A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

Makeup of a population El Tor vibrios according to their agglutinability by type-specific sera]

The authors studied population composition of 14 strains of EL Tor vibrios. Of 7 strains of Ogava serological type the population composition was homogeneous in 2, and heterogeneous in 5. All the 7 strains of EL Tor vibrios of Inaba serological type had homogeneous population composition. Changes of the existence conditions of EL Tor vibrios of Ogava serological type, containing in their population 5% cells of Hikoshima serological tupe, led to separation of the cells of Inaba serological type; under the same conditions, in El Tor vibrios of Ogava and Inaba serological type with homogeneous population the latter was stable.     

Purification of El Tor cholera enterotoxins and comparisons with classical toxin.

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.     

Long-term survival of E1 Tor cholera vibrios in naturally contaminated sewage]

El Tor cholera vibrios of Ogava serological type were revealed in the sewage of the locomotive shed for 15 months. In experiment with an oil catcher in naturally infected sewage El Tor vibrios survived 36 days, in storage of this sewage at the laboratory--39 days, in the artificially infected sewage of a settlement and of a milk plant--2 and 11 days, respectively, in the oil and disel fuel--14 months. Consequently, El Tor vibrio can survive in the sewage with a high oil product content for a long time.     

Detection of cholera toxin by a highly sensitive bead-enzyme linked immunosorbent assay.

A bead-enzyme linked immunosorbent assay (bead-ELISA) for detection and quantification of cholera toxin (CT) in broth cultures of Vibrio cholerae O1 has been developed. Under optimal buffer and pH conditions the bead-ELISA could consistently detect 40 pg/ml of CT. None of the ingredients of commonly used media for in vitro culture of V. cholerae O1 hindered the performance of the bead-ELISA. Evaluation of the sensitivity and specificity of the bead-ELISA against the commonly used reversed passive latex agglutination (RPLA) test for detection of CT was performed using a collection of 239 strains of V. cholerae O1 (including both biotypes and serotypes) which were examined by a gene probe encoding for the A1 subunit of CT. Although both the assays were highly specific, the bead-ELISA was more sensitive than the RPLA. Quantification of CT by the bead-ELISA revealed that the concentration of CT produced by the strains of V. cholerae O1 which were negative by the RPLA was lower than 1 ng/ml and therefore below the minimum detection ability of the RPLA. The bead-ELISA is a simple, specific and highly sensitive assay for routine detection of CT and is recommended for routine use in clinical microbiology laboratories.     

Epidemiological patterns of the import and spread of El Tor cholera]

The paper is a response to the paper by A. K. Akiev published in 1974 in the "Journal of Microbiology Epidemiology and Immunobiology": "Concerning the Epidemiology of El Tor cholera Abroad". The opinion of the author concerning the origin of El Tor infection in 1970, the sources of infection, and the factors of its transmission is critisized. Literature data and personal observations explaining the regularities of importation and spread of El Tor cholera as an intestinal infection are presented; these data are against the view of Akiev on El Tor cholera as a disease with a natural nidality caused by freely living vibrios.     

Expression and detection of different biotype-associated cell-bound haemagglutinins of Vibrio cholerae O1

The expression of two cell-bound haemagglutinins, one sensitive to L-fucose (FSHA) and the other to D-mannose (MSHA), on Vibrio cholerae O1 strains of both the classical and the El Tor biotypes was studied by (i) agglutination of chicken and human group O erythrocytes in the presence of L-fucose or D-mannose, (ii) binding of the bacteria to L-fucose- and D-mannose-coated agarose beads, and (iii) agglutination of the bacteria by 'biotype-specific' antisera. All of the 12 classical strains studied that were isolated before 1979 gave FSHA of human O erythrocytes whereas only 6 of 17 classical strains isolated during recent epidemics expressed FSHA; a few of the classical strains expressed MSHA in addition to FSHA. All the El Tor strains gave MSHA of chicken erythrocytes and one strain also expressed FSHA. Both the cell-bound HAs were optimally expressed during the exponential phase of growth; FSHA markedly decreased during the late exponential phase while the MSHA usually persisted into the stationary phase. The expression of FSHA and MSHA correlated very well with the direct binding of vibrios to fucose- and mannose-coated agarose beads, respectively. Antiserum 'specific' for classical vibrios agglutinated classical strains expressing FSHA and also the El Tor strain exhibiting FSHA. Similarly, the anti-El Tor serum agglutinated all El Tor strains and also classical strains expressing MSHA, suggesting that the 'biotype-specific' sera were specific for the biotype-associated cell-bound HAs.     

Interaction of cyclosporine A and calcitonin on bone resorption in vitro

Cyclosporine A (CsA), which is a potent immunosuppressive agent, inhibits bone resorption in vitro. The inhibition of bone resorption by CsA is sustained, unlike the transient inhibition of bone resorption produced by calcitonin (CT). These different patterns of inhibition were studied by examining the interaction between CsA and CT on stimulated bone resorption in the neonatal mouse calvarial resorptive system. "Escape" from the CT inhibition of PTH stimulated bone resorption occurred after 24 hr of organ culture. Coincubation with CsA (1 micrograms/ml) delayed the "escape" response of CT + PTH treated bones, so that the full "escape" response did not occur until after 48 hr of organ culture. Likewise, a pretreatment of 24 hr with CsA (1 micrograms/ml) was sufficient to delay "escape" from CT inhibition of PTH stimulated bone resorption until after 48 hr of organ culture. A higher concentration of CsA (10 micrograms/ml) completely prevented the "escape" response. Our data could indicate an interaction between the CsA and CT inhibitory effects on resorption.     

Variation in epitopes of the B subunit of El Tor and classical biotype Vibrio cholerae O1 cholera toxin

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.     

Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide

Lipopolysaccharide (10 micrograms/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 +/- 1.3 ng/10(6) cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was less than 1.6 ng/10(6) cells. Leukotriene C4 was characterized by reversed-phase high-performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5-60 micrograms/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C4 was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2 alpha by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.     

DNA damage and cell killing by nitrofurantoin in relation to its carcinogenic potential

Nitrofurantoin inhibited growth and produced loss of viability of Vibrio cholerae cells in a dose-dependent manner, the 10% (D10) and 37% (D37) survival doses being 18.0 and 5.5 micrograms/ml x hr. respectively. The drug also caused filamentation of the cells in a very significant manner. Ultraviolet absorption data and thermal chromatography through hydroxyapatite column revealed that nitrofurantoin treatment of Vibrio cholerae cells produced a maximum amount of 55% of DNA reversibly bihelical due to the formation of inter-strand cross-links. Helix-coil transition studies carried out by viscometric and also, spectrophotometric methods revealed that the nitrofurantoin-induced cross-links in Vibrio cholerae DNA, imparted to this DNA greater thermal stability than that of native DNA. The quantitative aspect and also the mode of nitrofurantoin action on DNA of Vibrio cholerae and Escherichia coli cells vis-à-vis the carcinogenic potential of the drug were discussed.     

Molecular-epidemiological characteristic and possible origin of Vibrio cholerae non O1/non O139 with complete and limited set of virulence genes

Study of molecular-epidemiological characteristics of Vibrio cholerae non O1/non O139 serogroup with complete and limited set of virulence genes was performed. Differences of their genes composition as compared to these of O1 serogroup (classic and El Tor biovars) were revealed, which points to their origin from avirulent environmental cholera vibrios.     

Expression of virulence factors by classical and El Tor Vibrio cholerae in vivo and in vitro

Abstract We examined the production of virulence factors of Vibrio cholerae O1 bacteria, and especially compared expression in vitro under near optimal growth conditions with that in vivo during experimental cholera infection. The results show that the in vitro formation of cholera toxin (CT), soluble hemagglutinin (SHA), colonizing pilus TCP, and the biotype associated hemagglutinins FSHA and MSHA, as well as of various cell envelope antigens often rather poorly reflected expression in vivo. For instance, production of CT by vibrios of classical biotype and of TCP by the El Tor biotype were enhanced in vivo, while production of SHA was instead suppressed. Likewise significant differences in cell envelope antigen composition were found between bacteria grown in vivo and in vitro. A more precise definition of the role of different postulated virulence factors in the processes of infection and immunity should include in vivo studies as outlined by this study.     

Variation in the genome of CTXphi prophage of Vibrio cholerae biovar El Tor caused by Tn5-Mob transposon

A key pathogenicity factor of the cholera etiologic agent is cholera toxin (CT) whose synthesis is encoded by the ctxAB operon forming apart of the CTXphi ptophage. Alterations in the virulent properties of the cholera vibrios are based on the variability of the CTXphi prophage containing the genes for ctxAB, zot, ace, cep, orfU, and psh in its core region. At the same time, the mechanism of the porophage genome reorganization needs further and more profound analysis. The goal of this work was to demonstrate that transposon Tn5-Mob (Kmr), when introduced into the chromosome of the V. cholera model strain MAK757 El Tor biovar containing two copies of the CTXphi prophage provoked a reorganization in the CTXphi prophage consisting in the deletion of zot, ace, cep, orfU genes. The level of the CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob still retaining only the ctxAB operon, increased more than 2000 times as compared to that of the original strain. The enhanced CT production was shown to be associated with the altered structure of the chromosomal DNA region containing one copy of the ctxAB operon encoding this protein biosynthesis. The mutation in the CTXphi genome induced by Tn5-Mob was unstable. Among 600 isolated colonies obtained after dissemination of the MAK757 chr::Tn5-Mob transposant capable of CT overproduction in the full medium with no antibiotics, 5.8% gave clones that in parallel to the loss of Kmr marker, appeared to be deprived of the ctxAB operon thus becoming non-toxinogenic. The observed formation of the V. cholerae insertion mutants both capable of CT overproduction and non-toxinogenic ones, may be indicative of an important role played in the evolution of the cholera pathogen by the CTXphi genome variability induced by Tn elements. The plasmidless V. cholerae El Tor strain characterized by type II CT hyperproduction thus obtained in our experiments could be used for the production of this protein routinely applied to construct efficient cholera diagnostic and prophylactic preparations.     

The fibrinogenolytic activity of purified tryptase from human lung mast cells

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.     

Electron microscopy of cholera vibrios from the intestinal contents of infected suckling rabbits]

Electron microscopic study of the cells of classic, El Tor and NAG-vibrios showed them to be no different by the structure type from the cells of ofter Gram-negative bacteria. A characteristic peculiarity of the cholera vibrios revealed after their passage through the intestine of nursling rabbits was the presence of microcapsules and protrusions of the areas of the wall membranous apparatus.     

Phage typing of cholera vibrios with different sets of phages]

A comparative study was made of two sets of Mukerjee and Drozhevkina-Arutyunova's bacteriophages in typing 514 strains of the El Tor vibrios and 45 strains of clasic biotype. It was shown that the Mukerjee or Drozhevkina-Arutyunova's phages could be used for the typing of cholera vibrios. The phages of the latter set prove to detect more phage types (18 against 11); they determine both the phage type and the biotype at the same time. The typing of cholera vibrios of both biotypes is possible, and the percentage of nontyping strains left is comparatively low (5.2 against 23.5 after Mukerjee). A table of the phage correspondence was made; it permits to obtain comparable data in using any set of the typing phages.     

Identification of Vibrio cholerae by Pyrolysis Gas-Liquid Chromatography

Cholera-like vibrios examined by pyrolysis gas-liquid chromatography could be distinguished from other common aerobic gram-negative bacilli, including oxidase-positive organisms, e.g., Aeromonas. Vibrios in Heiberg group I were subdivided into three types on the basis of differences in one complex in the chromatogram, and these closely corresponded with the identification as classical, El Tor, or "intermediate" biotypes of Vibrio cholerae by conventional methods.     

PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.