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

## TL;DR

This study found evidence of West Nile and Usutu viruses in dogs in Bulgaria's Danube region, showing these viruses are circulating and identifying risk factors for infection.

## Contribution

First serologic evidence of Usutu virus in Bulgaria and confirmation of WNV in dogs, along with risk factor analysis.

## Key findings

- WNV seroprevalence in dogs was 21.9% by VNT and 45.3% by ELISA.
- Usutu virus antibodies were detected for the first time in Bulgaria with a 6% prevalence.
- Region, district, and dog age were significant risk factors for WNV seropositivity.

## Abstract

West Nile virus (WNV) and Usutu virus are arboviruses primarily transmitted by mosquitoes. This study aimed to assess the seroprevalence of these viruses among the dog population in the Danube region of Bulgaria to confirm the results of ELISA against the reference method—virus neutralisation test (VNT)—and to analyse some risk factors associated with seropositivity in dogs. Two hundred one blood serum samples were collected from dogs in four districts in Bulgaria bordering the Danube River. Overall, 44 samples were confirmed positive for WNV, resulting in a seroprevalence of 21.9%. Antibodies against Usutu virus were detected for the first time in Bulgaria, with a prevalence of 6%. The region, the district and the dogs’ ages were risk factors associated with WNV seropositivity. This study demonstrates that WNV and Usutu viruses circulate among dogs in the Bulgarian Danube region and highlights a potential risk for susceptible hosts in the area.

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% (n = 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 (p < 0.0187), the district (p = 0.0258) and the ages of the dogs (p = 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.