[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.

[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.

[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.

[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.

[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.

[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.

[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.

[Comparative study of ribosomal proteins from Yersinia pestis and Escherichia coli: amino acid composition and electrophoretic mobility]. / Sravnitel'noe issledovanie ribosomnykh belkov Yersinia pestis i Escherichia coli: aminokislotnyi sostav i élektroforeticheskaia podvizhnost'.

The techniques of electrophoresis in polyacrylamide gel and amino acid analysis permitted to find the slight difference in the composition of ribosomal proteins from Yersinia pestis and Escherichia coli cells. Ribosomal proteins were mapped and classified on the basis of two-dimensional electrophoresis data. Protein "X" was registered in the total ribosomal protein due to its separation in course of ribosomal subunits dissociation.

[Cloning and study of the function of recA-like gene of Yersinia pestis in Escherichia coli cells]. / Klonirovanie i issledovnie funktsii recA-podobnogo gena Yersinia pestis v kletkakh kishechnoi palochki.

A 2 kb fragment of Yersinia pestis genome cloned in Escherichia coli cells of the strain HB101 contains a gene able to complement the recA-dependent deficiency of E. coli cells in UV-resistance, resistance to alkylating agents, UV- and MNNG-induced mutability. Cellular capability for homologous recombination in crosses with HfrH donor, derepressed synthesis of bacteriocins (colicin E1 and pesticin 1) is also complemented by the fragment in E. coli recA-strains. The obtained data suggest the functional homology of the cloned recA-like gene product with the product of E. coli recA-gene.

[Transcription map of a recombinant plasmid carrying a gene for the synthesis of pesticine I and a protein for pesticine I immunity in the plague microbe]. / Transkriptsionnaia karta rekombinantnoi plazmidy, nesushchei geny sinteza pestitsina I i belka immunnosti k pestitsinu I chumnogo mikroba.

Molecules of the plasmids pBR322 and pRD17 have been compared by electron microscopy technique of R-loops visualization. Comparative location of R-loops on the plasmids has been computerized on minicomputer HP9825A due to the program making possible to define the coordinates of the transcription start and direction. The 5.1 kb fragment coding for pesticin I and immunity protein to pesticin I and cloned in pBR322 vector plasmid is flanked by Bam HI-EcoRI sites. Five promoter regions and direction of transcription were localized on the fragment.

[DNA probe for detecting Yersinia pestis and serovariant I of Yersinia pseudotuberculosis by detecting specific DNA repeating sequences]. / DNK-zond dlia obnaruzheniia Yersinia pestis i I serovarianta Yersinia pseudotuberculosis metodom detektsii spetsificheskikh povtoriaiushchikhsiia posledovatel'nostei DNK.

In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.

[Restriction map of pesticinogenicity plasmid pYP1 of Yersinia pestis]. / Restriktsionnaia karta plazmidy pestitsinogennosti pYP1 Yersinia pestis.

The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.

[Genotyping of Yersinia pestis: variability of locus (CAAA)n in natural strains isolated in territory of former SSSR]. / Genotipirovanie Yersinia pestis: variabel'nost' lokusa (CAAA)n u prirodnykh shtammov, vydelennykh na territorii byvshego SSSR.

Genome polymorphism by the locus (CAAA)n was studied in 69 strains of Yersinia pestis isolated from natural foci of the former Soviet Union. The polymorphism was found to be represented by ten alleles in chromosomes, which could be regarded as evidence of variability of this VNTR-locus (diversity index, DI = 0.86). The value of DI was found to vary substantially: from 0.24 in a group of vole strains from seven isolates from the Transcaucasian highlands to 0.77 in nine strains from the Central Asia desert focus. The allele polymorphism of the variable locus (CAAA)n in natural strains of Y. pestis was suggested to be used as a possible genetic marker of the strain. It was concluded that the oligonucleotide primers used in polymerase chain reaction should be upgraded to the genotyping accuracy.

[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.

[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.

[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.