Evaluation of the recombinant 10-kilodalton immunodominant region of the BP26 protein of Brucella abortus for specific diagnosis of bovine brucellosis

Abstract

Brucellosis is a disease with worldwide distribution affecting animals and human beings. Brucella abortus is the causative agent of bovine brucellosis. The cross-reactions of currently available diagnostic procedures for B. abortus infection result in false-positive reactions, which make the procedures unreliable. These tests are also unable to differentiate Brucella-infected and -vaccinated animals. The present work is focused on the use of a nonlipopolysaccharide (LPS) diagnostic antigen, a recombinant 10-kDa (r10-kDa) protein of B. abortus, for specific diagnosis of brucellosis. The purified recombinant protein was used as a diagnostic antigen in plate enzyme-linked immunosorbent assay (p-ELISA) format to screen 408 bovine serum samples (70 presumptively negative, 308 random, and 30 vaccinated), and the results were compared with those of the Rose Bengal plate agglutination test (RBPT) and the standard tube agglutination test (STAT). Statistical analysis in presumptive negative samples revealed 100 and 98.41% specificity of p-ELISA with RBPT and STAT, and an agreement of 91.43% with the tests using Cohen's kappa statistics. In random samples, the agreement of p-ELISA was 77.92% and 80.52% with RBPT and STAT, respectively. p-ELISA investigation of vaccinated samples reported no false-positive results, whereas RBPT and STAT reported 30% and 96.6% false-positive results, respectively. The data suggest that p-ELISA with r10-kDa protein may be a useful method for diagnosis of bovine brucellosis. Furthermore, p-ELISA may also be used as a tool for differentiating Brucella-vaccinated and naturally infected animals.

Fig. 1.

SDS-PAGE analysis of 10-kDa protein expression in E. coli M15 cells. Lane 1, protein molecular mass marker (kDa); lane 2, uninduced cells; lane 3, IPTG-induced cells; lane 4, purified r10-kDa protein. The arrow on the right indicates the position of the r10-kDa protein.

Fig. 2.

Western blot of purified r10-kDa protein with anti-His-HRP conjugate. Lane 1, molecular mass marker; lane 2, purified r10-kDa protein. The arrow on the right indicates the position of the r10-kDa protein.