Compliance
This study was conducted in accordance with the guidelines for evaluating the efficacy of canine and feline parasiticides for the treatment, prevention, and control of flea and tick infestations set forth by the World Association for the Advancement of Veterinary Parasitology (WAAVP) [10] and the Good Clinical Practice document that provides guidance on the design and conduct of all clinical studies of veterinary products in the target species [11].
This study was conducted as a masked, randomized, complete block design positive- and negative-controlled laboratory efficacy study. Personnel involved in animal observations, infestation procedures, tick count, and live–dead assessment procedures were masked to the treatment groups. As previously described, personal protective equipment was worn by all personnel conducting tick infestations and tick counts to avoid skin contact with dogs and changed to ensure no possible transfer of any active ingredient when working with dogs in different groups [9]. Personnel that participated in treatment administration were not involved in conducting or recording tick counts, tick live–dead assessments, or dog health assessments. All animal work was conducted in full compliance with an approved Institutional Animal Care and Use Committee Protocol (IACUC Protocol 4677) on file with the University Research Compliance Office at Kansas State University (KSU) in Manhattan, KS.
Animals & allocation
A total of 24 purpose-bred beagles (> 6 months of age, 5.0–7.5 kg, 12F:12M) were enrolled in the study. All enrolled dogs met inclusion criteria: be clinically healthy with no preexisting conditions; greater or equal to 6 months of age; demonstrated susceptibility to tick infestation (> 25% recovery of live ticks from test infestation); amenable temperaments for handling; and have not been treated with an anti-tick or anti-flea products for the past 180 days. On the basis of descending live tick count, enrolled dogs were ranked and blocked into eight blocks of three dogs. Within each block, dogs were randomly assigned to one of three groups using the randomization function on a spreadsheet program (Microsoft Excel 2019, Redmond, WA). Dogs were housed individually in concrete runs that had solid side walls so that they could not physically contact each other. Dogs were fed a maintenance ration once daily and provided water ad libitum. General health observations were performed at least once daily for all dogs.
Tick infestations
Tick infestations were performed using laboratory-reared, adult Amblyomma americanum approximately 12 weeks post-molt (Ecto Services, LLC, Henderson, NC). Ticks were maintained in Dr. Kathryn Reif’s laboratory at Kansas State University for up to 2 weeks at room temperature and 92–94% relative humidity until dog infestation. Live ticks were sorted and counted into vials (50 ticks/vial) the day prior to dog infestation.
Tick infestations were conducted on dogs on days −2, 21, 28, and 35. At each infestation timepoint, each dog was infested with approximately 50 unfed adult A. americanum (25 female, 25 male). As in a previous study, infestations were conducted on non-sedated dogs and ticks were applied directly to the dog’s fur along dorsal side of the head, neck, and back [9]. Dogs were transferred to pet carriers within 5 min of the tick infestation and maintained in the carrier for 4 h to facilitate tick attachment. Following this 4-h period, dogs were removed from the carrier and returned to their individual pens. Ticks that failed to infest dogs were collected from pet carriers and disposed (not included in counts or analyses).
Treatments
On treatment day (day 0), dogs in group 1 (untreated) received no treatment; each dog in group 2 received a fluralaner chewable tablet (Bravecto® Chew, Merck Animal Health, Rahway, NJ, USA) on the basis of the dog’s individual body weight to achieve a minimum dose of 25 mg/kg fluralaner; and each dog in group 3 received a sarolaner-moxidectin-pyrantel chewable tablet (Simparica TRIO®, Zoetis Animal Health, Parsippany-Troy Hills, NJ) on the basis of the dog’s individual body weight to achieve a minimum dose of 1.2 mg/kg sarolaner, 24 µg/kg moxidectin, and 5.0 mg/kg pyrantel. All treatments were provided with a small spoonful of wet dog food approximately 30 min after the dogs’ morning meal. Untreated dogs were similarly handled but received only a small spoonful of wet dog food. Dogs were observed to assess whether the chewable tablets were spit or vomited out and monitored for adverse treatment reaction signs at 1, 2, and 4 h post-treatment. No dog experienced any adverse treatment reaction nor spat out or vomited the chewable tablet.
Tick counts
Tick assessment and counting procedures were performed as previously described [8]. Tick counts for initial speed of tick kill analysis were conducted at 8, 12, 24, 48, and 72 h (± 0.25-h) post-treatment (day 0). Residual speed of tick kill was assessed by conducting tick counts at 8, 12, 24, 48, and 72 h (± 0.25 h), post-infestation on days 21, 28, and 35. Ticks were left in situ on dogs following the 8-, 12-, 24-, and 48-h counts, but were removed with forceps after the 72-h tick count. Sex (male or female), attachment status (unattached or attached), and viability status (live or dead) was determined for each observed tick [8, 9].
Statistical analysis
Counts of live ticks were analyzed separately for each timepoint of every post-enrollment infestation using a linear mixed model. Treatment group was the fixed effect and block was the random effect. Error term variance was taken as heterogeneous with respect to treatment group. For efficacy assessment based on geometric means, counts of live ticks were subjected to (ln ({text{count}} + 1)) transformation before statistical modeling. Denote ({text{LSM}}_{ln } (i)) as the corresponding least squares mean of treatment group i, and efficacy of treatment group i relative to treatment group j was calculated as
$$1 – frac{{exp ({text{LSM}}_{ln } (i)) – 1}}{{exp ({text{LSM}}_{ln } (j)) – 1}},$$
where exp(LSMln(i)) − 1 represents the model-based estimate of geometric mean for treatment group i. For efficacy assessment based on arithmetic means, counts of live ticks were modeled directly without any transformation. Denote ({text{LSM}}(i)) as the corresponding least squares mean of treatment group i. Efficacy of treatment group i relative to treatment group j was calculated as
$$1 – frac{{{text{LSM}}(i)}}{{{text{LSM}}(j)}},$$
where ({text{LSM}}(i)) represents the model-based estimate of arithmetic mean for treatment group i. To assure convergence, variances for block and error term were bounded low by 10–3 for transformed counts and 10–1 for untransformed counts.
A tick-free dog was defined as a dog with no live ticks [i.e., an infested dog has one or more live tick(s)]. The percentage of tick-free dogs by treatment group was analyzed using the Fisher’s exact test for treatment group comparison at a given time when both groups were not 100% infested.
All tests were conducted at the 0.05 significance level. Pairwise comparisons were carried out using two-sided tests. No multiplicity adjustment was performed.
Statistical analyses were performed using Statistical Analysis Software (SAS version 9.4; Cary, NC) PROC MIXED.
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