Vol 86, No 3 (2009)

Aim. To study main biologic characteristics of C.diphtheriae strains circulating in North-West Region of Russia for the last 15 years. Materials and methods. Six hundred and fifty strains of C.diphtheriae isolated from ill persons and carriers in Saint-Petersburg, Leningrad region and Vologda region at various periods of time were studied. Identification of an infectious agent was performed according to methodic guidelines MU 4.2698-98. IHA-chromatographic test (ICS-test) on the basis of MKA, polymerase chain reaction, determination of adhesive activity and susceptibility to antibiotics were performed. Results. In recent years, circulation of C.diphtheriae strains with biologic characteristics similar to that observed in strains isolated during diphtheria epidemic and differed from that observed in strains isolated during the period of low incidence. Proportion of strains with «silent» gene between non-toxic in Elek-test C.diphtheriae increased. Decreasing of susceptibility to the range of antibiotics is observed in recent years. Conclusion. Revealed features of biologic characteristics of diphtheria agent circulating in post-epidemic period should be accounted during epidemiologic surveillance for diphtheria and choice of treatment of the infection.

Rfloxacin, gemifloxacin, levofloxacin and moxifloxacin were highly effective for urgent prophylaxis and treatment of glanders. Materials and methods. Golden hamsters of both sexes, with weight 80 — 100 g, were inoculated with 100 LD 50 of 48-hour agar culture of Burkholderia mallei (strain Ц-5). Commercial preparations of 2 — 4 th generations of fluoroquinolones: sparfloxacin (Sparflo, India), gemifloxacin (Faktiv, Russia), moxifloxacin (Avelox, Germany), pefloxacin (Abactal, Slovenia), levofloxacin (Eleflox, India), lomefloxacin (Lomeflox, India), ofloxacin (Russia). Urgent prophylaxis started 3 hours after inoculation with duration of 10 days, whereas treatment started 24 hours after inoculation with duration of 15 days. Dayly dose of pefloxacin, ciprofloxacin, ofloxacin was divided on 2 parts, which were administered with 12-hour interval; other drugs were administered once a day. Results. All studied drugs, excluding lomefloxacin, were highly effective for urgent prophylaxis and treatment of experimental glanders and provided 80 — 100% protection. Conclusion. Third-fourth generations of fluoroquinolones: sparfloxacin, levofloxacin, moxifloxacin, gemifloxacin were highly effective against agent of glanders in in vivo experiments. They are promising drugs for the development of schemes for urgent prophylaxis and treatment of glanders in humans.

Aim. To study effects of oxygen availability and presence of glucose in growth medium on adhesive and invasive properties of Yersinia pseudotuberculosis as well as its resistance to heat stress during sharp rise of temperature from 8°С to 37°C. Materials and methods. Yersinia pseudotuberculosis was grown on nutrient broth with or without glucose at 8°C and two regimen of aeration – during intensive stirring (180 rpm) and without it. Adhesive and invasive activities were studied on the model of HeLa human cell line. Effects of temperature stress on the bacterial growth were assessed from growth curves plotted on the basis of quantity of colony-forming cells. Morphology of bacterial cells was studied by electron microscopy. Results. It was shown that cultivation of Y.pseudotuberculosis at 8°C and low aeration increases its adhesive and invasive activity as well resistance to heat stress. Adding of glucose to growth medium decreases invasiveness of Y.pseudotuberculosis irrespective to aeration regimen. Conclusion. Oxygen deficiency during low temperature of growth promotes increasing of pathogenic potential of Y.pseudotuberculosis . Obtained data are useful for solving practical problems associated with development of prevention measures for pseudotuberculosis as well with food processing and storage.

Aim. To study protective activity of recombinant construction of heat-shock protein with lypopolysaccharide (rcHSP-LPS) as well as its variants (with destroyed protein or bounded LPS) against Salmonella typhimurium . It was also planned to study the ability of rcHSP-LPS to interact with tolllike receptors (TLRs) expressed on continuous cell lines. Materials and methods. One of the following preparations was administered to outbred mice: rcHSP-LPS; rcHSP-LPS treated by polymyxin B (PMB) for bounding of LPS — rc(HSP-LPS)PMB; rcHSP-LPS in which protein was treated by boiling during 30 min — rc(HSP-LPS)B; LPS ( E.coli K-235); polymyxin B (PMB). Twenty-four hours after single or last administration of rcHSP-LPS, each mice was intraperitoneally inoculated with 63 LD 5 0 of S.typhimurium 415 contained in 0.5 ml of physiologic solution. Antibody titer to LPS of Salmonella typhimurium was measured by immunoenzyme assay. Results. It was demonstrated that rcHSP-LPS administered 24 hours before inoculation induced resistance to S.typhimurium infection. Protection formed after 3 injections of rcHSP-LPS with 10 mcg in each or single injection with 100 mcg/mouse. Forty to eighty percent of immunized mice survived after challenge while 90% of control animals died. Destroy of the HSP by boiling of the construction led to loss of protective effect. Bounding of LPS by PMB did not lead to loss of protective properties of the construction but they expressed only after its multiple administration with 10 mcg per mouse. LPS of E.coli in dose 0.0266 mcg per mouse as well as PMB did not influence the course of S.typhimurium infection in mice. Conclusion. It was shown that rcHSP-LPS effectively protects mice from S.typhimurium infection by activating innate immunity; one of the possible mechanisms for such protection determined by interaction with TLRs 2 and 4 was considered. Other studies are needed in order to elucidate other mechanisms of innate immunity, which can be activated by rcHSP-LPS.

Aim. To develop infectious-toxic model of plague in mice and to assess perspectives of its use for selection of new vaccine preparations. Materials and methods. Cells of virulent strains of Yersinia pestis 231 and 231 FI incubated in lysates of human erythrocytes for their activation as well as suspensions of these strains in isotonic solution of NaCl were used for subcutaneous inoculation of infection-naпve and immune mice. Results. It was shown that activated cultures were characterized by maximal virulence (LD 50=1—3 CFU) and caused rapid infection — mean length of survival reduced on 1 — 3 days (P<0.01). Vaccine strain EV used by conventional way of inoculation (suspension in isotonic solution of NaCl) induced strong antibacterial immunity (index of immunity — 10 5), whereas activated (in lysate of erythrocytes) cells of Y.pestis 231 strain overcame it (index of immunity — 10 2). LD 50 value of Y.pestis 231 FI for immune and naпve animals was 3 m.c. (1 CFU), which demonstrates the absence of ability of EV strain to induce antitoxic immunity in the macroorganism. Conclusion. Use of two models of infection allows to make more adequate prognosis of efficacy for relevant vaccine preparations.

Aim. To experimentally assess activity and safety of anti-anthrax intravenous immunoglobulin manufactured on standard technology. Materials and methods. Plasma from selected donors vaccinated with combined anthrax vaccine was tested by enzyme immunoassay. Samples of plasma with increased titer of anti-anthrax antibodies were merged in one manufacturing load and fractionated in ethanol at negative temperature according to standard technology. Formulation of intravenous immunoglobulin was manufactured according to standard technology of acid-enzyme hydrolysis. Results. Proved medical technology of donors’ immune plasma fractionation provided 4 - 8-fold concentration of anti-anthrax antibodies. The finished product contained 5% of protein and was apyrogenic, non-toxic, thermostable, electrophoretically homogenous, had pH 6.65 and meet the requirements for manufacturing batches of human intravenous immunoglobulin. Conclusion. Protective effects of experimental human anti-anthrax immunoglobulin were comparable with control biological - equine anti-anthrax immunoglobulin for intramuscular use.

Aim. To compare results of mycobacteria identification by bacteriologic methods as well as by high-performance liquid chromatography (HPLC). Materials and methods. Two hundred and eighty strains of mycobacteria isolated from respiratory specimens and identified by bacteriologic methods and HPLC were studied. Results. It was established that results of HLPC use were highly correlated with results of microbiologic methods of mycobacteria identification: for identification of M.tuberculosis complex the correlation was 97.0%, for nontuberculous (NTM) slowly growing mycobacteria - 95.3%, for quickly growing NTM - 96.2% (overall - 96.1%). Results of identification of mycobacteria by HPLC were ready in significantly shorter time-frame (during 24 hours). Conclusion. HLPC method could be recommended for identification of mycobacteria in bacteriologic reference laboratories.

Aim. To assess sensitivity and specificity of phosphorescent immunochips developed by the authors on the basis of microplate phosphorescent assay (PHOSPHAN) for detection of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) in sera of patients and to compare results of PHOSPHAN assay with results obtained by lanthanide immunofluorescence assay (LIFA) and solid-phase enzyme immunoassay (SPEIA). Materials and methods. Two hundred sixty one serum samples were tested, including 155 samples from 74 patients with clinical diagnosis of TBE confirmed by serologic identification of IgM antibodies to TBEV. Sera were collected in 2003 in Perm region from persons, which fell ill during seasonal increased activity of ticks-vectors of TBEV, as well as from healthy blood donors. Phosphorescent immunochip corresponds 96-well plate with 4 active microzones formed on the bottom of each well, which are able to detect specific IgM and IgG antibodies to TBEV. Immune reaction was visualized by conjugate of streptavidin with Pt-coproporphyrin. Intensity of fluorescence was measured by scanning the bottom of previously dried microwell with scanner IFI-02. Results. Comparable sensitivity and specificity of POSHPHAN assay, LIFA and SPEIA was demonstrated for detection of IgM and IgG antibodies to TBEV in samples. Immunoluminescence-based PHOSPHAN assay and LIFA were more sensitive for analysis of sera with low titer of specific IgM antibodies. Conclusion. PHOSPHAN assay could be used for early serologic diagnostics of TBE as well as for assessment of antibody level for control of efficacy of treatment in patients with prolonged illness or level of protective immunity in vaccinees.

Aim. To develop rapid method of identification of mycobacteria based on laser fluorescence. Materials and methods. Characteristics of laserinduced fluorescence of 19 bacteria species, including 17 species of mycobacteria, were studied. Identification of microorganisms was performed using measurement of spectral-fluorescent characteristics. Results. Library of spectral-fluorescent characteristics of mycobacteria in different concentrations ratios and associations was created, which formed the basis of database for identification of mycobacteria by laser-fluorescent method. Principles of diagnostic algorithm of indication and differentiation of mycobacteria using this method were developed. Effect of myramistin for increasing the intensity of mycobacteria fluorescence, account of the diffracting characteristics of medium for adjustment of spectral characteristics of mycobacteria and processing of data by factor analysis are needed. Efficacy of the method was 80 — 90%. Conclusion. Principles of rapid identification of mycobacteria and their associations developed on the basis of laser-fluorescent method are experimentally founded and tested on unknown cultures of mycobacteria and objectively prove the possibility to apply this method for express identification of mycobacteria belonging to M. tuberculosis complex as well as non-tuberculous mycobacteria.

Aim. To study prevalence of hemolytic Escherichia in microbiocenosis of large intestine in 501 adult persons from Rostov-on-Don city. Materials and methods. Comparison of qualitative and quantitative composition of large intestine microbiocenosis in 248 persons with ultrasound examination of functional state of biliary tract was performed. Results. Statistically significant dependence of the rate of hemolytic Escherichia detection in microflora of large intestine from presence of dysfunctional disorders of biliary tract (biliary tract dysfunction) was revealed. Difference in character of microecological changes in large intestine of patients with such disorders and persons treated with wide-spectrum antibiotics was established. It was revealed that main feature of large intestine dysbiosis in patients with biliary tract dysfunction was the presence of significant quantity of hemolytic Escherichia as part of this compartment’s microflora. Conclusion. Hypothesis about possible role of functional disorders of biliary tract as a primary cause of microecologic imbalance was proposed.

Aim. To assessment acute and chronic toxicity and local irritative effect of antibacterial peptide complex (ABC) substance extracted from interferon preparations. Materials and methods. Preclinical assessment of ABC substance was performed according to «Requirements for preclinical study of overall toxicity of new pharmacological substances». Results. Series of experiments showed that single or prolonged (during 30 days) administration of ABC substance solution to mice intragastrically or rectally in maximal doses did not lead to death of experimental animals or pathological changes in their organs during pathomorphological and histological studies. Assessment of immune system state showed that administration of solution of studied substance during 1 month in dose 25 times higher than therapeutic leaded to stimulation of phagocytic activity of peripheral blood neutrophils. Conclusion. Assessment of acute and chronic toxicity showed that substance of ABC synthesized by virus-induced donor leukocytes during interferon synthesis is well tolerated by experimental animals, does not render toxic effects on their organisms, and possesses immunomodulating activity.

New class of regulatory molecules - microRNA - is of special interest now. Large amount of data about function of these molecules in immune response regulation is accumulated. Functions of several microRNA molecules (miRNA-146, miRNA-155, miRNA-223) contributing to such mechanisms of innate immunity regulation as transmission of signals from receptors, maturation, differentiation and proliferation of innate immunity cells are described in this review. It is necessary to underline the perspective of these molecules for development of drugs for correction of innate immunity abnormalities.

In 1970s enterovirus type 71 (EV71) caused several epidemics of poliomyelitis-like disease with severe neurologic sequelae. In the last 20 years EV71 was the cause of series of outbreaks and epidemics of foot-and-mouth disease-like conditions with neurologic sequelae in countries of South-East Asia. During the last epidemic of EV71 infection, which occurred in China in 2008, more than 60,000 cases was registered, 38 of which were lethal. Some aspects of epidemiology and laboratory diagnostics of disease caused by EV71 are considered in this review.