Each study was a completely randomized design with a fluralaner treated group and an untreated control group. All observations and procedures, including general health observations, tick infestations, and tick counts were performed by masked individuals. Both studies were conducted in accordance with Good Clinical Practices, and the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of parasiticides against flea and tick infestations of dogs and cats [17, 18].
Animals.
Prior to beginning each study, 24 dogs in Study 1 and 30 dogs in Study 2 were acclimated to kennel conditions beginning on Day − 10. To be enrolled in either study, dogs were required to be clinically healthy with no pre-existing conditions (e.g., injury, trauma, disease) that could have affected the outcome, at least 6 months of age, not treated with any long-acting anti-flea or anti-tick product within the previous 180 days and have demonstrated susceptibility to tick infestation based on retaining at least 13 live ticks or ≥ 25% of a pre-study infestation. In that pre-study period and then throughout the study dogs were housed in individual cages (except for removal for tick infestations) in a thermostatically controlled environment with an approximate 12-h light/12-h dark cycle, fed a commercial dog food per site practice and allowed ad libitum access to water. Cages were segregated by treatment group to avoid cross exposure of dogs to fluralaner. From the dogs presented for acclimation, in each study 20 were retained for experimental purposes. Dogs selected for Study 1 were Beagles, 11 males and 9 females, aged from 2 to 4 years and weighing from 10.2 to 14.6 kg. Study 2 dogs were Beagles and mixed breed, 7 males and 13 females, aged from 2 to 7 years and weighing from 10.2 to 13.3 kg.
Randomization and treatment
On Day − 3, study dogs were randomized to treatment groups in a 1:1 ratio using a computer-generated randomization table so that 10 dogs were included in each group. Dogs in Group 1 remained untreated but were handled in the same manner as Group 2 dogs to have a reference time for scheduling post-treatment activities. Group 2 dogs received a single treatment, on Day 0, with 13.64% w/w fluralaner chewable tablets (Bravecto®, Merck Animal Health, Madison, NJ, USA) based on body weights recorded on Day − 2. One or more whole chewable fluralaner tablets were administered, at doses as close as possible to the minimum recommended dose of 25 mg/kg without underdosing. Treatment was administered within 20 minutes after feed had been offered to ensure dosing within the fluralaner label dose (actual doses ranged from 25.2 to 35.9 mg/kg; chews were not shaved, split, or cut). Immediately after treatment, dogs were returned to their cages and observed for approximately 1-h for adverse events and to ensure the treatment was retained. Each dog was observed for general health on the day of treatment administration at 1-h, 3-h, and 6-h post-treatment. Post-treatment observations on all dogs were conducted in a random order of evaluation.
Tick infestations and counts
At the pre-study infestations on Days − 6 (Study 1) or -9 (Study 2) and on Days − 2, 28, 58 and 88 the 20 dogs included in the study were infested with approximately 50 adult, unfed female H. longicornis ticks. The H. longicornis isolate for Study 1 had been collected in New York during July, 2018 and for Study 2 the isolate was obtained from vegetation in Virginia in October, 2018. Prior to the infestation, each dog was sedated (dexmedetomidine hydrochloride, 0.5 mg/mL Dexdomitor®, Zoetis) and a vial containing the ticks was emptied onto the dog’s back. Each dog was then held individually in an infestation chamber (dimensions: Study 1, 0.9 x 1.1 x 0.9m; Study 2, 0.8 x 0.9 x 0.7m) for a period of up to approximately 4-h to ensure that ticks had established. After the infestation period, the dog was removed from the infestation chamber and returned to its cage. To avoid any cross-animal exposure to fluralaner, from Day 0 forward an infestation chamber was assigned to each dog and used only for that dog for the duration of the study. Before each use, infestation chambers were thoroughly washed with soap and water, rinsed with clean water, and double rinsed with isopropyl alcohol (with air drying between each alcohol rinse).
On each count day, each dog was examined for ticks by pushing the hair against its natural nap to expose the skin, covering the entire dog’s body. Any ticks found were gently removed using either forceps or fingers. After this examination was complete the dog was combed (tick comb, approximately 32 teeth per 2.5 cm) to recover any ticks that could have been missed during the visual inspection. Collected live ticks were classified as either free (on the host) or attached, and then discarded. The tick collection procedure on each dog lasted a minimum of 5 minutes.
Number of dogs
According to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guideline for studies investigating the efficacy of ectoparasiticides, a minimum of 6 dogs is recommended for each treatment group [18]. A sample size of 10 dogs per treatment group was used in this study to help ensure that an adequate infestation (≥ 13 ticks) was achieved in a minimum of 6 untreated control dogs.
Statistical analysis
The primary endpoint was based on between-group differences in live tick counts, attached and free on the host, with the experimental unit the individual dog. At least six dogs in the control group were required to be adequately infested (a minimum of 13 live ticks or 25% of the infestation dose) to allow comparisons at any time point. Tick counts were analyzed using a linear mixed model that included treatment group as a fixed effect. The fluralaner group and the control group were compared using a two-sided test at a 5% level of significance. Separate analyses were conducted at each tick infestation day. Arithmetic least squares means were used for treatment comparisons. The null hypothesis was that there was no significant difference between the treated group and the control group. The primary software used for analysis was SAS version 9.3 or higher.
Within each infestation schedule, the primary efficacy endpoint was calculated using the formula below, based on arithmetic mean tick counts:
Efficacy (%) = [(Group 1 mean counts – Group 2 mean counts)/ Group 1 mean counts] x 100