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

## TL;DR

This study shows that red foxes in Europe are important hosts for Sarcocystis parasites that infect wild and domestic ungulates, with new species identified in foxes from Croatia, Lithuania, and Portugal.

## Contribution

The study reports four Sarcocystis species for the first time in red foxes and confirms their role as key definitive hosts across European ecosystems.

## Key findings

- Twelve Sarcocystis species were identified in red fox fecal samples from Croatia, Lithuania, and Portugal.
- Four Sarcocystis species were newly reported in red foxes as definitive hosts.
- Genetic analysis showed high similarity among parasites with no geographic structuring.

## Abstract

Sarcocystis species are globally distributed protozoan parasites with a complex life cycle that requires two hosts. Sarcocysts develop mainly in the muscle tissues of intermediate hosts (prey), whereas oocysts and sporocysts occur in the intestinal tract of definitive hosts (predators and scavengers). One of the most widespread canid predators, the red fox (Vulpes vulpes), commonly acts as a definitive host, while various ungulates serve as intermediate hosts. This study investigated the diversity and prevalence of Sarcocystis species in red foxes from three European countries: Croatia, Lithuania, and Portugal. Overall, 164 faecal samples were analysed using molecular methods and identified using Sanger sequencing. A total of twelve Sarcocystis species were identified in the examined foxes, all using ungulates as intermediate hosts, with four species reported for the first time in the red fox as a definitive host. Genetic analyses indicated high similarity among detected parasites and no clear geographic structuring. These findings confirm the red fox as an important natural definitive host for multiple Sarcocystis species and highlight its role in the transmission of these parasites across different European ecosystems.

Sarcocystis 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 (Vulpes vulpes), often serve as DHs for species infecting various ungulates. This study identified Sarcocystis 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 cox1 gene. Twelve Sarcocystis species were detected: S. arieticanis, S. capracanis, S. capreolicanis, S. cruzi, S. gracilis, S. hjorti, S. iberica, S. linearis, S. miescheriana, S. morae, S. rossii, and S. tenella. 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 Sarcocystis species infecting European ungulates and underscore its epidemiological importance in parasite transmission across diverse ecosystems.