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 Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Bartonella spp., and Rickettsia helvetica. In conclusion, carcasses of accidentally killed urban wildlife provide a practical and valuable resource for TBP surveillance, complementing vector-focused methods. This approach supports One Health principles by integrating wildlife monitoring into urban disease surveillance efforts.

Centre for Monitoring of Vectors Netherlands Institute for Vectors Invasive plants and Plant health Netherlands Food and Consumer Product Safety Authority Geertjesweg 15 6706 EA Wageningen the Netherlands

Centre for Zoonoses and Environmental Microbiology Centre for Infectious Disease Control National Institute for Public Health and the Environment Antonie van Leeuwenhoeklaan 9 3721 MA Bilthoven the Netherlands

Department of Botany and Zoology Faculty of Science Masaryk University Kotlarska 2 611 37 Brno Czech Republic

Department of Data Science and Computing Systems Faculty of Agriculture and Technology University of South Bohemia Studentska 1668 370 05 Ceske Budejovice Czech Republic

Department of Parasitology Faculty of Science Charles University Vinicna 7 128 00 Prague Czech Republic

Department of Veterinary Sciences Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences Prague Kamycka 129 165 00 Prague Czech Republic

Department of Zoology Faculty of Natural Sciences Comenius University in Bratislava Ilkovicova 6 842 15 Bratislava Slovakia

Department of Zoology Faculty of Science Charles University Vinicna 7 128 00 Prague Czech Republic

Faculty of Tropical AgriSciences Czech University of Life Sciences Prague Kamycka 129 165 00 Prague Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences Branisovska 31 370 05 Ceske Budejovice Czech Republic

State Veterinary Institute Prague Sidlistni 24 165 03 Prague Czech Republic

University of Ostrava Dvorakova 7 701 03 Ostrava Czech Republic

Veterinary Research Institute Hudcova 70 621 00 Brno Czech Republic

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