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The expansion of urbanization in natural environments increases interactions between wildlife, domestic animals, and humans. In Italy, the red fox (Vulpes vulpes) is one of the most common wild carnivores. This species can serve as a reservoir and sentinel host for several infectious diseases. We aimed to improve knowledge about the exposure of red foxes to selected zoonotic (Anaplasma spp, Ehrlichia spp., Borrelia spp., and hepatitis E virus) and carnivore-specific pathogens (canine parvovirus, canine distemper virus, pseudorabies virus, and Dirofilaria spp.) through a retrospective survey performed in the Tuscany region during the spring season of 2013. Using specific ELISAs and serum samples (n = 38) collected during a culling campaign, a prevalence of 2.6% for canine distemper virus, 18.4% for canine parvovirus, 5.2% for Anaplasma spp., 2.6% for Ehrlichia spp., 7.9% for Dirofilaria spp., 21.05% for hepatitis E virus, and 10.5% for pseudorabies virus was observed. Conversely, antibodies against Borrelia spp. were not identified in any of the animals. Our results revealed no significant sex-related differences in seroprevalence and confirmed hepatitis E virus as the most common pathogen in the analyzed samples. All of the animals that tested positive for tick-borne zoonotic agents presented ticks at the time of sampling. Our study confirms the exposure of red foxes in the Tuscany region to viral and bacterial infections raising medical and veterinary concern and indicating the need for large-scale surveillance to fully assess the epidemiological significance of these findings. [ABSTRACT FROM AUTHOR]