Molecular Evidence of the Role of the Red Fox (Vulpes vulpes) in the Epidemiology of Ungulate-Related Sarcocystis Species in Croatia, Lithuania, and Portugal

<i>Sarcocystis</i> spp. (Apicomplexa: Sarcocystidae) are globally distributed protozoan parasites with an obligatory two-host prey-predator life cycle involving intermediate (IHs) and definitive hosts (DHs). Canids, including the red fox (<i>Vulpes vulpes</i>), often serve as DHs for species infecting various ungulates. This study identified <i>Sarcocystis</i> species in red foxes from Croatia, Lithuania, and Portugal. Between 2021 and 2024, 164 faecal samples (80 from Croatia, 50 from Portugal, and 34 from Lithuania) were analysed using nested PCR targeting the <i>cox1</i> gene. Twelve <i>Sarcocystis</i> species were detected: <i>S. arieticanis</i>, <i>S. capracanis</i>, <i>S. capreolicanis</i>, <i>S. cruzi</i>, <i>S. gracilis</i>, <i>S. hjorti</i>, <i>S. iberica</i>, <i>S. linearis</i>, <i>S. miescheriana</i>, <i>S. morae</i>, <i>S. rossii</i>, and <i>S. tenella</i>. The overall prevalence was highest in Croatia (78%) and Lithuania (62%) and lowest in Portugal (30%). Phylogenetic and haplotype analyses revealed high homogeneity and absence of geographic structuring. These results confirm the red fox as a key DH for multiple <i>Sarcocystis</i> species infecting European ungulates and underscore its epidemiological importance in parasite transmission across diverse ecosystems.