Serological and molecular insights into tick-borne pathogens in wild donkeys from an unexplored Mediterranean nature reserve.

The Island of Asinara (Sardinia, Italy) is a unique protected ecosystem in the Mediterranean basin. Its climate provides a favorable environment for a wide variety of flora and fauna, including ticks and tick-borne pathogens (TBPs) of medical and veterinary importance. In this context, wild donkeys (Equus asinus), i.e. the Sardinian grey donkey and the characteristic white Asinara donkey, serve as hosts for several tick species. This study aimed to increase the knowledge of the TBPs circulating on the Island of Asinara by assessing the seroprevalence and risk factors for Babesia caballi and Theileria equi infections in donkeys and the molecular prevalence of the aetiological agents of piroplasmosis, anaplasmosis, and rickettsiosis in ticks collected from donkeys. In this study, 110 wild donkeys and 197 adult ticks (Rhipicephalus bursa, Haemaphysalis punctata, Hyalomma marginatum) from the Asinara National Park were tested for the presence of TBPs. Serological analysis in donkeys revealed a seroprevalence of 23.6% for B. caballi and 39.1% for T. equi, with 11.8% of individuals showing co-infections. The likelihood of protozoan infection was associated with tick infestation for B. caballi and land cover type for T. equi. Molecular analysis of ticks detected T. equi in 15.7% of the samples and Babesia spp. in 3.5% of the samples. Rickettsia spp., including the zoonotic R. aeschlimannii, were identified in 8.1% of the ticks. No Anaplasma spp. positives were detected. In addition, 2% of ticks showed co-infections with multiple pathogens, highlighting the potential for co-transmission. An association with tick species and sex was demonstrated for Rickettia spp. infection. These findings confirm the circulation of piroplasms and zoonotic agents within the Asinara National Park, underscoring potential health risks for both animals and humans, particularly in a tourist-attractive area, and thus emphasize the need for integrated "One Health" surveillance strategies to monitor and manage TBPs.