Production of Salmonella serogroup D (O9)-specific enzyme-linked immunosorbent assay antigen

Serologic testing to detect persistent IgG titer directed at Salmonella lipopolysaccharide (LPS) has proven useful in detecting Salmonella carrier cattle without clinical signs of disease and in seroepidemiologic studies. Although little cross-reactivity exists between most Salmonella serogroups, groups B (O1, 4 [5], 12) and D (O1, 9, 12) share somatic (LPS cell wall) antigens O1 and O12, which results in some cross-reactions. This may be unimportant in most instances, because group-B and group-D carriers need to be identified and culled. It may be desirable in some situations, such as when trying to control S dublin, to determine which serogroup is present in a given herd. For this reason, a procedure to produce a pure O9 group-D antigen was developed. Salmonella dublin (group D) was grown by use of standard procedures, and LPS was extracted by use of the phenol-water method. The LPS was then oxidized with sodium periodate, dialyzed, reduced with sodium borohydride, cleaved with hydrochloric acid, and again dialyzed. This procedure successfully cleaved the saccharides comprising O antigens 1 and 12, leaving a pure O9 ELISA antigen. Sera from cattle vaccinated or naturally infected with S typhimurium, S agona, and S schwarzengrund (all group B), S montevideo (group C1), and S dublin (group D) were tested by ELISA, using modified and unmodified antigens. When the ELISA antigen used was the chemically modified (pure O9) group-D antigen, elimination of cross-reactions confirmed the structural loss of cross-reacting O1 and O12 antigens.