The paper presents the analysis of the current epidemiological situation from the viewpoint of identification of risks of emergency situation onset in the sphere of biological safety, which differs from the emergency situation of sanitary-epidemiological welfare in the character of after-effects that are comparable in the quality and the scale to the threats to national and international security. In reference to the parameters of emergency situation in the sphere of biological safety, epidemic of Ebola fever in West Africa countries (2013-2016) has been described. Biosafety emergency situation is most likely to be predicted in the following cases: occurrence of the flu pandemic of the novel subtype (International Health Regulations, 2005); intensification of unfavorable for public health tendencies against the background of Zika fever transmission; larde-scale failures in the provision of biological safety when working with pathogenic biological agents; foreseeable, hard to control consequences of onset and transmission of infectious diseases caused by microorganisms with synthetic genome; occurrence of epidemic events during international mass meetings; justified interpretation of emergency situation, applying IHR (2005), as an after-effect of violation of the Convention on the Prohibition of Biological and Toxin Weapons (CBTW) and the grounds for intervention into the internal affairs of offending countries with social-economic, geo-political aftermaths and damage to the national security.

Objective of the study was to develop the methodology for identification of administratively independent areas under potential risk of “external” epidemiological threat realization, by the example of cholera.

Materials and methods. Analysis was conducted using free software package with open source code (QGIS 2.8 and GRASS GIS 7.0) on the basis of the data received from Rosgranitsa and the Federal State Statistics Service. The construction of risk cartogram was performed on the base of Euclidean distance and estimation of nuclear density.

Results and conclusions. In accordance with the obtained results, the GIS containing information about the checkpoints on the Russian border, settlements, roads and railway lines was worked out. The method of identification of risk areas due to importation of infectious diseases based on the cholera model has been developed, and the total area of such territories was less than 1 % of the total area of the country. It was found that in some cases the risk area is located at a certain distance from the checkpoint, but the existence of checkpoint does not lead to the formation of risk areas. The developed GIS is available on the geo-information portal of FGHI Rostov-on-Don Research Anti-Plague Institute of Rospotrebnadzor. 

Currently existent procedure for free positioning and registration of sites for field sample collection results in chaotic concentration of visualized on electronic map icons of surveyed areas, which obstructs spatial analysis of the results on monitoring in a given period of time.

Objective of the study is to develop the system of standardization, positioning, and numbering of the points for epizootiological surveillance as part of monitoring over natural plague foci.

Materials and methods. Topographical maps, epizootiological mapping data and their analysis.

Results and conclusions. Optimum size of the icons corresponding to sites of epizootiological surveillance (SES) is 10 seconds in latitude and 15 seconds in longitude. One sector comprises 900 standard SESs. Put forward system of optimization is able to significantly ease epizootiological and epidemiological mapping, enhance visualization of monitoring data, as well as expand the capacities and improve the quality of spatial and retrospective analysis of epizootic activity of natural plague foci. 

The high-priority task of prospective preventive vaccination consists in the development of differentiated approaches to vaccination with the aim of generation of effective immunity in each immunized person, using various dosages and vaccination regimes as well as adjuvants and accessory means of immune response modulation. At present, multiple investigations are dedicated to the search of immunoadjuvants capable of inducing immune response even in individuals with compromised immune activity and immunodeficiency signs. The review exemplifies combined usage of different groups of immunobiological agents in domestic applied medicine – vaccines, immunomodulators, cytokines, as well as prospective preparations, prequalified and recommended for clinical trials. The review covers major objectives, additional approaches and focus areas in experimental immunology related to the advancement of vaccinal prevention of infectious diseases of different etiology applying preparations capable of stimulating the induction of postvaccinal immunity. Proper attention is given to the possibility of immunomodulator usage in the specific prophylaxis of particularly dangerous infections.

The article presents the results of bioassays investigation by the specialists of the Center for Special Laboratory Diagnostics of Particularly Dangerous and Exotic Infectious Diseases, isolated during 2002–2015, obtained from deceased people suspected for rabies virus infection. Outlined are the main challenges of working with the received samples of biological material from dead people and ways of handling these issues. Put forward are the pressing problems of epizootic and epidemiological bias that require comprehensive solutions on the part of healthcare authorities and the veterinary service of the Russian Federation. Given are the results of the laboratory-diagnostic activities of the Center for Special Laboratory Diagnostics of Particularly Dangerous and Exotic Infectious Diseases, which indicate that the applied methods of isolation and identification of the rabies pathogen allow for efficient and in-time diagnosis of the disease.

The review presents brief analysis of the published over the past decade results of investigations devoted to studies of currently known molecular action mechanisms of Yersinia pestis virulence factors. Analyzed are different Y. pestis components, synthesized by the bacteria at four stages of infectious process in case of bubonic plague: in derma, lymph nodes, parenchymal organs, and blood. Described are the factors and mechanisms that induce microbe protection from bactericidal action of humoral and cell factors of innate host immunity, which effect the organism of animals in different ways, stimulating pro- or anti-inflammatory reaction of a host to the infection, and contribute to the shift of bacterial life cycle inside the host, providing for the transfer from intracellular propagation in phagocytes at early stages to extracellular propagation in lymph node, spleen, liver and blood at later stages. Discussed are only those factors of Y. pestis the interaction of which with host molecules and cells at different stages of infectious process in case of bubonic plague is experimentally proved.

Objective of the study is in vitro investigation of mutual relations between Citellophilus tesquorum altaicus and Yersinia pestis with various plasmid composition: influence of the strain on flea alimentary activity and mortality rate, frequency and dynamics of biofilm formation.

Materials and methods. C. tesquorum altaicus were infected by three Yersinia pestis strains: virulent triple-plasmid I-3230 isolated in Mongolia, referential for the Tuva focus I-2638 carrying four plasmids (pYT, pYV, pYP, pTP 33) and also selected from it avirulent isogenic clone I-3480 that lost two plasmids (pYV, pYP). Peculiarities of interaction between fleas and Y. pestis strains were estimated through the lens of specimens with «conglomerates» and “blocks” for feeding, the period from infection prior to the beginning of conglomerates’ formation, alimentary activity, and mortality rate of the infected fleas.

Results and conclusions. It was revealed that alimentary activity of the infected insects was higher than that of the control group, and the highest – in fleas infected with I-2638 strain. Greater numbers of dead fleas at feeding was noted in specimens inoculated with I-3230 strain. Predominant significance of I-2638 strain was established in C. tesquorum altaicus biofilm formation both as «conglomerates» and “blocks”. I-3480 strain also formed the conglomerates in fleas more actively than I-3230 lacking pTP33 plasmid. Thus, four-plasmid I-2638 strain surpassed triple-plasmid I-3230 and two-plasmid I-3480 strains in reference to all tested indicators except flea mortality rates. It may testify to co-adaptation of Y. pestis and C. tesquorum altaicus from the Tuva plague focus and to the possibility of a pTP33 functional role in enhancement of a biofilm formation in vivo. 

Objective of the study was to develop peroxidase conjugate on the base of monoclonal antibodies (MCA) H2F6 and to study the possibility of its application for the detection of tcp+ Vibrio cholerae O1/O139 strains using direct ELISA methods.

Materials and methods. Utilized for the investigation was the hybrid H2F6 clone, which synthesized monoclonal antibodies specific to the outer membrane protein of cholera vibrio into culture medium.

Results and conclusions. Peroxidase conjugate was designed on the base of MCA which allows for the detection of tcp+ V. cholerae O1 and O139 strains in direct solid-phase enzyme-linked immunosorbent assay and dot-ELISA. The preparation was tested on a group of strains of V. cholerae and heterologous microorganisms and showed specificity in relation to V. cholerae O1 and O139. Monoclonal peroxidase conjugate H2F6 can be used for the detection of epidemically significant V. cholerae O1/O139 strains by means of immune-enzyme methods. 

Objective of the study was to model the interaction of Burkholderia pseudomallei with Tetrahymena pyriformis in vitro and investigate the changes in the population composition of the protozoa when co-cultured with a microorganism.

Materials and methods. B. pseudomallei 110, C141, 57576, 107 strains differing in virulence for BALB/c mice were used. The axenic culture of T. pyriformis was incubated with microorganisms in 100 to 1 ratio, at 28 °C, in LB. Samples of co-cultures were examined using light microscopy, by counting the number of trophozoites and cysts in the population. Dynamics of multiplication of B. pseudomallei cultures associated with T. pyriformis was determined through seeding bacteria on a dense nutrient medium to count the grown colonies.

Results and conclusions. B. pseudomallei in association with T. pyriformis is ingested by protozoan cells; it multiplies in them and stimulates protozoa encystment. Hereby virulent strain B. pseudomallei 110 induces encystment of T. pyriformis on days 2–4 and complete cell destruction within 7–8 days. Avirulent strain, B. pseudomallei 107, induces full encystment on day 7; significant part of the cysts remains intact on day10. Dynamics of B. pseudomallei growth, co-cultured with T. pyriformis is characterized on day 1 by distinct decrease in the number of viable bacterial cells and increase in it within following 24 hours. Bacteria concentration curves depend on the virulence of the strain: maximum level of B. pseudomallei 110 replication is observed after 48 hours, while that of B. pseudomallei 107 – not less than after 7–8 days. 

Objective of the study is to design the algorithm for assessment of expression of the structural and regulatory virulence Vibrio cholerae genes by the model of ctxA and toxR genes encoding and controlling biosynthesis of cholera toxin.

Materials and methods. Utilized were 10 strains of Vibrio cholerae, classical and El Tor biovars. Cloning of gene fragments was carried out through transformation and ligation. RT-PCR was done in “BIS M112” and “Rotor-GeneQ” amplifiers. Processing of the results was performed by means of the software package in set with Rotor-GeneQ (Software 1.8.17.5).

Results and conclusions. Developed has been the algorithm for evaluation of the level of expression of V. cholerae ctxA and toxR genes applying RT-PCR with real-time hybridization-fluorescent registration of results. The stated algorithm allows for rapid and effective statistically significant specification of the expression of structural and regulatory virulence V. cholerae genes and can be used for evaluation of newly discovered cholera vibrio strains. 

Objective of the study is to investigate genetic diversity of Brucella strains of various species, isolated from humans and small mammals in the territory of the North Caucasus Federal District using MLVA14, as well as to identify the correlation between the formed clusters and the place setting, the time setting, and object of isolation.

Materials and methods. Carried out has been MLVA14 genotyping of 91 Brucella spp. strains isolated from patients, small mammals and live-stock animals. The sizes of amplicons were compared with MLVA Bank 5.0 database, tutorial version 1.6. Based on the data obtained, constructed was a dendrogram of phylogenetic interrelations of the studied strains. Results and conclusions. Using the data on MLVA genotyping, the studied strains were subdivided into 61 MLVA14 genotypes. Strains of B. suis, B. abortus, B. melitensis form separate clusters in the dendrogram. Strains isolated during cluster cases of brucellosis in humans have similar MLVA14 B. melitensis types, B. suis strains are confined to the epizooties among small mammals that may occur in different administrative territories. The data can be used for monitoring of brucellosis agent, including as an effective tool for retrospective epidemiological analysis. 

Modernization of continuing education system in the sphere of healthcare and enhancement of training efficiency is associated with utilization of information and communication technologies, including remote and e-education.

Objective of the study is to assess the prospects of application of electronic and remote learning in case of vocational training for work with pathogenic biological agents.

Materials and methods. Using analytical methods, evaluated have been legislative, normative-methodological documents, publications in the sphere of information-communication technology application in educational activities, as well as safety of works with PBA of the I-IV groups of pathogenicity; professional competencies of the specialist allowed to handle PBA; advanced retraining and vocational programs realized at the premises of the plague-control institutions of the Rospotrebnadzor.

Results and conclusions. Holding vocational reorientation courses via remote education is considered to be impossible. It is reasonable, prospective, and upto-date to apply elements of electronic learning when realizing curricula for professional reorientation, and elements of remote and electronic education in case of advanced professional training. Modern information technologies can significantly enhance efficacy of further (continuing) vocational education at the premises of plague control institutions of the Rospotrebnadzor. 

Objective of the study is to investigate biological properties of avian influenza virus strains that caused the outbreaks in Russia in 2016–2017.

Materials and methods. The study was performed using advanced virological and molecular-biological methods in state-of-the-art equipment.

Results and conclusion. In 2016, the outbreaks among wild birds and poultry caused by highly pathogenic avian influenza H5N8 virus have occurred in the territory of the Russian Federation. In May, 2016 an outbreak of H5N8 among wild birds was registered in the territory of the Republic of Tyva. In October-November, 2016 influenza virus H5N8 was isolated in the territory of the Republics of Tatarstan and Kalmykia, Krasnodar and Astrakhan Regions of Russia. In 2017 avian influenza H5N8 has become widespread in European part of Russia and caused multiple outbreaks among wild birds and poultry. Results of the investigations of the isolated strains show that all of them are highly pathogenic and belong to the clade 2.3.4.4. Molecular-genetic and virological analysis has revealed the differences between the viruses isolated in 2016–2017 and the virus of the same clade 2.3.4.4 that was isolated in 2014. 

Objective of the study was to assess analytical and diagnostic sensitivity and specificity of the “Reagent kit. Erythrocytic coccidioidomycosal and histoplasmosal immunoglobulin dry diagnosticum”, designed for identification of causative agents of coccidioidomycosis and histoplasmosis in isolated cultures of micromycetes, as well as in clinical and biological samples using indirect hemagglutination test.

Materials and methods. The investigation included 264 positive samples (216 samples of micromycete suspensions, 48 samples of biological and clinical material) containing pathogens of histoplasmosis and coccidioidomycosis concentrated up to 3,12·10⁶ and 1,56·10⁶ cells/ml, respectively, and 128 negative samples containing heterologous microorganisms in concentrations equal to 5·10⁶ cells/ml. The study was carried out using biological samples that were artificially contaminated with stated pathogens of particularly dangerous mycoses and samples, obtained from bioassay animals with experimental infection.

Results and conclusions. It is established that diagnostic sensitivity of the reagent kit is not less than 99,0 %. The diagnostic specificity is not less than 98,0 %. Reproducibility of the results in all cases was 100 %. The results obtained testify to the prospect of introduction of the developed kit into the health care practice. 

Minimization of risks of infection when training the specialists to work with microorganisms – agents of particularly dangerous infectious diseases – is one of the key objectives for staff members of the Department for specialists training and curricula development at the FGHI RusRAPI “Microbe”. The paper discusses the ways to reduce the risks of infection with agents of particularly dangerous infectious diseases among the attendee and the tutors of qualification courses while studying how to work with this group of microorganisms. Outlined are priority areas: usage of attenuated, avirulent strains of the agents, as well as recombinant strains of nonpathogenic bacteria; exploitation of the state-of-the-art technical equipment; insure the skills of safe PBA handling and professionally significant traits of character in the process of realization of currently existing academic programs. Identified has been the optimum approach to the assessment of reliability and safety of professional activities among the personnel working with PBA of the I–II groups of hazard. Developed has been the algorithm for competence level evaluation in the specialists who are allowed to work with PBA of the I–II groups of hazard; job profile diagrams have been charted. Designed methods of expert evaluation of professionally significant skills and traits of the staff provide for personnel identification using a range of psychological tests and play an important role in vocational selection and orientation, both during the job placement and advanced training of the staff, as well as in the reduction of risks associated with the factor of human error.

Objective of the study is to investigate the role of resident plasmids pMT1, pCD1, and pPCP1 in the production of extracellular form of Yersinia pestis lipopolysaccharide (LPS).

Materials and methods. The experiments have been performed using Y. pestis strain EV76 (pMT1, pCD1, pPCP1), carrying the whole plasmid set, as well as plasmid-free Y. pestis variant EV76 (pMT1^-, pCD1^-, pPCP1^-), and isogenic clones, harbouring only one plasmid: Y. pestis EV76 (pMT1); Y. pestis EV76 (pCD1); Y. pestis EV76 (pPCP1). The presence of extracellular LPS in the incubation medium of Y. pestis EV76 cells has been confirmed by supernatant toxicity for laboratory animals and also by LAL-test reaction.

Results and conclusions. It has been established that LPS extracellular form is produced by 37 °C Y. pestis EV76 cultures of the initial strain and its variants, carrying pMT1 or pPCP1 plasmid. Plasmid-free cultures and variant harbouring pCPP1 plasmid are deprived of such ability. The results of LAL-test has shown that the process of LPS separation from cell wall membrane into the environment is associated with translocation of proteins encoded by pMT1 and pCD1 plasmids and constitutes a natural form of existence of Y. pestis cells. The involvement of pCD1 plasmid in realization of the toxic potential of Y. pestis LPS has been established for the first time ever. 

Objective of the study is to evaluate the effect of immunomodulators on the intensity of the post-apoptotic lysis of sensitized organism leukocytes in the presence of specific antigens of tularemia microbe in vitro.

Materials and methods. Flow cytometry method was used to determine the relative content of apoptotic and proliferating splenocytes obtained from mice, immunized against tularemia against the background of immunomodulation.

Results and conclusions. Obtained is the evidence that is consistent with modern data on the massive leukocyte apoptosis and post-apoptotic leukocyte autolysis (secondary necrosis) in case of tularemia infection. Given the important role of secondary necrosis in the systemic inflammatory response development, the use of immunomodulators suppressing macrophage apoptosis and dead leukocyte lysis, emerging in the course of interaction with Francisella tularensis antigens, may be promising in order to reduce the live tularemia vaccine reactogenicity.

Objective of the study was to determine allelic variants of HLA II class haplotypes in persons living in the Republic of Kalmykia in the territory of Pre-Caspian sandy natural focus of the plague, immunized for epidemic reasons with live plague vaccine and search for associations of HLA class II haplotypes with peculiarities of post-vaccinal immunity development.

Materials and methods. 20 individuals took part in the study. HLA typing was performed applying multiplex PCR. Production of immune-regulatory cytokines and antibody titers to fraction 1 of the plague microbe was determined using enzyme immunoassay. Statistical processing of the results was carried out using standard programs.

Results and conclusions. Allelic variants of haplotypes HLA-DQA1, HLA-DQB1 and HLA-DRB1 class II of the main histocompatibility complex of 20 persons residing in Lagansky and Chernozemelsky districts of the Republic of Kalmykia have been identified. Determined have been the differences in the ratio of allelic variants of HLA-DQA1 and cytokine production INF-γ, TNF-α, IL-4 and IL-10 by the areas of residence. Association of the HLA-DRB1*01 allele with a high level of spontaneous and induced IL-10 cytokine production has been revealed at various times after booster vaccination. Further study of genes that regulate the development of immunity, along with immunological methods will make it possible to personalize the use of the existing vaccine against plague, and predict the immunogenicity and effectiveness of preventive drugs under development. 

Objective of the study is to experimentally substantiate the possibility of production of anti-cholera immune-enterosorbent in the pharmaceutical form of a pill.

Materials and methods: experimental antitoxic immune-enterosorbent against cholera, stabilized using cool dehumidification. Residual moisture of enterosorbent lyophilizate was determined by means of Sartorius MA 150 moisture meter. Screen assay of the powders and granulates was performed applying the sieving of bulk material samples through screen set with sieve openings of various sizes. Specification of tap density was carried out with the help of SVM-10 unit. Granulation of specific enterosorbent was done using GPCG 2 LabSystem. The pills were manufactured in tablet press, MiniTabT. The hardness of the pallets was carried out by means of TBH 125 TD tester. The test for friability of the pills was performed in GTA 120.

Results and conclusions. Utilization of cellulose microcrystalline and polyvinyl pyrolidone as pharmaceutical aids for granulation of lactose monohydrate is experimentally substantiated. It is established that the obtained granulate with passable values of technological parameters can serve as the basis for pharmaceutical dosage form of specific enterosorbent. Identified has been optimum mass of anti-cholera enterosorbent tablet cores. Demonstrated has been the possibility of Acryl-eze enteric coating application as protective shell of the pills. Studied has been specific activity of the pelleted enterosorbent in vitro, verified is its resistance capacity under conditions modeling gastrointestinal tract. In consequence of the performed trials and tests, technology for the production of antitoxic enterosorbent against cholera – enteric-coated tablet – has been developed. Long-term trials: the storage of the constructed preparation at 4–8°C within 24 months period (the observation time) – have revealed retention of enterosorbent activity, which testifies to its stability and defined the shelf life of specific tableted anti-cholera immune-enterosorbent. 

Objective of the study was to optimize technological scheme for the production of preparation for gene diagnostics of plague with hybridization-fluorescent registration (HFR) of results, which is associated with the assessment and selection of synthesis conditions and method of oligonucleotide probe purification included into the kit and carrying fluorophore (6-carboxyfluorescein) and quencher (Black Hole Quencher-1) tags, as well as to evaluate their properties and implement this new technological line into manufacturing.

Materials and methods. The object of investigation was the reagent panel “Gene Yersinia pestis Identification-HFR” and its constituent elements, probes and primers, providing for hmsH gene amplification. Synthesis of the primers and probes was carried out in 8-well DNA synthesizer ASM-800 (Bioset, Russia), using solid phosphite amid method. For the experiments the probes obtained were purified either by means of electrophoresis in 20 % polyacrylamide gel (size 20×20) or (size 8×10) with further purification through RP-cartridges in semi-automated mode in OPS-201 system and manually, or on RP-cartridges only in manual mode; or applying highperformance liquid chromatography (HPLC). Specific activity of the produced probes was tested by polymerase chain reaction using the DNA isolated from bacterial suspensions of Yersinia pestis C-624 strain, in concentrations up to 1·10³ – 1·10⁶ mc/ml.

Results and conclusions. Optimized has been synthesis technology; selected has been the method for oligonucleotide probe purification, carrying phluorophore-6-carboxyphluorescin and Black Hole Quencher-1. Demonstrated is viability of applying the stated oligonucleotides or HPLC, or electrophoresis in 20 % polyacrylamide gel with 7 M urea and further purification through RP-cartridges in manual mode for purification purposes. Introduction of the optimized technological scheme in manufacturing of preparations for gene indication and identification of particularly dangerous pathogens with hybridization-fluorescent detection will allow for the reduction of lead time by 50 % and original cost – by 66 %.