[Epidemiological Surveillance of Cholera in Russia During the Period of the Seventh Pandemic].

In this work basic stages of formation of the epidemiological surveillance of cholera in Russia are described. In 1990-s for the first time zoning by epidemic manifestations of cholera was carried out at the level of subjects forming parts of Russia and other Republics of the Soviet Union with the introduction of differential tactics of epidemiological surveillance. Improvement of epidemiological surveillance of cholera was aimed at harmonization with the IHR (2005), integration of epidemiological surveillance of cholera and social-hygienic monitoring of water objects of I and II categories. Characterization of isolated Vibrio cholerae strains (1990-2014) on the genomic basis determined the emergence of new VNTR-genotypes of V. cholerae O1 ctxAB+ tcpA+, responsible for outbreaks, simultaneously with isolation of V. cholerae 01 ctxAB-tcpA-strains during monitoring of environmental objectsfor cholera. A viewpoint is considered of the beginning of the eighth cholera pandemic in the context of emergence of V. cholerae El Tor strains with CTXφ prophage carrying ctxB gene of cholera toxin of classical biovar. Main directions offurther enhancement ofepidemiological surveillance include the study of basic data structures used in the epidemiological surveillance system, the use of zoning of municipal units offederal subjects with corresponding surveillance tactics and expected economic effect.

[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.

[Study of Legionella pneumophila strains isolated from environmental objects].

The paper presents the results of studying the biological properties of Legionella pneumophila strains isolated from environmental objects. Elective legionellosis medium (ELM) has been found to be suitable for the isolation of the causative agent from the starting material and to be as sensitive as CYE (Oxoid company) containing growth and selective additives. Polymerase chain reaction (PCR) with a home-produced commercial test system used to detect L. pneumophila DNA enables identification of the causative agent, including its species. Hyperimmune sera against L. pneumophila 1-7 serogroups used in slide-agglutination and agglutination, as well as a series of co-agglutinating diagnosticums for legionellosis 1-7 serogroups make it possible to identify even the serogroups of L. pneumophilla. Comparative analysis of the virulence of L. pneumophila cultures in vivo and in vitro allows recommendation that practical laboratories should employ a simple NaCl resistance test, which can be used as a guide virulence test.

[Cholera caused by Vibrio cholerae O1 ctxAB- tcpA+].

Results of analysis of cholera outbreak during which V. cholerae O1 biovar El-Tor ctxAB- tcpA+ was isolated from 2 patients and 30 carriers are presented. Epidemic was caused by contamination of water source and water route of transmission. Strains identical to ones detected in humans were isolated from water of surface well in zone of water intake. Genome and VNTR-analysis of ctxAB- tcpA+ vibrios that caused outbreak in Rostov region in 2005 showed that they differed from ctxAB- tcpA- and ctxAB- tcpA+ vibrios isolated previously during and beyond of outbreaks from patients, carriers and environment and formed separate group with certain genotype. These results confirms conclusions of epidemiological analysis about imported cause of recent outbreak.

[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.

[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.

[Dynamics of the isolation and biological properties of Vibrio cholerae cultures, serogroups 01 and 0139, isolated from water reservoirs and sewage water on the territory of the city of Rostov-on-Don]. / Dinamika vydeleniia i biologicheskie svoistva kul'tur kholernykh vibrionov 01 i 0139 serogrupp, vydelennykh iz poverkhnostnykh vodoemov i stochnykh vod na territorii g. Rostov-na-Donu.

The dynamics of the isolation of V. cholerae cultures from various water objects on the territory of Rostov-on-Don during the period of 1994-2001 was analyzed and biological properties of 14 such cultures were studied. In the absence of epidemic complications during the above-mentioned period, a growth in the amount of V. cholerae isolates, serogroups 01 and 0139, including toxigenic V. cholerae 01, was registered. The microbiological and epidemiological aspects of the monitoring of surface reservoirs and sewage were considered and the expediency of the profound and systematic study of its results for epidemiological surveillance on cholera was emphasized.

[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.

[Characterization of Vibrio cholerae eltor in the city of Kazan in 2001]. / Kharakteristika kholernykh vibrionov él'tor, vydelennykh v g. Kazan' v 2001 g.

Information on V. cholerae eltor isolated in the focus of cholera in Kazan in 2001 at different periods of the outbreak is presented. The identity of strains isolated from patients, vibriocarriers and environmental objects, including their antibioticograms (sensitivity to cyprofloxacin and resistance to trimethoprim--sulfamethoxazole, streptomycin, furazolidone and nalidixic acid, which may be regarded as markers), is shown. Variable tandem repetitions in the DNA of 30 isolates strains of different origin have been determined. The results of this determination make it possible to classify all these strains as one genotype, which confirms the suggestion on the circulation of one subclone of the infective agent of cholera in the focus. As revealed in this investigation, the isolated strains are labile with respect to diagnostic phage eltor, while ctx+ strains are resistant to phage eltor ctx+.

[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.

[Vibrio cholerae O139 isolated from humans and the water from open reservoirs: a comparative genotyping]. / Vibrio cholerae O139, vydelennykh ot liudei i iz vody otkrytykh vodoemov: sravnitel'noe genotipirovanie.

The comparative study of the genomes of V. cholerae O139 isolated from humans and from water of surface reservoirs was carried out with the use of single- and double-primer polymerase chain reaction (PCR). The profiles of polymorphic DNA fragments obtained in this study made it possible to find out differences between groups of strains, as well as the individual features of some of them. The comparison of strains isolated from humans and from water in single-primer PCR revealed that they, in spite of the general similarity of their genomes, essentially differed, which was probably due to changes in the genome of this infective agent. Strains of aqueous origin lacked genes ctx and tcpA, which made them epidemiologically unimportant.

[Noncultivatable forms of Francisella tularensis]. / Nekul'tiviruemye formy Francisella tularensis.

Conditions for the appearance of F. tularensis uncultivated forms and for their reversion into the initial state have been studied. As revealed in this study, the combined influence of stress factors (starvation and low temperature) may result in the transition of F. tularensis into the uncultivated state in which it persists in the environment during the period between epidemics. The reversion of F. tularensis uncultivated forms into the initial state has been carried out with the use of sensitive animals. The uncultivated state of F. tularensis should be regarded as the actual form of the existence of the causative agent of tularemia in soil and water ecosystems.

[The laboratory diagnosis of an outbreak of hemorrhagic fever at Oblivskaya village, Rostov Province: proof of the etiological role of the Crimean-Congo hemorrhagic fever virus]. / Laboratornaia diagnostike vspyshki gemorragicheskoi likhoradki v stanitse Oblivskoi Rostovskoi oblasti: dokazatel'stva étiologicheskoi roli virusa krymskoi-kongo gemorragicheskoi likhoradki.

The results of the molecular biological detection of the etiologic agent of hemorrhagic fever in Rostov Province are presented. The role of the causative agents of Astrakhan rickettsial fever, hemorrhagic fever with the renal syndrome, Q fever, leptospirosis and listeriosis has been excluded by means of such immunochemical reactions as the direct and indirect immunofluorescent tests, the solid-phase immunoenzyme assay, the complement fixation test and the agglutination test. The relationship between the cases of hemorrhagic fever in the focus of the outbreak and Crimean-Congo hemorrhagic fever virus has been demonstrated due to the use of the polymerase chain reaction with preliminary reverse transcription.