This study was conducted in accordance with the VICH GL9 (June 2000) guideline on Good Clinical Practice [9], and Guideline on Statistical Principles for Veterinary Clinical Trials [10]. Ethical approval was obtained by the ClinVet Institutional Animal Care and Use Committee before beginning the study, and all owners signed informed consent forms after receiving written and verbal details of the study procedures and products to be administered.
Dogs and Management
To qualify for the study, dogs were required to be clinically healthy, except for clinical signs associated with generalized demodicosis; to present with clinical signs of infestation (including erythema, hair loss, comedones, follicular casts, scales and crusts) affecting more than 5 sites on the body, pododemodicosis involving at least 2 paws, or an entire body region). Dogs were excluded if they had received glucocorticoid therapy or any ectoparasiticide or endectocide within 12 weeks prior to Day 0, if known to be pregnant, or if fractious to the point of posing a danger to themselves or facility personnel.
Adult dogs were sourced from rural areas with limited access to animal health care. Following owner consent, 16 suitable dogs were transferred to the study site to undergo a 7-day acclimatization period, during which daily health observations were made, with clinical examinations and body weight measurements completed on Days -7 and -2. Starting on Day -7 all dogs received an appropriate antibiotic (Convenia®, cefovecin, Zoetis) as a treatment for pyoderma. The dogs also received a probiotic (Protexin® Soluble, Kyron Laboratories) from Day -7 until the end of the study on Day 84. Demodicosis was confirmed by identification of D. canis mites from deep skin scrapings completed on Day -2.
During the study, dogs were kept individually in concrete-floored pens under strict quarantine conditions. No physical contact between dogs was possible, but dogs had visual and auditory contact with conspecifics. The dog pens consisted of a 1.69 m x 0.7 m enclosed sleeping area, with panel heaters, plastic beds/rubber mats and an outside run of 1.69 m x 3.0 m. Standard commercially available diets that met the nutritional requirements of the dogs were fed at the recommended rates, and water was provided in stainless steel bowls and replenished at least twice daily. Food was removed in the afternoon prior to skin biopsies to facilitate anesthesia for biopsy procedures. At least one toy/chew was available to each dog (replenished weekly). A roof covering the kennels prevented exposure to rain and dogs were held in ambient temperatures with natural lighting.
Study Treatments
On treatment days, dogs consumed up to half of their daily food ration within approximately 20 minutes before treatment, after which they were offered the remainder of the ration so that all were in the fed state at the time of treatment. Dogs were ranked according to live mite counts and randomized to a treatment group. Dogs allocated to Group 1 were treated with a new formulation of fluralaner (5.46% w/w flavored chewable tablets) (Bravecto® 1-Month) on Days 0, 28 and 56 at a minimum dose rate of 10 mg/kg (actual dose rates 10.0 to 14.4 mg/kg). Group 2 dogs were treated with a commercially available topical formulation containing imidacloprid (100 mg/mL) and moxidectin (25 mg/mL) (Advocate®) administered according to label directions (10 mg imidacloprid/kg, 2.5 mg moxidectin/kg). Treatments with imidacloprid-moxidectin were applied directly to the skin at one spot between the shoulder blades. Dogs were restrained for approximately one minute following topical administration and any drip-off was re-applied. Although scheduled for applications on Days 0, 28 and 56, the frequency of treatments with this topical product could be increased to once weekly if the blinded attending veterinarian diagnosed the demodicosis as being severe. Appropriate dosing for study treatments was calculated according to each dog’s body weight, assessed on Days -2, 27 and 56. On Day -2 weights of dogs in Group 1 ranged from 9.7 to 17.6 kg (median 13.0 kg) and in Group 2 from 7.1 to 16.4 kg (median 9.0 kg). To prevent unmasking during assessments, all dogs in the fluralaner group received saline solution topically during the treatment process. If any dogs in the imidacloprid-moxidectin group required weekly treatment, all dogs in both treatment groups, excluding those receiving the active topical product, were treated topically with saline solution on the same days to maintain masking of study personnel. All dogs were observed hourly for up to 4 hours after treatment and full clinical examinations were completed at 2-week intervals from Days 14 to 84. No additional treatments with potential miticidal activity were used during the study on the dogs or their environment.
Mite counts, clinical assessments, and skin biopsies
On Days -2, 28, 56 and 84, deep scrapings were taken from 5 sites on each study dog using a scalpel blade to a skin depth at which capillary blood began oozing. Subsequent deep scrapings were made from the same sites or from sites of new lesions. Each scraping was transferred to a uniquely identified microscope slide containing mineral oil and examined under a stereo microscope to identify and count live D. canis mites.
Clinical signs and the extent of demodectic lesions on each dog were assessed on the days on which scrapings were made and sketched on a silhouette to assess the degree of body area covered by casts, scales, crusts, erythema and pyoderma, and to score body areas with hair loss (1 = slight thinning of hair; 2 = conspicuous hair loss; 3 = no hair). A semi-quantitative assessment of hair re-growth compared to the pre-treatment assessment was also completed (1 = 0 to 50%; 2 = > 50% to ≤ 90%; 3 = >90%). The overall severity of demodicosis (mild, moderate, severe) was evaluated by the blinded veterinarian investigator.
On Days -7 and 27, biopsies from affected skin areas were taken from each dog under general anesthesia (propofol 1%). Any dogs with clinical signs of pyoderma at the Day 27 skin biopsy examination were treated with an appropriate antibiotic (Convenia®), administered according to label. Antibiotic therapy was continued until receipt of the biopsy results, and if evidence of active infection was present the antibiotic treatment was continued in affected dogs for another four weeks. From Day 0 until the end of the study all clinical assessments and mite counts were completed by qualified personnel blinded to treatment group.
Statistical analysis
The European Committee for Medicinal Products for Veterinary Use (CVMP) guideline for evaluation of the efficacy of anti-parasitic substances for the treatment and prevention of tick and flea infestations in cats and dogs states that at least 6 animals should be used per group [11]. For this study the guideline minimum was exceeded with 2 additional dogs included in each treatment group.
Primary efficacy was based upon the percent reduction in arithmetic mean mite counts relative to pre-treatment counts.
Reduction [%] = | 100 X | Meanpre – Meanpost |
Meanpre |
where Meanpre was the Day -2 mite counts and Meanpost was derived from counts made on Days 28, 56 and 84.
Geometric mean mite counts were calculated using logarithm transformed counts (count +1) with 1 subsequently subtracted and analyzed using a Linear Mixed Model. The fixed factor in the model was the time point. An un-adjusted comparison of the pre-treatment mite counts versus the post-treatment mite counts was performed using a two-tailed test where the level of significance was set to α = 0.05.
The parasitological cure rate was defined as the percentage of treated dogs having two consecutive negative scrapings on Days 56 and 84. Non-inferiority and superiority of the proportion of cured fluralaner-treated dogs in comparison to the proportion of cured dogs in the imidacloprid-moxidectin group were investigated using the Farrington-Manning test of non-inferiority for the risk difference [12] with a level of significance of α = 0.025 and a tolerated difference of δ = 0.15. Both P-value and lower 97.5% one-sided confidence limits were calculated. If the lower confidence limit was above -0.15, non-inferiority was concluded. If the lower confidence limit was above 0, superiority was concluded.