[VNTR-genotyping of Vibrio cholerae strains isolated from objects in the territory of Russian Federation in 2012].

AIM: VNTR-typing of Vibrio cholerae strains isolated in the territory of Russian Federation in 2012. MATERIALS AND METHODS: 71 Vibrio cholerae O3 and 3 V cholerae O1/O139 strains were used in the study. Genotyping was performed by using PCR for 5 VNTR-loci. RESULTS: Multilocus VNTR-typing allowed to group the strains into 31 VNTR-genotypes. Genotypes were divided among 10 discrete clusters by results of a cluster analysis. The presence of tcpA gene is clearly linked with the presence of VcB locus. Each geographic region was characterized by their own VNTR-genotypes. CONCLUSION: In the course of the carried out VNTR-genotyping of V. cholerae isolated in 2012, 2 types of vibrio population formation were detected. A geographic attachment to specific regions was characteristic for most of the genotypes.

[System of activation of plasminogen in Vibrio cholerae].

AIM: Study system of activation of plasminogen in Vibrio cholerae. MATERIALS AND METHODS: 75 strains of V. cholerae of various origins were used in the study. Plasminogen was isolated from human plasma by using affinity chromatography on L-lysine sepharose, alpha-enolase activity was determined by a direct method assuming transformation of 2-phosphoglycerate into phopshoenolpyruvate. Vibrios were destroyed by ultrasound disintegrator to isolate membrane Omp protein, intact cells were discarded by centrifugation and cell lysate was centrifugated for 1 hour at 105000 g. The precipitate was solubilized in buffer with 1% triton X-100 and passed through a column with DE-52 cellulose. RESULTS: Vibrio cholerae O1 and O139 strains isolated from clinical specimens and water samples from open water bodies had the ability to bind by using alpha-enolase and transform human plasminogen into plasmin under the effect of outer membrane protein OmpT A protein with molecular weight around 40 kDa had proteolytic activity with a wide specter of substrate specificity, degraded fibrin, gelatin, collagen, protamine and activated plasminogen. Computer analysis showed that OmpT protein of cholera vibrion had a low degree of relation with Enterobacteriaceae omptins. CONCLUSION: The study carried out showed that vibrios have a system of activation of plasminogen that includes at least alpha-enolase and OmpT membrane protein. OmpT protein is assumed to belong to a new class of porins of Vibrionaceae family and its enzymatic activity may play a significant role in pathogenesis of infection.

[The detection of capability of Vibrio comma to activate human plasminogen in vitro].

The article discusses the technique of defining the strains of comma bacillus according their capability to convert human plasminogen into plasmin in vitro. This method can be implemented in applied and fundamental research concerning the study of subtle mechanisms of pathogenesis and colonization of intestines under cholera.

[Infectious-toxic model of plague in mice].

AIM: To develop infectious-toxic model of plague in mice and to assess perspectives of its use for selection of new vaccine preparations. MATERIALS AND METHODS: Cells of virulent strains of Yersinia pestis 231 and 231 FI- incubated in lysates of human erythrocytes for their activation as well as suspensions of these strains in isotonic solution of NaCl were used for subcutaneous inoculation of infection-nanve and immune mice. RESULTS: It was shown that activated cultures were characterized by maximal virulence (LD50 = 1-3 CFU) and caused rapid infection--mean length of survival reduced on 1 - 3 days (P < or = 0.01). Vaccine strain EV used by conventional way of inoculation (suspension in isotonic solution of NaCl) induced strong antibacterial immunity (index of immunity--10(5)), whereas activated (in lysate of erythrocytes) cells of Y. pestis 231 strain overcame it (index of immunity--10(2)). LD50 value of Y. pestis 231 FI- for immune and nanve animals was 3 m.c. (1 CFU), which demonstrates the absence of ability of EV strain to induce antitoxic immunity in the macroorganism. CONCLUSION: Use of two models of infection allows to make more adequate prognosis of efficacy for relevant vaccine preparations.

[Effect of serotonin and dopamine on growth of Yersinia pestis and Francisella tularensis strains].

AIM: To study the effects of serotonin and dophamine on the growth of Yersinia pestis and Francisella tularensis strains as well as ability of monoamines to change susceptibility of experimental animals to plague infection. MATERIALS AND METHODS: Effect of various doses of biogenic amines on the growth of Y. pestis and F. tularensis was studied by biophotometer "BIO-LOG II" (F ISABIO, France). When studying the effect of amines on LD50 value and mean survival time, serotonin and dophamine were administered to mice peritoneally in dose 25 mg/kg and 100 mg/kg respectively before their inoculation with Y. pestis suspension. RESULTS: It was shown that one-time addition of serotonin (2.5 - 40.0 mcM) to medium for cultivation of Y. pestis and F. ularensis strains did not significantly affect the bacterial growth both at cultivation temperature 28 degrees C and at 37 degrees C. At the same experimental conditions dophamine stimulated growth of bacterial cultures accelerating the onset of exponential phase of culture growth. Administration of serotonin for 1 hour before inoculation of mice with Y. pestis EV-76 strain increased LD50 value and decreased mean survival time; in contrast, administration of dophamine decreased LD50 value and increased mean survival time. CONCLUSION: Data on stimulating effect of dophamine on agents of transmissible infections allow to propose that physiological state of an organism as well as medical administration of catecholamines could influence on susceptibility of the host to infection and determine the septic course of the disease.

[Role of cholera toxin-induced apoptosis in alteration of macrophages in mice].

AIM: To study the role of active programmed cell death induced by Vibrio cholerae antigens in alteration of peritoneal macrophages of experimental animals. MATERIALS AND METHODS: Apoptosis was assessed by cytofluorometric analysis with propidium iodide using cytofluorometer "Coulter" as well as on characteristic morphological changes of cells in stained histological preparations. RESULTS: Performed experiments carried out by both methods provide evidence that V. cholerae and its antigens (cholera toxin, neuraminidase, chitinase, and lypopolysaccharide) cause apoptosis of mice peritoneal macrophages, which leads to their alteration. CONCLUSION: Our results demonstrate that programmed cell death of phagocytes is one of the causes of cytotoxic effect of V.cholerae and its antigens. Performed experiments show the role of apoptosis of macrophages in formation of postimmunization immunosuppression after vaccination against cholera.

[Genotypic heterogeneity and geographic diversity of collection strains of Francisella tularensis as determined using the VNTR variability analysis and DNA sequencing].

The analysis of the identification genotypes allowed the strains to be grouped into 61 variants from A to I with the incidence rate 0.002-0.142. The cluster analysis of the identification genotypes allowed the strains to be grouped into 9 clusters with different number of components. Actual existence of genotypic heterogeneity and geographic diversity of the F. tularensis strains was demonstrated in addition to territorial attribution of certain strains. The geoinformation system Tularemia was developed to provide spatioterritorial analysis of distribution of the genotypes of the strains. A specific feature of the geoinformation system is dynamic mode of its operation, which provides the ability of continuous addition of information not only by the expense of available data but also by the expense of creation of new layers. Any new information is automatically added to the geoinformation system, thereby providing both retrospective and operative analysis. The geoinformation system Tularemia should find promising application to the structure of the epidemiological method. The use of the system will bring the epidemiological control of tularemia to a qualitatively higher level.

[Vntr-genotyping of Francisella tularensis strains isolated in the former USSR territory and some European countries during epizootics in 1988 - 1989].

Retrospective VNTR-analysis of 159 Francisella tularensis subsp. holarctica strains isolated in December 1988 - February 1989 in former USSR and some European countries was carried out. Analysis of heterogenic genotypes of strains allow to subdivide them into 30 groups of variants by individual genotypes, while cluster analysis--to subdivide them in 7 clusters with different number of compositions. The predominance of genotype C1 strains isolated on the Rostov and Archangelsk regions and the Crimea was established. F. tularensis strains isolated in winter time 1988 - 1989 in different geographic regions were supposed to be resident cultures typical for their biotope in natural focus of disease.

[Apoptosis of phagocytes as one of the probable mechanisms of the pathogenetic action of Yersinia pestis "mouse" toxin].

Apoptosis was evaluated by characteristic morphological changes of cells in preparations stained with histological dyes and in live preparations, as well as by DNA degradation, colorimetrically detected with the use of the diphenylamine reagent. "Mouse toxin" (MT) was found to have a pronounced apoptogenic action with respect to the phagocytic cells of mice, but not guinea pigs. Macrophages were affected by this action stronger than neutrophils, and in both cases this effect was dose dependent. As the dose of MT decreased to 0.01 microg/ml, the proportion of cells dying as the result of apoptosis increased, the necrotic type of damage was almost absent. On the contrary, as MT concentration rose to 1.0 microg/ml and over, the proportion of phagocytes dying due to necrosis increased with a decrease in the number of cells in which the process of apoptosis started. The results of the study are indicative of the fact that the mechanisms programming the death of cells under the action of MT on macrophages and neutrophils took part in the process, which, in its turn, determined their role in the pathogenesis of plague.

[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 multi-locus VNTR-analysis in studies of the population structure of Yersinia pestis in natural foci]. / Mul'tilokusnyi VNTR-analiz v izuchenii populiatsionnoi struktury Yersinia pestis v prirodnykh ochagakh.

A collection of Yersinia pestis strains was investigated by the multi-locus VNTR analysis. All 9 used locuses were diverse, although they differed between themselves by the quantity of genotypes displaying 4 to 13 variations in the sample. The diversity index (DI) ranged from 0.18 (ms21) to 0.86 (ms46); 8 locuses had DI > 0.5. The statistical processing showed 55 individual genotypes in a group of 81 examined strains, which denoted a high discriminative potentiality of the typing system (DP = 0.98). On the basis of the cluster analysis, the genotypes were shared between 11 main groups. The strains belonging to one genotype group were found to originate, as a rule, from one natural focus. The suggested scheme of typing and of creating the databases of genotypes of plaque agent can be used to establish, with a high probability degree, the source of strains.

[Retrospective VNTR-analysis of genotypes of Vibrio cholerae 01 strains isolated, during the 7th cholera pandemic, in Rostov Region]. / Retrospektivnyi VNTR-analiz genotipov shtammov Vibrio cholerae 01, vydelennykh na territorii Rostovskoi oblasti v gody VII pandemii kholery.

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.

[Characterization of Vibrio cholerae O1 cultures isolated from environmental objects on the territory of the Russian Federation in 2002]. / Kharakteristika kul'tur kholernyk vibrionov O1, izolirovannykh iz ob"ektov okruzhaiushchei sredy na territorii Rossiiskoi Federatsii v 2002 g.

The biological properties of 46 V. cholerae O1 eltor cultures isolated in 2002 from water environment on the territory of Russia are presented. All isolated vibrios proved to be typical in their cultural, morphological, biochemical and serological properties. The atypical character of some of them was mainly linked with their phage resistance. The appearance of vibrios, sensitive to bacteriophage ctx+ and containing gene tcp in the absence gene ctx, was noted. Multilocus VNTR typing made it possible to group the cultures under study in 34 genotypes. The presence of toxin coregulated pili was found to be directly related to locus VcB. The necessity of the systematic study of the pheno- and genotypes of the isolated cultures with the aim of epidemiological surveillance of this infection is emphasized.

[Multilocus VNTR-typing of Francisella tularensis strains]. / Mul'tilokusnoe VNTR-tipirovanie shtammov Francisella tularensis.

In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis.

[Genotyping of the Francisella tularensis strains isolated from natural foci of tularemia in the Rostov region by multilocus VNTR analysis]. / Genotipirovanie metodom mul'tilokusnogo VNTR-analiza shtammov Francisella tularensis, vydelennykh iz prirodnykh ochagov tuliaremii Rostovskoi oblasti.

On the basis of an analysis of the VNTR alleles' distribution in 109 strains of F. tularensis it was established that 19 genotypes of the disease causative agent circulated in the Rostov Region from 1945 to 2002. The microbe-provoked infection episodes can be divided into polyclonal, monoclonal and cluster ones. A retrospective analysis of the genotypes' distribution is indicative of that strains of similar or of closely-related genotypes circulate simultaneously in the studied territory. All investigated F. tularensis strains could be differentiated into two groups; strains, whose genotypes are encountered almost evenly within the entire Region's territory, belong to group 1; and strains of group 2 displayed a trend towards being geographically bound. Isolations of cultures with similar (close) genotypic features made in prolonged time periods suggest that a part of F. tularensis clones can persist for a long time in environmental foci. A set of strains described by genotype can provide a foundation for a database of the tularemic microbe culture within the geo-information system of the South Federative Okrug of Russia.

[Variable nucleotide tandem repeats (VNTR analysis) in Vibrio cholerae 0139 isolated from humans and from water of surface reservoirs in Russia]. / Variabel'nye tandemnye nukleotidnye povtory (VNTR-analiz) u Vibrio cholerae 0139, vydelennykh ot liudei i uz vody poverkhnostnykh vodoemov Rossii.

The comparative study of variable tandem repeats (VNTR analysis) in genomes of V. cholerae 0139 isolated from humans and from water samples taken from surface reservoirs was carried out. The results of the study of the allele state of 5 loci of tandem repeats in 50 strains of vibrios, carried out in the double-primer polymerase chain reaction (PCR), as well as the earlier comparison of the same isolates in the single-primer PCR, showed essential differences and the absence of clonality in the cultures of the clinical and aqueous origin. The suggestion was made that vibrios with individual VNTR genotypes and having no genes ctx and tcpA, isolated from water samples, were epidemic unimportant representatives of the autochthonous microflora of water reservoirs.

[Variable tandem repeat VcA of Vibrio cholerae]. / Variabel'nyi tandemnyi povtor VcA Vibrio cholerae.

Computer analysis revealed seven potential variable-number tandem-repeat (VNTR) loci in the Vibrio cholerae genome. Specific primers were designed to amplify locus VcA located on chromosome 2 and containing a TGCTGT repeat. The locus was found in all tested strains from a V. cholerae strain collection, the repeat number varying 3 to 23. In total, 14 VcA alleles were observed. The VcA locus was proposed as a marker for the molecular typing of V. cholerae strains.

[Cholera toxin as Vibrio cholera superantigen]. / Kholernyi toksin--superantigen Vibrio cholerae.

Experimental data confirming our earlier suggestion, that cholerae toxin (CT) possesses superantigen (SA) properties are presented. When used in very small doses, CT has been found to induce polyclonal activation of T lymphocytes, essentially exceeding that observed in classical T mitogens characteristic of SA. CT, in contrast to mitogens and similarly to other SA, is shown to display this activity only in the presence of antigen-presenting cells. Experiments with the use of monoclonal antibodies to the variable region of the beta-chain of the T-cell receptor (V beta TCR) have demonstrated that CT, similarly to other SA, are capable of inducing expression of certain types of V beta TCR and causing polyclonal activation of T lymphocytes carrying these types of V beta TCR. The presence of these properties gives grounds for regarding CT as SA. The SA activity of CT has been found to be linked with its subunit A.