, 2014 and Shoop et al., 2014) against C. felis in dogs. Both studies were conducted with thirty-two healthy beagles of both sexes. The dogs in Study 1 included twenty-two males and ten females over twelve months of age which weighed between 9.1 and 19.1 kg. The dogs allocated to Study 2 were twelve males and twenty females over

6 months of age which weighed between 8.2 kg and 19.6 kg. The protocol see more of the studies was reviewed and approved by the Merial Institutional Animal Care and Use Committee. Dogs were handled with due regard for their welfare (USDA, 2008). All animals were housed individually. All dogs received commercial food, once daily, in a sufficient amount to maintain a healthy physical state, and water was provided ad libitum. The Everolimus dogs were not treated with ectoparasiticides (either topical or systemic) within three months prior to the start of the study. Dogs enrolled in the studies underwent a full physical examination by a veterinarian on Day −7 and

were examined once daily for health observations. The study designs were in accordance with the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestation on dogs and cats (Marchiondo et al., 2013), and was conducted in accordance with Good Clinical Practices, (VICH guideline GL9) (EMEA, 2000). The studies were blinded, negative-controlled clinical efficacy studies, utilizing a block design based on Day −6 flea counts. In each study, dogs were infested with 100 (±5) unfed adult C. felis on Day −7, which were removed and counted on Day −6. The C. felis strain used for infestations in Study 1 was a U.S. strain of second fleas that has been maintained for approximately 11 years from wild fleas captured in California. In Study 2, a flea strain that originated from Hanover University, Germany, was used. The experimental study unit was the individual dog, which was identified, treated and

assessed on an individual basis. In each study, the 32 dogs were allocated to 8 blocks of 4 animals each. Within each block, the dogs were randomly assigned to one of the 2 counting time-by-treatment combinations (i.e., control 12-h (Groups 1), treated 12-h (Groups 2), control 24-h (Groups 3) and treated 24-h (Groups 4). In each study, dogs were infested with 100 (±5) unfed adult C. felis once on Days −1, 7, 14, 21, 28 and 35. Fleas were removed, counted and categorized as dead/alive 12 h ± 30 min after treatment or challenge infestation for Groups 1 and 2 and 24 ± 1 h after treatment or challenge infestation for treatment Groups 3 and 4. Each dog was combed for a minimum of 10 min using a fine-toothed flea comb. If fleas were found during these 10 minutes, then the dog was combed for 15 min.