Characterization of smooth Brucella lipopolysaccharides and polysaccharides by monoclonal antibodies

Two mouse monoclonal antibodies (mAb) generated to the M antigen of Brucella melitensis 16M were analyzed. Binding profiles of both monoclonals were established by a competitive enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides, O polysaccharides and native hapten polysaccharides from B. melitensis 16M, B. abortus 544 and Yersinia enterocolitica O:9. Using this assay, significant differences in the reactivity of both antibodies were found with A and M antigens from Brucella spp. and the O polysaccharide from Y. enterocolitica O:9. These findings are consistent with the simultaneous expression of A and M epitopes on the lipopolysaccharide of all smooth Brucella strains. Quantitatively similar inhibitory powers were established for the native hapten and O polysaccharide from B. melitensis 16M. However, different behaviour was observed between both antigenic preparations obtained from B. abortus 544. The use of lipopolysaccharide-M-specific mAb in different serological tests instead of polyclonal antisera may be of great practical use for minimizing the risk of the appearance of cross-serological reactions between smooth Brucella strains and Y. enterocolitica O:9.     

Comparative analysis of Brucella serotype A and M and Yersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats.

Hapten polysaccharides of Brucella smooth M and A serotypes were prepared from Brucella sp. and Yersinia enterocolitica O:9 by previously described hydrolytic (O chain) or nonhydrolytic (native hapten [NH]) procedures. The purified polysaccharides differed only in the presence (O chain) or absence (NH) of lipopolysaccharide core sugars. The polysaccharides were compared by reverse radial immunodiffusion for the diagnosis of brucellosis in cattle (Brucella abortus biotype 1 [A serotype] and Brucella melitensis biotype 3 [AM serotype]), sheep (B. melitensis biotypes 1 [M serotype] and 3), and goats (B. melitensis biotype 1). The reverse radial immunodiffusion test with the NH from B. melitensis 16 M (serotype M) showed the highest sensitivity (89.6 to 97.3%), regardless of the host species and the serotype of the infecting Brucella sp. Y. enterocolitica O:9 NH (A serotype) was useful for diagnosing disease in cattle infected with B. abortus biotype 1, but not in cattle infected with B. melitensis biotype 3, sheep, or goats. The different results obtained with the serotype M and A polysaccharides and the sera from animals infected with M, A, and AM serotypes of Brucella spp. showed that in naturally infected animals, a large proportion of the antibodies are directed to or react with a previously defined common epitope(s) (J. T. Douglas and D. A. Palmer, J. Clin. Microbiol. 26:1353-1356, 1988) different from the A or M epitopes. By using the radial immunodiffusion test with B. melitensis 16M NH, it was possible to differentiate infected from vaccinated cattle, sheep, and goats with a sensitivity and specificity similar to that of the complement fixation test.     

Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9

Yersinia enterocolitica O:9 bears a smooth lipopolysaccharide (S-LPS) of Brucella sp. O-chain A+C/Y epitopic structure and is a cause of false-positive serological reactions (FPSR) in standard tests for cattle brucellosis. Brucella S-LPS, cross-reacting S-LPSs representing several O-chain epitope combinations, Brucella core lipid A epitopes (rough LPS), Brucella abortus S-LPS-derived polysaccharide, native hapten polysaccharide, rough LPS group 3 outer membrane protein complexes, recombinant BP26, and cytosolic proteins were tested in enzyme-linked immunosorbent assays (ELISA) and precipitation tests to detect cattle brucellosis (sensitivity) and to differentiate it from FPSR (specificity). No single serological test and antigen combination showed 100% sensitivity and specificity simultaneously. Immunoprecipitation tests with native hapten polysaccharide, counterimmunoelectrophoresis with cytosolic proteins, and a chaotropic ELISA with Brucella S-LPS were 100% specific but less sensitive than the Rose Bengal test, complement fixation, and indirect ELISA with Brucella S-LPSs and native hapten or S-LPS-derived polysaccharides. A competitive ELISA with Brucella S-LPS and M84 C/Y-specific monoclonal antibody was not 100% specific and was less sensitive than other tests. ELISA with Brucella suis bv. 2 S-LPS (deficient in C epitopes), Escherichia hermannii S-LPSs [lacking the contiguous alpha-(1-2)-linked perosamine residues characteristic of Y. enterocolitica S-LPS], BP26 recombinant protein, and Brucella cytosolic fractions did not provide adequate sensitivity/specificity ratios. Although no serological test and antigen combination fully resolved the diagnosis of bovine brucellosis in the presence of FPSR, some are simple and practical alternatives to the brucellin skin test currently recommended for differential diagnosis.     

Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides.

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and O polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica O:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A- and M-antigen structures, an interpretation supported by competitive binding studies with O polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous alpha 1,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by alpha 1,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element alpha-D-Rha4NFo(1----3)alpha-D-Rha4NFo bracketed by alpha 1,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated alpha 1,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.     

A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody.

A coagglutination test using monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 16M has been developed for the rapid identification of the smooth Brucella species. All reference strains of several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Brucella species, with the exception of B. abortus 45/20, were negative in the assay. Among the different organisms tested not belonging to the genus Brucella, serovar O9 of Yersinia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity, simplicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Brucella strains in diagnostic laboratories.     

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.     

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.     

Characterization of a native polysaccharide hapten from Brucella melitensis.

The 13C nuclear magnetic resonance spectrum of Brucella melitensis native polysaccharide hapten proved to be very similar to that generated by the O-specific chain (PS) isolated from B. melitensis lipopolysaccharide; that is, to a linear polymer in which the repeating unit is composed of five N-formylperosaminyl residues, one of them being substituted at position C-3 and the other four at position C-2. The serological analysis suggests that the so-called A determinant is present solely in Brucella abortus PS, the M determinant is only in B. melitensis PS, and the extensive cross-reaction observed is due to a determinant shared by both polysaccharides.     

Characterization of a Brucella species 25-kilobase DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide

In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysaccharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells.     

Molecular characterization of a Brucella species large DNA fragment deleted in Brucella abortus strains: evidence for a locus involved in the synthesis of a polysaccharide.

A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment.     

Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies.

Previously, four epitope specificities on the O chain of Brucella species were reported: M, A, C, and C/Y. In this work, according to monoclonal antibody binding to smooth lipopolysaccharides of Yersinia enterocolitica 0:9, Brucella abortus W99 (A-dominant strain), and B. melitensis Rev1 (M-dominant strain), seven O-chain epitope specificities were defined: M, A, C (M > A), C (M = A), C/Y (M > A), C/Y (M = A) and C/Y (A > M). Competitive binding assays between these monoclonal antibodies suggested that these different epitopes are probably overlapping structures.     

Analysis of Brucella lipopolysaccharide with specific and cross-reacting monoclonal antibodies.

Monoclonal antibodies which bind Brucella A lipopolysaccharide (LPS)-specific, M LPS-specific, or cross-reactive epitopes were used as reagents in quantitative dot blot, Western blot (immunoblot), and immunoprecipitation analysis of Brucella whole cells, whole-cell extracts, and purified LPS preparations. This set of monoclonal antibodies detected four unique epitopes on Brucella LPS. The specificity of monoclonal antibodies reactive with Brucella unique (A and M) and common (C and C/Y) LPS epitopes was demonstrated by blot analysis. The serotype specificity of monoclonal antibodies for A LPS of B. abortus 1119.3 or M LPS of Brucella melitensis 16M was confirmed. Type C monoclonal antibodies recognized epitopes on Brucella A and M LPS and did not cross-react with Yersinia enterocolitica O:9. In Western blots, type C monoclonal antibodies were bound by epitopes on Brucella A and M LPSs ranging in Mrs from 30,000 to 70,000, relative to marker proteins. Type C/Y monoclonal antibodies were cross-reactive with Y. enterocolitica O:9 and recognized Brucella A LPS epitopes with a restricted Mr ranging only from 40,000 to 50,000, relative to marker proteins. Type C/Y monoclonal antibodies also displayed a more restricted pattern of binding to Brucella M LPS. The monoclonal antibodies were able to detect 5 to 50 pg of a purified A LPS preparation in dot blots. The limits of detection by the monoclonal antibodies of a purified M LPS preparation ranged from 0.05 to 50 pg. Monoclonal antibody analysis of whole-cell preparations also demonstrated quantitative differences in the presence of the respective epitopes. The binding profiles of the monoclonal antibodies to Brucella whole cells varied between acetone- and chloroform-killed organisms as well as between species and strains. The lower limit of detection of any whole-cell preparation by the dot blot technique was 10(5) CFU. Binding profiles in Western blots and endotoxin activity of immunoprecipitates obtained with these monoclonal antibodies further defined the Brucella LPS antigens. These monoclonal antibodies and the techniques described may be useful in monitoring the antigenic content of Brucella vaccines and diagnostics.     

Rapid identification of smooth Brucella species with a monoclonal antibody.

A colony blot enzyme-linked immunosorbent assay was developed for the rapid identification of smooth Brucella species, i.e., Brucella abortus, B. melitensis, and B. suis. Bacterial colonies from plates were blotted onto nitrocellulose disks, lysed by immersion in chloroform, and reacted with BRU 38, a rat monoclonal antibody with specificity for the O side chain of B. abortus. Reaction with anti-rat immunoglobulin G conjugated to horseradish peroxidase and development in 4-chloro-1-naphthol resulted in colonies of naturally occurring smooth Brucella species staining purple. Results could be obtained within 4 h after colonies were visible on plates and individual colonies could be detected. Yersinia enterocolitica serovar O:9 strains were the only other organisms tested which showed cross-reaction by using this procedure. Because of its speed, sensitivity, and specificity, this technique should be very useful for identifying smooth Brucella strains in diagnostic laboratories.     

Immunochemical characterization of Brucella lipopolysaccharides and polysaccharides.

Purified lipopolysaccharide (LPS) extracted with phenol-water from smooth Brucella abortus was hydrolyzed with 1% acetic acid at 100 degrees C. The degraded polysaccharide (AH) released gave reactions of identity with the native polysaccharide hapten (NH) in phenol-water- or trichloroacetic acid-extracted endotoxin preparations of B. abortus and with the polysaccharide (poly B) extracted by trichloroacetic acid from rough B. melitensis strain B115. Poly B was present in the soluble cytoplasmic fraction but not in the membrane fraction, of disrupted B115 cells. It could not be extracted from three rough mutants of B. abortus or from B canis or B. ovis cells. Both AH and NH shared determinants present on smooth LPS and missing from poly B. Sugars found in purified LPS, NH, and AH included mannose, glucose, quinovosamine, glucosamine, and 2-keto-3-deoxyoctonate. Poly B contained only a trace amount of quinovosamine and no 2-keto-3-deoxyoctonate detectable by the thiobarbiturate assay. Sera from some rabbits immunized with pure smooth LPS and some, but not all, cows infected with field strains of B. abortus recognized the determinants missing from poly B. A subclass-specific enzyme-linked immunoassay showed that most of the antibody in sera from infected cows which binds to smooth LPS and to NH is of the immunoglobulin G1 subclass.     

Immunological identity of brucella native hapten, polysaccharide B, and yersinia enterocolitica serotype 9 native hapten.

Yersinia enterocolitica serotype 9 contained an antigenic component giving a reaction of total identity with Brucella native hapten and polysaccharide B. This component was present in a phenol-water extract (fraction 5; M. Redfearn, Ph.D. Thesis, University of Wisconsin, Madison, 1960) along with the smooth lipopolysaccharide. The native hapten could be purified free of lipopolysaccharide and proteins by gel filtration.     

Humoral immunity in mice mediated by monoclonal antibodies against the A and M antigens of Brucella

All smooth strains of Brucella bear two lipopolysaccharide (LPS) antigens in a ratio that defines the classification of strains in serovars, A (A greater than M), M (M greater than A) and A.M (A = M). Anti-LPS-A monoclonal antibodies (MAb-A) were previously shown to convey protection to mice against B. abortus (A) strain 544, as shown by lower spleen counts than in controls at days 7 and 21 after challenge. Anti-LPS-M monoclonal antibodies (MAb-M) were obtained and tested for M-specificity with LPS from reference strains by ELISA, by agglutination of LPS-coated latex particles, and by inhibition of this agglutination. Antigens A and M of three strains were quantified by a homologous LPS-latex and MAb agglutination inhibition assay. Protection conferred by MAb-A and MAb-M against three strains, B. abortus 544 (A), B. abortus 292 (M) and B. melitensis H38 (M), was tested at equivalent challenge and MAb doses: intravenous challenge was adjusted to give similar infection at day 7; MAb doses were adjusted to the same specific ELISA titre. Under these conditions, MAb-A and MAb-M conferred both early and late protection, as shown at days 7 and 21, against the strains that bore the homologous major antigen, i.e., strain 544 on one hand and strains H38 and 292 on the other. In contrast, MAb directed against the minor antigen of the challenge strain conferred significant protection at day 7 only with strains 544 and H38 and no or inconsistent protection against strain 292, which expressed the lowest amount of minor antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen

Brucella is a facultative intracellular parasite that causes brucellosis in animals and humans. The protective immune response against Brucella involves both humoral and cell-mediated immunity. In previous studies, we demonstrated that the T-dominant Brucella antigens bacterioferritin (BFR) and P39 administered either as CpG adjuvant recombinant proteins or as naked-DNA plasmids induced a specific Th1-biased immune response in mice. In order to improve the protection conferred by the BFR and P39 vaccines and to evaluate the additive role of antilipopolysaccharide (anti-LPS) antibodies, we used live attenuated Yersinia enterocolitica serotypes O:3 and O:9 as delivery vectors for naked-DNA plasmids encoding these BFR and P39 antigens. Following two intragastric immunizations in BALB/c mice, the Yersinia vectors harboring a DNA vaccine encoding BFR or P39 induced antigen-specific serum immunoglobulin and Th1-type responses (both lymphocyte proliferation and gamma interferon production) among splenocytes. Moreover, as expected, antibodies recognizing Brucella abortus 544 lipopolysaccharide were detected in O:9-immunized mice but not in O:3-treated animals. Animals immunized with O:9 organisms carrying pCI or with O:9 organisms alone were found to be significantly resistant to infection by B. abortus 544. Our data demonstrated that pCI plasmids encoding BFR or P39 and delivered with live attenuated strains of Yersinia O:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors induced a highly significant protective immunity against B. abortus 544 infection pointed out the crucial role of anti-LPS antibodies in protection. The best protection was conferred by a serotype O:9 strain carrying pCIP39, confirming the importance of the P39 T-cell antigen in this mechanism.     

Fine structure of A and M antigens from Brucella biovars.

Brucella A and M epitopes were found on single O-polysaccharide chains of all biotype strains of this species. Lipopolysaccharides from the type and reference strains of five of the six Brucella species, B. abortus, B. melitensis, B. suis, B. canis, and B. neotomae, were extracted and purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in conjunction with silver staining and immunoblotting developed by monoclonal antibodies, showed bands characteristic of A, M, or mixed A and M antigens. The A antigen previously described as an exclusively alpha 1,2-linked homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranose was shown by 1H and 13C nuclear magnetic resonance spectroscopy to possess a fine structure consistent with the low-frequency occurrence of alpha 1, 3-linked 4,6-dideoxy-4-formamido-D-mannopyranose residues. This feature was previously attributed only to the M antigen, which is also a homopolymer of the same sugar. B. melitensis biotype 3 and B. suis biotype 4 lipopolysaccharides showed characteristics of mixed A and M antigens. Immunoabsorption of these O polysaccharides on a column of immobilized A-antigen-specific monoclonal antibody enriched polymer chains with A-antigen characteristics but did not eliminate M epitopes. Composite A- and M-antigen characteristics resulted from O polysaccharides in which the frequency of alpha 1,3 linkages, and hence, M-antigen characteristics, varied. All biotypes assigned as A+ M- expressed one or two alpha 1,3-linked residues per polysaccharide O chain. M antigens (M+ A-) also possessed a unique M epitope as well as a tetrasaccharide determinant common to A-antigen structures. B. canis and B. abortus 45/20, both rough strains, expressed low-molecular-weight A antigen.     

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.     

Protective properties of rifampin-resistant rough mutants of Brucella melitensis

Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.