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.     

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.     

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.     

Development of antilipase immunoglobulin polymer diagnosticum for the detection of Vibrio cholerae eltor, possessing hemolytic and lipase activity

The possibility of using a heterogeneous, but structurally similar antigen--the commercial preparation of Pseudomonas sp. lipase (Sigma, USA)--for the development of polymer diagnosticum aimed at determination of lipase production in cholera vibrios was shown. The new diagnosticum (antilipase antibodies) on a polymer carrier was used in the serological volume agglomeration test for the detection of hemolytic atoxigenic V. eltor, obtained from environmental, objects, which produced lipase in 80% of cases. The differentiating capacity of the diagnosticum was confirmed on 120 V. eltor cultures isolated from environmental objects. The newly developed diagnosticum makes it possible to determine the lipase activity in cholera vibrios of different biovars and serovars.     

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.     

In Vitro and In Vivo Studies of Streptomycin-Dependent Cholera Vibrios

Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.     

Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera

Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i) death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii) flies harboring mutant alleles of either adenylyl cyclase, Gsalpha, or the Gardos K channel homolog SK are resistant to V. cholerae infection; and (iii) ingestion of a K channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 mug of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera.     

Prophage CTXphi genome variability and its role in alteration of Vibrio cholerae El Tor virulence characteristics

Comparative analysis of CTXphi prophage genome of 366 V. cholerae El Tor strains isolated from infected people and water was carried out using the polymerase chain reaction. Four groups of vibrios, which carry different combinations of ctxA, zot, and ace genes from core region of CTXphi prophage coding key (cholera enterotoxin) and accessory (Zot and Ace toxins) pathogenicity factors, were determined: ctxA(+) zot(-) ace(+), ctxA(-) zot(+) ace(+), ctxA(-) zot(+) ace(-), ctxA(-) zot(-) ace(+). Vibrios that had lost all tested genes were also revealed. Genomic rearrangements occurring in water environment in virulent V. cholerae strains, which acquired foreign pathogenicity genes necessary for their existence in human organism, were proposed as one of the mechanisms of formation of clones with an incomplete or no prophage. Infection process in model animals challenged with wild and isogenic strains of V. cholerae differing in the set of the phage genes (ctxA, zot, and ace) was comparatively analyzed. It was shown that variability of CTXphi prophage genome was an important factor of modification of cholera vibrios virulent characteristics. Obtained data point to usefulness of ctxA, zot, and ace phage genes detection in wild V. cholerae isolates as it could permit evaluation of their virulent potential determining the severity of the infection.     

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.     

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.     

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 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.     

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.     

Effect of sulfide water from natural springs on the properties of Vibrino cholerae

The authors studied the properties of E1 Tor cholera vibrios isolated from sulfide water of the natural sources. There was demonstrated under experimental and natural conditions the influence of ecological conditions of sulfide water on such vibrio properties as cholerogenicity, sensitivity to diagnostic and typing phages, hemolytic activity and the value of the hemolysin-destructive factor. A short-liver stay of cholera vibrios in sulfide water was accompanied by some reduction of their virulence.     

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.     

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 and NAG vibrio utilization of different C-containing substrates in Hiss media and a medium with a limited mineral content

A total of 55 V. cholerae strains and 175 NAG vibrio strains were studied with a view to establish their capacity for utilizing citrate in Simmons citrate agar or for growing in it in the absence of the source of carbon. The strains were divided into 3 groups, each containing approximately an equal number of cholera and NAG vibrios irrespective of their origin or serovars. None of 50 signs used in this investigation permitted the reliable differentiation of the cholera and NAG vibrio groups due to considerable differences between the strains within each group. The use of Hiss medium with starch instead of Kodam medium is proposed for the determination of the diastatic activity of cholera and NAG 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.