First Serologic Evidence of West Nile Virus and Usutu Virus Circulation Among Dogs in the Bulgarian Danube Region and Analysis of Some Risk Factors

This study aimed to assess West Nile virus (WNV) and Usutu virus seroprevalence among the dog population in the Danube region, Bulgaria, to confirm the results of ELISA by the virus neutralisation test (VNT), as well as to analyse several risk factors of seropositivity in dogs. To implement this, a total of 201 blood samples were collected from dogs in four districts bordering the Danube River. All the samples were tested for anti-WNV protein E antibodies using competitive ELISA. Neutralising antibodies against WNV and Usutu virus were tested in all the ELISA-positive samples. The results show a WNV seroprevalence of 45.3% (<i>n</i> = 91, CI = 36.45-55.59) by ELISA, whereas the virus neutralisation test indicated a seroprevalence of 21.9% (n = 44, CI = 15.91-29.39). Neutralising antibodies against Usutu virus were detected for the first time in Bulgaria, with a prevalence of 6% (n = 12, CI = 3.09-10.43). Compared to VNT, ELISA demonstrated 100.0% sensitivity and 70.1% specificity. The region (<i>p</i> < 0.0187), the district (<i>p</i> = 0.0258) and the ages of the dogs (<i>p</i> = 0.0180) were identified as statistically significant risk factors associated with WNV seropositivity. This study provides indirect evidence of WNV and Usutu virus circulation among dogs in the Danube region of Bulgaria, highlighting a potential risk for susceptible hosts in the area.