A serological survey for West Nile virus (WNV) infection involved 395 horses 
from 43 administrative districts of the Czech Republic (163 animals) and 29 
districts of Slovakia (232 animals), sampled between 2008 and 2011. Using a 
plaque-reduction neutralization microtest, antibodies to WNV were not detected 
in any horse from the Czech Republic, whereas 19 nonvaccinated horses from 
Slovakia had specific antibodies to WNV (no cross-reactions were observed with 
tick-borne encephalitis and Usutu flaviviruses in those animals). The 
seropositivity rate of nonvaccinated horses in Slovakia was 8.3% (95% confidence 
interval [CI] 4.7-11.9%), and autochthonous local infection with WNV occurred at 
least in 11, i.e., 4.8% (95% CI 2.0-7.6%) of the animals. All seropositive 
horses lived in six lowland districts of southern Slovakia; overall, 15.1% (95% 
CI 8.8-21.4%) of 126 nonvaccinated horses were seropositive in those districts, 
situated relatively closely to the border with Hungary, i.e., the country where 
WNV disease cases have been reported in birds, horses and humans since 2003.