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AIM: Study N-acetyl-ß-D-glucosaminidase (chitobiase) (EC 3.2.1.30) in strains of Vibrio cholerae of O1/non-O1 serogroups of various origin, that is a component of chitinolytic complex taking into account object of isolation and epidemiologic significance of strains. MATERIALS AND METHODS: Cultures of V. cholerae O1/non-O1 serogroup strains were obtained from the museum of live culture of Rostov RIPC. Enzymatic activity analysis was carried out in Hitachi F-2500 fluorescent spectrophotometer using FL Solutions licensed software. NCBI databases were used during enzyme characteristics. RESULTS: N-acetyl-ß-D-glucosaminidase in Vcholerae O1/non-O1 serogroup strains was detected, purified by column chromatography, studied and characterized by a number of physical-chemical and biological properties. Comparative computer analysis of amino acid sequence of N-acetyl-ß-D-glucosaminidases of V. cholerae (VC2217 gene), Serratia marcescens etc. has allowed. to attribute the enzyme from V. cholerae to glycosyl-hydrolases (chitobiases) of family 20 and classify it according to enzyme nomenclature as EC 3.2.1.30. CONCLUSION: N-acetyl-ß-D-glucosaminidase in V. cholerae of O1/non-O1 serogroups of various origin and epidemiologic significance, participating in chitin utilization was studied and characterized for the first time, and its possible role in biology of cholera causative agent was shown.

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Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.

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Wild-type representatives of Francisella genus (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia) produce S-type lipopolysaccharides (LPS) possessing different antigenic activity and common antigenic determinants in the core oligosaccharide. Electrophoretic analysis showed that F. philomiragia produced S-LPS containing two major molecular components with minor fractions between them, whereas S-LPS of F. tularensis, F. novicida, and F. novicida-like are characterized by the typical frequency distribution of molecules. A characteristic feature of Francisella LPS was the ability to form the dominant molecular components with similar electrophoretic mobility of major fractions of the original F. philomiragia LPS upon long storage in water solution. Natural virulent F. tularensis strains produce at least two types of S-LPS. Polysaccharide chains of type I S-LPS possess O-species-specific antigens, whereas the polysaccharide part of type II S-LPS has nonspecific antigenic epitopes. A decrease of F. tularensis virulence can be associated with impaired production of both S-LPS types or loss of S-LPS with O-species-specific antigenic activity.

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The comparative study of the immunogenic and protective properties of V. cholerae, grown in vivo and in vitro, and cell walls and lysates obtained from these organisms. In the mouse protection test the efficacy of preparations obtained from vibrios grown in vivo did not exceed that of the preparations obtained from V. cholerae agar cultures.

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In this investigation isogenic avirulent variants obtained from F. tularensis standard virulent strain 503 were used. The capsule-deficient variants (cap-) were shown to have no species-specific capsular antigens and to be capable of producing R-LPS having no the polysaccharide part of the molecules. The capsule-defective forms (cap +/- ) were found to synthesize capsular antigens and S-LPS whose polysaccharide part essentially differed from the O-lateral chains of LPS of the virulent strain. The study of bacterial dissemination revealed that virulent bacteria rapidly spread in the macroorganism, and their subsequent proliferation shortly led to the death of animals. Avirulent mutants (cap- and cap +/- ) appeared in the organs of animals later and proliferated slower, parasitizing in the macroorganism without fatal outcome. The cap- variants were not capable of inducing the synthesis of antitularemic antibodies and possessed no protective properties. The cap +/- mutants were capable of inducing the synthesis of antitularemic antibodies in mice. These antibodies facilitated the elimination of avirulent strains from the macroorganism, but did not ensure protection from infection with virulent strains.

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Lipopolysaccharide and outer membranes from the three virulent encapsulated (Cap(+)) strains of three subspecies of Francisella tularensis and their isogenic avirulent capsule-deficient (Cap(-)) mutants were isolated. It was shown that the Cap cells and their outer membranes almost completely consumed the available complement of normal human serum whereas Cap(-) LPS (R-LPS), Cap(+) cells and their components activated the complement less effectively. Absorption of normal human serum with Cap(-) strain dramatically reduced the complement consumption for homologous strain and its surface structures. This reduction reflected the loss of bactericidal antibodies. Addition of antibodies to whole cells of F. tularensis completely restored complement activity. The cross-absorbing experiments demonstrated that Cap(-) cells more effectively deplete bactericidal antibodies than homologous virulent strain. From these results it can be concluded that normal human serum is bactericidal for serum-sensitive Cap(-) F. tularensis strains through the action of complement initiated by the classical complement pathway and serum resistance of virulent strains is not due to absence of targets for bactericidal antibodies, but is due to their low accessibility because of O-side chains of lipopolysaccharide.

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The study of the sensitivity of F. tularensis to the bactericidal action of normal serum (NS) revealed that all virulent cultures were resistant, while avirulent cultures were highly sensitive to NS. The synthesis of R-lipopolysaccharide (LPS) by capsule-deficient (Cap-) clones or the synthesis of S-LPS by capsule-defective (Cap+/-) clones of the avirulent phenotype of these bacteria had no influence on the sensitivity of these forms to NS, but ensured longer survival of Cap+/- variants in the serum in comparison with Cap- cells. The antibacterial activity of NS with respect to avirulent strains was determined by the system of the complement, activated following the classical route. Only complete S-LPS characteristic of virulent strains was capable of preventing the sorption of components and the assembly of the complement on the surface of bacteria, while defective forms of LPS of avirulent strains did not protect serosensitive receptors from interaction with the complement. The opsonization of Cap+/- virulent and Cap- avirulent bacteria with tularemic antibodies did not alter [correction of after] their reaction to incubation with NS, but ensured the survival of Cap+ forms for more than 24 hours.

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The bacteriophage designated RD2 has been isolated from the sewage in Rostov-on-Don city and studied. The morphology of bacteriophage particles and the biological properties of the bacteriophage make it related to the plague bacteriophage isolated by D'Errel. The molecular masses of the compared bacteriophages are almost identical being 26.4 +/- 0.4 Md for RD2 and 24.7 +/- 0.2 Md for D'Errel bacteriophage. The DNAs of the bacteriophages share 80% of homology and possess 15 nonhomologous regions scattered along the genomes. The phages are serologically related. The DNAs of both bacteriophages give the similar pattern of hydrolysis by restriction endonuclease EcoRV, but have the different sensitivity to many other restriction endonucleases. The protein specter of bacteriophage RD2 contains 18 polypeptides (11 minor ones), while the one of D'Errel bacteriophage contains 7 polypeptides similar in molecular mass with the polypeptides of RD2. The bacteriophage RD2 cannot be considered one of the plague causative agents of bacteriophages since the region where it has been isolated has a long epidemiological and epizootical record of absence of plague.

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The use of different schemes of albino mice immunization either by living or by killed preparations of the vaccine strain of Francisella tularensis when obtaining monoclonal antibodies to the tularemia microbe made it possible to reveal definite regularities in the dynamics of antibody formation. The highest titres of antibodies in sera of animals-donors of splenocytes were obtained during the daily (for 3 days) intraperitoneal immunization of mice with living vaccine or with its thrice administration to the spleen thrice with the interval of 10 days. Revaccination against a background of high titres of antibodies decreased their quantity in blood serum of mice, while that against a background of low titres increased them.

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Molecules of the plasmids pBR322 and pRD17 have been compared by electron microscopy technique of R-loops visualization. Comparative location of R-loops on the plasmids has been computerized on minicomputer HP9825A due to the program making possible to define the coordinates of the transcription start and direction. The 5.1 kb fragment coding for pesticin I and immunity protein to pesticin I and cloned in pBR322 vector plasmid is flanked by Bam HI-EcoRI sites. Five promoter regions and direction of transcription were localized on the fragment.

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Four Y. enterocolitica strains (10166, 10373, 2119, 5513) have been studied for the presence of the enzymatic systems of modification-restriction (M-R). As revealed with the use of cross titration, strains 10166 and 10373 contain M-R systems, supposedly of type II.

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The restriction map of Yersinia pestis pesticinogenicity plasmid pYP1 has been constructed with the use of 18 restriction endonucleases. Plasmid dimensions (6.3 Md) have been specified, the genes for pesticin synthesis, for pesticin immunity protein, fibrinolysin and plasmocoagulase have been localized by molecular cloning of single plasmid DNA fragments in vector plasmid pBR322.

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Cobra poison at a dose of 4 microng/kg of body weight intensified the 3H-thimidine incorporation into DNA of mice liver tissue; 20-40 microng/kg of the poison inhibited the incorporation. Single intraperitoneal administration of high doses of cobra poison (50-150 microng/kg) decreased the incorporation of 14C-thimidine into DNA 1.2-1.9-fold. Little doses of the poison increased the incorporation of 14C-hydrolysate of Chlorella protein into proteins of liver cells and the high doses inhibited this process. Microdoses of cobra poison caused a distinct increase in weight of experimental mice.

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