This was a randomized, assessor-blinded, single-site study in which the efficacy of the deltamethrin collar (Scalibor® protector band, Intervet International B.V., The Netherlands) in preventing engorgement of P. perniciosus in Beagles was compared to a placebo control group at the Charles River Laboratory in County Mayo, Ireland. Study personnel carrying out general health observations, clinical assessments, and assessments of P. perniciosus exposure, engorgement, and counting were masked to the treatments.
Animal inclusion, randomization and treatment
Twenty Beagle dogs, individually identified by an implanted microchip, were acclimatized for eight days prior to the day of treatment. On Day -7, dogs were exposed to challenge with 60 ± 10 female and 10 ± 2 male, laboratory-bred adult P. perniciosus that were free of Leishmania. The 16 dogs (8 males and 8 females) with the highest sand fly engorgement rate were allocated to the study. Dogs were excluded if they had been treated with any product with insecticidal activity within three months prior to enrollment, or if there was evidence of skin disease on the dog’s neck at the intended site of application of the study product. The 16 selected dogs ranged in age from one to six years and on Day -8 weighed 9.8–13.4 kg.
The 16 qualifying dogs were ranked within sex from highest to lowest Day -7 sand fly blood engorgement rate and blocked. The first two females in each block of two dogs formed a replicate. Those in the first replicate were assigned to the two study groups (placebo group or treated group) using random order numbers derived from Fisher and Yates tables. This procedure continued until four females had been allocated to each group and was repeated for males until the 16 dogs (eight males, eight females) had been allocated to the two groups (n = 8 per group). An equal number of males and females were allocated per group.
Dogs assigned to Group 1 received a placebo collar; those assigned to Group 2 received a deltamethrin collar (Scalibor® protector band) at the label dose rate. The collars were placed on Day 0 and left in place for 365 days. Each collar was removed from the sachet directly before use. The length (not including buckle) and the weight of each collar were recorded and the collars were marked in permanent ink with each dog’s identification in case of accidental displacement during the study. The collar was fastened around the dog’s neck, adjusted until a comfortable fit was achieved so that two fingers could be inserted between the neck and the collar, and fastened. Excess collar was pulled through the buckle and any excess beyond 5 cm was clipped. To reduce the risk of collar displacement, a small cable tie was fastened at the end of the collar overlap and a second cable tie was fastened at the midpoint of the overlapping area.
Animal husbandry
From the start of acclimatization (Day -8) to Day 6, dogs were pair-housed in indoor pens measuring approximately 2.0 × 2.0 × 2.4 m (length × width × height), with the exception of Days 0 and 1 when they were individually housed for treatment and to facilitate clinical assessments. From Day -3 until Day 6, dogs of the same sex were pair-housed within treatment groups, unless circumstances such as veterinary care required single housing. Due to an observation of pen-mate damage to collars during the first days of the treatment period, study dogs were individually housed (pens approximately 2.0 × 2.0 × 2.4 m) from Day 7 until the end of the study, the damaged collars having been replaced with a new collar. The anti-feeding and insecticidal results obtained on day 7 for the 4 dogs with damaged collars were excluded from the statistical analyses for day 7.
Each pen contained a resting area, and a small amount of wood shavings was used as bedding. Each single-housed dog was provided a toy and received social interaction for at least two minutes daily until Day 15 when interaction was extended to at least 10 min daily.
Dogs were fed a standard commercially available dog food once daily at the recommended rate (approximately 300 g/dog/day). Food was removed from each pen on the evening prior to each administration of sedative for sand fly exposure. Potable water was available ad libitum via stainless steel bowls in each pen, except during periods when animals were sedated for exposure to sand flies. Water bowls were removed prior to sedation and replaced after dogs had fully recovered from sedation. For the duration of the study, the temperature in the dog pens was maintained between 17–19 °C and the humidity ranged between 38–71%. Photoperiod was controlled for approximately 10 h light and 14 h darkness per 24 h period. Dogs were weighed on Days -8, 54, 82, 110, 145, 173, 208, 236, 264, 299, 327 and 362, and examined by a veterinarian on Days -8, 54, 166, 222, 278 and 362. Daily general health observations were carried out on all dogs by a trained technician, except on Day 0 when multiple clinical assessments were performed.
Sand fly infestations and assessments
All sand flies, free of Leishmania, were obtained from a colony maintained since 2007 at Charles University in Prague, Faculty of Science, Department of Parasitology, Czech Republic. After receipt at the research facility the flies were maintained in the dark at 25 ± 3 °C, and at least 70% humidity.
Each dog was exposed to challenge with viable P. perniciosus on Days -7, 7, 28, 56, 84, 112, 140, 168, 196, 224, 252, 280, 308, 336 and 364. For the challenge procedure dogs were sedated with intramuscular medetomidine hydrochloride (1 mg/ml) which was reversed post-challenge by administration of intramuscular atipamezole hydrochloride (5 mg/ml). Sedated dogs were placed in an exposure chamber (approximate dimensions 0.6 × 0.6 × 0.9 m) in a room where temperature ranged between 22.7–28.6 °C and relative humidity ranged between 64–91%. To preclude any possibility of active product cross-contamination between groups, the exposure chambers used for each of the two groups were in separate sections of the animal housing unit. After the dogs had been placed in an exposure chamber, a container containing 85 (± 10) female and, to help stimulate female feeding behavior, 15 (± 5) male P. perniciosus was put into the chamber. Because sand flies are nocturnal feeders, the lights were turned off immediately after the lid of the container was removed [6]. After approximately 60 min, the lights were turned on and all live sand flies were collected using an aspirator. Disposable liner placed on the floor of each chamber was replaced after each dog had been removed. Where necessary, each chamber was wiped dry between dogs. Once all live flies had been removed, each dog was checked for dead and feeding flies prior to and after removal from the exposure chamber. All live feeding flies were aspirated from the dog and collected into the same vented container as the other live flies that had been collected. All dead and moribund P. perniciosus (on the dog and in the chamber) were collected using forceps and placed into a separate vented container. Prior to evaluation of their engorgement status all live flies were killed by freezing. One container at a time, the collected P. perniciosus were poured into a Petri dish or onto a white background and the engorgement status for each determined by inspection and, as required, use of a stereomicroscope to detect traces of a blood meal. All female sand flies (live, moribund and dead) were categorized as engorged or un-engorged. A fly was considered moribund if it did not show progressive movement but retained leg movement or twitching. All male sand flies were removed prior to counts/assessments of engorgement status and were not included in efficacy assessments.
Assessments and statistical methods
The experimental unit was the individual animal. Arithmetic and geometric mean engorged P. perniciosus counts were calculated for each group on each day of counting and used to calculate the percent anti-feeding efficacy. Anti-feeding efficacy was determined by comparing the number of engorged female sand flies in the deltamethrin collar group to the mean number in the placebo control group at each post-treatment assessment. Anti-feeding counts included live, moribund and dead engorged flies. An effective dose was expected to provide a > 80% reduction in engorged P. perniciosus counts (based on arithmetic means) compared to the control group (as per EU Guideline 7AE17a).
Insecticidal efficacy was determined by comparing the mean counts of live female P. perniciosus (engorged + unengorged) in the treated group versus those of the placebo control group at the end of exposure, for each timepoint after treatment.
For each end-point geometric means were also calculated. The formula for determining efficacy was:
$$ \mathrm{Efficacy}\ \left(\%\right)=\left[\left(\mathrm{Mc}-\mathrm{Mt}\right)/\mathrm{Mc}\right]\ast 100 $$
where Mc is the arithmetic/ geometric mean count of Group 1 (placebo control), and Mt is the arithmetic/geometric mean count of Group 2 (deltamethrin collar group).
The groups were compared at each time point by a one-way ANOVA with a treatment effect on the sand fly data. The data were log-transformed if the normality and equal variance assumptions were not violated. Otherwise, the groups were compared by a non-parametric analysis using the Kruskall-Wallis test. All analyses were performed using SAS®/STAT (version 9.4). All statistical tests were two-tailed with a level of significance of 5%.