The animals were not treated by any individual involved in performing the post-treatment assessments and observations. Study groups were coded to blind the assessors.
Dogs
Forty-eight healthy mongrel dogs (>6 months old, 1:1 sex ratio) were enrolled for the study and started an acclimation period of at least 7 days. Because removal and sampling of fleas was required through combing at 5- and 10-min post-infestation from untreated animals, an additional untreated group (n = 6, CQ) was specifically enrolled in parallel to quantify flea feeding. The dogs had not been treated with an acaricide or insecticide for 16 weeks. All dogs were identified with electronic transponders and were dewormed at the beginning of the study. Their body weight (BW) ranged from 10.21 to 22.86 kg, and their hair length ranged from 11 to 39 mm. The dogs were housed individually in an indoor animal unit, controlled for temperature with 12 h light:12 h darkness photoperiod. Humidity was recorded daily with a hygrometer. Dogs were fed commercial dog food once daily with water available ad libitum. No contact between dogs was possible during the study. All dogs were observed for general health status and adverse reactions to treatment once daily, from day −7 to day 28, except on day 0, where specific health observations were made hourly for 4 h after treatment. On day −7, 1 mL blood was collected in EDTA tubes from each animal prior to white blood cell counts. This protocol was approved by an independent animal ethics committee.
Allocation
The study followed a randomized block design. The 48 dogs were ranked, within gender, in descending order of individual day −5 flea counts. Within each gender, animals were blocked into blocks of 8 dogs each. Within each block, dogs were randomly allocated to one of the six groups.
Treatment
Each dog was treated with the allocated treatment on day 0. Dogs in groups C1 and C4 were untreated. Dogs in groups DPP1 and DPP4 were treated with the commercial formulation DPP containing the active ingredients dinotefuran (4.95 %, w/w), pyriproxifen (0.44 %, w/w) and permethrin (36.08 %, w/w). Dogs in groups S1 and S4 were treated with the commercial formulation S containing the active ingredients spinosad. DPP was administered topically, as a spot-on, according to the manufacturer’s label directions, at a rate of 3.6 mL per dog applied equally (1.2 mL per site) in three spots at the shoulder blades, the mid-back and the base of tail. S was administered orally: one 665 mg tablet was administered to the animals weighing 9.5 to 14.7 kg while one 1040 mg tablet was administered to the animals weighing 14.8 to 23.1 kg. Dogs were given the possibility to take the pill spontaneously. If the pill was not taken spontaneously, the dogs were force-fed. Dogs were fed immediately after dosing with S.
Flea infestations
A laboratory-bred strain of C. felis felis (European origin), routinely fed on cats, was used in the experimental infestation. The fleas were unfed and of mixed sex. Each dog was infested with 100 adult fleas on days −6, −2, 2, 7, 14, 21 and 28, except the CQ-untreated group which was infested once with 150 fleas prior to combing of at least 50 live fleas from each dog 5 and 10 min after infestation.
In the treated groups, dislodged dead and moribund fleas were collected at 5, 10, 15 and 60 min after flea infestations for DPP1 and S1 and at 5, 10, 30 and 240 min after flea infestations for DPP4 and S4. These fleas were collected on pans placed underneath the cages on days 2, 7, 14, 21 and 28. In all groups, fleas were counted and removed from dogs by combing, respectively, 1 h ± 5 min or 4 h ± 5 min post-treatment and subsequent infestations (Table 1). A fine-toothed flea comb was used to recover fleas from the animal’s fur. Several strokes of the comb were applied in each body area. This procedure was performed at least twice and until no more live fleas were found. At each assessment, fleas were classified as live, moribund or dead.
Table 1 Study design and time interval between flea infestations and assessments
Quantification of flea feeding
Real-time quantitative PCR analysis of the canine cytochrome b gene (Woods et al. 2009) was conducted on pools of ten randomly selected dislodged fleas collected from individual treated dogs at 5 (n = 16) and 10 min (n = 16) after each weekly infestation (Table 1). For optimal reliability, quantitative assessments must be performed shortly after infestation, before blood saturation of fleas and the excretion of bloody faeces which is expected to occur approximately 30 min after infestation (Wang et al. 2012). The analysis was also performed on pools of ten randomly selected live fleas combed from individual control (CQ) dogs 5 (n = 3) or 10 min (n = 3) after infestation. The number of canine gene copies was used as a marker of blood volume ingested by fleas. Fleas were preserved in sterile tubes containing 70 % ethanol before being washed in phosphate buffer saline prior to DNA isolation (Han et al. 2006).
Statistical analysis
The cumulative arithmetic means (CAM) of dead and moribund fleas dislodged from dogs were calculated for each treatment 5 and 10 min after infestations. The groups were compared by an ANOVA with a treatment effect. Statistical significance was declared at a two-sided p value of 0.05.
The geometric means (GM) of canine cytochrome b gene copies in fleas dislodged from dogs were calculated for DPP, 5 and 10 min after infestations. GM calculations were based on the means of the average canine cytochrome b gene copies in fleas (average + 1) data. One (1) was subsequently subtracted from the result to obtain a meaningful value for the GM of the study groups. Percent of feeding inhibition for each assessment time (5 min or 10 min) on each day was calculated as:
$$ \mathrm{Feeding}\ \mathrm{inhibition}\kern0.5em \left(\%\right)=100\times \frac{\left(\mathrm{MCcytb}-\mathrm{MTcytb}\ \right)}{\mathrm{MCcytb}} $$
where:
- MCcytb
:
-
Mean (GM or AM) of the average canine cytochrome b gene per fleas on dogs in the C1 and C4 groups and
- MTcytb
:
-
Mean (GM or AM) of the average canine cytochrome b gene per fleas on dogs in the DPP1 and DPP4 groups.
The AM and GM were calculated for live fleas collected from dogs in the respective groups 1 and 4 h post-treatment and subsequent infestations. Moribund fleas were considered as a failure and counted as live ones. Calculations of GM were based on the means of the live and moribund flea (count + 1) data. One (1) was subsequently subtracted from the result to obtain a meaningful value for the GM of the study groups. The groups were compared using a one-way ANOVA with a treatment effect after logarithmic transformation on the (flea count +1) data. Unadjusted pair-wise comparisons were performed between each pair of groups with ANOVA contrasts. Statistical significance was declared at a two-sided p value of 0.05. Groups were also compared by a non-parametric analysis using the Kruskal-Wallis test. Unadjusted pair-wise comparisons were performed between each pair of groups with the Mann-Whitney test. Percent of insecticidal efficacy for each assessment time (1 h or 4 h) on each day was calculated as:
$$ \mathrm{Efficacy}\kern0.5em \left(\%\right)=100\times \frac{\left(\mathrm{M}\mathrm{C}-\mathrm{M}\mathrm{T}\ \right)}{\mathrm{MC}} $$
where:
- MC:
-
Mean (GM or AM) of live and moribund fleas in the control groups and
- MT:
-
Mean (GM or AM) of live and moribund fleas in the respective treated groups.
Guidelines
The study was conducted according to the Good Clinical Practices (CVMP 2000). The study was conducted in accordance with the current and appropriate guidelines (CVMP 2007).