Rhamnose-Positive Strains of Plague Agent: Virulence and Epidemiological Significance

The aim of the review is to show the groundlessness of the unconditional assessment of rhamnose-positive strains of plague pathogen as avirulent for most species of carriers and humans and having no epidemiological significance. The main carriers of rhamnose-positive strains are several species of voles and the Mongolian pika. The vast majority of experts are of the opinion that rhamnose-positive (“vole`s” and “pika`s”) strains of Yersinia pestis are avirulent or weakly virulent for many species of warm-blooded animals and humans, and therefore have no epidemiological significance. However, in a series of experiments on infecting marmots, ground squirrels, and large gerbils with rhamnose-positive strains, some of the experimental animals fell ill acutely and died from the plague. In nature, rhamnose-positive strains have been isolated from carcasses of relatively resistant red marmots. When evaluating the epidemiological significance of rhamnose-positive strains, such an important criterion as the presence or absence of effective factors and pathways of pathogen transmission in foci of the vole and pika types is omitted. Voles and pikas are not eaten; therefore, the contact route of infecting humans in these foci is impossible. The second way of transmission of the pathogen to humans – vector-borne – is difficult due to the lack of migration of vole fleas from burrows to the surface and their low efficiency as vectors. Nevertheless, cases of human infection with rhamnose-positive strains of the plague agent in the Caucasus and Mongolia give grounds to assert that at least some rhamnose-positive strains have a sufficiently high virulence and are capable of causing infectious process in humans as well. Therefore, epidemiological surveillance in the foci of plague of the vole and pika types cannot be totally abandoned. It can be conducted according to an abbreviated scheme

Comparative Analysis of Expression of the Main Virulence Genes in Various <i>Vibrio cholerae</i> О1 Strains

The aim of the work was a comparative study of the expression of the main virulence genes in Vibrio cholerae strains of the classical biovar, typical and genetically modified strains of V. cholerae, El Tor biovar.Materials and methods. Natural toxigenic strains of V. cholerae O1, classical biovar (J89, Pakistan, 1969), typical (M-887, Astrakhan, 1970) and genetically modified (301, Taganrog, 2011) strains of the El Tor biovar were used as model ones. The strains were grown under optimum conditions for the production of cholera toxin and toxin-coregulated pili. The assessment of strain growth was carried out in LB broth at room temperature with determination of the cells number on a Biowave DNA spectrophotometer (Biochrome Ltd., UK). Determination of gene expression was performed using real-time PCR with reverse transcription.Results and discussion. The expression of structural (ctxA, tcpA) and regulatory (toxR, toxT, tcpP, tcpH) virulence genes has been investigated in V. cholerae strains of the classical biovar, typical and genetically modified strains of the El Tor biovar. Significant differences have been revealed in terms of time and level of maximum expression of these genes in strains of classical and El Tor biovars. It was found that ctxA and toxR genes expression in the genovariant strain reached its maximum 1–3 h earlier than in the other strains. At the same time, the level of ctxA gene expression corresponded to the level of the classical strain. The maximum expression of the toxR gene in the genovariant strain was higher than in typical El Tor and classical strains, and also had a clear inverse correlation with ctxA gene expression. Expression of the tcpA, toxT, and tcpH genes in the classical biovar strain reached its maximum 1–2 h earlier than in the El Tor biovar strains. These differences should be taken into account when conducting research work related to the study of the expression of the main virulence genes

Historical and Modern Views on the Problem of Specific Plague Prophylaxis

Objective of this review is to analyze diachronically paradigm shift as regards problems of specific plague prophylaxis and appreciate contribution of the present-day scientific discoveries in the sphere of plague agent investigations and peculiarities of its interaction with host organism to the solution of topical issues of vaccine development that will be safe and tangibly effective against this particularly dangerous disease. Outlined is the historical background of the conceptual evolution concerning specific plague prophylaxis and events that are landmarked with eminent scientific discoveries by A.Yersin, French researcher and microbiologist. Given are the data on the current state of plague immune-prophylaxis both in Russia and around the world. Through the prism of the latest researches that assume application of advanced technological resource of medical sciences (molecular biology, biotechnology, bioinformatics, molecular immunology) put forward is the prospective of search and construction of safe and effective anti-plague next generation vaccines

Variability of <i>pgm</i>‑Region Genes in <i>Yersinia pestis</i> Strains from the Caspian Sandy and Adjacent Plague Foci

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

New Method of Plague Agent Lipopolysaccharide Obtaining

Put forward are two alternatives of a new method for optimization of conditions of LPS obtaining and purification from Y. pestis strains; as well as for avoiding application of poisonous and hard-to-remove reagents; for simplification and cost-cutting of the technique; and for rationalization of production waste management. This method involves preliminary salt-water extraction of bacteria, for elimination of easy-dissolving substances, with the subsequent fracturing using ultrasound in lysing buffer (0,1 M Tris-HCl, pH 8,0; 10 mmol of EDTA, 1 % Triton X-100). The first alternative for deproteinization of non-purified endotoxin is the commercial preparation of proteinase K (Sigma), the second one - an enzyme complex - proteovibrin, isolated from waste material accumulated in the process of cholera chemical bivalent vaccine production. Apart from this, introduced has been a phase of sample acidification by applying glacial acetic acid up to pH 3,2-3,4 to decontaminate LPS from nucleic acids. These two variations of the method provide for enhancement of LPS preparation quality as compared to prototype method, and for obtainment of plague agent endotoxin that is hardly distinguishable in physical-chemical properties, homogeneity, immunochemical activity and specificity from the antigen, manufactured by means of water-phenol extraction following Westphal O. technique

Usage of nutrient Medium Based on Dry Hydrolysate of Casein in Manufacturing Bivalent Chemical Cholera Vaccine

Objective of the study was to select the standardized substrate containing dry hydrolysate of casein for preparation of nutrient medium utilized for manufacturing bivalent chemical cholera vaccine under submerged cultivation of cholera vibrio strains in fermenters. Materials and methods. We used Vibrio cholerae O1 strains of classical biovar: strain 569B Inaba and strain M-41 Ogawa. Examined were two dry substrates of the medium: enzymatic hydrolysate of casein, Type I Himedia (India) and pancreatic hydrolysate of casein, produced by the State Scientific Center of Applied Microbiology and Biotechnology (Russian Federation). Produced under laboratory conditions at the premises of the RusRAPI “Microbe” medium was used as a control. Submerged cultivation was conducted in bioreactors during (9±1) h with aeration and automatic feeding of glucose and ammonia. Production of protective antigens was measured applying immunochemical and biological methods. Results and discussion. It is demonstrated that submerged cultivation of cholera vibrio production strains on nutrient media under study provides for synthesis of protective antigens the parameters of which comply with the requirements of normative documentation. More standardized and higher indicator values of the target product are ensured by cultivation of producer strains on nutrient medium with a substrate from dry enzymatic hydrolysate of casein, containing (1.5±0.1) g/l of amino nitrogen for the strain V. cholerae M-41 and (2±0.1) g/l – for V. cholerae 569 B. Transition to the use of standardized dry protein components of cultivation media does not lower the quality of the chemical cholera vaccine, but allows for the reduction of cost price and duration of technological process

Effectiveness of the “Sterius 60” SHF Radiation Installation for Disinfection of Objects Contaminated with PBA of Groups I–IV, when Working with Infected Biomodels

The aim was to evaluate the effectiveness of using the “Sterius 60” microwave disinfection system (Russia) for decontamination of objects infected with PBA of groups I–IV emerging as a result of working with infected laboratory animals.Materials and methods. Effectiveness verification of disinfection of biological waste generated as a result of the life of laboratory animals by SHF radiation was carried out in the microwave system “Sterius 60”, recommended by the manufacturer for disinfection of epidemiologically hazardous and extremely dangerous medical waste, including biological ones (classes B and C), by volumetric SHF heating. Carcasses of uninfected laboratory animals (white mice, Guinea pigs, suckling rabbits), granulated feed and bedding material (wood shavings), which are objects directly in contact with biomodels, were used as vivarium waste to be decontaminated. The following microorganisms were utilized as model test ones: Bacillus subtilus VKM B-911, Bacillus stearothermophilus VKM B-718, Bacillus licheniformis G VKM B-1711-D, Alcaligenes faecalis 415, Yersinia pestis EV, Bacillus anthracis STI. Laboratory utensils (plastic Petri dishes, porcelain mortars and pestles) were used as a mock-up chamber filler for model test microorganisms.Results and discussion. As a result of the study, data were obtained indicating that the microwave system for disinfection of medical waste “Sterius 60” is ineffective for decontamination of biological waste in laboratories working with biomodels infected with PBA of groups I–II. The established standard mode of disinfection of this system was effective only for non-spore forms of microorganisms, pathogenicity groups III–IV. Therefore, in our opinion, it is advisable to use it for decontamination of laboratory utensils infected with PBA of groups III–IV, directly at sites of waste generation

Comparative Study of Some Physical-Chemical and Immunochemical Properties of Plague Microbe Lipopolysaccharide Preparations Obtained with the Help of Different Techniques

, and degraded polysaccharide (PS) are easily soluble in water and in 0,9 % NaCl solution. They are homogenous and characterized by an adequate degree of purity. Aside from that, it is demonstrated that potentially PS is the most productive molecule fragment of LPS for the construction of plague immunodiagnostic preparation, since despite its decreased cytotoxocity PS retains identity of chemical composition and immunechemical specificity of endotoxin

Molecular-Genetic and Phenotypic Peculiarities of Plague Agent Strains Isolated in Vietnam

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

Results of Modeling Experiments in Designing Immuno-Enzyme Test-System for the Detection of Antibodies to <I>Yersinia pestis</I> F1 (ELISA-Ab-F1 <I>Yersinia pestis</I>)

Designed is immuno-enzyme test-system for the detection of antibodies to Yersinia pestis capsular antigen F1 – “ELISA-Ab-F1 Yersinia pestis”. On the model of laboratory mice it is demonstrated that this test-system is highly specific, its diagnostic titer being 1/320.Diagnostic value of the test-system is 83.3–88.9 % as revealed through investigations of sera and blood suspension samples, swabs of thoracic organs of animals, inoculated with live plague vaccine, strains of plague microbe, containing and deprived of pFra, as well as with heterologous bacteria