Study design
In the present study, Bravecto™ administered as chewable tablets on a single occasion was the test product and Advocate®, administered three times at 28-day intervals was included as a positive control. This made it possible to compare the long term efficacy of 12 weeks of a single oral treatment with Bravecto™ chewable tablets against the efficacy of Advocate® administered at a 28-day interval according to the product label.
The study was designed as a parallel group, blinded, randomized, single centre, and positive controlled efficacy study. The study was conducted in accordance with the FDA Code of Federal Regulations: Good Laboratory Practice for Nonclinical Laboratory Studies 2009 [24], and all procedures were in compliance with South African National Standard “SANS 10386:2008: The care and use of animals for scientific purposes” [25]. The protocol was submitted to the ClinVet animal ethics committee that authorized the conduct of the study.
The test system was the individual dog. Dogs with natural infestations of Demodex spp. mites and presenting clinical signs of generalized demodicosis, e.g. erythema, hair loss, comedones, follicular casts and crusts were enrolled, with consent from their owners, in the study and were returned to their owners on completion of the animal phase. Dogs included in the study were mostly mongrels and of both sexes, older than 12 months, weighed between 3.5 and 13.7 kg, and except for clinical signs of generalized demodicosis, the dogs were healthy and as far as could be determined the dogs had not been treated with a glucocorticoid or any product with a miticidal effect for at least 12 weeks prior to inclusion. Additional requirements for inclusion were that deep skin scrapings performed before treatment had to be positive for Demodex spp. mites.
Sixteen dogs (7 male and 9 female), ranked within sex in descending order of individual pre-treatment mite counts were included in the study and allocated to two equal groups. Each dog was housed individually for the duration of the study in an indoor/outdoor run, without contact between animals, and was fed once a day according to the food manufacturer’s recommendations. Potable municipal water was available ad libitum.
Each dog was acclimatized to the housing and maintenance conditions for at least 14 days before treatment. As a precautionary measure all dogs were treated subcutaneously with an antibiotic (cefovecin), appropriate for the treatment of pyoderma on Days −14, −1, 13 and 27. Additionally, on Days −14 and 27, deep skin biopsies were taken from each dog after sedation. The biopsies indicated that exudative pyoderma was present in two dogs in each group on Day −14 and that it had cleared by Day 27. Chronic dermatitis, epidermal acanthosis and hyperkeratosis was present and unchanged in all dogs on both occasions. No inflammatory cells or bacteria were observed in the Day 27 biopsies and antimicrobial therapy was discontinued. Twice during acclimatization (Day −14 and Day −1) and on Days 27/28, 56 and 84 after treatment each dog was clinically examined by a veterinarian.
The dogs were weighed on a calibrated and verified electronic scale on Days −2, 13, 27, 41, 55, 69 and 84 for dose calculation for treatment, for the use of sedatives for skin scrapings and to document the body weight during the study period. General health observations were performed daily throughout the complete study period.
Treatment
On Day 0, dogs of one group were treated once orally with Bravecto™ chewable tablets (fluralaner, 13.64% w/w), based on the dog’s individual body weight, to achieve a minimum dose of 25 mg/kg body weight and an efficacy over 12 weeks following treatment. The tablet(s) was (were) administered 20 (±10) minutes after food had been offered by placement in the back of the oral cavity over the tongue to initiate swallowing. Also on Day 0, commercially available Advocate® (imidacloprid, 10%/moxidectin, 2.5% w/v) was administered topically to the other group of dogs (positive controls) according to the product label. Due to the 28 days efficacy duration of Advocate®, these dogs were re-treated on Day 28 and 56. With the dog in a standing position, the coat was parted until the skin was visible and the Advocate® was administered directly onto the skin. Both treated groups were observed prior to treatment and again hourly for 4 hours after treatment of the last animal, for possible adverse events. Personnel performing the post-treatment observations were blinded with respect to the treatment.
Mite assessments
Deep skin scrapings (~4 cm2), in which the skin was squeezed and scraped until capillary oozing was seen, were made from five sites on each dog on Days −4, 28, 56 and 84. Skin scrapings of the dogs treated with Advocate® were performed on Day 28 and Day 56, before the second or third treatment was applied, respectively. The same sites and/or sites of new lesions were scraped at each subsequent examination. Each scraping was transferred to a separate labelled microscope slide containing mineral oil and was examined under a stereomicroscope for the presence of Demodex spp. mites. The numbers of mites counted in each scraping were recorded separately.
Skin and hair assessments
The clinical signs and the extent of demodectic lesions on each dog were assessed on the days when skin scrapings were made, and recorded on a standardised form. The following parameters were assessed and sketched on a silhouette (left and right hand side) for each dog: body areas exhibiting erythema; body areas covered by casts, scales and crusts; body areas with hair loss. A semi-quantitative assessment of hair re-growth was performed, comparing hair coat before, within and after the 12 weeks study duration and assessed as percentage hair re-growth (0-50%, 50-90% or > 90%) defined as estimated percentage of hair cover growth compared to baseline total hairless area as assessed prior to veterinary product administration. Colour photographs illustrating the extent of lesions and their resolution were taken of each dog on Day −4 and subsequently at approximately monthly intervals up to Day 84 after treatment.
Efficacy evaluation
The primary assessment variable in the study was the decrease in total number of mites counted in skin scrapings following treatment.
Efficacy was calculated using geometric means with Abbott’s formula:
$$ \mathrm{Efficacy}\ \left(\%\right) = \left(\mathrm{Mpre}\ \hbox{--}\ \mathrm{Mpost}\right)/\ \mathrm{Mpre}\ \mathrm{x}\ 100 $$
where Mpre was the mean number of pre-treatment mite counts, and Mpost the mean number of post-treatment mite counts.
Additionally, the groups were compared using an ANOVA with a treatment effect after a logarithmic transformation on the mite (count + 1) data, for each study day.
On Day 50 one of the dogs in the group treated with Advocate® developed severe oedema of the left hind leg and prepuce and the swelling did not dissipate with antibiotic, diuretic and corticosteroid treatment. The dog was removed from the study on Day 59, sedated and upon laparotomy a large nodular tumour compatible with malignant lymphoma was found attached to the ventral spine, and the dog was euthanised during surgery. The results pertaining to this dog until Day 56, before its exclusion from the study on Day 59, have been included with those of the other dogs in the group treated with Advocate®.