S. Theodore Chester

The prevention of transmission of Babesia canis canis by Dermacentor reticulatus ticks to dogs using a novel combination of fipronil, amitraz and (S)-methoprene.

Four groups of seven dogs were treated topically with a novel combination of fipronil, amitraz and (S)-methoprene in a spot-on formulation (CERTIFECT™, Merial Limited, GA, USA) on 28, 21, 14 and 7 days prior to tick infestation, respectively and acaricidal efficacy and transmission blocking compared with an untreated control group (seven dogs). All dogs were infested with adult Dermacentor reticulatus ticks harbouring Babesia canis canis. Babesia canis canis was transmitted by D. reticulatus to all seven untreated control dogs, confirmed following demonstration of clinical signs, by the detection of B. canis parasites in thin blood smears and B. canis canis PCR-RLB DNA assay on blood and the development of B. canis canis antibody titres by 14-21 days after tick infestation. The majority of treated dogs remained sero-negative for 42 days after infestation. Therefore, the treatment of dogs with CERTIFECT applied up to 28 days prior to infestation with D. reticulatus harbouring B. canis canis, successfully prevented the development of clinical signs of canine babesiosis.

The ability of a topical novel combination of fipronil, amitraz and (S)-methoprene to protect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infections transmitted by Ixodes scapularis.

Healthy, purpose-bred laboratory beagle dogs that had not been exposed to ticks and were seronegative for Borrelia burgdorferi and Anaplasma phagocytophilum were randomly assigned to four groups of eight dogs each. Control group 1 was not treated. Groups 2, 3 and 4 were treated with a single topical application of a new formulation of fipronil, amitraz and (S)-methoprene (CERTIFECT™, Merial Limited, GA, USA) at 28, 21 or 14 days prior to tick infestation, respectively. Each dog was infested with 25 female and 25 male field-collected adult Ixodes scapularis ticks that had infection rates of 66% for B. burgdorferi sensu stricto and 23% for A. phagocytophilum, as determined by polymerase chain reaction. Two and five days after tick infestation, control dogs had an average of 9.5 and 13.9 attached adult female ticks, respectively, whilst the 24 treated dogs remained tick-free aside from a single tick on the 2nd day after infestation. Serial serological tests demonstrated that the ticks successfully infected 8/8 control dogs with B. burgdorferi and co-infected 6/8 with A. phagocytophilum. B. burgdorferi infection also was confirmed in most control dogs by culture (6/8) and PCR (7/8) of skin biopsies. In contrast, CERTIFECT protected all 24 treated dogs against infection by both B. burgdorferi and A. phagocytophilum, as demonstrated by their negative serological tests throughout the study and the absence of any positive skin biopsy culture or PCR in these dogs.

Efficacy of afoxolaner against Ixodes scapularis ticks in dogs.

Efficacy of afoxolaner, a novel isoxazoline insecticide/acaricide, against Ixodes scapularis was evaluated in a laboratory study. One day prior to treatment, beagle dogs (n=16) were infested with 50 unfed wild adult ticks. Repeat infestations were performed weekly for four additional weeks. The number of live ticks remaining on each dog was determined 48 h after treatment and after each subsequent infestation. A single oral treatment with a dose approaching the minimum effective dose of afoxolaner (2.5mg/kg) eliminated the pre-existing infestations of I. scapularis ticks and controlled weekly re-infestations, with efficacy between 98% and 100% recorded until Day 23 and 94% at Day 30.

Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against larval and adult stages of the cat lungworm, Aelurostrongylus abstrusus

The efficacy of a novel topical combination of fipronil 8.3% w/v, (S)-methoprene 10% w/v, eprinomectin 0.4% w/v, and praziquantel 8.3% w/v (BROADLINE(®),(1) Merial) against larval and adult Aelurostrongylus abstrusus lungworms in cats was assessed in a controlled laboratory study. The study included 48 purpose-bred, short-haired cats which were each inoculated with 225 infective A. abstrusus larvae. The cats were formed into eight blocks based on pre-treatment bodyweight and were then, within each block, randomly allocated to one of six treatment groups: untreated control; treated once when A. abstrusus were expected to be third-stage larvae (4 days post inoculation [dpi]), fourth-stage larvae (7 dpi), immature adults (14 dpi) or adult nematodes (32 dpi), or treated twice, once when A. abstrusus were expected to be third-stage larval and once again when A. abstrusus were expected to be adult nematodes (4 dpi+32 dpi). Cats weighing ≥ 0.8-2.5 kg received one 0.3 mL applicator and cats weighing >2.5-7.5 kg received one 0.9 mL applicator. For determination of the efficacy of treatments, lungworm larval counts were established on faecal samples collected from all cats 32, 39, 46, 53 and 60 dpi. At each occasion from 46 dpi on, cats treated with fipronil, (S)-methoprene, eprinomectin and praziquantel had significantly lower A. abstrusus larval counts than the untreated controls with percentage reductions of 91.6% (cats treated 14dpi; P=0.012), ≥ 98.9% (cats treated either 4 dpi, 7 dpi or 32 dpi; P<0.001) or >99.9% (cats treated 4 dpi+32 dpi; P<0.001) at 60 dpi. Thus, the novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel was highly effective in the prevention and treatment of A. abstrusus lungworm infection in cats.

The combined mode of action of fipronil and amitraz on the motility of Rhipicephalus sanguineus.

The motility of adult Rhipicephalus sanguineus was evaluated subsequent to treatments of amitraz, fipronil and the combination of fipronil plus amitraz against a vehicle control in a Petri dish assay using the LemnaTec Scanalyzer Imaging System. The assay was run using a fixed dilution of amitraz (0.32μg/cm(2)); serial dilutions of fipronil (1.3, 0.33, 0.08, 0.02, or 0.005μg/cm(2)); and the same serial dilutions of fipronil in combination with the fixed dilution of amitraz. Measurement of motility was made of unstimulated ticks and then after stimulation at 1, 4, 18-22, and 24h post exposure (hpe) of the Petri dishes. For the unstimulated ticks, there was no difference in motility between the amitraz treatment group and the fipronil plus amitraz treatment group at the early time points. However, these two treatment groups had significantly higher motility than the solvent control and fipronil treatment groups. The unstimulated ticks in the amitraz treatment group had significantly higher motility than the fipronil plus amitraz treatment group at the later time points. Measurements after stimulation demonstrated there was no difference in motility between the amitraz treatment group and the fipronil plus amitraz treatment group at the early time points. By 18 hpe, the fipronil plus amitraz treatment group had significantly lower motility than all other treatment groups and at 21-22 and 24 hpe the other treatment groups did not differ from the control group. The action could be divided in two phases in the combination experiment: phase 1: an early increase in motility due to amitraz is identified in both amitraz alone or fipronil plus amitraz groups; phase 2: the combination of fipronil plus amitraz caused a significantly greater reduction in motility, suggesting mortality of the ticks, compared to fipronil or amitraz alone. These results demonstrate a synergism resulting from the combination of fipronil plus amitraz.

Efficacy of afoxolaner plus milbemycin oxime chewable tablets against naturally acquired intestinal nematodes in dogs

The efficacy of oral afoxolaner plus milbemycin oxime combination chewable tablets (NexGard Spectra, Merial) against naturally acquired intestinal nematode infections in dogs was evaluated in six negative control, blinded studies including a total of 114 dogs. Dogs were selected based on a pre-treatment fecal examination indicating patent infections with hookworms (two studies), Toxocara or Toxascaris ascarids (one study each) or Trichuris whipworms (two studies). In each study, dogs were assigned to blocks of two animals each, based on decreasing pre-treatment body weight and were randomly allocated to one of two groups consisting of eight, nine or 10 dogs: untreated (control) or treated with the combination chewable tablet formulation. Chewable tablets were combined to provide doses of actives as close as possible to the minimum effective dose of afoxolaner and milbemycin oxime, i.e., 2.5 mg/kg body weight and 0.5 mg/kg body weight, respectively, once on Day 0. For parasite recovery and count, dogs were euthanized humanely and necropsied seven or eight days after treatment. A single treatment with afoxolaner plus milbemycin oxime chewable tablets provided 94.8% and 90.9% efficacy against adult Ancylostoma braziliense and A. caninum, respectively, 97.8% and 99.4% efficacy against adult Toxocara canis and Toxascaris leonina, respectively, and ≥98.3% efficacy against adult Trichuris vulpis. Compared to untreated controls, nematode counts of the treated dogs were significantly reduced (F-test; p<0.002). In addition, analysis of the pooled data across studies revealed that treatment with afoxolaner plus milbemycin oxime chewable tablets reduced adult Uncinaria stenocephala burdens by 74.9% (p=0.002). All dogs tolerated the treatment well based on clinical observations post-treatment and daily clinical observations. No adverse experiences or other clinical problems related to the treatment were observed throughout the studies. The results of this series of controlled studies demonstrated high efficacy and excellent acceptability and safety of the afoxolaner plus milbemycin oxime chewable tablets when administered for treatment of a broad range of canine intestinal nematode infections.

Efficacy of a novel topical fipronil, (S)-methoprene, eprinomectin and praziquantel combination against naturally acquired intestinal nematode and cestode infections in cats.

The efficacy of a novel topical combination formulation of fipronil, (S)-methoprene, eprinomectin and praziquantel against naturally acquired intestinal nematode and cestode infections in cats was evaluated in seven negative control, blinded studies. Cats were selected based on a pre-treatment faecal examination indicating a patent infection with at least hookworms (two studies), Toxocara ascarids (one study), taeniid cestodes (two studies) or Dipylidium cestodes (two studies). In each study, cats were assigned randomly to blocks of two animals each, based on decreasing pre-treatment body weight and were randomly allocated to one of two groups of six to 12 cats: untreated (control) or treated with topical fipronil (8.3%, w/v), (S)-methoprene (10%, w/v), eprinomectin (0.4%, w/v) and praziquantel (8.3%, w/v) (BROADLINE(®), Merial) at 0.12 mL/kg body weight (providing a minimum of 10mg fipronil+12 mg S-methoprene+0.5mg eprinomectin+10mg praziquantel per kg body weight). The topical treatment was administered directly on the skin in the midline of the neck in a single spot once on Day 0. For parasite recovery and count, cats were euthanized humanely and necropsied seven or ten days after treatment. A single treatment with the novel topical combination product provided 91% efficacy against Ancylostoma braziliense, ≥ 99% efficacy against Ancylostoma tubaeforme, and >97% efficacy against Toxocara cati. Similarly, excellent efficacy was established against Taenia taeniaeformis, Dipylidium caninum and Diplopylidium spp. as demonstrated by >97% and up to 100% reductions of cestode counts in the treated cats when compared to the untreated controls (P<0.01). All cats accepted the treatment well based on health observations post-treatment and daily health observations. No adverse experiences or other health problems were observed throughout the studies. The results of this series of controlled studies demonstrated high efficacy and excellent acceptability of the novel topical combination formulation of fipronil, (S)-methoprene, eprinomectin and praziquantel against a broad range of feline intestinal nematode and cestode infections.

Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against adult and larval stages of Toxocara cati in cats

The efficacy of a novel topical combination of fipronil 8.3% (w/v), (S)-methoprene 10% (w/v), eprinomectin 0.4% (w/v), and praziquantel 8.3% (w/v) (BROADLINE(®), Merial) was evaluated against adult and larval Toxocara cati in four controlled studies. All studies included experimentally infected, purpose-bred, short-haired cats. In two studies, 22 or 20 cats harbouring patent infections as confirmed by pre-treatment faecal examination, were included. Within each study, cats were allocated to one of two groups: control or treated. In a further two studies, 30 cats were included in each; cats were allocated to one of three groups: control, treated when T. cati were expected to be either migrating third and/or fourth-stage larvae, or treated when T. cati were expected to be fourth-stage larvae. Cats allocated to the treated groups received a single topical application of the combination product at 0.12 mL/kg bodyweight (10mg fipronil+12 mg (S)-methoprene+0.5mg eprinomectin+10mg praziquantel per kg). For parasite recovery and count, cats were euthanized humanely at different intervals after treatment. In the studies targeting adult T. cati, ascarids were recovered from all controls (range 1-150) while only two worms were isolated from one treated cat. Thus, the efficacy of the novel combination was 99.4% and 100% against adult T. cati. For studies targeting larval T. cati, up to 21 worms were recovered from each of seven or eight of the control cats per study. No T. cati were recovered from the treated cats in two studies, corresponding to 100% efficacy against both, migrating third and/or fourth-stage larvae and luminal fourth-stage larvae. All cats accepted the treatment well and no adverse experiences or other health problems were observed throughout the studies.

Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against adult and immature stages of the cat flea (Ctenocephalides felis) on cats

The efficacy of a novel topical combination of fipronil 8.3% (w/v), (S)-methoprene 10% (w/v), eprinomectin 0.4% (w/v) and praziquantel 8.3% (w/v) (BROADLINE(®)) was tested against adult and immature stages of Ctenocephalides felis fleas in six studies. For that purpose, fleas from different colonies from North America, Germany and South Africa were used to induce infestations in cats under laboratory conditions. In each study, between 12 and 16 cats were allocated randomly to 2 groups. Cats in Group 1 were not treated and served as controls. Cats in Group 2 were treated once on Day 0 with BROADLINE(®) at the minimum recommended dosage of 0.12 mg/kg body weight. In 4 studies, all animals were infested experimentally with unfed C. felis (100 ± 5) on Days 2 (or 1), 7, 14, 21, 28 and 35. Live fleas were counted 24h post-treatment or infestation. In 2 additional studies, animals were infested at the same frequency with gravid C. felis fleas (100 ± 5) that were fed previously on an untreated host. Forty-eight hours post-infestation, flea eggs were collected, counted and incubated for the evaluation of the reduction of emergence of adults. The combined curative efficacy against adult fleas at 24h after treatment was 94.3% and the combined preventive efficacy values remained greater than 95.9% at 24h after 5 subsequent weekly infestations. In addition, the product reduced dramatically the emergence of new adult fleas for at least 5 weeks (>98.1% for one month and 93.2% at 5 weeks after infestation), demonstrating its efficiency in preventing environmental contamination by immature stages.

Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against induced infections of Ancylostoma spp. nematodes of cats

Four studies were conducted to examine the efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin, and praziquantel (BROADLINE(®), Merial) against Ancylostoma tubaeforme and Ancylostoma braziliense hookworms of cats. In each study, purpose-bred cats were randomly assigned to treatment groups of 10 or 12 cats per group. In three studies the cats were inoculated with A. tubaeforme and in one study with A. braziliense. The inoculations were undertaken on a schedule which resulted in the hookworms reaching the fourth larval stage in two of the studies, or the adult stage in four of the studies, by the day of treatment. In each study there was also an untreated control and 1 or 2 groups treated with the novel combination. In the two studies where efficacy against the fourth larval stage of A. tubaeforme was tested, the efficacy recorded was 100%. In the three studies where efficacy against the adult stage of A. tubaeforme was tested, efficacy of 100% was also confirmed. In the study where efficacy against the adult stage of A. braziliense was tested efficacy was 99.5%.