Flea infestation and assessment

The C. felis used in the study were from a locally maintained laboratory colony of European origin. The colony was initiated in 2010 with fleas from Germany. Additional fleas obtained from Ireland were introduced into the colony in 2012 (approximately two years prior to the study). Flea infestations were performed on Days -7 (host suitability and allocation), -1, 14, 29, 44, 59, 74, and 90. At each infestation a pre-counted aliquot of 100 (±5) adult, unfed C. felis were directly applied to the animal which was gently restrained for a few minutes to allow the fleas to penetrate and disperse into the hair coat. Each dog was examined and combed to remove and count fleas at 24 h after the initial host suitability infestation, and at 8, 12, and 24 (±1) hours after treatment and each subsequent weekly re-infestation. Fleas were replaced on the dogs immediately after each 8 and 12 h evaluation, and discarded after the 24 h counts.

Flea counts were performed by personnel trained in the standard procedures in use at the test facility. Commercial fine-toothed flea combs were used. Dogs were combed using repeated strokes initially while standing, starting from the head, then proceeding caudally along the dorsum. The dog was then placed on each side and then on its back for combing of the sides and ventral surfaces. After a few combing strokes were completed, the comb was examined and hair and fleas were removed from the comb and all live fleas were counted. Dogs were repeatedly combed for a minimum of 10 min; if any fleas were recovered in the last minute, combing was continued in one-minute increments until no fleas were detected.