Vol 88, No 5 (2011)

Aim. Comparative analysis of DNA content in individual cells of Vibrio cholerae strains with various biological properties. Materials and methods. 24-hour agar cultures of 2 avirulent (lacking cholera toxin gene) and 2 virulent strains and their subcultures obtained by cultivation in 1% peptone water for 1, 3 and 5 hours were studied. DNA of the killed bacteria was dyed by a mixture of ethidium bromide and mitramycin. Ratio of cells with low, intermediate and high relative DNA content in conditional units of specific DNA fluorescence intensity was determined by flow cytofluorimetry method. The degree of inhomogeneity of the studied microbial population cells was evaluated by DNA histogram variation coefficient value. Results. At the level of major statistical samples of individual V.cholerae cells a principally different reaction pattern of the studied toxigenic and non-toxigenic strains on changes of cultivation conditions was registered. Conclusion. Populations of cells of toxigenic V.cholerae strains in contrast to non-toxigenic probably shift to polyploid state during starvation. This phenomenon may turn out to be a differential feature in determination of the risk group (hazard) of a strain.

Aim. Genotype characteristic and determination of serological properties of Vibrio cholerae nonО1/nonО139 strains that caused diseases in population of Rostov region from 2000 to 2009. Materials and methods. 15 clinical strains of V.cholerae nonO1/nonO139 were studied. Serotyping was performed by using a kit of monospecific typing sera against serogroup O2-O84 cholera vibrios obtained from Rostov Research Institute for Plague Control, PCR and VNTR-genotyping - by using specific primers described in scientific publications and constructed by us. Results. Serologic features of strains are very diverse and strains contain various combination of pathogenicity factor genes that seem to be interchangeable. Similar pattern was observed for VNTR-genotyping. Distribution of the examined strains by VNTR-genotyping did not correlate with either PCR-genotyping data or serotyping, or place and time of isolation. Conclusion. The data obtained indicates a lack of general source of human infection even in the same location and time period. On the other hand, serological and genotypic features of V.cholerae nonO1/nonO139 may undergo changes in the process of staying in the macro organism or environment due to high plasticity of their genome.

Aim. Study of possibility of existence of combined natural foci of spirochetoses (ixodes tick borrelioses and leptospiroses) in typical taiga forests, and their etiologic and reservoir-host structure. Materials and methods. Small mammals of 19 species were captured in 1992-2010 at a station in low-mountain southern taiga forests of Chusov area of Perm region. Borreliae were isolated by seeding urinary bladder or aural bioptates into BSK II medium, leptospirae - by seeding a suspension of kidney tissue into Vervoort-Wolf medium. 1350 animals were studied by seeding for borrelia infection and 1077 - for leptospira. 287 of those, small animals of 6 species, were simultaneously studied for borrelia and leptospira infection. Borrelia isolates were identified by using PCR and restriction fragment length polymorphism methods, and leptospirae - by using standard diagnostic agglutinating sera kit. Blood of 2893 rodents of 12 species and insectivorous of 7 species was studied in microagglutination reaction for the detection of antibodies against leptospirae. Results. Infection by Borrelia garinii and Borrelia afzelii or Grippotyphosa serogroup leptospira was detected in 6 most numerous species of forest small mammals. 3 root voles and 1 bank vole were simultaneously infected by borreliae and leptospirae. B.garinii and Grippotyphosa serogroup leptospira were simultaneously isolated from 2 root voles, and B.garinii and Javanica serogroup Leptospira interrogans - from 1 root vole. A bank vole was infected by B.afzelii and Javanica serogroup leptospira. Mixed-infected animals composed 1.4% of all animals of background species studied in parallel. Conclusion. The data obtained indicate a presence of natural foci of leptospiroses in the southern taiga forest pre-Urals. The data confirm the conceptions regarding a predominant presence in European forest ecosystems of foci with Grippotyphosa serogroup L.interrogans pathogen, and the main carrier of these leptospirae being bank vole. Combined natural foci of spirochetoses of two groups (ixodes tick borrelioses and leptospiroses) were detected.

Aim. Serological examination for leptospirosis of domestic and certain species of wild animals in Mongolia. Materials and methods. Collection of material from domestic and wild animals was performed in 2009-2010 in 7 aimags (regions) of Eastern, Central and Southern Mongolia. Serological study of filter paper dried blood samples obtained from 51 specimens of cattle and small cattle, camels, and 545 specimens of rodents of various species was performed in microagglutination reaction (MAR) of leptospirae with 13 reference strains. Results. There is a presence in certain regions of Mongolia of anthropurgic loci of leptospirosis infection including arid zones where ecological conditions do not favor the development of epizootic process. The results of the study indicate the epizootic significance of Tarassovi serogroup leptospirae in cattle and Sejroe serogroup (probably hardjo serovar) in goats, sheep and camels. Results of serological studies of desert and steppe specimens of wild fauna of Mongolia suggest a possibility of circulation of leptospirae in natural foci. Conclusion. Detection in a significant percent of cases in tarbagan and long tailed ground squirrel blood sera of agglutinins to Pomona (mozdok) leptospirae with negative MAR results for Pomonа (рomona) strain suggests a presence of a pathogen of a previously unknown serovar. However final conclusion could be made only after the isolation of cultures of the pathogen and their identification.

Aim. Comparative immunogenicity studies of experimental vaccines based on A/Aichi/2/68 neuraminidase peptide fragments (NA) and influenza virus A and B strains produced in MDCK cell culture. Materials and methods. Anti-hemagglutinin and virus neutralizing activity of mice sera was determined in MN and HI reactions in accorda- nce with the WHO recommendations. Results. Sera against peptides 136-147 and 154-164 from variable sites, as well as against peptide 314-328 from conservative region of the heavy chain of A/ Aichi/2/68 influenza virus NA showed distinct anti-hemagglutinin and neutralizing activity against homologous influenza virus. Anti-(314-328) serum was also active in HI and MN reactions against other strains of the H3N2 subtype. Combined administration of peptide sample with an im- munomodulator (Immunomax) increased the immunogenicity to the level of the cultural samples based on influenza A virus. Conclusion. The results show higher immunogenicity of cultural vaccines based on influenza virus in comparison to peptide samples. A possibility of peptide vaccine immunogenicity increase was demonstrated by combined administration with the immunomodulator.

Aim. Study of macrophage migration inhibiting factor (MIF) effect after intracerebral administration on the course of experimental infection induced in mice by tick borne encephalitis virus (TEV), and study of sodium polyprenyl phosphate (PPP) and/or antibodies against MIF on the course of this infection against the background of MIF administration. Materials and methods. Phosprenil preparation was used as a source of PPP. PPP was administered intracerebrally. MIF - human recombinant (R&D, USA), mice - Balb/c line. Results. In the sera of mice infected with TEV, MIF production stimulation was detected at days 8 through 10 after the infection - against the background of clinical signs presentation of tick borne encephalitis (TE). Administration of PPP to infected mice, on the contrary, resulted in MIF production suppression at the specified period. After administration of 20 ng of MIF to mice, lethality increased by 40% and average life span decreased by 2.3 days. Thus, MIF at high doses caused an increase of infection course severity, in- duced by TEV in mice, and administration of 60 μg of PPP resulted in the protection from infection in 100% of cases. Intracerebral administration to mice of antibodies against MIF resulted in a decrease of lethality indicator up to 26% as compared with control and an increase of average life span by 5.5 days. During simultaneous administration into the brain of infected mice of MIF, PPP and antibodies against MIF, prevention of MIF-induced increase of TE course severity was registered. Conclusion. The data obtained allow to conclude that MIF may serve as an indicator of TE course severity, and pos- sible prognostic indicator of meningo-encephalitic form development in humans.

Aim. Development of primers for the detection of leptospirae in clinical material including urine. Materials and methods. Study of specificity and sensitivity of primers complementary to colA gene in standard PCR by using DNA preparation of cultures of pathogenic and saprophytic leptospirae, biological materials from healthy humans and dogs, including contaminated with pathogenic leptospirae culture. Results. Specific interaction of these primers with DNA of pathogenic leptospirae of 14 serogroups was established. Sensitivity of the technique was 50 cells in 1 ml of sample. Conclusion. The primers described fulfill the requirements for the sensitivity and specificity and can be recommended for the detection of leptospirae in both serum and urine.

Aim. Determination of antibacterial activity of acid-soluble chitosan in bacterial vaginosis therapy in women of reproductive age and comparison of therapy results of traditional scheme and local administration of acid-soluble chitosan. Materials and methods. Vaginal microflora of 76 gynecologic and obstetrical female patients (18 - 43 years of age) with bacterial vaginosis diagnosis was examined by using bacteriological method. Control group consisted of 30 practically healthy women. Sensitivity of 145 bacteria strains from 14 genera to 2% acid-soluble chitosan solution (200 - 250 kDa molecular weight) was determined by using agar diffusion method. Results. During bacterial vaginosis members of the Gardnerella, Mobiluncus and Bacteroides genera dominated with a background reduction of lactobacilli quantity. An increase of population level of Staphylococcus, Enterococcus, Prevotella, Porphyromonas, Peptostreptococcus genera members, and Candida species was also observed. All the microorganisms were sensitive to 2% chitosan solution. Mycoplasma and Ureaplasma spp. were sensitive to chitosan in 82% of cases. Administration of chitosan in local therapy of vaginosis resulted in suppression of opportunistic microflora and clinical effect with a lack of adverse effects. Conclusion. Local administration of chitosan is perspective against bacterial vaginosis.

Aim. Establishment of presence of periodontopathogenic microorganisms in atherosclerosis plaque and surrounding tissues, and possible relation of development of atherosclerosis and TLR4 gene Asp299Gly polymorphism in ischemic heart disease patients (IHD). Materials and methods. Samples of coronary vessels obtained during autopsy of 31 individuals deceased from IHD and 5 individuals deceased due to reasons not related with IHD were studied. PCR was used to determine DNA of the microorganisms. TLR4 gene polymorphic segment was amplified by using specific primers. Results. Analysis of coronary vessel atherosclerotic plaques revealed presence of the studied periodontopathogenic microorganisms in 83.9% of cases. The most frequently detected were Porphyromonas gingivalis (64.5%), Treponema denticola (41.9%), Actinobacillus actinomycetemcomitans (32.3%), less frequently - Bacteroides forsythus and Prevotella intermedia (12.9% and 6.5% respectively). In 51.6% of cases 2 or more microorganisms were detected. Only in 11.1% of coronary artery samples, with plaques containing microorganisms, the microorganisms were detected in undamaged tissues. Patients deceased from IHD had TLR4 gene 299Gly allele significantly more frequently. Conclusion. The studied periodontopathogenic microorganisms can play an important role in the pathogenesis of atherosclerotic injury of coronary arteries in IHD. The presence of TLR4 gene allele 299Gly significantly contributes to these processes.

Scientific data and data obtained by us regarding new innate immunity receptor agonists from sea hydrobionts are presented. Highly efficient and low toxic biologically active substances isolated from sea organisms are inductors of dendritic cells maturation, activate neutrophils, monocytes/macrophages and NK-cells, have an anticomplementary (fucoidans) and pro- and antiinflammatory effects. Protective properties of biologically active substances are exhibited when various pathogens entering the organism - bacteria, viruses, protozoa, fungi. These substances may become a basis for the creation of new pharmaceutical preparations.

Evolution of key terms used in epidemiology of nosocomial infections is discussed. A modern point of view on terminology and comparison of Russian terms with foreign analogues are presented.