Molecular and Phylogenetic Characterization of Onchocerca flexuosa in Red Deer from South-Eastern Europe

Onchocerca flexuosa is a vector-borne filarial nematode infecting red deer (Cervus elaphus) throughout Europe. Despite numerous reports from Central, Northern, and Southern Europe, its occurrence in South-Eastern Europe has remained largely undocumented. This study provides the first molecularly confirmed report and the first systematic epidemiological assessment of O. flexuosa in red deer in Croatia. During the 2024-2025 hunting season, 110 legally harvested red deer from central Croatia were examined for subcutaneous nodules. Nodules were evaluated morphologically, and adult nematodes were identified and confirmed by sequencing of a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Subcutaneous nodules were detected in 53.6% (59/110) of examined animals. O. flexuosa was confirmed in 52 deer, corresponding to an overall prevalence of 47.3%. Co-infection with Hypoderma diana occurred in 21.2% of infected animals. Sequence similarity ranged from 96.37% to 99.85% compared to published European O. flexuosa isolates. Phylogenetic analysis placed Croatian sequences within the established European lineage, without evidence of regional genetic divergence. The observed prevalence falls within the intermediate range reported across Europe and indicates stable local transmission. These findings close an important geographical knowledge gap and demonstrate that nodular onchocercosis is established in red deer populations in South-Eastern Europe.