Repellency and Efficacy of 65% Permethrin and Selamectin Spot-on Formulations Against Ixodes ricinus Ticks on Dogs*

Veterinary Therapeutics Vol. 3, No. 1, Spring 2002 Repellency and Efficacy of 65% Permethrin and Selamectin Spot-on Formulations Against Ixodes ricinus Ticks on Dogs* Richard G. Endris, PhD a Dara Cooke, BSc b Deborah Amodie, BS c Diane L. Sweeney, PhD d Terry L. Katz, MS a a Schering-Plough Animal Health Corp. 1095 Morris Avenue Union, NJ 07083 b RKB House Irishtown Athlone County Westmeath, Ireland c Fort Dodge Animal Health P.O. Box 5366 Princeton, NJ 08543 d Schering-Plough Animal Health Corp. 21401 West Center Road Elkhorn, NE 68022 ABSTRACT Two topically applied spot-on products used for flea and tick control on dogs, 65% permethrin (Defend EXspot Treatment for Dogs, Schering-Plough Animal Health Corp., Union, NJ) and selamectin (Revolution [United States] or Stronghold [Europe], Pfizer Animal Health, New York, NY), were evaluated for repellency and efficacy against Ixodes ricinus, the primary vector of Lyme disease in Europe. Eighteen dogs were evenly and randomly allocated to the following treatments: 1) 65% permethrin, 2) selamectin, 3) untreated control. Dogs were treated by topical application of the assigned product in accordance with product label directions on Day 0. At 7, 14, 21, 28, and 35 days after treatment, each dog was exposed for 2 hours to 50 unfed, adult ticks in a cage with a carpet that covered approximately 70% of the floor area. After the *This study was sponsored by Schering-Plough Animal Health Corp., Union, NJ. exposure period, dogs were removed from the cages and live and dead ticks were counted on the dogs and in the cages. The number of live ticks recovered was reduced by 90.3% to 99.5% for dogs treated with 65% permethrin (P <.0001 versus controls and selamectin), compared with 10.9% to 31.1% for dogs treated with selamectin (P >.05 versus controls). The repellency of 65% permethrin was 63.4% to 80.2% against I. ricinus ticks (P <.0001 versus controls, P <.0007 versus selamectin), compared with 0% to 10.9% repellency for selamectin (P >.05 versus controls). INTRODUCTION Ticks of the genus Ixodes are distributed worldwide and generally exhibit cosmopolitan host preferences. Pathogens and toxins transmitted by Ixodes spp to dogs include Borrelia burgdorferi sensu lato 1 and the toxin responsible for tick paralysis in Australia. 2,3 In addition, the potential role of dogs as reservoir hosts for 64

R. G. Endris, D. Cooke, D. Amodie, D. L. Sweeney, and T. L. Katz other pathogens transmitted by Ixodes spp (e.g., Babesia spp, 4,5 Ehrlichia spp, 6 and the flaviviruses 7,8 of the tickborne encephalitis complex) has not been elucidated. Permethrin, a photostable synthetic pyrethroid, was first described in 1973. 9 Since that time, this insecticide has been developed for control of a wide variety of arthropod species. 10 In addition to its acaricidal properties, permethrin has been shown to repel ticks when applied to cloth or directly on the dog. 11 15 Selamectin, a 16-membered macrocyclic lactone (avermectins and milbemycins), was selected 16 and developed 17 for topical application on dogs and cats for the control of fleas (Ctenocephalides felis felis), ticks (Dermacentor variabilis), mites (Otodectes cynotis, Sarcoptes scabiei var canis), heartworm (Dirofilaria immitis), and helminths (Ancylostoma tubaeforme, Toxocara cati). The present study was designed to determine and compare the duration of repellency of two topically applied products, 65% permethrin and selamectin, against infestation of dogs by Ixodes ricinus, a representative species of the genus Ixodes. The study was conducted in accordance with guidelines for clinical trials established by the Committee for Veterinary Medicinal Products. MATERIALS AND METHODS Dogs Twenty-two beagles were selected from a laboratory animal colony and allowed 7 days to acclimate to the new environment and handling procedures. On two separate days during the acclimation period, between Days -7 and -2, all dogs were taken from their individual pens and placed in a tick-containment cage for approximately 2 hours each day. Dogs that did not acclimate well to the tick containment cages or those that demonstrated any health problems upon veterinary examination were excluded from the study. Of the original 22 dogs, 18 (9 males and 9 females) that acclimated well and were determined to be healthy and free of skin disease and ectoparasites were selected for inclusion in the study. The dogs ranged in age from approximately 8 months to 3 years and in weight from 8.2 to 13.7 kg. Each dog was individually identified by a number tattooed in the ear. Before participating in the study, dogs were washed with a noninsecticidal soap and had not been treated with any parasiticide for at least 28 days. Commercial dry dog food was fed either once or twice daily, and water was available ad libitum. Allocation and Treatments Dogs were evenly and randomly allocated by sex to one of three treatments, providing three males and three females for each of the following: 65% permethrin, selamectin, or untreated control. Treatments were applied once on Day 0 according to instructions provided on the respective product labels. In Group 1, all dogs weighed less than 15 kg each; therefore, in accordance with label directions, each dog was treated with 1.0 ml of the commercial formulation of 65% permethrin (Defend EXspot Treatment for Dogs, Schering-Plough Animal Health Corp., Union NJ) applied to the skin of the dog s back between the shoulder blades. In treatment Group 2, each dog was treated with a commercial product containing 120 mg selamectin per ml (Revolution, Pfizer Animal Health, New York, NY) applied to the skin of the dog s back between the shoulder blades. Total doses were adjusted as recommended on the label (i.e., one dog that weighed between 5.1 and 10.0 kg received 0.25 ml and 5 dogs that weighed between 10.1 and 20.0 kg each received 0.5 ml of the product). These doses provided approximately 6 mg selamectin/kg of body weight. 65

Veterinary Therapeutics Vol. 3, No. 1, Spring 2002 Tick Challenge Laboratory-reared adult I. ricinus ticks were obtained from the National Environmental Research Council, Institute of Virology and Environmental Microbiology, Oxford, UK (Days 14, 28, 35) and from Humboldt Universitaet zu Berlin Parasitologie, Institut fuer Pathologice, Berlin, Germany (Days 7 and 21). Dogs were exposed to ticks 7, 14, 21, 28, and 35 days after treatment in individual cages measuring 1.1 0.7 0.5 m and made of heavygauge plastic with smooth, impermeable surfaces. The lid included a window covered with a fine-mesh screen to allow gas exchange and to prevent ticks from escaping. Approximately 70% of the floor area was covered by a carpet of lightcolored nylon with a nap of 12 to 15 mm. Cages were thoroughly washed, and carpets were discarded after each use. Test cages were assigned to individual dogs for the duration of the study to preclude the possibility of cross contamination. On each challenge day, carpets were lightly sprayed with water and 50 unfed adult I. ricinus ticks (with an approximately equal distribution of males and females) were placed in each cage. After 15 minutes, the dogs were then placed in their assigned test cages with the ticks, where they remained for 2 hours with the cage lid securely fastened. After the 2- hour exposure period, the dogs were removed from the cages and transported to a counting table by two handlers wearing disposable, impermeable gloves and aprons. Each dog was thoroughly examined by two investigators, and the numbers of live and dead ticks on each dog were determined by parting the hair and then combing with a fine-toothed comb. Each cage was examined, and the numbers of live and dead ticks on the carpet and elsewhere in the cage were determined. Approximately 1 hour was required to count ticks on each dog and its respective cage. Live and dead ticks on the dogs or in the cages were destroyed, and cages were thoroughly washed to remove any potential insecticide residues. Statistical Analysis Using SAS Proc Mixed (SAS Institute, Cary, NC), tick counts were analyzed by one-way analysis of variance (ANOVA) to determine differences between treatments for the number of live ticks on the dog (efficacy) as well as the number of live or dead ticks in the cage plus the number of dead ticks on the dog (repellency). Tick counts were transformed to the log 10 (count + 1) before analysis to stabilize the variance. The differences between least squares means were compared by a two-sided (comparing treated groups) or one-sided (untreated control group compared to each treated group) using ANOVA type III sum of squares; differences were declared significant at the 5% level. Percent efficacy of each treatment 18 relative to the control group based on geometric mean tick counts was calculated for the number of live ticks recovered from each dog. Percent repellency based on geometric means was calculated as the number of ticks dead or alive in the cage plus the number of dead ticks on the dog. Efficacy and repellency were calculated as follows: % Efficacy = Geometric mean live ticks on controls Geometric mean live ticks on treated dogs 100 Geometric mean live ticks on controls % Repellency = Geometric mean ticks repelled on treated dogs Geometric mean ticks repelled on controls 100 (50 Geometric mean ticks repelled on controls) where 50 is the total number of ticks placed on the carpet in each cage. 66

R. G. Endris, D. Cooke, D. Amodie, D. L. Sweeney, and T. L. Katz TABLE 1. Live Ixodes ricinus Ticks on Dogs Following Application of 65% Permethrin or Selamectin Spot-on Formulations on Day 0 and Exposure of Dogs to Ticks for 2 Hours at Weekly Intervals Geometric Mean Live Ixodes ricinus Ticks per Dog Efficacy* Days After Untreated 65% 65% Treatment Control Permethrin Selamectin Permethrin Selamectin 7 26.6 a 1.0 b 23.7 a 96.3% 10.9% 14 23.1 a 0.1 b 19.9 a 99.5% 13.8% 21 22.3 a 2.1 b 18.7 a 90.7% 16.4% 28 20.5 a 2.0 b 17.1 a 90.3% 16.7% 35 19.2 a 1.7 b 13.2 a 91.3% 31.1% *% Efficacy = Mean live ticks in control group Mean live ticks in treated group 100 Mean live ticks in control group a,b Means in the same row with different superscript letters are significantly different (P.05). RESULTS The mean number of live ticks per dog and the percent reduction in live ticks for each product relative to the untreated control group are shown in Table 1 and Figure 1. Exposure of dogs to I. ricinus ticks at weekly intervals following treatment with 65% permethrin significantly (P <.0001) reduced the number of live ticks on the dogs by 90.3% to 99.5% versus untreated controls over the 5-week challenge and evaluation period. In contrast, exposure of dogs to ticks on the same schedule following treatment with selamectin reduced the number of live ticks by only 10.9% to 31.1% (P >.05). The number of live ticks recovered from dogs treated with 65% permethrin was significantly (P <.0001) less than the number recovered from dogs treated with selamectin. The mean number of ticks repelled and the percent repellency for each product relative to the untreated control group are shown (Table 2, Figures 2 and 3). The 65% permethrin spoton treatment repelled 63.4% to 80.2% (P <.0001) of the I. ricinus ticks placed in cages with treated dogs each week for 5 challenge weeks. Selamectin spot-on treatment exhibited % Efficacy 100 90 80 70 60 50 40 30 20 10 0 65% Permethrin Selamectin 0 7 14 21 28 35 Days After Treatment Figure 1. Comparative efficacy of Defend EXspot (65% permethrin) and Revolution (selamectin) in reduction of live ticks relative to untreated controls when treated dogs were exposed to 50 unfed, adult Ixodes ricinus for 2 hours in a closed cage on given days after treatment. little or no ability to repel I. ricinus ticks (0% to 10.9%). The level of repellency achieved by treating dogs with selamectin was not significantly different from the untreated control group (P >.05) at any time. Repellency was 67

Veterinary Therapeutics Vol. 3, No. 1, Spring 2002 TABLE 2. Repellency of 65% Permethrin and Selamectin Spot-on Formulations Against Ixodes ricinus Ticks Geometric Mean Ticks Repelled* Repellency Days After Untreated 65% 65% Treatment Control Permethrin Selamectin Permethrin Selamectin 7 11.3 a 38.8 b 10.6 a 71.1% 0% 14 16.6 a 43.4 b 18.7 a 80.2% 6.2% 21 16.8 a 41.9 b 18.8 a 75.6% 6.0% 28 17.0 a 42.5 b 20.6 a 77.3% 10.9% 35 23.1 a 40.1 b 21.6 a 63.4% 0% *Repelled is defined as ticks in cage (live and dead) plus unattached dead ticks on the dog. % Repellency = Mean ticks repelled in treated group Mean ticks repelled in control group 100 50 geometric mean ticks repelled in control group where 50 is the total number of ticks placed on the carpet in each cage. Indicates a numerically greater number of ticks present than on untreated control. a,b Means in the same row with different superscripts are significantly different (P <.05). significantly better for the 65% permethrin spot-on than for selamectin spot-on at each evaluation (P <.0007). DISCUSSION A comparison of the efficacy of 65% permethrin in the present study against I. ricinus with that previously observed 15 shows that efficacy in the two studies was relatively similar for the first 4 weeks. However, in the present study, efficacy of the 65% permethrin product was 91.3% in reducing numbers of live ticks on dogs 5 weeks after treatment compared with 74.1% in the earlier study. Comparison of the repellency between the two studies reveals a greater mean number of ticks repelled in this experiment 35 days after treatment (40.1) than in the previous study (32.4). Although ticks found either live or dead in the cages and dead on dogs were considered repelled, the survival of so few ticks placed in the cages 35 days after treatment suggests that a longer duration of repellency would have been observed had the present study been continued. Permethrin is a contact repellent such that ectoparasites must come in contact with the molecules in order to be affected. It is unlikely that any such repellent could provide sufficient vapor activity to be effective against ectoparasites without some direct contact. In the case of permethrin, there has been a substantial body of evidence accumulated over the past 7 years to support its contact repellency properties for a variety of arthropods. Ticks found in the cage (either dead or alive) and those found on the dogs (unattached but dead) were considered repelled due to the likelihood that ticks found dead on the dogs may have received a sufficient dose of material to be repelled but died before they could successfully leave the dog. Exposure to permethrin was up to 2 hours, but it is possible that only a few minutes of exposure were sufficient to cause the ticks to move away from the permethrin-coated surface, and many of the ticks acquired a lethal dose during this period. The acaricidal activity of selamectin has been demonstrated against ticks 19 and mites. 20,21 Consequently, some activity was anticipated for selamectin against I. ricinus; however, no acaricidal activity or repellency of 68

R. G. Endris, D. Cooke, D. Amodie, D. L. Sweeney, and T. L. Katz selamectin against I. ricinus was observed in this study. In a previous study, 19 maximum efficacy of selamectin against Rhipicephalus sanguineus and D. variabilis was demonstrated 2 weeks after treatment but only after ticks had been exposed to selamectin for 5 days. In the present experiment, I. ricinus ticks were exposed to selamectin-treated dogs for 2 hours. Therefore, if the selamectin spot-on formulation does demonstrate acaricidal activity against Ixodes spp, then the onset of activity against I. ricinus is likely to be much later than that of the permethrin spot-on formulation. There are few reports available on the duration of tick feeding on dogs necessary for successful transmission of tickborne pathogens. However, the duration of feeding required for Ixodes spp to transmit B. burgdorferi sensu lato to several mammalian species has been determined (Table 3). Lyme borreliosis can be transmitted by Ixodes spp to mice, hamsters, and humans after a 24-hour feeding period, and Ixodes scapularis was capable of transmitting Babesia microti to 9% of exposed hamsters after 36 hours of attachment. 22 31 Increasing the duration of tick feeding from 24 hours to 120 Tick Count hours resulted in increased rates of B. burgdorferi sensu lato transmission. Similar dynamics of Borrelia spp transmission would Mean number of ticks recovered per cage (live and dead) and per dog (live and dead) Mean number of dead ticks per dog Mean number of live ticks per dog 50 45 40 35 30 25 20 15 10 5 0 0 7 14 21 28 35 Days After Treatment Figure 2. Mean adult Ixodes ricinus ticks repelled and killed during 2- hour exposures at weekly intervals after treatment of dogs with 65% permethrin (Defend EXspot Treatment for Dogs) on Day 0. The shaded area represents the mean total ticks repelled. Tick Count Mean number of ticks recovered per cage (live and dead) and per dog (live and dead) Mean number of dead ticks per dog 50 Mean number of live ticks per dog 45 40 35 30 25 20 15 10 5 0 0 7 14 21 28 35 Days After Treatment Figure 3. Mean adult Ixodes ricinus ticks repelled and killed during 2-hour exposures at weekly intervals after treatment of dogs with selamectin spot-on (Revolution ) on Day 0. The shaded area represents the mean total ticks repelled. be expected to occur for dogs. Therefore, the high order of efficacy and repellency observed within 2 hours of exposing I. ricinus 69

Veterinary Therapeutics Vol. 3, No. 1, Spring 2002 TABLE 3. Duration of Ixodes spp Feeding Times Required for Transmission of Borrelia burgdorferi Duration of Host Species Feeding (hr) Transmission? Transmission* Reference Hamsters 24 Yes 16.7% (1/6) 22 (Mesocricetus auratus) 48 Yes 50.0% (3/6) 72 Yes 83.3% (5/6) White-footed mice 24 No 0.0% (0/8) 22 (Peromyscus leucopus) 48 Yes 25.0% (2/8) 72 Yes 100.0% (8/8) Rabbits 24 No 0% (0/3) 23 (Oryctolagus cuniculus) 36 No 0% (0/5) 48 Yes 66.7% (2/3) 120 Yes 100% (5/5) White-footed mice 36 Yes 7.1% (1/14) 24 (Peromyscus leucopus) 42 Yes 25.0% (3/12) 48 Yes 75.0% (6/8) Mice 24 No 0.0% (0/8) 25 (Peromyscus maniculatus) 48 Yes 11.1% (1/9) 72 Yes 25.0% (2/8) 96 Yes 80.0% (8/10) Humans 24 Yes 1 reported 26 (Homo sapiens) 24 Yes 5.9% (1/17 reported) 27 24 Yes 4.2% (2/48 reported) 28 <24 Yes 1/109 30 >72 Yes 3/109 8 12 Yes 3.7% overall 2 cases reported 29 *The percentage of animals fed upon by infected ticks that became infected with B. burgdorferi sensu lato. to dogs treated with 65% permethrin is expected to be sufficient to prevent transmission of tickborne diseases such as Lyme disease for which a minimum tick-feeding period of 24 hours is required for transmission. The apparent slow onset of acaricidal activity of selamectin suggests that treatment with selamectin would not be effective for prevention of Lyme borreliosis or any other pathogen transmitted by ticks 24 hours or more after attachment. These data confirm that I. ricinus ticks are highly susceptible to permethrin. 15 Furthermore, the data suggest that dogs treated with Defend EXspot Treatment for Dogs will be protected from infestation with I. ricinus for 4 to 5 weeks after treatment. The remarkably high level of protection afforded dogs treated with Defend EXspot Treatment for Dogs from infestation with I. ricinus should enhance the prevention of tickborne diseases and tick paralysis in dogs caused by Ixodes spp by repelling and killing the ticks before they can attach and begin feeding. 70

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