Blastocystis in free-ranging wild ruminant species across the Iberian Peninsula

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.