N N N No 5 P N N N No 6 N P N P No 7 N N N N Yes 8 N N N N Yes 9 N N N N Yes NorthSea 1 P P N P No 2 P N N N No 3 P N N N No 4 N N N N Yes White-beakeddolphin (Lagenorhynchus albirostris)

AtlanticOcean 1 P P P P Yes

2 P N N N No

3 P N N N No

4 N N N N Yes

NorthSea 1 P P N N No

2 N N N N Yes

MAT=modiﬁedagglutinationtest;ELISA=enzyme-linkedimmunosorbentassay;S/P=sample-to-positiveratio;IFA=immunoﬂuorescentassay;P=positive,N=negative, −=nottested.

coeruleoalba)(DiGuardoetal.,2010;Grattarolaetal.,2016),and withmorbillivirusinﬁnwhales(Balaenopteraphysalus)(Mazzariol etal.,2012).Additionally,researchonT.gondiiinmarinemammals isrelevantasT.gondiiactsasanindicatorformarinepollutionand marinemammalsaresentinelspeciesforthehealthofthemarine ecosystemstheylivein(deMouraetal.,2014;Gibsonetal.,2011; Moore,2008).

Samplingofwildlifeischallengingandcomeswithunknown factorsandbiases(Nusseretal.,2008).Convenience-based sam-pling is not a random selection of animals and therefore does notrepresentthecompletewildlifepopulation.Inthisstudyonly strandedanimalsweretested.Sealsaremore coastalspecies in comparisonwithmostcetaceansandcouldthereforebe overrepre-sented.Thesampledpopulationwaslargelydominatedbyharbour porpoises(Phocoenaphocoena),asthis isthemostabundantsea mammalspeciesintheinvestigatedareas.Theconvenience-based samplingfurtherexplains thedifferences in sampling methods usedacrossanimals.Indeed,thesetof samplesperanimal var-iedacrossinstitutesandfurtherdependedonfactorssuchasthe preservationstateoftheanimalcarcass–e.g.,fewersampleswere takenfromdecomposed animals.Asourstudycoveredsamples takeninatimespanof21years,notallnecropsiedanimalswere sampledspeciﬁcallyfor Toxoplasmaresearch; brainand muscle sampleswerethereforenotavailablefromallanimals.

Thediagnostictechniquesusedinthisstudyarenotvalidated for marinemammalsby themanufacturers and thereforetheir

diagnostictestcharacteristicsformarinemammalsamplesarenot exactlyknown.For detectingT. gondii inwildanimals, brainis demonstratedtobeausefulsamplingtissue(DeCraeyeetal.,2011). Outof193animalstestedbyPCR,twoharbourporpoise(Phocoena phocoena)cerebrumsamplesprovedpositiveforT.gondiiDNA.The cerebellumsamplesofthesametwoanimalstestednegativein PCR,whichmightbeduetotheheterogeneousdistributionofT. gondiiinthehost’sorgansortoalowintensityofinfection.Also thesamplesizeof400mgforDNAextractionislow.UsingPCRon marinemammalsamplestodetectT.gondiiDNAhasnotidentified manypositiveanimalsinotherstudieseither(Dubeyetal.,2009; Omataetal.,2006).Furthermore,alargediscrepancybetweenthe PCRandserologicresultswasfound, similartootherstudieson T.gondiiprevalenceinotherhosts(DeCraeyeetal.,2011;Wang etal.,2011;Prestrudetal.,2007).Thiscouldbedueto difficul-tiesindeterminingserologicalparameterssuchassensitivityand specificity.

Blanchetetal.(2014)testedtissuefluidandblood(thawedand centrifuged)withMATandELISA.Inthecurrentstudy,no chloro-formclean-upwasappliedontheserumsamples.Inaccordanceto thefindingsofBlanchetetal.(2014),theresultsobtainedbyIFA andELISAindeedgavealowernumberofpositiveanimals com-paredtotheMATresults.MATmightbeusefulasafirstscreening method,buttheresultsmustbeinterpretedwithcaution.

Inthecurrentstudy,usingMAT,51%and39%oftheserafrom marinemammalsstrandedonScottishcoastlinesalongtheAtlantic

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OceanandtheNorthSea,respectively,werefoundpositive com-pared to13% of thesera from animals strandedon the Dutch coastline.Extrapolatingthesedifferencesinseroprevalenceshould bedonewithcaution,becausethesamplingwasbiasedandnot representativeforthewholepopulation.Nevertheless,the differ-enceinseroprevalencebetweentheanimalsfoundonDutchand Scottishcoastlinesisstrikinganddeservesfurtherinvestigation.

WhileMATwasdesignedfortestingserumsamples,the Toxo-Screen DA® _test _kit _was_reported _to_give_reliable _results_when

usedonswinemusclefluid(Forbesetal.,2012).MAThasproven itsusefulnessasadiagnosticmethodinexperimentallyinfected seals(Gajadharetal.,2004).Inourstudy,nopositiveresultswere obtainedfromthemusclefluidsamplesata1/10cut-offvalueas describedbyForbesetal.(2012).Themusclefluidsamplesshowed variousdegreesofdegradation,whichmayhaveinterferedwith thereadingofMAT.

Acrossthethreeserologicalmethodsusedinthisstudy,more sampleswerefoundpositiveinMATthaninELISAandIFA.Amajor differencebetweenthesetestsistheuseofamultispeciesconjugate inELISAandspeciesspecificconjugatesinIFA.Finding appropri-atespecificconjugatesforallthetestedspeciesofseamammalsis notonlychallenging(asformostofthemtheyarenotcommercially available),itisalsoratherexpensiveandimpractical.Therefore,for theIFAwereliedonpreviouslypublishedcross-reactivitybetween species(Milleretal.,2002;Nollensetal.,2008)(Table5).Another waytoapproachthisproblemisusingamultispeciesconjugate consistingofproteinG,whichnon-specificallybindstotheFc por-tionofIgGantibodies.ThecommercialmultispeciesELISAusedin thisstudyreliesonsuchaproteinGconjugatefordetectingthe adsorbedIgG’s.However,it is alsopossiblethattheconjugates usedinIFAandthecommercialELISAarenotoptimal,leadingto possiblefalsenegatives.Thefactthatweobtainedsimilarresults withIFAandELISAwhenloweringtheS/Pcut-offratioofthe lat-terto≥20,couldbeexplainedbyproteinGhavinglessaffinityfor marinemammalantibodies.Indeed,differencesinbinding affini-tiesbetweenproteinGandimmunoglobulinshavealreadybeen describedforvariousotheranimalspecies(Phillips,2015).

Althoughitremainspoorlyunderstoodhowmarinemammals acquireT. gondiiinfection,it hasbeenshown that certainprey species,suchasanchovies(Engraulidaefamily),sardines (Clupei-daefamily)andbivalves,areabletoconcentrateviableT.gondii oocystsintheirdigestivetract(Massieetal.,2010).Astheseprey speciesarealsoconsumedbyhumans,T.gondiiinfectioninmarine mammalscouldbeanindirectindicatoroftheriskthesespecies poseforcausingT.gondiiinfectioninhumans.Ottersare hypothe-sizedtobeaninterestinglinkinthetransmissionofT.gondiifrom landtomarineenvironmentsandwerethereforeincludedinthis study(Conradetal.,2005).Ahigherpercentageofsamplestesting positiveonallthreetestswasfoundinotters(28%)comparedto marinemammals(2%),supportingthishypothesis.Thepresenceof T.gondiioocystsinthemarineenvironmentcouldbeanindicator ofmarinepollutioningeneral(Meirelesetal.,2004;Stewartetal., 2008).However,formarinemammalstobetrulyusedassentinels forboththezoonoticandecologicalimplicationsofT.gondii infec-tion,furtherresearchisneededtoovercomethecurrentdiagnostic difﬁculties.

5. Conclusions

ThisstudyhasconfirmedthepresenceofT.gondiiDNAand anti-bodiesinmarinemammalslivingintheNorthSea(andEastern AtlanticOcean), toour knowledge for thefirst time. The com-parisonbetweenthedifferentdetectionmethodshighlightedthe diagnosticdifficultieswhentestingmarinemammalsamplesfor thisparasite.Togainmoreinsightsinthesignificanceandpotential

threatofT.gondiiinmarinemammals,theserologicaltechniques fordetectingT.gondiiantibodiesinmarinemammalsshouldbe furtheroptimized.Despitethesediagnosticdifﬁculties,the differ-enceinseroprevalencebetweenanimalsfoundontheScottishand Dutchcoastlinesshouldbefurtherinvestigated.

Acknowledgments

WewouldliketothankZehraYildirimer,NabilaElMoussaoui, Mariel ten Doeschate, Kornelia Schmidt, Miriam Hillmann, Jur-genWillemsandLaurieFlamentfortheirkindcooperationduring thisproject.SamplesgainedfromUtrechtUniversitywere possi-blethroughagrantfromtheDutchMinistryofEconomicAffairs (140000353).Wearegreatlyindebtedtoallvolunteersspending theirtimeandefforttocollectthestrandedanimals.Withouttheir dedication,thisprojectwouldnothavebeenpossible.Themarine mammalswashedashoreinBelgiumwerecollectedbytheRoyal Belgian InstituteofNatural Sciences(RBINS). We wouldlike to thankandacknowledgeallmembersoftheFrenchstranding net-worksfortheircontinuouseffortincollectingstrandingdata.We speciﬁcallywouldliketothankthefollowing natureprotection associationsofNorthernFrance:OCEAMM(Observatoirepourla Conservationetl’EtudedesAnimauxetMilieuxMarins),LPA (Ani-malProtectionLeague)andCMNF(CoordinationMammalogique duNorddelaFrance)andtheObservatoirePELAGISinchargeofthe nationalstrandingnetwork.TheObservatoirePELAGISisfundedby theministryinchargeoftheenvironment,andbytheCommunauté d’AgglomérationdelaVilledeLaRochelle,withsupportfromthe RégionPoitou-CharentesandtheEuropeanUnion.

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