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Three antigens of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto–Slovak strain Ir 105, B. garini – Slovak strain K 24 and B. burgdorferi sensu stricto–American strain B 31) were compared by ELISA on a group of dogs from urban agglomeration of Kosice, eastern Slovakia. Of 256 serum samples from dogs examined for the presence of anti-Borrelia IgG antibodies, 128 (50%) were positive with Ir 105 antigen, 107 (41.7%) with K 24 and 74 (28.9%) with B 31. The seroprevalence between strains B 31 and K 24 and B 31 and Ir 105 differed statistically significantly (test chi2, p < 0.05), however, the difference between strains K 24 and Ir 105 was insignificant. A significantly higher seroprevalence of all the strains examined was detected in hunting dogs (test chi2, p < 0.05) when compared with service and pet dogs. The seroprevalence correlated with the frequency of outing the dogs in woody areas with the occurrence of borreliae in ticks (R = 0.5 or 0.7) as well as with the frequency of finding engorged ticks (R = 0.5). An epizootiological anamnesis showed a fair specificity in all the strains examined. The Slovak strains showed the higher consistency of positive and negative findings (70%) but statistically lower specificity than the American strain.