A SENSITIVE IMMUNOBLOTTING TECHNIQUE FOR THE SERODIAGNOSIS OF BRUCELLA-OVIS INFECTIONS

A simplified electrophoretic immunoblotting technique based on antigen extracted from ovis cells with sodium dodecyl sulfate/mercaptoethanol was compared with the complement fixation test (CFT), the enzyme-link ed immunosorbent assay, and the gel diffusion test. Sera from 89 chron ically infected, semen culture-positive rams, 378 sera from B. ovis-in fected flocks, 300 sera from accredited disease-free flocks, and 29 se ra from specific-pathogen-free sheep were used. The immunoblotting tec hnique had sensitivity and specificity comparable to those of the stan dard tests and was able to identify several CFT-negative or -borderlin e sera as positive. The major immunoreactive antigens of B. ovis had m olecular masses of 63, 29, 19 kD (proteins) and 8-12 kD (rough lipopol ysaccharide). 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 antibo dies to components other than the maj or antigens in 1% of CFT-negativ e sera from infected flocks and in 7.7% of the sera from flocks with a history of freedom from the disease. These reactions probably represe nt cross-reactivities with other microorganisms and were distinguishab le from truly positive reactions.