Turning urban wildlife mortality into a surveillance tool: Detection of vector-borne pathogens in carcasses of hedgehogs, squirrels, and blackbirds.

Tick-borne zoonoses pose a major challenge to human and animal health, driving efforts to monitor the distribution, intensity, and diversity of their causative agents. Within the One Health framework, which links human, animal, and environmental health, integrated surveillance strategies are increasingly needed. However, most studies focus on tick vectors, while vertebrate reservoirs are often overlooked due to labour-intensive sampling, the need for specialized skills, and legislative or species protection constraints. This study evaluated whether carcasses of accidentally killed wildlife (primarily roadkill) can serve as a source of biological material for vector-borne pathogen surveillance, with a focus on urban habitats due to their public health relevance. Hedgehogs, squirrels, and blackbirds were selected as synanthropic species that thrive in cities, are commonly infested by ticks, and act as hosts for zoonotic tick-borne pathogens (TBPs). A total of 268 carcasses (125 hedgehogs, 55 squirrels, and 88 blackbirds) were collected across multiple Czech cities with public assistance. Overall, 1836 tissue samples were analyzed using multiplex real-time PCR assays targeting over ten microorganisms. Detection efficiency was compared across tissues, with ear and skin consistently the most reliable and versatile sample types. Individual pathogen-host-tissue combinations reached 65-93% efficiency, highlighting the value of multi-tissue sampling. The most prevalent TBPs detected were