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Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA – tcpA + strains vary considerably and can be clustered into two separate groups, the CTXφ–RS1φ +VPI+VSP+/CTXφ–RS1φ–VPI+VSP+ isolates and the CTXφ–RS1φ–VPI+VSP– isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTXφ–RS1φ +VPI+VSP+/CTXφ–RS1φ–VPI+VSP+ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTXφ–RS1φ–VPI+VSP– strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTXφ–RS1φ–VPI+VSP– strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.

The study in question of the structure and content of international cooperation in the sphere of sanitary-and-epidemiological welfare of the population in its various formats – on the UN, WHO, “G-8”, CIS, EurAsEC, SCO, and BRICS levels, as well as within the frames of bilateral relations between the Russian Federation and Republic of Kazakhstan – has made it possible to develop a model of international intercourse organization characterized by such functional features as assessment of the epidemiological situation peculiarities and IHR implementation status, realization of global strategy for infectious diseases control, systemic modernization of methodology, technologies, materials-and-equipment resources as well as human assets and production of medical immune-biological preparations (MIBP), stationary network and mobile complexes for emergency response in order to level the interstate potentials for the provision of effective prevention, detection and monitoring over emergency situations in the sphere of sanitary-and-epidemiological welfare of the population (ES). Top-priority in the list of the ESs is given to novel emerging infectious diseases control as it primarily demands improvement of the informational–and–analytical prognosis–modeling base within the frames of epidemiological surveillance system and sanitary protection of the state territories. Great need in such type of projects is substantiated based on the examples of Crimean-Congo hemorrhagic and West-Nile fevers control. Nevertheless, for the realization of the model, coordinating functional authority/agency is required.

The results of epizootiological surveillance in 11 plague natural foci in the territory of the Russian Federation in 2010 are presented in this review. The short-term prognosis of foci epizootic activity is performed based on the analysis of data on epizootic activity, the conditions of plague carrier and vector populations, and abiotic factors influence on their living conditions.

Described is cholera epidemiological situation in the world and in the Russian Federation. One of the causes of its deterioration is pointed out to be the emergence, in the modern period, of altered Vibrio cholerae eltor variants with increased virulence, which displaced typical strains in many countries of Asia and Africa. Described are licensed oral cholera vaccines and their prophylactic efficiency. Considered is the WHO strategy in regard with oral cholera vaccines application in emergencies of different character. The prospects of new generation vaccines development are presented.

Suggested and substantiated is the integrated algorithm for diagnostics of dangerous infectious diseases that require measures on sanitary protection of territories using clinical, epidemiological and laboratory diagnostic standards. These standards are based on criteria for detection of suspected, probable and confirmed cases, with consideration for clinical manifestations, epidemiological anamnesis and laboratory analysis results. Distinguished and described are two stages for diagnosis of new infectious diseases that represent an emergency in the sphere of sanitary and epidemiological well-being of the population.

On the basis of research of statistic indices of custom-house, the population and geographic location of cities of Donetsk region, the method of calculation of optimum placing of the specialized transport-logistical centre of foodstuff is offered.

A unique, many-functional complex of mobile laboratories for SAET on the basis of cross-country chassis was created for the first time. The complex is designed for work in the areas of ES in the sphere of sanitary and epidemiologic welfare of the population. This unparalleled project was carried out in the course of step-by-step modernization of the specialized anti-epidemic teams (SAET) of the Rospotrebnadzor anti-plague institutes in the light of implementation of the conception of SAET modernization and main principles of SAET functioning. Presented are structural and functional characteristics of the complex of SAET mobile laboratories on the basis of cross-country chassis, and engineering and technical means used. Substantiated are relevant levels of biological safety provision for every laboratory. Qualitative advantages of the laboratories on the basis of cross-country chassis are marked, and algorithm of provision of constant readiness of SAET mobile complex is presented.

Detection of circulation of West Nile virus (WNV) on the territory of Saratov Region and prerequisites for formation of natural focus of West Nile fever (WNF), determination of the role of WNV in infectious pathology on the territory of the region.of organs of small mammals, birds, blood-sucking arthropods for the presence of WNV markers (antigens and/or RNA) were studied. Clinical material from patients with symptoms not excluding WNF was studied. Donor blood sera samples were analyzed with the aim of detection of immune layer against WNV in population of Saratov Region.In 2010 WNV antigens were detected by EIA in 12 samples (7.1%) of mammal organ suspensions. In 2012 by using RT-PCR and EIA, markers of WNV were detected in 6 samples of bird brain suspensions (6.3%) and 1 sample of mammal organ suspension. Immune layer of population against WNV was 4% in 2011, 2.8% in 2012. In 2012 in 11 of 27 examined patients IgM against WNV in diagnostic titers and/or serconversion of IgG in paired sera were detected. In addition in 5 individuals virus RNA was detected in blood. Based on clinical, laboratory data and epidemiologic anamnesis 11 patients were diagnosed with WNF.The results obtained give evidence on the circulation of WNV on the territory of Saratov Region in 2010 - 2012. With the development of complications of WNF epidemiologic situation in 2012 an expansion of WNV areal onto the territory of the region took place and the process of formation of WNF natural foci is ongoing.

Actual problems of biosafety are presented. Examination of biosafety as a subsystem of life safety of humans allows to attract methodical apparatus developed by taking into account general terms of safety theory applied to problems of ensuring biosafety. As an actual goal implementation of technologies of risk analysis in evaluation of potentially dangerous biological objects and territories of Russian Federation is determined. Analysis of legislation and normative-methodical documentation in the field of ensuring biological safety during work with pathogenic biological agents revealed a number of problems of technical, organizational and scientific nature. Proposals for their solution are given.

The comparative analysis of the gene sequences encoding the synthesis of enzymes responsible for the intermediary metabolism of methionine in Bacillus anthracis strains and in closely related bacterial species was carried out. Deletion of 42 nucleotides in the hom2 gene, which determines the homoserinedehydrogenase, is detected in all tested Bacillus anthracis strains. In the strains of other bacillar species hom2 gene mutation, which blocks up the tracts of methionine and threonine biosynthesis, was not identified. The single nucleotide polymorphism was determined in asd1, metX, and metH genes. It provides the identification of B. anthracis strains using sequencing technology.

Based on the results of computer analysis of nucleotide sequences in strains Yersinia pestis and Y. pseudotuberculosis recorded in the files of NCBI GenBank database, differences between genes argA, aroG, aroF, thiH, and thiG of strain Pestoides F (subspecies caucasica) were found, compared to other strains of plaque agent and pseudotuberculosis microbe. Using PCR with calculated primers and the method of sequence analysis, the structure of variable regions of these genes was studied in 96 natural Y. pestis and Y. pseudotuberculosis strains. It was shown that all examined strains of subspecies caucasica, unlike strains of plague-causing agent of other subspecies and pseudotubercolosis microbe, had identical mutations in genes argA (integration of the insertion sequence IS100), aroG (insertion of ten nucleotides), aroF (inserion of IS100), thiH (insertion of nucleotide T), and thiG (deletion of 13 nucleotides). These mutations are the reason for the absence in strains belonging to this subspecies of the ability to synthesize arginine, phenylalanine, tyrosine, and vitamin B1 (thiamine), and cause their auxotrophy for these growth factors.

Comparative molecular-genetic analysis of clinical Vibrio cholerae eltor biovariant strains isolated in Russia during various years.Microbiological and biochemical methods were used for studies of 25 clinical strains of classic and eltor biovariant cholera, PCR testing and sequencing of various genes was also performed.Phenotypic and genetic analysis of clinical V. cholerae strains isolated in Russia during 7th cholera pandemic has confirmed that they belong to biovariant eltor. PCR testing of 21 isolates obtained from patients in 1970 - 2010 has shown that epidemic complications in Russia from 1993 were caused by altered V. cholerae biovariant eltor. Presence of classic cholera biovariant ctxB coding gene in cholera toxin coding CTX prophage is the genetic alteration of these variants, ctxB sequencing in altered variants has confirmed PCR data and shown 2 ctxB gene alleles (ctxB1 and ctxB7). Altered variants produced significantly more cholera toxin than typical strains.In 1970 - 2010 67.6% of clinical isolates were altered V. cholera biovariant eltor variants. These new variants were genetically diverse. Alteration of cholera eltor biovariant genome caused toxigenicity increase.

Literature data on main immunobiological characteristics of 1st generation plague vaccines as well as ways of development of new tools for specific prophylaxis of plague: recombinant live, chemical, antiidiotypic, and DNA vaccines are presented in the review. Their expected advantages and disadvantages, perspectives of development and practical use in system of antiepidemic measures are assessed.

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and minor subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metB, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and minor subspecies revealed that the reason for the methionine dependence of strains belonging to the major subspecies is a deletion of a single nucleotide (-G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.

To study the ability of recombinant protective antigen (PA) to stimulate adaptive immune response in laboratory animals.Vaccine, recombinant, and reference strains of Bacillus anthracis were used in the study. Laboratory animals were immunized subcutaneously with two doses of antigenic preparation or one dose of B. anthracis strain. After inoculation with reference strain of B. anthracis, measurement of LD50 as well as indexes of immunity was performed by specified methods.It was revealed that asporogenic recombinant strain has stable biological characteristics during passages in vitro and is effective producer of PA. Using 2-stage chromatography, highly purified protein was obtained. Experiments on different biomodels--BALB/c mice, guinea pigs, and rabbits--demonstrated high protective activity of PA obtained from asporogenic producer. Increase of immunity index was noted when EA1 protein from S-layer was added to preparation for immunization.Immunity indexes determined in experiments on laboratory animals point to high protective efficacy of recombinant PA. Further studies of its interaction with macroorganism's innate and adaptive immunity systems are promising.

A study of the structural and regulatory genes, determining rhamnose fermentation, that are located in the rha locus of the chromosome of Yersinia pestis main and non-main subspecies and of Yersinia pseudotuberculosis of serogroups I-III was performed. The nucleotide sequence of Y. pestis main subspecies differs substantially from those of non-main subspecies and Y. pseudotuberculosis by the presence of a nucleotide substitution in 671 bp location of rhaS gene resulting presumably in the Y. pestis non-main subsp inability to utilize rhamnose. This results in incapability of Y. pestis non-main subspecies to utilize rhamnose. Other nucleotide substitutions found in Y. pestis non-main subspecies strains influence only upon expression efficiency of this diagnostic criterion.

Using developed scheme, complex study of protective properties of avirulent recombinant strain Vibrio cholerae El Tor Inaba KM 184 was performed. Necessity for broadening of standard procedure of testing of cholera vaccines protective properties by using of quantitative methods of assessment of morphological changes and state of biomodel's functional systems, which could increase the information value of assessment of studied vaccines, was experimentally substantiated.

Cholera, plague, and anthrax, the diseases that have accounted for millions of human victims, still endanger the entire mankind by possible development of epidemic outbreaks due to their spread or application as bioterrorist agents. Generalized results of research into the genomic features of the Vibrio cholerae, Yersinia pestis, and Bacillus anthracis are discussed. Despite different frequencies of evolutional transformations occurring in their genomes, that are likely to be associated with diverse life cycles of the pathogens, clones with altered diagnostic, and virulence characteristics were shown to have a fair probability of formation. Also presented in the review, are literature data concerning the main evolutional stages for any of these pathogens, determination of new genetic variants, consideration of the mechanisms facilitating maintenance of the microbial agents during the interepidemic periods.

The paper contains a survey of published data about the use of DNA-diagnostics in indicating and identifying the causative agents of highly dangerous infections like plague, cholera and anthrax. A discussion of data about the genetic relationship between strains of the mentioned causative agents isolated from different sources by using the molecular-typing methods as well as about the evolution ties between strains of different origins is in the focus of attention. Results of comparative studies of nucleotide sequences of genomes or of individual genomes in different Yersinia pestis, Vibrio cholerae and Bacillus anthracis strains, which are indicative of the evolution of their pathogenicity, are also under discussion.

This review is devoted to problems of regulation of the synthesis of the main virulence determinants encoded by a virulence plasmid of Yersinia pathogenic for humans. The principal attention is paid to differences in the mechanisms inducing the synthesis of Yersinia outer membrane proteins (Yop) in vitro and in vivo. A model of regulation of Yop expression and synthesis is offered. Modern data on the role of these proteins in virulence is discussed.

The genetic analysis of Y. pestis virulence factors accomplished in the 358 strain isogenic system allowed us to determine a minimal set of known factors providing pathogenicity. The combination of chromosomal marker Pgm+ and calcium dependence plasmid (pCad) is shown to be sufficient for preserving the virulence of Y. pestis. Experimental modelling of virulence in this microorganism by the genetic exchange methods was carried out. The reduced virulence of the strains Pgm+ and pCad+ for guinea pigs was detected.