Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.

The growing wild ungulate populations across Europe represents an increasingly important source for the spread of zoonotic pathogens. Blastocystis is a common intestinal protist observed in humans and animals worldwide. Studies on Blastocystis occurrence and subtype (ST) diversity in free-ranging wild ruminants are lacking globally, and more data are needed to understand the epidemiological scenario in wild European ruminants. We collected 833 faecal samples from free-ranging wild ungulates across Spain (n = 699) and Portugal (n = 134) between 1998 and 2021. Using conventional PCR and next-generation amplicon sequencing, Blastocystis was found in 13.8% (115/833; 95% CI: 11.5–16.3) of the wild ruminants analysed. Its occurrence was significantly higher in Portugal (38.1%, 51/134; 95% CI 29.8–46.8) than in Spain (9.2%, 64/699; 95% CI: 7.1–11.5). Fifteen Blastocystis STs, fourteen previously recognised (ST2, ST5, ST10, ST13, ST14, ST21, ST23–ST26, ST30, and ST42–ST44), and one novel (named ST49), were detected among the surveyed wild ruminant populations. Novel ST49 was described using Oxford Nanopore sequencing to produce full-length reference sequences of the small subunit ribosomal RNA gene. A greater ST diversity was observed in Spanish samples. Mixed infections were found in 58.3% (67/115) of the total Blastocystis-positive samples. Our results have enhanced the knowledge regarding Blastocystis occurrence and ST diversity and host preference present in wild ruminants from the Iberian Peninsula, which will assist in clarifying the relationships between the sylvatic and domestic cycles of this protist and may ultimately provide tools to help manage future public health epidemiological scenarios.

The Iberian lynx (Lynx pardinus) is one of the most endangered felid species in the world. Conservation efforts have increased its population size and distribution and reinforced their genetic diversity through captive breeding and reintroduction programmes. Among several threats that the Iberian lynx faces, infectious and parasitic diseases have underlined effects on the health of their newly reintroduced populations, being essential to identify the primary sources of these agents and assess populations health status. To achieve this, 79 fresh faecal samples from Iberian lynx and sympatric mesocarnivores were collected in the reintroduction area of Extremadura, Spain. Samples were submitted to copromicroscopic analyses to assess parasite diversity, prevalence, and mean intensity of parasite burden. Overall, 19 (24.1%, ±15.1–35.0) samples were positive for at least one enteric parasite species. Parasite diversity and prevalence were higher in the Iberian lynx (43.8%) compared with the others mesocarnivores under study (e.g., the red fox Vulpes vulpes and the Egyptian mongoose Herpestes ichneumon). Ancylostomatidae and Toxocara cati were the most prevalent (15.6%) parasites. Obtained results revealed that Iberian lynx role as predator control might have reduced parasite cross-transmission between this felid and mesocarnivores due to their decreasing abundances. Surveillance programs must include regular monitoring of this endangered felid, comprising mesocarnivores, but also domestic/feral and wild cat communities.