[The development and application of reagents kit for detection RNA of the causative agents of melioidosis and glanders by transcription-based amplification (NASBA) in real-time.]

The polymorphism of clinical manifestations of melioidosis and glanders and their high mortality require improvement of diagnostics for detection of this agents. The perspectivity of development of transcription-based amplification real-time NASBA diagnostic kits is determined by high analytical sensitivity and the opportunity to perfom the verification of the results of other methods for pathogenic Burkholderia species detection. The fragment of 23S rRNA gene was selected as the target for development of real-time NASBA kit. The high specificity of the constructed oligonucleotides was confirmed during the analysis of wide range of heterological strains of microorganisms and sequencing of amplified fragments of 23S rRNA gene. The analytical sensitivity of the developed kit allowed to detect Burkholderia pseudomallei and Burkholderia mallei in concentration of 1×101 microbial cells per ml. The high functional characteristics of developed kit as well as the possibility to use it in case of appereance of discordant result during the detection of pathogenic Burkholderia species were demonstrated while studying biological samples.

[Immunogenic properties of experimental production series of Staphylovac-2 vaccine].

AIM: Study experimental production series of Staphylovac-2 by accumulation of specific IgG and safety. MATERIALS AND METHODS: Experimental production samples of staphylococci vaccines were studied by the accumulation of specific IgG in sera of immunized BALB/c line mice in EIA. Safety was evaluated in tests of acute and chronic toxicity including pathomorphologic and histologic, hematologic and biochemical studies, studies of the effect on central nervous system. RESULTS: A statistically significant (2.6 - 3.0 times) increase of IgG levels in sera of immunized mice compared with control was noted. In the experiments studying acute and chronic toxicity the increase in body mass and mass of internal organs differed from data obtained from control animals at no observation periods. None of the studied methods of safety evaluation showed differences of the studied vaccine series from the control. CONCLUSION: The recommended dose for subcutaneous administration into human of 200 µg is experimentally justified and could be the basis for carrying out clinical studies of staphylococci vaccines in humans.

[Immunobiological properties of Bordetella pertussis lipopolysaccharide in the acellular pertussis vaccine].

AIM: Study of Bordetella pertussis lipopolysaccharide (LPS) immunobiological properties in the acellular pertussis vaccine. MATERIALS AND METHODS: Experimental series of acellular pertussis vaccines (APV), lyophilized LPS were used. Antibody titers against LPS in mice sera were evaluated by using EIA with peroxidase conjugate of anti-species antibodies against mice IgG. LPS activity in B. pertussis antigen complex preparations was determined in quantitative chromogenic LAL-test by end point. APV protective activity was determined in mice test during intracerebral infection by B. pertussis strain No. 18323 virulent culture. APV safety was determined in the mice body weight change test. RESULTS: The presence of LPS in APV was shown in immune electrophoresis with purified B. pertussis LPS preparation as a control. Formalin treatment changes immunochemical properties of APV LPS that lead to the shift of precipitation bands with pertussis agglutinating sera from the start zone into cathode. The quantity of LPS in pertussis culture supernatants was on average 49050 +/- 6774 endotoxin units per ml (EU/ml). In APV preparations the quantity of LPS was on average 906 +/- 90 EU/ml, i.e. decreased by more than 50 times. An increase of antibody titers against B. pertussis LPS in mice sera after the APV immunization was shown in EIA, which gives evidence of its presence in immunogenic form in the complex preparations. The preclinical studies carried out show protective activity and specific safety of the experimental APV series. CONCLUSION: Formalin-neutralized APV preparation is a complex of protein antigens in association with LPS. Formalin treatment results in modification of LPS molecule that retains antigenic properties but is significantly less toxic.

[The comparative evalution of informativeness of immunologic and molecular genetic methods and means during stages of specific indication of melioidosis agent].

The reference-center of monitoring of agents of glanders and melioidosis carried out testing of reagents kits for diagnostic of agent of melioidosis and other close-related species of Burkholderiae in vitro. At the stage of specific identification of pathogenic Burkholderiae the diagnostic possibilities of commercial and experimental kits of reagents for express- and rapid analysis were evaluated. The criteria of evaluation of diagnostic value of kits of reagents were sensitivity, specificity and time of implementation of studies. The analysis with application of mono- and multi-locus amplification systems, including real-time polymerase chain reaction permitted during 5-6 hours to implement identification and differentiation of Burkholderia pseufomallei, B. thailandensis and B. cepacia.

[Biochemical markers of virus cytopathogenicity in macrophages].

The results of macrophage metabolism studies at their infection by viruses differing in the level of virulence are presented. With the purpose of optimizing the estimation of viral cytopathogenic effects on macrophages, an index of cell reactions, which allows one to reveal the degree of virus influence in standard units, is offered. Generally, the application of high-sensitivity methods for functional activity determination and identification of the correlative communication between its changes and morphological features of cells can be prescribed to objective identification methods of not only viral reproduction, but also differentiation of types and the degree of their cytopathogenic effects.

[Preclinical studies of an adsorbed diphtheria-tetanus-pertussis vaccine (ADTP-vaccine) with acellular pertussis component].

AIM: Evaluate standardness of antigenic composition of pertussis component, completeness of sorption of pertussis, diphtheria and tetanus components, specific activity and safety of experimental series ofADTP-vaccine with acellular pertussis component (ADTaP-vaccine). MATERIALS AND METHODS: The content of separate antigens (pertussis toxin, filamentous hemagglutinin and agglutinogens 1, 2, 3) in samples of acellular pertussis component of ADTaP-vaccine and completeness of sorption of pertussis component of ADTaP-vaccine were evaluated by using enzyme immunoassay. Completeness of sorption of diphtheria and tetanus components were determined in flocculation reaction and antitoxin-binding reactions, respectively. Protective activity ofADTaP-vaccine was studied in model ofmeningoencephalitis development in mice infected with Bordetella pertussis (strain 18323) neurotropic virulent culture, protective activity oftetanus component - by survival of mice after administration of tetanus toxin, protective activity of diphtheria component - by survival of guinea pigs after administration of diphtheria toxin. Safety of preparations was evaluated in tests of acute and chronic toxicity with carrying out pathomorphologic studies including immature animals. RESULTS: All the studied experimental series ofADTaP-vaccine were standard by content of separate antigens of pertussis microbe. All the ADTaP-vaccine components were completely sorbed on aluminium hydroxide gel. By protective activity ADTaP preparations satisfied the WHO requirements. The preparations were non-toxic in acute and chronic toxicity and did not induce pathomorphologic changes including immature animals. CONCLUSION: Experimental samples of ADTaP-vaccine by specific activity and safety satisfied WHO requirements.

[Experimental study on chemotherapy of acute glanders].

Glanders is a zoonotic infection inducing acute forms of the disease (pneumonia, sepsis) in humans and animals under certain conditions, which even with the use of modern chemotherapy have unfavourable prognosis. Insufficient of efficacy of antibiotics with in vitro low MIC for planktonic bacterial suspension of Burkholderia mallei in chemotherapy of acute forms of glanders was due to the capacity of the pathogen for intracellular survival and formation of biofilms. Under such conditions the susceptibility of B. mallei to antibiotics lowered by several orders of magnitude. Chemotherapy of the glanders acute forms in animals usually provided only an increase of the lifespan, while among the survivors there was recorded a high relapse rate. More favourable outcomes were observed with the use of in vitro effective antibiotics in the form of clathrate compounds or especially liposomal forms. In the experiments with golden hamsters the survival rate reached 100% in 1000 Dlm infection even with the treatment onset by meropenem liposomal form 48 hours after the infection. Chemotherapeutics in the liposomal form significantly lowered resistance of B. mallei in both the experiments with a suspension of planktonic organisms and the use of bacteria interned in eukaryotic cells (Tetrahymena pyriformis).

[The use of polymerase chain reaction for detection of the agents of glanders and melioidosis using experimental infection].

Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material.

[Ultrastructural immunochemical study of pathogenic Burkholderia capsule].

The capsular structures of Burkholderia pseudomallei, B. mallei, B. cepacia and their avirulent noncapsular mutants were studied with the use of electron ahd immunocytochemical techniques. For this purpose, antimelio-idosis monoclonal antibodies (McAb) G11 and 1 G2, epitope-aimed at capsular glycopyotein of 200 kD and outer-membrane proteins of 42 and 39 kD, were used. As revealed in this study, the typical causative agents of melioidosis and glanders formed the capsule and exhibited high virulence due to the antiphagocytic activity of 200 kD glycoprotein, whose epitopes were found to be incorporated into the capsule, in contrast to avirulent variants and B. cepacia, found to have no such structure. The recognition of the membrane determinants of McAb 1 G2 on the outer-membrane surface of the non-capsular variants of microbes known to be the causative agents of melioidosis and glanders was indicative of absence of the capsule in these microbial cells. These data concerning the role of 200 kD antigen in virulence, its structural and functional characteristics may be efffectively used in the study of the pathogenetic mechanisms of melioidosis and glanders, as well as in the construction of preparations for their immunodiagnostics and prophylaxis.

[Use of infusoria Paramecium caudatum for the evaluation of the toxicity of antigens of different causative quarantine agents].

The results of the evaluation of the toxicity of bacterial antigens obtained from the causative agents of plaque, glanders, melioidosis, cholera on infusoria of the species P. caudatum, as well as on cell lines L-929, CHO K-1 and peritoneal macrophages of BALB/c mice, are presented. As revealed in this study, the method of toxicity determination on infusoria is similar in its sensitivity to the methods of testing on. CHO K-1 and L-929 cells, but the former is simpler, more available and permits the determination of toxic doses producing disturbances in the vital activity of the infusoria, but not leading to their death.

[Experimental study on the possibility of using live tularemia vaccine to increase resistance to heterologous infection disease]. / Eksperimental'noe obosnovanie vozmozhnosti primeneniia tuliaremiinoi zhivoi vaktsiny glia povysheniia rezistentnosti k geterologichnym infektsionnym zabolevaniiam.

In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B. mallei, Mycobacterium tuberculosis was studied. The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls. Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased. Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.

[Dependence of mode of DNA binding to benzo crown derivatives of actinocin on the size and distance from the chromophore of crown fragments]. / Issledovanie zavisimosti sposoba sviazyvaniia s DNK benzokraunsoderzhashchikh proizvodnykh aktinotsina ot razmera i udalennosti ot khromofora kraun-gruppirovok.

Complexes of DNA with benzocrown derivatives of actinocin were studied by viscometry and dynamic birefringence. Changes in the macromolecular structure of DNA caused by complex formation were determined. Models of DNA binding to the studied compounds were suggested on the basis of data obtained. The intercalation of actinocin chromophore of benzocrown derivatives of actinocin was shown to occur only when benzocrown groups were bound to the chromophore via glycine fragment. A change in the distance between the crown group and the chromophore prevents ligand intercalation. Increase in medium ionic strength results in appearance of new nonintercalational binding mode for crown-containing compounds with DNA caused by interaction of the crown groups with DNA.

[Burkholderia thailandensis: biological properties, identification and taxonomy]. / Burkholderia thailandensis: biologicheskie svoistva, identifikatsiia i taksonomicheskaia pozitsiia.

Burkholderia pseudomallei-like microorganisms have been isolated from soil and water in regions with endemic melioidosis. These strains have biochemical and antigenic profiles identical to melioidosis agents, except that they differ by virulence and L-arabinose (vir-, ara+). There are minor differences between these species by rRNA sequence. DNA hybridization and, more so, positive transformation of DNA auxotrophic mutants of B. pseudomallei by cell lysates of B. thailandensis and B. mallei confirmed the homology of these species' genomes. These members of the Burkholderia genus (pseudomallei, mallei, and thailandensis) can be regarded as a supraspecies taxon: pseudomallei group. B. thailandensis strains are not virulent for guinea pigs and slightly virulent for golden hamsters. Immunization with live cultures of B. thailandensis protected more than 50% guinea pigs challenged with 200 LD50 B. pseudomallei 100. B. thailandensis is suggested as a potential melioidosis vaccine.

[Immunotropic and immunogenic properties of Burkholderia pseudomallei surface and membrane antigens]. / Immunotropnye i immunogennye svoistva poverkhnostnykh i membrannykh antigenov Burkholderia pseudomallei.

The immunotropic and immunogenic properties of some chromatographic fractions of B. pseudomallei surface antigenic complex, as well as the preparations of B. pseudomallei outer and cytoplasmic membranes, were studied. The difference between the biopolymers under study in cytotoxicity, humoral and cell-mediated immunity characteristics, phagocytic activity were established. Some antigenic fractions (B, C, C1, H) showed perceptible protective activity (25-60%) in experiments on mice infected with B. pseudomallei virulent strain. One of the preparations of cytoplasmic membrane (CM-1) was also found to have protective properties (30%). Complex immunization with the antigenic complexes under study, introduced in combination with the immunomodulating agent Bromantan, was shown to enhance the protective effect.

[The design of a diagnostic test system based on magnetic sorbents and liposomes]. / Konstruirovanie diagnosticheskoi test-sistemy na osnove magnosorbentov i liposom.

The results obtained in the study of the possibility of using magnetic sorbents for the construction of a diagnostic assay system based on the antigen-antibody interaction are presented. As a model, Yersinia pestis capsular antigen and immunoglobulins to it have been used. A solid-phase immunofluorescent liposomal assay method has been developed; this method can be used for the detection of biopolymers in the sample under study and for the determination of their activity.

[Study of protective properties of antigen-containing liposomes of different lipid composition in plague]. / Izuchenie protektivnykh svoistv antigensoderzhashchikh liposom razlichnogo lipidnogo sostava pri chume.

Immunization of animals with a liposomal capsular and major somatic antigen of a plague causative agent enhanced their protective effects against plague infection. Lipopolysaccharide incorporation into the lipid membrane resulted in an increased protective effect of antigen-containing liposomes, which may be related to the single delivery and interaction with immune-competent cells. Incorporation of phospholipids with high phase transfer temperature and positively charged stearylamine by the membrane facilitates the immunogenicity of agents.

[Entrapping of hydrophobized plague capsular antigen into large unilamellar liposomes]. / Vkliuchenie gidrofobizovannogo kapsul'nogo antigena chumnogo mikroba v bol'shie oligosloinye liposomy.

The entrapping of a plague capsular antigen into the liposomes, prepared by the "reverse phase" evaporating method, was investigated. Both the native antigen and the antigen modified by palmitoylchloride were used. The entrapping of the modified antigen into the liposomes was greater than that of the native one: 46.1 +/- 5.4% and 3.7 +/- 2.7%, respectively. The highest content of the antigen on the membrane surface was observed when using palmitoylized protein and liposomes with egg lecithin, cholesterol and dicethylphosphate in the molar ratio 7:2:1.

[Various properties of urease encapsulated into liposomes]. / Nekotorye svoistva ureazy, inkapsulirovannoi v liposomy.

The enzymic activity of plant urease encapsulated into liposomes from egg lecithin was studied. Liposomes contained 3-5% of the initial enzymic preparation. Incorporation of urease into liposomes increases the permeability of the lecithin membrane for urea. The liposome membrane provides protection of the incorporated material from the inhibitory action of heavy metal ions. Kinetics of the reactions catalyzed by the free enzyme and encapsulated one is different. Km for the encapsulated enzyme is 1 X 10(-3) M and for free urease--4 X 10(-4) M, that is related to limited substrate mass transfer rate and as a result of it due to inhomogeneity of the catalysis proceeding in liposomes.

[Recanalization of the hepatic ducts in high obstructive blockage]. / Rekanalizatsiia pechenochnykh protokov pri ikh vysokoi obturatsionnoi neprokhodimosti.

Forty three patients were subjected to 48 operations for restoration of the passability of lobular ducts when the latter were obturated by tumor (36 patients) and by alveococcus (7 patients). Ten patients died immediately after operation. Death was caused by acute hepato-renal insufficiency and acute pancreatitis. Duration of life after operation was from 7 months to 3 years. Best results were obtained in patients with alveococcus of the portal fissure. The authors make a conclusion that recanalization of main biliary ducts in obturation ileus is less traumatic as compared with other operations, improves the general state of the patient and prolongs the duration of their life.