Temporal changes in tick-borne pathogens prevalence observed in questing Ixodes ricinus (Linnaeus, 1758) across different habitat types in north-eastern Italian Alps

Changes in land use, climate, and host community lead to increased complexity in ecoepidemiological relationships and zoonotic emergence. This study investigates the changes in the prevalence of Ixodes ricinus-transmitted pathogens in questing ticks over a 10-year interval in natural and agricultural habitats of the Autonomous Province of Trento (North-eastern Alps). We investigated three study sites classified as natural and agricultural by collecting questing ticks in 2011-2013, 2020 by tick-dragging along 100 m transects. The collected ticks were morphologically identified, while bacteria and protozoa were detected using PCR-based methods combined with sequencing. We investigated the effect of habitat type, tick stage and year on the proportion of infected ticks by using Generalized Linear Models (GLMs) and Two Proportion Z-test. In total 2652 ticks belonging to I. ricinus were collected and analysed. We found an overall prevalence of 27.1% in the study area, referred to Borrelia spp. (21.1%), Rickettsia spp. (8.4%) Anaplasma phagocytophilum (1.6%) and Babesia spp. (0.4%). We reported the circulation of 11 different zoonotic pathogen species, with varying infection rates across different years and habitats. In 2020, A. phagocytophilum, associated with agricultural habitats decreased, while Babesia spp. was found in all habitats. Among Borrelia burgdorferi s.l. complex, only B. burgdorferi s.s. and B. lusitaniae showed significant relationships in natural habitats, increasing in 2020. Rickettsia spp. did not show any significant habitat preference. Co-infections were identified in 8% of positivetested ticks with different spatio-temporal associations, primarily in natural settings. Our results provide new evidences that the risk of infection with tick-borne pathogens in the Alpine region varies over time and in different environments, broadening the current information on coinfection rates and circulation of zoonotic pathogens, previously not reported in this area.