Objective of the study is to sum-up the results of prophylactic (anti-epidemic) measures associated with elimination of epidemic focus in Kosh-Agach Region of the Republic of Altai in 2016. Materials and methods. Utilized were the data of reporting and source documentation from Altai Plague Control Station, Rospotrebnadzor Administration in the Republic of Altai, RusRAPI “Microbe”, and Irkutsk RAPI. Results and conclusions. It is pointed out that carried out in 2016 complex of organizational, anti-epidemic, and sanitary-prophylactic activities against plague is the importnant stage of rehabilitation of Gorno-Altai high-mountain natural plague focus, the final goal of which is maximally possible decrement of risks of primary human infection with plague, and in case of occurrence of such – performing of prompt response measures on localization and elimination of epidemic focus. It has been substantiated that for epidemic risk minimization in 2017 it is necessary to continue prophylactic vaccination, desinsection and deratization in the areas of predictive epizootic situation aggravation, which is contained in “Complex Action Plan of the Rospotrebnadzor Institutions for Sanitation and Rehabilitation of Gorno-Altai high-mountain natural plague focus in Kosh-Agach Region of the Altai Republic in 2017”. It is also necessary to provide for implementation of the Program on lowering risks of importation from cross-border Sailyugemsky natural focus and spread of plague in the territory of the Russian Federation, within the frames of RF Government Order No 1864-p, dated 05.09.2016. 

Objective of the study is to analyze the situation on anthrax in the Altai Territory and the Republic of Altai in 1985–2015. Materials and methods. Utilized were statistical and report forms provided by the Rospotrebnadzor, veterinary, and Phytosanitary Surveillance Service institutions in the Republic of Altai and Altai Territory, as well as by Altai Plague Control Station. Laboratory investigations of Bacillus anthracis cultures, field and clinical samples was conducted in accordance with Methodological Recommendations 4.2.2413- 08. Results and conclusions. Between 1985–2015, in the Altai Territory, an expressed unfavorable situation on anthrax was observed, defined by the large number of stationary potentially hazardous areas (SPHA) (1262 sites) and their high density (7.68); registration of six novel SPHA in five separate districts; and excess of long-term average annual morbidity rates among live-stock animals (0.111 per 100 thousand animals) and the population (0.022±0.001 о /оооо). The Republic of Altai is classified as a territory with relatively favorable epizootiological-epidemiological situation on anthrax. Current situation on anthrax in this region is characterized by domination of old non-manifesting SPHA; infections among cattle stock on farms and private subsidiary holdings; and registration of sporadic cases among non-vaccinated rural population. The major cause of human infection was participation in compulsory slaughtering, cutting, and skinning of cattle, as well as in cooking of the infected meat (88.2 %). It manifested itself in cutaneous anthrax (94.1 %). Indicated has been an ineffective organization of sanitary-veterinary measures in private holdings and on the farms. 

Objective of the study is to analyze epidemiological and epizootiological manifestations of natural-focal infections in the territory of the North-Caucasian Federal District (NCFD) in 2015. Materials and methods. Utilized were the reports of the Rospotrebnadzor Administrations, “Centers of Hygiene and Epidemiology” in the NCFD entities, Stavropol Research Anti-Plague Institute, Dagestan and Kabardino-Balkaria Plague Control Stations. Data processing was conducted using Excel Microsoft ware. Results and conclusions. Identified have been 8 nosological forms of natural-focal infections, including infections of bacterial etiology – 6 (75 %), and viral etiology – 2 (25 %). The majority of patients (164) are identified in the Stavropol Region: 67 (intestinal yersiniosis), 43 (Crimean–Congo hemorrhagic fever), 40 (Lyme borreliosis), 9 (leptospirosis), 2 (pseudotuberculosis), 1 (rabies), 1 (human granulocytic anaplasmosis) and 1 (human monocytic ehrlichiosis). The patients with 2 nosological forms of natural focal infections are identified in the Republic of Dagestan: 2 – Crimean–Congo hemorrhagic fever and one patient – tularemia. Also 2 nosological forms of natural focal infections are revealed in the Republic of Karachay-Cherkessia: one case of leptospirosis and one case of Crimean–Congo hemorrhagic fever (imported from the Stavropol region). 8502 field samples have been tested for the presence of markers of 12 nosological forms of natural-focal infections. Positive results for 10 of them have been obtained. The most comprehensive epizootiological monitoring has been carried out in the Stavropol Territory (10 nosological forms), whereat circulation of 8 pathogenic agents is registered. 

Objective of the study is to summarize long-term data on the dynamics of areal and abundance rates of the prairie tick, D. reticulatus in the territory of the Tula Region. Materials and methods. Processed and reviewed have been literature sources, archive documents, and results of personal observations over a period of 1949–2013. Applied have been computer-based software products Google Earth, MapInfo Professional v. 11.5. wrapped in GIS Software Package. Results and conclusions. It is determined that over the last decade D. reticulatus has been found not only in the forest and adjacent western forest-steppe zone, but also in the eastern and south-eastern forest-steppe area. Specified have been the territories with uniformly high density of population and sparse distribution of D. reticulatus ticks. It is established that nowadays abundance rates of the ticks show no substantial differences both in the forestmeadow areas of the forest zone, and similar areas of the forest-steppe zone. Identified has been correlation between the nature of F. tularensis culture isolation from D. reticulatus and intensity of epizootic process of tularemia infection, which allows for in-time targeted epizootiological survey and regulation of preventive activities. 

Objective of the study is to substantiate performance, assess the conditions, and analyze the results of activities on non-specific plague prophylaxis in the epizootic territory. Materials and methods. Utilized were archival data and operational reports of Altai Plague Control Station, Irkutsk RAPI, RusRAPI “Microbe”, Center of Hygiene and Epidemiology in the Republic of Altai, and Rospotrebnadzor Administration in the Republic of Altai, as well as literature sources. Spatial analysis was carried out using GIS-technologies. Results and conclusions. Concerted and coordinated activities of the Rospotrebnadzor institutions, medical and veterinary institutions, local executive authorities have allowed for complex prophylactic and anti-epidemic measures on prevention of plague dissemination and transmission in the territory of the Republic of Altai in 2016. Demonstrated has been the necessity to expand the scope and scale of operations and scientific research in the focus, deploying advanced toolkit of methods and means. 

Objective of the study is the retrospective evaluation of epidemic cholera manifestations in the Republic of Dagestan in 1994, taking into account VNTR-typing and studies of genome peculiarities in the isolated V. cholerae O1, Biovar El Tor. Materials and methods. Utilized have been the data on the infection rates in different periods of the epidemic. The strains have been investigated using VNTR-analysis and whole genome sequencing. Results and conclusions. Heterogeneity of the agent population has been established on the basis of the circulating VNTR-genotypes. The whole-genome sequencing has revealed the presence of B-subunit allele ctxB1 in CTXφ prophage of El Tor V. cholerae O1 strains. The strains isolated in the early and the following stages of epidemic development have been found to be closely related to strains from India, Bangladesh, and Nepal. Some strains possessed a cluster of multiple antibiotic resistance genes, SXTET: floR, strA, strB, sul2 – of “Indian” type – SXT-ICE-Ind, as well as ICE mobile elements, containing determinants of resistance to antibiotics of tetracycline group – tetA. Phylogenetic relatedness of the Dagestan strains to the strains from Asia indicates to the origin of the strains, as well as to the independent cholera importations that took place at different stages of the epidemic. The role of genetically altered V. cholerae O1 El Tor variants with identified peculiarities on molecular-genetic level, as a system-forming factor in the emergence and development of epidemic in the Dagestan Republic, was directly linked to the social conditions. 

In 2016, in the territory of Yamalo-Nenets Autonomous District an outbreak of anthrax took place, 2650 reindeers were infected. As a consequence of contacts with affected and fallen animals, 36 cases of human infection occurred, of which one was fatal. Performed full-extent complex of anti-epidemic, anti-epizootic, and preventive measures, prompt organization of operations at the regional level allowed for localization of large-scale anthrax focus within the minimum possible time. Based on the lessons learned, identified were the ways to further enhancement of epidemiological surveillance and prophylaxis of anthrax under current conditions. 

Objective of the study is to investigate natural focality of Ixodidae tick borreliosis, granulocytic anaplasmosis, monocytic ehrli-chiosis in humans in the Republic of Tatarstan. Materials and methods. Utilized were the data from tick studies conducted between 2010–2015. Applying immune-enzymatic analysis, investigated were the blood sera from residents (donors) of Kazan and municipal districts of the Republic of Tatarstan for the presence of specific antibodies to borreliosis, ehrlichiosis, and anaplasmosis pathogens. Results and conclusions. For the first time ever, the data on spontaneous carriage of Ehrlichiosis pathogens in Ixodidae ticks have been obtained. The information received is an indicative of the active circulation of Borrelia, Ehrlichia and Anaplasma in the territory of the region and of necessity to expand the research on the “novel” for the Republic nosological forms of natural-focal infections. 

Objective of the study was a complex pheno- and genotypic investigation of Yersinia pestis I-3595 and Y. pestis I-3596 strains, isolated from marmots that were the source of human infection in 2015. Materials and methods. A comprehensive microbiological, molecular-genetic and mass-spectrometric studying of Y. pestis ssp. pestis strains, isolated from two grey marmots that became a cause of the plague case in Gorno-Altai high-mountain focus in 2015, was performed using up-to-date methods. Results and conclusions. It was established that the studied Y. pestis strains belonged to the main subspecies. Results of plasmid screening, multilocus VNTR-and mass-spectrometric analyses showed that those strains were closely related to Y. pestis variant detected in the focus in 2012 and in 2014, and circulating in Northwest Mongolia and Tuva natural focus. 

Objective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the fliР gene of B. mallei and gp68 gene of B. pseudomallei, respectively. Species specificity was tested with a panel of 56 B. pseudomallei strains, 14 B. mallei strains and 34 strains of closely or distantly related species. To define the analytical sensitivity of the assay, the serially diluted bacterial suspension at concentrations of 109 –102 cells /ml was used. Conclusions. The multiplex PCR assay with two primer pairs and fluorescently-labeled probes, allowing for simultaneous detection and differentiation between B. mallei and B. pseudomallei was designed. Species-specific for glanders agent, B. mallei, fragment of fliP gene, which encodes protein of flagellin biosynthesis, and species-specific gene region of B. pseudomallei, encoding gp68 protein, were identified as DNA targets. Testing of Burkholderia and non-Burkholderia bacterial species revealed 100 % specificity of the amplification assay. The minimum detection limit of the designed multiplex PCR test-system was 1·103 cells/ml for B. mallei, and 1·104 cells/ml for B. pseudomallei. 

Objective of the study is to develop and validate the method for species-specific detection of cowpox virus (CPV). Materials and methods. Utilized were oligonucleotide primers and fluorescence-labeled probes for genus-specific detection of orthopoxviruses (OPV) and species-specific detection of cowpox and ectromelia viruses (EV). Pairs of fluorescent dyes and corresponding fluorescence extinguishers were embodied into probes in accordance with their specificity: OPV-specific probe contained FAM/BHQ1 pair, CPVspecific probe – JOE/BHQ1, and EV-specific – Cy5/BHQ3. For evaluation of sensitivity and specificity of the method for species-specific detection of CPV, investigated were samples of 68 different strains of orthopoxviruses. Results and conclusions. Implemented was complex approach to species-specific detection of CPV using multiplex real-time PCR variant for simultaneous multilocus detection on the basis of three independent target genes of CPV, to genus-specific detection for the exclusion of false-negative results, and additional oligonucleotide primers and probes, providing for specific detection of EV, aimed at the exclusion of false-positive results. 

Combination of multiplex microarray immunoassay tests and luminescent nanoparticle tags is considered as a promising approach to the development of highly sensitive, specific, and rapid methods of causative agent detection. Objective of this study was to develop and compare the sensitivity of the tests for simultaneous detection of five botulinic toxins (A,B,C,E,F) applying multiplex phosphorescence analysis (PHOSPHAN) using standard (Pt coproporphyrin tag-based) and modified (europium containing nanoparticles) systems of phosphorescent signal registration. Materials and methods. PHOSPHAN assay was performed in standard 96 well microplate. The toxoids added to the wells interacted with monospecific and polyvalent immunoglobulins printed as tiny spots on the bottom of each well, and with a mixture of the same antibodies conjugated to biotin. Analyzed anatoxin concentration range – 0.005 to 100 ng/ml. The reaction was manifested by streptavidin conjugated to either Pt coproporphyrin, or the luminescent nanoparticles. The luminescence of both tags was recorded in time-resolved mode by Biochip Analyzer. The limit of detection corresponded to a minimum toxoid concentration, at which the P/N ratio was equal or exceeded 2, while the number of such samples (in a series of 10-30 experiments) was no less than 50%. Results and conclusions. Both multiplex tests provided for simultaneous group-specific detection of five botulinum toxins with the option of type-specific (A, B, E) identification. No cross-reactivity was revealed. The use of phosphorescent nanoparticles allowed for the increase in detection sensitivity by an order of magnitude, up to 10 pg/ml. The tests developed could be recommended for specific detection and identification of botulinum toxins in clinical, environmental, and food samples. 

Objective of the study is to identify species appurtenance of Brucella spp. strains, using a complex of restriction and amplification techniques. Materials and methods. Carried out has been revision and clarification of species appurtenance of 17 reference and 48 natural strains, stored in the biobank of the State collection at the RusRAPI “Microbe”, using amplification (“Gen Brucella – identification – RGF“, AMOS-DEL, Bruce-Ladder, Suis-Ladder) and restriction analyses of omp25, omp2a, omp2b loci, applying AluI, EcoRI, Eco32I (EcoRV) ferments. Results and conclusions. Performed molecular identification has confirmed the stated in the profile taxonomic status of 50 Brucella strains, while for 12 isolates species appurtenance has been rectified. New profiles of amplification and restriction have been established for three cultures, which outlines the necessity to continue further investigations. The results obtained fully substantiate the prospects of the complex approach, which implies several amplification systems and preparations, as well as restriction analysis for determination of species and biovar of brucellosis agent strains. 

Objective of the study is to conduct comparative structural and functional analysis of the genes, encoding biosynthesis of man-nose-sensitive hemagglutinating pili in typical V. cholerae El Tor strains and genovariants. Materials and methods. Utilized were typical and genetically altered strains of cholera vibrio, biovar El Tor, isolated in the territory of the Russian Federation between 1970 and 2012 from ambient environment objects and from patients. Applied were microbiological and molecular-genetic methods, as well as analysis of the data on the whole genome sequencing. Results and conclusions. Using PCR assay the presence of mshA gene, encoding basic sub-unit of MSHA pili, has been detected in all of the tested strains. Comparative analysis of the nucleotide sequence of the genes, included into msh cluster, has revealed identity of the nucleotide sequence of mshA gene, both in typical V. cholerae strains and genovariants. Thereat, in strains of genovariants, isolated at the time of their emergence (1988, 1993, 1994), the structure of the other genes in msh operon is identical to typical El Tor vibrios. At the same time, since 1997, in strains of V. cholerae El Tor genovariants, in the sequence of the genes, involved in the assembly and secretion of MSHA pili, identified have been the SNPs, which do not influence biosynthesis of these pili, but their occurrence, probably, was due to adaptation of the genovariant strains to varying living conditions. 

Objective of the study is to assess diagnostic efficiency of PCR kits «Gen Francisella tularensis - REF» and «Gen Francisella tularensis - RGF», when performing analysis of biological samples from animals with experimental tularemia, as compared to other diagnostic methods. Materials and methods. Samples of lymphatic node, liver, spleen, lung and heart tissues, taken from animals with experimental tularemia with different infectious load, have been analyzed by PCR, ELISA, immune-chromatography, DFA, microscopy and culture cultivation. Results and conclusions. High diagnostic efficiency (98 %) has been determined for PCR kits «Gen Francisella tularensis - REF» and «Gen Francisella tularensis - RGF», used for detection of tularemia agent in biological samples taken from infected animals. For other diagnostic methods this index is: 70,4 % – for ELISA, 72,1 % – for DFA, 54 % – for microscopy and 64,3 % – for culture cultivation. Demonstrated has been the possibility and informational content of investigating lung samples for the presence of F. tularensis at early stages of infection in highly susceptible animals, using PCR and culture cultivation. 

Objective of this work was to determine the long-term preservation of Yersinia pestis DNA, detected by polymerase chain reaction (PCR) in the bone tissue of rodents, and to quantify content changes over time under the influence of biotic and abiotic factors. Materials and methods. After death of model animals infected with virulent Y. pestis strain, their skeletons were freed from soft tissues and placed in the environments where conditions were close to the natural ones. The study of bone remains for the presence of Y. pestis DNA fragments was held immediately after the death of the animals, and then, twice a year. Results and conclusions. The data obtained allow for dividing all the fossil remains of the fallen from plague infection mammals into three separate groups: with high, medium, and low content of bacteria. Further study demonstrates that reduction of Y. pestis DNA concentration in bone tissue of animals after six months of storage does not occur. After a year, a sharp decrease (100-fold) of observable material dilutions for which the identification of detected plasmids is still possible, is registered. After one and a half year of observation, the genes of plague microbe in the bones of experimental animals could not be found. In order to increase the information content of the study of this type of material put forward has been proposal to reestablish the system of observation sites to collect castings and bone remains of mammals. 

Objective of the study is to determine phylogenetic affinity of the Vibrio cholerae strains of classical biovar, isolated between 1937 and 1969 in Russia, to the strains from near and far abroad countries. Materials and methods. Utilized were 27 Vibrio cholerae strains of classical biovar. PCR was carried out applying “BIS M112” amplifier. Genetic analyzer ABI 3500xl was used for sequencing of the strains. MLVA-analysis was performed by reference to 5 MLVA-loci: VC0147, VC0436-0437, VC1650, VCA0171, and VCA0238. Nutrient requirement and soluble hemagglutinin/protease production was established using plate method. Results and conclusions. Identified have been 8 MLVA-clusters and 21 MLVA-types. It is determined that the strains, isolated during atypical cholera outbreak 1942–1943 in Russia, are inhomogeneous as regards phenotype and genotype and fall into two separate groups, one of which is related to the strain, isolated during cholera outbreak 1938 in Khabarovsk, and another group – to the strains from India and China, isolated in 1946 and 1949, respectively. 

Objective of the study is to conduct comparative assessment of specific activity of pancreatic hydrolysates of protein-containing products, both phytogenous and zoogenous, as regards Brucella test strains; and consequently, to develop nutrient medium for Brucella cultivation, to study its biological parameters. Materials and methods. Gelatin, soy, soy concentrate, corn gluten, fish meal, freshly frozen Caspian sprat, and bovine blood pancreatic hydrolysates, obtained using conventional methodology, served as the test objects. Determination of biological parameters of nutrient media, containing the studied hydrolysates was carried out using the test-strains of Brucella abortus 19 BA and B. melitensis Rev I. Results and discussion. Gelatin hydrolysate has the best biological parameters as regards the Brucella strains. Studied have been biological parameters of nutrient media, containing combination of gelatin hydrolysate and other selected hydrolysates. Experimental nutrient medium for Brucella cultivation has been developed. Comparative assessment of the experimental nutrient medium and two other commercial media used for cultivating Brucella has been performed. 

Objective of the study was to prepare hyperimmune sera to Brucella S- and L-form thermo-extracts. Materials and methods. Thermo-extracts from Brucella abortus I-206 strain in S- and L-forms were used. Chinchilla rabbits were used as animals, sera producers. Specific activity was determined by test-tube agglutination with Brucella L-form corpuscular experimental and commercial Brucella colored diagnosticums, while specificity – with heterologous strains: Francisella tularensis 15 NIIEG, Vibrio choleraе 5 (Ogawa), V. choleraе 35 А3 (Inaba), Yersinia enterocolitica О:9, Y. pestis EV NIIEG, Escherichia coli 3912/41. Protein content in the obtained extract was defined using Lowry method. Results and conclusions. Consequently, the antigen activity of the generated thermo-extracts has been shown, the immunization schemes have been developed permitting to obtain highly active and specific sera that could be used for identification and differentiation of brucellosis causative agent. 

Considered have been possible variants of emergencies, which may arise while working in BSC with pathogenic microorganisms. Put forward have been the algorithms for localization and control of the incident when working in BSC Safety Class II without spills; with spills within the working chamber; with spills and release of infectious material beyond the BSC working chamber; and incidents with compromised personal protection equipment. 

To the 100-th Anniversary of Masgut A. Aykimbaev.

To the 60-th Anniversary of Sergey V. Balakhonov.