Vol 87, No 2 (2010)

Aim. Analysis of genetic heterogeneity of Pseudomonas aeruginosa strains isolated in and out of hospitals. Materials and methods. To study the genetic diversity of 36 strains of P.aeruginosa plasmid analysis, random amplified polymorphic DNA (RAPD) technique as well as polymerase chain reaction for detection of virulence genes algD, lasB, toxA, plcH, plcN, exoS, nan1, and nan2. Results. Epidemically important strains were found in different ecological niches. It was shown that these virulence factors could play important roles in pathogenesis of infec- tion. Conclusion. RAPD technique was effective for analysis of P.aeruginosa isolates. Number of studied typing bands differed between related isolates for each random primer.

Aim. To study the characteristics of group A strep- tococcal infection epidemic process in children aged 12—14 years arrived to summer camp «Orlenok» (Tuapse) from different regions of Russia. Materials and methods. Epidemiological (retrospective analysis of incidence of acute respiratory infections, tonsillitis, and scarlet fever), microbiological (isolation and identification of group A streptococci [GAS]), and molecular biological (pulse-electrophoresis, analysis of spe and emm genes) methods were used for the study. Objects of the study were GAS strains isolated from patients and carriers. Results. Performed genotyping showed that cases of GAS infection in newly formed children collectives were caused by 2—3 epidemically important clones, which were genotypically heterogenous. Conclusion. Performed molecular biologic studies demonstrated polyclonal structure of GAS that determines the features of development of epidemic process.

Aim. To obtain recombinant atoxic form of Pseudomonas aeruginosa exotoxin A and assess its protective properties during simultaneous administration with recombinant protein F in experiment. Materials and methods. Genetic methods were employed for construction of deletion variant of P.aeruginosa exotoxin A gene, whereas Escherichia coli cells were used for transformation. Purification of proteins was performed by common method. White outbred mice were used for immunization and experimental infection was produced by intraperitoneal administration of live virulent culture of P.aeruginosa strain PA103. Results. The gene coding defect form of P.aeruginosa exotoxin A with lacked 106 C-terminal aminoacid residues was cloned. Synthesized protein was nontoxic and immunogenic. Recombinant variants of anatoxin A and outer membrane protein F of P.aeruginosa protected animals from experimental infection caused by toxigenic strain PA103 of P.aeruginosa if were administered separately or concomitantly. Nonetheless, the highest protective effect was observed after immunization with both proteins. Conclusion. Studied recombinant toxoid and OprF could be the candidates for inclusion in vaccines for prevention of pseudomonas infection.

Aim. Assessment of characteristics of antigenic complexes of Staphylococcus aureus vaccine strains in different cultivation conditions. Materials and methods. S.aureus vaccine strains (No. 5, 9, 1986, 1991) were grown in liquid nutrient media – full value and semi-synthetic – as well as on solid medium. Reactor cultivation was performed in the fermenter ANKUM-2M. Complex of antigens were obtained by water extraction method applied to staphylococcal biomass inactivated with acetone and assessed by common methods on protein and carbohydrate content; specific activity was assessed by minimal inhibitory dose in passive hemagglutination inhibition assay. Study of acute toxicity was performed on outbred mice. Results. Using strain no. 1991, model of reactor cultivation in full value medium with separation of biomass by microfiltration was validated on the basis of biomass and semiproduct of antigenic complex (acetone powder) yield as well as productivity of biomass cumulation. Study of antigenic complexes obtained from biomass of 4 strains during reactor cultivation compared with complexes extracted from cultures grown on solid medium revealed increased protein and decreased carbohydrate content but similar specific activity. It was demonstrated that complex of antigens obtained from cultures grown either by reactor cultivation or on solid medium were non-toxic. Conclusion. New technology for manufacturing staphylococcal complex of antigens with reactor cultivation of vaccine strains in full value medium with subsequent purification of antigenic complex from the biomass by microfiltration was developed. Results of the study demonstrated the usefulness of the developed technology for both further studies on acellular staphylococcal vaccine and manufacture of staphylococcal component of «Immunovac» ® vaccine.

Aim. Assessment of results of immunization against measles and mumps in children with recurrent respiratory infections. Materials and methods. Levels of IgG against measles and mumps viruses were measured using enzyme immunoassay. Two hundred and twelve serum samples obtained from 6 groups of children with 20 — 45 persons (boys and girls) in each were tested. Children of various ages were presented in each group. Also, immunologic parameters were measured in all children. Results. It was established that mean antibody titers to measles and mumps viruses did not change during 10 years. In more than 50% of children correlation between high and low titers of antibodies of different specificity was found. Conclusion. Recurrent respiratory illnesses determine complex regulation of transition of memory cells into cells secreting antibodies to measles and mumps viruses.

Aim. To study antiviral activity of metabolites of spore-forming strain «Pashkov» of B.pumilus on the model of enterovirus infection in vitro. Materials and methods. B.pumilus strain «Pashkov» isolated from environment and identified by common methods. Cell cultures: Vero-6, Vero-ECC, and Vero-E6. Enteroviruses: type 1 poliovirus, Coxsackie B virus (1—6), ECHO-3, and ECHO-6 viruses. Unfectious activity of viruses was evaluated according to their cytopathogenic effect on Vero-E6 cell line by method of serial dilutions. Cultural fluid (CF) for the study was obtained by centrifugation and sterilizing filtration of B.pumilus strain «Pashkov» biomass produced by cultivation during 72 hours on optimized nutrient medium. Cytotoxicity of CF (chronic and acute) and maximal tolerated dose were measured by effect on viability of Vero-E6 cells, which was assessed by trypan blue exclusion test of cell viability. For measurement of antiviral activity (AV-activity), two treatment schedules — therapeutic and prophylactic — were used. Results. The most sensitive cell lines were Vero-ECC and Vero-E6. Assessment of AV-activity showed that protective effect was observed for all dilutions of CF and lasted for 7 days from time of infection by used doses of virus. CF does not have acute and chronic cytotoxicity. CF studied in vitro with Vero-E6 cells infected with 4 types of enteroviruses provided protection against viruses and had prophylactic effect. Degree of effect of CF depended from type of enterovirus, dose used and CF dilution. Conclusion. For the first time effective antiviral activity of CF, which have low cytotoxicity for Vero-E6 cell culture in vitro and is produced by strain «Pashkov» of B.pumilus , was demonstrated. Obtained data open perspectives for development of medications against enterovirus infections.

Aim. To clone the DNA fragment encodi ng conservative domain of LigA protein of Leptospira interrogans into Escherichia coli and to investigate antigenic properties of constructed chimeric protein. Materials and methods. E . coli strain M15 [pREP4], recombinant plasmid pTT10 encoding cellulose-binding domain (CBD), restriction endonucleases BamHI, BglI, BglII, XbaI, T4 DNA-ligase, RNAse were used in the study. Molecular cloning of ligA gene fragment was performed using standard protocols, and expression of hybrid genes—according to «Qiagen» company’s protocols. Extraction and purification of proteins were performed using original method. Results. DNA fragment encoding immunoglobulin-like domain 5 of LigA was cloned in E.coli . Effective strain-producer of chimeric domain D5-CBD consisting of the immunoglobulin-like domain 5 of LigA, Gly-Ser spacer, and cellulose-binding domain (CBD) was obtained. The high-purity D5-CBD preparation was obtained using one-stage purification on cellulose. Antigenic specificity of this chimeric protein was studied and it was shown that it could be used as a marker for the development of diagnostic ELISA kit. Conclusion. Recombinant domain of LigA in chimeric protein produced in E.coli retains antigenic properties of native LigA protein. Obtained results confirm the feasibility to use recombinant antigen D5-CBD as a marker for development of diagnostic kits on the basis of ELISA.

Aim. To assess the role of thermolabile enterotoxin of Enterobacter cloacae on level of caspases 3, 7, and 10 in experiment. Materials and methods. Observation of apoptosis in mice splenocytes and peritoneal exudate cells. Experimental infection was created by intraperitoneal injection of live bacteria and cultural fluid of E.cloacae producing thermolabile enterotoxin. Results. The study showed that thermolabile enterotoxin of E.cloacae does not have equal effect on apoptosis of studied cells: it slows apoptosis of splenocytes and virtually does not have any influence on peritoneal phagocytes. Conclusion. Apoptosis of infected cells is a protective reaction of microorganism to invasion of infectious agent. Cell death leads to rapid elimination of pathogenic agent. Furthermore, cell death by apoptosis compared to necrosis is more favorable for bacteria because it is not induce inflammatory reactions. In our experiments thermolabile enterotoxin of E.cloacae had had antiapoptogenic effect on mice splenocytes that could be a key element in pathogenesis of diseases caused by enterotoxin-producing strains of Enterobacter.

Aim. To study the effects of sea microalgae exometabolites on reproduction of pathogenic bacteria. Materials and methods. Experiments were performed with unialgal cultures of sea microalgae Phaeodactylum tricornutum, Chlorella minutissima, Chroomonas salina. Strains of Lysteria monocytogenes 4b, Staphylococcus aureus, Salmonella typhimurium with typical cultural, morphologic, biochemical and antigenic characteristics were used as model bacteria. Cultivation of microalgae was performed in synthetic and natural sea water (salinity 32‰). The algae were grown on modified Goldberg medium. Natural sea water was taken in different seasons of year. Exometabolites were obtained by extraction from microalgae cultural fluid with solvents in the order of increase of their polarity (hexane, benzol, ethyl acetate) as appropriate fractions (HF, BF, EAF). Results. Obtained fractions of exometabolites of sea microalgae cultivated in synthetic sea water influenced weakly on the reproduction of pathogenic bacteria, which differed depending on type of both microalga and m icroorgan ism. However E A F fraction of exometabolites of P.tricornutum obtained during its cultivation in natural sea water (especially obtained in summer time) significantly stimulated growth of all tested microorganisms. Conclusion. It is possible that stimulation of growth of pathogenic bacteria could be caused by either changes of medium composition (and, consequently, appearance of other metabolites than in synthetic sea water) or enhancement of effect of microalgae’s own metabolites by metabolites of natural sea water, in which plenty organic compounds produced by numerous sea inhabitants exist.

Despite the numerous ways the host defended viral infections, which mediated through reactions of innate and adaptive immunity, viruses escape from immune surveillance by interacting with receptor molecules of immune system for penetration into a cell and activation of synthetic processes in it for realization of early stages of viral replication. Recently, data about signaling receptors of innate immunity — Toll-like receptors (TLRs), which participate in recognition of conservative molecules of pathogenic microorganisms including viruses, were published. In this review several variants of effects of viruses (paramyxovirus, cytomegalovirus, smallpox virus, measles virus, and others) on TLR-mediated mechanisms of innate immunity are presented. Obtained data can be used for development of new antiviral drugs as well as vaccines.

Two widely known antivaccine inventions are discussed: «vaccination is accompanied by adverse effects, which exceeded complications of respective infections on frequency and severity» and «vaccines represent appalling conglomerate of toxic substances, which is unnaturally to administer to children». Informational and psychological nature of dissemination of these inventions is analyzed. On the basis of recent literature data conclusion was made about the absence of real toxicity (including neurotoxicity), carcinogenicity, allergenicity and autopathogenicity of phenol, folmaldehyde, aluminium hydroxide, Twin 80, squalen (MF59) and ethylmercury in concentrations found in vaccines of national immunization schedule.