ENZYME-LINKED-IMMUNOSORBENT-ASSAY WITH PARTIALLY PURIFIED CYTOSOLUBLE28-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-reacting 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. meliten sis Rev.1-vaccinated ewes by enzyme-linked immunosorbent assay with th ree antigenic fractions: O polysaccharide, a cytosoluble protein extra ct (CPE) from the rough strain B. melitensis B115, and a partially pur ified cytosoluble protein of 28 kDa (CP28) from the CPE. Immunoglobuli n 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 B rucella-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 resul ts obtained with CP28 showed the potential usefulness of this antigen to permit the detection of B. melitensis-infected ewes and their diffe rentiation from B. melitensis Rev.1-vaccinated ones.