Evaluation of oral fluralaner (Bravecto®) for efficacy against nymphs of Amblyomma americanum and Rhipicephalus sanguineus (sensu lato)

Background Amblyomma americanum and Rhipicephalus sanguineus (sensu lato) nymphs commonly feed on and transmit pathogens to dogs (Canis familiaris). Control of immature and adult tick life stages is necessary to fully protect animals. We evaluated efficacy of oral fluralaner (Bravecto®) against induced infestations with A. americanum and R. sanguineus (s.l.) nymphs on dogs in two experiments. Methods In each experiment, 10 dogs were administered oral fluralaner chewable tablets one time on Day 0 at a targeted minimum dose of 25 mg/kg body weight and 10 dogs remained non-treated controls. Dogs were infested with two groups of 50 A. americanum nymphs and two groups of 50 R. sanguineus (s.l.) nymphs on Days -1, 6, 28, 56 and 84. At 48 h and 72 h post-infestation, nymphs were collected from dogs, assessed as live or dead, and enumerated into categories defining attachment and engorgement status. Fluralaner efficacy was determined in separate analyses against all live nymphs and against live-fed nymphs, i.e. live nymphs that were attached to dogs at the time of collection and/or were engorged. Fluralaner was considered effective when mean numbers of live ticks were reduced in fluralaner-treated dogs by ≥ 90%. Results Fluralaner efficacy against all live and live-fed A. americanum nymphs in the first experiment was > 94% on all collection days. Efficacy against all live R. sanguineus (s.l.) nymphs in the first experiment was  > 96% on all collection days excluding the 48 h counts for infestations on Days 28 (83.7%), 56 (82.9%) and 84 (86.7%); efficacy against live-fed R. sanguineus (s.l.) nymphs was > 95% on all 48 h/72 h count days. Fluralaner efficacy against all live A. americanum nymphs in the second experiment was > 93% on all collection days for 8 weeks excluding the 48 h count for infestation on Day 56 (87.8%); efficacy against live-fed A. americanum nymphs was > 91% on all count days for 8 weeks. Efficacy against all live R. sanguineus (s.l.) nymphs in the second experiment was > 91% on all 72 h collection days except for infestations on Days 28 (76.8%) and 56 (86.3%); efficacy against live-fed R. sanguineus (s.l.) nymphs was 100% on all 72 h count days. Conclusions A single administration of oral fluralaner to dogs is effective against A. americanum and R. sanguineus (s.l.) nymphs for up to 12 weeks.


Background
Deleterious effects of tick infestations on animals are numerous. Direct consequences as a result of tick feeding include anemia, dermal irritation, hypersensitivity, and toxicosis. Also, a variety of pathogenic agents are transmitted to animals by ticks during blood-meal acquisition, which may result in illness and death in infested hosts [1][2][3]. Control of immature stages as well as adults is important for complete protection of animals against tick infestations.
Control of immature ticks on dogs as well as adult ticks is important in reducing infestations, decreasing risk of pathogen transmission, and limiting tick populations in the environment. In vivo trials determining acaricidal compound efficacy against immature A. americanum and R. sanguineus (s.l.) are lacking. The isoxazoline fluralaner is a systemic compound that exerts acaricidal and insecticidal activity by blocking gamma-aminobutyric acid (GABA)-and glutamate-gated chloride channels [20]. Oral fluralaner (Bravecto ® ) is indicated for the treatment and control of tick infestations on dogs, including A. americanum for 8 weeks and R. sanguineus (s.l.) for 12 weeks, as demonstrated using adult ticks in both laboratory experiments and field trials [20,21]. Here, we present the results of two Good Clinical Practice (GCP) laboratory experiments evaluating the efficacy of oral fluralaner against A. americanum and R. sanguineus (s.l.) nymphs for 12 weeks after administration of the minimum commercial dose (25 mg/kg body weight).

Methods
The efficacy of 13.64% w/w fluralaner chewable tablets (Bravecto ® , Merck Animal Health, Madison, New Jersey) against A. americanum and R. sanguineus (s.l.) nymphs was evaluated in two separate randomized, blinded laboratory experiments with parallel-group designs. Each experiment was conducted in compliance with Good Clinical Practice (GCP) guidelines (VICH GL9) [22] and the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating parasiticide efficacy against flea and tick infestations on cats and dogs [23]. The duplicate experiments were conducted to document fluralaner efficacy against different genetic isolates of nymphal A. americanum and R. sanguineus (s.l.) to demonstrate the compound's utility in the field against immatures of the species.
Twenty laboratory reared neutered male or intact female beagles, each at ≥ 6 months of age, were used in the two experiments. To reduce the total number of animals used between both experiments, 8 non-treated control dogs from the first experiment were transferred to the second experiment and re-used in control or treated groups along with 12 newly acquired, non-treated dogs. The dogs were initially weighed and physically examined to ensure overall health, and allowed an acclimation period of at least 28 days prior to experiment commencement. Dogs were housed individually or in pairs inside raised kennels with plastic-coated expanded metal floors in designated climate-controlled rooms provided a 12:12 h L:D cycle.
Amblyomma americanum were obtained from colonies maintained by Oklahoma State University (OSU) (Stillwater, Oklahoma) and Texas A&M University (College Station, Texas) and R. sanguineus (s.l.) were from colonies maintained by OSU and El-Labs (Soquel, California) [24]. In both experiments, dogs were infested with 50 R. sanguineus (s.l.) nymphs on Day-28 for treatment group allocation. For all infestations, the hair on the lateral thorax was clipped and nymphs were applied directly to shaved skin beneath bandage pads ( to further contain nymphs and facilitate later recovery and accurate counting. Elizabethan collars (Well & Good E-Collar, Petco, San Diego, California, USA) were placed on dogs as needed to prevent removal of the tick containment materials. When infested with ticks, dogs were housed individually in kennels. After each counting event, we discarded all ticks.
Rhipicephalus sanguineus (s.l.) nymphs applied on Day-28 were removed after 72 ± 4 h (Day-25) and enumerated into the following 8 categories indicating vitality, attachment, and engorgement status: live, attached and engorged; live, attached and not engorged; live, free and engorged; live, free and not engorged; dead, attached and engorged; dead, attached and not engorged; dead, free and engorged; dead, free and not engorged. On all sampling days, nymphs were collected using fine forceps and examined using a stereomicroscope. Live status of ticks was determined by movement of legs. If no movement was observed, nymphs were gently probed with forceps and stimulated with CO 2 (exhaled air) to assess leg movement. Any tick exhibiting leg movement at the time of removal was considered as live. Nymphs were considered attached if mouthparts were partially to completely anchored within the canine dermis at the time of removal. Engorgement status of ticks was determined by inspecting the ventral idiosoma; nymphs that had ingested blood appeared distended and light brown in color while nymphs that had not fed were completely flat and dark in color. Numbers of live and dead nymphs were recorded according to attachment and engorgement status.
Dogs were ranked according to R. sanguineus (s.l.) live-fed nymph counts (i.e. live nymphs categorized as attached and engorged, attached and not engorged, and free and engorged) and animal identification number, then randomly assigned to treatment and control groups. After treatment group allocation, dogs were allowed to co-mingle with the other dogs of the same group. Observations and procedures after animals were randomized, including infestations, tick counts, and tick assessments, were performed by individuals masked to dog treatment status. Sterile gloves, surgical gowns, and shoe-covers were worn when treating and mock-treating dogs, infesting dogs with ticks, and collecting ticks from infested dogs to prevent product cross-contamination between animals. Experiment participants and personnel responsible for husbandry changed protective wear after handling each dog.
Dogs were treated or mock-treated on Day 0 according to assigned group. The number and size of 13.64% w/w fluralaner flavored chewable tablets was determined for each dog by body weight and the targeted minimum dose of 25 mg/kg. Animals were offered a small amount of food (~1/2 cup) and allowed 20 min to eat before treatment. The fluralaner dose was placed in the back of the dog's oral cavity over the tongue to initiate swallowing. Animals were monitored for 1 h after fluralaner administration for adverse reactions and to ensure that doses were retained.
To evaluate fluralaner efficacy against nymphal A. americanum and R. sanguineus (s.l.), dogs were infested 20 ± 2 h prior to and 1 week (Day 6), 4 weeks (Day 28), 8 weeks (Day 56), and 12 weeks (Day 84) after treatment administration. At infestation, each dog was challenged with two groups of 50 A. americanum nymphs and two groups of 50 R. sanguineus (s.l.) nymphs unless otherwise stated in results. Groups of ticks were applied to separate caudal and cranial locations along the lateral thoracoabdominal areas of dogs: left side (A. americanum); right side (R. sanguineus (s.l.)). Nymphs were collected from caudal locations 48 h after infestation and from cranial locations 72 h after infestation. At removal, nymphs were morphologically confirmed as A. americanum or R. sanguineus (s.l.) and enumerated into the aforementioned categories delineating vitality, attachment, and engorgement status.
Fluralaner efficacy assessment SAS version 9.3 was used as the primary software for data analyses [21,25]. Evaluations of fluralaner efficacy against each tick species were considered valid if the number of all live nymphs (attached and engorged, attached and not engorged, free and engorged, and free and not engorged) removed from individual dogs at each collection time was at least 12 ticks on at least 6 control dogs. The log-counts of all live nymphs from treated dogs were compared to the log-counts of all live nymphs from control dogs at each 48 h and 72 h post-infestation collection time. Additionally, the log-counts of live-fed nymphs (live nymphs that were attached to dogs at the time of collection and/ or were engorged) from treated dogs were compared to live-fed nymphs from control dogs at each 48 h and 72 h post-infestation evaluation time. Enumerated livefed nymphs were categorized as attached and engorged, attached and not engorged, and free and engorged (i.e. free and not engorged counts were excluded from the fluralaner efficacy evaluation). A linear mixed model which included treatment group as a fixed effect and block as a random effect was used for comparisons. In case of any zero count, the logarithm of (count + 1) transformation was applied to all observations prior to analysis [22,25].
For both tick species, geometric means of all live nymphs and live-fed nymphs from each count day were calculated; t-tests were performed (P < 0.05). Fluralaner control efficacy (%) was calculated against all live and Table 1 Percent efficacy of fluralaner based on all live and live-fed Amblyomma americanum nymphs from control and treated dogs (Experiment 1). Each group had 10 dogs unless otherwise noted a Only nine dogs available for infestation due to one dog having inflammatory tick attachment site reactions  live-fed nymphs at both post-infestation collection times (48 h and 72 h) using Abbott's formula [23]: where M c is the mean (geometric) live nymph count from the control group and M T is the mean (geometric) live nymph count from the fluralaner-treated group. Fluralaner efficacy against A. americanum and R. sanguineus (s.l.) nymphs was declared when live counts from treated dogs were reduced by ≥ 90% [23].

Results
All Due in part to a bandage breach on some dogs, 48 h R. sanguineus (s.l.) nymph infestations were not adequate in the second experiment and these data were not included or interpreted. When live-fed nymphs were assessed, adequacy of infestation was not achieved for R. sanguineus (s.l.) in the first experiment at the 48 h count on infestation Day 56, and in the second experiment at 72 h counts on infestation Days 28, 56 and 84. Because infestation adequacy was achieved for all live R. sanguineus (s.l.) nymphs on these specific count days however, the corresponding live-fed nymph efficacy data are also reported.

Experiment 1
All live and live-fed A. americanum nymph counts from fluralaner-treated dogs at 48 h and 72 h post-infestation were significantly lower (P < 0.001) than counts from control dogs for all tick challenge days. Fluralaner efficacy against all live and live-fed A. americanum nymphs was > 94% at 48 h and 72 h for all time points evaluated through infestation Day 84 (

Discussion
Although several compounds have been evaluated in vitro for efficacy against larval or nymphal stages of ticks [20,[26][27][28][29][30], reports of in vivo trials in dogs to determine efficacy against immature stages of ticks are sparse [25]. However, limited field trial data from dogs indicate efficacy [30]; data gleaned from in vivo experiments in other animals with other acaricides also indicate efficacy [25,27]. Oral fluralaner (Bravecto ® ) is labeled for the treatment and control of A. americanum for 8 weeks and R. sanguineus (s.l.) for 12 weeks based on experiments using adult ticks [20,21]. Although logical to deduce its efficacy against immature tick stages in the field, acaricidal activity of the systemic compound against nymphal A. americanum and R. sanguineus (s.l.) has not been experimentally demonstrated in vivo until now. Here, we document efficacy of oral fluralaner in dogs against nymphal A. americanum and R. sanguineus (s.l.) in two separate laboratory controlled experiments. Taken together, efficacy data against all live and live-fed ticks in both experiments demonstrated a single administration of oral fluralaner to dogs at the minimum approved dose was effective against A. americanum and R. sanguineus (s.l.) nymphs for as long as 12 weeks. Lower than expected (< 90%) fluralaner efficacy against all live nymphs on some tick challenge days in both experiments could be due to several reasons, or combinations thereof. First, the live unengorged nymphs that were not attached to dogs but entrapped in bandage adhesive were enumerated as live, free and not engorged, and were included in counts of all live nymphs used for efficacy calculations. However, these nymphs apparently had not attached to dogs and thus had not ingested the fluralaner. Feeding is necessary for exposure of ticks to isoxazoline acaricides [20]. This is demonstrated by the fact that, with the exception of Day 86 counts for A. americanum in the second experiment, fluralaner efficacy was restored to ≥ 90% for both species on all count days when geometric means of live-fed nymphs were analyzed. Additionally, the 12-week efficacy maintained against live-fed A. americanum nymphs in the first trial suggests that immature A. americanum may be more susceptible to fluralaner than adults, as has been demonstrated for other tick species in in vitro experiments [20,28].
Another explanation for the occasional decrease in fluralaner efficacy against all live counts on some sampling days in both experiments, as well as Day 86 livefed A. americanum counts in the second experiment, is that cohorts of nymphs used for successive infestations may have taken longer to attach and feed. Thus, these nymphs would have ingested less fluralaner before collection at 48 h and 72 h, and the fluralaner would have had less time to exert its acaricidal effects on nymphs prior to their evaluation. Also, in Experiment 2, the 8 dogs transferred from the first experiment may have developed some level of immunity to infestation, which may have decreased efficiency of tick feeding [31] and potential fluralaner ingestion. Still, despite challenges leading to occasional reductions in fluralaner efficacy, overall data indicated that the compound was highly efficacious against A. americanum and R. sanguineus (s.l.) nymphs.
Control of immature ticks is necessary for complete protection of animals from tick infestations. Amblyomma americanum are aggressive and feed on a variety of animals during all instars [5]. Because of this generalist feeding behavior, A. americanum are exposed to a wide variety of pathogens during all instars and can successfully vector numerous infectious agents to dogs and humans [4,5,9]. Rhipicephalus sanguineus (s.l.), although less diverse in feeding behavior, are well known to establish thriving infestations surrounding and within kennels and homes; infestations with R. sanguineus (s.l.) instars can be difficult to mitigate once established [4]. If R. sanguineus (s.l.) are feeding on dogs that are infected Table 4 Percent efficacy of fluralaner based on all live and live-fed Rhipicephalus sanguineus (sensu lato) nymphs from control and treated dogs (Experiment 2). Each group had 10 dogs with pathogenic agents, this creates a dangerous circumstance for other dogs and humans near or within infested environments [17,19]. The experimental demonstration of long term in vivo acaricidal activity of oral fluralaner against nymphal A. americanum and R. sanguineus (s.l.) indicates the isoxazoline is an appropriate treatment for dogs infested with immature and adult stages of these tick species in the field.

Conclusions
Empirical evidence from two separate GCP-governed laboratory experiments presented here demonstrates that oral fluralaner (Bravecto ® ) is effective in controlling infestations on dogs with A. americanum and R. sanguineus (s.l.) nymphs for as long as 12 weeks, indicating the compound would be efficacious in the treatment of dogs naturally infested with immature stages of these tick species. Eliminating feeding nymphs and adults on dogs will lead to fewer tick instars developing in the environment that will subsequently go on to feed on new hosts, and thus would be expected to reduce the risk of pathogen transmission.