[Preventive measures against infectious diseases brought back to Japan from abroad and caused by imported foods].
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Animals↗
Biomedical subjects
T Miwatani
Publications and source records attributed to T Miwatani.
Production of monoclonal antibody against a hemolysin (Vp-TRH) produced by Vibrio parahaemolyticus.
The antigenicity of a hemolysin (Vp-TRH: Vp-TDH related hemolysin) produced by Kanagawa phenomenon-negative clinical isolates of Vibrio parahaemolyticus was studied using monoclonal antibodies (MAbs). A total of 12 hybridoma clones which produced MAbs against Vp-TRH were established. All MAbs contained the Kappa light chain and were IgG type. These MAbs were divided into a minimum of 5 different specificity groups, including antibodies specific to Vp-TRH and common to both Vp-TRH and Vp-TDH, a possible pathogenic toxin of Kanagawa phenomenon-positive V. parahaemolyticus. These results clearly show the immunological similarity and dissimilarity (specificity) of Vp-TRH and Vp-TDH.
Antibodies, Monoclonal↗
Demonstration of enterotoxigenic Escherichia coli in diarrheic broiler chicks.
An investigation was made to survey the possible presence of enterotoxigenic Escherichia coli (ETEC) in the stools of diarrheal chicks. We analyzed two outbreaks of diarrhea in broiler chicks at two independent farms in the Philippines, from which no pathogens other than Escherichia coli were found. In one outbreak at Farm #1, all 42 isolates produced heat-labile enterotoxin (LT), with 3 of these isolates also producing heat-stable enterotoxin (ST). The O serotypes of 15 strains tested randomly could not be identified as any known serotype (0-antigen; 1-170). In another outbreak at Farm #2, 7 out of 52 isolates produced only LT, their subtypes being identified as O-149 or O-8, common serotypes in pig ETEC. Strains from Farm #1 did not produce any pili usually found in human ETEC. We believe this to be the first isolation of ETEC from diarrheal chicks.
Animals↗
Production of paralytic shellfish toxins by a bacterium Moraxella sp. isolated from Protogonyaulax tamarensis.
A bacterium Moraxella sp. isolated from Protogonyaulax tamarensis was cultured in various conditions. Changes of toxicity and toxin components of the cells during culture were analyzed by bioassay and HPLC-fluorometric analysis. Toxin productivity of Moraxella sp. increased when it was cultured in nutrition-deficient environments. The main toxins produced by Moraxella sp. in these conditions were gonyautoxins (GTXs), mainly GTX 1 and 4 which are major toxins of P. tamarensis.
Animals↗
Comparative analysis of the hemolysin genes of Vibrio cholerae non-01, V. mimicus, and V. hollisae that are similar to the tdh gene of V. parahaemolyticus.
The genes encoding the hemolysins similar to the thermostable direct hemolysin (tdh gene) of Vibrio parahaemolyticus were cloned from chromosomes of V. mimicus and V. hollisae. These cloned hemolysin genes and previously cloned tdh genes of V. parahaemolyticus and V. cholerae non-01 were compared by physical mapping and by hybridization with oligodeoxyribonucleotide probes. The nucleotide sequences in the coding regions of all the cloned hemolysin genes were very homologous and had only minor variations but the sequences flanking the homolysin genes were dissimilar, indicating that the hemolysin genes have a common ancestor and suggesting that they may have been transferred between Vibrio species as a descrete genetic unit.
Biological Evolution↗
A heat-labile enterotoxin (LT) purified from chicken enterotoxigenic Escherichia coli is identical to porcine LT.
We have previously reported that the heat-labile enterotoxin (LTc) isolated from a chicken enterotoxigenic Escherichia coli (ETEC) was identical to LTh produced by human ETEC (Tsuji et al. (1988) FEMS Microbiol Lett. 52, 79-84). In this study, we purified an LTc-like toxin (LTc') from another strain isolated from a chicken that developed diarrhea at a different place and time to the previously reported chicken. Its molecular weight and antigenicity were compared with those of purified LTs from porcine and human ETEC (LTp and LTh). The A subunit of LTc' was identical to those of the purified LTs in mobility on SDS-polyacrylamide gel electrophoresis. The Ouchterlony test demonstrated that LTc' was antigenically identical to LTp. The isoelectric point and amino acid composition of LTc' were also identical to those of LTp. These data suggest that chicken ETEC can be grouped with both the porcine and human types on the basis of the LTs produced.
Amino Acids↗
Bacterial aetiology of infantile diarrhoea in Papua New Guinea.
Bacterial examination of stools of children with diarrhoea was carried out at Port Moresby, Papua New Guinea, where little information is available concerning the causative bacteria of diarrhoea. Shigellae, enterotoxigenic Escherichia coli, Bacillus cereus, Staphylococcus aureus, Campylobacter jejuni, and Aeromonas hydrophila were isolated with the decreasing frequency in that order. Among these, enterotoxigenic E. coli and C. jejuni were isolated for the first time in this country. Although Vibrio parahaemolyticus was recovered from sea water, no cases suffering from this organism were found.
Bacterial Infections↗
[Development and application of enzyme-linked immunosorbent assay using monoclonal antibody against a hemolysin (Vp-TRH) of Vibrio paraheamolyticus--evidence that Vp-TRH producing-Kanagawa phenomenon-negative V. parahaemolyticus is a human pathogen].
Although it has been well established that Kanagawa phenomenon-positive Vibrio parahaemolyticus is a human enteropathogen, the Kanagawa phenomenon-negative one has been considered to be probably not pathogenic. We have found, however, an outbreak of gastroenteritis due to Kanagawa phenomenon-negative V. parahaemolyticus which produces a new toxin (Vp-TRH) resembling to Vp-TDH, a responsible toxin of Kanagawa phenomenon. In this study, we developed monoclonal antibodies against Vp-TRH which were used for development of an enzyme-linked immunosorbent assay (ELISA) for specifically detecting Vp-TRH. The ELISA was applied for analysis of production of Vp-TRH by various isolates of V. parahaemolyticus and we found that Vp-TRH-producing strains were derived mostly from human diarrheal stool, and not from the environment or sea foods. The results of the rabbit ileal loop test showed that Vp-TRH-producing (Kanagawa phenomenon-negative) strains, as well as Vp-TDH-producing (Kanagawa phenomenon-positive) strains could induce fluid accumulation. These results indicate the possibility that Vp-TRH-producing Kanagawa phenomenon-negative V. parahaemolyticus is a human enteropathogen.
Animals↗
Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.
Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.
Amino Acid Sequence↗
Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus.
A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.
Amino Acid Sequence↗
Characterization of Vibrio cholerae non-O1 hemagglutinin/protease.
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Binding Sites↗
Charge heterogeneity of the heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli.
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Animals↗
Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli.
We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the pI points of B subunits of the LThs were identical to each other, the pI points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli.
Bacterial Toxins↗
A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus.
A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus was isolated from the culture of a strain of this organism mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.
Amino Acids↗
Homogeneity of a hemolysin(Vh-rTDH) produced by clinical and environmental isolates of Vibrio hollisae.
The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.
Animals↗
A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation.
We isolated a mutant strain of enterotoxigenic Escherichia coli by nitrosoguanidine mutagenesis, which produces an immunologically altered B subunit of heat-labile enterotoxin. This mutant B subunit was detected as a monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis even without prior heating, suggesting a problem in oligomer formation. Furthermore, this mutant B subunit could not form holotoxin with the native A subunit, and the affinity to GM1-ganglioside receptor was 10-fold lower than that of the native B subunit. The amino acid sequence analysis of this mutant B subunit revealed only one amino acid substitution compared with the native B subunit, at the 64th position from the N terminus (valine instead of alanine). These data suggest that the alanine at position 64 from the N terminus is important for the native B subunit to form an oligomer structure and express its functions.
Alanine↗
Crystallization and preliminary X-ray diffraction study of the heat-labile enterotoxin B subunit produced by enterotoxigenic Escherichia coli.
Heat-labile enterotoxin LT produced by enterotoxigenic Escherichia coli is composed of A and B subunits. The A subunit is enzymatically active; whereas, through the action of the B subunit, the toxin binds to the receptor, a GM1 ganglioside present on the cell surface. Crystals of the LT-B subunit were formed at room temperature by vapor diffusion with polyethylene glycol in the presence of the non-ionic detergent beta-octylglucoside. The crystals were characterized by X-radiation as orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions of a = 224.1 A, b = 65.3 A, c = 118.4 A. They diffract X-rays to a resolution of at least 2.5 A and are stable to X-rays.
Bacterial Toxins↗
Purification of a TDH-related hemolysin produced by a Kanagawa phenomenon-negative clinical isolate of Vibrio parahaemolyticus 06: K46.
A hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus 06: K46 was purified by 55% ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q. The purified hemolysin was physicochemically and immunologically identical with the Vp-TRH (V. parahaemolyticus thermostable direct hemolysin related hemolysin) recently described in V. parahaemolyticus 03: K6 (Honda et al. Infect. Immun. 56: 961-965, 1988). This indicates that V. parahaemolyticus of Kanagawa-negative clinical isolates possessing not only 03: K6 but also different serotypes such as 06: K46 produce Vp-TRH. Production of Vp-TRH by most clinical isolates of Kanagawa-negative V. parahaemolyticus was also demonstrated. These results suggest the importance of Vp-TRH among clinical isolates of Kanagawa-negative V. parahaemolyticus.
Diarrhea↗