West Nile virus (WNV) infection, caused by a flavivirus, emerged in Europe and America in the past two decades. The etiological agent causes asymptomatic to life-threatening infection in humans and in some animal species. The objective of this study was to evaluate the seroprevalence of WNV among donkeys and mules in Bulgaria. A total of 200 archived serum samples were tested by competitive enzyme-linked immunosorbent assay (ELISA). Positive samples were additionally analyzed by virus neutralization assay. Seroprevalence of 7% (14/200) was established among tested animals by ELISA. Two samples were subsequently verified for the presence of virus neutralizing antibodies; thus, the seroprevalence against WNV was determined to be 1% (2/200 [confidence interval = 0.12-3.61]). Positive results among mules included in the study were not found. The findings in the present research demonstrate that donkeys are exposed to WNV infection and seroconvert, which adds to the understanding of virus circulation among donkeys in settlements in north and south Bulgaria.
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