Long-term Efficacy Against Fleas (Ctenocephalides felis, Bouché 1835) of Monthly Topical Treatments with Based Spot on Formulations Compared to a Flumethrin/ Impregnated Collar on Dogs Subjected to Regular Water Exposure Lénaïg Halosa* Josephus Fourieb Ina Besterb Matthias, Pollmeiera Frédéric Beugneta aMerial S.A.S., 29 Av Tony Garnier, 69007, Lyon, France. [email protected] bClinVet International (Pty) Ltd, Uitsig Road, Bainsvlei, 9321 Bloemfontein, Republic of South Africa. * Corresponding author

KEY WORDS: long term efficacy, amounts of fipronil and (S)-, and Ctenocephalides felis, fleas, topical are registered in Europe with a one-month treatment, fipronil/(S)-methoprene, fipronil/ flea control claim while the flumethrin/ (S)-methoprene/, collar, flumethrin/ imidacloprid impregnated collar has a flea imidacloprid, dog efficacy claim of 7 to 8 months. Eighteen dogs were followed during an 8 months ABSTRACT period. Six dogs remained untreated, 6 dogs The objective of this study was to compare were fitted with the imidacloprid/flumethrin the long term efficacy against fleasCteno ( - collar on Day 0, and 6 dogs were treated cephalides felis) of repeated monthly topical monthly for 3 months with fipronil-based treatments with Fipronil based spot-on formulations starting on Day 120 (Certifect® products (Certifect® followed by Frontline® on Days 120 and 150 followed by Frontline® Combo, both Merial) to the efficacy of a Combo on Days 181 and 210). Flea infesta- flumethrin/imidacloprid impregnated collar tions comprising 100 C. felis each were per- (Seresto®, Bayer Animal Health) on dogs formed twice a month starting on Day 134 exposed to simulated rain. The two fipronil and repeated through Day 231. Dogs were based spot-on products contain the same showered with water for 1 minute at fort-

101 Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med. nightly intervals starting the week after each nation products, which are appropriate and treatment throughout the study. The efficacy safe to treat domestic dogs, have been for- of monthly administered fipronil-based mulated for application either topically (spot spot on products was not impacted by the on, collar) or more recently orally (Rust, simulated rain as confirmed by reductions 2005; Horak et al, 2012; Beugnet and Franc, in C. felis counts between 96% to 100% at 2012). The impregnated collars all timepoints. The efficacy of the flume- claim to be effective for several months. thrin/imidacloprid impregnated collar was Although its long lasting efficacy has been markedly impacted by the simulated rain tested and validated before registration, the with reductions in C. felis counts of between duration of efficacy may be challenged un- 31.7% to 64.8% versus control following der conditions of normal use, including ex- challenge in months 4-8. The efficacy of the posure to factors like rain, water immersion, fipronil-based spot-on products was sig- UV degradation, etc. Spot on formulations nificantly higher (p<0.05) than the efficacy have a shorter duration of action, ie, gener- observed with the flumethrin/imidacloprid ally 1 month, and it could be hypothesized impregnated collar at all challenge times. that the efficacy would remain more stable INTRODUCTION in the face of environmental factors. Fleas, particularly those of the genus Cteno- The aim of the present investigation cephalides, are the most common ecto- was to evaluate the long-term flea efficacy parasites of dogs and cats worldwide. Flea of a flumethrin/imidacloprid impregnated ®, Bayer Animal Health) that infestation can cause considerable irritation collar (Seresto is applied once compared to the efficacy of to animals and humans, or can lead to severe fipronil-based spot on formulations (Certi- disorders such as anemia and dermatologi- fect® and Frontline® Combo, Merial) applied cal problems including flea allergic derma- monthly to dogs under experimental condi- titis (FAD). It is estimated that flea related tions mimicking natural water exposure diseases are responsible for almost 50% of (twice a month exposure to simulated rain). dermatological cases reported to veterinar- Certifect® and Frontline® Combo have been ians (Rust and Dryden, 1997; Guaguère and demonstrated to provide similar efficacy Beugnet, 2006). against fleas as the formulation contain- Many flea species are of veterinary and ing fipronil as well as the dose delivered to public health importance as they can be dogs is the same for both products. The two reservoirs and potential vectors for a variety spot-on products are registered in Europe of pathogens including zoonotic agents. The with a 1-month flea control claim while cat flea,Ctenocephalides felis, is a known the impregnated collar has a 7 to 8-months vector of Bartonella henselae, B clarridge- flea efficacy claim. Based on Seresto® s’ iae, and Rickettsia felis, which in humans claim that flea efficacy gradually decreased can cause cat scratch disease, endocarditis, following monthly shampooing or water and flea borne spotted fever, respectively. immersion starting in the 5th months, it was The fleas of dogs and cats are also known as assumed that intermediate hosts of Dipylidium caninum • the collar would be fully efficacious (Beugnet and Fourie, 2013; Dryden and for at least 3 months and that Hodgkins, 2010). • the efficacy of the spot-on products Because of animal and human welfare, would be comparable each month, controlling fleas is of great importance challenge flea infestations were there- worldwide. Despite the increasing number fore initiated from the fourth month. of flea control products and their use, flea -in festation of dogs remains common in Europe MATERIALS AND METHODS and other continents (Rust, 2005). The study was designed in accordance with Several , alone or in combi- the “World Association for the Advancement

Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 102 Figure 1: Study Design

of Veterinary Parasitology (W.A.A.V.P.) 150 and Frontline® Combo on Day 181 and guidelines for evaluating the efficacy of par- Day 210, according to the registered label asiticides for the treatment, prevention and instructions. Six dogs in Treatment Group 3 control of flea and tick infestations on dogs (flumethrin/imidacloprid impregnated collar) and cats” (Marchiondo et al, 2013), and was were fitted with the commercially available conducted in accordance with Good Clinical Seresto® collar following the label instruc- Practices as described in the International tions on Day 0. Cooperation on Harmonisation of Technical Following treatments, all dogs were Requirements for Registration of Veterinary observed hourly (±30 minutes) for 4 hours Medicinal Products (VICH) guideline GL9 after the last animal was treated. (EMEA, 2000). All dogs were managed Experimental Study Design similarly, with due regard for their well-be- The impregnated collar treated Group was ing and in compliance with Merial and local allocated on Day -5, the control and fipronil- Ethics Committee approvals. based spot-on product treated Groups were Dogs added to the study on Day 115 (and acclima- Eighteen healthy mixed breed dogs (12 tized from Day 110) (see Figure 1 for study males/6 females) aged at least 3 months old design). were enrolled in the study. Dogs treated with Flea Infestations and Counts ectoparasiticides (either topical or systemic) Adult C. felis were used for artificially within 3 months before the start of the induced infestations. These fleas were from study were excluded. Seresto® collar treated a South African strain that is not known to dogs had a physical examination on Day be resistant to any ectoparasiticide. All dogs -7 to ensure a healthy status. Untreated and were infested every 2 weeks with 100 adult fipronil treated dogs had a physical exami- C. felis fleas starting on Day 134 ( Days : nation on Day 113. None of the dogs were 134, 148, 161, 175, 189, 203, 217, and 231), debilitated, suffering from disease or injury, followed by comb counts 24 (±2) hours or otherwise unsuitable for inclusion in the post infestation (Figure 1). Each dog was study. None of the dogs included presented combed for a minimum of 10 minutes using abnormalities in the neck area where prod- a fine flea comb. If fleas were found during ucts were to be applied. these 10 minutes, then the dog was combed Treatment Administration for 15 minutes. However, if no fleas were Six dogs formed the untreated control Group found on the dog in these 10 minutes, then 1. Six dogs in Treatment Group 2 (fipronil- the count was stopped. The dog’s entire coat based spot on products) were treated with was combed thoroughly. To prevent cross- the appropriate commercially available pi- contamination between treatment groups, pette size of Certifect® on Day 120 and Day separate flea combs and lab coats were

103 Vol. 12, No.2, 2014 • Intern J Appl Res Vet Med. Table 1: Summary of mean flea counts and percent efficacies against fleas on dogs treated with Fipronil 10% w/v combination products or flumethrin/imidacloprid collar.

Dogs GROUP 1 Treated with Dogs Negative Certifect® on Day 120 and Treated with Seresto® at control Day 150 and Frontline® Day 0 n =6 Combo on Day 181 and n =6 DAY Day 210n =6 Efficacy (%) Efficacy (%) Flea counts Flea counts Flea counts Arithmetic Arithmetic (arithmetic (arithmetic (arithmetic mean (Geo mean (Geo mean) mean) mean) mean) mean) 135 74.0 0.0 100.0 (100) 42.3 42.8 (45.1) 149 66.7 4.2 93.8 (96.8) 47.2 29.3 (31.7) 162 60.2 0.0 100.0 (100) 33.8 43.8 (48.0) 176 67.2 2.2 96.8 (98.3) 40.2 40.2 (47.9) 190 62.3 0.0 100.0 (100) 39.2 37.2 (53.9) 204 39.8 0.8 97.9 (98.8) 20.7 48.1 (64.8) 218 59.3 0.0 100.0 (100) 39.0 34.3 (40.3) 232 67.7 3.3 95.1 (97.5) 44.2 34.7 (34.1) used for each treatment group. Gloves were the formula [(C - T) / C] x 100, where C was changed systematically between each dog. the geometric or arithmetic mean for the Rain Simulation untreated control group and T is the geomet- Water showering was carried out in a ric or arithmetic mean for Treatment Groups designated area by wetting dogs, including 2 or 3. Each treated Group was compared to the head, with a gentle spray from a bathing the untreated control group as well as other wand for one minute. The dogs were then treated group using Analysis of Variance on dried smoothly using a blow-dryer before re- log count (ANOVA). All testing was two- turning them to their cages. Dogs fitted with sided at the significance level α=0.05. the collar were showered every two weeks Results starting on Day 14 (Days 14, 28, 42, 56, 70, The adult flea counts at 24 hours post infes- 84, 98, 112, 126, 140, 155, 170, 184, 198, tation and efficacy results throughout the 212, and 226). Dogs treated with Fipronil study are summarized in Table 1. The arith- were showered at 14 days intervals starting metic mean flea counts of untreated dogs D126 through day 226 (Days 126, 140, 155, ranged from 39.8 to 74. The fipronil-based 170, 184, 198, 212, and 226) (Figure 1). spot on product treated dogs had statisti- Data Analysis cally significantly (p<0.05) lower flea counts The flea counts were transformed to the nat- (based on geometric means) compared to the ural logarithm of (count +1) for calculation negative control Group on all assessments of geometric means by treatment group at days. The flea counts were not significantly each time point. Arithmetic means were also different between the four spot on treatment calculated. Percentage reduction from the time points supporting the fact that the two untreated control group (Treatment Group 1) fipronil-based products provide similar ef- mean was calculated for Treatment Groups ficacy. The imidacloprid/flumethrin treated 2 and 3 at each counting time point using dogs had statistically significantly (p<0.05)

Intern J Appl Res Vet Med • Vol. 12, No. 2, 2014. 104 less fleas (based on geometric means) cloprid impregnated collars demonstrated compared to the negative control Group on 93.2% efficacy on Day 177 (6 months) Days 135, 162, 204 and 232 but not at the and 94.5% on Day 191 against fleas under other time points. The fipronil-based spot on experimental conditions without water product treated dogs had statistically signifi- exposure (Horak et al, 2012). In the present cantly (p<0.05) less fleas (based on geomet- study, dogs were exposed to simulated rain ric means) compared to the imidacloprid/ for one minute representing a non-severe flumethrin treated dogs on all assessment water exposure scenario. Nonetheless, the days throughout the study. efficacy decreased dramatically to level The fipronil-based spot on product treat- below 50% (arithmetic mean) after 4 months ment provided 96 to 100% efficacy against with exposure to simulated rain. In contrast, fleas (based on geometric means), whereas topical spot on products usually have a dura- the imidacloprid/flumethrin impregnated tion of prophylactic efficacy that extends to collar provided 31.7% to 64.8% efficacy 1 month or more. Fipronil translocates on against challenge starting on Day 134. No the skin within 24 hours and accumulates in adverse events or other health problems skin lipids. Its lipophilic properties and dose were observed throughout the study. were studied to provide a month of protec- tion against fleas and ticks under natural DISCUSSION conditions including water exposure and This study demonstrated the efficacy of shampooing (Beugnet and Franc, 2012). monthly applications of fipronil-based Water immersion is an important factor to be ® combination products, i.e., Certifect and considered when assessing the efficacy of an Frontline® Combo, in the control of flea ectoparasiticide in the field. Indeed, animals infestations in dogs subjected to simulated exposed to parasitic pressure usually have rain. Four successive monthly treatments an outdoor lifestyle and are therefore often ® ® either with Certifect or Frontline Combo subjected to natural conditions including offered a similar sustained flea efficacy of rain, swimming and bathing. This study un- >96% over a period of 4 months. Follow- derlines the need to choose the appropriate ing a monthly schedule of application, these antiparasitic treatment adapted to each dog’s spot on products were significantly more lifestyle and environment. effective against C. felis on dogs exposed to simulated rainfall twice a month than the Conflict of Interest flumethrin/imidacloprid impregnated collar The work reported herein was funded by exposed to simulated rain twice a month Merial SAS, France. All authors are current when efficacy was evaluated between 19 and employees of Merial or contractors 33 weeks after application. Acknowledgments Collars are often considered as inexpen- The authors gratefully acknowledge the ex- sive devices for the prevention of ectopara- pert contributions of all collaborators from site infestations, including fleas, due to their ClinVet International (Bloemfontein, 9321, long lasting activity, .ie, several month of Republic of South Africa). prophylactic efficacy. This long duration is Certifect® and Frontline® combo are a due to the slow release of the actives from registered trademark of Merial. All other the matrix of the collar. Nevertheless, under marks are the property of their respective natural conditions of use, many factors, such owners. This document is provided for as insufficient skin contact, wear by friction, scientific purposes only. Any reference to a regular water immersions, or mechanical brand or a trademark herein is for informa- deteriorations may impair the sustained ef- tional purposes only and is not intended for ficacy and lead to variable protection over a commercial purpose or to dilute the rights time. of the respective owner(s) of the brand(s) or In previous studies, flumethrin/imida-

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