Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle

Abstract

Background and Aim: An accurate diagnosis of Brucella-infected animals is one of the critical measures in eradication programs. Conventional serological tests based on whole-cell (WC) antigens and detecting antibodies against pathogen-associated lipopolysaccharide might give false-positive results due to the cross-reactivity with other closely related bacteria. This study evaluated the serological potential of Brucella spp. chimeric outer membrane proteins (Omps) as antigens in an indirect enzyme-linked immunosorbent assay (i-ELISA). Materials and Methods: The chimeric gene constructs of the most immunodominant regions of Brucella Omps 25+31, 25+19, and 19+31 were cloned into the pET28a expression vectors and transformed into Escherichia coli BL21 (DE3). The serological potential of chimeric proteins compared with single recombinant Omps (rOmps)19, 25, and/or 31 were studied on blood serum samples of (i) a rabbit immunized with killed Brucella abortus 19WC, (ii) mice immunized with single rOmps, (iii) cows seropositive for brucellosis by rose Bengal test, and (iv) cattle naturally and/or experimentally infected with brucellosis. Results: E. coli BL21 actively produced Brucella chimeric rOmps, the concentration of which reached a maximum level at 6 h after isopropyl-β-D-1-thiogalactopyranoside stimulation. Target proteins were antigenic and expressed in an active state, as recognized by rabbit anti-B. abortus antibodies in an i-ELISA and western blotting. Murine antibodies against the single rOmps reacted with chimeric antigens, and conversely, antichimeric antibodies found their epitopes in single proteins. Brucella chimeric rOmps showed higher antigenicity in blood sera of seropositive cattle kept in the hotbed of the infection and/or experimentally challenged with brucellosis than single proteins. Conclusion: Brucella chimeric recombinant outer membrane proteins could be a potential antigen candidate for developing an ELISA test for accurate diagnosis of bovine brucellosis.