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In this investigation the Western blot test was treated as a method verifying results of the IFA, commercial ELISA and standardized ELISA tests (described in Part I). The verifying investigations were performed on 82 serum samples, which in the commercial ELISA were positive in 36 cases, dubious in 31 cases and negative in 15 cases as well as on 5 serum samples obtained from horses infected with Leptospira spp., which in the ELISA commercial were dubious (total of 87 sera samples). The antigens, against which the immunological response in horses was directed, were also established. The Milenia–Blot–Borrelia IgG test (MIDBO IgG-Kit 30 TESTS: DPC Bierman GmbH) was used in the investigation. In view of species differences, rabbit anti-horse IgG (whole molecule) alkaline phosphatase conjugate, no A6063 SIGMA-ALDRICH was used interchangeably. Also the control sera were substituted with the horse control sera. It was demonstrated that the Western blot test is the most reliable in the serological diagnosis of B. burgdorferi infection in horses. The commercial ELISA and standardized ELISA tests represent a lower diagnostic value than the Western blot test, although similar to each other, while the value of the IFA is minimal. In the Western blot test antigens were established against which the immunological response in horses in mostly directed. In the sera evaluated in this test as positive the presence of antibodies, mainly against antigens with the following molecular weights: 41 kDa, 62/60 kDa, 93 kDa, 72 kDa, 34 kDa (OspB), 66 kDa was noted. At the same time, antibodies contained in the sera accepted as negative, in 55.5% cases also reacted with the antigen of 41 kDa. It points to its minimal specificity. On the basis of the results obtained it is recommended that serological examination of horses should be with the ELISA and that positive or dubious results should be verified with the Western blot test.