Efficacy of Broadline® spot-on against Aelurostrongylus abstrusus and Troglostrongylus brevior lungworms in naturally infected cats from Italy

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ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

Short

Communication

Efﬁcacy

of

Broadline

®

spot-on

against

Aelurostrongylus

abstrusus

and

Troglostrongylus

brevior

lungworms

in

naturally

infected

cats

from

Italy

夽

Alessio

Giannelli

a

_,

_Emanuele

_Brianti

b

_,

_Antonio

_Varcasia

c

_,

_Vito

_Colella

a

_,

Claudia

Tamponi

c

_,

_Giancarlo

_Di

_Paola

a

_,

_Martin

_Knaus

d

_,

_Lénaïg

_Halos

e

_,

Frederic

Beugnet

e

_,

_Domenico

_Otranto

a,∗

a_Dipartimento_di_Medicina_Veterinaria,_University_of_Bari,_Bari,_Italy

b_Dipartimento_di_Scienze_Veterinarie,_University_of_Messina,_Italy

c_Dipartimento_di_Medicina_Veterinaria,_University_of_Sassari,_Italy

d_Merial_GmbH,_Kathrinenhof_Research_Center,_Rohrdorf,_Germany

e_Merial_SAS,_Sanoﬁ_Pasteur_Conﬂuence,_Lyon,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24December2014

Receivedinrevisedform23February2015

Accepted28February2015 Keywords: Aelurostrongylusabstrusus Troglostrongylusbrevior Lungworms Broadline® Cats Treatment

a

b

s

t

r

a

c

t

TheincreasingreportsofAelurostrongylusabstrususinfectionandthenewinformationon Troglostrongylusbreviorhavespurredtheinterestofthescientiﬁccommunitytowardsthe researchofpharmaceuticalcompoundseffectiveagainstbothpathogens.Anovel topi-calcombinationofﬁpronil,(S)-methoprene,eprinomectinandpraziquantel(Broadline®_,

Merial)hasbeenreleasedforthetreatmentofavarietyoffelineparasiticinfections.The presentstudyreportstheefﬁcacyofthisspot-onintreatingcatsnaturallyinfectedbyfeline lungworms.Clientownedcats(n=191)wereenrolledfromthreegeographicalareasofItaly andfaecalsampleswereexaminedbyﬂoatationandBaermanntechniques.Twenty-three individualswerepositiveforL1ofA.abstrusus(n=18)orT.brevior(n=3)orforbothspecies (n=2)andtheyweretopicallytreatedwithBroadline®_._Seventeen_of_them_were_also

con-comitantlyinfectedbyotherparasites.Fourweeksaftertreatment,faecalsampleswere collectedandexaminedtoassesstheefficacyofasingleadministrationoftheproduct. Basedonlungwormlarvaecounts,theefficacyofthetreatmentwas90.5%or100%for A.abstrususorT.brevior,respectively.Catsreleasedsignificantlyloweramountsof lung-wormlarvaeaftertreatmentcomparedtopre-treatment(p<0.0001).Allbutthreecats werenegativeforothernematodesaftertreatmentandallcatsrecoveredfromrespiratory signs.Resultsofthisstudyindicatethatasingleadministrationofthetopical combina-tionfipronil,(S)-methoprene,eprinomectinandpraziquanteliseffectiveandsafeforthe treatmentofA.abstrususand/orT.breviorinfectionsincatslivingunderfieldconditions.

夽 Broadline®_is_a_trademark_of_Merial._All_other_marks_are_the_property

oftheirrespectiveowners.

∗ Correspondingauthor.Tel.:+390804679839;fax:+390804679839.

E-mailaddress:domenico.otranto@uniba.it(D.Otranto).

1. Introduction

Domesticcatsmay beinfected byseveral

endopara-sites,whichmaycauseavarietyofclinicalsigns,ranging

fromdigestivealterationstoanaemia,andrespiratory

dis-tress (Beugnet et al., 2014). Gastrointestinalhelminths,

protozoansand lungworms are accounted asthe major

http://dx.doi.org/10.1016/j.vetpar.2015.02.037

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causeofparasiticdiseasesincatsacrossEurope(Beugnet

et al., 2014).Some of these species (e.g., Toxocaracati,

Toxoplasma gondii, Giardia lamblia) represent a risk for

humanbeings(Traversa,2012),whereasothers(e.g.,

lung-worms), though are not of zoonotic concern, may be

highlypathogenicforcatsandcauseseverelife-threatening

conditions(Gerdinetal.,2011).Inaddition,some

intesti-nalparasitesmaycauseself-limited infectionsbut their

presenceisnotacceptablebyowners.Consequently,the

administrationofan efﬁcacioustreatmentagainst these

pathogens,aswellasthepreventionoftheinfection,

repre-sentseitheranactofresponsibilitytowardsanimalwelfare

andPublicHealth.

Among the lungworms infecting cats,

Aelurostrongy-lusabstrusus(Strongylida,Angiostrongylidae)isthemost

prevalentspeciesacrossEurope(Beugnetetal.,2014)and

severalﬁeld studies have provided data on the

useful-ness of anthelmintic treatments against this nematode

(Traversaetal.,2009;Ianninoetal.,2013).Nonetheless,the

recentreportoffelinetroglostrongylosisby

Troglostrongy-lusbrevior(Strongylida,Crenosomatidae),alongwiththe

little information available on its treatment and

biol-ogy,hasspurredtheinterestofthescientiﬁccommunity

towardstheresearchofpharmaceuticalcompounds

effec-tiveagainstbothpathogens(Briantietal.,2012).

Recently,anoveltopicalcombinationofﬁpronil8.3%

(w/v), (S)-methoprene 10% (w/v), eprinomectin 0.4%

(w/v) and praziquantel8.3% (w/v) (Broadline®, Merial),

hereinafterreferredtoastheproduct,hasbeenregistered

for the treatment of a broad spectrum of

parasites-causing infections in cats (e.g., Dipylidium caninum,

Taenia taeniaeformis, Echinococcus multilocularis,

Ancy-lostoma tubaeforme, Diroﬁlaria immitis, Ctenocephalides

felis, Ixodes ricinus) (Rehbein et al., 2014). The main

active ingredient, which is effective against nematodes

(i.e., eprinomectin), belongs to the avermectin family

and it affects pathogens by irreversibly opening the

glutamate-gatedchlorideion channelsandinducingcell

hyperpolarization(Wolstenholme,2012).Thisproducthas

beentestedindomesticcatsforsafetyandefﬁcacyagainst

natural nematode and tapeworm infections, and was

demonstrated highly efﬁcaciousagainst ascarids (i.e.,T.

catiandToxascarisleonina),hookworms(i.e.,A.tubaeforme

andAncylostomabraziliense)andCapillaria(syn.Eucoleus)

spp. (Rehbein et al.,2014).Moreover, in a single study

conducted under experimental conditions, the product

showedtobehighlyeffectiveforthetreatmentofdifferent

developmentalstagesofA.abstrususincats(Knausetal.,

2014a).However,thisformulationhasneverbeentested

in cats living under natural conditions, where multiple

infections may occur, as well as in T. brevior-infected

animals.Therefore,theaimofthepresentstudywasto

assesstheefﬁcacyofBroadline®spot-onincatsnaturally

infectedbyA.abstrususand/orT.brevior.

2. Materialandmethods

2.1. Prevalenceofendoparasiteinfectionsincatsatthe

enrolment

Aﬁeld trialstudy wasconducted inthree

geograph-icalsitesofItaly,(i.e.,Messina,Sassariand Bari),where

the occurrence of feline lungworm infections had been

previouslyreported(Briantietal.,2012;Annosciaetal.,

2014;Giannellietal.,2014a;Tamponietal.,2014).From

MaytoSeptember2014,clientownedcats,whichhad

reg-ular accessto theoutdoorenvironment, were clinically

examined.Followingthereceiptoftheowners’informed

consent, anamnesticdata (i.e.,age,gender,clinical

pre-sentationandweight)werecollectedandfaecalsamples

were examined for gastrointestinalparasites and

lung-wormnematodes.Inparticular,faeces(atleast10g)were

subjected toﬂotationwithZnSO4 (speciﬁcgravity=1.2)

andquantitativeBaermanntechnique,asdescribed

else-where(MAFF,1986;ZajacandConboy,2012).Brieﬂy,faecal

sampleswereplaced intoBaermannfunnels,ﬁlledwith

50mlwarmtapwaterand,after24h,theliquidwas

col-lectedintoatubeand centrifugedat2500rpm(600×g)

for5min.Finally,thesupernatantwasremovedandthe

sedimentwasexaminedonamicroscopeslide.Thewhole

numberoflarvaewascountedandlungwormswere

iden-tiﬁed at species level (Gerichter, 1949; Giannelli et al.,

2014b),thusassessingthenumberoflarvaepergram(Lpg)

andthepresenceofco-infections.Parasitesinthefaeces

wereidentiﬁedbasedontheirmorphology(Euzeby,1981;

MAFF,1986).

2.2. Evaluationoftreatmentagainstlungworms

Catspositiveforlungwormlarvaewereenrolledinthe

studyifownersconsentedtotreatmentwithBroadline®

spot-on and tosubsequent follow-up. The productwas

administeredtopicallyonthemidlineoftheneck,between

the base of the skull and the shoulder blades, in a

single spot, as described in the label. Speciﬁcally, cats

weighing 0.8–2.5kg received 0.3ml dose, while

ani-mals weighing2.5–7.5kgreceiveda doseof0.9ml.The

ownerswererequestedtoreportanyhealthproblemsor

adverse eventsduring thecourseof thestudyand

ani-malswerefollowed-up28±2daysaftertreatment,with

clinicaland faecalexaminationsperformedasdescribed

above.

2.3. Dataanalysis

A.abstrususand T.breviorLpgweretransformedinto

thenaturallogarithm(count+1)forcalculationof

geomet-ricmeansforeachspecies.Theefﬁcacyofthetreatment

wascalculatedaspercentage,usingtheformula:efﬁcacy

(%)=100[(C−T)/C],whereCwasthegeometricmeanof

lar-vaebeforethetreatmentandTwasthegeometricmean

followingtheadministrationoftheproduct.Thechange

in counts was analysed by modelling the logarithm of

the counts+1 by a repeatedmeasures analysisof

vari-ance,wherethemeasurementtime(pre-treatmentversus

post-treatment)wastherepeatedmeasurementwithinthe

individualanimal.ThemixedprocedureinSAS®Version

9.1.3 wasusedfortheanalysiswithmeasurement time

deﬁned,astheﬁxedeffectaswellasrepeatedmeasure,and

animaldeﬁnedasthesubject.Allanalysesweretwo-sided

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Table1

Numberandprevalenceofcatsinfectedbygastrointestinalparasitesandlungwormnematodesineachsamplingarea.

Pathogens Samplingareas

Messina(n=23) Sassari(n=111) Bari(n=57) Total(n=191)

Gastrointestinalparasites Toxocaracati 5(21.7%) 13(11.7%) 11(19.3%) 29(15.2%) Ancylostomatubaeforme 14(60.9%) 9(8.1%) 0 23(12%) Cystoisosporafelis 2(8.7%) 21(18%) 0 23(12%) Taeniataeniformis 0 4(3.6%) 0 4(2.1%) Dipylidiumcaninum 2(8.7%) 0 7(12.3%) 9(4.7%) Giardialamblia 0 9(8.1%) 0 9(4.7%) Strongyloidesspp. 0 1(0.9%) 0 1(0.5%) Lungworms Aelurostrongylusabstrusus 11(47.8%) 22(19.8%) 1(1.8%) 34(17.8%) Troglostrongylusbrevior 1(4.3%) 2(1.8%) 3(5.3%) 6(3.1%) Eucoleusaerophilus 1(4.3%) 0 0 1(0.5%) 3. Results

3.1. Prevalenceofendoparasiteinfectionsincatsatthe

enrolment

Results of faecal examinations are reported in

Tables 1 and 2. Out of 191 cats examined (i.e., 84

male, 107 female), 87 (45.5%) were positive for either

gastrointestinalparasitesand/orlungwormnematodes.In

particular,29cats(15.2%)wereinfectedbyT.cati,23(12%)

byA.tubaeforme,23(12%)byCystoisosporafelis,9(4.7%)

byG.lamblia,13(6.8%)bytapeworms(i.e.,9D.caninum

and4T.taeniformis)and1(0.5%)byStrongyloidesspp.In

addition,34animals(17.8%)wereinfectedbyA.abstrusus,

6 (3.1%) by T. brevior, and one cat (0.5%) by Capillaria

aerophila (syn. Eucoleus aerophilus). Mixed-infections

by twoor morepathogens werereportedin 35(18.3%)

animals (Table 2) and one cat was co-infected by the

threelungwormspecies(i.e.,A.abstrusus,T.breviorandE.

aerophilus),andtwocatswereinfectedbyA.abstrususand

T.brevior.

3.2. Evaluationoftreatmentagainstlungworms

Twenty-threecats(13 male and 10 female), ranging

from 2 months to 7 years of age, were positive for A.

abstrusus(n=18)orT. brevior(n=3)orfor bothspecies

(n=2)andwereenrolledintheefﬁcacystudy(Fig.1).In

particular,17(73.9%) wereco-infectedwithuptothree

gastrointestinalparasites:7animals(30.4%)werepositive

forA.tubaeforme;2(8.7%)fortapeworms(oneD.caninum

andoneT.taeniformis);1(4.3%)forT.cati,1forA.

tubae-formeandD.caninum;2forA.tubaeformeandT.cati,1for

T.taeniformisandT.cati,1forA.tubaeforme,T.catiandE.

aerophilus,2forA.tubaeforme,T.catiandC.felis.

Table2

Frequencyofdiagnosisofsingleormixedparasiteinfectionsincatsofdifferentagegroups.

Parasitespecies <6months(n=77) 6months–2years(n=59) >2years(n=55) Total(n=191)

Singleinfections Toxocaracati(T.c) 6(7.8%) 2(3.4%) 0 8(4.2%) Dipylidiumcaninum(D.c.) 0 1(1.7%) 2(3.6%) 3(1.6%) Taeniataeniformis(T.t) 0 0 1(1.8%) 1(0.5%) Ancylostomatubaeforme(HW) 1(1.3%) 1(1.7%) 1(1.8%) 3(1.6%) Cystoisosporafelis(C.f.) 11(14.3%) 0 1(1.8%) 12(6.3%) Giardialamblia(G.l.) 5(6.5%) 2(3.4%) 0 7(3.6%) Lungworms(LW) 5(6.5%) 7(9.1%) 4(7.3%) 16(8.4%) Strongyloidesspp. 0 0 1(1.8%) 1(0.5%) Mixedinfections T.c.+HW 0 1(1.7%) 1(0.5%) T.c.+TW 1(1.3%) 1(1.7%) 2(3.6%) 4(2.1%) T.c.+LW 1(1.3%) 1(1.7%) 2(1.1%) T.c.+C.f. 5(6.5%) 0 0 5(2.6%) G.l.+LW 1(1.3%) 0 0 1(0.5%) HW+LW 0 7(9.1%) 1(1.8%) 8(4.2%) T.t.+LW 0 0 1(1.8%) 1(0.5%) D.c.+HW 0 0 1(1.8%) 1(0.5%) T.c.+HW+C.f. 2(2.6%) 0 0 2(1.1%) T.c.+D.c.+LW 3(3.9%) 0 0 3(1.6%) T.c.+HW+LW 0 1(1.7%) 0 1(0.5%) D.c.+HW+LW 0 1(1.7%) 1(1.8%) 2(1.1%) HW+C.f.+LW 0 0 1(1.8%) 1(0.5%) T.c.+HW+C.f.+G.l. 1(1.3%) 0 0 1(0.5%) T.c.+HW+C.f.+LW 1(1.3%) 0 1(1.8%) 2(1.1%)

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Fig.1. First-stagelarvae(L1)ofTroglostrongylusbrevior(A)andAelurostrongylusabstrusus(B)(Scalebar=50␮m). Table3

Lugwormlarvalcountsbeforeand28±2daysaftertreatment,percentageefﬁcacyandresultsofdataanalysis.

Lungworms MeannumberofLpga_(range)

Pre-treatmentb _Day₂₈_(±2) _%_Efﬁcacyc _p-valued

NI/Ne _GMf_(range) _NI/Ne _GMf_(range)

A.abstrusus 20/23 43.9(1–1700Lpg) 8/23 4.2(0–1000Lpg) 90.5 <0.0001

T.brevior 5/23 23.9(3–85Lpg) 0/23 0 100 <0.0001

a_Lpg₌_larvae_per_gram_of_faeces.

b _Treatment₌_Broadline®_.

c _Percentage_efﬁcacy₌_100[(C₋_T)/C],_where_C_and_T_are_the_geometric_mean_faecal_larvae_counts_prior_to_treatment_and_at_Day₂₈_(±2),_{respectively.}

d _Probability_value_analysed_by_modelling_the_logarithm_of_the_counts₊₁_by_a_repeated_measures_analysis_of_variance_where_the_measurement_time

(pre-treatmentversuspost-treatment)wastherepeatedmeasurementwithintheindividualanimal.

e_NI/N₌_Number_of_cats_that_had_evidence_of_the_indicated_parasite_per_number_of_cats_treated.

f _GM₌_geometric_mean.

Faecalnematodelarvalcountsbeforeandafter

treat-ment(day28±2),percentageefﬁcacyandresultsofdata

analysisarereported inTable3.Attheenrolment, cats

shedameanof43.9or23.9LpgofA.abstrususorT.

brev-ior,respectively.Sevencats(30.4%)infectedbyA.abstrusus

displayedsevererespiratorysigns (i.e.,dyspnoea,

recur-rentcough,nasaldischarge,wheezing),whileoneanimal

co-infectedbyT.breviorandT.catihaddiarrhoea.

Post-treatmentlarvalcounts ofbothmetastrongyloid

speciesweresigniﬁcantlyreduced(p<0.0001) and

clini-calrespiratorysignsresolvedinalltreatedanimals.Fifteen

(65.2%)catstested negativefor lungwormlarvaeaftera

singletreatment.TheoverallefﬁcacywithBroadline®was

90.5%or100%forA.abstrususorT.brevior,respectively.In

addition,20(87%)catswerenegativeforgastrointestinal

parasiteswhereasthree(13%)shedeggsofA.tubaeforme,

E.aerophilus orT. cati,respectively.No healthproblems

relatedtothetreatmentwereobserved.

4. Discussions

Thehighprevalenceoftheinfectionforanyhelminthic

parasite (i.e., 45.5%) suggests that cats living in the

regions examined are exposed to the infection with

severalpathogenspeciesthroughouttheirlife,accordingto

theirage,lifestyleandtheenvironmentwheretheylive.

Indeed,kittenstendtobemoreofteninfectedby

proto-zoans(e.g.,C.felisandG.lamblia)andascarids(i.e.,T.cati),

than adultcats.Thistrendhasbeenconﬁrmedin

stud-iesonT.catiandC.felis,inwhichithasbeenshownthat

infectedcats≤9monthsofageareathigherriskforthose

pathogens(Beugnetetal.,2014;Knausetal.,2014b).

Con-versely,inthepresentstudytheageofanimalswasnot

a riskfactor for lungworminfection,sinceeitheryoung

oradultanimalswereinfected byA.abstrususorT.

bre-vior. However,theseverityoftroglostrongylosismaybe

correlatedwiththeageoftheindividual,withfatal

out-comesobservedinkittens(Briantietal.,2014;Giannelli

etal.,2014a,b).Furtherstudiesshouldassesswhetherthe

occurrenceofmixedinfectionsincreasethepathogenicity

offelinemetastrongyloids.However,thereisanindication

thatsimultaneousinfectionswithmorethanonespecies

of lungwormsmay worsentheseverityof clinicalsigns

(Varcasiaetal.,2015).

Results of this study indicate that a single

adminis-tration of the combination of ﬁpronil, (S)-methoprene,

eprinomectin andpraziquantelis effectiveforthe

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incatslivingunderﬁeldconditions.Inaddition,thesame

combination productwasconﬁrmedtobehighly

efﬁca-ciousagainstnaturallyacquiredgastrointestinalparasites

(e.g.,T.cati,hookworms,tapeworms),alsointhecaseof

multipleinfections,underﬁeldconditions(Rehbeinetal.,

2014).In thepresent study,the enrolledcats probably

harboureddifferentdevelopmentalstagesoflungworms,

since theywere infected under naturalconditions. This

complementstheresultsobtainedfromacontrolled

lab-oratorystudy,inwhichtheefﬁcacyoftheproductagainst

third-andfourth-stagelarvaeofA.abstrususwasassessed

(Knauset al., 2014a).Whether repeatedmonthly

treat-mentswouldincreasetheefﬁcacyoftheproductincats

infectedbyA.abstrususshouldbedeﬁned.

In conclusion, Broadline® spot-on was proven tobe

highly effective against both A. abstrusus and T.

brev-iorunderﬁeldconditions,byreducingsigniﬁcantlylarval

shedding.Alltreatedcatsrecoveredfromrespiratorysigns.

Conﬂictofinterest

TheworkreportedhereinwasfundedbyMerialLimited,

GA, USA. Some authors (i.e., MK, LH, FB) are currently

employedatMerial.

Disclaimer

Thisdocumentisprovidedforscientiﬁcpurposesonly.

Anyreferencetoabrandortrademarkhereinisfor

informa-tionalpurposesonlyandisnotintendedforacommercial

purposeortodilutetherightsoftherespectiveowner(s)

ofthebrand(s)ortrademark(s).

Acknowledgment

Theauthorsgratefullyacknowledgethestatistical

sup-portprovidedbyS.TheodoreChester.

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