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The aim of the work is a retrospective molecular examination of plague outbreaks that occurred on the northern coast of the Aral Sea in the middle of the past century, and a phylogenetic analysis of the Yersinia pestis strains that caused them. Materials and methods. Whole-genome sequences of 39 Y. pestis strains from foci of the Northern Aral Sea region, isolated in 1945–1974, were used. Whole-genome sequencing was performed in the Ion S5 XL System (Thermo Fischer Scientific, USA). Data processing and sequence assembly were carried out using the Ion Torrent Suite software package, v5.12, FastQC v0.12.0, unicycler v0.5.0. Phylogenetic reconstruction was carried out using the wholegenome SNP analysis method, the dendrogram was constructed using the Maximum Likelihood algorithm using the PhyML v3.1 program. Molecular typing of strains was performed using the MLVA25 method. Results and discussion. Y. pestis strains isolated in the Northern Aral Sea region between 1945 and 1974 belong to the phylogenetic branch 2.MED1 of the medieval biovar of the main subspecies. Plague outbreaks in the Northern Aral Sea region in 1945, 1955, 1966, and 1969 were caused by strains of the Caspian subbranch 2.MED1, and the outbreak of 1967 – by strains of the Central Asian subbranch, which successively reached this region from the Northern Caspian and Balkhash regions. It is shown that the sources of human infection were epizootics occurring on the northern coast of the Aral Sea. It is established that in the 1960s, Y. pestis strains of the Caspian and Central Asian subbranches 2.MED1 medieval biovar circulated simultaneously in the Northern Aral Sea region. A characteristic feature of the plague outbreak of 1945 was the presence of a large number of SNPs specific to each strain, possibly due to the process of rapid adaptation of the Caspian subbranche 2.MED1 strains to the conditions of the natural biocenosis of the Northern Aral Sea region. Subsequently, the rate of evolution of the emerging Northern Aral population 2.MED1 significantly decreased, which was accompanied by the formation of individual clusters of strains with a small number of individual SNPs.

The Aksai high-mountain focus is part of the Tien Shan focus, which has recently shown constant epizootic activity. In 2024, Yersinia pestis cultures were isolated from the Aksai focus for the first time in the 21st century. The aim of the study was molecular identification of Y. pestis strains collected in the Aksai focus in 2024, their phylogenetic analysis to establish their origin and obtain whole-genome sequences that are reference ones for this focus in the mo dern period. Materials and methods. The genomes of Y. pestis strains were sequenced on a MinIon genetic analyzer (Oxford Nanopore Technologies, UK). Sequencing libraries were prepared using the Native Barcoding Kit 24 v14 (SQKNBD114.24) and sequenced on an R10.4.1 flow cell. Core SNPs were identified applying pair wise alignment of the obtained whole-genome sequences to the genome of Y. pestis CO92 strain using Snippy v4.6.0 software. The Maximum Likelihood dendrogram was constructed using PhyML 3.1. Results and discussion. According to the phylogenetic analysis of whole-genome sequences, it has been established that the Y. pestis strains isolated in the Aksai focus in 2024 belong to the phylogenetic branch 0.ANT3 of antique biovar. The Y. pestis strains of 2024 fall under the 0.ANT3 polytomy, which also includes all 0.ANT3 strains of the 20th century from the Aksai, Upper Naryn and Alai high-mountain foci and strains from the Xinjiang province in China. The genetic differences between the 0.ANT3 strains of the current and last centuries are small, which proves the continuity of the preservation of this population in the 20th–21st centuries and the local origin of the 2024-strains. Strains 0.ANT3 were previously isolated across large areas of the Upper Naryn, Aksai, Alai plague foci and on the adjacent territories of China, which indicates the existence of a mega-focus of 0.ANT3 Y. pestis there. Strains 0.ANT3 are highly virulent and epidemically significant. The activation of the Aksai natural plague focus requires increased epidemiological surveillance of this autonomous plague focus in the Tien Shan.

The aim of the study is to obtain a comprehensive characterization of Yersinia pseudotuberculosis strains from the Aksai high-mountain and Balkhash desert foci of plague in Central Asia. Materials and methods. Investigation of the cultural, morphological and biochemical properties of the examined strains was carried out using conventional methods of laboratory diagnostics. Ion S5 XL System (Thermo Fischer Scientific) was used for whole-genome sequencing. Data processing and sequence assembly of raw reads de novo were performed using Ion Torrent Suite software package 5.12 and Newbler gsAssembler 2.6 (454 Life Sciences). To search for marker SNPs, the Wombac 2.0 program based on the BioLinux 8.0 operating system was applied. When constructing the dendrogram, the Maximum Likelihood method, the PhyML 3.1 program and the HKY85 model were used. Results and discussion. The studied strains from the Aksai high-mountain and Balkhash desert foci have properties characteristic of the Y. pseudotuberculosis species, are prototrophs and do not contain the pYV plasmid. According to phylogenetic analysis based on identified 109,641 core SNPs, they are closely related to strains of the pseudotuberculosis microbe from the Sarydzhaz high-mountain focus in the Kyrgyz Republic and the Republic of Kazakhstan, as well as to the O:3 serovar strain from Turkmenistan. The cluster of these Central Asian strains on the phylogenetic tree of Y. pseudotuberculosis is localized next to strains of the pseudotuberculosis microbe of O:3 serovar from various regions of the world. The results obtained testify to the circulation of a separate phylogeographic population of Y. pseudotuberculosis in the territory of natural plague foci in Central Asia. The characterized Y. pseudotuberculosis strains and their whole genome sequences can be used as reference ones for the Aksai and Balkhash natural plague foci of Central Asia.

The aim of the study was to assemble full-length nucleotide sequences of the chromosome and plasmids for 13 Yersinia pestis strains from 11 natural plague foci located in the Russian Federation, using data from two sequencing technologies.Materials and methods. Y. pestis strains were grown on Hottinger’s agar (pH 7.2) at 37 °C. DNA was isolated using phenol-chloroform extraction. For the MinIon genetic analyzer (Oxford Nanopore), DNA fragments were prepared by ligation according to a modified protocol. For the Ion S5 genetic analyzer (IonTorrent), sample preparation was carried out according to the standard protocol for obtaining a library with a DNA fragment size of 400 nucleotide pairs (bp). The resulting single reads were filtered by average quality Q30 for IonTorrent and Q7 for Oxford Nanopore.Results and discussion. DNA fragments containing 50 000 or more nucleotide pairs were prepared for subsequent sequencing using nanopore sequencing technology (Oxford Nanopore). The Trycycler algorithm was applied for hybrid assembly of the genome of Y. pestis strains and correction of errors arising during this process, allowing the obtainment of full-length nucleotide sequences of the chromosome and plasmids for each genome of the strain. The nucleotide sequences of the chromosomes of 13 Y. pestis strains from 11 natural foci of plague located in the Russian Federation have been deposited in the international genetic database NCBI GenBank. It has been established that to assemble full-length genomes of Y. pestis strains, a significant number of reads with a size of 50 000 bp or more is required, and the use of the Trycycler algorithm allows one to generate a more accurate assembly of complete bacterial genomes.

The review summarizes the data of modern domestic and foreign studies on the mechanisms of evolutionary adaptation of the plague pathogen to transmissive spread by arthropods. The data on the molecular basis of the rapid formation of a highly pathogenic bacterium due to the acquisition of new genetic information; structural and functional changes in the genome, causing the disruption of functionality of some genes that prevent survival in the vector are presented. The stage of the complex life cycle of the pathogen associated with the peculiarities of its stay in the vector’s organism and its tactics of evasion from the action of antibacterial substances formed by the flea is considered. The importance of biofilm formation for effective transmission of the pathogen is discussed. A complex cascade of transcriptional regulators of biofilm in Yersinia pestis is considered, which includes activators and repressors of biofilm formation, as well as regulators of synthesis or modification/transport of exopolysaccharide. The hms-dependent mechanism of biofilm formation in Y. pestis is described in detail, as well as the impact on biofilm formation through the regulation of LPS-related genes and its role in the modification and transport of biofilm exopolysaccharide. The data from our own studies on the variability of genes involved in biofilm formation in the main subspecies of the plague pathogen in comparison with non-main subspecies of the plague pathogen, as well as on the ability of strains of different subspecies to form biofilm not only in the proventriculus of the flea, but also on the cuticle of soil nematodes of the Tylenchida and Rhabditida orders and the genus Panagrolaimus are presented. The latter allows us to assume the possible participation of soil and entomoparasitic nematodes in the removal of Y. pestis biofilms from the soil to the above-ground biocoenosis of the natural plague focus.

The aim of the work was to study the genetic diversity and spatial-temporal structure of Yersinia pestis in the Caspian sandy plague focus using MLVA25 and CRISPR typing methods.Materials and methods. 98 Y. pestis strains isolated in the territory of the Caspian sandy plague focus in 1925–2015 were used in the study. Whole genome sequencing was performed on Ion GeneStudio S5 System (ThermoFisher Scientific) and MGI (DNBSEQ-G50RS) platforms. Fragment sequencing was conducted using “ABI PRISM 3500XL”. The search for VNTR and CRISPR loci with subsequent alignment of nucleotide sequences was carried out in the MEGA X program. The obtained sequences were entered into the created database in the Bionumerics v7.6 (Applied Maths) software. The phylogenetic tree was constructed using the UPGMA method.Results and discussion. According to the results of MLVA25 and CRISPR analysis, 98 Y. pestis strains are divided into 60 genotypes (CS1 – CS60). Variability by 23 VNTR loci has been discovered. 7 new CRISPR spacers are described: 5 in YPa and 2 in YPb (size 31–33 bp, GC composition 34–58 %). The described spacers are designated as a108, a109, a110, a111, a112, b53, b54. The interrelation of changes in the copy number of VNTR loci depending on the place and time of isolation of strains has been revealed. The data obtained can be used to carry out molecular-genetic certification of the territory of the Caspian sandy plague focus and to study the routes and patterns of evolution and spatiotemporal circulation of Y. pestis populations in the Caspian plague foci.

The aim of the work was to analyze the results of investigating the field material obtained from natural plague foci of the Kyrgyz Republic (KR) in 2023, using modern diagnostic technologies.Materials and methods. 1435 biological samples from the Tien Shan, Alai and Talas high-mountain foci of the Kyrgyz Republic were studied using conventional methods of laboratory diagnosis of plague: microbiological, immunological, biological; as well as modern molecular-genetic methods. Testing of the obtained samples for the presence of plague pathogen DNA was carried out using RT-PCR; and the presence of antibodies to the plague microbe was detected by enzyme-linked immunosorbent assay (ELISA). Molecular identification of Yersinia pestis strains according to their appurtenance to subspecies, biovars, and phylogenetic lineages was performed by RT-PCR using the method of identifying single-nucleotide substitutions based on the analysis of melting curves of products.Results and discussion. An approach to the molecular identification of Y. pestis strains from plague foci of the KR has been developed and validated through identifying singlenucleotide substitutions using the analysis of product melting curves (HMR-analysis) with a set of designed primers. It has been established that Y. pestis strains isolated in the Sarydzhaz autonomous focus of the Tien Shan high-mountain focus belong to the biovar antiqua of the main subspecies, phylogenetic branch 0.ANT5. The phylogenetic relation of Y. pestis strains isolated in 2023 was studied based on genome-wide SNP analysis. Areas of epizootic activity in Eastern Alai have been identified. The data obtained indicate the sustained activation of plague foci in the KR. Areas of the territory of the KR that are promising for paleogenomic research are also discussed.

The aim of the work was to study the growth dependence on amino acids in Yersinia pestis strains of different phylogenetic branches of the antiqua biovar and to determine the genetic basis of auxotrophy of these strains.Materials and methods. We used 38 strains of Y. pestis, the main subspecies of the antiqua biovar, isolated in various foci of the world in the period of 1928–2020. The nutritional requirements of the strains were determined by incubation on Difco minimal agar with different sets of amino acids. Phylogenetic analysis of the strains was carried out using the Wombac 2.0 and SeaView 5.0.5 programs. Comparative analysis of the nucleotide sequences of the genes was performed using the BLAST algorithm and the Mega 7.0 program.Results and discussion. The phylogenetic appurtenance of the investigated Y. pestis strains of antiqua biovar to the phylogenetic branches 0.ANT3, 0.ANT5, 1.ANT, 2.ANT3, 3.ANT2, 4.ANT has been determined. It is established that all studied strains have a common dependence of growth on three amino acids – phenylalanine, threonine, and methionine. In the majority of the strains of antiqua biovar of all phylogenetic branches, growth is also dependent on the presence of cysteine in the medium, except for a part of the strains of the phylogenetic branch 4.ANT. In 14 genes of sulfur and cysteine metabolism, 19 mutations have been identified. Each phylogenetic group of the antique biovar has a distinct profile of mutations in genes involved in cysteine biosynthesis. The leucine requirement of the strains belonging to phylogenetic branch 0.ANT5 has been established, possibly caused by a frame shift in the leuA gene. Strains of the 1.ANT branch isolated in the Democratic Republic of the Congo demonstrate an additional proline-dependent growth. The defined nutritional requirements in the strains and the genetic causes of auxotrophy complement the phenotypic and genetic characteristics of the phylogenetic branches of the antiqua biovar and can be used as genetic markers for the differentiation of these strains.

The review contains information on paleogenomic studies of the plague pathogen, Yersinia pestis, covering the prehistoric epoch, the periods of the first and second plague pandemics, epidemics and outbreaks of plague of the late XIX–XX centuries. We have summarized the data on the reconstruction of ancient Y. pestis genomes of the Late Neolithic, Bronze and Iron Ages, the Justinian Plague epidemic of the first plague pandemic, the Black Death epidemic and the subsequent epidemics of the second plague pandemic of the XIV–XVIII centuries, as well as on tracing the pathways of plague propagation waves in Eurasia and the course of plague agent evolution with the formation of a vector-borne transmission route with the help of arthropods. We present the results of our own research of Y. pestis genomes from the key sites of formation of etiological agents of the first and second plague pandemics in the Tien Shan Mountains, historical outbreaks in the Northern and North-Western Caspian sea region and other regions of Russia and adjoining countries in the late XIX–XX centuries. The paper discusses the areas of the Caucasus, Crimea, Northern Caspian, Siberia, and Tien Shan in the territory of Russia and neighboring states that are promising for national paleogenomic studies of plague.

The aim of the work was to study interaction of Yersinia pestis with soil nematodes isolated on the territory of the Gorno-Altai high-mountain plague focus. Materials and methods. We used the fluorescent Y. pestis strain KM2083, a derivative of the natural strain of the 4.ANT phylogenetic line, the antique biovar of the main subspecies, and a nematode culture isolated in the same area of the Gorno-Altai plague focus. The taxonomy of nematodes was determined by the region of the 18S rRNA gene; phylogenetic analysis was performed using the Maximum Likelihood method based on the Tamura-Nei model in the Mega 7.0 software. The interaction of the Y. pestis KM2083 strain and the nematodes was studied during cultivation on a solid NGM agar medium. Nematodes were observed using microscopes Stemi-2000C (Carl Zeiss, Germany) and Axio Imager Z2 (Carl Zeiss, Germany). Results and discussion. It has been established that the nematodes from the Gorno-Altai high-mountain plague focus used in the work belong to the genus Panagrolaimus. Cultivation of nematodes on the lawn of the Y. pestis strain of the main subspecies of antique biovar, the 4.ANT phylogenetic line for 24 hours did not lead to a reduction in the lifespan of nematodes compared to the control sample, which indicates the absence of toxicity of the used strain towards Panagrolaimus nematodes. On the cuticle of nematodes, the formation of a biofilm in the genital area and tail has been noted, and accumulations of fluorescent cells of the plague pathogen observed in the digestive tract. The data obtained can indicate the ability of nematodes to carry the plague pathogen in the soil biocoenosis.

The paper describes a developed comprehensive system for molecular-genetic identification of Yersinia pestis strains according to their appurtenance to certain subspecies, biovars, phylo-geographic populations, using realtime PCR (RT-PCR), allele-specific RT-PCR, and multiplex PCR with hybridization fluorescent registration of results on a solid substrate. Application of this system makes it possible to establish the appurtenance of Y. pestis strains to the following phylogenetic branches: 0.ANT1, 0.ANT2, 0.ANT3, 0.ANT5, 3.ANT, 4.ANT of antique biovar of the main subspecies; 2.MED0, 2.MED1, 2.MED2, 2.MED3, 2.MED4 of medieval biovar of the main subspecies; 1.IN1, 1.IN2, 1.IN3 of intermedium biovar of the main subspecies; 1.ORI1, 1.ORI2, 1.ORI3 of oriental biovar of the main subspecies; 0.PE3 (angolica subspecies), 0.PE7 (tibetica subspecies) and 0.PE10 (qinghaica subspecies). The first stage of the studies within the frames of the developed system is indication of plague agent using registered diagnostic drugs. The second stage is the determination of belonging to individual subspecies through RT-PCR or by the method of multiplex PCR system with hybridization-fluorescent registration of results on a solid substrate, which also allows for establishing to which biovars of the main subspecies and the main phylogenetic lines of the ancient biovar the strains belong. The third stage is the identification of strain appurtenance to phylogenetic branches by the AS-RT-PCR method. The designed complex system for molecular-genetic identification of Y. pestis strains can be applied at the regional and federal levels of the laboratory network of the Russian Federation for diagnostics of infectious diseases. Its use will considerably facilitate and increase the efficiency of intraspecific differentiation of Y. pestis strains within the framework of the epidemiological investigation of outbreaks or importation of strains of plague pathogen into the territory of the Russian Federation or during the certification of strains in collection activities.

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 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 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 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 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.

The purpose of the work was to analyze the phylogenetic relations and origin of Yersinia pestis strains isolated in different periods of epizootic activity of the Caspian sandy natural focus (CSNF) of plague in the XX–XXI centuries.Materials and methods. We used 40 Y. pestis strains from CSNF and adjacent plague foci, isolated in 1922–2015. Carried out was whole genome sequencing of 19 Y. pestis strains from CSNF. Phylogenetic analysis was performed using whole genome SNP analysis based on 1914 identified SNPs. The search for marker SNPs was conducted using the Snippy 4.6 software. The phylogenetic tree was constructed using the Maximum Likelihood algorithm, the GTR nucleotide substitution model.Results and discussion. The whole genome SNP analysis has revealed that Y. pestis strains of the medieval biovar from CSNF belong to 2.MED1 phylogenetic lineage and fall into two major branches. One of them circulated in the focus in the first half of the XX century, and the other – in the second half of the XX – early XXI centuries. It is shown that strains of the first branch were the cause of outbreaks and individual cases of plague in the CSNF in the first half of the XX century. They are closely related to strains from the Caspian North-Western steppe and Volga-Ural sandy natural plague foci, which caused numerous outbreaks with high mortality rate in the same period. Y. pestis strains from the CSNF of the second half of the XX and early XXI centuries belong to the second phylogenetic branch of the 2.MED1 line, at the node of which the strains from the Northern Aral Sea region of 1945 lay. The latter were the predecessors of all strains isolated in the CSNF after a long inter-epizootic period that occurred in the middle of the XX century. There can also be traced a genetic relation between the strains from CSNF and the Dagestan plain-foothill focus.

A comprehensive analysis of the accumulated epidemiological and epizootiological data in combination with results from phylogenetic analysis of Yersinia pestis strains creates the basis for establishing patterns of spatialtemporal distribution of the plague pathogen and opens up the prospect of long-term forecasting of natural plague foci activation. Previously, we traced the distribution pathways of Y. pestis, medieval biovar, in the plague foci of the Northern and Northwestern Caspian Sea regions in the 20th and early 21st centuries.The purpose of this work was to identify the regularities of circulation of Y. pestis, medieval biovar, in four natural plague foci located in the Eastern Caspian Sea region.Materials and methods. A complex study of the phenotypic and genetic properties of 16 Y. pestis strains isolated in the Ustyurt, Mangyshlak, Karakum and Kopetdag autonomous desert plague foci in 1926–1985 was carried out. They were compared with strains from other natural plague foci in Eastern Europe and Central Asia obtained in 1917–2003. Whole-genome sequencing of 12 of those strains was performed. Phylogenetic analysis included the genomes of other 19 Y. pestis strains that we had sequenced earlier. Based on the 1717 polymorphic nucleotides (SNPs) identified in the core genome, a dendrogram of the relations of the studied strains was constructed.Results and discussion. All 16 Y. pestis strains from the Ustyurt, Mangyshlak, Kopetdag, and Karakum desert foci belong to the 2.MED1 branch of the medieval biovar. All investigated strains from the first three foci and most of the strains from the Karakum focus are in the Caspian 2.MED1 branch, and three strains from the Karakum desert focus are included in the Central Asian one. We have revealed several waves of dissemination of the strains under the 2.MED1 phylogenetic branch of Y. pestis of the medieval biovar in the Eastern Caspian Sea region in the 20th century.

The aim of the work was to conduct phylogenetic analysis of Y. pestis strains, biovar altaica ssp. central asiatica, isolated in Gorno-Altai high-mountain and Sailyugem natural plague foci on the territory of Russia and Mongolia in 1965–2020, by full-genome sequencing data.Materials and methods. To determine the population structure of the biovar altaica ssp. central asiatica, 34 whole genome sequences were used (including 20 Y. pestis strains of the biovar altaica, 18 of which were sequenced by us). To isolate DNA from the Y. pestis strains, a PureLink Genomic DNA Mini Kit (Invitrogen, USA) was applied. Sequencing of the nucleotide sequences of Y. pestis strains was carried out in Ion PGM system Lifetechnologies. The analysis and processing of the obtained data were performed with the help of Newblergs Assembler 2.6 and IonTorrent Suite software package, 3.4.2. The search for SNPs was performed using the Wombac 2.0 program. The Maximum Likelihood dendrogram was built in the PhyML 3.1. The dendrogram was visualized using the FigTree 1.4.3 software.Results and discussion. Based on the data of whole genome analysis, taking into account the 1871 revealed polymorphic nucleotides, the spatial structure of the biovar altaica ssp. central asiatica has been determined. It includes several phylogeographic branches: the Kurai-Tarkhatinskaya (cluster 0.PE4a-1) and the Ulandryk-Mongolian (0.PE4a-2), which is in agreement with the geographical regions of the isolation of strains forming these branches in the Altai Mountains. The Kurai-Tarkhatinskaya branch is further divided into the Kurai (sub-cluster 0.PE4a-1-1, formed by the strains of 2009–2018) and Tarkhatinskaya (subcluster 0.PE4a-1-2, formed by the strains of 2012–2020) sub-branches, while the Ulandryk-Mongolian branch of evolution is split into sub-branches represented by strains from the Ulandryk meso focus (sub-cluster 0.PE4a-2-2, strains 1965–2010) and the Sailyugem focus of Mongolia (sub-cluster 0.PE4a-2-1, strains 1964–1990).

The aim of the study was to develop a test system that allows for detecting plague pathogen DNA in clinical and biological samples, environmental objects with the simultaneous determination of its appurtenance to the main and non-main subspecies, differentiation of the altai biovar central asiatica subspecies separately. Materials and methods. Primer sets for specific genetic markers have been selected using the VectorNTI 10 software, optimal conditions for PCR were determined for RotorGene devices. To assess the specificity and sensitivity of the developed set of reagents, 44 strains of microorganisms were used, of which 19 were Yersinia pestis strains and 25 strains of heterologous microorganisms. The diagnostic sensitivity of “GenPest-subspecies/Altai-RGF” is 98.6 % with a confidence level of probability of 91 %. The diagnostic specificity of “GenPest-subspecies/Altai-RGF” is ≥ 99 % with a confidence level of 91 %. Results and discussion. A medical product “A set of reagents for the detection and differentiation of plague pathogen strains of the main and non-main subspecies (altai biovar, subspecies central asiatica exclusively) by the polymerase chain reaction with hybridization-fluorescent registration of results in real-time mode (GenPest-subspecies/Altai-RGF)” has been developed. The set of reagents passed the state registration in accordance with the established procedure. The use of the developed set of reagents is relevant for the Gorno-Altai high-mountain plague focus of the Russian Federation and the adjacent part of Mongolia.

The aim of the study was to characterize and carry out molecular-genetic identification of Yersinia pseudotuberculosis strains isolated in the Sarydzhaz high-mountain focus of the Tien-Shan.Materials and methods. The Y. pestis and Y. pseudotuberculosis cultures were differentiated according to their sensitivity to diagnostic bacteriophages, motility, urea fermentation, and pesticin production. Whole genome sequencing was performed using the Ion S5 XL System (Thermo Fischer Scientific). Ion Torrent Suite software package, 5.12 and Newbler gsAssembler 2.6 were used for data processing and de novo assembly of raw read sequences. The dendrogram was constructed using the Maximum Likelihood method applying the PhyML 3.1 software.Results and discussion. Strains of Y. pseudotuberculosis were isolated from museum mixed with Y. pestis cultures obtained from marmots in the Sarydzhaz high-mountain focus. According to whole genome SNP analysis, they belong to the phylogenetic group O:3 serovar, which is widespread throughout the world. Y. pseudotuberculosis is often present in mixed with Y. pestis cultures, isolated from rodents, which should be taken into account when carrying out laboratory diagnostics of plague in natural foci.

The aim of the study was a comprehensive analysis of the phenotypic and genetic properties of Yersinia pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci of the Tien Shan in 2019–2020; determination of the present-day population structure and areal of these highly virulent strains of the plague pathogen.Materials and methods. Studies of biochemical properties (fermentation of carbohydrates, nutritional requirements), virulence (in vitro and in laboratory animals), molecular-genetic analysis and whole genome sequencing of Y. pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci in 2019–2020 have been carried out. We used Y. pestis strains from the foci of the Tien Shan and Pamir-Alai dated 1928–2016 for the comparison. Whole genome sequencing was performed using the Ion S5 XL System. Phylogenetic analysis was performed on the basis of 1443 identified core SNPs in 36 Y. pestis strains of various phylogenetic lines included in the analysis. The construction of dendrograms was carried out using the Maximum Likelihood algorithm, PHYML program, HKY85 model.Results and discussion. It is established that all Y. pestis strains isolated in the Sarydzhaz and Upper-Naryn high-mountain foci in 2019–2020 belong to the 0.ANT5 phylogenetic branch of the ancient biovar of the main subspecies. Genome-wide sequencing revealed the presence of two 0.ANT5 clones, the first of which consists of strains from the basin of the river Kooylu in the Sarydzhaz focus, dated 2020. The second powerful clone includes the strains of 2012–2020 isolated in the Sarydzhaz and Upper-Naryn foci. The high virulence of the isolated strains has been shown. It was concluded that further study of the territories of the highmountain foci of the Tien Shan and Pamir-Alai is necessary to establish the current boundaries of the 0.ANT5 areal, as well as to identify the circulation areas of Y. pestis of other phylogenetic lineages.

The aim of the work was to substantiate the forecast of the epidemiological and epizootiological situation in natural foci of plague in the Russian Federation, countries of the near and far abroad for the year of 2021. Characteristics of the distribution of Yersinia pestis strains of the main subspecies (subspecies pestis) of medieval and antique biovars, Caucasian (ssp. caucasica) and central Asian (ssp. central asiatica) subspecies by 45 natural foci of the CIS countries are presented in the paper. The persistence of a multidirectional trend in the dynamics of epizootic activity of natural foci of the CIS countries with the circulation of Y. pestis pestis strains of the medieval biovar of the 2.MED1 phylogenetic branch and the antique biovar of the 0.ANT5, 4.ANT phylogenetic branches in the current decade has been outlined. For the Russian Federation, the development of epizootics is predicted in the Gorno-Altai highland and Tuva mountain natural foci caused by the circulation of Y. pestis pestis strains of the antique biovar 4.ANT and Y. pestis of the Altai biovar of the Central Asian subspecies 0.PE4a in 2021. For the Republic of Kazakhstan, there is a high probability of preserving epizootic activity in the North Aral, Aral-Karakum, Balkhash, Mojynkum, Taukum desert and Ili intermountain natural foci with the circulation of Y. pestis pestis strains of the medieval biovar of the phylogenetic branch 2.MED1. For the Kyrgyz Republic, the forecast for the development of plague epizootics caused by Y. pestis pestis strains of the antique biovar 0.ANT5 phylogenetic branch in the Sarydzhaz and Upper Naryn high-mountain natural foci has been substantiated. A high epidemic risk of epizootic manifestations caused by highly virulent strains of Y. pestis pestis of antique biovars 0.ANT5, 4.ANT and medieval biovar 2.MED1 for the entire territory of the CIS countries is noted. The relevance of implementing forecasts of the epidemiological situation into practice, taking into account the molecular-genetic and epidemiological characteristics of Y. pestis strains circulating in areas of expected epizootic manifestations of plague, is highlighted.

The aim of the study was molecular-genetic identification and analysis of the phylogenetic relationship of Yersinia pestis strains isolated on the Ukok Plateau in 2020, in order to establish the current boundaries of the natural mega focus of plague in the Altai Mountains in Russia and Mongolia.Materials and methods. 37 strains of Y. pestis of the main subspecies isolated in the Tuva mountain and Gorno-Altai high-mountain plague foci and adjacent territories of Mongolia in 1971–2020 were studied. The whole genome sequencing of the strains was performed using the Ion S5 XL System (Thermo Fischer Scientific). Ion Torrent Suite software package 5.12 and Newbler gsAssembler 2.6 were used to process the data and assemble de novo the sequences of raw reads. The average size of the collected genome was 4.55 million base pairs. Core SNPs were detected by aligning the contigs of Y. pestis strains on the CO92 genome using the Snippy 4.6 program, then 28 SNP homoplasies were removed. The resulting set of SNPs contained only the core region of the genome (955 SNPs). The dendrogram was constructed using the Maximum Likelihood method applying the PhyML 3.1 program.Results and discussion. The current population structure of Y. pestis of the main subspecies, antique biovar, phylogenetic line 4.ANT, endemic to the foci of the Altai Mountains in Russia and Mongolia has been determined. The presence of 4.ANT-21 clone, which became widespread in the territory of these natural foci of plague at the begining of the XXI century, was revealed. It is shown that three strains isolated on the Ukok Plateau in 2020 belong to clone 4.ANT-21. According to phylogenetic analysis, evidence of 4.ANT circulation on the Ukok Plateau before 2018 was obtained. The lesson that has been learned is that it is necessary to study the territories of Mongolia, Kazakhstan and China bordering the Ukok Plateau in order to establish the current boundaries of the 4.ANT mega focus.

Objective of the study was to analyze phylogenetic relation of the strains and dissemination routes of Yersinia pestis in Kara Kum desert focus in XX century.Materials and methods. We have carried out whole genome sequencing of 8 Yersinia pestis strains isolated in Kara Kum desert focus between 1949 and 1985. We also used nucleotide sequences of 16 Y. pestis strains from neighboring foci of East and North Caspian Sea region dated 1917–2002, sequenced at the premises of RusRAPI “Microbe” on Genestudio Ion S5XL platform (Thermo Fischer Scientific). Phylogenetic analysis was performed based on 1720 identified core SNPs. Phylogenetic dendrogram was constructed using Maximum Likelihood algorithm, PhyML v. 3.1 software package, and nucleotide substitution model GTR.Results and discussion. On the basis of whole-genome SNP analysis of Y. pestis strains from Kara Kum desert focus, West and North Caspian Sea region foci analysis of plague agent populations circulating in Turkmenistan in XX century was conducted. It has been demonstrated that three separate waves of Y. pestis dissemination swept the territory of Kara Kum desert focus. The origin of the first one, registered in 1912, plague outbreak in south-eastern part of the focus and its etiological agent remain unspecified so far. The second wave is associated with distribution of North-Aral 2.MED1 population of medieval biovar in 1949–1970, which led to the emergence of epizootic activity in 1949, as well as subsequent cases and outbreaks of plague in this territory in the middle of the XX century. In the mid-second half of the past century, one more 2. MED1 dissemination wave from the North Balkhash region hit the Kara Kum desert focus. This central-asian population has also taken root in the natural biocoenosis of the Kara Kum focus. The data obtained testify to active processes of distribution of Y. pestis of medieval biovar in East Caspian Sea region in southern sub-zone of Eurasian deserts in XX century.

Objective of the work was to identify molecular-genetic peculiarities, to conduct whole genome sequencing and phylogenetic analysis of Yersinia pestis strains isolated inVietnam between 1962 and 1989.Materials and methods. We have studied the properties of 50 Y. pestis strains, carried out whole genome sequencing of 18 and fragment sequencing of 32 strains from Vietnam. Phylogenetic analysis was performed on the basis of whole genome SNPanalysis by 1391 identified SNPs. Whole genome SNP-analysis and search for marker SNPs were conducted applying Wombac 2.0 software package. Phylogenetic diagram construction was done using Maximum Likelihood algorithm.Results and discussion. Several phylogenetic branches and Y. pestis populations coinciding with geographical and historical dissemination of the strains have been distinguished. The major part of the strains from Vietnam falls under the branch designated by us as 1.ORI2v. Two strains form a separate branch together with the strain from India belonging to 1.ORI2 line, one more strain, 55-801 Saigon, is set among the strains of 1.ORI1 line. Based on the data obtained and evidence from the literature sources it can be assumed that introduction of plague into Vietnam occurred through several waves: Nha Trang city in 1898, by sea; north provinces of the country – 1908. The second wave of Y. pestis dissemination across the territory of Vietnam began in 1960s with the emergence of the strains from the natural plague focus in Yunnan province, China.

Objective of the investigation was to assess epidemiological and epizootiological conditions in natural plague foci of the Russian Federation, neighboring states and foreign countries in 2019. Negative effect of the current climate warming on the state of parasitic systems of natural foci with circulation of plague microbe of the main subspecies, medieval biovar, phylogenetic branch 2.MED1 is emphasized. In 2019, local plague epizooties were registered in the territory of two (Gorno-Altai high-mountain and Tuva mountain) out of 11 natural plague foci of the Russian Federation. The total area of epizooty covered 2248.5 km2 . All in all, 31 cultures of Y. pestis of the main subspecies, antique biovar, phylogenetic branch 4.ANT and 5 cultures of Altai biovar, central-asian subspecies, phylogenetic branch 0.PE4a were isolated. The forecast for continuing tense epidemiological situation, both for 2020 and for 2021–2025, has been substantiated for natural foci with circulation of the main subspecies of antique biovar, phyologenetic branch 4.ANT: Gorno-Altai high-mountain and Tuva mountain ones. In the remaining nine natural foci in the territory of the Russian Federation epizootic manifestations of plague in 2020 are highly improbable. This prognosis is an indication to optimize the deployment of human and logistical resources of plague control institutions through concentrated preventive activities in the territories of epizootically active natural plague foci. Otlined is the necessity to put a new enhanced classification of Y. pestis into practice.

Objective of the study was to conduct phylogenetic analysis of Yersinia pestis strains of the main subspecies, belonging to antique biovar, phylogenetic branch 0.ANT, isolated in XX – early XXI centuries in the foci of Tien-Shan and Pamir-Alay to identify the regularities of spatial-temporal circulation of plague agent in the territory of the foci. Materials and methods. We have carried out whole genome SNP-analysis of Y. pestis strains of antique biovar, isolated in natural foci of Kyrgyz Republic in 1928–2016. Phylogenetic investigation is based on 1646 identified core SNPs in 51 included in the analysis strains of different phylogenetic lines. Phylogenetic tree was constructed using Maximum Likelihood algorithm, PHYML software package, and HKY85 model. Results and discussion. All 29 studied Y. pestis strains isolated between 1928 and 2016 in the foci of Tien-Shan and Pamir-Alay fall under the phylogenetic branches 0.ANT3 and 0.ANT5 of antique biovar of the main subspecies. Strains of 0.ANT3 branch were collected predominantly in Aksay and Alay foci, while strains of 0. ANT5 – in Upper-Naryn and Sarydzhas foci of Tien-Shan. Strains of phylogenetic lines 0.ANT1 and 0.ANT2 were not found in the foci of Kyrgyz Republic. According to the results of whole genome SNP-analysis, Y. pestis strains isolated in the XXI century belong to phylogenetic branch 0.ANT5. This branch also comprises a strain obtained from a patient (lethal case) in Sarydzhas plague focus in 2013. Genetic homogeneity of the modern strains of 0.ANT5 branch and their wide dissemination testify to the extension of the areal of this population and activation of Tien-Shan foci, caused by climate warming. For the first time ever, genomes of Y. pestis 0.ANT5 branch, isolated in XXI century, including clinical strain dated 2013, have been sequenced. We have obtained the whole-genome sequence of Y. pestis strain, 0.ANT3 branch, which caused pneumonic plague outbreak in Tien-Shan in 1928.

Objective of the study was to determine the origin of Y. pestis strains that widely disseminated in natural plague foci of Northern, North-Western Caspian Sea region and Fore-Caucasus in the second half of the XX century. Materials and methods. We have carried out the investigation of properties and whole genome sequencing of 22 Y. pestis strains, isolated between 1923 and 2003 in five natural foci of the souslik type, situated in Northern and North-Western Caspian Sea region and Fore-Caucasus. Phylogenetic analysis was conducted using the data of whole genome SNP-typing, based on 1348 identified SNPs. The search of SNPs in the core genome was performed with the help of Wombac 2.0 software. Phylogenetic relations were analyzed using Maximum Likelihood dendrogram, GTR model. Results and discussion. All the studied strains from the foci of Northern, North-Western Caspian Sea region and Fore-Caucasus fall into phylogenetic branch 2. MED1of medieval biovar. On the basis of whole genome SNP-analysis, existence of two groups of closely related strains, comprising the strains dated 1923-1945 and 1962-2003, has been revealed. The predecessors of the 1962-2003 strains from the Northern, North-Western Caspian Sea region and Fore-Caucasus on the phylogenetic tree are the strains from Northern Aral Sea region that go back to 1945. It testifies to the fact that synchronized activation of a group of natural foci in Caspian Sea lowland and Fore-Caucasus in 1975-1979, after prolonged 20-37 year long breaks, could be caused by the dissemination of the strains from the Northern Aral Sea region. The data of epizootic observations indicate that activation of Volga-Ural sandy plague focus in 1960s preceded the start of registration of plague epizooties in the territory of natural foci of souslik type in Northern, North-Western Caspian sea region and Fore-Caucasus.

Objective of the study was to construct the recombinant Y. pestis strain producing fluorescent protein GFP and analysis of the prospects of its usage for investigation of interaction of the agent with the protozoa, and also macroorganism of mammals (rodents). Materials and methods. To create the strain producing fluorescent protein GFP, we used the natural Y. pestis strain isolated in 2016 in Gorno-Altai high-mountain plague focus. Fluorescent protein gen was inserted into commercial vector pTurboGFP-B via electroporation. Properties of the obtained recombinant Y. pestis strain 367 pTurboGFP-B were studied using microbiological, biological, and molecular-genetic methods. Results and conclusions. The plague agent strain, containing vector plasmid pTurboGFP-B which encodes synthesis of green fluorescent protein GFP, and being resistant to ampicillin was constructed applying electroporation. The designed Y. pestis strain, producing green fluorescent protein, does not differ from the stock strain by its cultural-morphological, biochemical properties, virulence and survivability in mixed culture with Acanthamoeba castellani. On solid nutrient media, recombinant strain and its subcultures, obtained from infected animals, formed colonies of greenish-yellow color, becoming fluorescent under UV-light. In preparations from animals and from co-cultures with amoeba, fluorescent cells of plague agent were observed. The constructed strain can be utilized as a model one for investigation of interactions between plague agent and cells of protozoa (soil amoeba, nematodes) and mammals under laboratory conditions.

Aim. The present paper provides a comparative analysis of the phylogenetic relationship between Yersinia pestis strains isolated in the Volga-Ural sandy natural focus during the periods of 1912-1945 and 1963-2003, which were characterised by different levels of epidemic activity, in order to identify the spatiotemporal patterns in the circulation of the plague pathogen in the North Caspian region. Materials and methods. We studied the properties and performed whole-genome sequencing of 18 Y. pestis strains from the Volga-Ural sandy focus, along with 12 strains from other foci in the North Caspian and North Aral regions, isolated from 1912 to 2003. The phylogenetic analysis was performed drawing on the whole-genome SNP analysis, which was conducted on the basis of 2188 SNPs identified in the core genome using the Wombac 2.0 program. Maximum Likelihood Dendrogram (GTR model) was used for the analysis of phylogenetic relationships between strains. Results and discussion. All studied strains from the foci of the North Caspian region belong to the main subspecies (biovar medievalis) of the plague pathogen. These are highly virulent and epidemiologically dangerous strains. The whole-genome sequencing and phylogenetic analysis of 30 strains from the Volga-Ural sandy focus, as well as adjacent plague foci, reveal that the strains (biovar medievalis) of two phylogenetic branches - 2.MED4 and 2.MED1 - were spread across the focus under study in the early 20th century. It is confirmed that 2.MED1 strains were the etiological agents of plague outbreaks in the Volga-Ural sandy focus during this period. The study revealed the presence of parallel evolutionary lines in 2.MED1 associated with plague outbreaks in the first half of the last century. In the second half of the 20th and early 21st centuries, the modern evolutionary line of 2.MED1 became widespread in the Volga-Ural sandy focus. The strains of this line are closely grouped, which indicates their close genetic relationship. Only sporadic cases of plague were recorded during this period. Modern strains from the Volga-Ural sandy focus (1963-2003), as well as the strains previously isolated there (1912-1945), do not originate from each other. These strains represent closely related, independent evolutionary branches, extending from the common trunk of 2.MED1. Modern strains originating from those of the North Aral desert focus (1945) form a separate cluster in the dendrogram. This suggests that, following a break in the 1950s, the Volga-Ural sandy focus was recolonised by closely related strains from the North Aral region.

Objective of the study – comparative phylogenetic analysis of Yersinia pestis strains, isolated in Precaspian North-Western steppe focus in 1924–1926, 1972, and 1986–1990 to understand the causes of focal reactivation during different time periods of the XX century.Materials and methods. The work included 30 strains of Yersinia pestis from Precaspian North-Western steppe natural focus and adjacent plague foci. Whole genome sequencing of eight Y. pestis strains from the former was carried out. Also whole-genome sequences of 16 strains from neighboring natural foci were used. Whole-genome sequencing of Y. pestis strains was conducted in Ion PGM system (Life technologies). SNPs search across the core genome was performed using software package Wombac 2.0. Tree diagram Maximum Likelihood, HKU85 model, was constructed to analyze phylogenetic relations.Results and discussion. It is established that in early XX century (1924–1926), strains of phylogenetic branches 2.MED4 and 2.MED1, belonging to medieval biovar, main subspecies, circulated on Ergenin Upland in the Precaspian North-Western steppe natural focus. Later on they became extinct in the territory. It is shown that the strains, isolated on Ergenin Upland in 1972, constituted a common subcluster on the dendrogram with the strains from low-mountain and piedmont plague foci of Caucasus and Transcaucasia, dated the same time period. It was inferred that epizootic manifestations on Ergenin upland in 1972, after a long recess since 1938, were caused by importation of Y. pestis strains from low-mountain natural plague foci of Caucasus and Transcaucasia. It was noted that expansion of Caucasian strains was of short-term character, and plague infected animals have not been found on Ergenin Upland since 1974 (including modern period). It is established that Y. pestis strains isolated in the eastern part of Precaspian North-Western steppe focus between 1986 and 1990, do not have close genetic relation to the strains that circulated on Ergenin Upland in 1924–1926 and 1972. It is determined that each epizootic period (1913–1938 and 1972–1973) in Precaspian North-Western steppe natural focus culminated in the elimination of the circulating Y. pestis strains and rehabilitation of the focal territory.

The paper presents the analysis of epidemic activity of natural plague foci around the world, including in CIS and the Russian Federation, over the period of 2000–2018. It was established that in 2010–2018, epidemic manifestations took place in natural plague foci where the main carriers are synanthropic (black, multinippled rats) and commercial (marmots, souslik, ground squirrels) species of rodents. Retaining of low epizootic activity of lowland natural plague foci in the territory of the Russian Federation is observed. In 2018, plague epizooties were detected in the territory of the Gorno-Altai high-mountain and Tuva mountain natural foci, the total coverage area being 2641 km2. 13 strains of the main subspecies and 6 strains of Altaic subspecies of plague microbe were isolated. Demonstrated were the prospects of GIS-technology usage for the enhancement of epidemiological surveillance over plague in Russia. Epizootiological forecast for persisting challenging epidemiological situation in Kosh-Agach district of the Republic of Altai and Mongun-Taiginsky, Ovyursky and Tes-Khemsky Kozhuuns of the Republic of Tuva was substantiated. Recorded was high probability of retention of strained epizootic and epidemiological situation in the south and central parts of the desert zone in the Republic of Kazakhstan, high-mountain areas of the Republic of Kyrgyzstan. Also conservation of epidemic risks for the territories of the countries in Africa (Republic of Madagascar), South (Peru, Bolivia), and North (USA) America, China, and Mongolia was noted.

Актуальність. Дистрофічні зміни у емалі та дентині виникають внаслідок порушення обміну у твердих тканинах зуба та характеризуються її недостатнім формуванням і мінералізацією або надмірним накопиченням пігментів у вигляді плям. Накопичення пігменту (якісне та кількісне) є незворотнім, тобто пігментна дистрофія не має зворотного розвитку і залишається в емалі на все життя. Метою роботи було проаналізувати структурні зміни в емалі зубів при пігментних дистрофіях. Матеріал та методи. Після екстракції зуби ущільнювали в епоксидну смолу епон-812. Після полімеризації отриманий блок розрізали сепарувальним диском на шліфи товщиною 0,25 мм, на препарати наносили шар алюмінію у вакуумі та досліджували на скануючому електронному мікроскопі РЭМ – 102Э з прискорювальною напругою 30КВ. Результати. Встановлено, що при набутих дисколоритах емаль частково фрагментована, визначаються явища лізису емалі. На емалево-дентинній межі спостерігаються лакуни. На препаратах емалі з вродженими дисколоритами визначені явища потовщення емалевих призм, мозаїчної виразності ліній Ретциуса. При екзогенній пігментній дистрофії основні зміни встановлені на поверхні емалі. Спостерігались явища вогнищевої фрагментації, дифузного стоншення емалевих призм (атрофічні зміни). При ендогенній пігментній дистрофії основні зміни визначені з боку емалево-дентинної межі. З боку призм встановлено їх стоншення та фрагментація. Підсумок. Вроджена пігментна дистрофія має дифузний характер і вражає всю товщу емалі, що обумовлено порушеннями в процесі гістогенезу. При набутих дисколоритах локалізація патологічних змін залежить від етіології – ендогенного походження або екзогенного. При ендогенній пігментній дистрофії морфологічні зміни виявляються в поверхневих шарах емалі, що розвиваються внаслідок порушення процесу третинної мінералізації, який створює сприятливі умови для проникнення пігментів за рахунок зменшення вмісту неорганічних речовин. При ендогенній пігментній дистрофії основні зміни розвиваються в глибоких шарах емалі, які прилягають до дентино-емалевої межі. Джерелом просочення пігментів у даному випадку є емалеві веретена, які мають низький вміст неорганічних речовин і проникають в емаль на 1/3 її товщини.

Objective of the study was to investigate entomoparasitic nematodes from rodent fleas, collected in Gorno-Altai high-mountain natural plague focus in 2016–2017, with identification of their taxonomic status. Materials and methods. Given are the results of morphological and genetic analysis of entomoparasitic nematodes – parasites of fleas Amphipsylla primaris primaris living on flat-headed high-mountain voles Alticola strelzovi, fleas Rhadinopsylla li transbaicalica of Daurian pika Ochotona dauurica, fleas Rhadinopsylla dahurica, collected at the burrows of the rodents. Results and conclusions. Identified have been nucleotide sequences of ITS2 spacer region of ribosomal operon in entomoparasitic nematodes of three flea species: Amphipsylla primaris primaris, Rhadinopsylla li transbaicalica, and Rhadinopsylla dahurica. Based on comparative phylogenetic analysis and identified high percentage of homology of the stated nucleotide sequences (92–99 %), appurtenance to Rubzovinema spp. species and close relation to earlier proposed by us multi-host species Rubzovinema polyxenica from C. tesquorum, A. rossica, and C. secundus fleas from Volga-Ural steppe plague focus has been established. For the first time ever separate branch of evolution of entomoparasitic nematodes – flea parasites, represented by Rubzovinema ssp., has been identified. Determined has been wide spread of Rubzovinema ssp. nematodes in biocenoses of steppe zone of Russia across Precaspian territory and Altai Mountains.

Objective of this study was to investigate the areal of Yersinia pestis strains of antique biovar, main subspecies, phylogenetic line 4.ANT, as well as to establish the borders and spatial structure of mega-focus in the territory of Gorny Altai. Materials and methods. Complex comprehensive analysis of the properties in Yersinia pestis strains of the main subspecies, circulating in Gorny Altai has been conducted. 33 out of them, isolated predominantly in 2012–2017 have been sequenced. Whole genome SNP analysis and search of marker SNPs was performed using Wombac 2.0 software package. Tree diagram was built applying Maximum Likelihood algorithm, using PhyML 3.1 software on the basis of HKY85 model. Results and conclusions. Based on the results of whole genome SNPs analysis of 33 endemic strains and creation of the tree diagram of Y. pestis strains, the presence of natural mega-focus of Y. pestis belonging to the main subspecies, antique biovar, phylogenetic line 4.ANT has been substantiated. Epizootic manifestations on multiple local areas, characterized by persistent autonomous nature of plague manifestations, are registered on an annual basis. Within the boundaries of the areal of Yersinia pestis main subspecies, antique biovar, phylogenetic line 4.ANT, existence of joint natural foci of Yersinia pestis belonging to non-main subspecies ssp. altaica and ulegeica is established. Location of natural foci of the main and non-main subspecies of Yersinia pestis in different altitudinal belts of the Altai Mountains Range on the whole provides for observed multi-host and multi-vector feature of epizootic manifestations. For the first time ever, the data on the areal of the main subspecies of plague microbe are used for setting the boundaries of its natural focus.

The causes of emergence and development of pneumonic plague outbreak in 2017 on Madagascar were analyzed. Summarized were the literature data, characterizing the spatial and biocoenotic structure of natural plague focus on Madagascar, assessed were the risk factors of plague infection among rural and urban population of the island, as well as the properties of Yersinia pestis strains isolated there in the XX century. It is substantiated that the distinctive type of plague outbreaks on Madagascar is a rural one. It is demonstrated that one of the main causes of wide spread of plague in 2017 is importation of the infection from central regions of the Republic of Madagascar into big cities with further formation of multiple epidemic foci. Confirmed was the circulation of Y. pestis strains that belong to the third branch of irradiation of oriental biovar (1.ORI3). Discussed are the issues of diagnostics and treatment of plague in epidemic foci in the territory of the Republic of Madagascar. The evidence is provided to the fact that the plague outbreak in 2017 on Madagascar is the consequence of a lower surveillance level, first and foremost undervaluation of specific and non-specific prophylaxis capacities regarding this infection.

Objective of the study is to assess the effectiveness of the methods for molecular diagnostics and identification of Yersinia pestis strains in the field material obtained from Sarydzhas high-mountain focus in Kyrgyz Republic. Materials and methods. Investigated were the samples of the field material, isolated in 2016 in Sarydzhas high-mountain plague focus, using conventional methods of laboratory diagnostics and PCR with hybridization-fluorescent and electrophoretic registration of results. Results and conclusions. It is demonstrated that in a number of cases molecular-genetic method has a higher resolution as compared to conventional methods of laboratory diagnostics of plague. It proves the necessity of wider usage of molecular-diagnostic methods in epizootiological monitoring of plague in natural foci in Kyrgyz Republic.

Objective of the study is to analyze the appurtenance, numbers, and dynamics of interaction of acanthamoeba from soils of GornoAltai plague focus with Yersinia pestis 367 strain, isolated in 2016 in enzootic territory of this focus. Materials and methods. Utilized were soil amoeba from Gorno-Altai high-mountain focus and the strain Y. pestis 367 of the main subspecies of antique biovar, isolated there in 2016. Determination of systematic relation of the isolated amoeba was carried out using PCR with genus specific primers and sequencing of the obtained PCR fragments followed by identification of nucleotide sequences against GenBank database. Localization of Y. pestis cells in acanthamoeba was performed using fluorescent antibody technique by means of Axio Imager Z2 (Carl Zeiss, Germany). Results and conclusions. For the first time ever established has been the presence of Acanthamoeba castellanii in soils of burrows of Marmota altaica in the numbers of up to 300000 cells/gr in Gorno-Altai high-mountain focus. Investigated has been the dynamics of interaction of these microorganisms. Preservation of the agent in vacuoles of endoplasmatic reticulum within 14 days has been revealed. It is an indicative of the possibility of Y. pestis persistence in amoeba of Acanthamoeba subspecies in soil biocoenosis of Gorno-Altai high-mountain plague focus.

Objective of the study is to investigate phenotypic and molecular-genetic features and perform whole genome sequencing of Y. pestis strains isolated in Vietnam. Materials and methods. Studied were phenotypic and genotypic peculiarities of 20 plague agent strains isolated in different prefectures of Vietnam. Carried out was SNP-analysis of the strains, sequenced were genomes of 8 Y. pestis strains. Results and conclusions. Based on the results of studies of differential biochemical characteristics all the investigated strains were attributed to oriental biovar of the main subspecies of plague agent, which was confirmed by the presence of marker indel mutation – deletion of 93 bps in glpD gene. Investigated was also the plasmid composition of the strains. On the basis of the conducted genome sequencing and SNP-analysis appurtenance of 19 out of 20 strains under examination was determined. They belong to 1.ORI2v phylogenetic branch, relative to the strains isolated in Yunnan Province, China, which points to their common origin. Identified was a marker SNP and developed the method of SNP-typing for 1.ORI2v strains from Vietnam.

Objective of the study is to design the DNA-microarray for differentiation of Y. pestis strains of the main and non-main subspecies and biovars of the main subspecies. Materials and methods. Efficiency analysis for the devised means was conducted using 62 Y. pestis strains of various subspecies and biovars, isolated in the natural foci of Russia and neighboring countries. Results and conclusions. Selected have been the DNA-targets, probes and primers – calculated. Enhanced is the method of sub-specific and biovar differentiation of Y. pestis strains by means of DNA-microarray. DNA-chip with “Med24”, “glpD(-93)”, and “45” targets allows for prompt differentiation of the strains of the main and non-main subspecies and biovars of the main subspecies based on the presence and absence of fluorescent signal by the specific for the main subspecies and its biovars DNA-targets.

Aim. Develop a method of differentiation of Y.pestis strains of different subspecies based on PCR with hybridization-fluorescent detection in real-time. Materials and methods. DNA target search for differentiation of subspecies of plague causative agent was carried out by Mauve 2.3.1, Mega 5.0 and BLAST algorithm based on comparison offull-genome sequences of Y.pestis strains. Primers and TaqMan probes were calculated for the DNA targets found, conditions of PCR with hybridization-fluorescent detection - optimized. Results. DNA targets carrying marker mutations for the caucasus, altai, gissar, ulegei subspecies, strains from Talass alpine plague reservoir were detected. The effectiveness of the DNA targets found and the developed approach of subspecies differentiation is confirmed on 101 Y.pestis strains of different subspecies, isolated from natural foci of Russia, near and far abroad. Conclusion. The developed approach based on PCR with real-time detection allows for a rapid and effective differentiation of Y.pestis strains of various subspecies.

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.

Entomoparasitic nematodes are the natural regulators of numbers of fleas – plague vectors in natural plague foci. They have an impact on the intensity of epizootic process, progressing in these foci.Objective of the study is to examine entomoparasitic nematodes from fleas of the souslik and vole, collected in the territory of Povolzhsky Region.Materials and methods. Presented are the results of morphological and genetic analysis of entomoparasitic nematodes, flea parasites – Citellophilus tesquorum, Amphipsylla rossica and Ctenophthalmus secundus, collected from the small ground squirrel Spermophilus pygmaeus and social vole Microtus socialis, respectively.Results and conclusions. Established has been morphological identity of nematode parasitic females isolated from all the three species of fleas, and a high degree of similarity to the previously described Rubzovinema ceratophylla, parasitizing on C. tesquorum fleas. Substantiated has been the belonging of parasitic nematodes isolated from fleas C. tesquorum, А. rossica and C. secundus, to one and the same species, specified as Rubzovinema sp., based on the high percentage of the homology of the nucleotide sequences of rRNA genes (99.3–100 %). This species differs from the previously described monoxenous R. ceratophylla in absence of strict host specificity.

Objective of the study is to explore the feasibility of the long-term persistence of Yersinia pestis strains in association with ameba - Acanthamoeba sp. Materials and methods. Investigated has been interaction of ameba - Acanthamoeba sp., isolated from rodent burrows in the Pre-Caspian sandy, Volga-Ural steppe, and Pre-Caspian North-Western steppe natural foci, with 4 strains of Y. pestis of the main subspecies, 1 strain of caucasica and 1 strain of altai subspecies. Results and discussion. It is established that the strains of the main subspecies survive in the cells of ameba at 26 °C and 20 % humidity (modeling of the drought conditions in the natural plague foci) within 2-4 months of experiment, and 10-20 times longer that in pure culture. Two strains of the non-main ssp. have not demonstrated an increase in persistency in association with Acanthamoeba sp., which may occur due to degraded resistance to phagocytosis in the ameba of this specie. Using fluorescent and transmission electronic microscopy, it is determined that the cells of plague agent persist in ameba cells in individual vacuoles, enclosed in endoplasmic reticulum. The data obtained may testify to the possible involvement of ameba Acanthamoeba sp. . into sustainment of Y. pestis in soil biocoenoses of natural plague foci.

Objective of the study is to conduct phylogenetic investigation of Yersinia pestis strains (medieval biovar) from plague foci of Russia and bordering countries, using SNP-analysis of the genome-wide sequences of these strains. Materials and methods. Carried out has been sequencing of 14 Y. pestis strains, medieval biovar, from 13 natural plague foci of Russia and neighboring states, as well as their comparison to 9 strains of the same biovar, contained in the NCBI GenBank database. Using software products - Wombac 2.0 and Bionumerics 7.1, revealed is the presence of 1875 core SNPs, on the basis of which a dendrogram of phylogenetic relations between medieval strains is constructed. Results and conclusions. In consequence of genome-wide SNP-analysis, it is established that Y. pestis strains, medieval biovar, from plague foci of Russia and bordering states are assigned to 2.MED1 phylogenetic line and fall under two major evolutionary branches, the first one of which includes strains from the Caucasus and Caspian-Sea regions, and the second one - from Central Asia and China. The data obtained can be used for the development of molecular-genetic methods for differentiation of Y. pestis strains, medieval biovar.

Carried out has been genome-wide sequencing of 20 Yersinia pestis strains from all 11 natural plague foci in Russia and some foci in the neighboring states. Phylogenetic analysis on the basis of 1918 core SNPs, identified in the genomes of these strains and 16 Y. pestis strains from NCBI GenBank, has revealed 5 clusters of closely related strains. Taking these clusters into account, up-dated has been subspecific classification of plague agent. New taxonomy has combined 5 subspecies: major (ssp. pestis ), Caucasian (ssp. caucasica ), ulegeisk (ssp. ulegeica ), and two novel ones - Central Asian (ssp. central asiatica ) and Angolan (ssp. angola ). Central Asian subspecie comprises evolutionally related strains, earlier classified as altaica and hissarica, as well as the strains from Talas high-mountain focus in Kirghizia and Uzbekistan, and microtus strains from China. Central asiatica is divided into three biovars - altai, hissar, and microtus. Set forward is a serviceable designation for the strains: 0.PE4a - for altai ones, 0.PE4h - for hissar, 0.PE4t - for talas, and 0.PE4m - for microtus, and also for ulegeica subspecie - 0.PE5.

Identified are the complete nucleotide sequences of two cryptic plasmids - pCKF from the Central-Caucasian high-mountain plague focus and pTP33 - from Tuva mountain plague focus in Russia. It is established that the size of the pCKF is 5.4 kbp, and its G-C pair composition accounts for 38.4 %. The plasmid contains 8 open reading frames, encoding transport and secretion functions, in particular, secretion systems of the VI type. The size of another cryptic plasmid, pTP33, is 33.8 kbp, and G-C pair composition accounts for 50.3 %. It contains 52 open reading frames, the majority of which have been classified as phage proteins. Hence, it is inferred that pTP33 is a ring genome of phage. It also has two genes of two-component protein system, toxin-antitoxin, YoeB/YefM, that affects replication apparatus of bacteria.

On the basis of comparison between the genome-wide sequences of Altaic and Hissar strains of non-main subspecies and strains from Talas high-mountain plague focus, identified have been two novel DNA-targets, the usage of which provides for easy rapid subdivision of these closely-related strains of non-main subspecies that fall into separate phylogenetic brunches of plague agent evolution, applying PCR assay. One of the targets is allocated to intergenic region between YPO3333 and YPO3332 genes. In strains of altaica ssp. it contains 122 bps deletion. The other target is YPO2412 gene region which contains 72 bps deletion in Talas strains. Special primers for DNA-targets have been designed. Established have also been test specifications. Efficacy of the method for differentiation between the strains of Altaic-Hissar group of non-main subspecies is validated on 97 Y. pestis strains of various ssp.

Laboratory diagnostics of plague was carried out in compliance with valid operational guidelines and regulations. But its peculiarity consisted in the performance of diagnostic investigations secondary to antimicrobial therapy with application of preparations characterized by the expressed activity towards gram-negative microorganisms, including the agent of plague (ceftriaxone, ciprolet, and amikacin). The studies revealed that under antibiotic treatment during the early phase of infection the most effective method for the laboratory plague diagnostics was PCR. Based on the results of the assay it was possible to establish not only provisional, but also the final diagnosis in a patient. Obtained was genetic characteristics of the strains isolated from the patient and the marmot, withdrawn at the patient’s place, using techniques of molecular-genetic analysis, in particular PCR, multilocus VNTR, and multilocus and genome-wide sequencing. Thereupon the strains were attributed to antique biovar of the main subspecies of plague agent. In addition, close relation to Y. pestis of the main subspecies isolated in the same focus in 2012 and to the strains from Mongolian Altai and Tuvinian mountain focus was determined based on phylogenetic analysis of the isolates.