The morphological heterogeneity of lipopolysaccharides (LPSs) among salmonella mutants with different LPS chemotypes was analyzed in silver-stained polyacrylamide gels. The biochemical differences in the LPS chemotypes were reflected in the unique profiles of the purified LPSs. The LPS profiles in the whole-cell lysates were also unique for each chemotype. (Whole-cell lysates were assessed by a method which preferentially silver stains LPS and by a proteinase K digest of whole-cell lysates. The silver-stained LPS profiles of proteinase K-digested lysates were similar to the homologous purified LPS and could be used to preliminarily characterize the LPS chemotype before purification.) In summary, biochemical variation in LPS composition can be detected in silver-stained polyacrylamide gels.

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae,Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of ≥2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.

We report the development and evaluation of a Salmonella O-group-specific Bio-Plex assay to detect the six most common serogroups in the United States (B, C 1 , C 2 , D, E, and O13) plus serotype Paratyphi A. The assay is based on rfb gene targets directly involved in O-antigen biosynthesis; it can be completed 45 min post-PCR amplification. The assay correctly and specifically identified 362 of 384 (94.3%) isolates tested in comparison to traditional serotyping. Seventeen isolates (4.4%) produced results consistent with what is known about the molecular basis for serotypes but different from the results of traditional serotyping, and five isolates (1.3%) generated false-negative results. Molecular determination of the serogroup for rough isolates was consistent with a common serotype in most instances, indicating that this approach has the potential to provide O-group information for isolates that do not express an O antigen. We also report the sequence of the O-antigenencoding rfb gene cluster from Salmonella enterica serotype Poona (serogroup O13). Compared with other, previously characterized rfb regions, the O13 rfb gene cluster was most closely related to Escherichia coli O127 and O86. The O-group Bio-Plex assay described here provides an easy-to-use, high-throughput system for rapid detection of common Salmonella serogroups.Serotyping of salmonellae is a valuable phenotypic subtyping tool for understanding the epidemiology of this important food-borne pathogen. Salmonella isolates are serotyped using the Kauffmann-White scheme according to their O, H, and Vi antigens (4, 40). The O antigen contains multiple repeats of an oligosaccharide unit (O unit), which, together with lipid A and core oligosaccharides, form the lipopolysaccharide present in the outer membranes of gram-negative bacteria (7). Many of the genes required for O-antigen biosynthesis are organized in a large regulon termed the rfb gene cluster (47). rfb gene clusters have been characterized from a growing number of gram-negative bacteria; this operon is located between galF and gnd in Salmonella enterica and Escherichia coli (49). In general, rfb genes have a low GϩC content (usually less than 40%); the deviation in GϩC content from that of typical S. enterica genes (51%) suggests that rfb DNA originated in species other than S. enterica and was captured by lateral gene transfer (46, 56). Typically, three classes of genes are found in rfb clusters: (i) genes for synthesis of nucleotide sugars specific to the respective O antigen, (ii) sugar transferase genes to build the O subunit, and (iii) the O-antigen polymerase (wzy) and transport protein (wzx) genes for assembly of the O subunit into the O antigen (49).There are 46 O serogroups described in the KauffmannWhite scheme; serogroups were originally designated by alphabetic letters, and then it was necessary to continue with numbers 51 to 67. For consistency in the scheme, all serogroups were given a number designation; however, the most common serogroups (A to E) are commonly designated by ...