Considered are the current views on the mechanisms and factors of bacterial cell degradation during lyophilization and storage of dry preparations. Given are the data on the most effective lyo-rotectors and mechanisms of their shielding action. Lyophilization or sublimation from the frozen state is the basic method of bacteria preservation in culture collections and biological resource centers. In the process of lyophilization cells are exposed to damaging stress factors. Low temperatures, water crystallization, osmotic process, pH alterations, and dehydration affect cell cultures and molecules. Oxidative reactions, running in dry cell preparations, change the composition and structure of lipids, proteins, nucleic acids, and, thereby reduce the number of living cells during the storage. One of the key factors that influences bacterial viability after lyophilization and storage is the composition of shielding medium, with which the cells are mixed up before conservation. Utilization of protective media, containing carbohydrates, amino acids, restored milk, gelatin and other components, decreases the probability of cell elements damaging and extends the assured storage life.

Yersinia pestis plasminogen activator, a surface protease (Pla) is encoded by species-specific plasmid pPla. This enzyme belongs to outer membrane protein family and has a ß-barrel structure. Pla protease interacts complexly with homeostasis system of a hostorganism and is an important factor of plague agent virulence. Represented are the literature data on the part of plasminogen activator in the development of bubonic and pneumonic plague. Described is the role of R-form lipopolysaccharide in protease Pla activity manifestation. Plasminogen activator directly contributes to fibrinolysis by activating plasminogen and inactivating plasminogen-1 activator inhibitor (PAI-1) and ά2-anti-plasmin, as well as promoted by thrombin fibrinolysis inhibitor (TAFI). Pro-coagulant Pla protein activity is caused by the destruction of tissue factor pathway inhibitor (TFPI). In addition, given is the information on non-proteolytic functions of plasminogen activator.

In 2009, a novel virus, named severe fever with thrombocytopenia syndrome (SFTS) virus, was isolated from a patient in China. The illness caused by this novel virus is characterized by a sudden onset of fever and respiratory or gastrointestinal disorders, followed by progressive thrombocytopenia and leucocytopenia, the case-fatality rate amounting to 6–30 %. Genomic sequencing of the isolated agent indicated that the SFTS virus constituted a new (third) group of Phlebovirus genus, Bunyaviridae family. Presently, different means for specific diagnostics of SFTS (real-time reverse transcription polymerase chain reaction, indirect fluorescent antibodies method, enzyme-linked immune-sorbent assay) are developed. Constructing of diagnostic kits, basic characteristics of methods for determination of causative agent of infection or specific antibodies against it are considered in this review.

Objective of the study is to analyze immunological structure of the mountain souslik, habitant in various zones of natural focality in the Central-Caucasian natural plague focus.

Materials and methods. The data on serological investigations conducted on the mountain souslik, within the periods of 1974–1988 and 2001–2004 have been obtained from FGHI “Kabardino-Balkaria Plague Control Station” of the Rospotrebnadzor. Applying passive hemagglutination test and indirect hemagglutination test, with the help of Excel Microsoft Office 2010, evaluated have been 63147 blood survey results.

Conclusions. It is established that the highest ratio of the souslik with low antibody titer in blood, which characterizes initial contact with plague infection, is observed at the peak of epizootic activity. In the mountain steppe of the eastern focal part, the apex coincides with June, in the western part, as well as in the Alpine and Subalpine zones of the eastern one – with August. Animals with high antibody titers, i.e. secondarily infected, emerge in vast numbers at the peak of mountain souslik lethality. In the mountain steppe this period falls upon June, i.e. simultaneously with the peak of epizootic activity. In the western part lethality apex is also observed in June, but maximum epizootic activity takes place in August. In Alpine and Subalpine zones of the eastern focal part, peaks of epizootic activity and lethality among the souslik populations concur and fall upon August. Isolation of plague microbe cultures alongside with positive serological tests is reasonably more often observed in the mountain steppe of the river Baksan, than in other areas of natural focality.

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 specify present-day locations and sizes of natural plague foci in the North-Western Pre-Caspian.

Materials and methods. Based on the results of field and office mapping of Pre-Caspian sandy and North-Western steppe plague foci, conducted in 2013–2014, designed have been electronic maps of the sectors, situated in the periphery of the foci.

Results and conclusions. Detected has been marked reduction in sizes of natural foci owing to plowing of arid pastures. Over a significant distance, new natural focal boundaries are represented by linear elements of hydrography. Wherein such elements are absent, sector frames, in which evidence of enzooty remains, are accepted as formalized external boundaries. The process of deep and irreversible anthropogenic transformation of landscapes has resulted in the reduction of enzootic as regards plague territories: the steppe focus area has decreased by 22 %, being 51152 sq. km, the sandy one – by 13 %, amounting to 62510 sq. km. Farming on the extensive territories as a means of radical alterations of the landscapes has made the lands unsuitable for habitation of the little souslik, midday and tamarisk gerbils, which is an evidence of a complete loss of plague enzooty factors in the territory. The survey of actual position and sizes of natural plague foci within the rigid bonds, plotted on topographic maps and easily identifiable afield, provides for substantiated planning and complex prophylactic plague-control measures. Novel spatial parameters of the natural foci are suggested for the inclusion into official regulatory-methodological documents, guiding performance of epidemiological surveillance over plague.

Objective of the study is to analyze the influence of vaccination against hepatitis A on the corresponding morbidity rates in the Republic of Sakha (Yakutia).

Materials and methods: statistical data contained in the statutory forms of the State statistical reporting on infectious diseases and preventive vaccination against hepatitis A in Yakutia, dated 1992 to 2015.

Results and conclusions. Following selective immunization of high-risk groups, performed during the last 14 years in the Republic of Sakha, the incidence of hepatitis A has decreased by 38 times. According to the results of 2015, registered were 15 cases of infection, the rate being 1.6 per 100 thousand of the population, which was below the national average by 2.8 times. Assessment of the long-term dynamics has demonstrated the impact of preventive vaccination on the epidemic process of hepatitis A. Morbidity rates fall down with the increment of the numbers of immunized persons. Effect of the vaccination among the age group of 7 to 14 years is especially strong.

Objective of the study is to construct a test-system allowing for specific identification of brucellosis agent, using real-time polymerase chain reaction.

Materials and methods. Carried out has been analysis of literature sources and nucleotide sequences of Brucella spp., applying the specialized software for DNA-target sequence selection, specific for each brucella species. Developed is methodological tool and reagent panel for identification of brucellosis agent species appurtenance by means of RT PCR using thermocycler with hybridization-fluorescent registration of results, RotorGene type.

Results and conclusions. Developed is the test-system, providing for differentiation of brucella species or group of species: B. abortus/B. ovis; B. melitensis; B. suis/B. canis; B. neotomae, applying real-time polymerase chain reaction. BR0262, BRA0541-0542, BMEII0711 genes are selected as DNA-targets, being completely or partially deleted in different species of brucella.

Objective is an experimental study of the low temperature and nutrient deficiency effects on the stability of Vibrio cholerae El Tor genetic loci complex.

Materials and methods. The stability of the genetic loci (i.e. the basic pathogenicity genes; genome fragments containing variable tandem repeats and sites of rare cutting restrictase recognition defining VNTR- and PFGE-profiles of the isolates, accordingly; V. cholerae «housekeeping» genes) of the four Vibrio cholerаe El Tor strains was investigated on the experimental microcosm at different stages of incubation.

Results and conclusions. The study demonstrated that clones with altered VNTR- and PFGE-profiles (the detection frequency was 4–8·10–2) were being formed when strains were cultivated under nutrient deficiency and low temperature conditions. Neither changes in the structure of «housekeeping» genes nor loss of pathogenicity genes of the mobile genetic elements were revealed. The findings affirm the probability of analyzed V. cholerae loci alteration occurrence under unfavorable environment conditions. Obtained results should be taken into consideration for interpretation of molecular typing data.

Automated Vitek 2 system, based on comparison of a biochemical profile of the studied bacterial cultures with the existing database, is widely used for B. pseudomallei and B. mallei identification in the laboratory practice.

Objective of the study is to conduct extended phenotypic characterization of the strains of glanders and melioidosis causative agents, stored in the biobank of the Volgograd Research Anti-Plague Institute and analyze variations in their biochemical profiles, using Vitek 2 system.

Materials and methods. Using Vitek 2 device, (bioMerieux, France) analyzed have been biochemical properties of 52 collection strains of B. pseudomallei and B. mallei grown on L-agar (Difco, USA) and trypticase-soy agar – TSA (HiMedia, India).

Results and discussion. Most of the investigated strains (31 out of 40 B. pseudomallei and 8 of 12 B. mallei) have been identified with an acceptable probability for determining certain specie appurtenance, amounting to 90–99 %. The percentage of correct identification of B. pseudomallei and B. mallei is higher when strains are cultured on L-agar, than when on TSA. Due to the variability of the biochemical features, some strains have showed non-typical for its species results in certain tests (for B. pseudomallei strains – the absence of enzyme activity of β-N-acetyl-glucosaminidase, β-N-acetyl-galactosaminidase and ability to utilize D-cellobiose; for B. mallei strains – the absence of enzyme activity of L-proline-aryl-amidase and tyrosin-aryl-amidase, existence of glycin-aryl-amidase activity and ability to utilize sucrose, D-trehalose), which has led to their mal-identification. The probability of error diagnostics of microorganisms belonging to Burkholderia species necessitates up-dating of the database built into Vitek 2 analyzer as regards biochemical characteristics of the strains which have peculiar profiles.

The aim of the work was to detect specific antibodies to West Nile, dengue, CCHF, and chikungunya viruses in blood sera of Guinean Kindia Province residents.

Materials and methods. The obtained sera were analyzed in ELISA to discover IgG antibodies to abovementioned viruses.

Results and conclusions. Detected were 267 (82 %) positive samples out of 326, containing immunoglobulins of G class to these arboviruses. The obtained data provide evidence for active circulation of dengue and West Nile fevers agents in this territory. Further studies of immune strata of the population, and possible carriers and vectors of arboviruses were demonstrated to be advisable for optimization of approaches to prophylactic (anti-epidemic) measures implementation.

Objective of the study is to evaluate applicability of different freeze-driers for lyophilization of pathogenic microorganisms of bacterial origin, and to perform comparative analysis of the quality of the strain formulations obtained using these devices.

Materials and methods. Four strains of pathogenic microorganisms, belonging to different species and applied as test ones for quality assessment of nutrient media, have been utilized as controls. Their lyophilization has been conducted on four different modules for sublimation, collector- and chamber-type. The quality of lyophilized strain formulations obtained has been evaluated via determination of their cell viability, as well as with the help of rapid survivability test on lyophilized bacterial cultures (thermo-stability assay).

Results and conclusions. Specified are the conditions for lyophilization of collection test-strains of pathogenic bacteria, falling under the category of the III–IV groups of hazard, using collector devices of the new generation, Martin Christ Alpha 1-4 LDplus and Heto Power Dry. Carried out has been comparative analysis of viability, survivability and prognosticated terms of storage for the strain preparations obtained with the help of the mentioned above modules and the like, lyophilized on the instrument based on K.E. Dolin technology and programmable drier of the chamber type, Martin Christ Epsilon 2-6D. It is revealed that microorganism preparation lyophilized on different sublimation devices are characterized with high viability of the contained bacterial cells and have prognosticated storage terms of 4–105 years, depending upon the microorganism species and the type of freeze-drier utilized.

The aim of the work was to develop a kit for detection of IgM and IgG antibodies to Ebola virus in ELISA.

Materials and methods. Ebola virus strain Zaire H.sapiens-wt/GIN/2015/Kalidie-Kindia-1022 grown on cell culture was used as antigen.

Results and conclusions. The kit was used by the Rospotrebnadzor SAET team in its work in Guinea while investigating cases during Ebola fever epidemics. It was established that specific IgG antibodies persisted in Ebola fever survivors for 2 years. Application of this kit in the laboratory diagnostics permits ELISA to become the main and confirmatory laboratory method of Ebola fever detection.

Objective of the study is to investigate the impact of cultivating temperature and medium on the terms of persistence, ctx gene retention, and enzymatic activity of V. cholerae O1 with various toxigenicity.

Materials and methods. Utilized were the strains of V. cholerae El Tor: P-5879, P-19613, and also the strain P-19787.

Results and conclusions. In the process of studying cholera vibrios El Tor with different genetic characteristics it was determined that the longest terms of persistence (19 days) on mineral substrates at 5 ºC were observed for toxigenic strains, while for non-toxigenic ones it made less than 17 days. At the same time cholera vibrios can persist continuously and even reproduce on mineral substrates under the conditions of subnormal lowered temperatures (not less than 10 °C). Toxigenic strains of Vibrio cholerae, irrespectively of cultivating medium and temperature, retained ctx gene in their genome and maintained enzymatic activity throughout the experiment. Such long-term persistence of cholera vibrios at low temperatures on mineral substrates may be regarded as possibility of preservation of V. cholerae toxigenic strains in case of import by the infected persons or vibrio-carriers.

Objective of the study was to develop and test new biotechnological approaches for live tularemia vaccine production.

Materials and methods: Francisella tularensis 15 NIIEG strain was used as producer-strain; Francisella tularensis 503 strain – as test infecting one. Producer strain was cultivated on solid and liquid nutrient media. Tangential ultrafiltration was performed with the help of microfiltration module “Viva-flow”. Lyophilization was conducted using drying installation – Free Zone 2.5 L.

Results and discussion: Application of the designed liquid nutrient medium on the basis of enzymatic fibrin hydrolysate and submerged cultivation of the producer-strain has allowed for a significant biomass yield increment. At the stage of tularemia microbe culture concentration via microfiltration through filtering membranes with pore size of 0.2 μm, in the mode of tangential liquid flow, increased has been the content of microbe cells; the nutrient media residues – removed. Comparative analysis of the obtained in accordance with experimental technique laboratory series of the vaccine and commercial preparation of live tularemia vaccine has demonstrated their conformity with the specific normative properties. It is established that application of modified liquid nutrient medium, submerged cultivation conditions, methods of biomass concentration and separation has no negative influence on the main properties of live tularemia vaccine and will provide for considerable produce-ability increase in the future.

Objective of the study was to develop and then conduct medical trials of the “Monoclonal immune-enzyme test-system for the detection of plague agent (“EIAPESTF1-M”).

Materials and methods. Utilized were materials for carrying out sandwich EIA. MCA 1B3 to Y. pestis F1 served as diagnostic immune-reagent. To determine sensitivity and specificity of the designed immune-enzyme test-system, investigated were samples of 24 natural and genetically modified pFra+ and pFra– Y. pestis strains and 56 strains of heterologous bacteria Enterobacteriaceae family.

Results and conclusions. Based on monoclonal antibodies to Yersinia pestis capsular antigen, constructed was “Monoclonal immune-enzyme test-system for the detection of plague agent (“EIAPESTF1-M”), which is characterized by high specificity and allows for the detection of encapsulated plague agent strains in biological samples and for identification of pure cultures with sensitivity of 5·105 – 1·106 mc/ml. It is highly competitive with comparator drug – “Diagnostic fluorescent absorbed equine plague immunoglobulins”, lyophilizate for diagnostic purposes, by RusRAPI “Microbe”. It possesses such advantages as objective result recording and capability of result documentation. “EIAPESTF1-M” is registered in the RF Federal Service for Surveillance in the Sphere of Healthcare and Social Development, reference No 2013/711, dated 31.05.2013 and approved for use in the territory of the Russian Federation.

Objective of the study is to investigate the impact of activator, oxidize, and unblocking solution on the quantitative yield of oligonucleotides during manufacturing of the test-system for Vibrio cholerae (ctxA+) DNA detection “GenChol”, using polymerase 
chain reaction.

Materials and methods. The subject of the study is ctx2 and ctx3 primers, incorporated in the test-system “GenChol”. Utilized have been experimental formulations of solutions for unblocking, activator, and oxidizer and “standard” solutions, recommended by the manufacturer “BioSet” Ltd. Specific activity of the synthesized primers ctx2 and ctx3 has been evaluated on three different strains: V. cholerae 569B, M-1298, 158 and E. coli 12226 O-55. Bacterial suspension from the pathogen with final concentration of 1·103 – 1·101 m.c./ml has been produced. The DNA has been isolated applying nucleo-sorption in presence of guanidinium isothiocyanate.

Results and conclusions. Demonstrated is the possibility of optimization of diagnostic preparation production via increment of oligonucleotide yield in the process of phosphoramide synthesis, due to application of 3% dichloroacetic acid in dichloromethane as unblocking solution and oxidizer – 0.1 M iodine in athetic acid and pyridine in the ratio of 1:9. Utilization of such reagents increases the yield of the end product (primers) by 6 and 95 %, respectively. Enhanced technology for oligonucleotide synthesis will provide for the reduction of costs, associated with expensive imported reagents and the raise in the numbers of gene-diagnostic preparations for the detection of particularly dangerous pathogens.

Species composition and contamination by the causative agents of natural focal infections, notably Ixodes ticks, collected in 2014–2015 on the Island Russky were studied. The total of 817 ticks was collected, including Ixodes persulcatus, I. pavlovskyi, Haemaphysalis concinna, H. japonica douglasi. The most of the nymphs (46 specimens) were observed in I. pavlovskyi. Using immune-enzyme analysis, reliably higher rate of positive findings was revealed in I. pavlovskyi (2.2 %) compared to I. persulcatus (0.5 %) while analyzing individual contamination of two Ixodes species. The pattern of inter-specific distinctions did not changed (I. pavlovskyi – 1.3 %; I. persulcatus – 0.5 %) when investigating tick virus burden by means of polymerase chain reaction. However, in case of comparative evaluation, using the same method, of their contamination with borrelia (I. pavlovskyi – 25.1 %; I. persulcatus – 46.4 %), agents of granulocytic anaplasmosis (I. pavlovskyi – 1.9 %; I. persulcatus – 5.7 %) and monocytic ehrlichiosis (I. pavlovskyi – 1.3 %; I. persulcatus – 7.2 %) I. persulcatus showed higher indicators. Only tick-borne encephalitis RNA and borrelia DNA were revealed in I. pavlovskyi nymphs, whereat their contamination by spirochetes (45.7 %) was authentically higher than of imagoes. The natural focus of tick-borne encephalitis on the Island Russky is estimated now as low-active one. At the same time, of Ixodes tick-borne borreliosis – as active, with significant contamination of the tick nymphs with spirochetes. In terms of high nymph contamination it is supposed that in 2016 the risk of Ixodes tick-borne borrelioses morbidity rate will increase for humans visiting the Island within the period of high vector activity (May– June).