Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev.1-vaccinated sheep.

The problem of differentiating sheep infected with Brucella melitensis from those vaccinated or exposed to cross-reaching organisms has not been resolved by conventional serological tests or through the use of the smooth lipopolysaccharide in primary binding assays. We therefore analyzed sera from ewes experimentally infected with B. melitensis H38, from ewes naturally infected with B. melitensis, and from B. melitensis Rev.1-vaccinated ewes by enzyme-linked immunosorbent assay with three antigenic fractions: O polysaccharide, a cytosoluble protein extract (CPE) from the rough strain B. melitensis B115, and a partially purified cytosoluble protein of 28 kDa (CP28) from the CPE. Immunoglobulin G anti-O polysaccharide and anti-CPE responses were detected in all groups of animals tested (Rev.1 vaccinated and B. melitensis infected). However, false-positive reactions with CPE occurred with sera from Brucella-free ewes. The use of partially purified CP28 abolished these false-positive reactions. Furthermore, no immunoglobulin G antibodies against CP28 were detected in sera from vaccinated ewes, whereas 80% (8 of 10) of ewes experimentally infected with B. melitensis H38 and 89% (25 of 28) of naturally infected ewes showed various degrees of anti-CP28 reactivity (absorbance values of between 0.5 and 2.5). The results obtained with CP28 showed the potential usefulness of this antigen to permit the detection of B. melitensis-infected ewes and their differentiation from B. melitensis Rev.1-vaccinated ones.