A novel 3R-compliant ex vivo approach to assess ectoparasiticide repellency against Phlebotomus perniciosus in dogs

Ectoparasiticides are essential for preventing insect bites and transmission of arthropod-borne pathogens such as Leishmania spp. Traditionally, their anti-feeding efficacy is evaluated in vivo using sedated dogs exposed to sand flies. To comply with 3R principles (replacement, reduction and refinement) and to avoid animal exposure, this study aimed to develop an ex vivo feeding model to assess the efficacy of a dinotefuran, permethrin, and pyriproxyfen (DPP) [Vectra ® 3D] combination against Phlebotomus perniciosus as an alternative to in vivo testing. Twelve dogs were assigned to either a DPP-treated group ( n = 6) or an untreated control group ( n = 6). On days -7, 1, 7, 14, 21, and 28, dogs were sedated and exposed for one hour to 50 (±5) female sand flies. In parallel, hair collected from each dog was used in an ex vivo artificial feeding system. After exposure, sand flies were assessed for feeding and survival rates. Anti-feeding efficacy was the primary criterion, while insecticidal efficacy was evaluated as a secondary outcome. In both models, control groups showed feeding and survival rates above 31% and 90%, confirming a reliable challenge. The treated group showed significantly lower feeding and survival rates than the control group ( p < 0.05). In both models, DPP demonstrated a strong anti-feeding effect in dogs for one month, with efficacy above 80%. This first direct comparison of in vivo and ex vivo models confirms DPP’s fast and lasting protection against Ph. perniciosus and positions the ex vivo model as a promising 3R-aligned alternative for studying vector-borne disease prevention.