The use of the gene hybridization method for the identification of epidemiologically dangerous strains of cholera vibrios

Using the labeled DNA fragments containing the genes for cholera toxin the strains of cholera vibrios were studied for the presence of cholera toxin genes. Vibrio cholerae strains isolated from natural water reservoirs under the favourable epidemic situation do not contain the cholera toxin genes. The DNA hybridization method was compared with other methods used in research and practical work for estimation of epidemic importance of cholera vibrios.     

Role of neuraminidase from cholera vibrions in the pathogenesis of experimental cholera]

A study was made of a possible effect of neuraminidase of cholera vibrios on cholera pathogenesis. It was shown that in intraintestinal injection of cholera vibrios of the El Tor biotype to nursling rabbits neuraminidase could be revealed in their intestine 5 to 8 hours after the infection. Addition of neuraminidase to the weakly cholerogenic strain cholera vibrios intensified its cholerogenic action in infection of the animals. The antineuraminidase serum administered to the infected rabbits prevented clinical manifestations of experimental cholera, although it failed to always eliminate the cholerogenic syndrome (revealed during autopsy). At the same time neuraminidase did not influence the capacity of cholerogen to produce the cholerogenic syndrome. The authors consider that the action of the enzyme should occur at the early stages of the pathogenic process, and could be associated with creation of conditions for the attachement of cholera causative agent to the intestinal wall or for the action of their exotoxin.     

Rapid Method of Determining Cholera Vibrio Biotype

The characteristic motility of cholera vibrios, as viewed through a dark-field microscope, and the adhesiveness of chicken cell-positive vibrios provide a means for rapidly identifying and biotyping cholera vibrios. Dilute suspensions of vibrios, such as one might find in a fresh rectal swab specimen from a cholera patient, when mixed with a 0.25% suspension of chicken erythrocytes in saline, can be used to biotype the cholera vibrios without prior isolation in pure culture. This is accomplished by using a dark-field microscope through which the chicken cell-positive cholera vibrios are observed to attach to the scattered erythrocytes and to propel them with a characteristic flipping motion.     

Detection of L forms of Vibrio cholerae in the water of open water reservoirs

L-forms of cholera vibrios were isolated from the river water for the first time. The presence of L-forms in water permitted to suppose that such variants served as one of the forms of cholera causative agent preservation in the external medium.     

Lipopolysaccharides of R mutants isolated from Vibria cholerae.

The chemical and serological properties of lipopolysaccharides isolated from the S form and from the R form of cholera vibrios were compared. It was found that the S-R mutation of cholera vibrios involves total elimination of the two component amino sugars of S-form lipopolysaccharides, i.e. quinovosamine and perosamine. This elimination resulted in the loss of O-specificity of S-form lipopolysaccharides and concomitant appearance of strong serological cross-reactivity, in the passive-haemolysis-inhibition test, among R-form lipopolysaccharides regardless of the serotypes (Inaba and Ogawa) of their S parent strains.     

Prevalence of type III secretion system genes in cholera vibrios from different serogroups

Prevalence of vcs genes coding the type III secretion system (T3SS) in cholera vibrios of different serogroups isolated in Russia and neighboring countries was studied for the first time. Virulent strains of O1 and O139 serogroups as well as toxigenic Vibrio cholerae strains of other serogroups contained no T3SS genes. Unlike mentioned strains, 29.2% of atoxigenic non O1/non O139 cholera vibrios isolated from patients in Russia and neighboring countries contained the T3SS genes cluster, which might contribute to the pathogenic properties of these strains.     

Houseflies (M. domestica L.) as transmitters of the agent of cholera

The authors present the experimental results of study of the role played by domestic flies in the spread of cholera causative agents. It was found that cholera microbes survived on the external surface of flies for 5 to 7 days, and in the insect organism--in the course of their whole life. Cholera vibrios underwent no sharp changes in the organism of flies. By means of individual infection method and keeping of flies excluding a possibility. By means of individual infection method and keeping of flies excluding a possibility of repeated autoinfection it was revealed that cholera vibrios could multiply in the organism of domestic flies. The infected insects can discharge cholera vibrios for a long time into the external environment and contaminate food.     

Determination of the degree of similarity of cholera and NAG vibrios

The numeric taxonomy taking into account 80 signs has demonstrated the similarity of NAG and cholera vibrios, the average similarity coefficient exceeding 80 %. Among NAG vibrios, 53 % of the strains have been found to deviate from the central strain of V. cholerae mainly with regard to their utilization of the sources of carbon. The use of the citrate sign for the study of the biological properties of NAG vibrios is proposed.     

The use of a CHO cell culture in studying Vibrio cholerae grown under different conditions]

Studies of biological activity of cholera vibrios in cultures of chinese hamster ovary cells (CHO) have revealed their strong dependence on culture conditions. Elongation of CHO cells is caused only by choleragenic strains. Under stationary conditions of culture the vibrios were found to release haemolisin into the medium and had a cytotoxic effect. Most of cytotoxic supernatants exhibited a neuraminidase activity. Proteolytic activity was less dependent on the vibrio culture conditions. Strains with a high proteolytic activity caused rounding of the CHO cells.     

Use of a new method for restoring the type properties of atypical Vibrio cholerae

Subcultures with a number of signs characteristic of epidemically significant strains have been isolated from cholera vibrios, nonpathogenic and atypical in a number of properties, by a new in vitro method developed by the authors. This method makes it possible to increase the virulence of poorly agglutinating cultures of V. cholerae O1 and their agglutinability with cholera antisera.     

Cholera vibrios lectins as main pathogenicity and persistence factors (biotechnological aspects of use)

Literature data and results of our studies of lectins are analyzed in the review. All the leading pathogenicity factors of cholera vibrios that possess enzymatic activity--cholera toxin, hemolysin, neuraminidase, chitinase have several lectin domains, that determine not only their pathogenetic role but also open perspectives for their use in medical practice. At the same time the variable receptor profile of cholera vibrios cells of various biovars and epidemical significance established with hemagglutination inhibition reaction by carbohydrates could be used to develop new principles of testing and typing of cholera vibrios.     

The agglutinability of cholera O-monoclonal immunoglobulins]

A set of 10 monoclonal antibodies specific for vibrio species and of 5 monoclonal antibodies specific for serovar (Ogawa) was studied. These monoclonal antibodies were directed toward V. cholerae O1 antigen in agglutination reaction and on slide plates. Monoclonal antibodies agglutinating typical strains of cholera vibrios with titration range from 1: 6000 to 1: 25,000 were selected. MA were revealed to interact with cholera vibrio strains with reduced agglutinability. The set of agglutinating O monoclonal immunoglobulins was developed for laboratory identification of cholera O1 vibrios.     

Model of Vibrio cholerae biofilm as a mechanism of its survival in surface water reservoirs

Vibrio cholerae eltor strains with different epidemic importance isolated from river water in the city of Vladivostok during a cholera outbreak (1999) and in the city of Irkutsk during a safe cholera period (2005) are used in the experiment. A biofilm structure consisting of a peripheral part, bundles, polysaccharide matrix, canals, and polymorphic vibrios is presented by light and luminescent microscopy. The metachromatic pink coloring of the matrix (crystal violet and toluidine blue) or fluorescent reddish orange color (acridine orange) are evidence of acid mucopolysaccharide (glucosaminoglycans) content. The biofilm of a toxigenic strain as opposed to a nontoxigenic one is formed much later, while the elements comprising its structure are more apparent. The viability of vibrio cells during the experiment (90 days) preserving the initial pathogenic potential testifies to the highly adaptable properties of the Vibrio cholerae eltor, which promotes its survival and existence in surface water reservoirs under favorable ecological conditions (optimal temperature, existence of chitin-containing substratum, etc.).     

Pathogenicity of nonagglutinating vibrios]

In studying the pathogenicity of nonagglutinating vibrios it was established that the majority of the strains isolated from the patients suffering from enteritis possessed enteropathogenic properties which were revealed in the trials on nursling rabbits and on the isolated intestinal loop a of an adult rabbit. In difference to the cholera vibryos, these microorganisms produced no typical cholerogenicity syndrome expressing, however, a number of enteropathogenic properties: caused diarrhea, overfilling of the intestine with fluid, etc. Autopsy showed a typical enterocolitis picture, confirmed by histological studies. Nonagglutinating vibrio cultures isolated from the water and from healthy persons possessed no enteropathogenic properties. An isolated intestinal loop of an adult rabbit proved to be the most sensitive experimental model.     

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.     

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.     

The isolation of Vibrio fluvialis on the territory of the USSR]

For the first time V. fluvialis strains were detected on the territory of the USSR. The taxonomic position of these vibrios was determined by their nucleotide DNA composition (the content of guanine + cytosine was 49.3-51.0 mole%) and the characteristic features of their phenotype. The individual features of the strains consisted in their capacity for agglutination with cholera antisera, groups 01 and Inaba, in diagnostic dilutions in the presence of differences in genomes and phenotypes with cholera vibrios. Molecular hybridization DNA-DNA also gave no confirmation of their relationship to cholera vibrios (23-26% homology). The comparative study of V. fluvialis strains from the USSR and other countries by a broader set of their phenotypical signs confirmed their identity.     

Retrospective VNTR-analysis of genotypes of Vibrio cholerae 01 strains isolated, during the 7th cholera pandemic, in Rostov Region

Antiplague Research Institute, Rostov-on-Don, Russia Retrospective multi-locus VNTR-analysis was made for 166 Vibrio cholerae strains isolated, 1967-2001, in Rostov Region from clinical samples (82 strains) and from water samples (84 strains). On the basis of cluster analysis of heterogeneous identification strain genotypes, 45 variations of individual strains were shared between 11 separate clusters, among which the F cluster vibrios were predominant. Having emerged, 1970, in the region, they were widely spread during the 1973-1975 cholera pandemic and were registered, among the isolated strains, till 1992 indicating the possibility of long persistence of V. cholerae 01 in the natural aquatic environment. Presumably, the ecosystem specificity contributed to the long-term vibrio persistence.     

Method for detecting small numbers of Vibrio cholerae in very polluted substrates.

A method is presented for the indirect detection of Vibrio cholerae by the multiplication of two specific bacteriophages: phiH74/64 for El-Tor vibrios, and phage group IV (Mukerjee) for classical vibrios. The product to be examined is seeded in alkaline tryptone water for enrichment, as in the classical method, and is then incubated for 6 h at 37 C. Thereafter, a loopful is transferred to each of two nutrient broth (pH 9) tubes. One of these receives a drop of phage phiH74/64; the other receives a drop of phage group IV. The stock phages are diluted so as to contain about 3,800 plaque-forming units in one drop; this is the maximum amount which, when added to 10 ml of broth, will not be detected in a loopful of 1 mm diameter. The tubes containing phage phiH74/64 are incubated at 42 C; those with phage group IV are incubated at 37 C. After 18 h the cultures are killed by agitation with chloroform, and a 1-mm loopful is deposited on a layer seeded with the detector strains: Makassar 757 for El-Tor phage and V. cholerae 154 for classical cholera phage. After 4 to 5 h at 37 C, lysis appears on the spot areas if there has been phage multiplication in the respective broth tubes. With experimentally contaminated sewage water, vegetables, or stools, 1 to 10 cholera vibrios were detected in every sample. In rare cases, false-positive results were obtained by multiplication of the phage on non-cholera vibrios.