SUBSPECIES DIFFERENTIATION OF YERSINIA PESTIS STRAINS BY PCR WITH HYBRIDIZATION-FLUORESCENT DETECTION.

Aim: Develop a method of differentiation of Y.pestis strains of different subspecies based on PCR with hybridization-fluorescent detection in real-time.

Materials And Methods: DNA target search for differentiation of subspecies of plague causative agent was carried out by Mauve 2.

[DETERMINATION OF VIRULENCE PROPERTIES OF PATHOGENIC MICROORGANISMS IN VITRO: STATE-OF-ART].

Various methods for evaluation of virulence properties of causative agents of infectious dis- eases in vitro were analyzed: molecular-genetic, cultural-biochemical, immunologic, physiologic. Predominant use of molecular-genetic methods, expediency of a complex approach, relevance of search of novel informative parameters of virulence are noted. Study of biological properties of pathogens in vitro is the first screening stage of evaluation of their virulence.

Effect of deletion of the lpxM gene on virulence and vaccine potential of Yersinia pestis in mice.

Yersinia pestis undergoes an obligate flea-rodent-flea enzootic life cycle. The rapidly fatal properties of Y. pestis are responsible for the organism's sustained survival in natural plague foci.

[The mechanism of interaction between Yersinia pestis and erythrocytes, and its importance for the pathogenesis of plague].

The ability of Yersinia pestis to get inside human and murine red blood cells (RBC) was found both in vivo and in vitro experiments. Due to oxidase and catalase activities, the microorganisms induced the denaturation of hemoglobin (Hb) through RBC oxidation to H2O2 in high concentration providing the formation of haemin and transformation of haem Fe2+ into the utilizable form, Fe3+. This phenomenon was found to be common in vitro for all Y.

[Development of an experimental immunoglobulin test system based on anti-idiotypic immunoglobulins for the determination of specific plague antibodies in serum of patients inoculated with the live plague vaccine EV NIIEG].

A new rapid, inexpensive, strictly specific, highly sensitive, and safe experimental ELISA test system based on rabbit anti-idiotypic immunoglobulins (anti-Id-ab) against Yersinia pestis lipopolysaccharide (LPS) was developed. Its efficiency was demonstrated, by using a panel of xenogenic antisera of biomodels immunized with the LPS extracted according to the Galanos procedure or the live plague vaccine EV NIIEG. In all cases, the similar proportions of positive reactions to the antigen itself or anti-Id-ab to LPS were registered and there was no significant difference in the detectable concentrations of both antigenic substances.

[Influence of cultivation conditions on the expression of Yersinia pestis YopE].

With the use three types of nutrient media made it possible to study the specific features of the biosynthesis of YopE, one of the main effector proteins, coded by Yersinia pestis virulence plasmid. This protein was proved to be produced practically at all stages of Y. pestis parasitism in the host body.

[Characterization of Yersinia pseudotuberculosis heat stable serovar specific polypeptides with the use of monoclonal antibodies].

The capacity of Y. pseudotuberculosis to express serovar specific polypeptides with different specificity of antigenic determinants was proved with the use of monoclonal antibodies (McAb). For the first time Y.

[Development of a competitive immunoassay based on monoclonal antibodies for the detection of specific antibodies to pseudotuberculosis pathogen].

A highly sensitive, effective and rapid method--a competitive alternative to ELISA--was developed for the detection of specific antibodies to Y. pseudotuberculosis. It is based on monoclonal antibodies (MAbs) recognizing the species- or serogroup specific epitopes of Y.

Heat-stable serogroup-specific proteins of Yersinia pseudotuberculosis.

A library of mAbs to the species- and serogroup-specific epitopes of Yersinia pseudotuberculosis serogroups I-VI was developed. These mAbs recognized linear sequential protein epitopes, as shown by ELISA and immunoblotting. Using the mAbs, Y.

[Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene].

The role of caf1M gene in biogenesis of Yersinia pestis capsule was studied in natural strains of the agent with Fra+/- phenotypes and recombinant variants with ycaA (caf1+;caf1M;caf1A+;caf1R+) locus defect. These bacteria did not form a clearly discernible capsule stained by classical methods but synthesized Cafl, whose content in the cells was many times higher than in lysates, in external cell wall, and in the medium with reference Y. pestis EV NIIEG culture (caf1+;caf1M;caf1A+;caf1R+).

The interaction of Yersinia pestis with erythrocytes.

Human and murine erythrocytes (RBC) were invaded by Yersinia pestis in vivo and in vitro during a short period and were probably used as an essential source of iron and porphyrin for survival, effective gross multiplication and rapid spread of these bacteria in the bloodstream of mammals. Both iron and porphyrin were extracted by Y. pestis from the RBC through oxidase-catalase activity which produced oxidation of the RBC glucose with generation of H2O2 in large concentration leading to oxidative transformation of haemoglobin into haemin.

[Study of protein and carbohydrate products, synthesized by Yersinia pestis under conditions imitating the internal environment of mammalian phagolysosomal macrophages].

Acid shift (pH 4.0) of liquid nutrient medium containing 20 mM Mg2+ created conditions in vitro simulating the internal environment of phagolysosome into which Yersinia pestis captured by a macrophage get in vivo. The capacity of Y.

Expression of acid-stable proteins and modified lipopolysaccharide of Yersinia pestis in acidic growth medium.

Growth medium simulating the phagolysosomal environment in which Yersinia pestis resides during its intracellular growth in vivo was made by acidification of Ca2+-deficient medium. When used for cultivation of Y. pestis EV-76 (pLCR+;pPst+;pFra+) and its isogenic derivatives--KM-217 (pLCR+;pPst-;pFra-) and KM-218 (pLCR-;Ppst-; pFra-)--this medium permitted survival and proliferation of viable bacteria without any growth restriction.

New genes involved in Yersinia pestis fraction I biosynthesis.

Antigenic and immunochemical properties of Yersinia pestis fraction I (FI) preparations extracted by different methods were studied with polyclonal and monoclonal antibodies. The existence of mature FI in a form of a complex antigen whose subunits have different genetic control was demonstrated. Galactolipid was shown, with caf1 product, to be the second species-specific component of the FI complex molecule and is probably encoded by chromosomal genes.

[Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis].

Hybridomas producing monoclonal antibodies (MAb) to Yersinia pseudotuberculosis, serovars I-IV, responsible for serovar appurtenance, were obtained. Virtually all MAbs reacted with protein antigens in immunoblotting. The only exclusion was MAb 3A2 presumably reacting with a glycoprotein epitope of complex structure.

Immunochemical characterisation of Vibrio cholerae O139 O antigens and production of a diagnostic antiserum without absorption.

Rabbits and mice immunised with chemically extracted O-antigens (O-Ags) of Vibrio cholerae O139 (O-AgB and O-AgD) developed antibodies (Abs) which appeared to be highly specific in ELISA for the relevant antigens and V. cholerae O139 strains without absorption, in contrast to the Abs against the heated O-Ag (O-AgH). An ELISA test based on the use of these Abs was shown to detect V.

[Synthesis of protective antigens during submerged cultivation of Vibrio cholerae].

The effectiveness of dot immunoanalysis for evaluating the dynamics of the synthesis of O-antigen, cholera toxin, neuraminidase, adhesin CFA1 in the process of the reactor cultivation of V. cholerae used for the production of oral chemical cholera vaccine is shown. The established regularities of the synthesis of the protective antigens of V.

Immunogeneity and structural organisation of some pLCR-encoded proteins of Yersinia pestis.

A novel method of cultivation of Yersinia pestis EV-76 and its isogenic strains KM-217 (pPst-;pCad+;pFra-) and KM-218 (pPst-;pCad-;pFra-) and careful extraction of Y. pestis proteins (YPPs) permitted isolation of >35 low Ca2+ response plasmid (pLCR)-encoded products, some of which are potentially new members of the LCR family. Immunisation with each YPP demonstrated that 25-, 54-, 72- and 87-kDa YPPs provided the highest level of protection in mice challenged with Y.

Development, characterisation and diagnostic application of monoclonal antibodies against Yersinia pestis fibrinolysin and coagulase.

A library of monoclonal antibodies (MAbs) which recognised different epitopes of Yersinia pestis fibrinolysin (Fib) was developed. These MAbs were species-specific and demonstrated no cross-reaction in indirect immunofluorescence tests (IIFT) with other gram-negative bacteria possessing plasminogen activator activity. All the MAbs provided equally high levels of immunofluorescence with pPst+ Y.

[The rapid diagnosis of toxigenic Vibrio cholerae strains in dot immunological analysis with monoclonal antibodies].

A method for identification of Vibrio cholerae toxigenic strains by dot immunoanalysis (DIA) with monoclonal antibodies to cholera toxin B-subunit is developed. It is based on analysis of lysates of isolated colonies of V. cholerae, grown in solid nutrient medium, broth cultures, and culture fluid.

[Characterization of Yersinia pestis fibrinolysin using monoclonal antibody].

Immunochemical identity of Y. pestis fibrinolysin and coagulase is demonstrated using monoclonal antibodies. These substances are proven to exist as complex proteins with two independent activities.

Adjuvant effect of anti-idiotypic antibodies to Yersinia pestis lipopolysaccharide.

Rabbit anti-idiotypic antibodies (anti-Id-ab) against Yersinia pestis lipopolysaccharide (LPS) were obtained with monoclonal immunoglobulins. Their complementary character to the original antigen was confirmed by immunohistochemical analysis and ELISA and gel precipitation tests. The anti-Id-ab were shown to possess all essential properties of Ab2beta subtype.

[Immunoenzyme test-system for the detection of Vibrio cholerae 0139].

On the basis of antibodies to the lipopolysaccharide (LPS) and capsular antigen of V.cholerae O139 two variants of the enzyme immunoassay (EIA) system permitting the detection of the infective agent at a concentration of 1 x 10(6) microbial cells/ml were developed. Antibodies to V.

[Study of antigenic determinants of Yersinia pestis lipopolysaccharide using monoclonal antibodies].

Species- and genus-specific antigenic determinants of total serological activity of plague agent lipopolysaccharide (LPS) were detected for the first time with a panel of monoclonal antibodies (MAb) in Y. pestis core polysaccharide in the R-form. MAb specifically bind radicals on one or several monosaccharides of core LPS.

[Comparative incidence of detection of specific antibodies to Yersinia pestis capsular antigen and lipopolysaccharide in humans immunized with pest vaccine].

Two to 8 years after vaccination, specific antibodies to capsular antigen (F1) and lipopolysaccharide (LPS) are found in the blood sera of but 10 to 20% of subjects 1-4 times epicutaneously immunized with live antipest vaccine. Only regular continuous (for at least 7-15 years) antipest vaccination leads to the production of antibodies to the main antigens of Yersinia pestis, detectable in remote periods in 75.9 +/- 4.