[The development of polymer immunoglobulin preparations to identify different serovars legionella pneumophilia in reaction of slide-agglutination].

The article deals with the results of study targeted to develop polymer diagnostic preparation to identify epidemically significant serogroups Legionella pneumophilia. The preparation combines rate of record (1-5 min) of reaction of paragglutinining preparations with color visualization and demonstrative of reaction of volume agglomeration with polymer diagnosticums. The specially synthesized polymer microspheres were sensibilized with serums enriched with antibodies to lipopolysaccharide of corresponding serovar L. pneumophilia. The derived immunoglobulin diagnostic preparations detect agent of legionellesis in the reaction of slide-agglutination on glass during 1-5 min. The polymer diagnostic preparations provide positive reaction with culture of corresponding serovar and no reaction with other gomologic and geterologic agents of infectious diseases.

[System of activation of plasminogen in Vibrio cholerae].

AIM: Study system of activation of plasminogen in Vibrio cholerae. MATERIALS AND METHODS: 75 strains of V. cholerae of various origins were used in the study. Plasminogen was isolated from human plasma by using affinity chromatography on L-lysine sepharose, alpha-enolase activity was determined by a direct method assuming transformation of 2-phosphoglycerate into phopshoenolpyruvate. Vibrios were destroyed by ultrasound disintegrator to isolate membrane Omp protein, intact cells were discarded by centrifugation and cell lysate was centrifugated for 1 hour at 105000 g. The precipitate was solubilized in buffer with 1% triton X-100 and passed through a column with DE-52 cellulose. RESULTS: Vibrio cholerae O1 and O139 strains isolated from clinical specimens and water samples from open water bodies had the ability to bind by using alpha-enolase and transform human plasminogen into plasmin under the effect of outer membrane protein OmpT A protein with molecular weight around 40 kDa had proteolytic activity with a wide specter of substrate specificity, degraded fibrin, gelatin, collagen, protamine and activated plasminogen. Computer analysis showed that OmpT protein of cholera vibrion had a low degree of relation with Enterobacteriaceae omptins. CONCLUSION: The study carried out showed that vibrios have a system of activation of plasminogen that includes at least alpha-enolase and OmpT membrane protein. OmpT protein is assumed to belong to a new class of porins of Vibrionaceae family and its enzymatic activity may play a significant role in pathogenesis of infection.

[The autoagglutination factor of plague pathogen--cross-reacting antigen].

An antigen similar to the autoagglutination factor (AF) of plague pathogen in immunochemical specificity was sought in 22 bacterial species. For this, an immunoglobulin anti-AF diagnosticum that is the sheep erythrocytes sensitized with rabbit immune serum to the AF preparation isolated from the plasmid-free variant of the Yersinia pestis strain EV76. The bacteriological study applying a passive hemagglutination assay revealed AF-like antigens not only in all study strains (n = 30) of Y. pestis, but also in Yersinia pseudotuberculosis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris. These bacteria were used to prepare AF = like antigen preparations that reacted with rabbit anti-AF serum in dot blot immunoassay. Also, as Y. pestis, AF-like antigens in polyacrylamide gel were detected as multiple protein bands that differed in mobility in Yersiniae and heterologous bacteria. Differences were found in the properties of AF-line antigens of 5 species of bacteria (sensitivity to temperature and formalin, binding to the cell surface, which enabled differentiation of serological reactions caused by AP-like antigens of other bacteria. Thus, Y. pestis AF is a cross-reacting antigen that, despite its immunochemical similarity with AF-like antigens of other bacteria, was ascertained to differ from them in properties. The findings are of interest for searching for new diagnostic tests to detect the Cafl- strains of the plague pathogen and for differentiating the causative organisms of plague and pseudotuberculosis.

[Immunochemical characteristics of the autoagglutination factor of plague pathogen].

Immunochemical studies were performed to characterize the autoagglutination factor (AF) isolated from the plasmid-free variant of Yersinia pestis EV76 strains. The preparation based on the capsule Cafl antigen isolated from the triplasmid variant of the same strain was used for comparison. AF and Cafl are the surface protein antigens of the plague pathogen, which are similar in their capacity for self-aggregation and in the molecular mass of a subunit (about 17 kDa). However, unlike Cafl, FA gives rise to protein aggregates resistant to 2-mercaptoethanol and sodium dodecyl sulfate. FA and Cafl were shown to be immunochemically different antigens, the antibodies to which were present in the plague hyperimmune equine serum. Thus, the new surface antigen of the plague causative organism, which induces the generation of antibodies during immunization with live bacteria of the vaccine Y. pestis strain, has been discovered.

[Study of Legionella pneumophila strains isolated from environmental objects].

The paper presents the results of studying the biological properties of Legionella pneumophila strains isolated from environmental objects. Elective legionellosis medium (ELM) has been found to be suitable for the isolation of the causative agent from the starting material and to be as sensitive as CYE (Oxoid company) containing growth and selective additives. Polymerase chain reaction (PCR) with a home-produced commercial test system used to detect L. pneumophila DNA enables identification of the causative agent, including its species. Hyperimmune sera against L. pneumophila 1-7 serogroups used in slide-agglutination and agglutination, as well as a series of co-agglutinating diagnosticums for legionellosis 1-7 serogroups make it possible to identify even the serogroups of L. pneumophilla. Comparative analysis of the virulence of L. pneumophila cultures in vivo and in vitro allows recommendation that practical laboratories should employ a simple NaCl resistance test, which can be used as a guide virulence test.

[Serologic diagnostics of Legionella infection].

Technical approaches to construction of preparations for serologic diagnostics of Legionella infection were presented in the article; antigenic- and immunoglobulin-based diagnostic kits with known characteristics were developed. Immunogenic properties of protein and lypopolysaccharide antigens, which have diagnostic value, were studied; similarity of protein antigens from 7 serogroups of L. pneumophila was demonstrated. Soluble antigen with known composition was obtained and used for the development of antigen-based polymeric kit for diagnostics of Legionella infection. On the basis of hyperimmune sera, immunoglobulin-based polymeric diagnostic kit and array of coagglutinating diagnostic kits for the mentioned 7 serogroups were developed. Antigen-based polymeric diagnostic kit was recommended for licensure.

[Approaches to species-specific typing of double mixed cultures comprising pseudotuberculosis bacteria and atypical plague bacillus strains].

The species relevance of atypical Yersinia strains was determined by various microbiological, immunological, and genetic (including polymerase chain reaction) tests. These strains were shown to represent mixed cultures of Y. pseudotuberculosis serovariant O1b and Y. pestis var antiqua. Identification-resistant cells with atypical properties and plasmid segregation were found in the populations of Y. pestis strains. Analysis of different diagnostic tests revealed the most reliable ones selected for the identification of atypical Y. pestis strains with unstable genome.

[Primary characterization of the properties and diagnostic value of the antigenic complex "fraction V" in a plague microbe].

The experimentally obtained antigenic complex isolated by extraction in the gradient surfactants from live and acetone-dried bacteria of the capsule-free vaccine strain EV76 of a plague microbe that had lost its ability to synthesize the diagnostic species-specific capsular antigen F1 was investigated. The antigenic complex fraction V (FV) was obtained after the fifth stage of extraction at a concentration of 1.28% of surfactants and after additional purification. The thermostable FV was found to consist mainly of protein. The protein having a molecular mass of about 43 kD predominates in the fraction. The latter is nontoxic for albino mice and antigenic. It forms a precipitate with commercial antiplague serum antibodies. FV antigenic sensitization of tanned sheep red blood cells gave rise to a diagnostic agent that specifically reacted with an antiplague serum rather than with heterologous sera against enterobacteria. The sera immunized with FV specifically reacted in the JDJFR with all the strains of the pathogen of plague irrespective of the temperature of their cultivation, including "fraction-free", which did not interact with a diagnosticum on F1. The animal sera immunized with capsule-free plague microbial strain reacted only with a FV-erythrocytic diagnosticum and they did not interact with F1 antigen-sensitized red blood cells. The erythrocytic FV diagnosticum was tested in ABNR with 130 typical and atypical plague microbial strains and with 133 strains of heterologous bacteria of different species of the family Enterobacteriaceae. The FV diagnosticum identified all the variants of a plague microbe, while the F1 diagnosticum revealed only its capsular variants. Among the heterologous bacteria, some strains of the closely related pathogen of pseudotuberculosis in those who were in the R form, rather than S form, positively reacted. The use of FV identified 2 groups of hybridomas obtained after immunization of albino mice with the capsule-free variant of a plague microbe. Some hybridomas reacted only with plague bacteria while others did with two above pathogens. The authors substantiate the expediency of using FV, its components, and obtained monoclonal plague pathogen antibodies to improve antiplague diagnosticums with an activity spectrum that exceeds that of the existing commercial F1 antigen-based diagnosticums. They also discuss the lines of further studies.

[The dynamics of antibody formation to a vaccinal strain of Francisella tularensis in different immunization regimens]. / Dinamika obrazovaniia antitel k vaktsinnomu shtammu Francisella tularensis pri razlichnykh skhemakh immunizatsii.

The use of different schemes of albino mice immunization either by living or by killed preparations of the vaccine strain of Francisella tularensis when obtaining monoclonal antibodies to the tularemia microbe made it possible to reveal definite regularities in the dynamics of antibody formation. The highest titres of antibodies in sera of animals-donors of splenocytes were obtained during the daily (for 3 days) intraperitoneal immunization of mice with living vaccine or with its thrice administration to the spleen thrice with the interval of 10 days. Revaccination against a background of high titres of antibodies decreased their quantity in blood serum of mice, while that against a background of low titres increased them.