Animals and husbandry
Purpose-bred Beagle dogs approximately 29 to 44 months of age and weighing 9.5 to 13.2 kg were included in the study. Twenty-four dogs, 11 females and 13 males, that were vaccinated against canine distemper, infectious hepatitis, parvovirosis, infectious laryngotracheitis, parainfluenza, leptospirosis, and rabies were dewormed prior to initiation of the study and did not harbor resident tick or flea infestations at the initiation of the study. Dogs had not been treated with a long-acting topical or systemic acaricide/insecticide whose claimed duration of efficacy had not expired for at least 2 months. Dogs were identified by electronic transponders with unique alphanumeric codes.
Dogs were generally housed in pairs within groups, except for days between tick infestation and tick counts, where the dogs were individually housed in indoor enclosures that conformed to accepted animal welfare guidelines and ensured no direct contact between dogs of different groups. During periods when not infested with parasites, dogs of the same sex and from the same group were allowed to access and interact in a social exercise area. Dogs were acclimated to these conditions for at least 7 days prior to treatment. Commercial, dry, canine feed was offered once daily for the duration of the study according to manufacturer’s recommendation. Water was available ad libitum from stainless steel bowls that were replenished at least twice daily. All dogs were given a physical examination to ensure that they were in good health at enrollment and suitable for inclusion in the study. General health observations of each dog were performed daily throughout the study.
Design
This study was a negative controlled, randomized, non-blinded efficacy study against ticks and was conducted in accordance with the EMEA antiparasitic guideline for dogs and cats [16] as well as Good Clinical Practice [17].
Dogs previously assessed for their suitability to harbor ticks were included in the study and randomized based on body weight within sex into three groups of eight dogs each. Two of the groups were treated with collars at different time points: at the first tick infestation, dogs in group 1 had already worn collars for 92 days, while dogs in group 2 had received collars only on the previous day, thus allowing evaluation of two different treatment durations at the same point in time. Infestation of the treated groups was conducted at 1, 7, 28, and 56 days (group 2) and 92, 119, 147, 168, 196, 227, and 238 days (group 1) after collar placement (Fig. 1). Group 3 served as untreated control and was infested whenever the dogs of the other two groups were infested. Tick counts were conducted 48 h after infestations. Dogs in group 1 covered efficacy from 3 to 8 months after collar placement (persistent efficacy), whilst dogs in group 2 covered efficacy from collar placement up to the time where efficacy evaluation of group 1 started (persistent efficacy).
Treatment
The dogs either received a collar (groups 1 and 2, imidacloprid 10% w/w/flumethrin 4.5% w/w, Seresto®) or remained untreated (group 3). Collars were placed and fastened around the dog’s neck according to label instructions, so that it was possible to insert only two fingers next to each other between the collar and the neck when fastened. Excess collar was pulled through the collar’s loops, and any excess length extending beyond 2 cm was clipped.
During the study, the dogs were observed daily during facility cleaning activities for correct collar fit. In case a collar was accidentally lost by a dog, it was immediately reapplied. If a dog’s collar was completely lost or destroyed, the dog would have been removed from the study.
Tick infestations and assessments
Each dog was artificially infested with 50 viable female H. longicornis on the days as set out in Fig. 1. Ticks were adult, unfed, and had molted at least 1 week prior to being used. A laboratory-bred parthenogenetic strain originally sourced from Virginia (USA) in October 2018 was used for the artificial infestations.
To facilitate tick infestation, dogs were sedated with xylazine (Xylased®, Bioveta) administered intramuscularly at a dose rate of 2 mg/kg body weight and placed in a dark infestation chamber (50 cm × 50 cm × 90 cm) for at least 4 h following infestation. Ticks were placed on the lower chest of dogs in lateral recumbency and not further manipulated. To facilitate darkness, each infestation chamber was covered with a drape, made from breathable material.
Counting and removal of ticks were performed at 48 (± 2 h) after infestation. Ticks were found by direct observation following parting of the hair coat and palpation of the entire body. Following this, each dog was combed to ensure that all ticks were counted and removed. Ticks were counted and categorized as dead or live, attached or free.
Statistical analysis
The individual dog was the experimental unit. The efficacy against ticks was calculated based on percent reduction in the arithmetic mean (AM) live (female and male) tick counts relative to control using Abbott’s formula:
$$ {\text{Efficacy}}\left( \% \right){\text{against}}\;{\text{ticks}} = 100 \times \left( {{\text{Mean}}\;{\text{count}}\;{\text{control}}{-}{\text{ Mean}}\;{\text{count}}\;{\text{treated}}} \right)/{\text{Mean}}\;{\text{count}}\;{\text{control}}{.} $$
To statistically support the validity of the efficacy results, the mean post-treatment H. longicornis tick counts were compared between treatments, using an analysis of variance (ANOVA) with a treatment effect untransformed tick count. Comparison was pairwise to the control group for each time point, at a two-sided 5% level of significance.
Body weight (BW) and hair length obtained during acclimation were compared between the groups using a one-way ANOVA (Proc GLM procedure in SAS version 9.4) with a treatment effect and assuming a normal distribution of the data, to evaluate their homogeneity at the time of inclusion.
Efficacy was claimed if the following criteria were met: (1) at least six adequately infected dogs in the control group (> 25% retention for ticks [≥ 13 live ticks] [16]), (2) adulticidal efficacy ≥ 90%, and (3) significant difference between the treated and control groups. SAS version 9.4 software was used for all analyses.