Large genetic diversity of Blastocystis and identification of novel subtype 50 in farmed red deer (Cervus elaphus) from southern Spain

Blastocystis is a common intestinal protist characterised by remarkable genetic diversity and a broad host range; however, its prevalence and subtype distribution in farmed wild red deer remain poorly understood. A total of 319 faecal samples collected from 247 farmed red deer in southern Spain (2012–2014) were analysed. DNA was extracted and screened for Blastocystis by PCR targeting the small subunit ribosomal RNA ( ssu rRNA) gene followed by Sanger sequencing. Positive and suspected samples were further characterised by next-generation amplicon sequencing, while selected samples harbouring potentially novel subtypes were analysed by full-length ssu rRNA gene sequencing using Oxford Nanopore technology. Phylogenetic and pairwise distance analyses were performed to validate novel subtypes, and prevalence estimates and associated factors were assessed using appropriate statistical tests. The overall prevalence of Blastocystis in the farmed red deer analysed was 14.7% (47/319; 95% CI 11.0–19.1). Thirteen previously described subtypes (ST5, ST10, ST14, ST21, ST23–ST26, ST30, ST32, ST42–ST44) and one novel subtype (ST50) were identified. Mixed colonisations were common and genetically diverse, comprising up to 44 unique subtype combinations, and high intra-subtype genetic variability was observed. Longitudinal analysis of five deer revealed both persistent and intermittent colonisation patterns across different subtypes. ST10a, ST14, ST21, ST25, ST43, and ST44 were present in 90–100% of the samples longitudinally analysed. The validity of the novel subtype ST50 and the subgroups ST10g and ST24d was confirmed via phylogenetic and pairwise distance analyses of full-length ssu rRNA gene sequences obtained using Oxford Nanopore sequencing. These findings underscore the extensive diversity of Blastocystis in farmed wild red deer and suggest potential persistent colonisation and complex transmission dynamics. They also raise important questions regarding potential zoonotic transmission and the ecological role of Blastocystis within the cervid gut microbiome.