Immunochemical properties ofVibrio cholerae LPS

Immunochemical analysis of LPS isolated fromVibrio cholerae O1 and non O1 showed that this macromolecular complex shares common antigenic epitopes in the sugar moiety. The epitopes can be detected after mild alkaline hydrolysis of LPSin vitro. Membrane-associating activity of both O1 and non O1 LPS did not indicate any differences of the species.     

Mechanism of action ofVibrio cholerae enterotoxin

Summary The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30°C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximateK _m for ATP, Mg⁺⁺ increases from about 1.8 to 3.4mm in the toxinstimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg⁺⁺ concentrations, from 20% at low levels (0.2mm) to 500% at high concentrations (greater than 3mm). Addition of GTP, Mg⁺⁺ (0.2mm) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation ofV _max. GTP enhances the toxin-treated enzyme activity two-to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6mm ATP and 5mm MgCl₂ the apparentK _a for GTP, Mg⁺⁺ is 5 to 10m. The control (unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide. 5-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg⁺⁺, decreasing the apparentK _a from about 25 to 5mm. (–)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparentK _a for free metal. Sodium fluoride, which cause a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparentK _a for Mg⁺⁺; moreover, it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three- to fivefold stimulation by toxin becomes a 30 to 60% inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (–)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.Part of this work was reported at the 1974 meeting of the Federation of American Societies for Experimental Biology (Bennett & Cuatrecasas, 1974).     

Ion-swimming speed variation ofVibrio cholerae cells

In the present work we report the variation in swimming speed ofVibrio cholerae with respect to the change in concentration of sodium ions in the medium. We have also studied the variation in swimming speed with respect to temperature. We find that the swimming speed initially shows a linear increase with the increase of the sodium ions in the medium and then plateaus. The range within which the swimming speed attains saturation is approximately the same at different temperatures.     

Lytic effect ofVibrio cholerae elastase on Gram-positive and-negative bacteria

Elastase ofVibrio cholerae caused the lysis of freshly grown cells of Gram-negative (Pseudomonas aeruginosa, Proteus vulgaris, Salmonella paratyphi A andKlebsiella pneumoniae) bacteria. Gram-positive (Staphylococcus aureus andS. epidermidis) organisms were resistant to this enzyme. Heat killed and lyophilized Gram-positive and-negative bacteria (exceptS. aureus andS. epidermidis) showed higher sensitivity to elastase. Both Gram-negative and-positive bacteria were lyzed maximally by elastase at pH 8.0. At this pH, lytic activity of elastase was maximum in Tris-HCl and glycine-NaOH buffers followed by Tris-maleate and cacodylate buffers.     

Ecology ofVibrio cholerae in the freshwater environs of Calcutta,India

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater accumulations, located in Calcutta.V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August–September and a secondary peak in May–June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities ofV. cholerae. In the lake, sediment samples harbored high densities ofV. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts ofV. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures ofV. cholerae, indicating that the rabbit intestine selects outV. cholerae from a mixed flora. Uniformly high isolation rates ofV. cholerae were observed from brackish water and freshwater species of export quality prawns.V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded thatV. cholerae non-01 is common in the freshwater environs of Calcutta.     

Phenotypic characterization of clinical and environmental isolates ofVibrio cholerae from Australia

One hundred fifty-seven isolates possessing the biochemical traits associated withVibrio cholerae were submitted to an extensive phenotypic characterization. A numerical analysis of the results suggested that isolates presently assigned to the biotypescholerae, eltor, andalbensis ofV. cholerae do not possess consistent phenotypic differences supporting their separation into distinct biotypes. The results further indicated that clinical and environmental isolates of non-O1 serotypes ofV. cholerae are phenotypically indistinguishable from strains ofV. cholerae serotype O1. This study also confirmed the persistent presence ofV. cholerae in the Australian environment.     

Direct immunofluorescence assay for rapid environmental detection ofVibrio cholerae O1

An immunofluorescence assay for direct detection of V. cholerae O1 was developed using polyclonal antibodies raised against outer membrane proteins (OMPs) of V. cholerae O1. Production of OMPs varied with growth media used; maximum production was found in tryptic soy broth. The detection system was specific because no cross-reactivity was observed with other bacteria including V. cholerae O139, E. coli, S. dysenteriae and Salmonella enterica subsp. enterica serovar Typhi. The technique was able to detect 240 CFU/mL of V. cholerae O1 suspended in phosphate-buffered saline. The assay coupled with bacterial enrichment in APW for 6 h detected as few as 5 CFU of V. cholerae in spiked samples. Moreover, a 2-h incubation of enriched bacterial cells in 0.1% yeast extract with 10 ppm nalidixic acid enhanced the bacterial size and helped in morphological identification of V. cholerae. Among 32 potable water samples from afflicted hand pumps and wells collected from a cholera-plagued area 12 were found to be contaminated with V. cholerae by immunofluorescence assay as well as by conventional culture methods. The proposed method could thus be employed in environmental surveillance of V. cholerae O1.     

Genetic relationships among clinical and environmental isolates ofVibrio cholerae from Australia

DNA-DNA homology among twenty-nine isolates having the phenotypic properties ofVibrio cholerae was studied using the S1 endonuclease method. Ten strains ofV. cholerae O1 isolated from patients and from the environment in Australia showed greater than 88% homology with the neotype strain ofV. cholerae NCTC 8021. Strains of the non-O1 serotype isolated from a variety of clinical and environmental sources also showed a high level of relatedness, including four luminescent strains and a reference strain of the biotypealbensis. A group of sucrose-negative strains showed a low level of homology (40 to 43%) withV. cholerae, but 75% and 82% homology within the group.     

Effects of furazolidone on the mutation ofVibrio cholerae cells to streptomycin resistance

Furazolidone induced the streptomycin-resistant (Str-r) forward mutation ofVibrio cholerae (classical) OGAWA 154 cells. The induced mutation frequency increased up to the furazolidone dose of 7.0 g/ml×h and then gradually declined. Statistical analysis (t-test and variance analysis) revealed that the furazolidone-induced mutation ofV. cholerae cells at any of the doses studied was highly significant.     

A phenotypic and genetic study of sucrose nonfermenting strains ofVibrio mimicus andVibrio cholerae

Sixty-six strains unable to ferment sucrose and resemblingVibrio mimicus andV. cholerae were submitted to an extensive phenotypic characterization. DNA-DNA homology among selected strains and the type strain ofV. cholerae was studied by the S1 endonuclease method. Seven sucrose-negative strains were shown to have the phenotypic properties of and a high percentage DNA relatedness toV. cholerae and a low level of homology withV. mimicus. Eight luminescent strains phenotypically most closely resembledV. mimicus; however, two of these were shown to have a high level of DNA homology withV. cholerae and a low level of relatedness toV. mimicus. A single strain was found to be phenotypically and genetically unrelated to eitherV. cholerae orV. mimicus and may represent a new species. The remaining strains were phenotypically shown to beV. mimicus, and selected strains were shown to have a high percentage DNA homology withV. mimicus and a low level of homology withV. cholerae. Problems associated with the identification of these strains and differential traits are discussed.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Copepod succession in two South African estuaries

The seasonal succession of copepod species was studied fromNovember 1976 through October 1978 in the Swartkops and Sundaysriver estuaries. South Africa. Acartia natalensis appeared inthe plankton in spring and reaches maximum abundance duringsummer and autumn. It was replaced by A. longipatella in lateautumn which reached maximum abundance in winter and spring.Cycles of dominance are regulated by the interaction of temperature,salinity and competition between the two species. A natalensis a more tolerant of low salinity and the replacementof A. longipatella by A. natalensis starts in the upper estuaryand spreads seawards. Maximum abundance of A. natalensis isattained in water of lower salinity than in the case of A. longipatella.In the Sundays estuary A. natalensis appeared briefly in thelatter half of the study and in the upper and middle estuaryonly. In the lower estuary A. longipatella was present duringall seasons. This was due to the absence of competition fromA. natalensis, high salinity and lower summer temperatures dueto marine influence. A third species of copepod, Pseudodiaptomushessei functions as a pioneer species, exploiting "new water"after flooding or strong fresh water inflow. High abundancemay therefore occur during any season and no competition betweenP. hessei and either species of Acartia was observed.     

Occurrence of Vibrio cholerae serotype O1 in Maryland and Louisiana estuaries.

Vibrio cholerae serotype O1 has been isolated from Chesapeake Bay in Maryland and estuaries and sewers in Louisiana. The occurrence of V. cholerae O1 in the aquatic environment in the absence of human disease suggests that this organism survives and multiples in the natural environment.     

Lipopolysaccharides ofVibrio fluvialis 181-86 Kobe possessing an antigenic factor in common with O1Vibrio cholerae

The chemical and serological characteristics of lipopolysaccharides (LPS) isolated fromVibrio fluvialis 181-86 Kobe were compared with those of LPS isolated from O1Vibrio cholerae and Vibrio bio-serogroup 1875 (Original and Variant), a marine vibrio possessing an antigenic factor in common with O1Vibrio cholerae. Two kinds of LPS (S-type LPS and R-type LPS) were extracted fromV. fluvialis 181-86 Kobe. The S-type LPS is different from LPS of the other serotypes (O1–O18) ofV. fluvialis in that the former was found to contain the pair perosamine and quinovosamine, a characteristic component of O1V. cholerae LPS. Furthermore, the sugar composition of the S-type LPS is close to that of Vibrio bio-serogroup 1875 LPS because both LPS contained mannose and the same two unidentified neutral sugars. Definite serological cross-reactivity in the passive hemolysis and the passive hemolysis inhibition tests were demonstrated between LPS fromV. fluvialis 181-86 Kobe and that from Vibrio bioserogroup 1875 Variant.     

Incidence of Vibrio cholerae from estuaries of the United States West Coast

The incidence of Vibrio cholerae in shellfish, sediment, and waters of California, Oregon, and Washington was determined during the summer of 1984. Samples from 24 distinct estuaries were analyzed qualitatively. V. cholerae non-O1 was found in 23 estuaries and in 44.6% of the 529 samples examined. V. cholerae O1 Inaba was isolated from water samples in Morro Bay, Calif. Vibrio mimicus was found in 2.3% of the samples. Cholera enterotoxin was not found in cell-free filtrates of the 100 isolates tested in the Y-1 mouse adrenal cell assay, but heat-labile cytotoxic activity was observed with 3% of the isolates.     

Studies on the elimination of non-specific inhibitors in sera against influenza viruses with the aid of filtrates ofVibrio cholerae

Summary In the sera of humans and various animals there are two different non-specific inhibitors (α-and β-inhibitors) which may be specifically abolished by two substances (α-and β-“enzymes”) both present in filtrates ofV. cholerae. This neutralization is necessary for the rapid classifying of influenza virus strains with the aid of the haemagglutination inhibition test. The egg line strains of the A- and A′-groups are only sensitive to β-inhibitor, while the mouse line strains of the same groups are only sensitive to the α-inhibitor. This does not apply to strains of the B group, where mouse as well as egg lines are sensitive to both inhibitors. With the aid of isolated β-inhibitor (easily to be prepared from cattle serum), it is possible to decide in a quick manner whether or not a strain from the A- or A′-group has previously been adapted to the mouse. With the technical assistance of Miss I.de Nooyer and with the financial support provided by the Institute for Preventive Medicine, Leyden; N.V. Philips Roxane, Weesp; the State Department of Science; the Jan Dekker Fund, and the Curacao Fund for Preventive Medicine.     

Isolation of Vibrio cholerae Serotype Ogawa from a Florida Estuary

Vibrio cholerae serotype Ogawa was recently isolated from the estuarine waters of Apalachicola Bay, Fla., in areas that are subject to consistent fecal contamination and in areas that are remote from any apparent source of contamination. The significance of these organisms in the environment has not been determined.     

Incidence of Vibrio cholerae from estuaries of the United States West Coast.

The incidence of Vibrio cholerae in shellfish, sediment, and waters of California, Oregon, and Washington was determined during the summer of 1984. Samples from 24 distinct estuaries were analyzed qualitatively. V. cholerae non-O1 was found in 23 estuaries and in 44.6% of the 529 samples examined. V. cholerae O1 Inaba was isolated from water samples in Morro Bay, Calif. Vibrio mimicus was found in 2.3% of the samples. Cholera enterotoxin was not found in cell-free filtrates of the 100 isolates tested in the Y-1 mouse adrenal cell assay, but heat-labile cytotoxic activity was observed with 3% of the isolates.     

Structural organization of the transfer RNA operon I ofVibrio cholerae: Differences between classical and El Tor strains

Nine major transfer RNA (tRNA) gene clusters were analysed in variousVibrio cholerae strains. Of these, only the tRNA operon I was found to differ significantly inV. cholerae classical (sixth pandemic) and El Tor (seventh pandemic) strains. Amongst the sixteen tRNA genes contained in this operon, genes for tRNA Gln3 (CAA) and tRNA Leu6 (CUA) were absent in classical strains as compared to El Tor strains. The observation strongly supported the view that the above two pandemic strains constitute two different clones.     

Seasonality and toxigenicity ofVibrio cholerae non-01 isolated from different components of pond ecosystems of Dhaka City,Bangladesh

Diarrhoea due toVibrio cholerae non-01 is common in Bangladesh. Four hundred and eighty samples, including plants, water, phytoplankton and sediment, were collected from five ponds in Dhaka every 15 days for one year.V. cholerae non-01 was isolated from 181 (38%) of the samples. Two peaks were evident: one in April and the other in August/September. Forty-three (23%) of the 181 isolates were examined for toxigenicity and 19% were cytotoxic to Y1 adrenal cells. This study provides evidence of the likely infectious nature of some ponds and may have relevance to the epidemiology of diarrhoea caused byV. cholerae non-01 in Bangladesh.