[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 2.3.1, Mega 5.0 and BLAST algorithm based on comparison of full-genome sequenc- es of Y.pestis strains. Primers and TAqMan probes were calculated for the DNA targets found, conditions of PCR with hybridization-fluorescent detection - optimized. RESULTS: DNA targets carrying marker mutations for the caucasus, altai, gissar, ulegei subspecies, strains from Talass alpine plague reservoir were detected. The effectiveness of the DNA targets found and the developed approach of subspecies differentiation is confirmed on 101 Ypestis strains of different subspecies, isolated from natural foci of Russia, near and far abroad. CONCLUSION: The developed approach based on PCR with real-time detection allows for a rapid and effective differentiation of Ypestis strains of various subspecies.

[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.

[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. pestis strains used in the study independently of Pgm phenotype and plasmid content, including vaccine Pgm(-) Y. pestis EV NIIEG and plasmidless Pgm(+) Y. pestis PKR-133 stains. This, probably, allows the bacteria to use Hb as an essential source of iron and porphyrins for de novo synthesis of DNA followed by effective multiplication in the mammalian organism. A correlation between the loss of the ability of RBC to transport O2 to organs and tissues and the development of progressive tissue hypoxia with specific clinical features of metHb accumulation and haemorrhagic syndrome was shown. The participation of Y. pestis phospholipases (A and D) in the destruction of RBC membranes and translocation of plague bacilli into RBC, as well as the phenomenon of polysaccharide chain lengthening depending on cultivation conditions of Y. pestis bacteria, are discussed.

[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. The same data were obtained in ELISA with antisera from the human beings immunized with the vaccine irrespective of the time of vaccination or the number of injections. Whether the use of ELISA is promising in the development of a diagnostic immunoglobulin antiplaque test system is discussed.

[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. The above-mentioned antigen was found capable of being synthesized, depending on the conditions of Y. pestis cultivation, in the form of membrane-linked (extracellularly and under phagosomal conditions) or secreted substance, mainly in phagolysosome. In the latter case the maximum level of its expression was registered. The experimental confirmation of YopE localization in the form of superficially localized antigen/receptor at the period of the extracellular growth of bacteria is presented, which suggests its important role in the realization of the virulent properties of Y. pestis and, together with the known data on the protective properties of the antigen, indicates the prospects of its use as the basis for the creation of new chemical antiplague vaccine.

[Characterization of Yersinia pseudotuberculosis heat stable serovar specific polypeptides with the use of monoclonal antibodies]. / Kharakteristika termostabil'nykh serovarospetsificheskikh polipeptidov Yersiniia pseudotuberculosis s pomosh'iu monoklonal'nykh antitel.

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. pseudotuberculosis O antigens were found to have heat stable protein components carrying linear epitopes complementary to serovar specific MaAb and ensuring the serological specificity of the infective agent. The possibility of improving intraspecific classification of Y. pseudotuberculosis and their differentiation from other pathogenic Yersinia on the basis of the capacity of these bacteria for synthesizing species and serovar specific proteins is substantiated.

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. pseudotuberculosis was found to produce serogroup-specific proteins, whose synthesis was dependent on cultivation temperature. These proteins appeared to be parts of heat-stable O-antigens prepared by heating Y. pseudotuberculosis serogroups I-VI at 100 degrees C for 2 h, and are responsible for the protein serotype specificity of these bacteria. The high specificity of serogroup- or species-specific mAbs obtained in ELISA suggests that they may be effective for serotyping of Y. pseudotuberculosis strains or differentiation from other pathogenic yersiniae.

[Development of a competitive immunoassay based on monoclonal antibodies for the detection of specific antibodies to pseudotuberculosis pathogen]. / Razrabotka konkurentnogo immunofermentnogo analiza na osnove monoklonal'nykh antitel dlia vyiavleniia spetsificheskikh antitel k vozbuditeliu psevdotuberkuleza.

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. pseudotuberculosis. The competitive ELISA was used in testing the murine hyperimmune sera and human antisera sampled from patients with different infectious intestinal diseases including several cases of pseudotuberculosis. The use of the MAbs-based ELISA in the laboratory diagnostics of pseudotuberculosis is under discussion.

[Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene]. / Immunokhimicheskaia kharakteristika fraktsii I shtammov Yersinia pestis, defektnykh po genu CAF1M.

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+). However Caf1 was not detected on the surface or culture fluid of natural and mutant Y. pestis cells. Exclusive role of Caf1M in Caf1 delivery to Y. pestis cell surface, but not in F1 monomer folding, was proven. Retention of lipopolysaccharide (LPS), a typical SR-LPS configuration and epitope specificity of its components was demonstrated, ensuring similar reactivity in solid-phase enzyme immunoassay with a panel of monoclonal antibodies to Y. pestis LPS. Study of immunochemical properties of antigenic substances derived from caf1M-defective Y. pestis cells by isolation of F1 showed that these substances represent an envelope protein involved in the caf1+ strains (together with Caf1) in assembly of "mature" F1 molecule as a result of posttranslation modification of various genes products. Variants of identification of Y. pestis with Fra+ phenotype by means of monoclonal antibodies to F1, fibrinolysis/coagulase, or LPS in solid-phase enzyme immunoassay are discussed.

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. Furthermore, some mainly chromosomally encoded effector proteins were implicated in this process because all were synthesised by Y. pestis grown in media simulating the intracellular conditions of mammalian RBC. Damaged RBC lost the ability to transport O2 in the mammalian organism. As a result, significant oxygen deficiency developed in host tissues providing specific clinical disease features of plague which are similar to characteristic symptoms of poisoning with haemotoxic substances when the transformation of the haemoglobin Fe2+ to Fe3+ occurs.

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. It, like caf1 product, was expressed in higher quantities at 37 degrees C than at 28 degrees C. Among FI subunits there were at least two proteins of 28 +/- 2 kDa and 43 +/- 2 kDa which were not specific for Y. pestis but were found also in all Yersinia spp. and some other bacteria. These proteins were synthesised independently of the incubation temperature (4 degrees-40 degrees C) and are possibly encoded chromosomally but outside the caf operon and galactolipid-encoding genes. Both proteins together with galactolipid comprise an envelope antigen found in pFra- or plasmidless Y. pestis strains. Organisation of Y. pestis FI (mature capsular antigen) in the form of a complex of the envelope antigen and the caf1 product is discussed.

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. Moreover, a correlation between the pH of growth medium and bacterial yield was established. Acidification completely inhibited fibrinolytic (pla protease) activity (PAA) of Y. pestis carrying pPst and allowed synthesis of specific outer-membrane proteins (Yops) without any degradation by the pla protease. Comparison of whole-cell lysates of the strains tested in PAAG-SDS showed that, in addition to previously described Yops, Y. pestis synthesised new acidic proteins which appeared only under acidic conditions and were encoded by pLCR or chromosomally. Some changes in O-specific polysaccharide chains of Y. pestis LPS that were dependent on cultivation temperature and pH of the medium were also demonstrated.

[Monoclonal antibodies for studying antigens of protein origin in serotyping of Yersinia pseudotuberculosis]. / Monoklonal'nye antitela dlia izucheniia roli antigenov belkovoi prirody v serotipirovanii bakterii 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. Variability of Y. pseudotuberculosis antigenic structure, depending on culturing temperature, was confirmed. Polypeptides with mono- or polydetermined antigenic specificity were determined using MAbs.

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. cholerae O139 strains down to concentrations of (9.4 x 10(4))-(7.5 x 10(5)) vibrios/ml and demonstrated no cross-reaction with other vibrios including representatives of serogroup O22. Native and proteinase K-treated O-AgB, O-AgD, O-AgH, as well as whole-cell lysates of V. cholerae O139 strains of different origin were used in immunoblotting with these Abs. Clear differences in the patterns of zones of specific reaction between chemically extracted and heated O-Ags and between lipopolysaccharide profiles of the V. cholerae O139 strains of different origin were observed. Serogroup-specific protein bands in the native O-AgB and O-AgD preparations were defined. The approach described for obtaining serogroup-specific Abs against vibrios and other bacteria seems to be promising for the development of specific diagnostic tests and further investigation of bacterial antigenic structure.

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. pestis virulent strain 231. Their immunological relationship was established with monoclonal antibodies (MAbs) and revealed several common properties, including oligosaccharide binding with specificity for N-acetylglucosamine. Affinity chromatography with MAb to the 25-kDa YPP permitted purification of the relevant antigen and its precursor. Their existence in the form of a complicated protein molecule was shown.

[Study of protein and carbohydrate products, synthesized by Yersinia pestis under conditions imitating the internal environment of mammalian phagolysosomal macrophages]. / Izuchenie produktov belkovoi i uglevodnoi prirody, sinteziruemykh Yersinia pestis v usloviiakh, imitiruiushchikh vnutrenniuiu sredu fagolizosom makrofagov mlekopitaiushchikh.

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. pestis to survive and multiply under these conditions irrespective of the plasmid composition of strains was confirmed experimentally. Y. pestis possesses a specific mechanism of fibrinolytic activity inhibition, preventing proteolytic degradation under the effect of Ca-dependent polypeptide (Yops) fibrinolysin and potentiating, in addition to these latter, the production of the so-called "acid" proteins by Y. pestis, coded for by pCad2+ or chromosome, including the potentially new members of LCR family. The culturing conditions affect the length of O-specific lateral chains of Y. pestis lipopolysaccharide (LPS), which corresponds to LPS SR, but not R form.

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. pestis strains cultivated at 37 degrees C and at 28 degrees C. In all cases, the MAbs inhibited both fibrinolytic and coagulase (Coag) activities of Y. pestis in Fib-activity inhibition and coagulase-activity inhibition reactions, and reacted with 35- and 37-kDa proteins of Y. pestis in immunoblotting, demonstrating bifunctional activity possibly similar to the properties of MAbs produced by hybrid hybridomas. On the basis of these and earlier studies, the immunochemical identity of Fib and Coag, two distinct subunits of a bifunctional fusion protein whose specific functional activity depends upon the temperature factor, was established. A new rapid, cheap, strictly specific and safe dot-ELISA based on the use of MAb against Y. pestis Fib (MAb-Fib) for reliable identification of Y. pestis strains was developed. This technique has great advantages over monoclonal diagnostic kits based on the use of MAb against Y. pestis fraction I (FI) because it allows detection of plague bacilli grown at 37 degrees C as well as at 28 degrees C. This dot-ELISA will be valuable as a clinical diagnostic tool and might be applicable to field studies and plague surveillance.

[Synthesis of protective antigens during submerged cultivation of Vibrio cholerae]. / Dinamika sinteza protektivnykh antigenov v protsesse glubinnogo kul'tivirovaniia 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. cholerae in the process of scaled-up cultivation are discussed.

[The rapid diagnosis of toxigenic Vibrio cholerae strains in dot immunological analysis with monoclonal antibodies]. / Ekspress-diagnostika toksigennykh shtammov Vibrio cholerae v dot-immunologicheskom analize s pomoshch'iu monoklonal'nykh antitel.

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. The analysis takes no more than 1-1.5 h, is highly sensitive, simple, and requires no special equipment and high qualification of specialists, which is of special importance in overall screening of humans and the environment for cholera.

[Characterization of Yersinia pestis fibrinolysin using monoclonal antibody]. / Kharakteristika fibrinolizina chumnogo mikroba s pomoshch'iu monoklonal'nykh antitel.

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. Possible causes of this phenomenon are discussed. Coagulase antigenic determinants are involved in specific fluorescence of Y. pestis cells grown at 28 degrees C. A new original method for screening the hybridomas producing monoclonal antibodies is proposed, based on inhibition of the functional activity of antigen.