[The analysis of possibility to apply new preparations in serologic diagnostic of agent of cholera in working activity of specialized anti-epidemic brigades].

The approbation of diagnostic preparations on the substrate of monoclonal antibodies developed in the institute was carried out during tactical specialized exercise with building up of units on the basis of mobile complex of specialized anti-epidemic brigades. It is established that diagnostic agglutinating and fluorescent monoclonal immunoglobulins by their sensitivity are equal to polyclonal commercial preparations and can be used at the stages of laboratory diagnostic of cholera both in conditions of stationary laboratory and mobile complex of specialized anti-epidemic brigades. The method of dot immunoanalysis on the substrate of monoclonal antibodies can, on a par with such common methods as immunofluorescence, slide-agglutination and polymerase chain reaction, be applied in complex of methods of express-diagnostic of cholera.

[Phylogenetic analysis of genomes of Vibrio cholerae strains isolated on the territory of Rostov region].

AIM: Determination of origin of 2 Vibrio cholerae strains isolated on the territory of Rostov region by using full genome sequencing data. MATERIALS AND METHODS: Toxigenic strain 2011 EL- 301 V. cholerae 01 El Tor Inaba No. 301 (ctxAB+, tcpA+) and nontoxigenic strain V. cholerae O1 Ogawa P- 18785 (ctxAB-, tcpA+) were studied. Sequencing was carried out on the MiSeq platform. Phylogenetic analysis of the genomes obtained was carried out based on comparison of conservative part of the studied and 54 previously sequenced genomes. RESULTS: 2011EL-301 strain genome was presented by 164 contigs with an average coverage of 100, N50 parameter was 132 kb, for strain P- 18785 - 159 contigs with a coverage of69, N50 - 83 kb. The contigs obtained for strain 2011 EL-301 were deposited in DDBJ/EMBL/GenBank databases with access code AJFN02000000, for strain P-18785 - ANHS00000000. 716 protein-coding orthologous genes were detected. Based on phylogenetic analysis strain P- 18785 belongs to PG-1 subgroup (a group of predecessor strains of the 7th pandemic). Strain 2011EL-301 belongs to groups of strains of the 7th pandemic and is included into the cluster with later isolates that are associated with cases of cholera in South Africa and cases of import of cholera to the USA from Pakistan. CONCLUSION: The data obtained allows to establish phylogenetic connections with V cholerae strains isolated earlier.

[Characteristics of Vibrio cholerae Cef (CHO cell elongating factor): bioinformatics analysis and experimental data].

Bioinformatics analysis of the primary and secondary structure of the Vibrio cholerae Cef (CHO cell elongating factor) protein was conducted. Similarity with triacylglycerol lipases and cytotonic toxins of other bacterial species was observed. Cef was predicted to be a heat-tolerant serine lipase with the Kunitz domain and leucine zipper. These data were confirmed experimentally. The Cefs of the two biotypes of V. cholerae O1, as well as O139 and nonO1/nonO139 serogroups, were purified from the recombinant Escherichia coli strains carrying corresponding cloned genes, and their physicochemical properties, biochemical and biological activities in vitro and in vivo were characterized. Biological activity against the cultured cells was not associated with estherase activity. Evidently, Cef is a bifunctional protein contributing both to pathogenicity of the cholera agent and to its competitive ability in different ecological niches.

Ultrastructural changes in cultured cells under the effect of Vibrio Cholerae hemagglutinin/protease.

Electron microscopic study of changes in cultured cells caused by Vibrio cholerae recombinant hemagglutinin/protease (HA/P) showed significant structural changes, most pronounced in HeLa and L-929 cells not forming a compact monolayer with tight junctions between the cells: formation of numerous vesicles on the cell surface and clasmatosis, vacuolation of the cytoplasm, swelling of mitochondria, clarification of their matrix and crist distortions, and increase in the number of lysosomes. Cytoplasm vacuolation predominated in MDCK culture, while clasmatosis was less intense. Addition of HA/P to CaCo2 cells forming a differentiated polarized monolayer, led to extension of cell-cell spaces not impairing tight junctions, swelling of mitochondria, cytoplasm vacuolation, and clasmatosis on the apical surface. These changes virtually completely coincided with those caused by the so-called NMDCY factor (non-membrane-damaging cytotoxin), described as new Vibrio cholerae toxin. These findings confirm our previous hypothesis about the identity of these factors.

[Use of various methods to isolate exopolysaccharide of eltor cholera Vibrios and its immunochemical characteristics].

AIM: Isolation of Vibrio eltor exopolysaccharide and study of its immunochemical properties. MATERIALS AND METHODS: Rugose variants of strains V. eltor 18895 and V. eltor 18843 obtained by us by selection in M9 medium were used in the study. Exopolysaccharides (EPS) were isolated by K. Kierek (2003), S.P. Zadnova (2004), N.P. Elinova (1984) methods and analyzed for carbohydrate, protein, nucleic acid content and lipopolysaccharide impurity. EPS, LPS, R-LPS structure was compared by high-pressure chromatography. Neutral sugars and amino sugars were identified by thin layer chromatography. Polyclonal antibodies were produced against EPS preparation isolated by N.P. Elinova (1984) method. Specific activity of obtained mice sera was tested by DIA method. RESULTS: EPS isolated by N.P. Elinova method (1984) was shown not to contain extraneous impurities. V. eltor EPS structure differs from LPS and R-LPS. Monosaccharide composition of EPS from ctx+ V. eltor 18895 strain is presented by a wider specter of carbohydrates including glucose, mannose, rhamnose, galacturonic acid. Use in DIA of specific sera produced against EPS from toxigenic strain did not reveal general epitopes with capsule polysaccharides of V. cholerae O139, V. parahaemolyticus and V. vulnificus. CONCLUSION: Use of EPS as an immunogen promoted production of sera that are specific against EPS and rugose variants of Vibrio cholerae eltor that can be used for their detection or characterization.

[Point mutation of the Virbrio cholerae O139 cef (CHO cell elongating factor) gene alters the substrate specificity of its product].

Sequencing of the cef (CHO cell elongating factor) of Vibrio cholerae serogroup O139 revealed one nucleotide substitution (C for T in position 2015) in comparison with classical V. cholerae O1 and two substitutions (AC for GT in positions 2014-2015) in comparison with V. cholerae O1 E1 Tor. A comparative bioinformatic analysis showed that the substitution determines a threonine residue in position 672 of the Cefprotein, while the position is occupied by an isoleucine residue in the classical strains and a valine residue in the El Tor group. The last two amino acids are hydrophobic, while threonine is hydrophilic, having a polar R group. The non- synonymous substitution affects the predicted secondary and, probably, tertiary structures of the Cef-O139 protein and explained our previous finding that the protein fails to degrade tributyrin, while retaining the tweenase activity spectrum and all other characteristics. It cannot be excluded that the inability of Cef-O139 to cleave triglycerides, along with other genetic specifics, contribute to the fact that the O139 serogroup has been displaced from a dominating position in etiology of cholera by the El Tor genotype. The nucleotide sequence of the V. cholerae O139 cefgene and the deduced amino acid sequence of its product are reported for the first time and were deposited in GenBank under accession nos. JF499787 and AEC04822.1, respectively.

[Effectiveness of expression of tdh gene of Vibrio parahaemolyticus depends on two point mutations in promoter region].

A molecular-biological study of the clinical strains of Vibrio parahaemolyticus that contain genes of thermostable direct hemolysin Tdh) and Tdh-related hemolysin (Trh). Using Southern blot hybridization, it is shown that genomes of strains that carry determinants of both hemolysins (tdh(+)-trh+) represent a single copy, whereas in tdh2+RH+ strains, there are two copies (tdh1 and tdh2). All of the examined tdh+trh+ and some of the tdh+trh strains either did not express the tdh gene or did not express the tdh gene (Kanagawa negative or KP-) or expressed it weakly and not often (Kanagawa intermediate, KP+), unlike several Kanagawa positive tdh+trh- strains. To establish the reasons for KP -/+ phenotypes, tdh, tdh11, and tdh2 genes of 13 strains isolated in Russia and neighboring foreign countries were sequenced, followed by the biotransformation analysis of the obtained sequences, as well as a comparison with those of a number of strains presented in GenBank. The results revealed that the weak expression of the tdh gene depends, not only on one point mutation in the promoter region (substitution of A for G in the -35 region), as was thought previously, but also on the second substitution (G for A in the -3 position relative to the -10 sequence), which is quite sufficient when the former is absent. Therefore, the reversion of KP -/+ strains that contain one of these substitutions can take place as a result of a single reverse point mutation, and they should be considered potentially dangerous. Strains that contain both substitutions may revert with lesser probability because, in this case, both mutations are necessary.

Ultrastructural changes in the small intestine of suckling mice, caused by vibrio cholerae hemagglutinin/protease.

Suckling mice aged 4-5 days were injected with Vibrio cholerae hemagglutinin/protease and ultrastructural changes in their small intestine were studied after 5 h. The preparation caused a statistically significant accumulation of fluid in the intestine, appearance of large gaps along cell-cell spaces in the villi and crypts, intense production and secretion of the mucus by goblet cells. The formation of interepithelial cavities was paralleled by vascular changes, supplemented by extravasal disorders caused by mast cell reaction. The role of enterochromaffin cells and lipofibroblasts, modulating the secretion in the intestine, is confirmed.

[Toxins of Vibrio cholerae].

Surveyed in the paper are published data on properties, biological activity, genetic determinants and action mechanisms of recently known toxins produced by different strains of Vibrio cholerae irrespectively of their capacity for the synthesis of choleric toxin--the main virulence factor. Their possible importance both for the general clinical pattern of cholera provoked by cholerogenic agents and as independent virulence factors causing diarrhea without cholera is elucidated. The sets and levels of expression of additional toxins can differ for different pathogenic clones and they can correspondingly condition degrees of their epidemic and etiological safety.

[Cloning and expression of Vibrio cholerae zonula occludens toxin (zot) gene in Escherichia coli].

Two recombinant plasmids containing the cloned PCR-amplifled Vibrio cholerae zonula occludens toxin (zot) gene was constructed in orientation providing its transcription from lac-promoter. One of them contained also its own zot promoter. The third plasmid was obtained by subcloning a Vibrio cholerae DNA fragment including intact zot and ace (accessory cholera enterotoxin) genes. The expression levels of the cloned genes in Escherichia coli varied depending on a promoter type, host strain and culture conditions. The human intestinal cell line CaCo2 appeared to be a suitable model for assessing the biological activity of toxin preparations. The product of zot gene possessed a marked activity in respect to CaCo2 in spite of the lack of the molecule cleavage and transport of its toxic C-terminal part from alien host cells into the culture media. The constructed recombinant plasmids can be used as a source of molecular hybridization probes; and E.col transformants carrying those plasmids can serve in zot purification both for the scientific and practical purposes.

[The genome polymorphism of Vibrio cholerae ctxAB(-) strains, containing the proximal part of the CTX element]. / Izuchenie genomnogo polimorphizma ctxAB(-)-shtammov kholernykh vibrionov, soderzhashchikh proksimal'nuiu chast' CTX-elementa.

The comparative analysis of the hybridization patterns of DNA restricts for 20 V. cholerae, groups 01 and non-01 (non-0139), containing the incomplete CTX element (ctxAB-) was carried out with the use of probes, complementary to the genes of the proximal part of the virulence cassettle and flanking its RS1 sequences. This group was found to be heterogeneous both in the number of copies of "truncated" CTX prophage and their localizations in the genome, as well as in the position of the sites of restriction endonucleases HindlII and BglII. Among 17 clinically noncholerigenic isolates, 5 etiologically dangerous clones were found, each of them characterized by the definite time and place of isolation. At least one of them proved to be the causative agent of the local outbreak of diarrheal diseases in Uzbekistan.