Detection of IgM antibody against region IV flagellin of Salmonella paratyphi A.

Salmonella paratyphi A is a pathogenic bacterium that causes paratyphoid fever. The current laboratory diagnostic techniques are unsatisfactory. To improve diagnosis, a plasmid (pSK-8E) encoding phase 1 flagellin gene nucleotide position 452-890 from S. paratyphi A has been constructed. The recombinant protein expressed from the plasmid has been used to develop an indirect ELISA for IgM antibody detection. Sera from patients with hemoculture positive for S. paratyphi A, S. typhi, other gram-positive and gram-negative bacteria, and dengue hemorrhagic fever as well as from healthy control subjects were tested. Sensitivity, specificity, positive and negative predictive values of the test were 56.9%, 98.8%, 90.6% and 92.1%, respectively. Since the sensitivity was low, the explanation for this result was investigated. It was found that the sensitivity of the test could be increased to 83.3% if the sera were obtained 9-12 days after onset of fever. The sera obtained earlier or later gave only 33.3% and 66.6% sensitivity, respectively. This result suggests that the IgM antibody detection assay which we have developed is a valuable tool for diagnosis of S. paratyphi A infection when the serum samples are taken at the appropriate time.

Molecular cloning and expression of Salmonella paratyphi A 52 kDa specific protein gene.

Monoclonal antibodies (MAbs) specific to Salmonella paratyphi A have been established by our group in 1989. These MAbs were proven to be species-specific for 52 kDa protein of S. paratyphi A but the nature of this protein is unknown. However, our group have proved that the 52 kDa protein which is specific to S. typhi was flagellin. This present study has characterized the 52 kDa protein of S. paratyphi A and identified its encoded gene. The plasmid containing the specific 52 kDa antigen gene was cloned from the S. paratyphi A genome, herein designated pSKA-4. Partial nucleotide sequences from this clone was analysed by computer program and found to be phase 1-a flagellin gene of S. paratyphi A. In addition, the nucleotide sequence analysis from such clone also showed that the structural gene for phase 1 flagellin has amino acid sequences conserved at the terminal whereas the central region is variable among Salmonella spp. Therefore, the central portion of flagellin which highly polymorphic in amino acid sequences would be the most specific to S. paratyphi A, thus, should be used as specific antigen for developing specific diagnosis of S. paratyphi A infection. Using the PCR technique, an expression plasmid containing the antigen gene producing only the variable region in the central portion of flagellin from S. paratyphi A, namely pSKA-7, has been established. The recombinant protein produced by the established plasmid has a MW 33.5 kDa as detected by immunoblotting using specific MAbs. Further study by using this specific flagellin protein for immunodiagnosis of S. paratyphi A infection is being carried out in our laboratory.