A sensitive immunoblotting technique for the serodiagnosis of Brucella ovis infections

A simplified electrophoretic immunoblotting technique based on antigen extracted from Brucella ovis cells with sodium dodecyl sulfate/mercaptoethanol was compared with the complement fixation test (CFT), the enzyme-linked immunosorbent assay, and the gel diffusion test. Sera from 89 chronically infected, semen culture-positive rams, 378 sera from B. ovis-infected flocks, 300 sera from accredited disease-free flocks, and 29 sera from specific-pathogen-free sheep were used. The immunoblotting technique had sensitivity and specificity comparable to those of the standard tests and was able to identify several CFT-negative or -borderline sera as positive. The major immunoreactive antigens of B. ovis had molecular masses of 63, 29, 19 kD (proteins) and 8-12 kD (rough lipopolysaccharide). Antibodies against these antigens were present in 96% of CFT-positive sera from infected flocks and in 100% of sera from semen culture-positive rams. However, immunoblotting also identified antibodies to components other than the major antigens in 1% of CFT-negative sera from infected flocks and in 7.7% of the sera from flocks with a history of freedom from the disease. These reactions probably represent cross-reactivities with other microorganisms and were distinguishable from truly positive reactions.