The objective of both studies was to evaluate the effectiveness of topical solutions of fluralaner as a single entity product, and fluralaner in combination with moxidectin in cats infested with H. longicornis ticks. Each study was randomized with an untreated control group. All observations and procedures, including general health observations, tick infestations, and tick counts were performed by masked individuals. The same protocol was used in each study, both of which were conducted in accordance with Good Clinical Practices, and with the guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP) for evaluating the efficacy of parasiticides against flea and tick infestations of dogs and cats [9, 10].
Animals
On or before Day − 8 in each study, 36 Domestic shorthair cats were bathed in a non-medicated shampoo and moved to cages for an acclimation period. The floor size of each cage was approximately 1.1 m x 1.2 m and at least 2.0 m high. To be included in either study cats had to be clinically healthy with no pre-existing conditions (e.g., injury, trauma, disease) that could have affected the study, at least 6 m of age, not treated with any long-acting anti-flea or anti-tick product within the previous 120 days and have demonstrated susceptibility to tick infestation based on retaining at least 13 live ticks (at least 25% of a pre-study infestation) placed on Day − 7. Cats were excluded if showing evidence of any concurrent disease, including skin conditions such as lesions or poor hair coat, if lactating or pregnant, or if fractious to a point that behavior could affect handling for study procedures. From placement for acclimation and throughout the study, cats were housed in individual cages in a thermostatically controlled environment with an approximate 12-h light/12-h dark cycle, fed a commercial cat food per site practice and allowed ad libitum access to water. Cages were segregated by treatment group to avoid cross exposure of cats to fluralaner. From those presented for acclimation, 30 cats in each study were retained for experimental purposes. Cats selected for Study 1 were 8 males and 22 females, aged from 17.0 to 41.2 months (median age in each group was approximately 28 months) and weighing from 2.5 to 9.1 kg (median weights in each group from 3.5 to 3.8 kg) on Day − 2. Study 2 cats were 7 males and 23 females, aged from 12 to 84 months (median age in each group was approximately 28 months) and weighing from 2.3 to 4.5 kg (median weights in each group from 2.9 to 3.2 kg).
Randomization and treatment
On Day − 5 or -4, 30 cats that satisfied the inclusion criteria were selected for each study. On Day − 5 or -3 cats were randomized, using a computer-generated randomization table, to treatment groups in a 1:1:1 ratio, so that 10 cats were included in each group. Cats in Group 1 remained untreated but were handled in the same manner as cats in groups allocated to one of the fluralaner-treated groups. Group 2 cats received a single application of a topical formulation of fluralaner, 280 mg/mL, at the minimum label dose of 40 mg/kg (Bravecto topical solution for Cats; Merck Animal Health, Madison, NJ, USA). Cats randomized to Group 3 were treated at the minimum label dose with a single application of the topical combination product of fluralaner (280 mg/mL; dose rate 40 mg/kg) and moxidectin (14 mg/mL; dose rate 2.0 mg/kg) (Bravecto Plus for cats, Merck Animal Health, Madison, NJ, USA). With doses calculated from the Day − 2 body weights, treatments were applied on Day 0, by parting the hair and placing the tip of a disposable syringe at the base of the skull and squeezing the plunger to apply the contents, taking care to avoid any run-off. Administered fluralaner dose rates in Group 2 cats ranged from 39.6 to 40.3 mg/kg and in Group 3 cats from 39.8 to 40.4 mg/kg. Each cat (all treatment groups) was kept on the treatment table for approximately 3 minutes following treatment administration before being returned to its cage. Any abnormal observations such as solution drip-off or shake-off, and cat behavioral changes were recorded. Any abnormal observations were recorded as an adverse event, and any run-off or product loss through head shaking was recorded, but no further treatment was applied to replace the estimated loss of dose.
Immediately after the treatment procedure cats were returned to their cages and observed for approximately 1 h for adverse events and to ensure the treatment was retained. Each cat was observed for general health by masked personnel on the day of treatment administration at 1 h (± 15 m), 3 h (± 30 m), and 6 h (± 30 m) post-treatment and then daily through the end of the study. Treatment sites of all cats were also examined on Days 1, 2, 3, 7 and 14 post-treatment. Post-treatment observations on all cats were conducted in a random order of evaluation.
Tick infestations and counts
At the pre-treatment infestation on Days − 7, and on Days − 2, 28, 58, and 88 each of the 30 cats included in each study was infested with approximately 50 adult, unfed H. longicornis ticks. The H. longicornis isolates in Study 1 had been collected in New York during January, 2020 and those in Study 2 from a colony originally collected from vegetation in Virginia, USA in October 2018. Prior to placing the infestation, each cat was sedated (dexmedetomidine hydrochloride, 0.12 mL/kg Dexdomitor®, Zoetis) and a vial containing the ticks was emptied onto its back. After placing the ticks, an Elizabethan collar was fitted to each cat to prevent self-grooming and to allow the ticks to find attachment sites, and the cat was then moved into an individual infestation chamber for a period of up to 4 h to ensure that ticks could establish. The collars remained in place until the tick counts for each infestation were completed. The exception was the Day − 2 infestation when collars were removed prior to treatment on Day 0 to avoid interference with the treatment retention. Collars remained off the cats for the subsequent 48 hours prior to the Day 2 tick counts. 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) and collars were thoroughly washed with soap and water, rinsed with clean water, and doubled rinsed with isopropyl alcohol (with air drying between each alcohol rinse). Each collar and each infestation cage were used for the same cat for the duration of the study.
Tick counts were completed on Day 2 and on Days 30, 60 and 90 (at 48 ± 4 h post-infestation). For each cat, after the head, dorsal and dorso-lateral aspects were visually examined the cat was turned onto its back for ventral and ventro-lateral examination. The cat was then thoroughly combed using a tick comb (at least nine teeth per cm) to remove any ticks that might have been missed during the visual examination. Each examination and combing procedure lasted for at least 5 minutes. Collected ticks, whether attached or free on the cat, were counted and classified as live or dead.
Number of cats
According to guidelines issued by the World Association for the Advancement of Veterinary Parasitology (WAAVP), for studies investigating the efficacy of ectoparasiticides, a minimum of six cats is recommended for each treatment group [10]. A sample size of 10 cats per treatment group was used in this study to help ensure sufficient data were available to justify a statistical assessment.
Statistical analysis
The primary endpoint in each study was based on arithmetic mean live tick counts. For between-group comparisons to be valid, on each tick challenge day an infestation rate of at least 25% of the infesting burden (at least 13 live ticks) was required to be collected from at least 6 control cats. Live tick counts of each treated group were compared to those of the control group using a linear mixed model that included treatment group as a fixed effect. Testing was two-sided with a 5% level of significance. The null hypothesis was that there was no significant difference between each treated group compared with the control group. Separate analyses for each fluralaner treatment group hypothesis were conducted (fluralaner formulation compared to the control group) at each tick count day. Least square means from the model were used for percent effectiveness calculation. The primary software used for analysis was SAS version 9.4. or higher.
Within each infestation schedule, the primary efficacy endpoint was calculated using the formula below, based on arithmetic mean tick counts:
Efficacy (%) = [(mean Group 1 counts – mean Group 2 counts)/mean Group 1 counts)] x 100.