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 ...

Very high vaccination coverage is required to eliminate measles, but achieving high coverage can be constrained by the logistical challenges associated with subcutaneous injection. To simplify logistics of vaccine delivery, a patch containing micron-scale polymeric needles was formulated to encapsulate the standard dose of measles vaccine (1000 TCID50) and the immunogenicity of the microneedle patch was compared with subcutaneous injection in rhesus macaques. The microneedle patch was administered without reconstitution with diluent, dissolved in skin within 10 minutes, and caused only mild, transient skin erythema. Both groups of rhesus macaques generated neutralizing antibody responses to measles that were consistent with protection and the neutralizing antibody titers were equivalent. In addition, the microneedle patches maintained an acceptable level of potency after storage at elevated temperature suggesting improved thermostability compared to standard lyophilized vaccine. In conclusion, a measles microneedle patch vaccine was immunogenic in non-human primates, and this approach offers a promising delivery method that could help increase vaccination coverage.

Measles vaccination programs would benefit from delivery methods that decrease cost, simplify logistics, and increase safety. Conventional subcutaneous injection is limited by the need for skilled healthcare professionals to reconstitute and administer injections, and by the need for safe needle handling and disposal to reduce the risk of disease transmission through needle re-use and needlestick injury. Microneedles are micron-scale, solid needles coated with a dry formulation of vaccine that dissolves in the skin within minutes after patch application. By avoiding the use of hypodermic needles, vaccination using a microneedle patch could be carried out by minimally trained personnel with reduced risk of blood-borne disease transmission. The goal of this study was to evaluate measles vaccination using a microneedle patch to address some of the limitations of subcutaneous injection. Viability of vaccine virus dried onto a microneedle patch was stabilized by incorporation of the sugar, trehalose, and loss of viral titer was less than 1 log10(TCID50) after storage for at least 30 days at room temperature. Microneedle patches were then used to immunize cotton rats with the Edmonston-Zagreb measles vaccine strain. Vaccination using microneedles at doses equaling the standard human dose or one-fifth the human dose generated neutralizing antibody levels equivalent to those of a subcutaneous immunization at the same dose. These results show that measles vaccine can be stabilized on microneedles and that vaccine efficiently reconstitutes in vivo to generate a neutralizing antibody response equivalent to that generated by subcutaneous injection.

These results show, for the first time, that MR vaccine delivered by MN patch generated protective titers of neutralizing antibodies to both measles and rubella in infant rhesus macaques and afforded complete protection from measles virus challenge.