Genetic variations in Aeromonas hydrophila isolates from clinical and environmental sources in Thailand.

Aeromonas hydrophila, a widely distributed human pathogen causing a variety of diseases, can be isolated from clinical and environmental sources. Analysis in Thailand of 110 isolates of Aeromonas hydrophila by randomly amplified polymorphic DNA-PCR (RAPD-PCR) revealed one specific RAPD pattern group (G) that was associated only with strains from environmental sources. Cytotoxic activity, adhesion to epithelial cells and exoenzyme secretions of A. hydrophila were also investigated. A comparison of isolates with pattern group G with a set of isolates derived from human blood showed low induction of cytotoxicity from those with RAPD pattern group G suggesting low virulence of these strains.

Nucleotide sequence of the small subunit ribosomal RNA-encoding gene from Opisthorchis viverrini.

The complete nucleotide (nt) sequence of the small subunit ribosomal RNA-encoding gene of Opisthorchis viverrini reported in this study is the first nt sequence reported for a trematode. The gene is 1992 nt long and has a G + C content of 50.94%. It is made up of alternated constant and variable regions that are similar to the gene organization of other eukaryotes. It is also of interest to note an unexpectedly high degree of sequence homology between O. viverrini and human genes.

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.

Monoclonal antibodies against protein antigens of salmonellae causing paratyphoid fever and their diagnostic application.

Hybrid clones producing monoclonal antibodies (MAbs) specific for Salmonella paratyphi A (72 clones), S. paratyphi B (9 clones) and S. paratyphi C (8 clones) were produced by using the affinity purified Salmonella protein (Bp) as immunogens. MAbs to S. paratyphi A and S. paratyphi B reacted specifically with the 52 kDa homologous flagellin protein components while those to S. paratyphi C reacted with a 61 kDa flagellin protein component. The MAbs against S. paratyphi A and S. paratyphi B were used to establish a double antibody sandwich ELISA for detection of the 52 kDa flagellin antigens in serum samples from patients with acute paratyphoid A and paratyphoid B fever. With this assay system, 6.25 ng per ml of flagellin antigens of S. paratyphi A and S. paratyphi B could be detected. However, the assay system could not detect the flagellin antigens in patients' sera. The presence of IgM antibodies to the 52 kDa antigens of S. paratyphi A and S. paratyphi B in the acute sera from paratyphoid A or paratyphoid B patients suggested that the 52 kDa protein components of both salmonellae are good immunogens for human and might be used as antigens for early diagnosis of paratyphoid A and paratyphoid B fever.

Characterization of a phase 1-d epitope on Salmonella typhi flagellin and its role in the serodiagnosis of typhoid fever.

A monoclonal antibody (MAb) directed against Salmonella typhi 52 kDa flagellin protein has been previously produced by our group. In this study, we have demonstrated that the epitope specific to the MAb is unique to phase 1-d. To map the epitope, plasmids encoding different regions of S. typhi flagellin gene were constructed. Analysis of protein produced from each recombinant plasmid indicated that the epitope specific to the MAb resided within amino acids 171-303 (region IV) of S. typhi flagellin protein. The recombinant region IV flagellin was used to develop an ELISA for the detection of IgM antibody to S. typhi in serum. In the hemoculture-positive typhoid group, the developed ELISA was positive in 77 of 92 cases. In patients with non-typhoidal Salmonella, gram-positive and gram-negative bacteria or dengue virus, the ELISA was negative in all 78 cases. Two from 116 healthy control subjects had positive reactions with the assay. The calculated sensitivity, specificity, positive and negative predictive values of the test were 83.7%, 99.0%, 97.5% and 92.8%, respectively. With such high validity together with the requirement of only a single serum specimen and one day for performing the test, the developed ELISA should become a valuable diagnostic test for typhoid fever.

Immunological detection of Salmonella paratyphi A in raw prawns.

A slot blot enzyme-linked immunosorbent assay, using monoclonal antibodies specific only for Salmonella paratyphi A, to detect S. paratyphi A contamination in raw prawns has been established. When artificially contaminated prawn samples were tested. S. paratyphi A contamination could be identified correctly within 20 h. No false positives from samples artificially contaminated by other microorganisms were obtained. The sensitivity was such that as few as 1 S. paratyphi A organism per g of raw prawn could be detected. Therefore, the assay constituted a promising test for the rapid and specific detection of S. paratyphi A in prawns.

Cloning and characterization of a nonhemolytic phospholipase C gene from Burkholderia pseudomallei.

We cloned and characterized a phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) gene from Burkholderia pseudomallei. DNA sequence analysis of the gene indicated an open reading frame coding for 700 amino acids with a 34-amino-acid signal peptide. When cleaved, this yields a secreted 73-kDa mature protein. The deduced amino acid sequence exhibited 48% similarity to that of a nonhemolytic PLC from Pseudomonas aeruginosa. The expressed PC-PLC was heat stable, nonhemolytic for sheep erythrocytes, and active between pH 2 and 8. Western blot analysis with sera from melioidosis patients indicated that they produced immunoglobulin M antibodies against this PC-PLC protein.

Cloning and characterization of ribosomal RNA genes from Opisthorchis viverrini.

The ribosomal DNA (rDNA) unit of the liver fluke Opisthorchis viverrini has been cloned and characterized. The results demonstrated that the total length of this unit is approximately 13 kb, containing 4.2 kb of large subunit (LSU) rDNA, 2.0 kb of small subunit (SSU) rDNA, 1.0 kb of transcribed spacer DNA and 5.8 kb of non-transcribed + external transcribed spacer DNA. Examination of the non-transcribed spacer region between different rDNA units showed variation in the restriction sites rather than in the length. Judging from hybridization of the rDNA plasmid to the restriction endonuclease digest of genomic DNA, rDNA units represent 6.1% of the total genomic DNA. At the RNA level, the LSU rRNA of O. viverrini and Fasciola gigantica contained a hidden break. The molecule consisted of two fragments co-migrating with a SSU rRNA, when electrophoresis was carried out under denaturing conditions. Ribosomal RNA sequence comparison has been previously used to determine phylogenetic classification of parasitic organisms. The sequence of 381 nucleotides at the 5' terminus of the LSU rRNA gene was determined and compared with those from species previously reported by other investigators. Phylogenetic classification of O. viverrini, as determined by rRNA gene sequence comparison, is comparable with the conventional taxonomic classification.

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli.

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of delta-aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5'-phosphate is conserved.

Rapid detection of Salmonella enterica serovar Choleraesuis in blood cultures by a dot blot enzyme-linked immunosorbent assay.

A dot blot enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific to phase1-c Salmonella was developed for the direct detection of Salmonella enterica serovar Choleraesuis in blood cultures. This system was applied to the identification of serovar Choleraesuis, and the results were compared with those obtained by a conventional biochemical method. It was revealed that all 12 samples identified to be infected with serovar Choleraesuis were positive on testing by the ELISA. In contrast, 77 samples infected with bacteria commonly isolated from the blood were not reactive by the ELISA. The calculated sensitivity and specificity of the established assay are 100%.