Seroprevalence of West Nile virus and related orthoflaviviruses in some animal species in Bulgaria

Orthoflaviviruses are a notable threat to public health owing to their capacity to induce severe neuroinvasive disease. The present study aimed to synthesise seroprevalence data concerning West Nile virus (WNV) and Usutu virus (USUV) among selected animal species in Bulgaria. A total of 779 serum samples were collected: 578 from equids and 201 from dogs, spanning various districts across both northern and southern Bulgaria. Equine samples were obtained in 2015 and 2022, while canine samples were collected in 2024. All sera were subjected to competitive enzyme-linked immunosorbent assay (ELISA) and confirmatory virus neutralisation tests (VNT). ELISA screening revealed seroprevalence rates of 7.0% in 2015, 9.26% in 2022, and 45.3% in 2024. Upon VNT confirmation, WNV seroprevalence was adjusted to 1.0% in 2015, 3.97% in 2022, and 21.9% in 2024. Neutralising antibodies against USUV were identified in 12 dogs (6.0%) from three districts situated along the Danube River. No IgM antibodies were detected in the equine samples, suggesting the absence of recent infections and the likelihood of prior exposure. This study provides serological evidence for the ongoing circulation of WNV and the closely related USUV in Bulgarian ecosystems. The findings underscore the necessity for sustained surveillance efforts targeting both animal reservoirs and vector populations to mitigate the risk of zoonotic transmission and associated public health implications.