Vol 92, No 3 (2015)

Aim. Determination of sensitivity of V. cholerae O1 serogroup El Tor biovar and O139 sero-group strains to antibiotics and determination of the presence of antibiotics resistance genes in their genome. Materials and methods. The studies were carried out in 75 V. cholerae О1 and О139 serogroup strains. Sensitivity of cultures to antibiotics was determined by disc-diffusion method. DNA isolation was carried out in the presence of 6M guanidine thiocyanate. PCR was carried out in multi-channel amplificator Tercyc. Results. A multiplex PCR was constructed, that includes 5 primer pairs for the detection of O1 and O139 serogroup resistance genes of vibrios to sulfame-thoxazolum, streptomycin B, trimethoprim, the presence of SXT element, an amplification program was developed. Using the developed PCR, V. cholerae O1 serogroup El Tor biovar strains with multiple drug resistance were established to be imported into Russia in 1993. The presence of SXT elements with genes of resistance to 4 antibiotics simultaneously was detected precisely in these strains, that belong to toxigenic genovariants of V.cholerae El Tor biovar. All the El Tor vibrio strains imported in the subsequent years were shown to stably preserve SXT element, this indicates its important role in biology of cholera vibrios. O139 serogroup strains with intact SXT element and having a deletion of the gene coding trimethoprim resistance were isolated. Conclusion. The data obtained may be used to establish molecular-genetic mechanisms of emergence of antibiotics resistant strains of cholera vibrio, construction of novel gene diagnostic test-systems and carrying out passportization of strains that are stored in the State collection of pathogenic bacteria.

Aim. Justification of the use of immune chromatographic analysis for diagnostic of pneumococcal pneumonia. Materials and methods. Sensitivity and specificity of an immune chromatographic method (Binax Now test system) was studied for verification ofpneumococcal pneumonia. Approbation of this method for etiologic deciphering of pneumonia in 260 patients hospitalized in infectious hospital was carried out. Results. A high sensitivity (84.8%) and specificity (90.5%) of the chromatography test was established. Streptococcus pneumoniae antigen in urine of patients with community acquired pneumonia was determined 3.6 times more frequently when immune chromatographic test was used compared with bacteriological study of nasopharyngeal swab and 1.8 times more frequently compared with sputum bacteriological study. Conclusion. Application ofimmune chromatographic express test Binax Now could be recommended for timely verification of pneumococcal pneumonia along with bacteriological methods of study.

Aim. Evaluation of an antiviral effect of miRNA in the nanoparticles of a polycationic compound against mRNA of vp16 protein (UL48 gene) of herpes simplex virus type 2 (HSV-2) in vitro. Materials and methods. 50% aqueous solution of polyethyleneimine (BDH, Great Britain), chitosan, containing approximately 15% of N-acetylated glucosamine chains (Sonat, Russia), hydrazine-hydrate and other chemical reagents (Chimmed, Russia); Vero continuous cell line, MS HSV-2 virus were used. Vero cells were cultivated in DMEM medium supplemented by 10% fetal bovine serum at 37°C in the atmosphere of 5% CO2. Cell viability was evaluated by using Neutral Red vital stain and MTT-test. Primers and probes for RT-PCR were modeled in Vector NTI 8.0 computer program according to the mRNA sequences of the studied genes (the sequences were obtained from GenBank) and synthesized in Sintol (Russia). RT-PCR tests were set using a standard procedure. Synthesis of PEI-PG-chitosan was carried out by Krivtsov G.G. et al. (2010). Results. A design and synthesis of nucleotide sequences, that have interfering activity against this virus, was carried out to study the effect of siRNA on HSV-2 virus replication. During simultaneous addition of HSV-2 and specific siRNA to Vero cells in cell culture, a significant (by 4 lg) reduction of virus yield was observed. A level of UL48 mRNA expression level was determined after the influence of various siRNA variants. A S2 siRNA variant was shown to cause the most pronounced virus-inhibiting effect, aiming for the center of RNA-target (the level of expression of the studied gene decreased by 0.5 lg). Conclusion. siRNA in the PEI-PG-chitosan complexes were established to possess in vitro pronounced suppressive HSV-2 replication activity. The results obtained could be used in creation of new therapeutic preparation against herpes viruses.

Aim. Study epitopic specificity of synthetic disaccharide, recurring link of serotype 3 S. pneumoniae, conjugated with bovine serum albumin (BSA). Materials and methods. Conjugate of the synthetic disaccharide with BSA was obtained by squarate method. Antigenic activity of the conjugate was studied in competitive EIA. Titers of IgG against capsule polysaccharide of serotype 3 S. pneumoniae were determined in EIA by using sera of mice immunized twice with disaccharide conjugate sorbed onto aluminum hydroxide. Results. Disaccharide conjugate used as a well-covering antigen (4 ^g/well) in EIA was characterized by a high degree of specificity and interacted only with IgG against serotype 3 S. pneumoniae in antimicrobial sera of animals without reacting with antibodies (ABs) against other pneumococcus serotypes (6B, 10A, 19A, 19F, 23F). Disaccharide conjugated with BSA was determined in competitive EIA to inhibit bonding ofABs to disaccharide by 78.8%, bacterial capsule polysaccharide by 56.9%, BSA did not inhibit the sera activity. The study of sera of mice immunized by serotype 3 S. pneumoniae disaccharide conjugate in EIA, where capsule polysaccharide was used as a plate-sorbed antigen, has established the presence of IgG against capsule polysaccharide at a titer of 1:1600. Conclusion. The disaccharide that is a single recurring link of serotype 3 S. pneumoniae contains a key epitope of capsule polysaccharide. The synthetic disaccharide could be used as a component of multivalent conjugated pneumococcal vaccines and for development of diagnostic test-systems.

Aim. Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). Materials and methods. 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tul4GF/R+tul4-PR2. Results. Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4GF/R+tul4-PR2 combination - 92% ofsera. The data were obtained when DNA was isolated from sera using «Proba Rapid» express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3 - 4 weeks after the onset of the disease. Conclusion. RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3 - 4 weeks after the onset of the disease.

Aim. Examine features of natural, natural-anthropourgic and anthropourgic foci of hemorrhagic fever with renal syndrome (HFRS) in various, mostly forest, biotopes of typical barrens due to differences of non-specific HFRS prophylaxis in foci of various types. Materials and methods. Epizootological and epidemiologic data from 1998 to 2012 were analyzed, gathered in HFRS foci of all types in Saratov area of Saratov Region (typical barrens). 14 606 trap-nights were worked off and 2669 small mammals were procured. The most significant population-ecologic and eco-logic-epizootological methods and criteria were used for comparative analysis of differences for 3 types of foci. Results. Based on analysis of multi-year data seasonal differences for HFRS foci of various types were shown by 10 population-ecologic and ecologic-epizootologic criteria. Conclusion. The results obtained allow to state that modern means and methods of non-specific prophylaxis of HFRS and other zoonoses in foci of various types different significantly. This allows the most rational use of material and financial resources.

Aim. Study the effectiveness of preventive vaccine prophylaxis of chicken pox in military collectives. Materials and methods. In the focus of chicken pox, 200 servicemen of the new addition by conscription were immunized once against chicken pox; 97 servicemen by conscription of the new addition (comparison group) were not vaccinated. Epidemiologic and immunologic effectiveness of conduction of preventive vaccine prophylaxis in chicken pox focus were studied. Results. In the group of 200 soldiers, that were present in the focus of infection and were immunized once against chicken pox, only 2 cases of this disease were registered (10%o). In the comparison group, that consisted of 97 unvaccinated servicemen, chicken pox disease was registered in 7 individuals (72%o). Epidemiologic effectiveness of preventive vaccine prophylaxis of chicken pox amounted to 86%. Immunologic effectiveness of vaccination 2 - 3 weeks after the immunization was 42%, and 2 months after - 44%. Local reactions in the form of hyperemia (up to 1.5 cm) and edema were noted in 10% of the vaccinated at the location of preparation administration; in 1.7% - general reaction in the form of temperature increase to 37.8°C was observed. Post-vaccinal complications in the immunized group were not detected. Conclusion. Preventive vaccination of servicemen allows to minimize the spread of chicken pox, however can not serve as means of complete elimination of the infection from military collectives.

Aim. Study of antimicrobial activity of a polymer compound - polyazolidinammonium, modified with hydrate-ions of iodine. Materials and methods. Antimicrobial activity of polyazo-lidinammonium, modified with hydrate-ions of iodine, against reference strains and clinical isolates of Gram positive and negative bacteria, microscopical fungi, as well as RNA viruses was studied. Results. High antibacterial activity of the studied compound was established, especially against Gram positive bacteria. A higher concentration of the preparation (125 - 250 pg/ml) was characterized by anti-fungal effect. A high sensitivity to polymer of swine transmissible gastroenteritis virus was noted. Conclusion. The polymer compound, based on the results of the studies, is a perspective antiseptic and etiotropic means for control of infectious disease causative agents.

Vaccination remains the most effective method of control of spread of a whole range of infections of both viral and bacterial nature. Many bacterial pathogens (Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae) carry polysaccharide capsule on the surface, that is one of the elements of protection from host organism immune system. At the same time, vaccination with bacteria exopolysaccharides (EPS) ensures infection neutralization. Effectiveness of such vaccine prophylaxis is limited by age of the vaccinated, intensity and duration of the immunity, development of secondary immune response. EPS conjugation with protein antigens was known for a long time to ensure activation of T-cell immunity against EPS and formation of secondary immune response. However, detailed studies of mechanism of immunity modulation by a protein partner as part of a glycoconjugate has not been carried out. T-lymphocyte activation was traditionally thought to occur exclusively due to peptide presentation, that are products of processing of protein component of the conjugate. Recently, information, accumulated in the field of natural carbohydrate, glycolipid and glycoprotein antigen presentation to T-cells, has generated interest in studying mechanisms of cell immunity activation by conjugated vaccines. Progress in this field, as well as development of novel chemical and biochemical, including combinative technologies of synthesis and study of these molecules, opens new opportunities for detailed understanding of mechanism of action for conjugated vaccines and creation of glycoconjugates with increased effectiveness of protective action.

An importance place in the system of prophylaxis measures against plague is allotted to vaccination of population contingents, that belong to risk groups for infection. The whole arsenal of accumulated knowledge on structure, properties, molecular nature, genetic determination, synthesis pathways, regulation and mechanisms of interaction with macroorganism of pathogenicity factors and immunogenicity of the infectious disease causative agent is used in the creation of new generation of vaccines. Contemporary technologies - genomics, proteomics, reverse vaccinol-ogy facilitate detection of protective antigens and help determine rational design of the vaccines. Main tendencies in development of recombinant live and chemical vaccines for specific prophylaxis of plague are presented in the review. Constructive approaches, that allow to produce highly effective and safe preparations are isolated.