Comparison of seroreactivity of rams with brucellosis in a complement fixation test, whole cell ELISA and by immunoblotting.

In rams with ovine brucellosis, a high degree of serological correlation exists between the complement fixation (CF) test which utilises antigen extracted from bacteria with hot saline, and the ELISA reactivity using methanol-fixed Brucella ovis as the assay reagent. Since the whole cell ELISA (CELISA) detects mainly antibodies against surface antigens of B. ovis, it was concluded that the similar findings of the two serological tests is due in part to the presence of membrane antigens in the CF test antigen following hot saline extraction of intact bacteria. Immunoblots with pooled sera representing different CF titres confirmed that the major immunoreactive antigens of B. ovis were located in four zones: alpha, beta, gamma 1 and 2 with corresponding apparent molecular masses of 55 and 60 kDa; 27 and 29 kDa; 18.5-20 kDa and 17-18 kDa, respectively. These zones of reactivity were consistently present in immunoblots when assayed against different B. ovis isolates even though Coomassie brilliant blue staining of SDS-PAGE gels revealed some differences in polypeptide banding patterns. However, these intensely-stained CBB bands located at 38 and 40 kDa which distinguished three of the seven B. ovis isolates were considerably less reactive in immunoblots compared to polypeptides that were located at positions equivalent to alpha, beta or gamma reactivities. Intensity of immunoblot reactivity against polypeptides located in the alpha, beta and gamma zones intensified with increasing CF titre. Sera with CF titres greater than 32 also tended to react against bands of higher apparent molecular masses located at 65, 70, 73, 78, 80 and 86 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay.

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities.

Smooth (S)-lipopolysaccharide (LPS) preparations from reference and field strains of several biovars of Brucella abortus, B. melitensis, and B. suis were prepared by (i) the hot phenol-water method, (ii) hot sodium dodecyl sulfate extraction and proteinase K digestion, or (iii) dimethyl sulfoxide extraction. These S-LPS-enriched fractions were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining after periodate oxidation. Immunoblots were developed by using either monoclonal antibodies specific for Brucella A or M antigens or polyclonal polyspecific or monospecific sera from rabbits, cattle, and goats. The specificity of monoclonal antibodies reactive with Brucella unique (A or M) epitopes was demonstrated by enzyme-linked immunosorbent assay, LPS latex agglutination, or agglutination inhibition. The most-represented subunits of S-LPS ranged in Mr from 30,000 to 70,000 relative to marker proteins. According to A or M immunodominance, two sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns were clearly distinguished among biovars, whatever the fraction tested: a close succession of regularly spaced narrow bands for A greater than M strains and regularly spaced triplets of bands including either (i) a first thin band followed by two thick bands for B. abortus M greater than A strains or (ii) one thick band between two thin bands for B. melitensis or B. suis M greater than A strains. Moreover, A and M specificities were reaffirmed by sandwich enzyme immunoassay and latex agglutination inhibition with monoclonal antibodies and polyclonal sera.

[A study on identification of atypical and R phase strains of Brucella].

This paper reports a comprehensive study on taxonomy of Brucella. In which 17 atypical and R phase strains were identified by using the routine methods, 6 groups of phages and oxidative metabolic tests. 4 McAb of Brucella were developed and applied for identification. Meanwhile OMP profiles of S phase of B. abortus, B. melitensis and B. suis were compared during identification of them. The results show that a comprehensive identification with routine methods, phage-typing and oxidative metabolic tests are able to determine these strains if they are species or biotypes. Several strains of Brucella McAb and OMP profiles of Brucella were firstly applied for identification of atypical and R strains, and provided some possibility in the application of identification of Brucella by using these two methods.

Immunochemical studies of oligosaccharides obtained from the lipopolysaccharide of Brucella ovis.

Brucella ovis rough lipopolysaccharide (R-LPS) was studied with respect to its heterogeneity, chain length, sugar composition and immunological activity. R-LPS was mildly hydrolysed and oligosaccharides were recovered in the upper phase after partition with chloroform-methanol. Gel-filtration of the upper phase in a column of Bio-Gel P-2 yielded oligosaccharides of 2, 4, 6 and 7 monosaccharide units, 2-keto-deoxy-octulosonic acid (KDO), and monosaccharides. Strong acid hydrolysis followed by paper chromatography showed that the hexa- and heptasaccharides are both composed of glucose, KDO and an unidentified sugar while tetrasaccharide is composed of glucose, mannose and glucosamine. These three oligosaccharides were able to inhibit the LPS-antibody reaction in a solid phase radioimmunoassay, suggesting the oligosaccharides bear antigenic determinants of LPS.

Comparison of protein components of different species and strains of Brucella.

The proteins constituents of Brucella abortus, Brucella melitensis and Brucella ovis were analyzed SDS-PAGE. From the comparison appears that the three species of Brucella studied shows a different electrophoretic pattern specially at the level of small peptides. On the contrary when two strains of B. abortus are analyzed no differences can be noticed.

Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis.

Sheep infected with Brucella ovis produce antibody responses to the rough lipopolysaccharide and to proteins present in hot saline (HS) extracts of B. ovis (J. I. Riezu-Boj, I. Moriyón, J. M. Blasco, C. M. Marín, and R. Díaz, J. Clin. Microbiol. 23:938-942, 1986). The distribution and antigenic relatedness of proteins in HS extracts and in outer membrane blebs were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting for 41 strains of B. ovis and 26 strains of Brucella melitensis of diverse geographic origin. Five major groups of proteins were identified in HS extracts of B. ovis that had been freed of rough lipopolysaccharide: proteins of 43 kilodaltons (kDa), group A (25.5 to 32.0 kDa), group B (21.5 to 22.5 kDa); group C (18.0 to 19.5 kDa), and group D (13.0 to 15.5 kDa). Group A, B, C, and D proteins were also present in blebs. The profiles of proteins in HS extracts or blebs from strains of both Brucella species were very similar. Cross-reactions were demonstrated among HS extracts and blebs of all strains tested in immunoblots performed with an antiserum against the HS extract of a reference strain of B. ovis. Evidence was also provided of an antigenic relationship between group 3 proteins of the outer membrane and some of the proteins in groups A, B, and C. The conservation of these antigens and their immunogenicity in infected animals provide promise that they may serve as components of an effective subcellular vaccine for ovine brucellosis.

[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.

Characterization of Brucella polysaccharide B.

Polysaccharide B was extracted from Brucella melitensis 16M and from a rough strain of Brucella abortus 45/20 by autoclaving or trichloroacetic acid extraction of whole cells and by a new method involving mild leaching of cells. The material obtained by either of the established procedures was contaminated by O polysaccharide. The new leaching protocol eliminated this impurity and provided a pure glucan, which was regarded as polysaccharide B. This polysaccharide was found by high-performance liquid chromatography separations, chemical composition, methylation, and two-dimensional homo- and heteronuclear magnetic resonance experiments to be a family of nonreducing cyclic 1,2-linked polymers of beta-D-glucopyranosyl residues. The degree of polymerization varied between 17 and 24. Polysaccharide B was essentially identical to cyclic D-glucans produced by Rhizobia, Agrobacteria, and other bacterial species. Pure polysaccharide B did not precipitate with Brucella anti-A or anti-M serum and did not inhibit the serological reaction of Brucella A or M antigen with either bovine or murine monoclonal Brucella anti-A or anti-M serum. Previously described serological reactions of polysaccharide B preparations with Brucella anti-A and anti-M sera are related in this study to the presence in crude extracts of contaminants with the antigenic properties of Brucella lipopolysaccharide O polysaccharides.

Isolation and antigenic reactivity of Brucella ovis outer membrane proteins.

Brucella ovis cell membranes were isolated from fractured and lysozyme-treated cells by ultracentrifugation. These preparations appeared to consist largely of outer membranes, as judged from the results of ultracentrifugation experiments in sucrose density gradients under conditions that are widely used to separate inner and outer membranes of gram-negative bacteria. The sequential detergent extraction of cell membranes yielded mainly lipopolysaccharide and three groups of outer membrane proteins. In immunoblotting, lipopolysaccharide had good antigenic reactivity with all sera from rams exposed to B. ovis (vaccination or natural infection), but some outer membrane proteins reacted strongly only with sera from immune (vaccinated) rams, not from infected rams, suggesting a possible diagnostic role for such proteins in predicting immunity or infection.

The structure of the lipopolysaccharide O-chain (M antigen) and polysaccharide B produced by Brucella melitensis 16M.

The surface M antigen of Brucella species has been identified as the lipopolysaccharide O-polysaccharide component composed of a repeating pentasaccharide unit containing a sequence of one 1,3- and four 1,2-linked, 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. A neutral polysaccharide produced by Brucella species and referred to as polysaccharide B (poly B) has been identified as a family of circular 1,2-linked polymers of beta-D-glucopyranosyl units ranging in ring size from 17 to 24 glucosyl units.

Release of outer membrane fragments by exponentially growing Brucella melitensis cells.

Rough and smooth strains of Brucella melitensis released a membranous material that was devoid of detectable NADH oxidase and succinic dehydrogenase activity (cytoplasmic membrane markers) but that contained lipopolysaccharide, proteins, and phospholipids. This material was composed of two fractions that had similar chemical compositions but that were of different sizes which were separated by differential ultracentrifugation. Electron microscopy showed that both fractions are made of unit membrane structures. The membrane fragments were released during the exponential phase of growth, and no leakage of malic dehydrogenase activity (cytosol marker) was detected. Thus, the fragments were unlikely a result of cell lysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that, although group 2 Brucella outer membrane proteins and lipoprotein were not detected, the proteins in the membranous material were outer membrane proteins. Gas-liquid chromatography analysis showed a similar fatty acid profile for the cell envelope and the outer membrane fragments of the smooth strain B. melitensis 16M. In contrast, the outer membrane fragments from the rough 115 strain were enriched in palmitic and stearic acids. With respect to the unfractionated cell envelope, outer membrane fragments were enriched in phosphatidylcholine, a phospholipid that is unusual in bacterial membranes.

Evidence of heterogeneity of lipopolysaccharides among Brucella biovars in relation to A and M specificities.

Sixteen smooth Brucella strains were lysed and digested by proteinase K, and the LPS fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Two profiles were distinguished after periodic acid oxidation and silver staining. The first, present in biovars A greater than M, was a close succession of regularly spaced narrow bands; the second, present in biovars M greater than A, showed regularly spaced doublets separated by a barely visible band (B. abortus = A, melitensis = M).

Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp.

The sodium dodecyl sulfate (SDS) extraction-trypsin digestion protocol used by Braun and Sieglin (V. Braun and U. Sieglin, Eur. J. Biochem. 13:336-346, 1970) to show the peptidoglycan-linked lipoprotein of Escherichia coli was applied to both Brucella abortus and E. coli. Whereas a single polypeptide of 8,000 molecular weight was obtained from E. coli, several proteins of apparent molecular weight lower than 35,000 were demonstrated by SDS-polyacrylamide gel electrophoresis in B. abortus. These results did not change when the trypsin digestion conditions were modified. On the other hand, when the SDS extractions were performed under conditions more stringent than those used for other gram-negative bacteria, only a polypeptide fragment of apparent molecular weight of 8,000 was obtained from B. abortus. This polypeptide was similar to the trypsin fragment of the E. coli lipoprotein with respect to its behavior in SDS-polyacrylamide gels, isoelectric point in urea, molecular weight, and presence of both ester- and amide-linked fatty acids. Moreover, the amino acid analysis showed an overall similarity with respect to the amino acid composition of E. coli lipoprotein. A polypeptide of the same molecular weight, isoelectric point, and amino acid composition was also obtained from Brucella ovis by the same method. These results demonstrated that B. abortus and B. ovis cell envelopes contain a lipoprotein and strongly support the hypothesis that it is the only major protein covalently linked to the peptidoglycan.

[Comparative studies of the properties of Br. ovis strains 03, 303 and 100671 to those of local strains for the production of antigen]. / Sravnitelni prouchvaniia na kachestvata na shtamove Br. ovis 03, 303 i 100671 s mestni shtamove za poluchavane na antigen.

Colorimetric analysis, biochemical methods, and the complement-fixation test were employed to study 3 Australian and 5 Bulgarian strains of Brucella ovis. Determined were some biochemical manifestations, the adaptation capacity with regard to aerobic conditions of cultivation without serum, the amount of polysaccharides contained in the strains, and the properties of the antigens obtained as checked via CFT. Milanov's method was employed for antigen production. Three of the Bulgarian strains showed definite advantages in the commercial production of antigens for the serologic diagnosis of the Brucella ovis infection in sheep.

Characterization of Brucella ovis lipopolysaccharide and its use for diagnosis of ram epididymitis by enzyme-linked immunosorbent assay.

Rough lipopolysaccharide, extracted by a mixture of phenol, chloroform, and petroleum ether from freeze-dried Brucella ovis cells with a yield of 0.71%, contained relatively small amounts of protein and nucleic acid contaminants as compared with lipopolysaccharides from other Brucellae. The crude lipopolysaccharide was suitable as a diagnostic antigen in an enzyme-linked immunosorbent assay for the sensitive and specific detection of ram epididymitis caused by B. ovis infection. In comparative serological tests, the enzyme-linked immunosorbent assay with B. ovis lipopolysaccharide gave better identification of infections and fewer false-negative results than the enzyme-linked immunosorbent assay with sonicated antigen or the complement fixation test.