[The problems of identification of various strains of vegetative form of Bacillus anthracis using MALDI-ToF MS technique.]

The article considers experience of application of mass-spectrometry with matrix-activated laser desorption/ionization for fast and reliable identification of Bacillus anthracis and heterologous species of microorganisms. The particular interesting characteristics and difficulties occurred during identification are covered.

[The diagnostic significance of particular immune-dominating proteins of isogenic variants of Bacillus Anthracis.]

The polyclonal mono-specific sera to proteins of toxin and antigens of S-layer of Bacillus Anthracis separated in preparative electrophoresis. While fluorescent antibody technique was applied, immunoglobulins of sera to protein of S-layer of Bacillus Anthracis m.m. 94 kDa had specificity and specific activity in presented number of strains. In the soil samples spores in concentration of 1x104 KOE/ml were detected that permits to recommend the given immunoglobulins for indexation of Bacillus Anthracis. The proteins m.m. 90 kDa relevant to antigens of toxin of Bacillus Anthracis reacted in immuno-blotting with sera from patients with skin form of anthrax and guinea pigs immunized and survived after infection. Thew sera to them are species specified and can be applied for diagnostic of disease (detection of protective antigen) and detection of production of proteins in reaction of immunodiffusion with growing cultures.

[Characteristics of isogenic variants of Bacillus anthracis with various content of virulence plasmids].

AIM: Production and characteristics by main cultural-morphologic and antigenic properties of isogenic variants Bacillus anthracis, that differ by the presence of virulence plasmids. MATERIALS AND METHODS: B. anthracis 81/1, 575/122 virulent and B. anthracis STI, 55, Sterne vaccine strains were used in the study. Isogenic variants, that differ by the presence of virulence plasmids, were obtained by temperature elimination of plasmids, as well as during cultivation of anthrax strains in medium with kanamycin. The strains were characterized by cultural-morphologic, biochemical properties. The presence of virulence plasmids was determined by polymerase chain reaction method. Antigenic properties were studied in immune diffusion reaction with growing cultures with sera against protective antigen and S-layer proteins, electrophoresis, immune blotting. RESULTS: Isogenic variants were produced from virulent strains B. anthracis 81/1, 575/122 and vaccine strains STI, 55, Sterne: mono-plasmid toxin-producing (81/1 R01, 575/122 R01) and capsule-containing (81/1 R02, 575/122 R02), and plasmid-less (81/1 R00, 575/122 R00, STI R00, 55 R00, Sterne R00), that differ by the presence of virulence plasmids. Strains had typical cultural-morphologic properties, differed by biochemical and antigenic properties. Cultural filtrates of toxin-producing strains had protein of anthrax toxin; plasmid-less strains--had proteins, that had molecular masses corresponding to molecular masses of S-layer EA1 and Sap proteins. CONCLUSION: These strains may be used to study variability and proteomic analysis of anthrax causative agent, as well as for isolation of antigens with the aim of evaluating their immune diagnostic significance.

[Species-specific sera against surface antigens of Bacillus anthracis strains].

The species-related specificity of sera against 94-KD proteins isolated from culture filtrates of B. anthracis strains with different levels of virulence plasmids was studied to determine whether they might be used to identify the pathogen of anthrax. Sera against fractions 1 of culture filtrates of B. anthracis strains CTI (pXO1+ pXO2-), 81/1TR (pXO1- pXO2-), Davies (pXO1- pXO) separated by gel chromatography on Sephacryl S-300 were examined. In the gel immunodiffusion test with growing cultures, the sera exhibited non-identical antigens and differed in the presence of antibodies to antigens of related bacilli. The sera against fractions 1 of culture filtrates of toxin-producing and plasmidless strains displayed antigens produced only by B. anthracis strains into nutrient agar. Electroimmunotransblotting revealed that they contained antibodies mainly to 94-kD proteins and failed to react with B. cereus proteins with a molecular weight of 94 kD and with B. thuringiensis proteins with a molecular weight of 97 kD, which were extracted from autonomous cells. In the immunofluorescence test, immunoglobulins of sera against fractions 1 of culture filtrates of three strains stained autonomous cells and spores of 23 B. anthracis strains with different levels of virulence plasmids. In working dilutions, they did not react with antigens of 18 strains of related bacilli, which presents a possibility of using them for species identification of B. anthracis.

[The characteristics of the interaction of Bacillus anthracis with host phagocytes in relation to the plasmid spectrum of the causative agent]. / Osobennosti vzaimodeistviia Bacillus anthracis s fagotsitami khoziaina v zavisimosti ot plazmidnogo spektra vozbuditelia.

The study revealed that after the intraperitoneal inoculation of spores of B. anthracis strains with different plasmid composition into guinea pigs the active germination of the spores both outside and inside the cells of the host occurred as early as on hour 2 of their interaction with the macroorganism. The further fate of the infective agent and the character of its interaction with peritoneal exudate cells depended on the plasmid composition of the bacilli. Thus, the presence of toxin-formation plasmid PXO1 and capsule-formation plasmid PXO2 in B. anthracis permitted the successful adaptation of these bacilli to the conditions of the macroorganism, while the absence of such plasmids made them nonviable in this environment. The presence of plasmid PXO1 in B. anthracis permitted the manifestation of their cytopathic effort on the cells and the presence of plasmid PXO2 only gave the bacilli incomplete protection from phagocytosis.

[Antigenic properties of the electrophoretic fractions of the causative agent of melioidosis]. / Antigennye svoistva élektroforeticheskikh fraktsii vozbuditelia melioidoza.

Antisera to the antigens of 5 fractions, isolated as the result of the separation of P. pseudomallei aqueous saline extract by continuous electrophoresis in the vertical block of granulated gel, have been obtained. Immunoelectrophoresis with the use of P. pseudomallei aqueous saline extract has revealed that antisera to electrophoretic fractions contain antibodies mainly to the antigens of the corresponding fractions, which shows that this technique ensures the effective separation of P. pseudomallei biopolymers by their electrophoretic motility and molecular weight. These antisera differ in their species specificity. Thus, antisera to antigens with anode motility have been found to contain antibodies mainly to P. pseudomallei antigens and antisera to electroneutral antigens or to those with cathode motility, to P. pseudomallei and P. mallei antigens.

[The nature of changes in the antigenic spectrum of the causative agent of melioidosis during animal passage]. / Kharakter izmenenii antigennogo spektra vozbuditelia melioidoza pri passirovanii na zhivotnykh.

The comparative analysis of the antigenic spectra of Pseudomonas pseudomallei museum and subcultured strains, carried out by the method of immunoelectrophoresis, has revealed that, along with an essential increase in the virulence of P. pseudomallei for white mice and changes in the morphology of colonies, a decrease in the amount of detected precipitinogens occurs in the process of subculturing. The immunoelectrophoregrams of the subcultured variants show the absence of antigens 5, 6 and the simultaneous increase of the production of antigen 8, one of the components of mucoid (in the pseudocapsule).