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Various methods for evaluation of virulence properties of causative agents of infectious dis- eases in vitro were analyzed: molecular-genetic, cultural-biochemical, immunologic, physiologic. Predominant use of molecular-genetic methods, expediency of a complex approach, relevance of search of novel informative parameters of virulence are noted. Study of biological properties of pathogens in vitro is the first screening stage of evaluation of their virulence.

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AIM: Comparative evaluation of the effect of exogenic serotonin on the development of apoptosis and proliferative activity of immune system cells of biomodels in vivo and in vitro in the dynamic of immunity forming against plague and tularemia. MATERIALS AND METHODS: Analysis of relative content of immune competent cell DNA of unlinear and BALB/c mice was carried out after staining of the samples with mithramycin and ethidium bromide by flow cytometry. RESULTS: Administration of serotonin into biomodels before immunization with vaccine strains of Yersinia pestis and Francisella tularensis was established to increase in vivo proliferative activity of immune system cells, without a significant effect on their death by apoptosis. Serotonin inhibited in vitro the development of apoptosis of mice blood leukocytes in response to both the vaccine Y. pestis EV strain and tularin. CONCLUSION: Biogenic amine serotonin shows equivalent modulating effect on both anti-plague and anti-tularemia immune response in vivo and in vitro, without disrupting immune system homeostasis.

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AIM: Compare the content of bactericidal granules (BG) in blood phagocytes of animals, that differ by species sensitivity to plague infection, under the conditions of measuring, that ensure automatic differentiating by this parameter of monocytes and granulocytes of human blood. MATERIALS AND METHODS: Human whole blood leukocytes were studied, as well as from 7 animal species: mice, guinea pigs, golden hamsters, white rats, rabbits, dogs and horses. Acridine orange (AO) was used for supra-vital staining in primary (bactericidal) granule cells. Relative BG content was measured in separate cells in conventional units of red fluorescence intensity by flow cytofluorometry. RESULTS: Deficiency of AO molecules in BG, that correlates with deficiency of leukocyte elastase in cells, that is most pronounced in mice and lest pronounced in rabbits, was established to be characteristic for all the blood phagocytes of all the laboratory animal species sensitive to plague. Blood phagocytes of dogs and horses, that were non-sensitive to plague, differed by high heterogeneity by the studied parameter, and in horse blood innate immunity cells were detected, that contained 2.5 times higher amount of BG, than blood granulocytes of humans. CONCLUSION: Leukocyte BG, that have enzyme cationic proteins: elastase, cathepsin G, protease 3 and myeloperoxidase, play and important role in protection of organism from plague infection.

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AIM: Evaluation of immune stimulating and toxic effects of a vaccine prototype protein components. MATERIALS AND METHODS: Linear mice, guinea pigs and rabbits were immunized subcutaneously once or twice by recombinant protective antigen (rPA), S-layer protein (EA1) or their complex. Innate immunity structure activation was registered by changes in Toll-like receptor (TLR) expression. Adaptive immune response parameters were determined by established methods. Toxicity of the preparations was determined using flow cytofluorometry and densitomorphometry. RESULTS: The ability of rPA and EA1 to activate structures of innate immunity - TLR 2 and 6 - was established. Features of anti-PA antibody titer dynamics for each of the animal species was determined, a comparison with antibody formation during immunization with Bacillus anthracis STI- 1 was carried out. 2 immunizations ofbiomodels with a complex preparation combined with an adjuvant provides protection from infection by a test-strain that is comparable with protectivity of a live vaccine. Evidences regarding damaging effect of rPA and EAI on cells and tissues of macro organism were not detected throughout the study. CONCLUSION: Aprototype of a chemical anthrax vaccine under development has high immunogenicity and its protein components are not toxic for laboratory animals based on the results of complex testing.

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AIM: Comparative analysis of DNA content in individual cells of Vibrio cholerae strains with various biological properties. MATERIALS AND METHODS: 24-hour agar cultures of 2 avirulent (lacking cholera toxin gene) and 2 virulent strains and their subcultures obtained by cultivation in 1% peptone water for 1, 3 and 5 hours were studied. DNA of the killed bacteria was dyed by a mixture of ethidium bromide and mitramycin. Ratio of cells with low, intermediate and high relative DNA content in conditional units of specific DNA fluorescence intensity was determined by flow cytofluorimetry method. The degree of inhomogeneity of the studied microbial population cells was evaluated by DNA histogram variation coefficient value. RESULTS: At the level of major statistical samples of individual V. cholerae cells a principally different reaction pattern of the studied toxigenic and non-toxigenic strains on changes of cultivation conditions was registered. CONCLUSION: Populations of cells of toxigenic V. cholerae strains in contrast to non-toxigenic probably shift to polyploid state during starvation. This phenomenon may turn out to be a differential feature in determination of the risk group (hazard) of a strain.

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AIM: Comparative analysis of Yersinia pestis strains with various biological properties by DNA content in individual cells. MATERIALS AND METHODS: Virulent strain 231, avirulent strain KM 260 (12) [231], that is its isogenic (no-plasmid) derivative, and vaccine strain EV NIIEG were used. 48-hour agar cultures of the studied strains reproduced at 28 degrees C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37 degrees C were prepared. DNA of the fixed bacteria was dyed by a mixture of ethidium bromide and mitramycin, and then the bacteria were studied by using flow cytofluorimeter for the determination of rates of cells with relatively low or high DNA content in the studied bacterial populations. The degree of inhomogeneity of a bacterial population was evaluated by DNA histogram variation coefficient value. RESULTS: In 6 hours of growth at 37 degrees C in optically non-dense bacterial cultures a high degree of DNA content per cell inhomogeneity was established that is related to the activation of DNA replication process in bacteria. In 48 hours of growth this inhomogeneity completely disappeared in the virulent strain cultures and remained in the avirulent strain cultures of the plague pathogen. Based on the studied parameters the vaccine strain held an intermediate position. CONCLUSION: Further studies of the plague culture DNA content per cell inhomogeneity may become a base for the operative strain differentiation based on pathogenicity level (hazard) for humans, and therefore the requirements for the management of safe working conditions with this microorganism.

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Using developed scheme, complex study of protective properties of avirulent recombinant strain Vibrio cholerae El Tor Inaba KM 184 was performed. Necessity for broadening of standard procedure of testing of cholera vaccines protective properties by using of quantitative methods of assessment of morphological changes and state of biomodel's functional systems, which could increase the information value of assessment of studied vaccines, was experimentally substantiated.

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Effect of biogenic amine serotonin on the development of human blood leukocytes during interaction with species from Yersinia genus (Y. pestis EV, Y. pseudotuberculosis serovars I and IV, Y. enterocolitica serovars 09 and 03) was studied in model system in vitro using flow cytofluorometry. Serotonin in concentration 10(-5) M had differently marked effects on Yersinia spp.-induced apoptosis of leukocytes. Pattern of the observed changes depended from species and serovar of Yersinia. Serotonin inhibited development of early (after 6 hours) apoptosis of leukocytes induced by Y. pestis and Y. pseudotuberculosis serovar I.

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The influence of Y. pestis phospholipase D on the physiological state of leukocytes in the blood of guinea pigs was studied in vivo by flow impulse fluorometry with the use of fluorochrome acridine orange. During the first hours of observation the intensity of leukocyte fluorescence increased due to a rise in the number of polymorphonuclear leukocytes and changes in the permeability of cell membranes. Further changes in the intensity of the fluorescence of the material under study after 24 hours of observation occurred due to the appearance of activated lymphocytes in the blood stream. The processes normalized by day 21. The reaction of blood leukocytes to phospholipase D was specific in comparison with the reaction to capsular antigen, "mouse" toxin, lipopolysaccharide and the main somatic antigen.

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The steps of human spermatozoa treatment for flow cytofluorometric analysis of their DNA content are described in detail. The authors demonstrate the possibility of simultaneous measurement of haploid, pathologically altered, and immature spermatozoa in the ejaculate and estimation of their ratio. The diagnostic value of the method is assessed.

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The work presents the results of the study (carried out by the method of continuous flow cytofluorometry) of changes in the distribution of lymphocytes and their populations (obtained by means of distributing cell electrophoresis) according to the phases of the cell cycle (G0 + G1; S; G2 + M) in the blood and spleen of guinea pigs, as well as in the blood of humans, before and after immunization with cholera vaccine. The results of the determination of DNA-synthesizing lymphocytes in the blood of immunized humans and animals have been shown to serve as an objective characteristic for the complex evaluation of the biological activity of cholera vaccines.

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