Vol 88, No 4 (2011)

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°C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37°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°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.

Aim. Study of wound microbial landscape in suppurative necrotic complications in patients with diabetic foot syndrome (DFS), and a possibility of suppression of bacterial biofilm development in these wounds by ultrasound cavitation (USC). Materials and methods. 2 randomized groups of patients were included in the study: main group of 145 individuals with suppurative necrotic complications of DFS, who received ultrasound cavitation therapy, and comparison group of 86 patients who received basic therapy. In the main group Sonoca-180 (Söring, Germany) unit, that allows to combine the process of mechanical wound treatment with antibacterial action of low frequency ultrasound, was used for wound treatment. Quantitative and qualitative composition of wound microflora was studied by using bacteriological method with parallel utilization of transmission and scanning electron microscopy in the dynamic of USC treatment. Results. In patients with DFS bacterial biofilms containing congestions in the polysaccharide matrix and fixed at the surface of dense structure of periostenum wound were detected. In the microbial landscape in DFS patients in 21% of cases aerobic-anaerobic microflora was detected, while associations included mostly from 2 to 5 bacteria species — members of the Peptococcus, Peptostreptococcus, Prevоtella, Bacteroides genera. Bacteria of Staphylococcus, Acinetobacter, Pseudomonas, Escherichia and Klebsiella sp. genera were detected less frequently. Application of USC in the main group resulted in a decrease of microbial contamination of wounds in the short term. Conclusion. USC method, when compared with classical therapy approach in DFS complications, in patients with suppurative necrotic complications allows not only to remove mechanically the necrotic tissues but also to effectively suppress the bacterial biofilms that have formed in the wounds, this promotes the acceleration of recovery process and preparation of the patients for further plastic surgery intervention.

Aim. Studies of cultural, virologic, antigenic properties of 89 samples of pandemic influenza A(H1N1) 2009 virus isolated in Russian Federation from May 2009 to March 2010. Materials and methods. Properties of isolated samples were compared with those of the reference strain A/ California/04/2009 (H1N1). Results. Studies of biological properties and analysis of genome nucleotide sequences of the isolated samples showed that those strains are closely related to the reference strain. Conclusion. Monitoring of genetic, virologic and antigenic properties of pandemic influenza A(H1N1) 2009 virus isolates carried out from May 2009 to March 2010 did not reveal significant changes in the abovementioned properties of the virus or emergence of mutations that can lead to such changes.

Aim . Mechanism of virus inhibiting action against measles virus of polyelectrolytes (PE) polystyrolsulfonate (PSS) of various polymerization degree and 60 kDa molecular weight polyallylamine (PAA) was studied. Materials and methods . Measles virus Leningrad-16 strain was used for the study. Virus infectious titer reduction kinetics after interaction with PSS with the degree of polymerization of 8 (PSS 8), 31, 77, 170, 360, 430 and PAA were determined by titration method with cytopathogenic effect detection in Vero continuous cell line. Circular dichroism and fluorescence spectra of viral proteins were obtained by using Zenith 200st spectrophotometer (Russian Federation) and Jasco J-810 dichrograph (Japan). Results . A significant decrease of measles virus infectious titers after interaction with PSS with the degree of polymerization of 8 and PAA in concentration of 30 mM was detected. Analysis of circular dichroism spectra and protein fluorescence allowed to determine the mechanism of interaction of the indicated PE with measles virus surface proteins. The secondary structure of viral proteins is damaged by hydrophobic polar frame of these PE, polyanion PSS 8 also interacts with positive charges of protein groups that leads to the formation of loops and tails that disrupt α-spirals. Conclusion . The studied PE could be considered as potential antiviral preparations, and methods of circular dichroism and protein fluorescence could be used to detect damage of viral protein secondary structure by agents of different kinds.

Aim. Evaluation of alterations of immune response regulation and possible risk of antenatal development of fetus in postvaccination period in pregnant women immunized against influenza A (H1N1). Materials and methods. Women were vaccinated with MonoGrippol plus vaccine in the II trimester of physiological pregnancy. At certain intervals of the vaccination period (before the vac- cination, 7 and 30 days after the vaccination) major biochemical markers in blood sera (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, creatinine, urea) and levels of key cytokines in spontaneous and stimulated test (IL-1α, IL-1RA, IL-2, IL-4, IL-10, IFNγ, TNFα) were evaluated. Vaccination safety for the fetus and trophoblast development was evaluated by using human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP) and trophoblasitc beta-1-glycoprotein (TBG) levels. Results. During vaccination in 13% of cases mild local reactions were noted, in 26.1% — general systemic reactions in the form of weakness, dizziness and headaches. Levels of major biochemical markers at days 7 and 30 after the vaccination did not have any significant difference from the initial values (p>0.05). Cytokine levels in spontaneous and stimulated tests also did not change significantly. Markers of the course of pregnancy and fetus development (HCG, AFP and TBG) in the two groups observed had comparable values. Conclusion. Vaccination of pregnant women against influenza A (H1N1) by Russian subunit formulation (MonoGrippol plus) showed reactogenicity comparable to control group by the level of influence on general metabolic and immunologic homeostasis and on the course of pregnancy, which is an evidence of its safety.

Aim. Development of a method of determination of anti-cytokine activity (ACA) of microorganisms, study of the prevalence and intensity of ACA to pro- and anti-inflammatory cytokines in pathogenic and opportunistic bacteria. Materials and methods. ACA was determined in 72 strains of microorganisms including members of the intestinal microflora and strains of pathogenic bacteria (salmonellae and gonococci). Study of the ability of supernatants and live cell cultures of microorgan- isms to induce changes in pro- (IFN-γ, TNF-α, IL-6) and anti-inflammatory cytokines (IL-4, IL-10) was performed by using co-incubation of exometabolites and live cell bacteria, fungi with recombinant cytokines. Results. A methodological approach allowing the determination of ACA, the prevalence of which among studied microorganisms was 50-62%, was developed. A decrease of cytokine concentration in the medium was registered in co-incubation of them with supernatants (in 56% of cases) and to a lesser degree - with live cell cultures (44%) of the studied bacteria and fungi. Expression of anti-cytokine activity was the most pronounced to TNF-α (Bifidobacterium spp. and Lactobacillus spp., Neisseria gonorrhoeae), IFN-γ (N.gonorrhoeae and Salmonella enterica) and IL-10 (Bifidobacterium spp. and Lactobacillus spp.). Conclusion. The data obtained expand the conception of modification of cytokine dynamic by pathogenic and opportunistic microorganisms, that can influence the course and outcome of an infectious process.

Aim. Study of the ability of Western Siberian Borrelia garinii 20047 isolate recombinant pro- tein FlaA to react with sera antibodies of ixodes tick borreliosis patients. Materials and methods. Recombinant antigen FlaA, sera of ixodes tick borreliosis patients, genetic engineering methods, solid phase EIA, and parametric and nonpara- metric statistical methods were used in the study. Results. Recombinant form of mature flagellar protein FlaA of B.garinii 20047 was obtained. In EIA study of sera of ixodes tick borreliosis patients with migrating erythema and without it, IgM or IgG against FlaA antigen were detected in more than 30% of sera. Indicator of the detection of IgM against FlaA antigen in sera of ixodes tick borrelio- sis patients with migrating erythema and without it was 43.3% and 33.3% respectively. Conclusion. The results obtained show a significant antigenic activity of recombinant protein FlaA of Western Siberian B.garinii isolate and the perspectives of its use for serodiagnostics of ixodes tick borreliosis.

Aim. Evaluation of postvaccination immunity against influenza in patients with bronchopulmonary pathology. Materials and methods. 22 patients with broncho-pulmonary pathology vaccinated against influenza with Grippol plus preparation participated in the study. Rates of influenza and acute respiratory illnesses (ARI), rates and duration of exacerbations of the underlying disease were tracked based on medical documents. Hemagglutination inhibition reaction in paired sera before and 6 and 12 months after the vaccination was used to evaluate vaccine immunogenicity. Results. None of the patients had influenza diagnosis for one year. A reduction of ARI rate of 37% was detected. Rate of exacerbation of the underlying disease decreased by 1.9 times, duration of exacerbations - by 2 times. Data on immunogenicity against A/H1N1/ Brisbane/59/07, A/H3N2/Uruguay/716/2007, B/ Florida/4/2006 are presented. Immunogenicity against B/Brisbane/60/2008 strain not contained in the vaccine was insufficient to induce adequate protection. Statistically significant differences were not detected during comparison of immunogenicity in patients with broncho-pulmonary pathology and healthy people. Conclusion. Grippol plus vaccine in patients with respiratory tract diseases is immunogenic and renders clinical effect by reducing influenza morbidity and rate of exacerbation of the underlying disease.

Aim. Studies of Helicobater pylori infection rate among population of North-western federal district including St.Petersburg in 2007 - 2009. Materials and methods. 1021 individuals with various digestive tract pathologies as well as healthy individuals and blood donors were examined. 378 children and adolescents up to 19 years of age, and 643 adults 20 - 80 years of age were among the examined individuals. IgG to H.pylori complex antigen and IgG to its CagA toxin were determined in blood sera. Results. Infection rate among children and adolescents was 40.48%, CagA positive strain infection rate - 34.92%. Among adults these parameters were 63.61% and 49.61%, respectively. During the 3 year study a tendency of CagA positive strain rate increase was noted among children and adolescents. Prevalence of H.pylori infection in subjectively healthy individuals, intestine oncology disease and gastric cancer patients was 55.1%, 67.16% and 83.33%, respectively. Conclusion. High rate of infection and CagA positive strain prevalence among children and adolescents can be considered as a possible factor of gastritis and ulcer rate increase in these age groups.

Aim. Evaluate reactogenicity, safety and immunogenicity in phase 2 clinical trials of 2 immunization schedules with Ultragrivac - an allantoic intranasal life influenza vaccine based on A/17/ duck/Potsdam/86/92 [17/H5] reassortant strain. Materials and methods. 4 groups of volunteers participated in the study: group 1 - 40 individuals were vaccinated twice with a 10 day interval; group 2 - 40 individuals were vaccinated twice with a 21 day interval; group 3 (control) - 10 individuals received placebo twice with a 10 day interval; group 4 (control) - 10 individuals received placebo twice with a 21 day interval. Local (secretory IgA), cellular and humoral immune response were evaluated. Humoral immunity was evaluated by the intensity of increase of geometric mean antibody titers against 2 influenza virus strains A/17/ duck/Potsdam/86/92 [17/H5] and А/chicken/ Suzdalka/Nov-11/2005 (H5N1), and by the level of significant (4 times or more) antibody seroconversions after the vaccination. Results. After the use of Ultragrivac the level of secretory IgA in the nasal cavity of vaccinated volunteers in the groups with revaccination intervals of 10 and 21 days increased significantly. The second immunization with 10 or 21 day intervals significantly increased postvaccinal humoral immune response. Humoral immune response induction after 2 vaccinations with 10 day interval was no less effective than with 21 day interval. Conclusion. Ultragrivac allantoic intranasal live influenza vaccine is areactogenic, harmless for vaccinated individuals, safe for those around, and has immunogenic properties against not only homologous virus A(H5N2), but also against influenza strain A(H5N1).

Prophylaxis of infectious diseases transferred by ticks is an important problem of contemporary medicine. One of the perspective approaches to solve this problem is the creation of vaccines against tick bite (anti-tick vaccines). Contemporary methods of the control of infectious diseases transferred by ticks are described in the review. Features of naturally and artificially acquired immunity against ticks are examined. Candidate tick antigens for the construction of vaccines against genus Ixodes tick bite are described. Perspectives of use of anti-tick vaccines against tick vector borne diseases are evaluated.

Scientific data is presented and problems of influenza prophylaxis in various age groups are discussed. Influenza prophylaxis in neonates is possible by inducing maternal antibodies, this dictates the necessity of influenza vaccination in pregnancy. Problems of influenza prophylaxis are most pressing in the group of children from 6 months to 2 years of age. More effective vaccines that do not cause adverse reactions are necessary for the children of this age group. Influenza prophylaxis in healthy working adults is most important for reducing economical impact during influenza epidemics. Influenza prophylaxis in the elderly is reasonable by using novel and more effective vaccines with adjuvants. The optimal method for influenza prophylaxis in the population in general is mass vaccination of children (80%), when, besides the induction of protection in children, influenza morbidity may decrease up to 80% in the other age groups of unvaccinated population.