Tularemia Microbe Research Articles

ROLE OF VARIOUS ANTIGENIC PREPARATIONS OF FRANCISELLA TULARENSIS IN FORMATION OF ALLERGY REACTION IN HUMANS AND ANIMALS

Study the role of LPS in induction of anti-tularemia immunity in humans and animals. Activity of various antigenic preparations of tularemia microbe, including highly purified from protein and S- and R-LPS, was studied using leukocytolysis reaction with blood of vaccinated humans and guinea pigs and skin allergy test (guinea pigs). Only the whole cells of Francisella tularensis, killed in protein non-denaturating conditions and conserving full S-LPS structure (tularin⁺) were shown to be inductors of delayed-type hypersensitivity reaction. Alterations in LPS structure (tularin⁻) results in a significant decrease, and denaturation of bacterial proteins (during boiling) results in a complete loss of immune stimulating properties of the preparations. Purified LPS preparations and O-polysaccharide fraction of S-LPS are not able to activate cell-mediated immunity. The presence of LPS with the full structure affects the ability of antigenic preparations of F. tularensis to cause allergic reactions, and thus, form cell-mediated antitularemia immunity. LPS of F. tularensis can not be excluded as an adjuvant and provides the most effective presentation of epitopes of protein molecules for interaction with receptors of T-lymphocytes.

Enhancement of the Technology for Live Tularemia Vaccine Production

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.

Characteristics of Phenotypic and Genetic Properties of <i>Francisella tularensis</i> 15 NIIEG Vaccine Strain with an Extended Storage Period

Investigated have been cultural-morphological, biochemical and genetic properties of lyophilized cultures of F. tularensis 15 NIIEG vaccine strain, accumulated within 60-years term and deposited at the State Collection of Pathogenic Microorganisms of Scientific Center on Expertise of Medical Application Products. The studies undertaken have demonstrated that storing of the strains in such a form at low temperatures, does not prevent changes of their genetic and phenotypic properties to the full extent. It is established that F. tularensis 15 NIIEG strain lyophilized in 1953, 1966, 1969, 2003 and 2012 maintains its immunogenic properties when cultivated on nutrient media Ft-agar with or without addition of blood, based on dissociation rates (87-99 %) of SR-colonies. While F. tularensis 15 NIIEG strain 1990 contains specified amounts (not less than 80 %) of immunogenic colonies if cultivated on nutrient media with the addition of blood, and fails to meet the requirements - if cultivated without. Identified in F. tularensis 15 NIIEG strain 1987 SR-colony decrement of 70-75 % in case of cultivation with or without addition of blood testifies to the deterioration of its immunogenic properties. RAPD and ERIC typing has showed high stability of the genome of F. tularensis 15 NIIEG cultures lyophilized at different times. Tularemia microbe vaccine strain has unique RAPD and ERIC profiles, insignificant alteration of which is observed upon storage of pathogen subculture in the dried from.

Development of Amplification Test-Systems for Tularemia Agent Detection

Developed is the method for tularemia microbe DNA detection using PCR with electrophoretic and hybridization-fluorescent registration of results. iglBC genes have been chosen as DNA-matrixes, being species-specific ones for tularemia agent. Based on the results obtained constructed have been preparations for tularemia microbe DNA detection in biological material and environmental samples applying PCR with electrophoretic registration of results and real-time PCR: “Gene Francisella tularensis - REP” and “Gene Francisella tularensis RHF”, respectively. Identified are the package contents to be included into the test-systems. Sensitivity and specificity of the designed panels are validated through investigations of tularemia agent bacterial emulsions and suspensions from small mammals’ organs, from ticks, fleas and mosquitoes, as well as through studies of soil and surface water samples, sputum and human blood probes, experimentally contaminated with tularemia agent. Test-systems demonstrate high sensitivity (1·10 3 microbe cells/ml) and specificity (100 %), irrespective of the type of test material.

Studies of Immunobiological Properties in <i>Francisella tularensis</i> Vaccine Strain 15 NIIEG under Extended Storage Conditions

Investigated have been 8 cultures of Francisella tularensis strain 15 NIIEG (lyophilized in 1953, 1966, 1969, 1987, 1990, 2003, 2012, and 2013, respectively) stored at the State Collection of Microorganisms of the Scientific Center on Expertise of Medical Application Products. It is established that the majority of cultures has maintained their immunobiological properties. However, it is of note that liophilization does not prevent F. tularensis strain 15 NIIEG from changes in its residual virulence under extended storage. Revealed is the fact that LD 50 for 7 cultures of tularemia microbe strain is within the limits of 100-250 microbial cells (m.c.). At the same time, residual virulence for the strain which dates back 1966 is 7.3·10 5 m.c. Immunogenic activity rates in F. tularensis 15 NIIEG strain cultures range within specified limits. Apart from this, F. tularensis 1987 strain does not comply with the established requirements to the “specific safety”, as subcutaneous inoculation with 5·10 9 m.c./ml caused death of Guinea pigs within the scheduled observation time. Demonstrated is the necessity in maintaining constant stability of the original immunobiological properties in Francisella tularensis strain 15 NIIEG under extended storage conditions.

Characteristics of Tularemia Agent Strains Isolated from Patients and Small Rodents during Tularemia Epidemic in Khanty-Mansiisk in 2013

The paper contains the data on isolation of Francisella tularensis cultures from humans and rodents during tularemia epidemic in Khanty-Mansiisk in August-September, 2013. All the obtained F. tularensis strains (six cultures of tularemia microbe from patients, and four cultures - from small rodents caught in the territory of the city and its suburbs) fall under Holarctic subspecies, are highly virulent for the laboratory mice, resistant to erythromycin, ampicillin, and cephalosporin, but sensitive to amikacin, tetracycline, doxycycline, and gentamycin. Using 25-locus-MLVA typing it is demonstrated that the strains under study form a common cluster on the phylogenetic tree along with F. tularensis strains previously isolated in the territory of the Southern Urals and Kazakhstan. All the strains from the foci are closely related ones, and based on the investigations of hyper-variable Ft-M3 locus, fall into four groups. The strains, isolated from humans, combine into three groups, and the strains isolated from rodents - into two, and notably that one of the groups of Khanty-Mansiisk cluster comprises the strains isolated from both humans and rodents.

Peculiarities of the Conjugative pSa Plasmid Transfer from <I>Escherichia coli</I> into <I>Francisella tularensis</I>

per a recipient cell). pSa′ plasmid molds several topological shapes with variable electrophoretic mobility in E. coli C600, while in tularemia microbe, this plasmid has only one shape. After the transfer of the plasmid from E. coli cells into F. tularensis cells each individual clone of tularemia microbe possesses a plasmid which differs in its electrophoretic mobility from plasmids of other clones.

Experimental Preventive Anti-Tularemia Preparation

Designed is an experimental preparation of a prototype chemical tularemia vaccine (PCTV). It is composed of protective antigenic complex (PAC) of tularemia microbe and S-layer protein (Slp) of plague microbe. Determined is optimum ratio of these components in the preparation and schedule of its administration. Displayed are the results of its testing as regards physical-chemical properties, reactogenicity, specific activity and impact on the immune system of laboratory animals. It is found out that preparation of the prototype is non-toxic for white mice and Guinea pigs and has no damaging effect on their immune systems. Single-stage subcutaneous immunization with PCTV induces elaboration of high-level adaptive immunity in laboratory animals within 14–21 days: specific antibody generation and stimulation of immune system cell component. PCTV protective index for white mice in case of experimental tularemia, caused by Francisella tularensis subsp. holarctica , is 87,5 % on average; in case of infecting with F. tularensis subsp. nearctica – 50 %; and high-level immunity in both cases. High potency of the experimental preparation against tularemia caused by subsp. holarctica (protective index is 75 %) and high-grade immunity persistence is verified on the model of Guinea pigs too.

Constructing and Medical Trials of a Monoclonal Dot-Immuno-Enzyme Test-System “DIATul-M” for Tularemia Microbe Detection

mc/ml. Additionally, this test-system has been proving for acquisition of sustainable results after 6 months of storing (the observation period). Medical trials of the panel of reagents “Monoclonal dot-immuno-enzyme test-system for tularemia microbe detection” have shown it to be a prospective preparation for implementation into the national healthcare practices both under stationary and field conditions.

Estimation of the Severity of Experimental Tularemia against a Number of Morphometric Characteristics

Research objective consisted in the development of a unified approach to the evaluation of severity of experimental tularemia disease based on the morphometric analysis. Experimental tularemia infection was morbidized in Guinea pigs, both female and male, using subcutaneous inoculation with virulent tularemia microbe cultures. Subsequent to the results of morphometric analysis, selected was a range of indicators characterizing intensiveness of dystrophy, necrobiotic and necrotic processes, infiltrate changes within cells and tissues of the infected animals to fullest extent, and reflecting a life-support system functioning state adequately. Thus, application of quantitative counting to distinguish the changes in experimental animals, while modeling specific infectious process, allowed for standardization and verification of severity evaluation approach and facilitated increase in quality of experimental investigations.

Immuno-Chromatographic Test System Application for Rapid Detection of <I>Francisella tularensis</I> Lipopolysaccharide in Monitoring of Natural Foci

Immuno-chromatographic test systems are shown to be applicable for rapid detection of tularemia microbe lipopolysaccharide for identification of Francisella tularensis strains isolated in natural foci, and in epizootiologic survey – for the analysis of suspensions of ixodic ticks and internal organs of dead animals.

Peculiarities of the Effect of the Lipopolysaccharide of Tularemia Microbe of Different Subspecies upon Metabolic Activity of Phagocytes in Vitro

Peculiarities of the Effect of the Lipopolysaccharide of Tularemia Microbe of Different Subspecies upon Metabolic Activity of Phagocytes <i>in Vitro</i>

DNA Cytometry of Immunocompetent Cells of Biomodels under Conditions of Immunization by S-Complex Preparations of Different Tularemia Microbe Subspecies

The effect of S-complex preparations of tularemia microbe upon apoptosis and proliferative activity of cells of central (thymus) and peripheral (spleen) organs of immune system has been studied in dynamics on the outbred white mice model. S-complex preparations were isolated from vaccine strain 15 NIIEG of holarctica subspecies, virulent strain 503 of the same subspecies, strain Acole of nearctica subspecies and strain A 179 of mediasiatica subspecies. The ratio of cells being at different stages of cell cycle was shown to vary in different periods of time after immunization with the studied preparations. That can be associated with the structure of preparations depending on the source they were obtained from. All preparations demonstrated the lack of harmful effect upon genetic apparatus of white mice' immune cells. The application of the present antigenic complex in the construction of chemical vaccines of new generation is thought to be promising.

Influence of Tularemia Microbe of Different Subspecies on the Functional Activity of Immunocompetent Cells of Experimental Animals

Influence of Tularemia Microbe of Different Subspecies on the Functional Activity of Immunocompetent Cells of Experimental Animals