Ultrastructural Changes of Bacteria in Static Cultures of Yersinia pseudotuberculosis under Long Storage under Conditions of Low Temperature.

Electron microscopy study revealed changes in the ultrastructure of bacteria of Yersinia pseudotuberculosis strains characterized by significantly reduced reproductive ability and virulence potential after long-term storage at low temperature of 4-8°C. Most bacterial cells contained dark cytosol with reduced cellular material or empty cytosol, while the cell wall was preserved. The revealed ultrastructural changes in the bacterial cells of the static culture of Y. pseudotuberculosis suggest that storage of strains under low positive temperatures could induce the transition of the majority of bacterial cell population to a dormant, non-cultivated state with a decrease in their virulence. This fact is of great scientific and applied importance in studies of causative agents of saprozoonoses, including pseudotuberculosis, which has the etiopathogenetic background of persistent infection.

[BIOLOGICAL ACTIVITY OF YERSINIA PSEUDOTUBERCULOSIS TOXINS].

AIM: Study of effect of heat-labile (HLT) and thermostable (HST) lethal toxins of Yersinia pseudotuberculosis on the development of embryos of sea urchin Strongylocentrotus intermedius, processes of biosynthesis of nucleic acids and protein in embryo cells and activity of nucleoside- kinases of sea urchin. Materials-and methods. Y pseudotuberculosis strains 2517 (pYV-) and 512 (pYV48MD, pYV82MD) were used for isolation of HLT and HST Gametes and embryos of sea urchin S. intermediuswere used to carry out the experiments and isolate nucleoside-kinases. RESULTS: , Both of the studied toxins of Y pseudotuberculosis possessed, spermiotoxic effect and reduced fertilizing ability of sea urchin spermies. HLT LD50 was 1 µg/ml, and HST - 2 µg/ml. Toxins affected the development of embryos of sea urchin resulting in severe morphologic damages, cessation ofthe development of embryos at early stages of embryogenesis, destruction of cells and death of embryos. Wherein; damaging effect of HLT was observed at lower concentrations compared with HST HLT inhibited DNA and RNA biosynthesis at concentrations of 1-2 µg/ml. HST did not affect biosynthesis of nucleic acids even at high concentrations, but inhibited protein biosynthesis in sea urchin embryos. HLT did not reduce the level of inclusion of labeled amino acids into embryo cells. HLT had inhibiting effect on the activity of thymidine- and uridine-kinase of sea urchin, whereas HST did not affect these enzymes. CONCLUSION: Both of Y pseudotuberculosis protein toxins affect the development of sea urchin embryos, however, mechanisms of action of HLT and HST on embryos and processes occurring in them differ.

[CHARACTERISTICS OF FORMATION, INHIBITION AND DESTRUCTION OF YERSINIA PSEUDOTUBERCULOSIS BIOFILMS FORMING ON ABIOTIC SURFACES].

AIM: Detection of conditions of Yersinia pseudotuberculosis biofilm formation, their quantitative testing. MATERIALS AND METHODS: Y. pseudotuberculosis strains, nutrient media, standard 96-well polystyrene plates, crystal violet dye as well as bacteriologic, spectrophotometric, statistical methods were used. RESULTS: All the studied Y pseudotuberculosis strains formed a well expressed biofilm on abiotic surface during cultivation of bacteria in 200 µl of a plate well at a temperature of 20-22°C for 4-7 days. Bacteria CFU number in biofilm reduced by day 10 of incubation. DNAse I was found to inhibit biofilm formation, and also partially destroyed mature Y. pseudotuberculosis biofilm. The presence of DNA in extra-cellular matrix of biofilm was shown. CONCLUSION: An ability of Y. pseudotuberculosis to form biofilm on abiotic surface was established. The conditions of biofilm formation were determined. Inhibiting effect of DNAse I on Y. pseudotuberculosis was shown.

Effect of thermolabile toxin from Yersinia pseudotuberculosis on functions of innate immunity cells.

The thermolabile toxin of Yersinia pseudotuberculosis produces a selective dose-dependent stimulating effect on functional activity of innate immunity cells. Prolonged apoptosis-inducing action of the toxin was associated with activation of enzymes of the oxygen-dependent system (LDH and myeloperoxidase) at the early terms of observation (up to 3 h). In turn, increased number of macrophages with apoptotic changes was noted at the early stages of contact with the thermolabile toxin (5 h), and its further growth was observed against the background of activation of mitochondrial enzymes and production of NO metabolites.

[Efficiency of a new synbiotic drink in treatment of chronic diseases of gastrointestinal tract and concomitant dysbacteriosis].

The efficacy of a novel synbiotic drink in the complex therapy of patients withchronic diseases of the gastrointestinal tract and concominant intestinal dysbacteriosis was investigated in a randomized trial. The synbiotic drink contains a probiotic strain of bifidobacteria and Fucus evanescens polysaccharides with prebiotic activity and broad spectrum of the biological action on the patients. The use of the synbiotic drink provided more evident reduction of the clinical symptomes, more efficient recovery of the intestinal microflora and higher percentage of the patients cure vs. the routine therapy and the therapy with inclusion of sour milk bifidobacterin.

[Immunogenic and protective properties of nanosized constructs based on tubular immunostimulating complexes and pore forming protein of Yersinia pseudotuberculosis].

AIM: Evaluation of immunogenic and protective properties of constructs based on subunit porin antigen from Yersinia pseudotuberculosis, immunostimulating complexes (ISCOM) and tubular immunostimulating (TI) complexes. MATERIALS AND METHODS: Porin antibodies and blood serum cytokines were determined by using EIA. Porin-specific cell immunity was evaluated by DTH reaction inflammation index. Protective activity of porin formulations was determined by measuring specific gravity of animals surviving Yersinia pseudotuberculosis lethal challenge. RESULTS: Porin in TI complexes develops higher immunogenicity when compared with individual protein or protein with complete Freunds adjuvant. Porin in TI complexes develops higher protective activity, inhibits interferon synthesis in mice. Incorporation of porin into TI complexes results in neutralization of porin suppressive activity against DTH mechanisms and interferon system. CONCLUSION: TI complexes may be used as perspective carriers for bacterial antigens. TI complexes have adjuvant properties and can provide protective properties to porin vaccine constructs.

[Variability of the INV gene fragment encoding a functionally important domain of Yersinia pseudotuberculosis invasin].

A total of 84 Y. pseudotuberculosis isolates were studied. The isolates were obtained in Russian Federation in 1967-2008. The majority of Y. pseudotuberculosis isolates (n = 55) were of clinical origin and were isolated from feces of patients with the clinically and serologically proved diagnosis of pseudotuberculosis/Far East scarlet-like fever. These isolates included 18 isolates obtained from 3 outbreaks. Nine isolates were isolated from the internal organs of wild rodents. Other isolates were obtained from environmental sources. Ten Y. pseudotuberculosis isolates belonged to the serovar III and the other isolates belonged to the serovar I. The sequences of 600 b.p. fragment of the inv gene that encodes 667 through 866 invasin amino acids were determined for all isolates. Totally, 3 allelic variants were found. The most abundant allele 1 was found in 76 isolates. The allele 1 is represented in the database Genbank by the strain IP31758 isolated in the Far East of Russia (Eppinger et al., 2007). The allele 2 differed from allele 1 in 3 positions: G,2299N, O2300N, and O2302N. Substitutions in positions 2299 and 2302 were non-synonymous and resulted in amino acid substitutions Ser768 Thr and Val769 Ala. Six isolates carried allele 2. Allele 3 was found in two isolates different from allele 2 by a synonymous substitution G2324O. This allele is similar to the sequence found in Y. pestis strains, represented in the GenBank. The allelic distribution was not serovar specific: Y. pseudotuberculosis of serovar III and majority of serovar I isolates carried allele 1. The analysis of the allelic distribution among subpopulations formed on the base of a source of isolation revealed a statistically significant difference in spreading of alleles among clinical and wild rodent isolates (p < 0.05). Allele 1 prevailed over clinical isolates (95%), while allele 1 and allele 2 were disseminated equally among rodent isolates (55 % and 45 %, respectively).

[Influence of thermolabile lethal toxin of Yersinia pseudotuberculosis on development of sea urchin embryos and biosynthesis of DNA and RNA in embryonic cells].

Influence of thermolabile lethal toxin of Y. pseudotuberculosis on the development of embryos of sea urchin (Strongylocentrotus intermedius) and on biosynthesis of nucleic acids in embryonic cells was studied. Thermolabile lethal toxin affected metabolic processes of cells by inhibiting DNA and RNA synthesis. It had damaging action on developing embryos of sea urchin causing morphological changes and, as a consequence, death of embryos.

[Biological properties of heat-labile lethal toxin of Yersinia pseudotuberculosis].

The aim of the study was to assess the biological properties of heat-labile lethal protein toxin of Y. pseudotuberculosis. Toxin was extracted from Y. pseudotuberculosis strain 2517 type III serovar pYV-. The toxin killed mice 1-3 days after intraperitoneal administration (LD50=0.3 mcg of the protein). Heating at 56 degrees C during 30 min inactivated lethal activity of the toxin. It had a dose-dependent dermonecrotic effect during intracutaneous administration in rabbits. Hyperimmune rabbit serum to the toxin was obtained. Incubation of the toxin (LD100=1.2 mcg of the protein) with the serum at 37 degrees C during 30 min resulted in neutralization of lethal and dermonecrotic effects. The toxin did not have the cytotoxic effect on HeLa, Hep-2, and SPEV cells, but showed hemolytic activity to human and animal erythrocytes, and weak mitogenic activity to splenic cells of CBA line mice compared with control mitogen (concanavalin A).

[Yersinia pseudotuberculosis toxins].

The review of publications about protein toxins Y. pseudotuberculosis are presented. It includes the main data obtained by domestic and foreign investigators as well as the results of our own elaboration in the study of Y. pseudotuberculosis protein toxins. The guestions of isolation, purification, characterization of physico-chemical and biological properties, the mechanism action and role of toxins on pathogenesis of infection were discussed.

[Influence of glucose and galactose on the morphology and biological properties of Yersinia pseudotuberculosis].

When cultivated in the presence of glucose, irrespective of temperature and the degree of aeration, Y. pseudotuberculosis cells have the ovoid form, constant size and low hydrophobic properties of their surface. Meanwhile the characteristics of the bacteria grown in the medium, carbohydrate-free or with galactose added, essentially depend on the conditions of medium aeration. Under the conditions of intensive stirring at both temperatures these bacteria acquire the coccoid form, not typical for Yersinia, they have a smaller area (approximately 2 times) and more hydrophobic surface in comparison with the cells grown in the presence of glucose. Under stationary conditions the differences between the cells, cultivated in the presence of galactose and glucose, in form and area disappear, but the differences in the hydrophobic properties of the surface are retained. As revealed in this study, the cells grown in the presence of galactose and under the conditions of intensive medium stirring, in contrast to those grown with glucose, have 1.5-fold greater invasive activity, irrespective of aeration conditions, eightfold greater resistance to ampicillin and twofold greater resistance to streptomycin and erythromycin.

[Yersinia pseudotuberculosis nucleoside-kinase].

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.

[Experimental morphological study of the therapeutic effect of shark cartilage preparation in a model of infective allergic arthritis].

The therapeutic effect of a Polar shark cartilage preparation which is an enzymatic hydrolysate was studied in a rabbit model of infective allergic pseudotuberculous arthritis. Characterization of the chemical composition of the preparation designed by an original method is presented. Improvement of the general state of the affected joints and development of tissue immunomorphological responses were shown.

[The action of Yersinia pseudotuberculosis heat stable lethal toxin on the biosynthesis of protein in vitro]. / Deistvie termostabil'nogo letal'nogo toksina Yersinia pseudotuberculosis na biosintez belka in vitro.

The results of the in vitro action of Y. pseudotuberculosis heat stable lethal toxin on the biosynthesis of protein are presented. The toxin was shown to inhibit the inclusion of exogenous amino acid into newly synthesized peptides. The degree of the inhibition of the biosynthesis of protein in the in vitro system depends on the amount of the toxin added to the incubation mixture. The use of the conjugated transcription and translation system confirms our earlier data on the influence of the lethal toxin on the biosynthesis of protein in eukaryotic cells in vivo.

[Experimental study on the effect of low molecular DNA from salmon milt in pseudotuberculosis infection]. / Izuchenie deistviia nizkomolekuliarnoi DNK iz molok lososevykh ryb pri psevdotuberkuleznoi infektsii v éksperimente.

The effect of low molecular DNA from salmon milt (nDNA) in experimental pseudotuberculosis in mice was studied. When nDNA was admiministered orally, dissemination of the organs by Yersinia pseudotuberculosis lowered and the survival of the animals infected with 100-percent lethal dose of the bacteria increased. nDNA decreased contamination of the epithelial cells by the microbe in vitro and prevented the lethal effect of the Y. pseudotuberculosis toxins on the mice.

[Interactions of certain sapronotic infection pathogens in mixed cultures on a solid medium at different temperatures]. / Vzaimodeistvie vozbuditelei nekotorykh sapronozov v smeshannykh kul'turakh na plotnoi srede pri raznykh temperaturakh.

Under experimental conditions within the time limit of 21-35 days the causative agents of sapronotic infections in binary cultures, grown on a solid medium at 37 degrees C, 25-27 degrees C and 6-8 degrees C, interacted with one another transbiotically and through contact, their interactions having the character of amensalism, commensalisms-amensalism, competitive equilibrium, antibiosis. Irrespective of the initial density, a change in the species composition was observed, one of them playing the dominating role. At 37 degrees C mutual antagonism of Yersinia pseudotuberculosis and Pseudomonas aeruginosa killed both cultures. P. aeruginosa cells were also killed when cultivated at 37 degrees C jointly with Listeria monocytogenes, the most resistant species under experimental conditions. While studying the character of microorganisms interactions the method of contacting cultures on a solid medium was shown to give more information in comparison with the "cross-strip" method. Possible interspecific relationships between the causative agents of sapronotic infections under natural conditions are discussed.

[Use of immunomodulators isolated from marine invertebrates for reducing the toxic effects of thermostable toxin and lipopolysaccharides from Yersinia pseudotuberculosis on a macroorganism]. / Ispol'zovanie immunomoduliatorov iz morskikh bespozvonochnykh dlia snizheniia toksicheskogo deistviia termostabil'nogo toksina i lipopolisakkharidov Yersinia pseudotuberculosis na makroorganizm.

The possibility to use immunomodulators isolated from marine invertebrates for the lowering of the toxic effects caused by Yersinia pseudotuberculosis thermoresistant toxin and lipopolysaccharide was investigated. Effects were evaluated by the animals survival rate in per cent and mice average lifetime after toxin lethal dose injection. It was shown that polypeptide gangleen when compared to timalin as well as glycanes mitilane and strombus had dose-dependent protective effect. These substances increased animals survival rate to 15-17 per cent and prolonged life period for about two times when compared to control group. These results demonstrates the possibility to use investigated immunomodulators is clinical practice at the treatment of the patients with pseudotuberculosis.

[Effect of peptide immunomodulator gangleen on the activity of Yersinia pseudotuberculosis thermostable toxin in oxidant and antioxidant systems of human leukocytes]. / Korrektsiia vozdeistviia termostabil'nogo toksina Yersinia pseudotuberculosis na okislitel'nuiu i antiokislitel'nuiu sistemy leikotsitov krovi cheloveka immunomoduliatorom peptidnoi prirody gangliinom.

The ability to correct activity of Y. pseudotuberculosis thermoresistant toxin on antioxidative enzymes and on active oxygen forms in neurophiles and mononuclears was investigated. Toxin at concentration 0.5 and 2.5 mcg/ml did not change O2- production and activity of superoxidedismutase (SOD) and glutathione peroxidase (GP), but significantly enhanced catalase and glutathiont reductase (GR) activity. Gangleen at concentration 0.0002-0.2 mcg/ml, when added to incubation medium with toxin, stimulated production of active oxygen forms and activity of SOD, catalase, GP in both types of leucocytes, but decreased activity of GR in mononuclears. The results of investigation proves the ability to use gangleen in correction of immune system disorders caused by Y. pseudotuberculosis thermoresistant toxin.