Efficacy of slow-release tags impregnated with aggregation-attachment pheromone and deltamethrin for control of Amblyomm...

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Title:
Efficacy of slow-release tags impregnated with aggregation-attachment pheromone and deltamethrin for control of Amblyomma variegatum on St. Kitts, West Indies
Physical Description:
Book
Language:
English
Creator:
Kelly, Patrick J.
Lucas, Helene M.
Randolph, Craig M.
Ackerson, Kate
Blackburn, Jason K.
Dark, Michael J.
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BioMed Central (Parasites & Vectors)
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Abstract:
Background: Amblyomma variegatum is an important cause of morbidity, mortality and economic losses in Africa and the West Indies. Attempts to control and/or eradicate the tick from the Caribbean have largely been unsuccessful because of difficulties relating to the biology of the three-host tick and problems with applying acaricides on a regular basis to free-ranging domestic ruminants. While plastic collars impregnated with insecticides are widely and effectively used in companion animals to control external parasites there is little information on this technology in ruminants. Methods: Over 21 months we tested the efficacy of slow-release plastic tags impregnated with deltamethrin (7%) and aggregation-attachment pheromones (DPITs) in controlling A. variegatum on free-ranging cattle on two farms on St. Kitts. The tags were replaced every three months or when found to be lost. Results: On sentinel animals fitted with tags containing only aggregation-attachment pheromones there were an average of 23.1 ticks per semi-monthly visit although this number varied considerably, peaking in the dry season around May and being lowest in August to October during the wet season. Significantly fewer ticks (3.5 on average) were found on cattle with DPITs at each visit (P < 0.001). Although the DIPTs provided good control (92% on average), they did not significantly reduce A. variegatum in the environment with tick numbers on sentinels being higher in the second year of the study, despite up to 44% of animals being fitted with DPITs. The tags were economical, costing 0.2% of the 1% flumethrin pour-on treatment widely recommended for A. variegatum control in the Caribbean. The major problem encountered was that 38% of tail tags were lost before they were due for replacement every three months. Conclusions: Our study has shown that DPITs are cheap to produce, easy to place, only require handling of animals every three months, and are very effective in protecting cattle from A. variegatum. Before DPITs can be considered for eradication programs the problems needing to be addressed include loss of tail tags, particularly in thick vegetation, and the optimum number of animals that must be treated to reduce numbers of ticks in the environment. Keywords: Deltamethrin, Slow-release tag, Tick control, Amblyomma, Aggregation-attachment pheromone
General Note:
Kelly et al. Parasites & Vectors 2014, 7:182 http://www.parasitesandvectors.com/content/7/1/182; Pages 1-8
General Note:
doi:10.1186/1756-3305-7-182 Cite this article as: Kelly et al.: Efficacy of slow-release tags impregnated with aggregation-attachment pheromone and deltamethrin for control of Amblyomma variegatum on St. Kitts, West Indies. Parasites & Vectors 2014 7:182

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RESEARCHOpenAccessEfficacyofslow-releasetagsimpregnatedwith aggregation-attachmentpheromoneand deltamethrinforcontrolof Amblyomma variegatum onSt.Kitts,WestIndiesPatrickJKelly1*,HeleneMLucas1,CraigMRandolph1,KateAckerson1,JasonKBlackburn2,3andMichaelJDark2,4AbstractBackground: Amblyommavariegatum isanimportantcauseofmorbidity,mortalityandeconomiclossesin AfricaandtheWestIndies.Attemptstocontroland/oreradicatethetickfromtheCaribbeanhavelargelybeen unsuccessfulbecauseofdifficultiesrelatingtothebiologyofthethree-hosttickandproblemswithapplying acaricidesonaregularbasistofree-rangingdomesticruminants.Whileplasticcollarsimpregnatedwithinsecticides arewidelyandeffectivelyusedincompanionanimalstocontrolexternalparasitesthereislittleinformationonthis technologyinruminants. Methods: Over21monthswetestedtheefficacyofslow-releaseplastictagsimpregnatedwithdeltamethrin(7%) andaggregation-attachmentpheromones(DPITs)incontrolling A.variegatum onfree-rangingcattleontwo farmsonSt.Kitts.Thetagswerereplacedeverythreemonthsorwhenfoundtobelost. Results: Onsentinelanimalsfittedwithtagscontainingonlyaggregation-attachmentpheromonestherewerean averageof23.1tickspersemi-monthlyvisitalthoughthisnumbervariedconsiderably,peakinginthedryseason aroundMayandbeinglowestinAugusttoOctoberduringthewetseason.Significantlyfewerticks(3.5onaverage) werefoundoncattlewithDPITsateachvisit(P<0.001).AlthoughtheDIPTsprovidedgoodcontrol(92%onaverage), theydidnotsignificantlyreduce A.variegatum intheenvironmentwithticknumbersonsentinelsbeinghigherin thesecondyearofthestudy,despiteupto44%ofanimalsbeingfittedwithDPITs.Thetagswereeconomical, costing0.2%ofthe1%flumethrinpour-ontreatmentwidelyrecommendedfor A.variegatum controlintheCaribbean. Themajorproblemencounteredwasthat38%oftailtagswerelostbeforetheyweredueforreplacementevery threemonths. Conclusions: OurstudyhasshownthatDPITsarecheaptoproduce,easytoplace,onlyrequirehandlingof animalseverythreemonths,andareveryeffectiveinprotectingcattlefrom A.variegatum .BeforeDPITscanbe consideredforeradicationprogramstheproblemsneedingtobeaddressedincludelossoftailtags,particularly inthickvegetation,andtheoptimumnumberofanimalsthatmustbetreatedtoreducenumbersofticksin theenvironment. Keywords: Deltamethrin,Slow-releasetag,Tickcontrol, Amblyomma ,Aggregation-attachmentpheromone *Correspondence: pkelly@rossvet.edu.kn1SchoolofVeterinaryMedicine,RossUniversity,Basseterre,St.KittsandNevis, WestIndies Fulllistofauthorinformationisavailableattheendofthearticle 2014Kellyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Kelly etal.Parasites&Vectors 2014, 7 :182 http://www.parasitesandvectors.com/content/7/1/182

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BackgroundThetropicalbonttick, Amblyommavariegatum ,wasintroducedontotheCaribbeanislandofGuadeloupeon cattleimportedfromSenegalintheearly1800s[1].The tickiswelladaptedtotheclimaticconditionsinthe Caribbean[2]and,bythe1990s,ithadspreadto19 Caribbeanislandsandcausedsubstantialeconomicloses [3].Onereasonforthelossesisthat Amblyommavariegatum haslongmouthpartsandattachmentandfeeding resultsinlocalinfections,abscessesandhidedamage. Also,infectionswith Dermatophiluscongolensis areassociatedwiththepresenceof A.variegatum andmajor epidemicsofdermatophilosiswithupto90%mortality havebeendescribedintheCaribbean[4,5].Further, A.variegatum isthevectorof Ehrlichiaruminantium ,a Gram-negativerickettsiathatisthecausativeagentof heartwater,anacutediseaseofdomesticruminantscausingmortalitiesofupto90%.Thediseaseisknownto occuronatleastthreeCaribbeanislands[6]andmight spreadtotheAmericaswithdisastrouseffectsontheir livestockindustries[7]. Amblyommavariegatum isalso importantinhumanmedicineasitisthevectorof Rickettsiaafricae ,theagentofAfricantick-bitefever, aspottedfeverrickettsiosisaffectinglocalpeopleandtouristsintheCaribbean[8]. Becauseoftheeconomicimportanceof A.variegatum intheCaribbeanandpossiblytheAmericas,various attemptshavebeenmadebylocalandinternationalauthoritiestocontrolanderadicatethetick[9,10].Local controlprogramshaveoftenbeenineffective[11]and apparenteradicationof A.variegatum fromsomeislands hasbeenshort-lived[4,10,12].Therearemanyreasons forthelackofsuccessincludingownernon-compliance [4,11],reintroductionofticksbyanimalmovementor migratorycattleegrets( Bubulcusibis )[13],refugiaof ticksonferallivestockorwildlifehosts[14,15],andthe difficultiesofcatchingandtreatinganimalskeptunder free-rangingconditions.Currently,themostcommon methodforcontrolof A.variegatum intheCaribbean isthebi-weeklyapplicationof1%flumethrinpour-on acaricide(BayticolW,Germany).Althougheffective,ithas significantlimitationsintheCaribbeanwherelivestock aregenerallykeptunderfree-rangingconditionsandare difficulttocaptureandhandle.Also,theprocedureis laborintensiveandexpensive,makingregulartreatment ofallanimalsunlikely.Intermittentacaricidetherapy mayleadtothedevelopmentofresistanceandenable A.variegatum topersistatlowlevelsthataredifficult todetect. Theidealmethodfor A.variegatum controlshouldbe cheap,easytoperform,requireminimaltraining,and haveaprolongedacaricidaleffecttominimizethenumberoftimesanimalshavetoberestrainedandtreated. Inthe1990s,slow-releaseplasticcollarandtailtagswere developedwhichdeliveracaricidesthatspreadwidely overthecoat,reachinglevelsthataretoxicforticksfor atleastthreemonths[16].Toincreasethenumberofticks killed,thetagscanalsobeimpregnatedwithaggregationattachmentpheromonesthatalsospreadaroundthehair coatandattractmale,femaleandunfednymphal A.variegatum ontothehostoveraperiodofatleastthreemonths [16,17].InapilotstudyonGuadeloupe[17],cattlewith tagscontainingtheaggregation-attachmentpheromone anddeltamethrinhadsignificantlyfewer A.variegatum (80%initially,then60-70%)thancontrolanimals.Ina similarstudyonthecloselyrelatedspecies Amblyomma hebraeum inZimbabwe,controlvariedfrompoor(5074%)toexcellent(over95%)usingtagscontainingthe aggregation-attachmentpheromoneandalphacypermethrinandcyfluthrin,respectively[16].Inbothstudies thecattlewereconfinedtopaddocksandonlymonitoredforthreemonths. Toprovidemoredataontheefficacyofslow-release tagsunderfieldconditionsintheCaribbeanandtodeterminetheirpracticality,wecarriedouta21-monthstudy oncattlekeptundertraditionalCaribbeanfree-ranging conditions.Ourfindingsarereportedbelow.MethodsStudysiteSt.KittsisanislandinthenortheasternCaribbean(17.2N; 67.1W)about2,100kilometerssoutheastofFlorida.Ithas anareaof168squarekilometersandconsistsofthemain islandandadryandsparselypopulatedsoutheastpeninsula.Theislandhasatropicalclimatewithtemperatures varyingfrom23Cto31Candhumidityfrom63%to 91%[18].Rainfallaveragesapproximately1.14meters/ yearandislowestinFebruary-Marchandpeaksin September.Themiddleoftheislandismountainouswith peaksreaching1,300meters.Betweenthebaseofthe mountainsandthecoasttherearegentlyslopinggrasslandsandabandonedcanefieldsthataregraduallyrevertingtonativebush;thesearetheprimarylivestockareason theisland. Totallivestocknumberspeakedinthemid-1980sat 22,000(6,000cattle,9,000sheep,and7,000goats),but declinedtoatotalof2,200(400cattle,800sheep,and 1,000goats)in1990when A.variegatum anddermatophilosisbecameprevalent[4].Thistickwasprobably introducedfromthesisterislandofNevisin1978 andthereafterspreadwidelytooccupyallareasapartfrom thecentralmountainousareasandthedrysoutheastpeninsula.FollowingtheCaribbeanAmblyommaProgramme, whichinvolvedblankettreatmentoflivestockwithbiweeklyflumethrin1%pour-on(BayticolW,Bayer,Germany), St.Kittswasdeclaredprovisionallyfreeofthe A.variegatum in2001[10]whenlivestocknumbershadrecovered. SubsequenttodiscontinuationofmandatoryflumethrinKelly etal.Parasites&Vectors 2014, 7 :182 Page2of8 http://www.parasitesandvectors.com/content/7/1/182

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treatment A.variegatum reappearedandbecameprevalent inallareas,apartfromthesouthernpeninsula.Further, outbreaksofdermatophilosisoccurredand,althoughno censusdataisavailable,localexperiencesuggestedthatcattlenumbershaddecreasedsubstantiallywhenourstudy beganin2011. Farmswereconsideredforthestudyif A.variegatum werepresent;theirownerswerewillingtoparticipate; therewereatleast15cattlepresentandnootherlivestock;thecattlecouldmostlikelyberoutinelypresented forexaminationeachmonth.Slow-releasetagsTwotypesofslow-releasetagswereusedintheproject; onecontainedaggregation-attachmentpheromonesalone (PITs)andwasusedonsentinelanimalswhiletheother containedpheromonesand7%deltamethrin(DPITs) (We-YoungIndustrialandTradingCo.,Nanjing,China) andwasusedontreatedanimals.Thetags(65x90mm) weremadefromliquidpolyvinylchloride(B.F.Goodrich, Avon,OH),plasticizers(WitcoCorporation,Avon,OH) andstabilizers(WitcoCorporation,Avon,OH)asdescribedpreviously[16].Thepheromonewasformulated fromo-nitrophenol,methylsa licylate,6-dichlorophenol andphenylacetaldehyde(Sigma-AldrichChemicalCo.,St. Louis,MO)inproportionsfoundinnaturaltickextracts [19,20],mainly1%,1%,0.2%and0.1%of100gPVC,respectively.ThetagsweremadeattheUniversityofFlorida andimmediatelysealedinaluminiumfoilpouchesbefore beingshippedtoSt.Kittswheretheywerestoredatroom temperature(around27C)untiluse. Thetailtagswereattachedtoabout100tailhairs asclosetotheendofthelivingtailaspossibleusing cyanoacrylateadhesive(Bondini-2,Pro-TelInc,Santa Monica,CA)andabouttenheavyduty1centimeter metalstaples(Staplex,Brooklyn,NY)asdescribedpreviously[17].Thecollartagswereattachedtopolyurethanecoatedneckcollars(BiothaneW,Bio-Plastics,North Ridgeville,OH)usingcyanoacrylateadhesive(Bondini-2, Pro-TelInc,SantaMonica,CA)andabouttenheavy duty1centimetermetalstaples(Staplex,Brooklyn,NY). Astagshavebeenfoundtoreleasepheromoneanddeltamethrinforprolongedperiods[17]theywerereplaced every3months,orsooneriftheywerefoundtobe missing. Whiletherearenodataonthepercentageofanimals thatneedtobetreatedforeffectivetickcontrolinthe herd,previousexperimentssuggest10%asareasonable startingestimate[17].Thetaggedanimalsareexpected toassimilatewiththefree-roamingpopulationandattractandkill A.variegatum inthearea.Monitoringthe populationoveraroughlytwo-yearperiodwouldberequiredtoassumethatalladult A.variegatum hadbeen removed[12].AnimalsApprovalofthestudywasobtainedfromtheInstitutionalAnimalCareandUseCommitteeofRossUniversitySchoolofVeterinaryMedicineandtheUniversityof Florida.Cattleusedinthestudywerealladultsthat couldbecaughtbythefarmersforstudyvisits.Theanimalsallhadneckcollarsmadefromcanvasstrapsto whichlengthsofchainorrope(around20meters)were tied.Beforeastudyvisittheanimalswererestrainedby attachingtheendsoftheirchains/ropestotreesorstout bushes.Toimmobilizethecattleforinspection,thefree chain/ropeattachedtothecollarwasprogressivelytaken inastheanimalwasgentlydriventowardsthetree/bush towhichitwasattached.Whentheanimalwasclosely tiedtothetree/bushitwassedatedandgivenanalgesiafor tickremovalbyinjectionofxylazine(0.1to0.2mg/kgIM) withablowdart(Dan-InjectW,Austin,TX;Pneu-DartW, Williamsport,PA).Followingdarting,theanimalswereleft undisturbedforaround15minutesuntilsufficientlysedatedtobehandledwithoutstress. Studyvisitstothefarmsweremadeapproximately monthly,dependingonavailabilityoftheownersand water.Althoughthecattlewerefreetoroamtheygenerallyreturnedtothefarmseverydayortwoforwater, whichwasnotreadilyavailableonsurroundinglands, especiallyiftherewasnorain.Bywithdrawingwaterfor adaybeforevisitsthefarmerscouldgenerallyensure manyofthestudyanimalswouldbeavailable.TicksamplesDuringtheapproximatelymonthlystudyvisitsthesedated animalswereexaminedthoroughlyfor A.variegatum .All ticksfoundwereremovedandstoredin100%ethanoluntil theiridentitywasconfirmedusingstandardmorphological criteria.ControlofticksonanimalswithDPITswascalculatedusingtheformula: Control% 100 mc– mt = mcwheremcisthemeannumberofliveticksonthecontrolanimalswithPITsandmtisthemeannumberof liveticksonanimalswithDPITs[ 17 ].StatisticalanalysisStatisticalanalysiswasp erformedusingSPSSV.20 (IBMCorp.,Armonk,NY).AP 0.05wasconsidered significant.ResultsStudysitesTwofarmswerefinallyselectedforthestudy,one(Farm A;1720 25.66 Nand6243 53.27 W)onthenorthofthe centralmountainrangeandone(FarmB;1718 36.10 N and6247 14.67 W)onthesouth.FarmAcoveredaboutKelly etal.Parasites&Vectors 2014, 7 :182 Page3of8 http://www.parasitesandvectors.com/content/7/1/182

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12hectaresandwassituatedatthebaseofthecentral mountainsontheAtlanticsideoftheislandabout120m abovesealevel.Ithadadeepimpassableravineontheeast sideandanincomplete2meterdiamondwirefence aroundtheothersideswhichdidnotpreventcattlestrayingupintothetropicalforestofthemountainsordown intograsslandsandabandonedcanefieldstowardsthe coastinsearchofgrazing.Pipedwaterwasavailableat threetroughsinthecenterofthefarmwhenconsiderednecessarybythefarmer.Thevegetationonthe farmconsistedofpatchesofgrasses( Cynodondactylon and Panicummaximum ),acaciascrub,avarietyofmaturetrees,andpatchesof Crotalariapallida,Xanthium stumarium ,and Phyllanthusamarus .FarmBwason theCaribbeansideoftheislandmidwaybetweenthe baseofthecentralmountainsandthecoast,approximately60mabovesealevel.Itcovered21hectaresand wasunfenced.Thevegetationconsistedofgrassland, predominantly Cynodondactylon and Panicummaximum aswellasscatteredacaciascrub, Solanumtorvum andvariousmaturetrees.AnimalsInitiallytherewere18mixedbreedcattleonFarmA, butoverthecourseofthestudythreediedandonewas sold.OnFarmBtherewere17mixedbreedcattlefor theduration.TherewerenolivestockpermanentlysituatedonthelandsimmediatelysurroundingFarmsAand Bandnoanimalsstrayedontothefarmsduringthe study.Priortothecommencementofthestudy,tick controlonbothfarmshadbeenwithtopicalapplications of1%flumethrin(BayticolPour-onW)whenanimals werethoughttohavelargetickburdensbytheowners. Notreatmentshadbeengivenonthefarmsforatleast twoweekspriortothestartofourexperiments. Fromtheoutset,dermatophilosisoccurredcommonly onthecattle,particularlyonA,withatotaloftwo deathsand19animalsrequiringonetothreetreatments. Althoughtherewasnoobviouscorrelationbetweentick numbersorpresenceofDPITsorPITsontheincidence ofdermatophilosistherewasastrongbeliefamongstthe farmersthatthepresenceof A.variegatum wasdirectly linkedtothedisease.Therewasthusstrongpressurefor moreanimalstobetreatedwithDPITs.Initiallythere weretwosentinelanimalswithPITsandtwocattle treatedwithDPITsoneachfarmbut,duetothedermatophilosis,weincreasedthenumberofcattlewithDPITs tosevenonFarmAandfiveonFarmBbymonth10of thefirstyearofthestudy.Overthesameperiod,weincreasedthenumberofsentinelanimalswithPITstofour oneachfarm. Ateachvisit,variablenumbersofanimalsweresecuredbythefarmownersforexamination(Table1). Dataonanimalsrequiringtreatmentfordermatophilosis werenotincludedinanalysesastheeffectsofthediseaseonticknumberscouldnotbedetermined. Duringeachfarmvisitthatwasmade(32;FarmA17, FarmB15),weexaminedanaverageof2.5(81/32;Farm A40,FarmB41)sentinelanimalswithPITs,1.5(48/32; FarmA20,FarmB28)animalswithDPITs,and1(33/32; FarmA22;FarmB11)animalthathadlostitstagsbefore theyhadbeeninplaceforthreemonths.TagsThetagswerecheaptoproduce,costing0.22US$fora PITand0.46US$foraDPIT,excludingequipment costs.TheDPITscouldprovidethreemonthsofprotectionforapproximately0.2%ofthecostofsixgovernmentsubsidizedBayticolWtreatmentsonSt.Kitts. InitiallyanimalsweresedatedusingPneu-DartWdarts butduetothelargenumberofdartsthatfailedtodetonate(upto50%onaparticularday)thesimplerand cheaperDan-InjectWdartswereusedwithalmost100% efficacy.Oncetheanimalswereimmobilizedandsedated,thetagsweregenerallyeasytoplaceandnoneck collarswerelostduringthestudy.Althoughnotags werelostfromthecollars,only62%(60/97)ofthetail tagswereretainedfor3months.Losswasgreateston FarmA(48%;24/50),whichhadconsiderablydenser scrubvegetationthanFarmB(28%;13/47)( 2=3.43, P=0.064).Thetailtagsappearedtobelostmostfrequentlytowardstheendofthethreemonthperiod(data notshown).Therewasnopredispositionforspecificanimalstolosetailtags.Examinationoflosttailtagsindicatedthiswasnotduetoglueorstaplefailure.Rather, whentagsbecametrappedinfencesordensefoliage, theyandthehairtowhichtheywereattachedwere wrenchedfromthetail. Thepurityofthedeltamethrinusedinthetagswas confirmedbytheCaliforniaAnimalHealth&Food SafetyLaboratorySystem(CAHFSL,Davis,CA)priorto studyinitiation.Tagstestedfordeltamethrinwhenthey wereremovedattheendofthe3monthperiodonthe animalshaddeltamethrinlevelsrangingfrom4.5-5.9%.TicksDataonticksonanimalsrequiringtreatmentfordermatophilosiswerenotincludedinanalysesastheeffectsof thediseaseonticknumberscouldnotbedetermined. Fromthehealthyanimalswecollectedatotalof2,318 A.variegatum withthemajority(1,527;66%)being foundonsentinelcattlewithPITsandonlyasmallpercentage(183;8%)onanimalswithDPITsthathadbeen placedwithintheprevious3months(Table1).The remaining25%ofticks(608)camefromanimals(33)that hadlosttheirtailtagwithinthepreviousthreemonths. Overall,wecollectedsubstantiallymoremalethanfemale A.variegatum withtheoverallmaletofemaleratiobeingKelly etal.Parasites&Vectors 2014, 7 :182 Page4of8 http://www.parasitesandvectors.com/content/7/1/182

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2.7:1(623/1695).Thiswasalsothecasefortheindividualfarms(FarmA[3.1:1;1234/399]andFarmB [2.1:1;461/224])andfortreated(2.1:1;123/60)anduntreatedcattle(2.7:1;1109/418). ThereweremoretickscollectedonFarmA(1633) thanFarmB(685)andFarmAhadagreatertickpressurewithanaverageof31.4tickspersentinel(973ticks in31sentinelexaminations)comparedto15.9onFarmB (554ticksin35sentinelexaminations)(t-test,P=0.05). Numbersofticksonsentinelanimalsonbothfarms peakedaroundMayandwereattheirlowestlevelsin AugusttoOctober(Table1).Thisapparentcycleoccurredonbothfarmsinbothyearsofthestudy.The tickchallengeonthefarmsinthesecondyearofthe studywasgreaterwithanaverageof30.2ticks/sentinel (FarmA433/23andFarmB202/21)inyearoneandan averageof30ticks/sentinel(FarmA328/8andFarmB 264/14)inyear2.Thisdifference,however,wasnot significant(t-test;P=0.064).Theincreaseinticksinthe secondyearoccurreddespiteDPITsbeingusedonhigher percentagesofanimalsonbothfarms(FarmA:from11% (2/18animals)to44%(7/16animalsonaverage);FarmB: from12%(2/17animals)to29%(5/17onaverage). PlacingDPITsoncattlegreatlyreducedinfestation with A.variegatum onbothfarmswithanaverageof only3.5ticksfoundonatreatedanimalatanyexamination(183/53)comparedtoanaverageof23.1ticksona sentinelanimal(1527/66)(t-test,P<0.001).Overall,significantlyfewertickswerefoundoncattlewithDPITs onFarmB(0.7;19/29)comparedtothoseonFarmA (8.4;167/20)(t-test,P<0.001).Cattlethathadbeen treatedwithDPITsforsomeoftheprecedingthree months,buthadlosttheirtagsbeforebeingreexamined, hadloweraverageticklevelsthansentinelsonboth FarmA(24.6;492/20)andFarmB(10.5;116/11).These levels,however,werehigherthanthoseofanimalsthat receivedthefulleffectoftheDPITtreatment. TheoveralltickcontrolefficacyoncattlewithDPITs was92%butthisvariedwidelyfrom64to100%duringanysinglevisit(Table1).Averagetickcontrolwas Table1Tickcollectiondataonsentinelanimalswithpheromoneimpregnatedslow-releasetags(PITs)andanimals treatedwith7%deltamethrinandpheromoneimpregnatedslow-releasetags(DPITs)onfarmsonthenorth(A)and south(B)ofSt.KittsFarmAFarmB DateAverageticks on(N)animals withPITs Averageticks on(N)animals withlosttags Averageticks on(N)animals withDPITs ControlAverageticks on(N)animals withPITs Averageticks on(N)animals withlosttags Averageticks on(N)animals withDPITs Control 11-Feb 46(2)3(2) 11-Mar 22(2)4(2) 11-Apr 9.5(2)np*0(1)100%1.5(2)np0(2)100% 11-May 107(1)46.3(3)9(2)92%28(2)np0(2)100% 11-Jun 35(1)np12.4(5)65%23(2)0(1)0(1)100% 11-Jul 27(1)np7.3(3)73%9(2)np0(2)100% 11-Aug 1.5(2)np0(1)100% 11-Sep 11-Oct 7(2)13.5(2)2.5(2)64%3(2)np0(2)100% 11-Nov 11(1)np0(1)100%4.5(2)0(1)0(4)100% 11-Dec 7(5)8.5(2)np6(2)5(1)0(2)100% 12-Jan 11.75(4)13.25(4)np12.7(3)np3(4)77% 12-Feb 16(4)14(1)0(2)100% 12-Mar 37.5(2)19.5(2)np 12-Apr 49(2)30(4)3(1)94%41.7(3)7.3(3)2(2)95% 12-May 101(1)24(1)15(3)86% 12-Jun 30(1)19.5(2)np15.5(2)17(1)1(2)94% 12-Jul 16(1)np4.3(3)73% 12-Aug 6.5(4)5(2)0(2)100% 12-Sep 12-Oct 8(1)np0(2)100%18(1)1(1)0.5(2)97%*noanimalspresentedwithlosttags.Kelly etal.Parasites&Vectors 2014, 7 :182 Page5of8 http://www.parasitesandvectors.com/content/7/1/182

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higheronFarmB(97%)thanonFarmA(86%)(t-test, P=0.022).DiscussionOurstudyshowedthat A.variegatum iscommonon thefarmswestudiedonthenorthandsouthsidesof St.Kittsandalsointhesurroundinglandswherethe animalswerefreetoroam.Localexperiencesuggests thefarmswestudiedwerenotexceptionalandthatthe tickisaproblemwhereverlivestockarekeptthroughout themainisland.Itisofnotethatin2001,nineyears beforeourstudybegan,St.Kittswasdeclaredtobeprovisionallyfreeof A.variegatum followingaperiodof mandatory1%flumethrintopicaltreatmentofalldomesticruminantsaspartofanationaltickeradicationproject whichwassupportedbytheCaribbeanAmblyomma Programme[10].By2006,however,thetickhadreappeared,andtheprovisionallyfreestatusoftheislandwas revokedwhen76 A.variegatum werefoundduring4,201 farminspectionvisitsonSt.Kitts[12].Duringourstudy fiveyearslater,wefoundanaverageof72 A.variegatum perfarmvisitwithanaverageof23ticksoneachuntreatedsentinelanimal.Thefigureforouruntreatedanimalsisconsiderablyhigherthanthemeanofthreeto fourperinfectedanimalreportedfromtheEnglishLesser Antilles,whichincludesSt.Kitts,upuntil2006[12].Itis alsohigherthanthatreportedpreviouslyfortheFrench islandsofGuadeloupe(6.1)andMarieGalante(6.7-11.5) whichhadawidelyadoptedprogramofbiweeklyamitraz treatments[21]. Whilethereappearanceof A.variegatum onSt.Kitts mightbetheresultofreintroductionofthetick,itseems morelikelythatthetickwasnevereliminated.InreviewingtheCaribbeanAmblyommaProgramme,Ahoussou etal. [12]pointedoutthatcatchingandtreatingallof thepredominatelyfree-ranginglivestockontheCaribbean islandswasnotpracticalandthattherewerelikelyisolated populationsoffree-ranginganimalsthatwerenottreated andthusabletoharborandspread A.variegatum .Thisis particularlyproblematicwith A.variegatum becauseitisa threehosttickwithalonglifecycleofsixmonthstoover threeyears[22,23].Thetickcanthussurviveinisolated areasonlyoccasionallyvisitedbyfree-ranginglivestock andtherebyappearstobeeradicatedwhennotseenon animalsduringroutineinspections.Further,femaleslay verylargenumbersofeggs(around20000)withhigh hatchingsuccessratesofabout60%[23],whichfacilitates reinfestationofpastures. Resurgenceof A.variegatum wasalsopromotedbya numberofotherfactors.Firstly,thecessationofmandatory andfreeflumethrintreatmentsoflivestockthatfollowed theawardofprovisionallyfreestatusin2001resultedin administrationoffeweracaricidetreatmentsandhencethe abilityofrefugiaof A.variegatum toexpandtheirrange. Secondly,theuneconomicalsugarindustryonSt.Kitts closedin2005andthesugarcanefields,whichoccupied largeareasoftheisland,wereabandonedandnolongeran illegalsiteforlivestock.Thefieldsthereforebecameadditionalgrazingareasthatenabledlivestocktorangeeven morefreelythanbefore,makingtheanimalsevenmore difficulttofind,monitorandtreatforticksonaregular basis[9].Further,itexpandedtheareawhere A.variegatum couldsurviveintheenvironmentandfindsuitable hosts.Finally,theCaribbeanclimateisideallysuitedtothe tick[22],whichenablesittosurviveforlongperiodsand tomaximizeitsreproductivepotential. Ourstudyshowedthenumbersof A.variegatum variedseasonallywhichisconsistentwithpreviousreports. PeaknumberswerearoundMayinbothyearsandon bothfarmswhichissimilartothesituationdescribed inGuadeloupein1985to1987[22].ThisperiodcorrespondstothestartoftherainyseasononSt.Kitts (WeatherSpark.com).ThesituationissimilarinAfrica whereadult A.variegatum numbersalsopeakinthe rainyseason[24],presumablyasthisensuresconditions ofhumidityandtemperatureidealforsurvivalofthe ticksandtheirprogeny. Ourfindingthatmoremalethanfemale A.variegatum (3.1:1)attachedtobothtreatedandsentinelanimalsis consistentwithpreviousreportsofratiosof4:1for Guadeloupein1992[25]and2.3:1forSt.Kittsbetween 1998and2002[9].AstudyinBurkinaFasoinAfrica, whereannualrainfallissimilartotheCaribbeanbutoccursprincipallyinafive-monthrainyseason,demonstratedrelativelylowpercentagesofattachedfemales comparedtomales(from10%)duringthebeginningof therainyseason[26].Attachmentratesofalmostequal numbersofmalesandfemalesoccurredlaterintherainy season,however,andthiswasassumedtobebecausefemalesrequiregreaterhumidityornumberofrainydays beforefemalehost-seekingactivityisactivated.Thiswould preventengorgementandegglayingwhenrainwassparse andsurvivalofeggsandlarvaewouldbecompromised.It maybethattheCaribbeanistoodryformaximumattachmentratesoffemalesandthislimitsthepopulation of A.variegatum intheregion.Thefemalesthatdo attach,however,havenormalfertility,whichispreserved throughsexratiosof4:1to1:4[22]. Ingeneral,thetickchallengeinourstudy(anaverage of23tickspersentinelpervisit)wasgreaterthanthatreportedinthetwopreviousstudiesontheefficacyof theslow-releasetags,whichwas,onaverage,2 – 15per animalperweekinZimbabwe[16]and5 – 14peranimal perweekinGuadeloupe[17].Therewassignificantly moretickpressureonthefarmonthenorthsideofthe island.Althoughthismighthavebeenasaresultofpreviouslylowertickcontrolordenservegetationwhich mightbemorefavorabletosurvivalof A.variegatum ,itKelly etal.Parasites&Vectors 2014, 7 :182 Page6of8 http://www.parasitesandvectors.com/content/7/1/182

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couldalsobeduetoweatherfactorswiththesouthside oftheislandbeingsomewhatdrierasitisintherain shadowareacreatedbythecentralmountains.Wewere unabletocollectweatherandgeographicaldata,which couldhavehelpedresolvetheissue. Thedifferenceintickpressurebetweenthefarms couldalsohavebeenbecauseofresistancetodeltamethrinandthiscouldaccountforthesignificantlylower averagecontrolseenonFarmA(86%)ascomparedto FarmB(97%).Althoughresistancetodeltamethrinhas beenreportedinticks[27]thereisonlyreferencetodeltamethrinresistanceinonespeciesof Amblyomma A.cajennense inBrazil[28].In1991,GarrisandBarre [29]reportedtherewasnoevidenceofresistancein A.variegatum fromGuadeloupeorPuertoRicoatthe time.ThetagswetestedfrombothfarmsAandBafter 3monthsusagestillcontainedsignificantdeltamethrin levelsindicatingtherewerenoenvironmentalfactors, suchasUVdamageorexposuretowater,whichmight haveledtodifferentialleachingorinactivationofthe acaricidebetweenfarmsandreducedefficacy.Ourdata suggeststheremightberesi stancetodeltamethrinby A.variegatum and,ifDPITsareconsideredforfurther studies,thispossibilityshouldbeexplored. Incommonwiththepreviousreportsontheslowreleasetags[16,17],wefoundtheDPITsprovidedvery goodcontrolof A.variegatum ,significantlyreducing theirnumbersontreatedanimalsandprovidinganaverageof92%controlthroughoutthe21monthstudy.Ina shortthreemonthstudyonGuadeloupe,cattlehad62 to85%fewer A.variegatum aftertheyweretreatedwith DPITs[17].InZimbabwe,therewasover92%controlof A.hebraeum withslowreleasetagsimpregnatedwith cyfluthrinorflumethrin,butonly50-75%withalphacypermethrin[16]. Similartopreviousstudies,wefoundtheslow-release tagswererelativelyeasytomake,onlyrequiringanoven andstandardlaboratoryequipment(scale,glassware,etc.). Theywerealsoeconomical,costingconsiderablylessthan treatmentswithcommerciallyavailablepour-onformulations,evenwhenthepriceofthelatterwassubsidizedby government. AlthoughtheDPITswereveryeffectiveincontrolling A.variegatum onthecattletowhichtheywereattached, therewasnoevidencetheydecreasedthefree-living populationsof A.variegatum intheenvironmentduring thecourseofthe21monthstudy(Table1).Insteadof ticknumbersfallinginthesecondyearofthestudy, therewasratheranincreaseinticksonsentinelanimals, althoughthiswasnotstatisticallysignificant.Thiswas despitethefactthatthepercentageofanimalswith DPITswasincreasedonbothfarmsinthesecondhalfof theproject.Further,over2,000 A.variegatum wereremovedfromthepopulationpoolsduringthestudywhen ticksweremanuallycollectedfromthecattleinthe study.Totalnumbersof A.variegatum inanareaare particularlysusceptibletoacaricideuseoncattleas95% ofallstagesofthetickarefoundonungulates,inparticularcattlewhicharefarmorefrequentlyinfestedthan sheeporgoats(25:1)[14].Ithasbeenestimatedthat, withoutreinfestation,regularacaricideapplicationto cattlewithastockingdensityof2 – 4animalsperhectare wouldremoveall A.variegatum in3 – 5weeks[22].The failureofDPITsinourstudytosignificantlyalterthe free-livingpoolof A.variegatum wasprobablybecause untreatedcattlewerefreetowanderintoareasofpoor tickcontrolwheretheyacquiredticksthatwerecarried backtothestudyfarmswhencattlereturnedforwater. Theoptimalpercentageofanimalstobefittedwith DPITstobringaboutsignificantreductionsinthefreelivingtickpopulationsneedstobedeterminedbutwill probablybemorethanthe29%to44%usedinour study. Themajorproblemweexperiencedwiththeslowreleasetagswastherelativelyhighrateoftailtagloss (38%)beforetheyweredueforreplacementatthree months.Similarproblemswereencounteredinastudy onpasturedcattleinGuadeloupewhere45%ofthetail tagswerelostandneededreplacingduringthethree monthstudy[17].Initiallossoftagswas3%perweek butthisroseto9%oftagslosteachweekinthefinal monthofthestudy.Whenthetagswereappliedas describedinourstudyandtestedondairycattlein Zimbabwe,15%werelostinthefirstmonth[16].Theloss oftagsreducestheoverallefficacyoftickcontroland meansincreasedworkandexpenseforfarmerstoreplace them.Inallpreviousreports,andinourstudy,tailtags werelostbecausethetagandhairwerepulledfromthe tailratherthanduetolossofadhesionofthetagstothe tailhair.Methodsto  streamline thetagsintothetailare requiredtopreventthemfromcatchingandsnaggingon vegetationandobjectsandbeingwrenchedfromthetail togetherwiththetailhairs.ConclusionsInconclusion,ourstudyhasshownthatDPITsare cheaptoproduce,easytoplace,andveryeffectivein controlling A.variegatum oncattle.Theyare,however, relativelyeasilylostfromthetails,particularlyinthick vegetation,and,whenplacedonupto44%ofanimals, theydonotsignificantlyreducethetotalenvironmental loadof A.variegatum .Abbreviations PITs: Slow-releaseplastictagsimpregnatedwithaggregation-attachment pheromones;DPITs:Slow-releaseplastictagsimpregnatedwithdeltamethrin (7%)andaggregation-attachmentpheromones. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterest.Kelly etal.Parasites&Vectors 2014, 7 :182 Page7of8 http://www.parasitesandvectors.com/content/7/1/182

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Authors ’ contributions PKwasinvolvedinthestudydesign,projectmanagementonStKitts,data collectionandprocessing,andhedraftedthemanuscript.HLwasinvolved inthestudydesign,managedthetechnicalaspectsoftheprojectonStKitts, andwasinvolvedindatacollectionandprocessing,andmanuscript preparation.CRandwasinvolvedinprojectmanagement,datacollection andsampleprocessing,andreviewingthemanuscript.KAwasinvolvedin projectmanagement,datacollectionandsampleprocessing,andreviewing themanuscript.JBwasinvolvedinthestudydesign,dataprocessingand draftingthemanuscript.MDwasinvolvedinthestudydesign,project managementinFlorida,dataprocessinganddraftingofthemanuscript.All authorsreadandapprovedthefinalmanuscript. Acknowledgements FundingwasprovidedbytheUSDANIFATSTARprogram(2010-34135-21018) andtheEmergingPathogensInstituteattheUniversityofFlorida.Thestudy sponsorshadnoroleinthestudydesign,inthecollection,analysisand interpretationofdata,thewritingofthemanuscript,orinthedecisionto submitthemanuscriptforpublication.WethankMr.L.Cranston,Mr.Williams, andMr.S.Mortonforallowingaccesstotheirfarmsandtheirhelpwiththe project.WealsothankAntoinetteMcIntoshfortechnicalassistanceand Drs.T.ChallengerandL.HenryandMr.L.ClarkeoftheDepartmentof VeterinaryServices,St.Kitts,fortheirinput. Authordetails1SchoolofVeterinaryMedicine,RossUniversity,Basseterre,St.KittsandNevis, WestIndies.2EmergingPathogensInstitute,UniversityofFlorida,Gainesville, FL32611,USA.3DepartmentofGeography,UniversityofFlorida,Gainesville, FL32611,USA.4DepartmentofInfectiousDiseasesandPathology,Collegeof VeterinaryMedicine,UniversityofFlorida,Gainesville,FL32611,USA. Received:27February2014Accepted:8April2014 Published:14April2014 References1.BeatiL,PatelJ,Lucas-WilliamsH,AdakalH,KandumaEG,Tembo-MwaseE, KrecekR,MertinsJW,AlfredJT,KellyS,KellyP: Phylogeographyand demographichistoryof Amblyommavariegatum (Fabricius)(Acari: Ixodidae),thetropicalbonttick. Vector-BorneZoonoticDis 2012, 12: 514 – 525. 2.BarreN,GarrisGI: Biologyandecologyandmanagementofthetick Amblyommavariegatum intheCaribbean. In TechnicalCenterfor AgriculturalandRuralCooperation(CTA)SeminarProceedings:Cowdriosisand DermatophilosisofLivestockintheCaribbeanRegion:12-14thNovember1990; StJohn,Antigua. EditedbyWalmsleyD.StAugustine,TrinidadandTobago: CaribbeanAgriculturalResearchandDevelopmentInstitute;1990:56 – 77. 3.BarreN,GarrisG,CamusE: Propogationof Amblyommavariegatum inthe Caribbean. RevSciTechOfIntEpiz 1995, 14: 841 – 855. 4.StClairPhillipK: Tropicalbonttick( Amblyommavariegatum )eradication intheCaribbean.TheSt.Kittsexperience. 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MedVetEntomol 2006, 20: 317 – 324. 27.Rosario-CruzR,GuerreroFD,MillerRJ,Rodriguez-VivasRI,TijerinaM, Dominguez-GarciaDI,Hernandez -OrtizR,CornelAJ,McAbeeRD, Alonso-DiazMA: Molecularsurveyofpyrethroidresistancemechanismsin Mexicanfieldpopulationsof Rhipicephalus ( Boophilus ) microplus ParasitolRes 2009, 105: 1145 – 1153. 28.FreitasEdeP,ZapataMT,FernandesF: Monitoringofresistanceor susceptibilityofadultsandlarvaeofAmblyommacajennense(Acari: Ixodidae)tosyntheticacaricidesinGois. BrazilExpApplAcarol 2011, 53: 189 – 202. 29.GarrisGI,BarrN: Acaricidesusceptibilityof Amblyommavariegatum (Acari:Ixodidae)fromPuertoRicoandGuadeloupe. ExpApplAcarol 1991, 12: 171 – 179.doi:10.1186/1756-3305-7-182 Citethisarticleas: Kelly etal. : Efficacyofslow-releasetagsimpregnated withaggregation-attachmentpheromoneanddeltamethrinforcontrol of Amblyommavariegatum onSt.Kitts,WestIndies. Parasites&Vectors 2014 7 :182.Kelly etal.Parasites&Vectors 2014, 7 :182 Page8of8 http://www.parasitesandvectors.com/content/7/1/182