The paper contains the data on international cooperation of the Republican Center of Quarantine and Particularly Dangerous Infections of the Ministry of Health of the Kyrgyz Republic and the Russian Research Anti-Plague Institute “Microbe” of the Rospotrebnadzor to combat plague and other dangerous infections over the period from 2016 to 2022. Areas of cooperation include conducting joint epidemiological monitoring of plague foci in the Kyrgyz Republic; exchange of up-to-date information on the state of natural foci of the two countries; equipping the anti-plague service of the Republic with modern equipment and mobile laboratories, diagnostic tools and technologies; conducting joint exercises to ensure biological safety and prompt response to emergencies; provision of advisory and methodological assistance; training and strengthening of professional personnel; conducting joint scientific researches, conferences; publication of scientific works. Data on the complex characterization of properties and phylogeographic analysis of Yersinia pestis strains isolated during field studies in the Kyrgyz Republic in 2012–2020 are summarized. The prospects for carrying out joint cartographic, molecular-genetic and paleomicrobiological work in the natural foci of the Kyrgyz Republic are outlined.

The review presents the data on domestic and foreign phenotypic classifications of Yersinia pestis strains developed in the XX century; genetic classifications of the XXI century; as well as on the genealogy of ancient strains of the plague microbe, reconstructed using paleogenomic technologies. Since the discovery of the plague agent in 1894, many classifications were created that corresponded to the level of development of microbiology at that time. The intraspecific classification schemes of the XX century were based on three principles: phenotypic differences between strains, features of the species composition of carriers, and geographical affiliation. With the development of molecular microbiology early on in the XXI century, a genetic nomenclature of the branches of the pathogen evolution was developed and a number of classifications based on the analysis of the population structure of Y. pestis were created. Through the prism of the genetic diversity of Y. pestis strains from natural plague foci in Russia, near and far abroad countries, an improved classification with a division into seven subspecies has been developed: pestis, tibetica, caucasica, qinghaica, angolica, central asiatica, ulegeica, which allocates the subspecies according to the phylogenetic principle and epidemic significance. With the advancements in paleomicrobiology, prehistoric lineages of evolution have been included in the genealogy of Y. pestis, which expand the data on the intraspecific diversity of the plague microbe.

The study of freshly isolated cultures is necessary to form an objective idea of the properties of plague microbe natural populations. The analysis of the levels of investigating the properties of strains has been carried out and the characteristics of Yersinia pestis in Kazakhstan are presented. The results of studying the phenotypic and genetic properties of plague microbe natural strains are provided. Following the epizootiological survey of natural plague foci, the museums of live cultures at plague control stations annually receive strains of plague pathogen, which are transferred to the National Collection of Microorganisms of the National Scientific Center of Particularly Dangerous Infections (NSCPDI). One of the main points of Y. pestis strains analysis is the determination of their typicality/atypicality. The study of strains begins at the moment of their isolation by anti-epidemic units. The primary identification of strains is carried out in laboratories of anti-epidemic units by morphology, sensitivity to plague and pseudotuberculosis bacteriophages, fermentation of glycerol, rhamnose and sucrose. In the laboratories of plague control stations and departments, fermentation of maltose and arabinose, denitrification, amino acid requirements, virulence, sensitivity to antibiotics are additionally investigated. Analysis of strains virulence includes determination of calcium dependence, the presence and amount of F1, pesticinogenicity and sensitivity to pesticin 1 and virulence for white mice. The assessment and preservation of the collected gene pool in the NSCPDI National Collection includes various activities, one of the main ones is an in-depth study of all features using standard microbiological methods, molecular methods for complete identification and creation of a data bank containing information about the genome of strains at different intensity of the epizootic process. The NSCPDI has a digital database on the registration and movement of strains, equipment for molecular research. The collection evaluates properties, systematizes information, and ensures the viability of plague pathogen strains for longterm storage.

The review provides an analysis of the literature data on the use of various modern molecular-genetic methods for the indication and identification of Yersinia pestis strains with different properties and degree of virulence, which is caused by the diverse natural conditions in which they circulate. The methods are also considered from the perspective of their promising application at three levels (territorial, regional and federal) of the system for laboratory diagnosis of infectious diseases at the premises of Rospotrebnadzor organizations to solve the problem of maintaining the sanitary and epidemiological well-being of the country’s population. The main groups of methods considered are as follows: based on the analysis of the lengths of restriction fragments (ribo- and IS-typing, pulse gel electrophoresis); based on the analysis of specific fragments (DFR typing, VNTR typing); based on sequencing (MLST, CRISPR analysis, SNP analysis); PCR methods (including IPCR, SPA); isothermal amplification methods (LAMP, HDA, RPA, SEA, PCA, SHERLOCK); DNA-microarray; methods using aptamer technology; bio- and nano-sensors; DNA origami; methods based on neural networks. We can conclude that the rapid development of molecular diagnostics and genetics is aimed at increasing efficiency, multi-factorial approaches and simplifying the application of techniques with no need for expensive equipment and highly qualified personnel for analysis. At all levels of the system for laboratory diagnosis of infectious diseases at the Rospotrebnadzor organizations, it is possible to use methods based on PCR, isothermal amplification, SHERLOCK, biosensors, and small-sized sequencing devices. At the territorial level, at plague control stations, the use of immuno-PCR and SPA for the indication of Y. pestis is viable. At the regional level, introduction of the technologies based on the use of aptamers and DNA chips looks promising. For the federal level, the use of DNA origami methods and new technologies of whole genome sequencing is a prospect within the framework of advanced identification, molecular typing and sequencing of the genomes of plague agent strains.

The SNP-typing method based on the detection of stable genetic markers in the genome, i.e., single nucleotide polymorphisms, is successfully used for genotyping of pathogenic microorganisms and can be applied for SNP-profiling of Yersinia pestis strains and molecular-genetic certification of focal areas. The aim of the study was to determine the SNP profiles of Y. pestis strains of the medieval biovar isolated in the Caspian Sea region plague foci in 1912–2015 and to develop a method for identifying unique SNPs using the Sanger sequencing for molecular-genetic certification of these territories. Materials and methods. A comprehensive study of the phenotypic and genotypic properties of 190 Y. pestis strains from plague foci in the Caspian Sea region was carried out. Phylogenetic reconstruction by the Maximum Likelihood method (GTR model) in the SeaView 5.0.4 software was performed on the basis of 1621 SNPs identified among 50 Y. pestis strains according to WG-SNP analysis in the snippy 4.6 program. Primers for PCR amplification of the SNP loci selected as target were calculated using the Vector NTI program. Sanger sequencing of SNPs loci was conducted on an ABI PRISM 3500XL genetic analyzer (Applied Biosystems, USA). Results and discussion. According to phenotypic characteristics, all studied strains from the Caspian foci belonged to a highly virulent and epidemically significant medieval biovar of the main subspecies of Y. pestis. According to the results of the WG-SNP analysis, 9 SNP genotypes were identified based on the polymorphism of single nucleotides of 24 genes characteristic of the main phylopopulations, which include strains isolated during various periods of epidemic and epizootic activity in the Caspian plague foci. Determining of SNP genotypes of Y. pestis strains of the medieval biovar, obtained over a hundred years in the Caspian foci, creates the prerequisites for defining the canonical SNP profile (canSNP) and for developing an algorithm for molecular epidemiological monitoring of the foci in which this highly virulent biovar circulates.

Designing of new means for the specific prevention of plague, especially protein subunit vaccines, is impossible without studying the role of individual antigens in the manifestation of the pathogenic and immunogenic properties of Yersinia pestis. The aim of the present study was to determine the antibody levels to Y. pestis antigens in guinea pigs that survived infection with sub-lethal doses of virulent plague agent strains using enzyme immunoassay (ELISA). Materials and methods. Guinea pigs were inoculated subcutaneously with 30 CFU of the wild type Y. pestis subsp. Pestis strain 231 or non-capsular Y. pestis subsp. pestis Caf1-negative strain 358/12. Blood samples from sick or recovered guinea pigs were collected on day 15, 30, 60, and 90 after infection. The antibody response was assessed by 18 recombinant Y. pestis proteins in ELISA. Results and discussion. Heterogeneity of the antibody responses to the majority of the antigens with variation of IgG titers from animal to animal has been revealed. We observed increase in antibody titers by day 90 for the most analyzed antigens in the sera of the guinea pigs injected with wild type Y. pestis 231. On the contrary we found reduction in antibody titers by day 90 in case of inoculation with Y. pestis 358/12. The preservation of antibodies to Y. pestis proteins of different localization in the organism of the guinea pigs, as well functional activity, and the degree of representation on the surface of bacterial cell for a prolonged period of time indicates the multiplex nature of the plague immunity formation. Our findings are significant for the future design and development of effective vaccines against plague and the search for new targets for diagnostics of this disease.

The aim of the study was to compare the nucleotide sequences of pgm‑region genes in Yersinia pestis strains isolated on the territory of the Caspian sandy and adjacent plague foci in 1925–2015. Materials and methods. 65 Y. pestis strains from the Caspian sandy and adjacent plague foci were used in the work. DNA isolation was performed using the PureLink Genomic DNA Mini Kit. Whole genome sequencing was conducted in Ion S5 XL System (Thermo Fischer Scientific). Data processing was carried out using Ion Torrent Suite software package 3.4.2 and NewblerGS Assembler 2.6. To compare the obtained sequences with the NCBI GenBank database, the Blast algorithm was used. The phylogenetic analysis was performed according to the data of whole genome SNP analysis based on 1183 identified SNPs. The search for marker SNPs was performed using the Snippy 4.6 program. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model. Results and discussion. The nucleotide sequences of pgm‑region genes of 65 Y. pestis strains from the Caspian sandy and adjacent plague foci have been assessed. Single nucleotide substitutions have been identified in Y. pestis strains from the Caspian sandy and Kobystan plain-foothill foci in the hmsR, astB, ybtS, ypo1944, ypo1943, ypo1936 genes, as well as a deletion of 5 bp in the ypo1945 gene, which is characteristic of strains of one of the phylogenetic lines of Y. pestis from the foci of Caucasus and Transcaucasia, isolated in 1968–2001. The data obtained can be used to differentiate Y. pestis strains from the Caspian sandy focus, as well as to establish the directions of microevolution of the plague pathogen in this region and adjacent foci.

The dependence of the incidence of various nosologies on the epidemic situation in neighboring countries has been a feature of the epidemic process in recent years. In this regard, it is of particular importance to carry out joint anti-epidemic and preventive measures in border areas in order to prevent the importation of dangerous infections into the territory of neighboring states. The aim of the work was to analyze the results of international cooperation in the prevention of particularly dangerous infections. Presented are the areas of cooperation and outcomes of joint research activities. Measures for cooperation between the relevant institutions of the Ministry of Health of the Republic of Kazakhstan and the Rospotrebnadzor on the operational exchange of information in case of emergencies, joint research work on the monitoring of particularly dangerous and other natural-focal infectious diseases in the border areas, joint seminars, scientific and practical conferences on the introduction of modern methods of laboratory diagnostics into practice, internships on the exchange of experience in epizootiological survey in foci of particularly dangerous infections are described in the paper. Examples of Russian-Kazakh cooperation are provided. The results of a joint epizootiological survey of the territory of the Kazakh part of the Altai Mountains are presented. On a global scale, cases of plague and other particularly dangerous infections in any geographic region can constitute international public health emergencies and this type of threat requires international cooperation.

The aim of the work was to put forward the methods for direct quantitative determination of the content of Yersinia pestis and Rickettsia raoultii protein antigens in preparations and various prototypes of subunit vaccines. Materials and methods. Y. pestis LcrV and Caf1 antigens enclosed in the substance of the molecular microencapsulated plague vaccine (MMPV) and separately, in microcrystals of amino acids co-precipitated with plague proteins were used as model antigens. R. raoultii Adr2, OmpB24, and YbgF antigens were adsorbed on the prototype substance of the rickettsia vaccine. The release of plague antigens from MMPV microcapsules was carried out through successive treatment of the latter with organic solvents, methylene chloride and methanol, respectively; the carrier microcrystals were dissolved in 0.1 M sodium citrate buffer at pH 6.0. The antigen content in the prototype substance of the rickettsial vaccine was determined by measuring the amount of proteins not bound to the alumogel. Quantitative parameters characterizing the content of antigens in the substances and prototypes of vaccine preparations were calculated by processing digital images of polyacrylamide gels obtained by electrophoresis of protein antigen fractions extracted from carriers. Results and discussion. Methods for direct extraction and subsequent quantitative analysis of Y. pestis LcrV and Caf1 antigens from subunit vaccine preparations based on amino acid microcrystals and polylactide microcapsules that do not cause protein degradation have been studied. A different nature of the binding of LcrV and Caf1 in the substances of microcrystals has been established, while the proportion of antigens released from microcrystals has been quantified only in case of their complete dissolution. It was found that at low concentrations of LcrV and Caf1 proteins extracted from microcrystals, it is necessary to concentrate the extracts with subsequent removal of salts for their reliable visualization. It has been confirmed that 10 μg of plague antigens and proteins of R. raoultii in a dose volume of 200 μl of suspension is sufficient for quantitative analysis using electrophoretic method. The prospects of other physicochemical methods alternative to direct extraction of antigens for evaluating the composition and quality of vaccine preparations are discussed.

Pathogenic bacteria use low-molecular-weight iron chelators – siderophores – to assimilate iron in the host body. Being recognized as virulence factors, these molecules, differing in structural and functional properties, are the subject of the most intensive research in medical microbiology. The present study is devoted to the investigation of yersiniachelin siderophore (Ych) found in the causative agent of plague, Yersinia pestis. The aim of the work was to clarify the role of Ych in the physiology of Y. pestis by comparing the properties of three strains of the plague microbe, differing in Ych production. Materials and methods. Three variants of Y. pestis EV76 strain were used in the experiments: parent strain Y. pestis EV76, its mutant that does not produce Ych due to deletion of three siderophore biosynthesis genes (analogues of ypo1530–1532 in Y. pestis CO92 strain) and a complemented mutant that was transformed by a recombinant pSC-A-5EV plasmid containing Ych biosynthesis genes cloned into the high-copy plasmid vector pSC-A-amp/kan. Comparative analysis of the three strains was carried out in terms of colony morphology, siderophore activity, growth rate, and sensitivity to hydrogen peroxide. Results and discussion. The comparison of these strains has revealed that the secretion of Ych by bacteria at 26 °С ensures the assimilation of iron. At 37 °С, Ych is not secreted into the medium and protects bacteria from the bactericidal action of reactive oxygen compounds. Thus, the study shows that yersiniachelin is able to stimulate the assimilation of iron by bacteria under iron-deficit conditions and has antioxidant properties.

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods.

The purpose of the study was to assess the current epizootic potential of the Transcaucasian high-mountain and Pre-Araks low-mountain natural plague foci on the territory of the Republic of Armenia using GIS technologies. Materials and methods. We used the data from an epizootiological survey, records of the abundance and species composition, spatial distribution of rodents and ectoparasites in the plague-enzootic territories of the Republic of Armenia in 2021. Results and discussion. Based on the results of the research, an electronic database of carriers and vectors of pathogens of natural-focal zoonotic infections in the plague-enzootic territories of the Republic of Armenia has been created. Applying GIS technologies, an assessment of the spatial distribution of carriers and vectors of plague has been made and areas of circulation of tularemia and leptospirosis pathogens identified. The results obtained serve as the basis for improving the efficiency of planning and carrying out preventive measures aimed at ensuring the epidemiological welfare as regards natural-focal infectious diseases in the territory of the Republic of Armenia.

Entomoparasitic nematodes are supposed to be a link between parts of Yersinia pestis population in the environment and the flea vector. The aim of the study was to assess the prevalence and intensity of infestation in the fleas of the long-tailed souslik with entomoparasitic nematodes on the territory of Mongun-Taiginsky station in the Tuva natural plague focus. Materials and methods. Fleas were collected during the scheduled epizootiological surveys in 2019–2021. In the course of taxonomic identification the presence of parasitic nematodes was registered. In order to evaluate the intensity of nematode invasion, a total of 190 fleas were dissected. The number of adult parasitizing females and presence of larvae was recorded. Statistical processing of the data was performed with the help of conventional methods using the Excel software. The criterion χ2 was applied; the influence of various factors (species, gender of fleas) on the studied parameters was assessed through single- and two-factor analysis of variance. Results and discussion. During three years of observations, entomoparasitic nematodes were found in six species of fleas: Citellophilus tesquorum, Frontopsylla elatoides, Rhadinopsylla li transbaikalica, Frontopsylla hetera, Oropsylla alaskensis, and Neopsylla mana. The differences in infestation with nematodes between the species are presented. The highest invasion rate – 25.1–25.6 % – is observed in Rh. li transbaikalica. The gender of leas does not influence their infestation. It is established that invaded fleas are more often found in the nest than in the fur of animals, they are less actively migrate to the burrow entrance compared to not invaded ones. Evaluation of infestation prevalence has revealed that fleas Rh. li transbaikalica are the hosts for nematodes of mono- or oligoxenic species, which do not occur in other fleas.

The aim of this study was to develop a new method of intraspecific genetic differentiation of Yersinia pseudotuberculosis, based on the detection of INDEL-markers using PCR. Materials and methods. Analyzed were 308 strains from the NCBI database and 15 strains sequenced within the frames of this study. The nucleotide sequences of the strains were determined using the MiSeq technology platform. The genomes of the strains sequenced in the work, as well as genomes from the NCBI database, were assessed using in silico PCR with 7 pairs of primers designed in the study. As a result of a comparison of genome-wide sequences of 22 Y. pseudotuberculosis strains from the NCBI database, using the author’s software (GenExpert), 7 INDEL-markers were selected that make it possible to effectively distinguish between strains of the causative agent of pseudotuberculosis. Based on these markers, 7 pairs of primers were designed and synthesized for the analysis of different strains using PCR. Analysis of 323 strains in PCR in silico and 70 strains in PCR in vitro allowed for dividing them into 30 genetic groups. Comparison of the results of PCR in silico and in vitro confirmed the possibility of using the proposed primers for intraspecific differentiation of Y. pseudotuberculosis. Based on the data obtained, a dendrogram reflecting the phylogenetic relations of different strains of Y. pseudotuberculosis was constructed. When analyzing the distribution of Y. pseudotuberculosis strains by various clusters and genetic groups, a number of patterns were revealed. Conducted in silico and in vitro PCR show that the proposed method of INDEL-typing can be used for intraspecific genetic differentiation of the causative agent of pseudotuberculosis.

Advanced molecular-genetic methods for the diagnosis and typing of Yersinia pestis ssp. pestis in the field and clinical material are used for epidemiological surveillance of plague in the Saylyugem natural focus. The aim of the work was to study the spatial genotypic structure of Y. pestis ssp. pestis in the transboundary Saylyugem natural plague focus using MLVA25 typing. Materials and methods. The MLVA25 typing of 160 strains of Y. pestis ssp. Pestis isolated in the Saylyugem natural plague focus in 2012–2021 was carried out. Phylogenetic tree construction was performed with the help of UPGMA and MST methods. Results and discussion. The Y. pestis ssp. pestis strains isolated from the Saylyugem natural plague focus were differentiated into 15 MLVA types by the 25 VNTR loci cluster analysis. The studied strains form a homogeneous complex of MLVA25 types without marked geographical distribution across seven spatial groups. The analysis of the frequency of occurrence of the tandem repeats number for three variable loci of Y. pestis ssp. pestis strains shows the significant differences between the samples from the Mongolian and Russian parts of the Saylyugem natural plague focus. The most pronounced differences in spatial genotypic structure are traced through the yp4280ms62 locus.