Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand.

BACKGROUND & OBJECTIVES: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. METHODS: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. RESULTS: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. INTERPRETATION & CONCLUSIONS: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).

Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity.

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

The significance of Ser1029 of the heat-stable enterotoxin receptor (STaR): relation of STa-mediated guanylyl cyclase activation and signaling by phorbol myristate acetate.

To characterize Ser1029 in STaR at a consensus sequence of phosphorylation site by PKC, two mutants of mS1029A with replacement of Ser1029 to Ala1029 and C delta 1029 lacking 22 amino acids including Ser1029 were prepared. Preincubation of the wild type-STaR (wt-STaR) transfectant with 1 microM PMA caused additional STa-mediated guanylyl cyclase (GC) activation compared to control, whereas the mS1029A- and C delta 1029-transfected cells did not show a similar enhanced GC activation by PMA. After metabolic labeling with [32P]phosphate, transfected cells with wt-STaR and mutants were incubated with 1 microM PMA. Subsequent 32P-radiolabeled proteins were immunoprecipitated using anti-STaR antibody, and analyzed by autoradiography after separation on SDS-PAGE. The immunoprecipitated wt-STaR but not mS1029A and C delta 1029 had a significant radioactivity. These results suggest that the effect of PMA on wt-STaR transfectants may be caused by phosphorylation of Ser1029. The C delta 1012 mutant, with further truncation (Gln1012-Phe1050) of the carboxy terminus, did not show STa-mediated GC activation. Based on these data, these 17 amino acids (Gln1012-Ala1028), essential for signaling of GC activation by STa, have an abundance of basic amino acids which might be functionally influenced by phosphorylation of Ser1029.

Production of beta-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein.

We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis toxin. The FliC-induced increase in hBD-2 promoter activity via nuclear factor kappaB (NF-kappaB) was also inhibited by chelation of intracellular Ca(2+) or by U73122. We conclude that FliC increased [Ca(2+)](in) via inositol 1,4,5-trisphosphate, which was followed by up-regulating hBD-2 mRNA expression via an NF-kappaB-dependent pathway.

Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin).

The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17. Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin. A PCR-amplified 185 bp DNA fragment was sequenced. This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end. This fragment did not contain the stop codons. It was further extended by a gene walking method using Eco RI cassette and its primers. Finally a 660 bp fragment was obtained and sequenced. This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids. The deduced amino acid sequence of the polypeptide encoded by the gene, designated fimA, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.

Distribution of the zot (zonula occludens toxin) gene among strains of Vibrio cholerae 01 and non-01.

The distribution of the zot gene that encodes the zonula occludens toxin, a newly described toxin of Vibrio cholerae, among clinical, environmental and food isolates of V. cholerae 01 and non-01 was investigated. Both the zot gene and the ctx gene that encode cholera toxin were found in 247 of 257 clinical strains and 62 of 415 environmental or food isolates of V. cholerae 01. The zot gene, but not the ctx gene was found in 37 strains (one clinical strain and 36 environmental or food isolates). In addition, two of 31 clinical strains and six of 98 environmental or food isolates of V. cholerae' non-01 possessed both the zot gene and the ctx gene. These results demonstrated the predominantly concurrent occurrence of the zot gene and ctx genes among strains of V. cholerae 01 which suggests a possible synergistic role of ZOT in the causation of acute dehydrating diarrhea produced by V. cholerae 01.

Structural and sequence diversity of the pathogenicity island of uropathogenic Escherichia coli which encodes the USP protein.

A total of 321 uropathogenic Escherichia coli (UPEC) strains and 12 strains of E. coli isolated from stool samples of healthy individuals, which were previously shown to be positive in colony hybridization test using the usp (encoding for the uropathogenic-specific protein) DNA probe, were examined by PCR amplification to determine the size of the usp gene and the pathogenicity island (PI). Three types of size variation were observed for the usp gene and four types for the PI. Sequencing analysis of the PIs from seven representative strains (six UPEC and one from a normal healthy individual) revealed that the usp genes can be classified into two groups, each having different sequences in the 3'-terminal region. The peptides encoded by the three open reading frames (ORFs) downstream of usp had identical 23 amino acid residues in the C-terminal region. The subregion encoding these small ORFs has a mosaic structure constituted of six segments. The positions of these segments vary from strain to strain, and in some strains, two to four segments are deleted. This indicates that rearrangements occur frequently in this region and the mosaic arrangement apparently contributes to the size variation observed in the PCR examination of the usp genes and PIs.

Virulence patterns of Vibrio cholerae non-O1 strains isolated from hospitalised patients with acute diarrhoea in Calcutta, India.

A collection of 28 strains of Vibrio cholerae non-O1 isolated during a 3-year period (1989-1991) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as co-cultures from cases infected with V. cholerae O1. Of the strains isolated in this study, 82% could be serotyped, with serovars O5 (32.1%), O11 and O34 (14.3% each) predominant. Serovars O7, O14, O34, O39 and O97 were associated exclusively with sole infections. Two strains of V. cholerae non-O1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-O1 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4-512. Fewer strains produced a cytotonic effect, with end-point titres of 2-16. Of the 28 strains of V. cholerae non-O1 examined, 75%, 75%, 25% and 14.3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35.7%). Ten (35.7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction.

Primers to amplify the genes encoding the virulence factors of uropathogenic Escherichia coli, such as pilus associated with pyelonephritis (pap), haemolysin (hly), aerobactin (aer) and cytotoxic necrotizing factor 1 (cnf1) genes, were designed. The above primers along with previously reported primers for S fimbriae (sfa) and afimbrial adhesin I (afaI) genes were combined to develop a multiplex polymerase chain reaction (PCR) for detection of the respective virulence factors and for the identification of uropathogenic E. coli. The multiplex PCR to detect pap, sfa, afaI, hly, aer and cnf1 genes was highly specific and the sensitivity was found to be about 5 x 10(3) colony forming units of E. coli per ml. A total of 194 E. coli strains isolated from patients with simple acute cystitis were examined by the multiplex PCR and the results were in complete agreement with that obtained by DNA colony hybridization test. The multiplex PCR developed was, therefore, concluded to be a useful, sensitive and rapid assay system to identify uropathogenic E. coli.

Genotypes and phenotypes of Shiga toxin producing-Escherichia coli isolated from healthy cattle in Thailand.

Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1+/stx2+ (22 strains), stx2+ (22 strains), stx1+ (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae+ and tir+ (4.1%), and katP+ (6.12%). The most predominant profile (22 isolates) was stx1+/stx2+, eae-, tir-, etpD-, hlyA+, katP-. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand.

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.

Estimation of vacuolating cytotoxin secreted by different strains of Helicobacter pylori using bead enzyme-linked immunosorbent assay & its correlation with bacterial genotype.

BACKGROUND & OBJECTIVES: A highly sensitive bead enzyme-linked immunosorbent assay was applied for the quantitative determination of vacuolating cytotoxin (VacA) released in the culture supernatant of 40 well characterized Helicobacter pylori strains in order to clarify the significance of allelic combination of the vacA gene as the predictor of the level of toxin secretion and also to determine the most appropriate genotype of H. pylori associated with high VacA release. Attempts were also made for the detection of VacA in the gastric juice of patients for the rapid diagnosis of H. pylori infection. METHODS: The genotypes of 40 H. pylori strains cultured from the gastric biopsy samples were determined by specific PCRs. The cell-free culture supernatant of the strains as well as the gastric juice of the patients were used for bead-ELISA and the purified VacA from the H. pylori strain ATCC49503 was used as positive control. RESULTS: Ninety per cent of the strains with vacAs1m1 allele combination secrete on an average 146.4 ng/ml of VacA while the corresponding value was 19.1 ng/ml for s1m2 strains. None of the s2m2 as well as the ice negative H. pylori strains produced detectable VacA in the medium while strains expressed the toxin irrespective of the presence or absence of cagA gene. Fifteen of 22 gastric juice samples yielded positive bead-ELISA results. INTERPRETATION & CONCLUSION: vacAs1, vacAm1 and iceA1 could be considered as the determinants of high VacA secretion. Also, the detection of VacA by bead-ELISA in the gastric juice could be considered as an alternative approach in the diagnosis of H. pylori infection.

Fluid accumulation in the ligated intestinal loop and histopathological changes of the intestinal mucosa caused by Clostridium botulinum C2 toxin in the pheasant and chicken.

Fluid accumulation was induced in ligated intestinal loops of both pheasants and chickens injected with crude Clostridium botulinum C2 toxin. Necrosis of the epithelium and lamina propria of the duodenum and jejunum occurred in both species of bird after intraduodenal administration of crude C2 toxin. The severity of such reactions depended upon the dose and the period after administration of C2 toxin up to eight hours, and such reactions were suppressed specifically with rabbit anti-C2 toxin. These results confirm that C2 toxin is one of the causes of diarrhoea in avian botulism.

Botulism among penned pheasants and protection by vaccination with C1 toxoid.

Fifty to 100 per cent of about 8000 penned pheasants raised on a farm in Hiroshima Prefecture died annually for three years. Deaths were ascribed to type C botulism. Vaccination of four groups of pheasants with partially purified C1 toxoid effectively protected them against type C botulism. The protective efficacy of the toxoid was emphasised by the relatively high susceptibility of the pheasant to C1 toxin.

Effects of rearing conditions on the colonization of Salmonella enteritidis in the cecum of chicks.

Salmonella enteritidis is the cause of human salmonellosis associated with contaminated eggs. In this study, we artificially challenged S. enteritidis to chicks just after hatching, and the effects of breeding conditions on the intestinal carriage of S. enteritidis were examined. S. enteritidis was not directly detected from spleen, liver and blood, but were constantly isolated from the cecal contents throughout the experiment. When chicks were reared in the unsanitary conditions and in the high housing density, the numbers of S. enteritidis increased. The subsequent experiment was undertaken to examine whether the antibacterial additive in a feed would have any impact on S. enteritidis colonization in chicks. Some antibiotic effective on the growth promotion had an influence on S. enteritidis colonization.

Establishment of the PCR system specific to Salmonella spp. and its application for the inspection of food and fecal samples.

We established the PCR detection system specific to Salmonella species using Salmonella enterotoxin gene (stn). The detection limit was one bacterial cell per one gram of fecal and minced-meat samples using enrichment procedure by Tripticase soy broth or Salmonella enrichment broth, respectively. We concluded that this PCR system is useful for the practical application in the field of the public hygiene.

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Validation of salmonellosis and shigellosis diagnostic test kits at a provincial hospital in Thailand.

Rapid Diagnosis of salmonellosis and shigellosis was performed using six different diagnostic test kits which recently have been made available commercially. They were Salmo-Dot, Typhi-Dot, Shigel Dot A, B, C, and D test kits for detection of Salmonella spp., group D salmonellae, and groups A, B, C, and D Shigella spp., respectively. The principle of all test kits is a membrane (dot) ELISA using specific monoclonal antibodies to the respective pathogens as the detection reagents. The present study was designed to validate the accuracy of the test kits, at a laboratory in a provincial hospital in Thailand, in comparison with the conventional bacterial culture method alone or with the combined results of the culture and the Western blot analysis (WB) for detecting the respective bacterial lipopolysacchharides (LPS) in specimens. Five hundred rectal swab samples of patients with diarrhea who seeked treatment at the hospital, were evaluated. The diagnostic accuracy of the Salmo-Dot was 91.0% when compared with the conventional bacterial culture method alone but was 100.0% in comparison with the combined results of the culture and the WB. The Typhi-Dot and the Shigel-Dot A, B, C, and D showed 100%, 99.2%, 95.0%, 94.0% and 96.4%, respectively when compared with the culture alone and all were 100% in comparison with the combination of the results of the bacterial culture and the WB. The Shigel-Dot A revealed antigen of type 1 Shigella dysenteriae in several specimens in which the bacteria could not be recovered by the culture method. This difference is important as type 1 Shigella dysenteriae have high epidemic potential and often cause severe morbidity. Unawareness of their presence by the conventional culture may have great impact on disease surveillance for public health. The pathogen detection using the six diagnostic test kits is sensitive, specific, rapid, and relatively simple and less expensive. Several specimens can be tested at the same time without much increase in turn around time. Moreover, these kits produce no contaminated waste as compared with the bacterial culture method. The test kits should be used for rapid screening of specimens of patients with diarrhea especially in areas where culture facilities are inadequate.

Detection of Salmonella contamination in food samples by dot-ELISA, DNA amplification and bacterial culture.

A dot-blot enzyme-linked immunosorbent assay (dot-ELISA) employing a genus Salmonella specific monoclonal antibody (MAb) was used for detection of the bacteria in food samples in comparison with the conventional culture method and the DNA amplification. Among the 200 chicken and pork samples (100 each) tested, 9% and 33%, 7% and 20% and 7 and 23% were positive for salmonellae by the dot-ELISA, the culture method and the DNA amplification, respectively. Statistical analyses revealed that the sensitivity, specificity, efficacy, and positive and negative predictive values of the detection of Salmonella in the food samples by dot-ELISA compared with the culture method were 93.33%, 91.76%, 92%, 66.66% and 98.73%, respectively. Comparison of the DNA amplification and the culture method revealed the sensitivity, specificity, efficacy, and positive and negative predictive values of 100%, 91.58%, 92%, 65.21% and 100%, respectively. The dot-ELISA and the DNA amplification results were in a better agreement when the two assays were compared. The sensitivity, specificity, efficacy, positive and negative predictive values of the dot-ELISA compared to the DNA amplification were 91.3%, 100%, 98%, 100% and 97.5%, respectively. From this study, the dot-ELISA is rapid, simple, sensitive, specific at low cost with limited amount of infectious waste to be disposed and offers another advantage in that it detects only the smooth LPS of Salmonella which implies the possible presence of the virulent organisms.

[Detection of bacterial protein toxins by a bead-ELISA].

A highly sensitive bead-enzyme-linked immunosorbent assay to detect bacterial protein toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as a substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The sensitivities of the bead-ELISA for various bacterial protein toxins were as follows: less than 40 pg/ml for cholera enterotoxin (CT), less than 20 pg/ml for VT1 and less than 6 pg/ml for VT2 of enterohemorrhagic Escherichia coli. The bead ELISA was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea. Of the 75 stool samples examined, 59 yielded biochemically and serologically confirmed strains of Vibrio cholerae O1. The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V. cholerae O1 was isolated. In addition, the bead ELISA was positive for three stool specimens which were negative by culture. These data indicate that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples.