Vol 86, No 2 (2009)

Aim. To study ultrastructural changes of hemolytic and enterohemorrhagic Escherichia coli during interaction with metabolites of Lactobacillus fermentum . Materials and methods. Strains of pathogenic hemolytic (Hly) and enterohemorrhagic Escherichia coli (O157:H7) as well as symbiotic bacteriocinogenic strain Lactobacillus fermentum 97 were used. Inhibition of growth of viable cells was performed by delayed antagonism method. Using electron microscopy, assessment of ultrastructural changes of hemolytic and enterohemorrhagic E.coli under the influence of diffusing in MPC-agar metabolites of lactobacilli. Results. Changes pointing to profound destructive processes in bacterial cells were detected on ultrathin sections. Under the influence of diffusing metabolites of lactobacilli, the following changes were observed: destabilization of cell wall, expansion of periplasmatic space, and emergence of low electron density areas of cytoplasm in polar sections of cells with visualization of floccular material. Emergence of elongated paracrystallic packings and filamentous structures of different length, which deserve special study, was observed in cells of hemolytic E.coli . Conclusion. Bacteriocin-like products of lactobacilli during interaction with pathogenic E.coli cause profound destructive changes in the latter which lead to destruction of target cells.

Aim. To study the ability of polyoxydonium (PO) to influence on adhesive properties of C.diphtheriae . Materials and methods. Ability of PO to influence on adhesive properties of toxigenic and nontoxigenic strains of C.diphtheriae isolated from patients, carriers, contact persons as well as from healthy persons during prophylactic examinations of population in Rostov-on-Don and Rostov region was studied. Results. It was established that addition of PO in cultivation medium always suppressed adhesive activity of C.diphtheriae . In experiments on breaking of adhesion the blocking effect of PO on adhesive characteristics of bacteria had dose-dependent pattern and was pronounced when PO was used in concentration of 1200 mcg/ml. Conclusion. Obtained data prove that PO has marked antiadhesive activity against both toxigenic and nontoxigenic strains of C.diphtheriae , which could be used for the treatment of diphtheria infection.

Aim. Molecular analysis of Streptococcus pyogenes types emm1 and emm12 genomes on the presence of genes belonging to prophage DNA. Materials and methods. Thirty-one strains of S. pyogenes type emm1 and 22 strains of S. pyogenes type emm12 were objects of the study. Polymerase chain reaction with special primers for detection of 24 phage genes was used in the study. Results. Prevalence of phage genes in strains of different emm types was assessed. The following phage genes were most frequently detected: speA, speC, ssa genes for emm1 type and speC and speH for emm12 type. On the basis of the study, 19 different profiles of phage genes were identified. Conclusion. The study allowed to identify not only differences but also similarities between strains of emm1 and emm12 types on prophage gene repertoire in bacterial genome. Presence of similarities allows to assume the possibility of circulation in the studied region similar or identical bacteriophages between streptococci group A (SGA) strains of different emm types.

Aim. To study features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modelling antigenic determinants of hepatitis A virus (HAV) and to evaluate perspectives for use of heterogeneous tetrameric multipeptide antigens for detection of HAV serological markers. Materials and methods. Linear peptides VP1 and VP3 were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid phase method. MAP 4(VP1+VP3) was synthesized according to 9-Fmoc strategy. Interaction of these peptides with anti-HAV IgM positive sera from patients with HA was studied by noncompetitive and competitive methods of immunoenzyme assay. Results. Using immunoenzyme assay, high heterogeneity of immune response in patients with HA (62 and 67% in two groups) was shown. MAP 4 (VP1+VP3), unlike the combination of linear peptides VP1 and VP3, interacted with anti-HAV IgM in 41 — 45% of sera and, at the same time, did not lead to false positive results. Conclusion. Population of HAV is not so uniform which is usually assumed. It could be reasonable to use heterogenous multipeptide antigens, including those containing VP1 (11 — 25 a.r.) and VP3 (110 — 121 a.r.), for the development of new assays for HA diagnostics.

Aim. To study chitozan as an adjuvant for inactivated vaccines against A/H5 influenza viruses. Materials and methods. Avian A/H5 influenza viruses were grown on chicken embryos or on MDCK cell line; viruses-containing fluid was inactivated with formalin. Mice were vaccinated intramuscularly with inactivated avian influenza virus mixed with chitozan and then levels of hemagglutination-inhibiting and neutralizing antibodies as well as protective efficacy against both homologous and drifted strains of avian influenza viruses A/H5 were measured. Results. Addition of chitozan to inactivated preparations of A/H5 avian influenza viruses for immunization of mice significantly increased levels of hemagglutination-inhibiting and neutralizing antibodies to both homologous and drifted variants of A/H5 influenza viruses, including those containing neuraminidase from other subtype as well as strains isolated 10 — 20 years earlier than virus used for vaccination. Chitozan significantly improved protective efficacy of inactivated avian influenza vaccines against infection with both homologous and drifted variant of the virus. Vaccination with inactivated avian influenza viruses A/H5 and chitozan induced high levels of antibodies even after single immunization as well as after administration of 8-fold reduced dose of preparation. Conclusion. Chitozan is a perspective adjuvant for inactivated vaccines against avian influenza viruses, which could significantly improve immune response and protective efficacy against both homologous and drifted variants of avian influenza viruses A/H5.

Aim. To study the influence of vaccination against pneumococcal and influenza infections on the levels of autoantibodies to DNA, pancreatic and adrenal tissues as well as on populations of lymphocytes and levels of immunoglobulins in children and adolescents with type I diabetes mellitus (DM1). Materials and methods. One hundred and thirty children and adolescents 2 — 18 years of age were followed during 12 months. One hundred participants were vaccinated against pneumococcal infection with Pneumo 23 vaccine. Twenty-eight of them (28%) were also vaccinated against influenza with subunit vaccine Grippol. Vaccination was performed during intensified insulin treatment. Unvaccinated group consisted from 30 children of the same age. During vaccination 59 (59%) children were in phase of diabetes compensation and 41 (41%) — in phase of subcompensation. Results. In 5 out of 10 participants with high level of antibodies (Abs) to native DNA (n-DNA) before vaccination with Pneumo 23, normalization of this level 1 year after vaccination was noted. Normalization of level of Abs to denaturated DNA (d-DNA) was observed in 2 out of 7 patients with high levels to d-DNA before vaccination. Normalization of level of Abs to n-DNA was observed in 2 out of 3 patients after combined vaccination. It is important to note that vaccination of children in subcompensation phase of DM1 did not result in changes of levels of autoantibodies. One year after vaccination decrease of IgG, IgA, and IgM levels to normal range was noted. Absence of increase of HLA-DR and CD16 levels in postimmunization period in children with DM1 vaccinated even in subcompensation phase is also an indirect evidence of absence of autoimmune process activation. Conclusion. Use of Pneumo 23 vaccine or its combination with Grippol vaccine in patients with DM1 did not result in increase of levels of autoantibodies to n-DNA, d-DNA and pancreatic tissue, was not able to initiate or lead to disease progression as well as positively influenced on the immune response with tendency to normalization of the several arms of the immune system and, at the same time, did not result in activation of autoimmune process.

Aim. To study changes in expression of TLR9, NFκB, TNFα genes in cervical canal mucosa in pregnant women with herpesvirus infection. Materials and methods. Using previously developed real-time polymerase chain reaction assays, levels of expression of genes, which corresponding proteins play important role in TLR9-dependent signaling pathway, was quantified. Results. Two-fold, 4.4-fold, and 3-fold rise in expression of TLR9, NF-κB, and TNFα genes respectively was revealed in pregnant women with herpesvirus infection compared to women with physiologically occurring pregnancy. Conclusion. Obtained results demonstrated the activation of TLR-mediated mechanisms of innate immunity on the level of cervical canal mucosa during pregnancy complicated by viral infection.

Aim. To study the role of neutralizing antibodies (NA) on the model of SHIV-infection in pig-tailed macaques immunized by different methods. Materials and methods. Titers of NA were determined using TZM-bl cells; titers corresponding to 50% neutralization were determined from serum dilution — relative luminescence graph. We have proposed to use index of integral neutralizing activity, which mathematically expressed as integral of neutralization function in serum dilutions interval from 1 (or first dilution used) to that giving selected titer (we selected 50% neutralization). Results. On the basis of analysis of data on course of SHIV-infection in previously immunized macaques it was concluded that more clear correlation exists between results of infection and integral neutralizing activity of serum compared with NA titers corresponding to 50% neutralization. Conclusion. Proposed method could be useful for analysis of immunization efficacy against HIV infection.

Aim. To assess efficacy of using the method of quantitative detection of Legionella in objects of the environment by real-time polymerase chain reaction (RT-PCR). Materials and methods. For the development of the assay, genus-specific primers from gene coding 16S rRNA as well as species-specific primers for detection of Legionella pneumophila on the basis of mip gene sequence. For quantitative detection of L . pneumophila calibration samples of pGEM plasmid containing fragment of the mip gene in known concentration were used. Samples of water and biofilms obtained from cooling stacks of production plants, systems of autonomic water supply, humidification blocks of centralized systems of air conditioning were studied. Results. Correlation of results obtained with RT-PCR and bacteriologic methods was shown during monitoring of potentially dangerous water objects as well as during epidemic outbreak of Legionella infection. Importance of samples preparation stage, during which considerable losses of DNA and inhibition of reaction could occur, is underlined. Disinfection measures on the studied objects significantly influenced on the results of the RT-PCR and can lead to false positive results. Conclusion. Obtained results confirm usefulness of testing of potentially dangerous water objects on the presence of Legionella based on the preliminary screening with RT-PCR for the 24 hours followed by bacteriologic testing of samples for 8 — 12 days.

Aim. To choose lactobacilli-contained probiotic for complex treatment of acute enteric infection caused by Klebsiella in infants. Materials and methods. On the basis of bacteriological analysis the group consisting of 40 infants with acute enteric infection caused by Klebsiella was formed. Efficacy of three probiotic preparations — lactobacterin, vitaflor, and biobacton — was assessed depending on biological features of causative agents and contents of lactobacilli and bifidobacteria in obligate gut microflora. Intraspecies antagonistic characteristics of manufacturing strains of lactobacilli against 9 clinical isolates of K.pneumoniae and 8 strains of indigenous lactoflora, as well as interspecies antagonism between Klebsiella and probiotic and indigenous strains of lactoflora were studied. Results. It was shown that complex therapy of Klebsiella infection in infants using vitaflor or biobacton promoted elimination of K.pneumoniae and restoration of indigenous microflora which became apparent in significant increase of titer of lactobacilli. In vitro maximal antagonism to K.pneumoniae was noted for vitaflor, lactobacterin and heteroenzyme autostrains of ill children’s lactoflora. Contrantagonism to lactoflora was typical for slowly growing strains of Klebsiella . Conclusion. It is therapeutically rational to use vitaflor, which promotes rapid elimination of infectious agent, in initial phase of acute infection caused by Klebsiella , and biobacton, which increases the titers of indigenous lactoflora, in phase of convalescence.

Aim. To study the effects of serotonin and dophamine on the growth of Y ersinia pestis and Francisella tularensis strains as well as ability of monoamines to change susceptibility of experimental animals to plague infection. Materials and methods. Effect of various doses of biogenic amines on the growth of Y.pestis and F.tularensis was studied by biophotometer «BIOLOG II» (F ISABIO, France). When studying the effect of amines on LD 50 value and mean survival time, serotonin and dophamine were administered to mice peritoneally in dose 25 mg/kg and 100 mg/kg respectively before their inoculation with Y.pestis suspension. Results. It was shown that one-time addition of serotonin (2.5 — 40.0 mcM) to medium for cultivation of Y.pestis and F.ularensis strains did not significantly affect the bacterial growth both at cultivation temperature 28°C and at 37°C. At the same experimental conditions dophamine stimulated growth of bacterial cultures accelerating the onset of exponential phase of culture growth. Administration of serotonin for 1 hour before inoculation of mice with Y.pestis EV-76 strain increased LD 50 value and decreased mean survival time; in contrast, administration of dophamine decreased LD 5 0 value and increased mean survival time. Conclusion. Data on stimulating effect of dophamine on agents of transmissible infections allow to propose that physiological state of an organism as well as medical administration of catecholamines could influence on susceptibility of the host to infection and determine the septic course of the disease.

Aim. To reveal factors related with presence of human papillomavirus in women. Materials and methods. Nine hundred and fifty women of childbearing age from 18 to 45 years old (30 of them — almost healthy) were examined. Samples of epithelium of urogenital tract obtained by scrapings were tested for the presence of urogenital infections by real-time polymerase chain reaction. Colposcopy, cytologic study, and ultrasound examination of pelvic organs were conducted. Results. Human papillomaviruses types 16 and 18 were detected in 41% of studied women. Of them, persisting form of papillomavirus infection was identified in 38%. Following factors promoted the development of persisting papillomavirus infection in women: age under 21 years, presence of pelvic inflammatory disease (endometritis, salpingoophoritis), cervical ectopy, use of untrauterine devices, oncologic diseases in family history, and presence of pointed condylomas. Signs specific for chronic inflammatory disease of cervix were observed significantly more frequently in women with persisting papillomavirus infection. Conclusion. Association between presence of chronic inflammatory diseases of female reproductive system, related with different adverse factors, and persistence of human papillomavirus of high oncogenic risk in women.

Innate immunity is the first line of host defense against infection including those caused by herpesviruses. Toll-like receptors (TLRs) are the main pathogen-recognizing receptors. TLR2 binds to glycoproteins of herpesvirus virion; intracellular TLR9 recognizes nonmethylated CpG repeats of herpesvirus' DNA, which lead to activation of receptor signaling pathways and synthesis of proinflammatory cytokines, chemokines, antimicrobial peptides and other molecules, which participate in induction of innate immunity mechanisms. Many viruses (measles virus, cytomegalovirus etc.) activate expression of TLRs for more effective replication of the virus in cells.

Literature data about mechanisms of interaction between viruses and bacteria in circumstances of associative symbiosis are summarized. Interactions of influenza viruses, adenoviruses, Coxsackie viruses with different taxons of pathogenic and opportunistic bacteria are showed. Influence of these interactions on pathogenicity factors of viruses and bacteria is multidirectional (induction, suppression) which could determine the course of infectious process.