Immunoselection and structural evaluation of Brucella O-polysaccharide epitopes and their application to the serodiagnosis of bovine brucellosis

Brucellosis is one of the world’s most significant zoonosis and is caused by infection with members of the genus Brucella. These have a cell wall characteristic of Gram-negative bacteria including lipopolysaccharides and, in the most significant Brucella species, O-polysaccharide (OPS). The major economic and health impacts of the disease arise from livestock, in particular ruminants and swine, where the main clinical feature is reproductive failure. The principle source of infection in the general human population is most often via ingestion of unpasteurised dairy products. Serology is the most cost effective means of disease detection but has significant imperfections including false positive serological reactions (FPSRs) due to antibodies that are raised against other Gram-negative bacteria in possession of OPS structures similar to that of Brucella. Serology with non-OPS antigens has been largely ineffective and alternative approaches such as bacterial culture, PCR or measurements of cell mediated immunity are impractical, ineffective or unproven. The OPS from Brucella is an unbranched homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyls (D-Rha4NFo) that are variably -(12) and -(13) linked. This structure contains some epitopes that are shared with the OPS from other organisms, notably Yersinia enterocolitica O:9, and some that appear to be unique. Previous attempts to harness the unique epitopes using competitive ELISA failed to resolve FPSRs because the unique and common epitopes overlap causing steric hindrance of specific antibody binding. Oligosaccharides were derived from the OPS of B. abortus, B. melitensis and Y. enterocolitica O:9 by partial acid hydrolysis. These were separated and analysed by chromatography with on-line ESI-QqToF and QqQ. OPS specific antibodies were used to select from this pool of oligosaccharides and those captured were evaluated by graphitised carbon chromatography with on-line ESI-QqQ. On the basis of the mass spectrometry evidence the synthesis of a D-Rha4NFo tetrasaccharide comprised of a single -(13) link flanked on either side by single -(12) links was commissioned. This tetrasaccharide was used to develop an indirect ELISA for the detection of specific antibodies. Equivalent indirect ELISAs were also developed using native OPS and from synthetic penta- and nonasaccharide antigens, received from a collaborating laboratory. The tetrasaccharide ELISA was the most effective assay for discriminating between bovine FPSRs and true positives. The diagnostic capability of this ELISA was significantly superior (P < 0.05) than all others except the pentasaccharide ELISA (P = 0.159). The results show that the iELISA developed with the tetrasaccharide may effectively detect antibodies from animals infected with Brucella strains with low or high abundance of -(13) links within their OPS and has a significantly improved capability to resolve FPSRs compared to antigens that include common OPS epitopes.Open Acces