Diagnosis of Babesia caballi infections in horses by enzyme-linked immunosorbent assay (ELISA) and western blot

Abstract

From Babesia caballi in vitro cultures a preparation of 100% infected erythrocytes was obtained. From this, B. caballi antigens were extracted with the detergent 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and used as ELISA antigens. A control antigen of normal erythrocytes from the same donor horse was prepared in an identical manner. The ELISA and Western blot were validated by testing of sera from horses experimentally infected with B. caballi or B. equi or not infected with Babesia spp. ELISA and Western blot results were compared with those obtained by the immunofluorescence antibody test (IFAT) and complement fixation test (CFT). The sensitivity of the ELISA of 98.3% obtained for sera from day 14 after infection was superior to the Western blot (94.9%), the IFAT (96.6%) and the CFT (28.8%). No positive results were obtained in the ELISA and Western blot with 106 sera from horses not infected with Babesia spp. resulting in a calculated specificity of 100% for both tests. Cross reactions of B. equi-positive sera did occur to a larger extent in the ELISA (20%) than in the IFAT (4%). No cross reactions were observed with the Western blot and the CFT. The higher sensitivity of the ELISA was also demonstrated by testing of 132 field sera: more positive results were obtained by ELISA (112) as compared to IFAT (92) or CFT (41). The validity of these results was confirmed by testing of sera by Western blot. The ELISA as the most sensitive test provides the best method for the identification of carrier horses to prevent the introduction into non-endemic areas (export testing). Positive ELISA results can be confirmed by Western blot, if a species-specific diagnosis is required.