Abstract
Members of the genus Brucella have cell wall characteristics of Gram-negative bacteria, which in the most significant species includes O-polysaccharide (OPS). Serology is the most cost-effective means of detecting brucellosis, as infection with smooth strains of Brucella leads to the induction of high antibody titers against the OPS, an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyl residues (D-Rha4NFo) that are variably α(1→2)- and α(1→3)-linked. Six d-Rha4NFo homo-oligosaccharides were synthesized, each containing a single α(1→3) link but with a varied number of α(1→2) links. After conjugation to bovine serum albumin (BSA), glycoconjugates 1 to 6 were used to develop individual indirect enzyme-linked immunosorbent assays (iELISAs). The diagnostic capabilities of these antigens were applied to panels of cattle serum samples, including those falsely positive in conventional assays, and the results were compared with those of the complement fixation test (CFT), serum agglutination test (SAT), fluorescent polarization assay (FPA), smooth lipopolysaccharide (sLPS) iELISA, and competitive enzyme-linked immunosorbent assay (cELISA) methods. Results from field serum samples demonstrated that all of the synthetic antigens had excellent diagnostic capabilities. Assays developed with the α(1→3)-linked disaccharide conjugate 1 were the best at resolving false-positive serological results. This was supported by the results from serum samples derived from experimentally infected cattle. Data from synthetic trisaccharide antigens 2 and 3 and tetrasaccharide antigen 4 identified an OPS epitope equally common to all Brucella abortus and Brucella melitensis strains but unique to Brucella. Synthetic oligosaccharide conjugates function as effective surrogates for naturally derived antigens. The creation of discrete OPS epitope antigens reveals not only the previously untapped diagnostic potential within this key diagnostic structure but also holds significance for the design of brucellosis vaccines and diagnostics that enable the differentiation of infected from vaccinated animals.