Babesia ovata: isolation from erythrocytes and development of an enzyme-linked immunosorbent assay for detection of antibodies.

A method of isolating Babesia ovata merozoites from infected erythrocytes and an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-B. ovata antibodies were developed. After B. ovata-infected erythrocytes had been lysed using the nitrogen cavitation method, the merozoites were separated from erythrocyte components by differential centrifugation and density-gradient centrifugation. The light microscopic examination showed that the purified merozoites were morphologically intact and contained few contaminants. Sodium dodecyl sulfate-polyacrylamide electrophoretic (SDS-PAGE) analysis revealed that the merozoite fraction contained very little contamination with erythrocyte components. The merozoites thus obtained were sonicated and treated with Triton X-100 and then used as an antigen to measure anti-B. ovata antibodies in ELISA. The ELISA was more sensitive in detecting anti-B. ovata antibodies than was either the indirect fluorescent antibody test or the complement fixation test on sera from cattle infected with B. ovata.