REVIEWS
ORIGINAL ARTICLES
Objective – analysis of epizootiological manifestations of natural focal infections in the territory of the south of the European part of the Russian Federation in 2017.
Materials and methods. Statistical documentation data from the Rospotrebnadzor Administrations, Centers of Hygiene and Epidemiology in the constituent entities of the Russian Federation, and Plague Control Research Institutes and Stations were used. The information was processed using Microsoft Excel 2010 software.
Results and discussion. Epizootiological survey for 19 nosological forms of natural focal infections in the territory of the south of the European part of the Russian Federation was conducted. The total of 70155 samples of field material was tested; markers of 14 pathogens of natural focal infections were identified. The circulation of Crimean-Congo hemorrhagic fever virus was revealed in 11 constituent entities, tularemia and Lyme borreliosis pathogens – in 8 entities, West Nile virus – in 7. Markers of leptospirosis, Q fever, human granulocytic anaplasmosis and human monocytic ehrlichiosis pathogens were detected in 6 constituent entities, markers of the agent of hemorrhagic fever with renal syndrome – in 5 entities; markers of intestinal yersiniosis pathogen – in 3 constituent entities of the Russian Federation, pathogens of tick spotted fevers group, tick-borne viral encephalitis and pseudotuberculosis – in 2. The circulation of the virus Sindbis was identified in the Rostov Region.
Objective was to assess the effect of specific bacteriophages and gentamycine on the morphological-functional properties of bacteria in the vaccine strain Yersinia pestis EV.
Materials and methods. The vaccine strain Y. pestis EV, Pokrovskaya bacteriophage and the pseudotuberculous diagnostic bacteriophage were used for the study. The microbial culture was grown on solid and in liquid growth media at 27 °C for 20–24 h. The co-incubation of bacteria and bacteriophage or gentamycine was carried out at 27 °C for 20 minutes or at 37 °C for 2 hours, respectively. Culture preparations were examined by transmission electron microscopy.
Results and discussion. The influence of cultivation conditions and various stress factors on the vesicle production by the vaccine strain Y. pestis EV cells was evaluated. The nature and intensity of morphological-functional changes in Y. pestis EV cells in response to the effect of bacteriophages (plague Pokrovskaya and pseudotuberculous bacteriophages) or an antibiotic (gentamycine) were determined. It was established that co-incubation of Y. pestis EV with Pokrovskaya bacteriophage or gentamycine for 20 min leads to the increase in the production of extracellular vesicles and is accompanied by the development of degenerative changes in bacterial cells.
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.
Objective of the study was to determine the mechanisms of acetoin biosynthesis change in genetically altered El Tor V. cholerae strains in Voges-Proskauer test.
Materials and methods. We used nine genetically altered V. cholerae O1 strains, biovar El Tor, imported in the territory of the Russian Federation and Ukraine between 1993–2011, and four typical strains isolated in 1970–1972. When assessing acetoin production in V. cholerae strains in Voges-Proskauer test, the strain V. cholerae 569B O1 serogroup, classical biovar served as negative control of the assay. Relative gene expression was studied using real-time RT-PCR. Protein model construction was performed by means of automated server SWISS – MODEL.
Results and discussion. It has been demonstrated that diagnostically significant feature – VogesProskauer reaction, utilized for V. cholerae O1 biovar differentiation, is changed in all investigated genetically altered strains of cholera agent, isolated in different periods of the current seventh pandemic (66.7 % of the strains show weakly positive reaction, 33.3 % – negative one). The data obtained testify to the reduction or absence of acetoin production in the investigated strains. Analysis of four structural genes of als operon, as well as expression of regulatory genes alsR and аphA, responsible for acetoin biosynthesis, has revealed that changes in acetoin production in the genovariants stem from the deletion of a single nucleotide (T in the position 315) in the structural gene alsD, encoding acetolaktat decarboxylase, and also from high levels of negative acetoin biosynthesis regulator expression – аphA gene. Modeling of the spatial (3-D) structure of AlsD protein in the genovariant M1293 and the reference-strain N16961 has shown that AlsD protein of the genovariant is, indeed, considerably reduced. However, spontaneous decarboxylation is possible in the absence of acetolaktat decarboxylase, which phenotypically manifests itself in borderline positive Voges-Proskauer test.
Objective of this work was to determine epidemiological significance of Hantaviruses, circulating in Crimea, on the basis of retrospective data and taking into account the results obtained over the monitoring period of 2015–2018.
Materials and methods. The study of small mammals and blood sera of donors was carried using enzyme-linked immunosorbent assay.
Results and discussion. Held in 1985–1989 studies of hemorrhagic fever with renal syndrome (HFRS) suggested that Hantaviruses, circulating among the voles of the genus Microtus in the territory of Crimea, do not have etiological significance in the structure of HFRS incidence. A 2008 study found the circulation of Hantaviruses of the Tula serotype among Microtus arvalis (obscurus). Studies on the natural focality of Hantavirus infection in Crimea during 2015–2018 showed circulation of Hantaviruses among small mammals: Microtus socialis, Mus musculus, Sylvaemus witherbyi, Crocidura suaveolens, which are not the main reservoirs of pathogenic Hantaviruses. Also, seropositive to hantavirus donors were detected – 0.4 %. Local cases of HFRS infection for the entire observation period were not registered. To confirm the positive findings and determine the epidemiological significance of circulating pathogens, samples with positive for Hantaviruses findings were sent to the reference center for HFRS in 2017. The results of the investigation did not confirm the presence of antibodies to human pathogenic Hantaviruses in the blood sera of donors; in the biological material from mouse-like rodents, the antigen of pathogenic for humans serotypes of Hantavirus was not detected. Thus, in the natural foci of Crimea in 2015–2018, the circulation of Hantaviruses which do not belong to pathogenic for humans serotypes of Hantavirus Puumala, Hantaan, Dobrava was registered. Detection of seropositive donors testifies to natural immunization of the population through circulation of Hantaviruses that are non-pathogenic for humans or Hantaviruses of other serotypes.
Objective – risk-oriented assessment of the current epidemiological situation on West Nile fever in the Astrakhan Region.
Materials and methods. Utilized were the data collected by the Astrakhan Plague Control Station, Rospotrebnadzor Administration in the Astrakhan Region, and A.M. Nichoga Regional Infectious Clinical Hospital. The key method of study was epidemiological analysis of West Nile fever incidence among the population of the Region during the period of 2000–2016. 145 case records were investigated.
Results and discussion. Retrospective analysis provided for identification and featuring of the main categories of epidemiological risk of infection with West Nile fever in the Astrakhan Region in 2000–2016. It was established that men of 19 to 70 age range ( 82.1 %) are infected more often (95 out of 145 – 65 %). WNF infections in women occur among the same age group (75.8 %), and also among children aged below 6 years old (9.0 %). Analysis of the risk territories showed that the level of risk is high in one district, medium – in one district, low – in four districts, and very low – in six. When investigating the conditions of infection (risk factors) with WNF, it was determined that in the majority of cases (107 – 73.8 %) the risk factors were not specified. Out of those that were identified, one should pinpoint the bite of mosquito inside the households, basements, while fishing (16.3 %), as well as the bite, removal or squashing of a tick with unprotected hands (6.9 %). The period of the highest risk is from May to October with the maximum values of incidence in August (55.1 %).
Objective of the study was monitoring of epizootic situation in the Mongolian part of the trans-boundary Sailugem natural plague focus through 2018 for optimization of preventive and anti-epidemic activities to decrease the level of risk of human plague cases among the population in the border areas of Mongolia and Russia.
Materials and methods. Epizootic survey was conducted across the area of 2668 km2 ; 282 mammals, 261 ectoparasites, including 257 fleas, were tested for plague. All laboratory investigations of the field material were carried out in the mobile laboratory for monitoring and diagnostics, mounted on the platform of KAMAZ. Field samples were subjected to molecular-genetic (PCR) and serological tests. Fresh and mummified pickings of birds of prey, corpses, caught rodents and lagomorphs, fleas collected from corpses, were tested using immune-chromatographic method (ICM) to detect capsular antigen (F1) of plague microbe. PCR and ICM positive samples were investigated applying bacteriological method. In the course of epizootiological survey, GIS-tools were employed. All the results obtained were plotted on electronic maps using QGIS 2.12.3 software package.
Results and discussion. The total of 47 Yersinia pestis ssp. pestis strains were isolated from grey marmots and their fleas. Y. pestis DNA was detected in 60 objects. Serological testing showed 60 positive results. Contamination of the caught souslik with plague agent reached 4.5 % (n=22), fresh corpses and picking of predatory birds – 63.4 % (n=41), mummified corpses and leftovers, skeletal remains – 10.0 % (n=140). It was established that in the border territory, adjacent to Russia, an intensive diffused plague epizooty, caused by the agent of the main subspecies, takes place. All epizootic manifestations were revealed at the altitudes of 2400–2800 m above sea level, in densely populated grey marmot settlements. The epizooty was registered in most of the southern macro-slope of Sailugem ridge, throughout 100 km and along the whole Karalakhtu ridge – throughout 30 km. The epizooty area, confirmed by plague agent isolation, amounted to 1207 km2 (45.2 % of the examined territory).
Objective of this work was cloning of the Vibrio cholerae nanH gene as part of a plasmid vector, providing expression of foreign genes under the control of T5 promoter, and construction of a E. coli strain – producer of V. cholerae recombinant neuraminidase.
Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplifi ed, the cloning was carried out by means of conventional methods, performance of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Neuraminidase activity was identifi ed by fl uorescence in ultraviolet light after incubation with specifi c substrate (4-methylumbelliferyl-N-acetylneuraminic acid).
Results and discussion. Recombinant plasmid pNanH, containing the cloned gene nanH V. cholerae, has been constructed. The gene is inserted into BamHI-PstI sites of the polylinker of pQE30. Expression of the cloned gene in the producer strain E. coli JM103pNanH occurs under the control of T5 promoter after isopropyl-1-thio-β-D-galactopyranoside (IPTG) induction. The strain shows neuraminidase activity. The recombinant NanH protein is accumulated in the producer’s cells in two forms. The fi rst form, with molecular mass (MM) of 89.5 kDa, is an unprocessed protein with the hexahistidine block (6His-tag) at its N-terminus, it is located in inclusion bodies. Its percentage is 5.6–6.6 % of the total cell proteins. The second one, with MM of 83 kDa, is found in the periplasmic space and corresponds to the mature NanH, its percentage being 3.4–3.8 %. The total percentage of both forms is 9–10 % of total cell proteins, which allows to consider the strain E. coli JM103pNanH to be a super-producer of the required protein. The strain may be used for purifi cation of NanH preparations for construction of specifi c diagnostic, therapeutic and pharmaceutical preparations as well as for investigation of the protein as a virulence/persistence factor of the pathogen.
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.
Abstract. Despite the decreasing TBE incidence trend in Russia, the disease is considered an ongoing challenge for the state’s public health and economics. Objective of our study was to describe the algorithm of short-term incidence forecast of TBE, to evaluate the conformity of these data to factual incidence and the results of annual strategic seasonal monitoring which takes place across all the entities of Russia. In the paper, we described the procedure for providing short-term extrapolation of TBE incidence forecast onto the Russian territories, depending on the absence or presence of incidence change trends.
Materials and methods. Utilized were the State statistics, “The data on infectious and parasitic diseases” (Form No 2), as well as the information on strategic monitoring for a period of 2007–2018. In order to determine the multi-year trend of epidemic process development, regression analysis was applied. If the trend was identified, predictions were made on its basis, if not – through calculating the long-term annual average. In all the cases, 95 % confidence interval for incidence trend deviation was considered. Comparative analysis of actual morbidity rates and predicted ones and the data on strategic monitoring was conducted by Student’s criterion.The commutations were performed using Excel software tools.
Results and discussion. The study has demonstrated that the expected rates of TBE incidence are not statistically different from the actual incidence or the data from strategic monitoring. The underestimation of the epidemiological risk is found only in 4 out of 49 entities (8,2 %), and it is of note that in 3 of them it was less than 16 %. The data from operational monitoring are downward biased by reference to actual incidence, which is probably due to inclusion of TBE cases confirmed and/or manifested upon termination of incubation period after expiration of terms of weekly observations. The unified and simple approach that we proposed to TBE-incidence forecasting within the territory of Russia provides for correct information on expected epidemiological risk assessment and timely planning of required preventative measures.
Objective of the study was epizootic-epidemiological zoning of the area of Krasnodar Territory and the Republic of Adygea by manifestations of tularemia to determine the level of epidemic hazard of each zone.
Materials and methods. Utilized were archival data of the Black Sea Plague Control station over the period of 1946–2017 and plague Control Center of the Rospotrebnadzor. With the help of GIS software packages, MapINFO 10.5 and ArcGIS 10.2, the data bases containing the point-like layers of the sites of infection with tularemia (49), isolation of tularemia agent (195), and the layer of landscape-geographical regions in the Krasnodar Territory and the Republic of Adygea were created.
Results and discussion. Usage of the geo-information technologies allowed for detailed consideration of tularemia manifestations in different parts of the region. The prospects of applying Arc GIS and MapINFO for geoencoding, processing and creation of geo-information pool of tularemia manifestations over a long period was shown. Vector data of landscapes and sites of epidemics and epizootic manifestations of tularemia on different species of mammals and ticks were generated. The conversion of the database to Microsoft Excel made it possible to make full use of statistical capabilities for epidemiological analysis. The work on epidemiological zoning carried out in the Krasnodar region and the Republic of Adygea starkly illustrated the feasibility of using GIS technologies for those purposes. The results of the analysis allowed for optimization of the mode of epizootiological survey in different parts of the studied region. Advisability of epizootiological inspection and monitoring of the territories with identification of geographical coordinates for epizootic manifestation sites was proved.
Objective of this work was to search for genome loci of various types of foot and mouth disease (FMD) virus, characterized by the lowest variability, to be used as genetic markers in the PCR-indication of the virus.
Materials and methods. The resources of the National Center for Biotechnology Information (NCBI) and BLAST and Vector NTI 9.1.0 software utilities were employed in the research. Plasmid DNA with marker insertion was utilized for PCR amplification.
Results and discussion. The nucleotide sequences of FMD virus genomes, the types A, Asia-1, C, O and SAT (1, 2 and 3), were analyzed. In the process of aligning of isolate genomes of each type, potentially conservative sites were identified. The comparison between these loci has revealed one most conserved locus, and the subsequent BLAST analysis has established its high specificity to FMD virus genome. Primers and a probe were selected for this locus. In addition, the oligonucleotide primers were selected for the three genes included in the cattle genome that are least homologous to the specific oligonucleotides. The primers/probe were used as internal control of amplification. To control the progress of amplification, a positive control has been developed that has a nucleotide sequence of the marker region of FMD virus genome. It was found out that genomes of certain virus isolates show high level of polymorphism in relation to PCR-probe (12 isolates by A, Asia-1, SAT1, and SAT2 serotypes). However, modifications of the PCR-probe (Pas FMDV and Psat FMDV) allow for elimination of the effect of such variability on the number of virus isolates identification. Nucleotide sequences of the primers, probes and positive controls are presented in the tables.
ISSN 2658-719X (Online)