Ethical approval and study outline
This study was conducted in compliance with the Good Clinical Practice (GCP) guideline (Veterinary International Conference on Harmonization GL9) [28] and the European Medicines Agency guidelines regarding testing of anti-parasitic substances for treatment and prevention of tick and flea infestation in dogs and cats [29]. Ethical approval for conduct of the study was obtained from the “Clinvet Committee for Animal Ethics and Welfare” ethics body prior to conduct of the study.
The study was performed using purpose-bred dogs belonging to Clinvet, at the Clinvet study site near Bloemfontein, Free State, South Africa. The study was randomised, blinded and employed a parallel group design. Inclusion criteria for dogs into the study were: clinically healthy, older than six months, not clinically pregnant, not treated with any ectoparasiticide for at least 12 weeks prior to the start of the study, sero-negative for E. canis by immunofluorescence assay (IFA) and negative for E. canis deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR). Thirty-two purpose-bred beagles and mongrels belonging to Clinvet that complied with these inclusion criteria were divided into weight ranges (< 10 kg; > 10 kg to 20 kg; and > 20 kg). Health status at inclusion was confirmed by a veterinarian during clinical examination. Dogs were ranked within sex (14 males and 18 females) in descending order of individual live tick counts prior to veterinary product or placebo administration, and subsequently blocked into eight blocks of four dogs each. Within blocks, dogs were randomly allocated to four groups using Microsoft Excel software. A non-blinded person randomly assigned the groups to four coded groups using the same software package. Blinded personnel only had access to group codes and not group numbers. The study was thus conducted on four groups of eight dogs each. All dogs, identifiable by a microchip number, were individually housed in tick-proof kennels and observed daily throughout the duration of the study. In order to eliminate possible bias, additionally to the use of a placebo spot-on product, persons involved in the post-treatment observations were different from those that performed group allocations and treatments.
The study was laid out over a timeframe of three months (84 days), with the actual challenge periods in month two (Days 28 to 56) for E. canis transmission blocking and months two and three (Days 28 to 84) for tick efficacy assessments.
Treatments and rescue treatments
Dogs allocated to group 1 served as negative control and received a placebo spot-on compound (mineral oil), dogs in group 2 were treated with Advantix® spot-on solution for dogs (50 % permethrin/10 % imidacloprid), those in group 3 received NexGard™ chewable tablets for dogs (afoxolaner) and those in group 4 received Bravecto™ chewable tablets for dogs (fluralaner). Treatment regime is shown in Fig. 1 including placebo compound administrations for orally treated groups. Bravecto™ chewable tablets were administered on Day 0 only, based on the up to three month efficacy against ticks registered label claim of this product. All products were administered within weight classes as per label instructions. The placebo treatment applied to dogs in the Control group as well as the orally treated NexGard™ and Bravecto™ groups consisted of mineral oil only and was applied in three or four spots along the midline of the back. In the Advantix®-treated group between 10.42 mg/kg and 24.51 mg/kg imidacloprid and between 52.08 mg/kg and 122.55 mg/kg permethrin were applied by parting the hair and applying the product directly onto the skin in three or four spots along the midline of the back. In the NexGard™-treated group between 2.54 mg/kg and 5.48 mg/kg afoxolaner were administered orally. In the Bravecto™-treated group between 25.25 mg/kg and 47.62 mg/kg fluralaner were administered orally.
Animals were screened weekly using PCR and IFA. A positive PCR result provided confirmation of clinical diagnosis and hence the necessity to perform rescue treatment. Animals that tested negative (both PCR and IFA) on the last day of the study were not rescue-treated. Six animals in control group 1, no animals in Advantix® group 2, four animals in NexGard™ group 3 and two animals in Bravecto™ group 4 were rescue-treated with a commercial product containing doxycycline (Doxydog 100 mg and 50 mg, registration numbers G2636 and G2688 respectively) at the recommended dose rates and treatment regime.
Tick efficacy assessments
Efficacy assessments focused on early time points after tick infestation as defined in the following sections:
Tick infestations for efficacy assessments
A laboratory-bred strain of pathogen-free R. sanguineus (European origin, French strain) was used for artificial infestations. Each dog was infested with 50 ticks on days indicated in Fig. 2. Dogs (not sedated) were placed in an infestation crate, followed by the placement of 50 ticks on the dogs. The dogs were subsequently restrained inside the crates for 10 min before closing the mesh cover to confine the animal in the crate for a period of 12 h. Ticks, which dropped off the dogs during the first 10 min, were not placed back, unless shaken off by the dog.
Tick counting procedures
The tick counting procedures were designed to allow calculation of speed of kill, immediate drop-off and anti-attachment efficacy as described in the sections to follow, which in turn aided in the interpretation of efficacy in preventing E. canis transmission. On animal tick counts for speed of kill assessment at 3 h, 6 h and 12 h after infestation were performed on Days 30, 35, 42, 49, 56, 63, 70, 77 and 84. In situ thumb counts were performed 3 h (± 15 min) and 6 h (± 30 min) after each infestation. During these in situ counts, sexes were not distinguished, but ticks were categorised as live or dead. Calculation was performed according to the most current guidelines [30] and as a result engorgement status was not considered during efficacy calculations. Tick removal counts were performed 12 h (± 30 min) after each infestation. During removal counts ticks were counted within sex (male or females) and same general status as defined for the in situ counts. On the days and time points specified for in situ counts above, the ticks that dropped off the dogs were collected from the infestation crates. Collection took place during the time the dog was removed from the infestation crate for tick counts.
Methods for calculating efficacy and comparing groups
All efficacy results reported were based on arithmetic means as requested by current guidelines [30].
The speed of kill efficacy was calculated as the acaricidal efficacies [30] for the treated groups at the different assessment time points (3 h and 6 h in situ and 12 h removal counts). Speed of kill efficacy calculations were based on arithmetic mean tick counts using Abbott’s formula:
$$ \mathrm{Speed}\ \mathrm{of}\ \mathrm{kill}\ \mathrm{efficacy}\ \left(\%\right) = 100\kern0.5em \times \kern0.5em \left(\mathrm{M}\mathrm{c}\hbox{--} \mathrm{M}\mathrm{t}\right)/\mathrm{M}\mathrm{c} $$
where Mc = Arithmetic mean number of live ticks on dogs in the control group at a specific time point and Mt = Arithmetic mean number of live ticks on dogs in the respective treated groups at a specific time point.
The immediate tick drop-off rate was calculated based on the number of ticks recovered off the animal (i.e. free in the infestation crate) within 3 h of infestation as follows:
$$ \mathrm{Immediate}\ \mathrm{drop}\hbox{-} \mathrm{off}\ \mathrm{rate}\ \left(\%\right)=\left[\left(50\hbox{--} \mathrm{M}\mathrm{c}\right)\hbox{--} \left(50\hbox{--} \mathrm{M}\mathrm{t}\right)\right]/\left(50\hbox{--} \mathrm{M}\mathrm{c}\right)\times 100 $$
where Mc = Arithmetic mean number of total ticks collected off animal in the control group at the 3 h time point and Mt = Arithmetic mean number of total ticks collected off animal in the respective treated groups at the 3 h time point.
The anti-attachment efficacies at 6 h and 12 h post-infestation were calculated based on attached tick counts on dogs only. The aim was to evaluate if ticks that remained on the animals at 3 h after infestation actually attached to the animals by 6 and 12 h, respectively as follows:
$$ \mathrm{Anti}\hbox{-} \mathrm{attachment}\ \mathrm{efficacy}\ \left(\%\right)=100\kern0.5em \times \kern0.5em \left(\mathrm{T}\mathrm{m}\mathrm{c}\hbox{--} \mathrm{T}\mathrm{m}\mathrm{t}\right)/\mathrm{T}\mathrm{m}\mathrm{c} $$
where Tmc = Total attached (arithmetic mean of live and dead) ticks on the dogs in the control group at the respective time point, and Tmt = Total attached (arithmetic mean of live and dead) ticks on the dogs in the respective treated groups at the respective time point.
The groups were compared using an ANOVA after a logarithmic transformation on the tick (count + 1) data. The proportion of animals in each group was also compared. SAS Version 9.3 TS Level 1 M2 was used for all the statistical analyses. The level of significance was set at 5 %; all tests were two-sided.
Tick infestations to assess Ehrlichia canis blocking efficacy and monitoring of infection
Ticks used in this study derived from the same laboratory-bred strain of R. sanguineus used for the acaricidal efficacy determination and were artificially infected with a South African strain of E. canis using methods previously published [16, 31]. In order to simulate environmental tick challenges, ticks were released into the sleeping kennels of dogs on days indicated in Fig. 2. Ticks used were unfed, at least two weeks old and had a balanced sex ratio. The average infection rate of the tick batch used was 3.8 % and 80 ticks were released into each kennel to ensure an adequate environmental challenge.
Following environmental challenges ticks were removed after four days on Days 35, 42, 49 and 56. Moreover, each sleeping kennel was visually inspected for detached ticks, which were removed and the kennel cleaned.
Infection with E. canis was monitored by clinical examinations, rectal temperature records, platelet counts, as well as by testing blood samples by PCR and IFA.
All animals were observed daily for general health. Also clinical examinations (all dogs) were performed during acclimatisation for inclusion purposes, as well as on Days -1, 27, 34, 41, 48, 55, 62, 69, 76 and 83. Rectal body temperatures were recorded at least three times per week from Day 35 to Day 84. When dogs displayed abnormally high body temperatures (> 39.4 °C), a further measurement was taken the following day to evaluate for persistent pyrexia.
Blood specimens were collected for E. canis DNA detection by PCR and for platelets counts on Days -8, 30, 35, 42, 49, 56, 63, 70, 77 and 84. As PCR target, a specific fragment of the dsb gene of E. canis was amplified according to conditions previously published [23]. Conventional PCR was employed for detection of E. canis in animal blood and quantitative real-time PCR was employed for parasite load detection. Platelets counts were conducted by PathCare Veterinary Laboratory. Platelet concentration was evaluated as part of the complete blood count (CBC). The concentration was determined using a laser optic method. Smear examinations were performed on all abnormal platelet concentrations.
Serum was collected on Days -6, 35, 42, 49, 56, 63, 70, 77 and 84 and frozen at -20 °C until assayed for the detection of specific E. canis antibodies using a commercial IFA test kit (Megascreen® Fluoehrlichia c. test kit manufactured by MegaCor Diagnostik, Hörbranz, Austria). IgG titres of 1:40 and greater were considered to reflect infection (i.e. positive result).
Additional clinical examinations to that specified on the before mention days were conducted on any animal that displayed signs associated with ehrlichiosis. These signs included, but were not limited to, persistent pyrexia, thrombocytopenia and lethargy. For all animals with suspected ehrlichiosis, additional blood specimens for PCR were collected as needed to confirm the diagnosis.
Whilst a positive PCR result provided confirmation of clinical diagnosis and hence the necessity to perform rescue treatment, an efficacy failure (successfully infected with E. canis as employed in blocking and risk reduction calculations as described below) was defined as a dog that was found positive for E. canis DNA by PCR analysis and also seroconverted (tested positive for E. canis antibodies).
Methods for calculating the Ehrlichia canis blocking efficacy
The blocking efficacy per infected animal considers the number of animals that were successfully infected. Blocking efficacy per infected animal for the treatment group was calculated as follows:
$$ \mathrm{Blocking}\ \mathrm{efficacy}\ \left(\%\right)=100\times \left(\mathrm{T}\mathrm{c}-\mathrm{T}\mathrm{t}\right)/\mathrm{T}\mathrm{c} $$
where Tc = Total number of infected dogs in the negative control group and Tt = Total number of infected dogs in the respective treatment group.
Whilst this is the simplest calculation method, the underlying assumption is that all animals were exposed to the same challenge pressure, which is not always the case.
Navarro et al. (2015) [32] first employed the number of infective challenges in their calculation method, as deduced from one of the result tables. Jongejan et al. (2015) [23] first defined a formula for this calculation method, that considers not solely number of infected animals but calculates the percentage of protection in comparison to the number of infective challenges.
Percentage of protection was defined and calculated as follows:
$$ \mathrm{Protection}\ \left(\%\right)=100\kern0.5em \times \kern0.5em \left(\mathrm{I}\mathrm{c}\mathrm{C}-\mathrm{I}\mathrm{c}\mathrm{T}\right)/\mathrm{I}\mathrm{c}\mathrm{C} $$
where IcC = the “infection proportion” calculated as the number of infected animals in the control group divided by the total number of pre-infection challenges with ticks from a batch infected with E. canis in the control group and IcT = the “infection proportion” calculated as the number of infected animals in the respective treatment groups divided by the total number of pre-infection challenges with ticks from a batch infected with E. canis in the respective treatment groups.