Robert H. Six 1*, William R. Everett 2, Melanie R. Myers 1 and Sean P. Mahabir 1

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1 Six et l. Prsites & Vectors (2016) 9:93 DOI /s z RESEARCH Comprtive speed of kill of srolner (Simpric ) nd spinosd plus milbemycin oxime (Trifexis ) ginst induced infesttions of Ctenocephlides felis on dogs Robert H. Six 1*, Willim R. Everett 2, Melnie R. Myers 1 nd Sen P. Mhbir 1 Open Access Abstrct Bckground: Fles re ubiquitous ectoprsite infesting dogs nd cuse direct discomfort, llergic rections nd re responsible for the trnsmission of severl pthogens. The rpid speed of kill of prsiticide is importnt to llevite the direct deleterious effects of fles, reduce the impct of llergic responses, nd brek the fle life cycle. In this study, the speed of kill of novel orlly dministered isoxzoline prsiticide, srolner (Simpric ) ginst fles on dogs ws evluted nd compred with spinosd in combintion with milbemycin oxime (Trifexis ) for 5 weeks fter single orl dose. Methods: Twenty-four dogs were rndomly llocted to tretment with single orl dose per product lbel of srolner (2 to 4 mg/kg), spinosd/milbemycin oxime (30 to 60 mg/kg / 0.2 to 0.4 mg/kg), or plcebo bsed on pretretment fle counts. Dogs were combed nd live fles counted t 8, 12, nd 24 h fter tretment nd subsequent re-infesttions on Dys 7, 14, 21, 28, nd 35. Efficcy (reduction in live fle counts) of ech tretment ws determined t ech time point reltive to counts for plcebo dogs. Results: There were no dverse rections to tretment. A single orl dose of srolner provided 94.0 % efficcy (bsed on geometric mens) within 8 h of tretment or subsequent weekly re-infesttions of fles to Dy 35. By 12 h, fles were erdicted from ll dogs nd they remined fle free t 24 h. Significntly greter numbers of live fles were recovered from spinosd/milbemycin oxime-treted dogs t 8 h from Dy 21 to Dy 35 (P 085), nd t 12 nd 24 h on Dy 35 (P 002). Conclusions: In this controlled lbortory evlution, dogs treted with srolner hd significntly fewer live fles thn spinosd/milbemycin oxime- treted dogs t 8 h fter re-infesttion from Dy 21 fter single orl dose. The rpid nd consistent kill of fles fter single orl dose of srolner over 35 dys indictes tht this tretment should provide highly effective control of fle infesttions, relief for dogs fflicted with fle llergy dermtitis, nd lso reduce the risk of trnsmission of fle-borne pthogens. Keywords: Ctenocephlides felis, Srolner, Simpric, Spinosd, Milbemycin oxime, Trifexis, Speed of kill, Fle, Dog, Orl, Isoxzoline * Correspondence: robert.six@zoetis.com 1 Zoetis, Veterinry Medicine Reserch nd Development, 333 Portge St., Klmzoo, MI 49007, USA Full list of uthor informtion is vilble t the end of the rticle 2016 Six et l. Open Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Six et l. Prsites & Vectors (2016) 9:93 Pge 2 of 7 Bckground The ct fle, Ctenocephlides felis felis, is the most common ectoprsite of dogs nd cts worldwide [1], fle infesttion of pets nd the home is common nd control cn be expensive nd time consuming [2]. Fle bites cn elicit n llergic response, fle llergic dermtitis (FAD), cusing intense pruritus nd severe skin inflmmtion [3]. Fles cn lso trnsmit number of bcteril pthogens nd re the intermedite host for filrid nd cestode prsites [4 9]. Fles feed lmost immeditely on ttining host [10], nd direct irrittion nd llergic rections re dependent upon the frequency nd durtion of feeding [11]. Thus, the rpid kill of fles is desirble to llevite both the immedite irrittion cused by fles s well s to reduce ssocited llergenic responses nd the risk of fle-borne pthogen trnsmission; this my include on-host fle tretment s well s environmentl tretment to reduce the household infesttion [2]. The speed of kill of dult fles on the host is lso n importnt fctor in the control of infesttions, s femle fles do not begin producing eggs until 24 to 48 h fter they strt feeding [12]. Killing fles before they ly eggs will, over time, effectively control the environmentl infesttion. The use of highly effective prsiticides hs llowed the primry mens of fle control to be vi direct tretment of the pet. Their use, s host-trgeted therpies, mrkedly reduces the severity nd prevlence of FAD nd hs reduced the need to tret indoor nd outdoor environments [1]. Orlly dministered compounds hve been introduced tht provide rpid systemic control of fles for up to 24 to 48 h like nitenpyrm [13] or for up to month, spinosd [14]. These products hve been widely ccepted by veterinrins nd dog owners for their efficcy, ese of use nd, since these gents re not topiclly pplied, the potentil lower exposure of the ownerorchildrentoresidues.newerisoxzolinecompounds (e.g. flurlner nd foxolner) hve demonstrted efficcy ginst fles nd lso ticks for month or longer following single orlly dministered dose [15, 16]. Srolner (Simpric, Zoetis) is new isoxzoline effective ginst fles nd ticks for t lest one month following single orl dose. A lbortory study ws conducted to compre the speed of kill of single dose of srolner (Simpric, Zoetis) nd spinosd/milbemycin oxime (Trifexis, Elnco) ginst n existing fle (C. felis) infesttion nd subsequent re-infesttions for period of 5 weeks fter tretment. Methods Ethicl pprovl This lbortory efficcy study ws msked, negtive controlled, rndomized complete block design conducted in Arknss, USA. Procedures were in ccordnce with the World Assocition for the Advncement of Veterinry Prsitology (WAAVP) guidelines for evluting the efficcy of prsiticides for the tretment, prevention nd control of fle nd tick infesttion on dogs nd cts [17] nd complied with the principles of Good Clinicl Prctices [18]. The protocol ws reviewed nd pproved by the locl Institutionl Animl Cre nd Use Committee. Msking of the study ws ssured through the seprtion of functions. All personnel conducting observtions or niml cre or performing infesttions nd counts were msked to tretment lloction. Animls Twenty-four, mle nd femle, purpose-bred mongrel dogs from 7 to 35 months of ge nd weighing from 5.4 to 14.6 kg were used in the study. All dogs hd undergone n dequte wsh-out period to ensure tht no residul ectoprsiticide efficcy remined from ny previously dministered compound. Ech dog ws individully identified by electronic trnsponder or er tttoo. The dogs were cclimtized to study conditions for minimum of 14 dys before tretment on Dy 0. Dogs were individully housed in indoor runs such tht no physicl contct ws possible between them. Dogs were fed n pproprite mintennce rtion of commercil dry cnine feed, nd wter ws vilble d libitum. All dogs were given physicl exmintion to evlute generl helth nd suitbility for inclusion into the study. Generl helth observtions were performed t lest once dily from the strt of the cclimtion period to the end of the study. Design The study followed rndomized complete block design. Dogs were rnked ccording to decresing fle counts into blocks of three nd within ech block dog ws rndomly llocted to tretment with plcebo, srolner, or spinosd/milbemycin oxime. There were eight dogs per tretment group. Dogs were infested with fles prior to tretment nd then weekly following tretment for 5 weeks. Fle counts were conducted t 8, 12, nd 24 h fter tretment nd fter ech subsequent weekly re-infesttion. Dy -2 bodyweights were used to determine the pproprite dose to be dministered. On Dy 0, dogs received either plcebo tblet, srolner chewble tblet (Simpric ) to deliver srolner t the minimum lbel dose of 2 mg/kg (rnge 2 to 4 mg/kg), or Trifexis per US lbel directions (spinosd t 30 to 60 mg/kg plus milbemycin oxime t 0.2 to0.4mg/kg).alldoses were dministered by hnd pilling to ensure ccurte nd complete dosing. Ech dog ws observed for severl minutes fter dosing for evidence tht the dose ws swllowed, nd for generl helth t 1, 4, nd 24 h

3 Six et l. Prsites & Vectors (2016) 9:93 Pge 3 of 7 fter tretment dministrtion. In order to comply with Trifexis lbel requirements, ll dogs were offered their regulr rtion within 30 min of tretment. Fle infesttion nd ssessment The C. felis used in the study were from loclly mintined lbortory colony initited in 2004 with fles from lbortory colony in North Crolin, USA. Wild cught fles loclly obtined in Arknss were introduced into the colony pproximtely six months prior to study initition. Fle infesttions were performed on Dys -7 (host suitbility nd lloction), -2, 7, 14, 21, 28 nd 35. At ech infesttion pre-counted liquot of (±5) dult, unfed C. felis were directly pplied to the niml which ws gently restrined for few minutes to llow the fles to penetrte nd disperse into the hir cot. Ech dog ws exmined nd combed to remove nd count fles t 24 h fter the initil host suitbility infesttion, nd t 8, 12, nd 24 (±1) hours fter tretment nd ech subsequent weekly re-infesttion. Fles were replced on the dogs immeditely fter ech 8 nd 12 h evlution, nd discrded fter the 24 h counts. Fle counts were performed by personnel trined in the stndrd procedures in use t the test fcility. Commercil fine-toothed fle combs were used. Dogs were combed using repeted strokes initilly while stnding strting from the hed, then proceeding cudlly long the dorsum. The dog ws then plced on ech side nd then on its bck for combing of the sides nd ventrl surfces. After few combing strokes were completed, the comb ws exmined nd hir nd fles were removed from the comb nd ll live fles were counted. Ech niml ws combed for minimum of 10 min; if ny fles were recovered in the lst minute, combing ws continued in oneminute increments until no fles were detected. Sttisticl nlysis The individul dog ws the experimentl unit nd the primry endpoint ws the live fle count. Dt for posttretment fle counts were summrized with rithmetic (AM) nd geometric (GM) mens by tretment group nd time point. Fle counts were trnsformed (log e(count + 1)) prior to nlysis to stbilize the vrince nd normlize the dt. Using the PROC MIXED procedure (SAS 9.2, Cry NC), trnsformed counts were nlyzed using mixed liner model. The fixed effects were tretment, time point nd the interction between time point nd tretment by time point. The rndom effects included block, block by tretment interction, nd error. Testing ws two-sided t the significnce level α = 5. The ssessment of efficcy ws bsed on the percent reduction in the rithmetic nd geometric men live fle counts reltive to plcebo clculted using Abbott s formul: % reduction ¼ men count ð plceboþ men count ð treted Þ men count ðplceboþ Results There were no tretment-relted dverse events during the study. Plcebo-treted dogs mintined good fle infesttions throughout the study nd these counts were mintined even following the combing nd reinfesttion procedures t 8 nd 12 h (Tbles 1, 2 nd 3). At the 8-h time point, both tretments resulted in significntly lower fle counts thn plcebo-treted dogs (P 021) throughout the study (Tble 1). with srolner resulted in significntly lower fle counts thn spinosd/milbemycin oxime t 8 h on Dys 21, 28, nd 35 (P < 085). The srolner tretment provided greter nd more consistent efficcy t 8 h, with efficcy >96.9 % (GM nd AM) from tretment through Dy 28. Efficcy for spinosd/milbemycin oxime ws <95 % on Dys 21 to 35 (AM) nd on Dys 28 nd 35 (GM) (Tble 1). At the 12-h time point, both tretments resulted in significntly lower fle counts thn plcebo-treted dogs (P 001) throughout the study (Tble 2). Fle counts were significntly lower for srolner-treted dogs thn spinosd/milbemycin oxime-treted dogs on Dy 35 (P < 001). Efficcy ws very high for srolner with ll dogs being fle-free on ll study dys, while live fles were detected on spinosd/milbemycin oxime treted dogs from Dy 21 through Dy 35. Efficcy of spinosd/milbemycin oxime declined s the study progressed (Fig. 1); t Dys 21 to 35 efficcy for spinosd/ milbemycin oxime-treted dogs ws <95 % (AM), nd ondy35(gm)(tble2). At the 24-h time point, both tretments resulted in significntly lower fle counts thn plcebo-treted dogs (P 001) throughout the study (Tble 3). Fle counts were significntly lower for srolner-treted dogs thn spinosd/milbemycin oxime-treted dogs on Dy 35 (P = 002). Srolner remined % effective t 24 h post tretment, with ll dogs fle free from tretment through Dy 35, while live fles were detected on spinosd/milbemycin oxime-treted dogs from Dy 21 onwrds. Efficcy for spinosd/milbemycin oxime declined below 95 % by Dy 28 bsed on AM nd on Dy 35 bsed on GM (Tble 3, Fig. 1). Discussion A single orl dose of srolner provided rpid reduction of n existing infesttion of fles s well s subsequent weekly re-infesttions for 35 dys; efficcy ws 99.1 % bsed on GM ( 96.9 %, bsed on AM) from

4 Six et l. Prsites & Vectors (2016) 9:93 Pge 4 of 7 Tble 1 Men live fle counts nd efficcy reltive to plcebo t 8 h fter tretment nd post-tretment re-infesttions for dogs treted with single orl dose of srolner or spinosd/milbemycin oxime on Dy 0 Dy of tretment or re-infesttion Plcebo Rnge A. men G. men Srolner Rnge A. men Efficcy (%) G. men 1 b 0.1 b b 0.1 c 0.8 c 5.2 c Efficcy (%) P-vlue vs.plcebo <001 <001 <001 <001 <001 <001 Spinosd/milbemycin oxime Rnge A. men Efficcy (%) G. men 1 b b b 2.6 b 5.3 b 23.1 b Efficcy (%) P-vlue vs. plcebo <001 <001 <001 <001 < P-vlue vs. srolner Geometric mens within columns with the sme superscript re not significntly different (P > 5) Tble 2 Men live fle counts nd efficcy reltive to plcebo t 12 h fter tretment nd post-tretment re-infesttions for dogs treted with single orl dose of srolner or spinosd/milbemycin oxime on Dy 0 Dy of tretment or re-infesttion Plcebo Rnge A. men G. men Srolner Rnge A. men Efficcy (%) G. men 1 b b b b b c Efficcy (%) P-vlue vs. plcebo <001 <001 <001 <001 <001 <001 Spinosd/milbemycin oxime Rnge A. men Efficcy (%) G. men 1 b b b 0.6 b 1.3 b 8.2 b Efficcy (%) P-vlue vs. plcebo <001 <001 <001 <001 <001 <001 P-vlue vs. srolner <001 1 Geometric mens within columns with the sme superscript re not significntly different (P > 5)

5 Six et l. Prsites & Vectors (2016) 9:93 Pge 5 of 7 Tble 3 Men live fle counts nd efficcy reltive to plcebo t 24 h fter tretment nd post-tretment re-infesttions for dogs treted with single orl dose of srolner or spinosd/milbemycin oxime on Dy 0 Plcebo Dy of tretment or re-infesttion Rnge A. men 1 Srolner Spinosd/milbemycin oxime G. men Rnge A. men Efficcy (%) G. men1 b b b b b b Efficcy (%) P-vlue vs. plcebo <001 <001 <001 <001 <001 <001 Rnge A. men Efficcy (%) G. men1 b b b 0.3b 0.7b 4.3c Efficcy (%) P-vlue vs. plcebo <001 <001 <001 <001 <001 <001 P-vlue vs. srolner Geometric mens within columns with the sme superscript re not significntly different (P > 5) Fig. 1 Percent efficcy bsed on geometric men counts reltive to plcebo t 8,12 nd 24 h fter tretment nd weekly post-tretment re-infesttions of fles for dogs treted with single orl dose of srolner or spinosd/milbemycin oxime on Dy 0

6 Six et l. Prsites & Vectors (2016) 9:93 Pge 6 of 7 tretment through Dy 28 nd 94.0 % (GM) or 82.8 % (AM) on Dy 35 t 8 h fter tretment or reinfesttion. By 12 nd 24 h fter tretment or reinfesttion, efficcy of srolner ws % (ll dogs were free of fles) for the entire 35 dy period. A similr speed of kill ws ttined for only 14 dys t 8 h fter tretment or re-infesttion with single orl dose of spinosd/milbemycin oxime, nd from Dy 21 onwrds its speed of kill ws significntly slower thn tht of srolner. Even t 12 nd 24 h fter reinfesttion, significntly more live fles were found on spinosd/milbemycin oxime-treted dogs on Dy 35 nd efficcy t 12 h declined below 95 % from Dy 21 onwrds (AM) nd on Dy 35 (GM), nd t 24 h efficcy ws <95 % from Dy 28 onwrds (AM) nd on Dy 35 (GM). In contrst srolner-treted dogs were free of fles ( % AM nd GM) t 12 nd 24 h for the entire 35 dys. A rpid onset of ctivity nd consistent speed of kill for ny prsiticide providing fle control is essentil to ensure tht ny newly cquired fles re rpidly eliminted before they cn reproduce to help eliminte the environmentl infesttion, decrese the likelihood of trnsmission of vector-borne disese nd ssist in the mngement of fle llergic dermtitis. This provides the pet with rpid relief from the irrittion nd debilitting effects of the existing infesttion nd protects it from new infesttions. A single orl tretment of srolner t the proposed commercil dose of 2 to 4 mg/kg resulted in the rpid reduction of n existing fle infesttion s well s rpid kill of newly infested fles for t lest 35 dys, nd efficcy ws more consistent over the full month with significntly fster kill of fles thn spinosd/milbemycin oxime from Dy 21 onwrds. Conclusions Both products resulted in rpid control of n existing fle infesttion. Fles were eliminted from ll dogs within 8 h nd dogs remined fle-free t 24 h. Aginst re-infesttions, efficcy of srolner t 8 h ws significntly superior to spinosd/milbemycin oxime on Dys 21 to 35 nd t 12 nd 24 h on Dy 35. The efficcy of spinosd/ milbemycin oxime wned t the end of the month long tretment intervl, while srolner mintined high efficcy with ll dogsbeingflefreeby12hfromtretmentthrough Dy 35. The rpid nd consistent speed of kill of fles over period of 35 dys mkes srolner chewble tblets (Simpric ) n excellent option for monthly fle control tht will reduce the direct irrittion cused by fle infesttion, ssist in the prevention of FAD, nd reduce the risk of fle-borne diseses. Competing interests The study ws funded by Zoetis, Florhm Prk, NJ. RHS, MRM nd SPM re current employees of Zoetis. WRE is current employee of BerTek Inc. nd ws contrcted by Zoetis to conduct the study. Authors contributions All uthors prticipted in study nd protocol design nd reviewed nd pproved the mnuscript. WRE conducted the study which ws monitored by MRM. SPM conducted the sttisticl nlyses. Acknowledgements The uthors would like to thnk Dougls Rugg for his ssistnce in prepring this mnuscript. Author detils 1 Zoetis, Veterinry Medicine Reserch nd Development, 333 Portge St., Klmzoo, MI 49007, USA. 2 BerTek, Inc, PO Box 606, Greenbrier, AR 72058, USA. Received: 14 December 2015 Accepted: 26 Jnury 2016 References 1. Rust MK. Advnces in the control of Ctenocephlides felis felis (ct fle) on cts nd dogs. Trends Prsitol. 2005;21: Blgburn BL, Dryden MW. Biology, tretment nd control of fle nd tick infesttions. Vet Clin Smll Anim. 2009;39: Scott DW, Miller WH, Griffin CE. Muller nd Kirk s smll niml dermtology. Phildelphi: Sunders; p Breitschwerdt EB. Feline brtonellosis nd ct scrtch disese. Vet Immunol Immunopthol. 2008;123: Kmrni A, Prreir VR, Greenwood J, Prescott J. The prevlence of Brtonell, hemoplsm, nd Rickettsi felis infections in domestic cts nd in ct fles in Ontrio. Cn J Vet Res. 2008;72: Woods JE, Brewer MM, Hwley JR, Wisnewski N, Lppin M. Evlution of experimentl trnsmission of Cndidtus Mycoplsm hemominutum nd Mycoplsm hemofelis by Ctenocephlides felis to cts. Am J Vet Res. 2005; 66: Eisen RJ, Borchert JN, Holmes JL, Amtre G, Vn Wyk K, Enscore RE, et l. Erly-phse trnsmission of Yersini pestis by ct fles (Ctenocephlides felis) nd their potentil role s vectors in plgue-endemic region of Ugnd. Am J Trop Med Hyg. 2008;78: Pugh RE. Effects on the development of Dipylidium cninum nd on the host rection to this prsite in the dult fle (Ctenocephtides felis felis). Prsitol Res. 1987;73: Mrshll AG. The ct fle, Ctenocephlides felis felis (Bouche, 1835) s n intermedite host for cestodes. Prsitology. 1967;57: Dryden MW, Gfr SM. Blood consumption by the ct fle, Ctenocephlides felis (Siphonpter: Pulicide). J Med Entomol. 1991; 28: Dryden MW. Fle nd tick control in the 21st century: chllenges nd opportunities. Vet Dermtol. 2009;20: Rust MK, Dryden MW. The biology, ecology, nd mngement of the ct fle. Ann Rev Entomol. 1997;42: Rust MK, Wggoner MM, Hinkle NC, Stnsfield D, Brnett S. Efficcy nd longevity of nitenpyrm ginst dult ct fles (Siphonpter: Pulicide). J Med Entomol. 2003;40: Snyder DE, Meyer J, Zimmermnn AG, Qio M, Gissendnner SJ, Cruthers LR, et l. Preliminry studies on the effectiveness of the novel pulicide, spinosd, for the tretment nd control of fles on dogs. Vet Prsitol. 2007;150: Rohdich N, Roepke RKA, Zschiesche E. A rndomized, blinded, controlled nd multi-centered field study compring the efficcy nd sfety of Brvecto (flurlner) ginst Frontline (fipronil) in fle- nd tick-infested dogs. Prsit Vectors. 2014;7: Shoop WL, Hrline EJ, Gould BR, Wddell ME, McDowell RG, Kinney JB, et l. Discovery nd mode of ction of foxolner, newisoxzoline prsiticide for dogs. Vet Prsitol. 2014; 201: Mrchiondo AA, Holdsworth PA, Fourie LJ, Rugg D, Hellmnn K, Snyder DE, et l. World Assocition for the Advncement of Veterinry

7 Six et l. Prsites & Vectors (2016) 9:93 Pge 7 of 7 Prsitology (W.A.A.V.P.) 2nd. ed.: Guidelines for evluting the efficcy of prsiticides for the tretment, prevention nd control of fle nd tick infesttions on dogs nd cts. Vet Prsitol. 2013;194: EMEA. Guideline on good clinicl prctices. VICH Topic GL9. em.europ.eu/docs/en_gb/document_librry/scientific_guideline/2009/10/ WC pdf. 2001; Accessed 23 Aug Submit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-submission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online submission Thorough peer review Inclusion in PubMed nd ll mjor indexing services Mximum visibility for your reserch Submit your mnuscript t

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