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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 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 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 aim was to present the experience of using mobile laboratory for monitoring and diagnostics (MLMD) during the epizootiological monitoring of the northern provinces of Vietnam. MLMD was transferred by Federal Service for Surveillance in the Sphere of Consumers Rights Protection and Human Welfare to the Socialist Republic of Vietnam as part of implementation of cooperation programs on combating infectious diseases. The use of MLMD made it possible to obtain new information on the circulation of pathogens of natural-focal infectious diseases on the territory of Vietnam. It also provided the necessary conditions for conducting research using methods of express diagnostics, bacteriological analysis, performing a full cycle of work – from the receipt of samples to the disinfection and destruction of infected material in compliance with the requirements of biological safety in the field. The effectiveness of using mobile laboratories in response to the emergencies of sanitary and epidemiological nature, both to strengthen stationary laboratory bases and to organize diagnostic studies in remote regions, has been shown. The use of MLMD for the diagnosis of COVID‑19 has been an effective component of countering the new coronavirus infection in Vietnam and significantly increased the volume of testing in the country.

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 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 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 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 is to detect circulation of plague agent and agents of other zoonotic infections (tularemia, pseudotuberculosis, leptospirosis, West Nile fever, Lassa fever, Dengue fever, Chikungunya fever, CrimeanCongo hemorrhagic fever, Q fever, Hantaviruses, tick-borne encephalitis, human monocytic ehrlichiosis, granulocytic anaplasmosis, and borreliosis) among small mammals and their ectoparasites in the territory of seven northern provinces of the Socialist Republic of Vietnam. Materials and methods. We have carried out epizootiological survey of seven northern provinces of the Socialist Republic of Vietnam (Dien Bien, Lai Chau, Lào Cai, Hà Giang, Lạng Sơn, Cao Bằng, and Quảng Ninh). Over the period of the study, 3400 trap/nights were accumulated, 179 specimens of small mammals caught, belonging to 10 species, 213 fleas of seven different species – collected, and 143 specimens of gamaside ticks falling under two species. The material gathered was investigated using enzyme immunoassay and polymerase chain reaction at the premises of mobile laboratory for monitoring and diagnostics. Results and discussion. Two-fold testing of 136 blood samples from small mammals revealed antibodies to F1 of Y. pestis in 14 (10.3 %) of them. Investigation of 158 samples of lung and kidney suspensions of small mammals showed that 22 (13.9 %) samples contained 16S rRNA of pathogenic leptospira, Leptospira spp. Analysis of 60 brain samples for the presence of Leptospira spp. revealed three (5.0 %) positive ones. 25 samples of gamaside ticks were tested for the presence of the DNA of Q fever, plague, tularemia and granulocytic anaplasmosis agents, and for the RNA of tick-borne encephalitis, human monocytic ehrlichiosis, and borreliosis agents. One sample (4 %) of gamaside ticks, Laelaps echidninus, contained RNA of Borrelia.

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.

Objective of the study. This work was carried out to identify markers (antigen and RNA) of CrimeanCongo hemorrhagic fever (CCHF) virus in samples from ticks, collected in all landscape-geographical areas of Guinea: Lower, Middle, Upper and Forest, to obtain up-to-date data on the distribution of the pathogen in the country.Materials and methods. Total of 4276 specimens of 8 species of ticks collected in 2016–2019 in the territory of the Republic of Guinea were studied, which were compiled into 1406 samples. Ectoparasites were collected from livestock animals, dogs, and small mammals. Viral antigen was detected using enzyme immunoassay (ELISA). The presence of RNA of the CCHF virus was determined by reverse transcription polymerase chain reaction (RT-PCR).Results and discussion. As a result of the studies, the antigen of the CCHF virus was detected in 21 samples (1.5 %), and RNA – in 37 (2.6 %). All samples, in which the viral antigen was detected, contained RNA of the CCHF virus. Positive results were obtained in samples from all geographical areas of the country. The main vectors and reservoirs of the pathogen in Guinea are ticks of the species Rh. sanguineus, Rh. geigyi, Rh. annulatus and Am. variegatum. The data obtained confirm the previously available information on the possibility of the pathogen circulation in this region and determine the need for further study of the spread of the CCHF virus in the territory of the Republic of Guinea.

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 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.

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 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.

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.

For the first time ever developed has been the method of identification of genovariants in Yersinia pestis strains belonging to the main subspecies using PCR with hybridization-fluorescent registration of results. A complex of DNA-targets, the most of which are newly detected ones, have been utilized to differentiate between genovariants of antique (0.ANT, 1.ANT, 2.ANT, 4.ANT), medievalis (2.MED, 2.MED0), and orientalis (1.ORI) biovars. Application of these targets in PCR with hybridization-fluorescent registration of results provides for a rapid, effective and reliable classifying of the strains of the genovariants, circulating in various geographical regions. Efficacy of the method has been validated via analysis of 110 Y. pestis strains, isolated in the territory of the Russian Federation, neighboring countries and beyond, including 37 strains of antique, 53 strains of medievalis, and 20 strains of orientalis biovars.