[Cholera Vibrio biofilm: production, characterization and role in reservation of causative agent in water environment].

AIM: Experimental production, characterization and evaluation of the role of cholera vibrio biofilm. MATERIALS AND METHODS: 33 strains of Vibrio cholerae eltor O1 and V. cholerae O139 of various epidemic significance and origin were studied in a series of experiments by bacteriologic, microscopic (light-optic, luminescent, scanning electron microscopy), molecular genetics, spectrophotometric and statistical methods. RESULTS: Formation of a biofilm involving inter-cellular bonds, pili and extracellular material and variability of the microorganism (RO-phenotype and transition into uncultivable forms) was shown at various temperature and substrate conditions. A more pronounced ability to form biofilms was detected for strains isolated from environmental samples compared with isolated from clinical material regardless of their epidemic significance. Toxigenic strains of eltor biovar (from surface reservoirs during cholera outbreaks) have demonstrated the highest parameters of optical density compared with toxigenic clinical isolates and non-toxigenic O1 and O139 serogroup cultures. The presence of mbaA1 and mbaA2, vpsR, toxR, hapA genes is common for strains that form a biofilm. CONCLUSION: The data obtained confirm the role of biofilm in reservation of cholera vibrio strains of various epidemic significance in saprophytic phase of microorganism existence.

[Membrane-bound proteases of ompT+ and ompT- Vibrio cholerae strains].

AIM: Detection ofproteases in outer membranes (OM) of ompT+ and ompT- Vibrio cholerae strains of O1 and O139 serogroups. MATERIALS AND METHODS: Specific sterile preparations of OM were obtained by lysis of live V. cholerae cells by 4.5 M urea solution with subsequent differential centrifugation and treatment by nucleases. Extraction of OM proteins previously treated by sodium sarcosinate was carried out by Triton X-100 in the presence of EDTA. Protease and polypeptide spectra were studied in substrate and SDS electrophoresis. Sensitivity of proteases to inhibitors was determined in diffusion test in agarose gel containing substrate by using soy trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF). The presence of ompT was determined in PCR by using specific primers. RESULTS: According to PCR data 13 Vibrio cholerae O1 strains and 3 V. cholerae O139 strains isolated from clinical material as well as 22 V. cholerae O1 strains isolated from environmental objects contained ompT gene. 2 V. cholerae O1 human isolated strains, 9 V. cholerae O1 strains and 2 V. cholerae O139 strains isolated from the environment did not have ompT gene. By using SDS- and enzyme-electrophoresis in polyacrylamide gel quantitative and qualitative differences in composition of polypeptides and proteases of OM ompT+ and ompT- V. cholerae strains that hydrolyze gelatin, casein and protamine sulfate were detected. Inhibition of OM by STI and PMSF resulted in a decrease of their proteolytic activity. CONCLUSION: In preparations and extracts of ompT+ and ompT- V. cholerae OM up to 3 proteases some of which may be related to ompT-like were detected.

[Detection of Brucella antigens by dot immunoassay using specific antibodies labelled with colloid gold particles]. / Vyiavlenie antigenov Brutsell metodom dot-immunoanaliza s ispol'zovaniemspetsificheskikh antitel, mechennykh kolloidnym zolotom.

Dot immunoassay was developed to improve the quality of laboratory diagnosis of brucellosis. Particles of colloid gold were used as a marker of specific antibodies. The method was used for detecting Brucella antigens in artificially contaminated environmental objects (soil and water) and in biological material (milk, blood serum, and visceral homogenates of animals). The sensitivity of the test system was 19.5.10(3)-62.5.10(4) CFU/ml. Specificity of the assay was tested with 10 heterologous antigenically closely related bacterial species. The proposed test system is simple, economic, highly sensitive and specific, and requires no expensive equipment and reagents.

[The use of gel chromatography for the isolation of biologically active toxin complex from Bacillus anthrax]. / Primenenie gel'-khromatografii dlia vydeleniia biologicheski aktivnogo toksinnogo kompleksa sibireiazvennogo mikroba.

The multistep fractioning of the protein components from cultural filtrate of B. anthracis grown on casaminoacids containing medium was done. The scheme for preliminary purification of a toxin complex of B. anthracis against low and high molecular mass contaminants has been elaborated and includes ultrafiltration, gel chromatography in ultragel AcA-202 and TSK-gel toyopal HW-55. Biological activities of the complex,toxicity for laboratory animals and adenylate cyclase activity characteristic of B. anthracis toxin, are shown to remain intact in course of preliminary purification. Molecular masses of protein subunits from the fraction containing anthracis toxin activity reach 85-90 kD, 0.3-0.5 mg of toxin complex protein is yielded from 1 l of B. anthracis cultural filtrate.

[Use of specific antibodies, labeled with colloidal gold particles, for the detection of Brucella antigens using dot-immunoassay]. / Ispol'zovanie spetsificheskikh antitel, mechennykh chastitsami kolloidnogo zolota, dlia obnaruzheniia antigenov brutsell metodom dot-immunoanaliza.

The method of dot immunoassay with the use specific antigens, labeled with colloidal gold particles, for the detection of brucellar antigens was developed and tested under laboratory and field conditions. In this work soluble antigens isolated from different Brucella species and corpuscular antigens (13 strains belonging to 7 species of the genus Brucella, most pathogenic for humans and animals in the S- and R-forms) were used. The method was tested in the study of pathological material obtained from sick animals and humans in a farm with unfavorable situation for brucellosis in the Irkutsk Region. The sensitivity of the proposed assay system was found to be high and constituted 10 pg/ml to 1 ng/ml for soluble brucellar antigens and 200 CFU/ml to 13.5 x 10(6) CFU/ml for corpuscular antigens of Brucella S- and R-forms. The specificity of the method was tested with the use of 10 heterologous microorganisms. False positive results were observed only with Yersinia enterocolitica 0:9 at a concentration of 1 x 10(6) CFU/ml due to similarity of their polysaccharide-containing surface antigens. The newly developed dot immunoassay is simple for use, rapid and does not require expensive reagents and equipment.

[Detection of brucella antigens with colloid metal particles as markers for specific antigens]. / Obnaruzhenie antigenov brutsell s pomoshch'iu chastits kolloidnykh metallov v kachestve markerov spetsificheskikh antitel.

Gold and silver sols were comparatively approved as markers of specific IgG isolated from hyperimmune Brucella antisera for the detection of brucellar antigens. The sensitivity of the test system using gold immunosol proved to be some higher (3.1-9.8 ng/ml of soluble and 2.0 x 10(4)-5.3 x 10(6) CFU/ml of corpuscular brucellar antigens) than that achieved with the use of silver immunosol (5.7-18.4 ng/ml of soluble and 6.1 x 10(4)-8.0 x 10(6) CFU/ml of corpuscular brucellar antigens). At the same time silver sol was a cheaper and more available marker. Both test systems were found to be highly specific. False positive results were observed only with Yersinia enterocolitica O:9 at high concentrations due to the fact that they had common polysaccharide haptens incorporated into lipopolysaccharides of these microorganisms. The proposed test systems with colloid metal particles used as markers of specific antibodies for the detection of Brucella antigens are technologically simple, economic, rapid, highly sensitive and specific. Their use in combination with other serological methods will make the results of analyses more informative, thus improving the quality of laboratory diagnostics of brucellosis.

[The outer membranes of Vibrio cholerae as a potential component in a chemical vaccine]. / Naruzhnye membrany kholernogo vibriona kak potentsial'nyi komponent khimicheskoi vaktsiny.

The results of the study of the preparation of V. cholerae eltor membrane, obtained by the lysis and inactivation of microbial cells with urea and the subsequent differential centrifugation and nuclease treatment. As revealed in this study, the outer membrane preparation, when introduced parenterally and orally to mice, induced pronounced immunity to experimental cholera infection and the production of vibriocidal antibodies in high titers. The treatment of V. cholerae eltor membranes with trypsin led to further increase of the immunogenic potency of the preparation. The protective action of V. cholerae eltor outer membranes considerably exceeded the protective effect of currently used whole-cell eltor vaccine. This opens prospects for using the above-mentioned preparation for the improvement of chemical vaccine as a component ensuring the formation of antibacterial immunity.

[The characteristics of the protectiveness of a preparation of Vibrio cholerae outer membrane based on the data from a study using the ligated intestinal loop]. / Kharakteristika protektivnosti preparata naruzhnoi membrany kholernogo vibriona po dannym issledovaniia pereviazannoi kishechnoi petli.

The study aimed at finding out the antiadhesive capacity of antigenic preparation, earlier obtained from V. cholerae outer membrane and highly effective with respect to cholera infection, was undertaken. The study was made on previously immunized adult rabbits who had been subjected to laparotomy under anesthesia and the ligation of intestinal loops, subsequently inoculated with the broth culture of V. cholerae eltor (P-3122, serovar Ogawa). The intestinal loops were studied histologically and bacteriologically with the calculation of the number of vibrios, the deduction of the adhesion index and the coefficient of the efficacy of immunization. The data thus obtained indicated that the specific immunization of rabbits with their subsequent inoculation with V. cholerae virulent strain suppressed the adhesive activity of the infective agent which was more pronounced in rabbits immunized with the preparation of V. cholerae outer membrane.

[Experimental non-culturable Vibrio cholerae eltor and its biological properties]. / Eksperimental'noe poluchenie nekul'tiviruemykh form Vibrio cholerae eltor i kharakteristika ikh biologicheskikh svoistv.

In experiments with the cultivation of V. cholerae eltor under the conditions of high salt concentration, as well as low temperature and deficiency in nutrient substances, uncultivable forms (UF) of toxigenic and nontoxigenic vibrios were obtained. The absence of growth of seeded vibrios after the filtration of samples (with a filter of 0.22 micron), the preservation of specific antigenic determinants and the initial set of genes, changes in the morphology of cells (small size, coccoid form with the flagella retained) confirm the transition of V. cholerae eltor under study into the uncultivable state which, under unfavorable conditions, more rapidly develops in toxigenic vibrios than in nontoxigenic ones. The analysis of the INT-reductase activity of UF disintegrates revealed that they had endogenic respiration whose activity increased (4.5- to 6.5-fold) in the presence of the exogenic intermediates of the Krebs cycle. The uncultivable forms of the vibrios retain genes responsible for pathogenicity, as well as their antigenic determinants.

[Use of protein-polysaccharide Brucella antigen labeled with colloidal gold for detection of specific antibodies by the dot-immunoassay method]. / Ispol'zovanie belkovo-polisakharidnogo antigena brutsell, mechennogo chastitsami kolloidnogo zolota, dlia obnaruzheniia spetsificheskikh antitel metodom dot-immunoanaliza.

Antibrucella antibodies were detected by dot-immunoassay with colloid gold label of antigen. The specific antigen was protein-polysaccharide complex (PPC) isolated from vaccine strain Brucella abortus 19 BA by acetic acid hydrolysis of bacterial cells. Dot immunoassay with PPC labeled with colloid gold was effective in testing cattle, rabbit, and human sera for antibrucella antibodies. The proposed test system is simple, economic, highly sensitive and specific, and requires no expensive equipment and reagents, which are its advantages over routine serological tests.

[Use of specific antibodies labelled with colloid silver particles for detection of Brucella antigens with dot immune analysis]. / Ispol'zovanie spetsificheskikh antitel, mechennykh chastitsami kolloidnogo serebra, dlia vyiavleniia antigenov brutsell metodom dot-immunoanaliza.

Fitness of dot immuno-analysis for detection of Brucella antigens labeled with colloid silver is evaluated. Soluble lipopolysaccharides and protein-saccharide antigen and corpuscular antigens of 22 Brucella strains (7 species) pathogenic for humans and animals in the S and R forms were used. The specificity of the method was tested on 10 heterologous microorganisms whose antigens were closely related. The suggested test system is simple, economic, highly sensitive (from 62 thousands to 8 million CFU/ml) and specific, requires no expensive equipment, and is an alternative to enzyme immuno-assay and dot immuno-analysis with gold immunosole.