CHAPTER 3 - 2019 ENVIRONMENTAL SCIENCES KAPOSVÁR UNIVERSITY FACULTY OF AGRICULTURAL AND GÁBOR N

5. CHAPTERS

5.3. CHAPTER 3

Benzimidazole resistance within red deer, roe deer and sheep populations within a joint habitat in Hungary

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SmallRuminantResearch

j o u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / s m a l l r u m r e s

Benzimidazoleresistancewithinred deer,roedeerand sheep populationswithinajointhabitatinHungary

GáborNagy^a,∗,ÁgnesCsivincsik^b,LászlóSugár^c,AttilaZsolnai^d

aKaposvárUniversity,DepartmentofAnimalNutrition,GubaS.u.40.,H-7400Kaposvár,P.O.Box16.,Hungary

bKaposvárUniversity,InstituteofDiagnosticImagingandRadiationOncology,H-7400Kaposvár,GubaS.u.40.,Hungary

cKaposvárUniversity,DepartmentofPhysiologyandAnimalHygiene,H-7400Kaposvár,GubaS.u.40.,Hungary

dKaposvárUniversity,DepartmentofGameBiologyandEthology,H-7400Kaposvár,GubaS.u.40.,Hungary

a r t i c l e i n f o

Articlehistory:

Received22December2016

Receivedinrevisedform8February2017 Accepted9February2017

Theanthelminticresistanceofgastrointestinalnematodesisoneofthemostimportant,economicrisk factorsingrazingruminantsystems,allovertheworld.Wehaveinﬁnitesimalinformationaboutthe resistancestatusofnematodesindeerspecies.OuraimwastodeterminethepresenceofBZresistance inthegeneralistworm,Haemonchuscontortusinpasturedsheepandfreerangingredandroedeerby RFLP-PCRmethodbasedonthedetectionPhe200Tyrsinglenucleotidepolymorphism.Byinvestigation of70wormsfromeachhostspecies,thehomozygoussusceptiblegenotypewasthemostrepresentative inthereddeer(100%),thehomozygousresistantgenotypewasmostprevalentinthesheep(68.6%)and moderateintheroedeer(17.1%),whiletheheterozygousgenotypewasobservedinequalproportionin thesheepandroedeer(28.6%).Ourresultssuggestthatoverlappinghabitatsofsheepﬂocksandroedeer couldcontributetotheoccurrenceandspreadofresistantallelewithinwildlife.

1. Introduction

Theanthelminticresistance(AR)ofgastrointestinalnematodes isoneofthemostimportanteconomicriskfactorsingrazing rumi-nantsystemsallovertheworld(SutherlandandLeathwick,2011;

Papadopoulosetal.,2012;Roseetal.,2015).Theﬁrstappearingof resistanceagainstbroadspectrumdrugsdatesbacktothe1960’s andits spreading couldbeobservedin everyclass (FAO, 2004;

Kaplan,2004).

Although,thephenomenonofARiswellknowninmajor domes-ticatedruminantspecies,asinthecaseofsheep(Barrereetal., 2013;Pe ˜na-Espinozaetal.,2014;Chaudhryetal.,2015);within theminorfood-producingspecies(eg.deerspecies),wehavemuch less information about the AR status of theirnematodes. In a study(Mackintoshetal.,2014),theresistancetomoxidectinand abamectinwasdemonstratedbyfaecaleggcountreduction test (FECRT)inreddeer.TheauthorssuggestedthatARof Ostertagia-type species was in the background of drug ineffectiveness. A similarresultwasshowedin thecase of albendazoleand iver-mectin (Mylrea et al., 1991) in fallow deer. Both studies were carriedoutunderfarm circumstances.It washypothesizedthat

∗Correspondingauthor.

E-mailaddress:nagy.gabor@ke.hu(G.Nagy).

failureoftreatmentswasaffectedbyseveralfactorsincludingthe ARofgastrointestinalnematodes.Chintoan-Utaetal.(2014)tested andconﬁrmedthecrosstransmissionofHaemonchuscontortusand its benzimidazole(BZ) resistancebetween thefallow deer, red deer,roedeer,cattle,and sheep.Bothoftheirresistance detec-tionmethods(egghatchtestandmoleculartest)demonstratedthe presenceofARinthewormsderivedfromroedeer.Threesingle nucleotidepolymorphisms(SNPs)inthe␤-tubulingeneisotype1 havebeenassociatedwithbenzimidazoleresistanceinH. contor-tus.ThePhe200Tyrmutationhasbeenfoundineverycountryata relativelyhighfrequency.TheSNPsatcodons167and198have alsobeenreportedin multiplecountriesbuthave a more vari-ableoccurrenceand aregenerallypresent ata lowerfrequency thanthePhe200Tyrmutation(Chaudhryetal.,2016).Sincethe mostcommonmolecularmechanismthat confersBZresistance intrichostrongyles insmallruminantsinvolvesa phenylalanine totyrosinemutationatcodon200of ␤-tubulingene isotype1;

therefore,moleculardiagnosticmethodsbasedonthedetection ofthisSNPareusedmostfrequentlytoidentifyBZresistanceinH.

contortus(Colesetal.,2006).

A Hungarian study (Nagy et al., 2016)demonstrated a high homozygousresistantgenotype(70%)andresistanceallele(85%) proportioninfarmedreddeer.Theresultsofamolecular diagnos-ticmethodshowedthatroutine,long-termadministrationofthe

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samedrugcouldcontributetotheappearanceofARinH.contortus inthereddeer.

Recently,anewapproach,refugiatheory,aroseduringthe inves-tigationof ARin natural environment. It hypothesizes thatthe presenceofwildruminantsonpasturesoflivestockprovidesa sig-niﬁcantpercentageofhomozygoussensitiveonesamongthetotal amountofinfectiouslarvae.Regularinfectionbythismixed pop-ulationimpedestheunlimitedspreadofresistancewithinaworm populationinfectingalivestockﬂockconcerned.(VanWyk,2001).

In this study, we aimed to determine the presence of BZ resistance in the generalist worm, Haemonchus contortus in a microregion,wheretheresourcespartlysharedbysheepandfree rangingredandroedeer.

2. Materialsandmethods 2.1. Studysite

Our investigationwascarried out in southwesternHungary, withinacontiguousareawithoutanynaturalorman-built isolat-ingelementsthatimpedemovementsofanimals(Fig.1).Thestudy site(approximately30km²)characterizedby145–276maltitude abovesealevel,sub-Mediterraneanclimate,withsome submon-taneoushabitatpatches,with10^◦Cannualmeantemperatureand 630–800mmannualprecipitation.Theproportionofforestsand agriculturalareasonstudysiteare about55%and 45%, respec-tively.Inthecoreofthesite,a6000-hamonoblockforestexists withthedominationofoaks(Quercusspp.),limes(Tiliaspp.), horn-beam(Carpinusbetulus)andEuropeanbeech(Fagussylvatica).The agriculturalareas(viz.grasslands,pastures,arablelands,andold orchards)arethemosttypicalontheperipheryofthestudysiteand theyprovideaheavilyfragmentedlandscapestructure.The aver-agedensityofreddeer,roedeer,andsheepare1.71,0.84and0.17 animal/km²,respectively.Ourdatawerebasedonhunting statis-tics(hunteddeerspecimen/km²);whileinthecaseofsheep,itwas calculatedbytheofﬁcialregistryoftheHungarianSheepandGoat BreedersAssociation.

2.2. Wormcollectionandmoleculardiagnosticprocedure

DuringtheintervalfromSeptemberof2013tillAugustof2015, wecollected38abomasifromreddeer(N=14),roedeer(N=14) andsheep(N=10from5differentﬂocks).Theorgansofdeerwere collectedfromhuntingbagsinregularhuntingseasons,whilesheep weresampledataregionalabattoir.Afterevisceration,each abo-masumwasplacedseparatelyintoaplasticbagimmediately,and eachwasstoredat −18^◦Cuntilexamination.Aftermelting,the organswereopenedalongsidethebigcurvature,andthecontent wasplacedintoaplasticjar,whilethemucosawaswashed thor-oughly.Inthecaseofdeerspecies,5Haemonchuscontortusmales werepickedrandomlyfromeachorgan,while7specimenswere collectedfromeverysinglesheepabomasum.Formorphological identiﬁcation,weusedLichtenfels’setal.(1994)work.Thatmethod isbasedonthelengthoftheleftandrightspiculebarbs.

Inordertogenotypethecollectedworms,weusedthe detec-tionofPhe200Tyrsingle-nucleotidepolymorphismsoncodon200 of␤-tubulingeneisotype1,whichisthemostcommonmolecular mechanismconferringBZresistanceintrichostrongylesofsmall ruminants(Colesetal.,2006).DNAlysatesweremadeseparately from210 adultmale worms. The appliedRestriction Fragment Length Polymorphism-PCR method and the primer sequences (AvikaF:5-CTACCCTTTCCGTCCATCAA-3 andAvikaR:5- TGAA-GACGAGGGAATGGAAC -3) were previously detailed by Tiwari etal.(2006).PCRreactionswereperformedinatotalvolumeof 10␮l,containing200␮MofeachdNTP,0.2␮Mprimers,10×PCR

buffer,0.5unitDynazymeDNApolymerase(FinnzymesOy,Espoo, Finland)and 100ng genomicDNA.ThePCR cyclingproﬁle con-sistedofdenaturationat94^◦Cfor3min,45cyclesofdenaturationat 94^◦C(for30s),annealingat56^◦C(for30s),andextensionat72^◦C (for30s),followed byaﬁnalextensionat 72^◦C for5min.After ampliﬁcation,1␮lTaaIrestrictionendonuclease(5U/␮l;Thermo FisherScientiﬁcInc.,Waltham,Massachusetts,USA),and1.22␮l 10×digestionbufferwereaddedtothetotalPCRvolume. Diges-tionwascarriedoutat65^◦C,overnight.Digestedfragmentswere resolvedon4%agarosegelstainedwithGRGreenNucleicAcidGel StainandvisualizedunderUVlight.Genotypedeterminationwas basedonthefragmentlengthssuchas305bpforSalleleand257bp forRallele.

2.3. Statisticalmethods

In this survey, the nomenclature of genotype and allele frequency,wefollowed,weredetailedbyPierce(2012).The pro-portionsweredeterminedwith95%conﬁdenceinterval (CI95%) (Reiczigel et al., 2010). For comparison of genotype and allele frequencyintheruminantpopulations,chi-squaretestwith sig-niﬁcancelevelof0.05wasperformedwithBonferronicorrection usingRstatisticalsoftwarei3863.3.0version.

3. Results

Inthisstudy,weexaminedaltogether210maleworms,which wereidentiﬁedasHaemonchuscontortus,todeterminethe propor-tionof Phe200TyrSNPoncodon200of␤-tubulingene isotype 1 within three different ruminant species. Among the worms, 110weregenotypedashomozygoussusceptible(SS),40as het-erozygous (RS) and 60 as homozygousresistant (RR), thus the totalfrequencyofSS,RSandRRwas52.4%(CI95%=45.5–59.1%), 19%(CI95%=14.1–24.9%)and28.6%(CI95%=22.7–35.2%), respec-tively. Considering thealleles,the proportionof susceptible (S) andresistant(R)allelewas61.9%(CI95%=57.2–66.5%)and38.1%

(CI95%=33.5–42.8%), respectively. Distribution of the different genotypewasshowedawidevarietyinhosts.TheSSwasthemost representativeinthereddeer,whereallofthewormsbelongedto thisgenotypegroup.TheRRwasmostprevalentinthesheepandit wasmoderateintheroedeer,whiletheRSwasobservedinequal proportioninthesetwohosts(Table1).Differenceofallele fre-quenciesbetweenthehostpopulationswasconﬁrmedsigniﬁcant bychi-squaretest.

4. Discussion

AlthoughseveralstudieswereconcernedwithARinsmall rumi-nants(Papadopoulosetal.,2012;Roseetal.,2015),ourknowledge isverydeﬁcientinconnectionwithwildruminants(Chintoan-Uta etal.,2014;Mackintoshetal.,2014)WeinvestigatedtheBZ resis-tancestatusofH.contortus,collectedfromsheepandtwosympatric deerspeciesand ourresultsconﬁrmedconsiderabledivergence betweenthehosts. InHungarianveterinary practice,oneof the mostpreferreddruggroupsisBZs;therefore,theirusageinparasite managementisconsideredverygeneral.Ourresultssuggestthat thehighlevelofRalleleproportioninsheepinthisregionisderived fromthehightreatmentfrequencyandthelong-termunrotated drugapplication;asourpreviousworkconﬁrmedit(under publi-cation).Ontheotherhand,inHungary,theusageofanthelmintics andotherprescriptionmedicinesforhuntingpartiesarerestricted bythefoodsafetylaw,becausetheconsumptionofthe therapeu-ticdosecannotbeensuredandthewithdrawalperiodcannotbe controlledinwildlife.Inconsequence,thehuntingpartiesofthe studiedareahaveneverappliedanyanthelmintics.Basedonthese

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Fig.1.LocalizationofcollectedHaemonchuscontorusfromdifferenthosts(circleswithx=reddeer;triangles=roedeer;blackdots=sheep).(Forinterpretationofthe referencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

Table1

Genotypeandallelefrequencies(CI95%)inHaemonchuscontortus(S=susceptible,R=resistant)collectedfromdifferentruminanthosts.

Host Genotypefrequency(%) Allelefrequency(%)

homozygous

facts,wesuggested,theresistantH.contortusrecoveredfromroe deermightderiveratherfromcrossinfectionfromsheep,thanfrom thefailureofhuntingmanagement.

OurobservationcouldnotexplainexactlytheabsenceofRallele inthereddeer.Wehypothesizedthatthepresenceofsheepon pasturesindicatedavoidancebehaviourinthereddeer.Somefree rangestudies(Osborne1984;DeGabrieletal.,2011)showedthe absenceof sheepin a given habitat induced a higher reddeer density.AccordingtoCuartasetal.(2000),thisphenomenonwas attachedtothedisturbanceeffectofsheephusbandryratherthan anydirectorindirectcompetitionbetweenthetwospecies.During ourinvestigation,manysheepﬂocksusedregularlythepastures ofthestudyarea,thustheirpresencemightbeaffectedthered deeraggregation;however,thisstudydidnotcoverdetailedﬁeld observationofallhostspecies.TheresultofaNorwegianstudy (Mysterudetal.,1999)suggestedthatroedeerreservedits ter-ritorybya trade-offmechanismbetweenshelterandforage. In conclusions,theauthorsfoundthattherewasnoanyevidencefor avoidancebetweenthetworuminants.

Thehomerangeofroedeercanvarywidelyinscale(Jeppesen, 1990; Maillard et al., 2002) and it can be affected by habitat resources.Somestudiesconﬁrmthat theroedeeradjusthome rangesizeinresponsetolandscapestructure.Fragmented land-scapescanprovideabundant,high-qualityfood,whichincreases deerhabitatcarryingcapacity(Reimoser,2003;SaïdandServanty 2005).Thesevaluable,fragmentedlandscapescanalsobe utiliz-ableforsheepgrazing;asitwasobservedinthestudysite.We

supposedthattheoverlapinhabitatusebetweensheepﬂocksand roedeercouldcontributetotheoccurrenceandspreadofresistant allelewithinwildlife.Ourresultsraisedanissueabouttheroleof roedeerinAR;whetherroedeerisapotentialtransmitterofthe problemorthepotentialsolutionforit,asasourceofSSgenotyped wormssupportingrefugiaformationonpastures(VanWyk,2001).

Toanswerthisquestion,weneedtoaccomplishmoreregulated andcomplexstudiestoknowtheexacteffectsofthishostspecies.

Ourstudywasbasedonthemoleculardiagnosticdetectionof Phe200TyrSNPoncodon200of␤-tubulingeneisotype1;while SNPs on codon 167and 198were not investigated. Therefore;

thetrueprevalenceofBZresistancewithinthestudiedH. contor-tuspopulationmightbeunderestimated;notwithstanding,inall threehostspecies;thereby thosewerecomparable.Thesample collectionperiodwasratherlongbecausedeerforpost-mortem examinationcouldbereachedduringtheirhuntingseasonsand inalimitedcount.Thisfactcouldaffectourresultsthroughthe annualcircleofwormburdenincertainhosts.Sheepsamplescould betakenallyearround;therefore,thesedatarepresentmostlythe averageprevalenceofresistance.Roedeersampleswerecollected mainlyin spring and summerwhen theintensity of this para-siteisgenerallyratherhighinruminants.Ontheotherhand,red deerwereharvestedduringautumnandwinterwhenthelevelof infectionislargelymoderateorlow.Consideringthesefacts,our deerdatacannotdescribethestatementofresistanceaccurately.

Theremarkabledifferencebetweenthetwodeerspeciescanbe explainedevenbydifferentsamplingperiods.Roedeerwere

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pledinspringandsummerwhenthesheepwithlambsattheirfeet areonpasturesandshedalotofeggs.Underthesecircumstances, wormsthatinfecttheroedeermightoriginatepartlyfromsheep.

Ontheotherhand,thesamplingofreddeerwascarriedoutafter themainwormburdenofsheep,whenfewlarvaecanbeingested bygrazing.During this period,theresearchercanmeeta natu-rallyselectedpopulationofworms;whichwereaffectedbyalot ofenvironmentalfactors,exceptanthelmintics,inawildruminant stomach.Consideringtheabove,ourresultsalsosupporttherefugia hypothesisifweassumethatabomasalcircumstancesofwild rumi-nantsevolveasortofselectionpressuretoparasites.Amongthese conditions,theselective-advantageattributedtoARmightprove tobeuseless;andthoseworms,whichcarryRallele,willperishin largeramountundertransferredselectionpressure.AsLeathwick (2013)conﬁrmedinhisstudy,wormscarryinganthelmintic resis-tancemechanismarelikelytobelessﬁt.Forscientiﬁcjustiﬁcation ofthishypothesis,furtherinvestigationswillbeneeded.

Conﬂictofinterest

Theauthorsdeclarenoconﬂictofinterest.

Acknowledgements

Theauthorsarevery thankfultotheprofessionalhunters of SEFAGPlc.fordeersamplecollection,totheseniorlecturerofthe KaposvárUniversity,GáborCsizmadiaforhisprofessional linguis-ticproposals;andtotheanonymousreviewersfortheirhelpful criticism.

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