[Comparative analysis of antigens from different Brucella species using immunoblotting with antisera from immunized rabbits]. / Sravnitel'nyi analiz antigenov razlichnykh vidov Brucella metodom immunoblota s antisyvorotkami immunizirovannykh krolikov.

Brucella antigens recognized by IgG antibodies in cell lysates from various Brucella species differing by the origin, biological, and virulent properties (including the reference, vaccine, and newly isolated strains) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in SDS-cell lysates were separated by 12% SDS-PAGE and protein gels were stained with Coomassie brilliant blue R-250 and Silver reagent. SDS-PAGE showed differences in the protein profiles of 15 strains of different species. Immunoblotting revealed that rabbit S-antisera contained IgG reacting with S-LPS and identical proteins of 90 to 16 kDa belonging to B, melitensis, B. suis, B. abortus, and B. neotomae strains. B. canis strains had 4 antigens reacting with these antisera, whereas B. ovis had none. No agglutinating antibody were detected by the standard tube agglutination test with smooth Brucella strains in rabbit R-antisera. By contrast, immunoblotting analysis with these sera demonstrated common 90-16 kDa antigens in the strains of B. melitensis, B. suis, B. abortus, B. neotomae, and B. canis. B. ovis possessed none of these antigens. These results confirm that all Brucella species except B. ovis possess common protein antigens reacting with IgG.

[Comparative study of the antigenic composition of Brucella canis strains by immunoblotting]. / Sravnitel'noe izuchenie antigennogo sostava shtammov Brucella canis metdom immunoblottinga.

Brucella cultures isolated from sick dogs have not been properly studied in Russia up to the present time. In 1994 a culture has been isolated from aborted fetus of a dog. Investigations by the traditional methods referred it to Brucella genus, species canis (strain K-01). Reference strain B. canis RM6/66 and B. canis K-01 were identical by the profiles of protein antigens in immunoblotting with a set of antibrucellosis sera. B. canis, B. suis, B. abortus, and B. melitensis. However, immunoblotting with sera to B. canis showed the similarity of B. canis cultures with the reference strain B. suis 1330, and use of sera to B. suis, B. abortus, and B. melitensis helped differentiate between the reference B. suis 1330 strain and B. canis strains. All the antisera used permitted the differentiation of Brucella strains from Yersinia enterocolitica 0:9, Escherichia coli 0:157, and Salmonella typhimurium cross reacting with Brucella in serological tests. Immunoblotting is a promising taxonomic criterion for identification of newly detected representatives of the Brucella genus.

[Isolation and purification of Brucella antigens synthesized by Escherichia coli K12 cells]. / Vydelenie i ochistka antigenov Brucella, sintezirovannykh kletkami Escherichia coli K12.

The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents. The 18 + 38 and 38 kDa antigens were obtained. The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens. The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation. Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells.

[Sensitizing properties of Brucella protein antigens synthesized in K12 Escherichia coli cells]. / Sensibiliziruiushchie svoistva belkovykh antigenov brutsell, sintezirovannykh v kletkakh Escherichia coli K12.

The sensitizing properties of brucella 31 kD and 31+15 kD protein antigens produced by Escherichia coli cell carrying and expressing the corresponding brucella genes were compared. In experiments evaluating the test of mouse feet oedema in CBA line animals the property of the 31 kD antigen preparation to induce the specific oedema effect was demonstrated on the level comparable with the one produced by the brucella outer membrane proteins preparation. The obtained data were confirmed in the experiments on adoptive transfer of prolonged type hypersensitivity via the spleen cells from the sensitized donors to intact recipient animals. The future of molecular cloning technique usage for obtaining the homogeneous stable preparations of brucella antigens with low reactivity and high specificity is discussed.

[Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]. / Klonirovanie genov Brucella v kletkakh Escherichia coli K12 i analiz produktov klonirovannykh genov.

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.

[Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods]. / Immunokhimicheskaia kharakteristika i serologicheskie svoistva lipopolisakharidov, vydelennykh iz raznykh vidov brutsell razlichnymi metodami ékstraktsii.

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.

[Phospholipids of bacteria of Brucella genus]. / Fosfolipidy bakterii roda Brucella.

The phospholipid composition of 6 Brucella species (B. melitensis, B. abortus, B. suis, B. ovis. B. canis, B. neotomae) and Australian mouse-derived strains of Brucella N 4, 11, 12 were studied. Comparison of phospholipid composition of Brucella cells with that of serologically related microorganisms revealed that all Brucella biotypes contain phosphatidyl-(N-methyl)ethanolamine and phosphatidylcholine while Y. enterocolitica, Sh. disenteriae, E. coli cells do not contain these two substances. It is concluded that the specific phospholipid pattern of Brucella biotypes may be useful in typing of new Brucella strains.

[Effect of gamma irradiation on the immunological properties of the brucellar protective antigen]. / Vliianie gamma-oblucheniia na immunologicheskie svoistva brutselleznogo protektivnogo antigena.

It was shown that the splenocytic response to mitogens in guinea pigs was activated 7 days following immunization thereof with a gamma-irradiated brucellar protective agent (gamma-BPA), while nonirradiated BPA inhibited lymphocyte proliferation under the effect of mitogens. gamma-BPA, as compared with BPA, circulated in blood for a longer time, induced a more rapid and prolonged synthesis of antibodies, and provided the development of a more intensive immunity.

[Free and bound Brucella antigen and the level of circulating immune complexes in brucellosis patients]. / Svobodnyi i sviazannyi antigen brutsell i uroven' tsirkuliruiushchikh immunnykh kompleksov u bol'nykh brutsellezom.

The aggregate hemagglutination test has been shown to be a highly sensitive and specific method for the detection of infectious antigenemia in different forms of brucellosis, permitting the determination of free Brucella antigen in 34% of patients with the acute form of the disease and in 53% of patients with the chronic course of brucellosis. The results of the determination of the quantitative level of circulating immune complexes (CIC) indicate that in the acute form of the disease their level exceeds the CIC level observed in the chronic course of brucellosis. The preliminary dissociation of CIC in the patients' blood sera has been found to increase the overall release of Brucella antigen to 80%.

[Structural changes in brucella chemical vaccine after gamma-irradiation]. / Strukturnye izmeneniia brutselleznoi khimicheskoi vaktsiny posle gamma-oblucheniia.

It was shown that gamma-irradiation of Brucella strain chemical vaccine stimulated phospholipid peroxidation therein: the content of extractable total phospholipids in the exposed vaccine decreased mainly due to diminution of phosphatidylcholines and phosphatidyl-ethanolamines. A relative content of high- and low-molecular weight protein components increased in the gamma-irradiated vaccine.

[Incorporation of the protective antigen of Brucella abortus into liposomes and the immunogenic properties of the antigen-containing liposomes]. / Vkliuchenie protektivnogo antigena Brucella abortus v liposomy i immunogennye svoistva antigensoderzhashchikh liposom.

The incorporation of B. abortus protective antigen into liposomes and its localization in liposomes have been found to depend on the lipid composition of liposomes. After the injection of the protective antigen conjugated with negatively charged liposomes humoral response is more pronounced than after the injection of the protective antigen incorporated into neutral liposomes. The immunization of guinea pigs with antigen-containing liposomes ensures the production of "incomplete antibodies" in the animals in high titers.

[Comparative study of the safety and reactogenicity of different doses of brucellosis chemical vaccine in human revaccination]. / Sravnitel'noe izuchenie bezvrednosti i reaktogennosti razlichnykh dozirovok brutselleznoi khimicheskoi vaktsiny pri revaktsinatsii liudei.

The period of 11-12 months has been found to be the optimal interval, among other experimentally tested periods of time, between primary immunization and booster immunization with chemical brucellosis vaccine. The safety and low reactogenicity of different doses (1 mg, 0.75 mg and 0.5 mg) of the vaccine have been established. The occurrence and intensity of local and systemic reactions to this vaccine depend neither on the dose of the preparation, nor on previous immunization.

[Comparative study of the antigenic activity and allergic transformation of the body in human revaccination with different doses of a chemical brucellosis vaccine]. / Sravinitel'noe izuchenie antigennoi aktivnosti i allergicheskoi perestroiki organizma pri revaktsinatsii liudei raznymi dozami brutselleznoi khimicheskoi vaktsiny.

The study of different doses of chemical brucellosis vaccine (0.5 mg, 0.75 mg and 1 mg), used for the booster immunization of persons who had received primary immunization with chemical and live brucellosis vaccines, revealed that the characteristics of its antigenic potency were somewhat higher after primary immunization with the live vaccine. When used for booster immunization, the tested doses showed no differences in their antigenic potency irrespective of prior immunizations received by the immunized persons. In booster immunization chemical brucellosis vaccine was found to have poorly pronounced allergenic properties irrespective of its booster dose with the tendency towards greater sensitization to brucellosis allergen in persons primarily immunized with the live vaccine. When considering the possibility of using brucellosis chemical vaccine in medical practice, the complex evaluation of the characteristics showing its reactogenicity and antigenic potency, as well as the allergic transformation of the body induced by the injection of the vaccine, was carried out, which made it possible to recommend 1 mg of the vaccine as the optimal booster dose.

[Somatic antigens of the Brucella genus. The structure of the O-specific polysaccharide chain of Brucella melitensis lipopolysaccharide]. / Somaticheskie antigeny roda Brucella. Stroenie O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Brucella melitensis.

The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.

[Use of a Brucella protein-polysaccharide antigen for the allergen diagnosis of brucellosis]. / Ispol'zovanie belkovo-polisakharidnogo antigena brutsell dlia allergodiagnostiki brutselleza.

The experimental and clinical study of the allergen diagnostic properties of new Brucella protein-polysaccharide antigen in comparison with brucellin has been made in the leukocyte migration inhibition test, and its working doses have been determined. The results thus obtained suggest that Brucella protein-polysaccharide antigen has considerable diagnostic advantages over commercial brucellin, which presents vast possibilities of using this antigen for the allergen diagnosis of brucellosis in the leukocyte migration inhibition test.

[Comparative study of the safety, reactogenicity and antigenic activity of chemical and live brucellosis vaccines in a controlled epidemiological trial]. / Sravnitel'noe izuchenie bezvrednosti, reaktogennosti i antigennoi aktivnosti brutselleznoi khimicheskoi i zhivoi vaktsin v usloviiakh kontroliruemogo épidemiologicheskogo opyta.

The complex evaluation of the reactogenicity characteristics revealed that after immunization with chemical brucellosis vaccine systemic reactions observed in most of the vaccinees were mildly pronounced and local reactions, mildly and moderately pronounced, their duration not exceeding 48-72 hours. During 4 months after immunization the antigenic and immunogenic potency of chemical brucellosis vaccine was no different from that of live brucellosis vaccine; seropositive persons immunized with chemical and live brucellosis vaccines showed no statistically significant differences in the geometric mean of their antibody titers, as determined in a number of serological tests, for a year after immunization. The examination of the vaccinees 4 and 12 months after immunization revealed that the sensitizing activity of chemical brucellosis vaccine was, respectively, 12.2 and 2.5 times lower than the allergenic action of live brucellosis vaccine.