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HeartwormandWolbachia:TherapeuticimplicationsJ.
W.
McCalla,*,C.
Genchib,L.
Kramerc,J.
Guerrerod,M.
T.
Dzimianskia,P.
Supakorndeja,A.
M.
Mansoure,S.
D.
McCalle,N.
Supakorndeje,G.
Grandie,B.
CarsoneaDepartmentofInfectiousDiseases,CollegeofVeterinaryMedicine,UniversityofGeorgia,Athens,GA,UnitedStatesbDipartimentodiPatologiaAnimale,IgieneeSanita`PubblicaVeterinaria,SezionediPatologiaGeneraleeParasitologia,Universita`degliStudidiMilano,Milano,ItalycDipartimentodeProduzioneAnimali,Universita`diParma,43100Parma,ItalydDepartmentofPathobiology,SchoolofVeterinaryMedicine,3800SpruceStreet,UniversityofPennsylvania,Philadelphia,PA,UnitedStateseTRSLabs,Inc.
,295ResearchDrive,Athens,GA,UnitedStatesAbstractAsafer,moreeffectiveadulticidaltreatmentandasafemethodforreducingmicrolaremiaandbreakingtransmissionofheartwormdiseaseearlyinthetreatmentareneeded.
Thepresentstudyevaluatedefcacyofivermectin(IVM)anddoxycycline(DOXY)aloneortogether(withorwithoutmelarsomine[MEL])indogswithinducedadultheartworminfectionandassessedtheabilityofmicrolariaefromDOXY-treateddogstodeveloptoL3inAedesaegyptimosquitoesandsubsequentlytobecomereproductiveadultsindogs.
Thirtybeagleswereeachinfectedwith16adultheartwormsbyintravenoustransplantation.
Sixweekslater,dogswererankedbymicrolarialcountandrandomlyallocatedto6groupsof5dogseach.
BeginningonDay0,Group1receivedIVM(6mcg/kg)weeklyfor36weeks.
Group2receivedDOXY(10mcg/(kgday))orallyWeeks1–6,10–11,16–17,22–25,and28–33.
Groups3and5receivedIVMandDOXYaccordingtodosesandschedulesusedforGroups1and2.
AtWeek24,Groups3and4receivedanintramuscularinjectionofMEL(2.
5mg/kg),followed1monthlaterbytwoinjections24hapart.
Group6wasnottreated.
Bloodsampleswerecollectedforperiodicmicrolariacountsandantigen(Ag)testing(andlaterimmunologicevaluationandmolecularbiologyprocedures).
Radiographicandphysicalexaminations,hematology/clinicalchemistrytesting,andurinalysisweredonebeforeinfection,beforeDay0,andperiodicallyduringthetreatmentperiod.
At36weeks,thedogswereeuthanizedandnecropsiedforwormrecovery,collectionoflung,liver,kidney,andspleensamplesforexaminationbyimmunohistochemistryandconventionalhistologicalmethods.
AlldogstreatedwithIVM+DOXY(withorwithoutMEL)wereamicrolaremicafterWeek9.
MicrolarialcountsgraduallydecreasedindogstreatedwithIVMorDOXY,butmosthadafewmicrolariaeatnecropsy.
MicrolarialcountsfordogstreatedonlywithMELweresimilartothoseforcontrols.
AntigentestscoresgraduallydecreasedwithIVM+DOXY(withorwithoutMEL)andafterMEL.
AntigenscoresforIVMorDOXYaloneweresimilartocontrolsthroughoutthestudy.
Reductionofadultwormswas20.
3%forIVM,8.
7%forDOXY,92.
8%forIVM+DOXY+MEL,100%forMEL,and78.
3%forIVM+DOXY.
MosquitoesthatfedonbloodfromDOXY-treateddogshadL3normalinappearancebutwerenotinfectivefordogs.
PreliminaryobservationssuggestthatadministrationofDOXY+IVMforseveralmonthspriorto(orwithout)MELwilleliminateadultHWwithlesspotentialforseverethromboembolismthanMELalone.
#2008ElsevierB.
V.
Allrightsreserved.
Keywords:Canineheartworm;Dirolariaimmitis;Ivermectin;Doxycycline;Melarsominedihydrochloride;Wolbachiawww.
elsevier.
com/locate/vetparAvailableonlineatwww.
sciencedirect.
comVeterinaryParasitology158(2008)204–214*Correspondingauthor.
Tel.
:+17065425684/+17065428449;fax:+17065425771.
E-mailaddress:jwmccall@uga.
edu(J.
W.
McCall).
0304-4017/$–seefrontmatter#2008ElsevierB.
V.
Allrightsreserved.
doi:10.
1016/j.
vetpar.
2008.
09.
0081.
IntroductionAllcurrentevidencesuggeststhatWolbachia(Sironietal.
,1995),whicharebacteriafoundinallindividualsofthelarialspeciesthatareknowntoharborthesebacteria,areendosymbionts(i.
e.
,thebacteriaisessentialforthelarialworms'survival)(Tayloretal.
,2005a).
Tetracyclinesinhibittheearlydevelop-mentoflarialinfectionsinanimalmodelsandblockembryogenesisinhumanlariaeandheartworms,presumablybyeliminationoftheWolbachiafromtheworm(Bosshardtetal.
,1993;Bandietal.
,1999;Hoeraufetal.
,1999,2001,2000a,b,2003a,b;McCalletal.
,1999;Langworthyetal.
,2000;Townsonetal.
,2000;Genchietal.
,2001;Casiraghietal.
,2002;RaoandWeil,2002;Raoetal.
,2002;Chirgwinetal.
,2003;Volkmannetal.
,2003;Tayloretal.
,2005a,b;SmithandRajan,2000).
Incontrast,larialspeciesthatdonotharborWolbachiaarenotaffectedbytetracyclines(Hoeraufetal.
,1999;McCalletal.
,1999).
Wolbachia-derivedproteinshavebeenimplicatedintheinam-matoryresponse(Brattigetal.
,2001,2004;Crossetal.
,2001;Tayloretal.
,2001;Bazzocchietal.
,2003;Grandietal.
,2005;Hiseetal.
,2004;Krameretal.
,2005),innateandadaptiveimmuneresponses(Bazzocchietal.
,2000,2007;Punkosdyetal.
,2003;Simonetal.
,2003;Brattigetal.
,2004;Krameretal.
,2003,2005;Morchonetal.
,2004)andtolerizedimmunologicalphenotype(O'Connoretal.
,2003;Brattigetal.
,2004;Turneretal.
,2006;Morchonetal.
,2007a,b)associatedwithhumanlariasisandheartwormdiseaseindogs.
Protractedmonthlyprophylacticdosesofivermectin(IVM)provide''safety-net''activityagainstdevelopingheartwormsanda''slow-kill''effectonadultheart-worms,withearlierinhibitionofembryogenesis(McCall,2005;McCalletal.
,1995,1996,1998,2001).
TheuniquedrugeffectsofIVMon3–8-month-oldheartwormsarerelatedtotheageofthewormsatinitiationoftreatment.
Theearliertreatmentisstarted,theshorterthesurvivaltimeofthewormsandthemorestuntedaretheworms.
Wormsthatarefullygrownwhentreatmentisstartedarenotshorterafterprolongedmonthlytreatment,butwormmassisreducedbyatleast20%duetothedeathofalluterinestagesofmicrolariaeandthe''wastingaway''conditionoftheworms(McCall,1993,unpublisheddata).
Melarsominedihydrochloride(MEL)istheonlydrugapprovedintheUnitedStatesbytheFoodandDrugAdministrationasanadulticideforheartworms.
Twointramuscularinjections(2.
5mg/kggiven24hapart)orthreeinjections(2.
5mg/kgfollowed1-monthlaterbytwoinjections24hapart)arethetwotreatmentprotocolsavailableforMEL.
TheAmericanHeartwormSociety(AHS)recommendsthethree-injectionprotocolbecauseitissaferandmoreefcacious.
TheAHSfurtherindicatesthatitmaybebenecialtoadministeramacrocycliclactonepreventativeforupto6monthspriortotheadulticide,whenthepresentationdoesnotdemandimmediateattention(Nelsonetal.
,2005).
Deathofadultlarialworms,whetherduetotreatmentornaturalcauses,resultsinanexacerbatedinammatoryresponse.
WehypothesizethatthisheightenedresponseisdueinparttothereleaseofWolbachiafollowingdeathanddisintegrationoftheworms.
ThestudypresentedheredescribesanevaluationoftheefcacyofIVMandDOXY,aloneortogether(withorwithoutMEL)indogswithinduced,adultinfectionwithDirolariaimmitisandassessmentoftheabilityofmicrolariaefromDOXY-treateddogstodevelopto3rdstageinA.
aegyptimosquitoesandthentobecomereproductiveadultsindogs.
Thisarticleispartofacomplexstudythatisstillongoingandotherdatafromthestudyisbeingpublished.
2.
Materialsandmethods2.
1.
StudydesignAnoverviewofthestudydesignfor,30-dogexperimentandispresentedinTable1.
J.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214205Table1TimingoftreatmentsandevaluationsindogswithinducedinfectionswithadultDirolariaimmitis.
TreatmentTimingoftreatmentsandevaluationsIvermectinWeeks1–36DoxycyclineWeeks1–6,10–11,16–17,22–25,28–33Ivermectin+Weeks1–36Doxycycline+Weeks1–6,10–11,16–17,22–25,28–33MelarsomineWeek24,28a/baMelarsomineWeeks24,28a/baIvermectin+Weeks1–36DoxycyclineWeeks1–6,10–11,16–17,22–25,28–33ControlNotreatmentEvaluationsMicrolarialcounts/AgWeeks–6,0,6,10,12,16,18,24,26,28,32,36RadiographsWeeks–6,0,12,24,28,32,36Hematology/clinicalpathology/physicalexam/urinalysisWeeks0,12,24,28,32,36aTwoinjectionsofmelarsominewereadministered24hapartatWeek28.
2.
2.
AnimalsAtotalof30purpose-bredbeagles,including23malesand7females,12–16.
5monthsofage,weighing15.
1–34.
2kg,wereusedinthestudy.
Thedogswerehousedinmosquito-proofindoorpensinapurpose-builtbuilding,withcontrolledtemperatureandventilationsystems.
Thedogswerefedatleastoncedailyanappropriatequantityofcommerciallyavailablemain-tenancedietandwaterwassuppliedadlibitum.
Treateddogswerehousedindividuallyinruns,andnontreateddogswerehoused1–3ofthesamesextoarun.
Theanimalsweremaintainedwithdueregardfortheirwelfareandinaccordancewithapplicablelaws,regulationsandguidelines.
TheprotocolwasapprovedbytheInstitutionalAnimalCareandUseCommitteepriortoinitiationofthestudy.
Anadditional11heartworm-naivebeagleswereobtainedfromthecommercialsupplierforthemosquitostudies(M-1,M-2,andM-3).
Twoofthesedogswereexperimentallyinfectedwithheartworms,becamemicrolaremic,andwereusedasdonorsinstudyM-3.
Theremaining9dogswereusedasrecipientsinthemosquitostudies.
Allpersonnelmakingobservations,performingtestsandprocedures,andcollectingdatawereblindedinregardtowhichweretreatedandcontrolanimals.
Groupswerecolor-codedforidenticationbylabora-torypersonnelthroughoutthestudy.
2.
3.
ParasiteEachofthe30dogswasinfectedwithnineadultfemaleandsevenadultmaleheartwormsbyintravenoustransplantationviaajugularvein(Dzimianskietal.
,1989).
Theheartwormsrangedinagefrom10to21monthsandrepresentedthreedifferentlaboratorystrainsoftheparasite(Butch,Lady,andMissouristrains).
2.
4.
DogtreatmentstudyApproximately6weeksafterinfection,the30dogswererankedbymicrolariaecountwithingender,andthenrandomlyallocatedtoanontreatedcontrolgroupofvedogsandvetestgroupsofvedogseach.
Eachgroupcontainedatleastonedogofeachsex.
2.
5.
StudydrugsCommerciallyavailablechewables(Heartgard1Plus,MerialLimited,Duluth,GA)containingIVMandpyrantelpamoatewereadministeredorallyaccord-ingtoinstructionsonthelabel,withIVMgivenataminimumdosageof6mcg/kgofbodyweightandpyrantelgivenataminimumdosageof5mg/kgofbodyweight.
Doxycyclinehyclate(West-wardPharmaceuticals,Inc.
,Eatontown,NJ)wasgivenorallyforvaryingperiodsthroughoutthestudyatadosageequivalentto10mgdoxycycline/(kgday).
Melarsominedihy-drochloride(Immiticide1,MerialLimited,Duluth,GA)wasadministeredasintramuscularinjectionsintheepaxialmusclesat2.
5mg/kgofbodyweightperinjection.
Thethree-injectionprotocol(alternatesche-dule)wasused:oneinjectionfollowed1monthlaterbytwoinjections24hapart.
AlltreatmentswithIVMandDOXYwerestartedonDay0(Table1).
Group1dogsweregivenIVMweeklyfor36weeks.
Group2dogsweregivenDOXYdailyduringWeeks1–6,10–11,16–17,22–25,and28–33.
Group3dogsweregivenbothIVMplusDOXYasforGroups1and2andMEL(IVM+DOX-Y+MEL)asforGroup4.
Group4dogsweregivenoneinjectionofmelarsomine(MEL)at24weeks,followed1monthlaterby2injections24hapart.
Group5dogsweregivenIVMplusDOXYasforGroups1and2.
Group6servedasthenontreatedcontrol.
WholebloodandserumsampleswerecollectedformicrolariaeandD.
immitisantigen(Ag)testing,immunologicevaluationandmolecularbiologyproce-duresbeforeIVMandDOXYdosingwasinitiatedandagainatWeeks6,10,12,16(microlariae/Agonly),18(microlaria/Agonly),24,26(microlariae/Agonly),28,32,and36.
Radiography,physicalexaminations,hematology/clinicalchemistrytestingandurinalysiswereperformedbeforeinfection(radiographyonly),beforeDay0,andagainatWeeks12,24,28(Groups3and4),32(Groups3and4)and36(Table2).
Allofthedogswerehumanelyeuthanizedandnecropsiedat36weeks.
2.
6.
MicrolarialcountingandantigentestingAteachbloodcollection,a20-mLsamplewasremovedtomakeaGiemsa-stainedpreparation(Schlotthaueretal.
,1986),and1mLwasretainedforlaterexaminationbythemodiedKnottmethod.
ForthemodiedKnottmethod,the1-mLsamplesweremixedwith2%formalinandkeptuntilafterthe20-mLslidepreparationswereexamined.
Ifmicrolariaewerenotseenonthispreparation,theentire1-mLsamplewasexamined.
J.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214206Ateachsampling,wholebloodsampleswerecollectedinEDTAforlateruse,andserumwascollectedandstoredat708Cuntilneeded.
SerumwasexaminedforadultD.
immitisAgusingacommerciallyavailablekit(DiroCHEK1,SymbioticsCorporation,SanDiego,CA).
Asubjectivescoringsystem,basedontheintensityofthecolorreaction,wasusedtoexpresstheresultsofeachtestasnegative(,0);veryweakpositive(vwk+,1),weakpositive(wk+,2);positive(+,3);strongpositive(++,4);orverystrongpositive(+++,5)(McCalletal.
,1996).
2.
7.
NecropsyJustpriortonecropsy,eachdogwasgivenapproximately2mLofheparin(1000USPunits/mL)intravenouslyandtheappropriatedoseofabarbiturateeuthanasiasolution.
Theheartandlungswereexaminedgrosslyforpathologicalchanges.
ThepleuralandperitonealcavitieswereexaminedforD.
immitisandtheanteriorandposteriorvenaecavaewereclampedbeforeremovaloftheheartandlungs.
Therightatrium,rightventricle,andpulmonaryarteries(includingthosecoursingthroughthelungs)weredissectedandexaminedforworms.
Thewormsfromeachdogwererecordedaseitherdeadoralive;livewormswerefurtherclassiedbymotilityandappearancecomparedwiththosefromcontroldogs.
Wormswerethensexedandcounted.
Forlaterstudies,somefemalewormsfromeachdogweredissectedandtheremovedintestinesandreproductivetractswereplacedinEMxative,someentirewormswereplacedinEMxative,somewormswerefrozen,somewereplacedinRNAlater,andsomewereplacedin10%formalin.
Alsoforlaterstudies,lung,liver,kidney,andspleensamplesweretakenfromeachdogforexaminationbyimmunohistochemistry,conventionalmicroscopy,transmissionelectronmicro-scopyandmolecularbiologytechniques.
2.
8.
StatisticalanalysisThenumbersoflivemaleworms,livefemaleworms,andtotallivewormsweredeterminedforeachdog.
Thenumbersofwormsrecoveredforeachdogweretransformedtothenaturallogarithm(count+1)forcalculationofgeometricmeansandanalysis,usingone-wayanalysisofvarianceforcompleterandomizeddesign.
Eachgroupwascomparedwiththecontrolgroup,usingasingledegreeoffreedomcontrast.
GroupswerecomparedwitheachotherusingDuncan'smultiplerangetest.
Efcacyagainstmale,female,andtotalwormsineachtestgroupwascalculatedasthepercentreductions,usinggeometricmeans,comparedwiththecontrolgroup.
2.
9.
MosquitostudiesForM-1andM-2experiments,pooledbloodsamplesfromseveraldogsineachofGroups1(IVMonly),2(DOXYonly),and6(nontreatedcontrol)weremembrane-fedtoadultfemaleA.
aegypti(LiverpoolBlack-eyedstrain)mosquitoes(McCall,1981).
Sixteendayslater,L3fromeachofthethreesetsofmosquitoeswerecollectedinapproximately1mLofHanks'balancedsaltsolution(pH7.
0)containingpenicillin(0.
4units/mL)andstreptomycin(0.
4mg/mL)andinjectedsubcutaneouslyintotheinguinalareaofeachofthreedogsusingatuberculinsyringewitha20-gaugeneedle.
FortheM-3experiment,bloodfromadonordogtreatedonlywithDOXY(Ladystrain)asforM-1andbloodfromadonorcontroldog(Ladystrain)weremembrane-fedtotwogroupsofmosquitoes.
Infective-stagelarvaefromtheDOXYmosquitoeswereinjectedsubcutaneouslyintotworecipientdogs,andonerecipientdogwasgivenL3fromthemosquitoesfedonthebloodfromthedonorcontroldog.
J.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214207Table2Efcacyofivermectinanddoxycycline,givenaloneortogetherwithorwithoutmelarsomine,beginning6weeksafterinfectionatnecropsy36weeksafterintravenoustransplantationofliveadultDirolariaimmitis.
TreatmentMeanno.
liveadultheartwormsPercentefcacyMaleFemaleTotalRangeWeeklyivermectin(6mg/kg)4.
2a6.
8a11.
0a5–1420.
3Intermittentdoxycycline(10mg/(kgday))4.
6a8.
0a12.
6a9–158.
7Weeklyivermectin+intermittentdoxycycline+melarsomine(threeinjections)0.
8b0.
2c1.
0b,c0–492.
8Melarsomine(threeinjections)0b0c0c–100Weeklyivermectin+intermittentdoxycycline0.
4b2.
6b3.
0b0–678.
3Nontreatedcontrol5.
4a8.
4a13.
4a12–16NADifferentsuperscriptsinacolumnindicateasignicantdifferencebetweengroups(p0.
01),NA=notapplicable.
ThemosquitoesfortheM-1experimentwereinfectedwithmicrolaremicbloodonDay66(Week9,3daysafterthelastdoseofIVMand25daysafterthelastdoseofDOXY).
Sixteendayslater,thetotalofsixL3collectedfromtheIVMmosquitoeswereinjectedsubcutaneouslyintoRecipientdog1,40oftheL3collectedfromtheDOXYmosquitoeswereinjectedsubcutaneouslyintoRecipientdog2,and40oftheL3collectedfromthecontrolmosquitoeswereinjectedintoRecipientdog3.
MicrolariaeandAgtestingwasperformed5.
8,6.
7,7.
1,8.
0,8.
8,9.
4,and10.
4monthsafterinfectionandthethreedogswerenecropsiedforrecoveryofadultwormsat10.
8months.
ThemosquitoesfortheM-2experimentwereinfectedonDay129(Week18).
Atthispointinthestudy(i.
e.
,3daysafterthelastdoseofIVMand4daysafterthelastdoseofDOXY),microlariaecountsfortheIVM-andDOXY-treatedgroupshadalreadydecreasedsubstantially.
Thetotal(3)L3collectedfromtheIVMmosquitoeswereinjectedintoRecipientdog4;thetotal(5)L3collectedfromtheDOXYmosquitoeswereinjectedintoRecipientdog5;and40ofthosefromthecontrolmosquitoeswereinjectedintoRecipientdog6.
MicrolariaandAgtestingwasdoneat5.
0,5.
9,6.
7,7.
3and8.
3monthsafterinfectionandthedogswerenecropsied8.
7monthsafterinfection.
FortheM-3experiment,themosquitoeswereinfectedwithmicrolaremicbloodonDay54(Week8,14daysafterthelastdoseofDOXY).
Sixteendayslater,Recipientdogs7and8wereeachinjectedwith40L3larvaefromtheDOXYmosquitoesandRecipientdog9received40controllarvae.
Recipientdog7wasnecropsied4.
2monthsafterinfection,whichwastooearlyformicrolariaeandAgtesting.
MicrolariaeandAgtestswereperformedforthetworemainingRecipientdogs(8and9)justpriortonecropsyat6monthsafterinfection.
Alldogsinfectedbyeitherintravenoustransplanta-tionorsubcutaneousinjectionofL3werenegativeforheartwormmicrolariaeandAgpriortoinfection.
3.
Results3.
1.
DogtreatmentstudyWeeklyadministrationofIVMplusintermittentDOXY(IVM+DOXY)for36weekswas78.
3%effectiveinreducingtheadultwormburden(Table2).
Oneofthevedogsinthisgroupwascompletelyclearedofworms,andmostoftheremainingwormsintheotherfourdogswereabnormalinappearance,withintermittenttranslucentareasinthebodyanddarkanteriorends.
Incomparison,treatmentwithonlyIVMwasonly20.
3%effective.
Alldogsinthisgrouphadadultworms,andmostofthewormswereabnormalinappearance,withintermittenttranslucentareasinthebodyanddarkanteriorends.
TreatmentwithonlyDOXYwasonly8.
7%effective.
TreatmentwithIVM+DOXY+MELwas92.
8%effectiveagainstadultworms.
Threeofthevedogsinthisgroupwereclearedofworms,andthetworemainingdogshad1maleand4(3males,1female)liveworms,respectively.
Allofthesewormswereabnormalinappearance.
MELalonewas100%effectiveinreducingthewormburden;onedeadfemalewormwasrecoveredfromonedogandfourofthevedogshadfragmentsofworms.
Incomparison,allnontreatedcontroldogshadliveworms,withanaverageof13.
4perdog(range=12–16).
Allexcepttwoofthewormsfromthesenontreatedcontroldogswerenormalinappearance.
3.
1.
1.
MicrolarialcountsGroupmeanmicrolarialcountsarepresentedinFig.
1.
Alldogswerenegativeforcirculatingmicrolariaepriortoinfection.
AlldogsalsowerepositiveformicrolariaejustpriortoDay0,exceptonedog,whichbecamemicrolaremicbyWeek6.
ForbothgroupsthatweregivenIVMplusDOXY(withorwithoutMEL),meanmicrolariaecountsdroppedrelativelysoonaftertreatmentstarted,andnomicrolariaewereseeninanyofthese10dogsafterWeek9.
BeginningapproximatelyWeek10,meanmicrolariaecountsfordogsthatreceivedIVM+-DOXYdroppedgraduallytonegativeornear-negativevalues(generally10microlariae/mL)justpriortonecropsy.
Atnecropsy,allveDOXYdogshadmicrolariae.
TwoofthevedogstreatedwithIVMwereamicrolaremic,andthreeofthemhad2,4,and100microlariae/mL,respectively,justpriortoJ.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214208Fig.
1.
Meanmicrolarialcountsfordogsgivenweeklyivermectin(IVM)orintermittentdoxycycline(DOXY),aloneortogether(withorwithoutmelarsomine[MEL]later),beginning6weeksafterintrave-noustransplantationofadultheartworms.
necropsyat36weeks.
MeanmicrolariaecountsintheMELandnontreatedcontrolgroupsremainedrelativelyhighthroughoutthestudy.
3.
1.
2.
AntigenscoresGroupmeanAgscoresforthenontreatedcontrolandtreatedgroupsarepresentedinFig.
2.
AlthoughthescorefortheIVM+DOXY+MELgroupspikedatWeek32,thescoresforbothgroupsthatreceivedIVMplusDOXY(IVM+DOXYandIVM+DOXY+-MEL)graduallydroppedafterWeek26,andthescoreforbothgroupswas2.
2justpriortonecropsy.
TheAgscorefortheMEL-treatedgroupdidnotdropuntiljustpriortonecropsy(score=1.
8).
ThemeanscoresforthegroupsthatreceivedonlyIVMorDOXYweresimilartothosefornontreatedcontrolsthroughoutthestudy(range3.
8–4.
2).
3.
2.
Mosquitostudies3.
2.
1.
M-1studyRecipientdog3thatwasgiven40infectivelarvaefrommosquitoesthathadfedonbloodfromnontreateddogswaspositiveformicrolariaeandAg6.
7monthspost-infectionandremainedpositiveonbothteststhroughtothetimeofnecropsyat10.
8monthsPI.
Atnecropsy,thisdoghadatotalof27adultworms.
Recipientdog1thatwasgiven6infectivelarvaefromthemosquitoesthathadfedonbloodfromthedogstreatedwithIVMwasnegativeformicrolariaeandAgthroughoutthestudyandhadtwoliveadultmalewormsatnecropsy.
Recipientdog2thatreceived40infectivelarvaefrommosquitoesthathadfedonbloodfromdogstreatedwithDOXYwasnegativeformicrolariaeandAgthroughoutthestudyandhadnolivewormsatnecropsy.
3.
2.
2.
M-2studyRecipientdog6thatwasgiven40larvaefrommosquitoesthathadfedonbloodfromthenontreateddogswaspositiveformicrolariaeandAgat5.
9monthsafterinfectionandremainedpositiveonbothteststhroughtothetimeofnecropsyat8.
7monthsafterinfection.
Atnecropsy,thisdoghadatotalof26adultworms.
Recipientdog5thatreceivedveinfectivelarvaefrommosquitoesthathadfedonbloodfromdogstreatedwithDOXYwasnegativeformicrolariaeandAgthroughoutthestudyandhadnolivewormsatnecropsy.
Recipientdog4thatwasgivensixinfectivelarvaefrommosquitoesthathadfedonbloodfromdogstreatedwithIVMwasnegativeforbothmicrolariaeandAgthroughoutthestudybuthadoneliveadultfemalewormatnecropsy.
3.
2.
3.
M-3studyNolivewormswererecoveredfromRecipientdog7necropsied4.
2monthsafterinfectionwith40larvaefrommosquitoesthathadfedonadogtreatedwithDOXY.
Justpriortonecropsyat6monthsafterinfection,Recipientdog8,whichwasalsoinfectedwithlarvaefrommosquitoesthatfedonadogtreatedwithDOXY,wasnegativeformicrolariaeandAgandhadnowormsatnecropsy.
Controldog9waspositiveforbothmicrolariaeandAgandhadatotalof21liveadultwormsatnecropsy.
4.
DiscussionWeeklyadministrationofprophylacticdoses(6mcg/kg)ofIVMresultedinagradualreductioninmicrolarialcounttonegative(fortwodogs)ornear-negative(2–100microlariae/mL)valuesbytheendofthestudy.
TheseresultsaresimilartothoseobtainedinotherstudieswhereIVMwasgivenmonthlyatthesamedosage(McCalletal.
,1998,2001;Bowmanetal.
,2001).
IntermittentadministrationofdailydosesofDOXYcausedagradualreductioninmicrolariaecount,withapatternsimilartothatoftheIVM-onlygroup,butallofthevedogshadafewmicrolariae(10microlariae/mL)justpriortonecropsyat36weeks.
StartingtreatmentwithDOXYandIVMtreatmentsapproximately6monthsbeforethersttreatmentwithMELwastocleartheWolbachiafromthewormsasmuchaspossiblebeforeinitiatingtreatmentwithMEL.
Inourpreviouslaboratorystudies,wehaveseenareboundinWolbachiaatvaryingtimesaftertreatment.
Givingintermittenttreatmentsoverthecourseofthestudywasexpectedtoprecludethatrebound.
J.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214209Fig.
2.
Meanheartwormantigentest(DiroCHEK1)scoresfordogsadministeredweeklyivermectin(IVM)orintermittentdoxycycline(DOXY),aloneortogether(withorwithoutmelarsomine[MEL]later),beginning6weeksafterintravenoustransplantationofadultheartworms.
ThesegradualreductionsinmicrolariaecountsduringadministrationofIVMorDOXYonlystronglysuggestthatinhibitionofembryogenesis(DOXY)anddeathofdevelopingmicrolariaestagesinutero(IVM)ledtomicrolariaereductionduetoattritionratherthantoadirectmicrolaricidaleffectofthedrugs(Loketal.
,1989;McCalletal.
,2001;Tayloretal.
,2005a).
ThecompletedisappearanceofcirculatingmicrolariaeafterWeek10inall10dogstreatedwithDOXY+IVMsuggeststhatthiscombinationofthedrugshadadirectmicrolaricidaleffectaswell.
ThelowermeanAgscoresinthedogsthatweregivenMELonly,IVM+DOXYorIVM+DOXY+-MELgenerallyreectedtheirlowerwormburdens(alongwiththepresenceorabsenceofwormfragmentsasasourceofAg),comparedwiththeothergroups.
ThegradualreductioninaverageAgscoresforthegroupsofdogsthatreceivedIVM+DOXY(withorwithoutMEL)indicatesthatsomeofthewormsdiedrelativelyearlyduringthetreatmentperiodandonlyafewwormswerealiveatnecropsy,asevidencedbythe78.
3%efcacyintheIVM+DOXYgroupandthe92.
8%efcacyforIVM+DOXY+MEL.
ThespikeinAgscorefortheIVM+DOXY+MELgroupat32weekswasprobablyduetothereleaseofAgfromwormskilledbyMEL,andthedramaticreductioninAgscoreintheMELgroupbetweenWeeks32and36wasprobablyduetothedeathandclearanceofAg,sinceefcacywas100%forthisgroup.
The20.
3%reductioninwormburdenfollowingweeklyadministrationofprophylacticdosesofIVMforabout8.
5–9monthswasnotsurprising.
Inanearlierstudy,16monthlydosesofthedrugwereonly56%effectiveagainstadultheartworms(McCalletal.
,1998),whichsuggeststhatweeklydosingonlywithIVMattheprophylacticdosageisnomoreeffectiveinkillingadultheartwormsthanmonthlydosing.
ThelackofearlyadulticidalefcacyofDOXYaloneagainstheartwormsisconsistentwithndingsforotherlariae,whichusuallyrequireatleast1.
5–2yearsforadulticidaleffectstobeevident(Langworthyetal.
,2000;Gilbertetal.
,2005;Tayloretal.
,2005a,b).
IVM+DOXYhadsubstantial(78.
3%)adulticidalefcacy,anditseemsreasonabletoassumethattheremainingwormswouldhavediedifthedogshadbeenheldafewmoremonthsbeforenecropsy.
ThishighlevelofadulticidalactivityofIVM+DOXYstronglysuggestsasynergisticeffectofthesetwodrugsandwarrantsfurtherattention.
Inthecurrentstudy,treatmentwithIVMandDOXYwaspurposefullyexcessiveinanattempttoobtainnearmaximaleffect.
Follow-upstudieswillfocusonincreasingthedailydosageofDOXYandshorteningtheinitialtreatmentperiodto4weeks,followedbytreatment4–6monthslaterandadministeringIVMnomorefrequentlythanevery2weeks.
LimitingDOXYtreatmenttooneperiodof4–6weekshasbeeneffectiveintreatmentpatientswithhumanlariasis(Tayloretal.
,2005b).
Thethree-injectionprotocolforMELwas100%effectiveagainstadultheartworms,whereasIVM+-DOXY+MELwas92.
8%effective.
Thismightsuggestdruginterference,however,MELisnotalways100%effective(Dzimianskietal.
,1989;DiSaccoandVezzoni,1992;Keisteretal.
,1992;Vezzonietal.
,1992).
Conrmationofdruginterferencewouldrequireastudywithalargerdogpopulation.
PopulationdynamicsinrelationtotheroleofWolbachiainvariouslife-cyclestageshasbeenstudiedinlariae,particularlyBrugiamalayi(McGarryetal.
,2004;FennandBlaxter,2004;Kozek,2005;Tayloretal.
,2005a).
Individualwormsappeartovarywidelyintheirbacterialload,whichmayreectadynamicchangeofpopulationsizeovertimeorifconstant,thepotentialforaselectiveadvantageintermsoflongevityorfecundityinwormscarryingmorebacteria.
However,atthepopulationlevel,thenumbersofbacteriaremainstaticinmicrolariaeandthemosquito-bornestages(L2andL3),withthelowestratiosofWolbachia/nematodeDNA.
Withintherstweekofinfectionofthemammalianhost,bacterianumbersincreasedramati-callyandthebacteria/wormratioisthehighestofalllife-cyclestages.
TherapidmultiplicationcontinuesthroughoutL4development,sothatthemajorperiodofbacterialgrowthoccurswithintherstmonthofinfection.
Therearefewbacteriainmosquito-derivedL3butmany,inlargegroups,inL4collected1–3weeksafterinfection.
Itappearsthatthelargeclustersofbacteriaobservedthroughoutthehypodermalcordofadultwormsoriginatefromthisrapidperiodofdivisionandthereafteraremaintainedatthatlevel,asseeninadultmaleworms.
Infemales,bacterialnumbersincreasefurtherasthewormsmatureandastheovaryandembryoniclarvalstagesbecomeinfected.
Thus,populationdynamicsisconsistentwith(1)thetwoprocessescompromisedmostby''tetracycline''treat-ment(i.
e.
,moltsfromL3–L4andL4–juvenile;micro-lariaeproduction)and(2)evasionofmammalianimmunity(i.
e.
,L3–L4andL4–juvenilemoltsandadultwormsurvival)allowingforlong-termsurvivalofadultlariae.
Inthepresentstudy,thesurvivalofmicro-lariaeafterDOXYtreatmentofHW-microlaremicdogs,followedby''normal''developmentofthesemicrolariaetoL3andtheinabilityoftheseL3todeveloptoadultsinrecipientdogsstronglysuggestthatJ.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214210relativelyfew(ifany)WolbachiaarerequiredformicrolariaetosurviveanddevelopintoL3,butsubstantialnumbersareneededfordevelopmentofL3intoadultsinthemammalianhost.
Arsenicaldrugshavebeenthemainstayforheart-wormadulticidaltherapyforthepastfourtovedecades.
However,treatmentstrategieshavechanged,andindeed,continuetoevolvesinceMELreplacedthiacetsamidesodiumintheearly1990s.
Themanu-facturerprovidestwotreatmentprotocolsforMEL:the''standard''two-injectionprotocolforClassI(mildclinicalsigns)andClassII(moderateclinicalsigns)dogsandthe''alternative''three-injectionprotocolforClassIII(severeclinicalsigns)disease.
Generally,thiswasfollowed3–4weekslaterbyadministrationofamicrolaricide,whichoftenhadtoberepeatedonceortwicetoclearthedogofcirculatingmicrolariae.
Duringthepastseveralyears,thismicrolaricidaltreatmentgenerallyhasbeenreducedtoaby-productofmacrocycliclactonechemoprophylaxis(Nelsonetal.
,2005).
Startingin1995,severalstudieshavedemonstratedthatprolongedadministrationofmonthlyprophylacticdosesofamacrocycliclactone,particularlyIVM,killsolderlarvae,immatures(juveniles),youngadultsandmatureadults(seeMcCall,2005).
Moreover,ahighpercentageofthedogsbecomeamicrolaremicwithin6–9monthsafterdosingisstarted.
Therateofkillwiththisslow-killtreatmentisdependentontheageoftheheartwormswhentreatmentisstarted,with3-month-oldlarvaerequiringupto1yearandmatureadultsneeding2.
5yearstoprovideefcacyofatleast95%.
AlthoughmonthlyIVMisnotanapprovedalternativetoMELtherapyanditisparticularlyriskyinveryactiveandsymptomaticdogs(Vencoetal.
,2004),itclearlyprovidespotent''safety-net''activityagainstolderlarvae,immatures(juveniles),andyoungadultsincasesofownercompliancefailure,evenwhentheownerandveterinarianarenotawarethattheanimalisinfected.
TheAmericanHeartwormSociety(Nelsonetal.
,2005)recognizesthesafety-netandadulticidalproper-tiesofsomeofthemacrocycliclactones,particularlyIVM.
Their2005canineguidelinesstatethatadmin-istrationofachemoprophylacticdoseofamacrocycliclactoneshouldbeginassoonasthedogisdiagnosedwithaheartworminfection.
Whilecontroversialduetothetheoreticalriskofinducingresistancetomacro-cycliclactones,itmaybebenecialtoadministeramacrocycliclactoneforupto6monthspriortoadministrationofMELwhenthepresentationdoesnotdemandimmediateintervention.
Thereasoningforthisapproachistogreatlyreduce,ifnotcompletelyeliminate,circulatingmicrolariaeandkillmigratingD.
immitislarvaeand(inthecaseofIVM)tostuntimmatureD.
immitis(!
20%)(McCall,1993,unpub-lisheddata)andreducefemalewormmass(!
20%)(McCall,1993,unpublisheddata)byinhibitingthereproductivesystem.
Moreover,administrationfor3monthsalsowillallowimmaturewormstoreachanageatwhichtheyareknowntobesusceptibletokillingbyMEL(Keisteretal.
,1992;AtkinsandMiller,2003)andadministrationforlongerthan3monthsshouldresultinreducedantigenicmass,whichinturnmayreducetheriskofthromboembolism.
ByeliminatingtheWolbachiaendosymbionts,tetracyclineshavebeenshowninseveralspeciesoflariaetopreventdevelopmentofthelarvalstages,inhibitembryogenesisinadultworms,andeventuallykilladultlariae,usually1.
5–2yearsaftertreatmentisstartedinlaboratoryanimals,largeanimals,andhumans(Tayloretal.
,2005a;Krameretal.
,2007).
Inthepresentstudy,IVM+DOXYhadthesynergisticeffectsofcompletelyclearingdogsofcirculatingmicrolariaebyWeek10andkilling78.
3%oftheadultwormsbyWeek36.
Interestingly,WolbachiawerestilldetectedatnecropsyinwormsfromdogstreatedonlywithDOXYbutwerevirtuallyeliminatedinthosereceivingbothDOXYandIVM(Bazzocchietal.
,2008).
Grosspathologyandhistopathologicobservationsofthelungsofthe30dogsrevealedthemostseverepathologyinthedogsreceivingonlyMELandtheleastevidenceofpathologyinthosereceivingIVM+DOXY+MEL(nogrosslyvisiblelesionsin3of5dogs).
Theselatterdogsalsoshowedsignicantlyfewerseverearteriallesionsandavirtualabsenceofthrombi(Krameretal.
,2008).
ClinicalndingsofsignicanceincludedeosinophiliainmostdogstreatedwithIVMorDOXYaloneat12weeksfollowingaprecipitousdeclineinmicrolariae;onlydogsthatreceivedIVMdidnothaveelevationsinserumalaninetransaminase;andMELhadnoapparenteffectonserumchemistries(Dzimianskietal.
,2006).
Inthepresentstudy,L3collectedfrommosquitoesfedonmicrolaremicbloodfromdogstreatedwithDOXYwerenormalinappearanceandmotilitybutwerenotabletodevelopindogs.
Thus,treatmentwithDOXYpreventedfurthertransmissionofthediseaseevenwhenmicrolariaewerepresent.
TheseandotherobservationsstronglysuggestthatadministrationofbothIVMandDOXYforseveralmonthspriortoMELorpossiblywithoutMEL,willeliminateadultheartwormswithlesspotentialforseverethromboembolismthanMELaloneandwillblocktransmission.
Therefore,itislikelythatDOXYJ.
W.
McCalletal.
/VeterinaryParasitology158(2008)204–214211eventuallywillbeincludedinheartwormadulticidetherapyfordogs.
Furthermore,thepotentiallylife-threateninginfectionsandhighriskassociatedwithMELtreatmentstronglyencouragethetestingofDOXYplusIVMasanalternativeadulticidaltherapyforheartworminfectedcatsandferrets(McCalletal.
,2008).
ConictofinterestJ.
W.
McCall,Genchi,L.
Kramer,Guerrero,Dzi-mianski,P.
Supakorndej,Mansour,S.
D.
McCall,N.
Supakorndej,Grandi,andCarsonhavenopersonalornancialrelationshipwithotherpersonsororganizationsthatcouldinappropriatelyinuenceorbiasthisstudy.
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