Epitope Mapping of the Brucella melitensis BP26 Immunogenic Protein: Usefulness for Diagnosis of Sheep Brucellosis

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.     

Molecular characterization, occurrence, and immunogenicity in infected sheep and cattle of two minor outer membrane proteins of Brucella abortus.

Screening of a Brucella abortus genomic library with two sets of monoclonal antibodies allowed the isolation of the genes corresponding to two minor outer membrane proteins (OMP10 and OMP19) found in this bacterial species. Sequence analysis of the omp10 gene revealed an open reading frame capable of encoding a protein of 126 amino acids. The nucleotide sequence of the insert producing the OMP19 protein contains two overlapping open reading frames, the largest of which (177 codons) was shown to encode the protein of interest. Analysis of the N-terminal sequences of both putative proteins revealed features of a bacterial signal peptide, and homology to the bacterial lipoprotein processing sequence was also observed. Immunoblotting with monoclonal antibodies specific for OMP10 or OMP19 showed that both proteins are present in the 34 Brucella strains tested, representing all six Brucella species and all their biovars. The OMP19 detected in the five Brucella ovis strains examined migrated at an apparent molecular weight that is slightly higher than those of the other Brucella species, confirming the divergence of B. ovis from these species. OMP10 and OMP19 were produced in recombinant Escherichia coli and purified to homogeneity for serological analysis. A large fraction of sera from sheep naturally infected with Brucella melitensis were reactive with these proteins in an enzyme-linked immunosorbent assay, whereas sera from B. abortus-infected cattle were almost completely unreactive in this assay.     

Minor nucleotide substitutions in the omp31 gene of Brucella ovis result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella species

The gene coding for the major outer membrane protein Omp31 was sequenced in five Brucella species and their biovars. Although the omp31 genes appeared to be highly conserved in the genus Brucella, nine nucleotide substitutions were detected in the gene of Brucella ovis compared to that of Brucella melitensis. As shown by differential binding properties of monoclonal antibodies (MAbs) to the two Brucella species, these nucleotide substitutions result in different antigenic properties of Omp31. The antigenic differences were also evidenced when sera from B. ovis-infected rams were tested by Western blotting with the recombinant B. melitensis or B. ovis Omp31 proteins. Twelve available sera reacted with recombinant B. ovis Omp31, but only four of them reacted with recombinant B. melitensis Omp31. These results validate previous evidence for the potential of Omp31 as a diagnostic antigen for B. ovis infection in rams and demonstrate that B. ovis Omp31, instead of B. melitensis Omp31, should be used to evaluate this point. The antigenic differences between the B. melitensis and B. ovis Omp31 proteins should also be taken into account when Omp31 is evaluated as a candidate for the development of subcellular vaccines against B. ovis infection. No reactivity against recombinant B. melitensis Omp31 was detected, by Western blotting, with sera from B. melitensis-infected sheep. Accordingly, Omp31 does not seem to be a good diagnostic antigen for B. melitensis infections in sheep. Two immunodominant regions were identified on the B. ovis Omp31 protein by using recombinant DNA techniques and specific MAbs. Sera from B. ovis-infected rams that reacted with the recombinant protein were tested by Western blotting against one of these immunodominant regions shown to be exposed at the bacterial surface. Only 4 of the 12 sera reacted, but with strong intensity.     

Antibody reactivity to Omp31 from Brucella melitensis in human and animal infections by smooth and rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Immunochemical characterization of rough Brucella lipopolysaccharides

Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its reaction of partial identity in immunodiffusion. Monospecific mouse sera against B. ovis R-LPS agglutinated only the homologous bacteria but not R cells of other species of Brucella. B. ovis R-LPS contained more 2-keto, 3-deoxyoctonate, and glucosamine as a percentage of dry weight than any other R-LPS tested. B. abortus R-LPS was identified by the absence of an unidentified sugar present in the other R-LPS molecules, and B. melitensis R-LPS could be differentiated from B. canis R-LPS by its higher content of fatty acids. In contrast to S-LPS, all of the R-LPS studied lacked quinovosamine. In electron micrographs, Brucella R-LPS had a granular appearance, in contrast to typical lamellar structures formed by Brucella S-LPS and Escherichia coli R-LPS.     

Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep.

Previously a Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and B. melitensis Rev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in Escherichia coli in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (n = 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (n = 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (n = 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (n = 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with B. melitensis H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and B. melitensis Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that a B. melitensis Rev.1 bp26 gene deletion mutant should be constructed to ensure this differentiation.     

Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp.

Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

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.     

Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis.

Brucella group 3 antigens (Ags) are outer membrane proteins (OMPs) with a molecular mass ranging from 25 to 30 kDa. The OMPs are of interest partially because of their potential use as vaccine and diagnostic reagents. We used human convalescent antibody (Ab) to clone a gene that encoded a 28-kDa protein from a lambdagt11 library of Brucella melitensis 16M genomic DNA. DNA sequence analysis revealed a single open reading frame that would encode a protein of 26,552 Da. The 28-kDa protein had a primary amino acid sequence that was 43% similar to a previously described Brucella abortus group 3 Ag, Omp25 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The similarity to a known group 3 OMP, immunoreactivity with Ab prepared against B. abortus group Ags, immunolabeling of whole cells, and Southern hybridization led to our conclusion that the B. melitensis 28-kDa protein was a group 3 protein distinct from B. abortus Omp25. We designated the B. melitensis protein Omp28. Human convalescent sera from patients infected with B. abortus and Brucella suis as well as rabbit antisera prepared against killed B. abortus whole cells recognized B. melitensis Omp28 on Western blots (immunoblots). Furthermore, mice and goats infected with smooth strains of B. melitensis produced Abs against Omp28. Our results may begin to explain the variability in molecular weight seen in Brucella group Ags and point toward their possible use in vaccination against infection as well as diagnosis of the disease.     

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.     

Outer membrane proteins from rough strains of four Brucella species.

Outer membrane proteins from 15 rough strains of Brucella abortus, B. ovis, B. canis, and B. melitensis were extracted with a dipolar detergent, and outer membrane proteins from selected strains were purified by anion exchange chromatography and gel filtration (Verstreate et al., Infect. Immun. 35:979-989, 1982). Outer membrane proteins produced two types of profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One type, demonstrated by B. abortus, B. ovis, and B. canis strains, contained the three predominant protein groups present in smooth B. abortus strains (Verstreate et al., Infect. Immun. 35:979-989, 1982): groups 1, 2 (porin [Douglas et al., Infect. Immun. 44:16-21]), and 3. B. melitensis strains demonstrated the second profile type, in which there was an additional band between groups 1 and 2. The relative proportion of porin was considerably lower in B. ovis, B. canis, and B. melitensis than in B. abortus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles could be used to distinguish B. abortus and B. melitensis from each other and from B. canis and B. ovis. The amino acid compositions of groups 2 and 3 from rough strains of B. abortus, B. canis, and B. melitensis were similar to those of corresponding proteins from smooth B. abortus strains. Zwittergent-soluble fractions from most rough strains contained antigen [b], which cross-reacted with group 2 from smooth B. abortus strains, and antigens [c] and [d], which cross-reacted with group 3 from smooth B. abortus strains. Antigen [a], shared by groups 2 and 3 (D. R. Verstreate and A. J. Winter, Infect. Immun. 46:182-187, 1984), was detected in most rough strains. None of these antigens were related to either rough or smooth lipopolysaccharide.     

Cloning and sequence analysis of a newly identified Brucella abortus gene and serological evaluation of the 17-kilodalton antigen that it encodes.

A thus far unknown gene encoding a Brucella abortus protein has been isolated from a lambda gt11 expression library probed with sera from Brucella-infected sheep. Sequence analysis of the cloned gene revealed the presence of an open reading frame of 158 amino acids encoding a protein of 17.3 kDa (calculated molecular mass). The recombinant B. abortus protein, expressed in Escherichia coli, and the corresponding Brucella melitensis protein migrated at the same apparent molecular masses as shown by Western blotting (immunoblotting). Among a series of serum samples from B. melitensis- or B. abortus-infected sheep and cows, 51 and 39%, respectively, showed a signal at 17 kDa on Western blot analysis of total protein extract from Brucella bacteria. These figures amount to 70 and 61% for sheep and cattle, respectively, in a competitive enzyme-linked immunosorbent assay with a specific monoclonal antibody. These data indicate that the 17-kDa antigen may be useful for serological diagnosis of Brucella infection.     

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.     

Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene.

The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.     

Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis.

Brucella spp. are the causative agents of brucellosis in many different hosts, including humans. Most of the serological methods of diagnosis are based on the detection of antilipopolysaccharide antibodies, which makes the differentiation of vaccinated animals from infected animals difficult. By using molecular biology techniques, a gene that encodes a 26-kDa protein (BP26) was isolated from a Brucella abortus S19 genome lambda gt11 library. This protein is in the periplasm of B. abortus and in transformed Escherichia coli. It is exported to the periplasm via a preprotein of 29 kDa with a signal sequence of 28 amino acids. The nucleotide and amino acid sequences of this gene and protein did not show any similarity with those of previously sequenced genes. The use of this protein in Western blotting allowed the differentiation between vaccinated bovines from infected bovines and the detection of infected rams: on the other hand, sera from human patients with active brucellosis were positive, while sera from human patients with chronic brucellosis or without clinical signs were nonreactive. BP26 might be of value as an antigen for serological diagnosis of brucellosis in different mammals.     

Cloning, Nucleotide Sequence, and Expression of the Brucella melitensis sucB Gene Coding for an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein

The Brucella melitensis sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced. The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from Brucella abortus and Escherichia coli, respectively. Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein.     

Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry.

Seven surface-exposed outer membrane proteins (OMPs) in Brucella supp. have been previously described (A. Cloeckaert, P. de Wergifosse, G. Dubray, and J. N. Limet, Infect. Immun. 58:3980-3987, 1990). OMPs were shown to be more accessible to monoclonal antibodies (MAbs) on rough (R) Brucella melitensis and B. abortus strains than to MAbs on their smooth (S) counterparts. In this work, we have extended this study to representatives of the main Brucella species, using MAbs specific for OMPs and S and R lipopolysaccharides (S-LPS and R-LPS). Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoelectron microscopy showed important differences between strains in the binding of OMP- and R-LPS-specific MAbs which were in part related to the particular expression of S-LPS, irrespective of the species. Results indicated that both the amount and the length of O polysaccharide on S-LPS greatly influenced the accessibility of OMP and R-LPS epitopes to MAbs. S-R B. melitensis EP and S B. suis 40, for instance, which express O-polysaccharide chains in small amounts and with short mean length, respectively, bound a greater number of OMP- and R-LPS-specific MAbs than the other S Brucella strains. The major 31- to 34-kDa OMP was the most exposed OMP on S strains of B. melitensis and B. suis. In most cases, flow cytometry results agreed with those of ELISA and supplied additional data, such as the homogeneity or heterogeneity of OMP expression at the strain level. However, there were some discordances between flow cytometry and ELISA results concerning the surface exposure of the 25- to 27-kDa and 31- to 34-kDa OMPs on S strains and that of minor OMPs in vaccine strain B. melitensis Rev.1. Immunoelectron microscopy confirmed the poor accessibility of OMPs to MAbs on the surface of S Brucella strains. The naturally R pathogenic species B. ovis and B. canis bound the majority of OMP-specific MAbs as well as the R-LPS-specific MAbs. Therefore, the conserved OMP and R-LPS epitopes could play a role as targets of protective antibody-mediated immunity in infections caused by naturally R B. ovis and B. canis.     

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.     

Use of an enzyme immunoassay test for characterizing the A and M antigens of Brucella.

An enzyme immunoassay test was developed for detecting the A and M antigens of brucellae. One hundred fifteen isolates, including 96 strains of Brucella abortus, 5 strains of B. suis, 1 strain of B. melitensis and 1 strain of B. neotomae, were accurately serotyped with the enzyme immunoassay test. No reactions were observed with 11 isolates of B. canis and 1 isolate of B. ovis. This rapid serological test provides for considerable savings in time and materials as compared with the standard tube agglutination test.