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A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia bigemina infection in cattle was standardized. Acetone-fixed whole Babesia bigemina-infected erythrocytes on micro-slides were immunoreacted with bovine serum samples followed by antibovine horseradish peroxidase conjugate and developed using diaminobenzidine tetrahydrochloride as a substrate. The positive immunoreactivity (staining pattern) was visualized in the form of dark brown piroplasms. Using the laboratory-standardized SELISA with a sensitivity of 94.4%, the seroprevalence of babesiosis was studied in cattle from two endemic areas of the disease. In comparison to IFAT, SELISA detected higher number of serum samples positive for bovine babesiosis.