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A Cloeckaert

Publications and source records attributed to A Cloeckaert.

At least 37 records · Page 2Linked to original sources
PubMed2000

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies.

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.

Animals↗
PubMed2000

DNA polymorphism in the genus Brucella.

The genus Brucella has been described as consisting of six species, three of them including several biovars, which display a high degree of DNA homology by DNA-DNA hybridization. However, DNA polymorphism able to differentiate the six Brucella species and some of their biovars has been shown to exist. This work reviews the DNA variability in the genus Brucella and discusses the relationships between its members according to this genetic variability and a proposal for their evolution based on genetic diversity of the omp2 locus.

Animals↗
PubMed2000

Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar typhimurium.

The occurrence of active efflux and cell wall modifications were studied in Salmonella enterica serovar Typhimurium mutants that were selected with enrofloxacin and whose phenotypes of resistance to fluoroquinolones could not be explained only by mutations in the genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a decreased susceptibility to ciprofloxacin (MIC = 0.125 microg/ml) but did not have a mutation in the gyrA gene. Mutants BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but exhibited different levels of resistance to ciprofloxacin (MICs = 2 and 8 microg/ml, respectively). None of the mutants had mutations in the parC gene. Evidence for active efflux was provided by a classical fluorimetric method, which revealed a three- to fourfold decrease in ciprofloxacin accumulation in the three mutants compared to that in the parent strain, which was annulled by addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In mutant BN18/71, a second fluorimetric method also showed a 50% reduction in the level of accumulation of ethidium bromide, a known efflux pump substrate. Immunoblotting and enzyme-linked immunosorbent assay experiments with an anti-AcrA antibody revealed that the resistance phenotype was strongly correlated with the expression level of the AcrAB efflux pump and suggested that decreased susceptibility to ciprofloxacin due to active efflux probably related to overproduction of this pump could occur before that due to gyrA mutations. Alterations were also found in the outer membrane protein and lipopolysaccharide profiles of the mutants, and these alterations were possibly responsible for the decrease in the permeability of the outer membrane that was observed in the mutants and that could act synergistically with active efflux to decrease the level of ciprofloxacin accumulation.

Anti-Infective Agents↗
PubMed2000

Occurrence of a Salmonella enterica serovar typhimurium DT104-like antibiotic resistance gene cluster including the floR gene in S. enterica serovar agona.

Recently a chromosomal locus possibly specific for Salmonella enterica serovar Typhimurium DT104 has been reported that contains a multiple antibiotic resistance gene cluster. Evidence is provided that Salmonella enterica serovar Agona strains isolated from poultry harbor a similar gene cluster including the newly described floR gene, conferring cross-resistance to chloramphenicol and florfenicol.

Animals↗
PubMed2000

An IS711 element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals.

DNA polymorphism of the bp26 gene, coding for a diagnostic protein antigen for brucellosis, was assessed by PCR and restriction fragment length polymorphism analysis using primers to amplify the bp26 gene with its flanking regions. Surprisingly, whereas PCR performed on DNA of the reference strains of the six recognized Brucella species produced a product of the expected size (1,029 bp), PCR performed on DNA of three representative strains from marine mammals (from a seal, a dolphin, and a porpoise) produced a larger product, of about 1,900 bp. Nucleotide sequencing of the 1,900-bp PCR products revealed the presence of an insertion sequence, IS711, downstream of the bp26 gene and adjacent to a Bru-RS1 element previously described as being a hot spot for IS711 insertion. PCR performed on a large number of field strains from different geographic origins and from marine mammal isolates indicated that the occurrence of an IS711 element downstream of the bp26 gene was a feature specific to the marine mammal Brucella strains. Thus, this PCR assay is able to differentiate Brucella terrestrial isolates from marine mammal isolates and could be applied for diagnostic purposes.

Animals↗
PubMed2000

Characterization of heat, oxidative, and acid stress responses in Brucella melitensis.

Brucella melitensis is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells. Therefore, it has to adapt to a range of different hostile environments. In order to understand the mechanisms of intracellular survival employed by virulent B. melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used. Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis. On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified. Some of them were previously reported for Brucella, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD). Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit. Results indicated that B. melitensis could bring specific regulatory mechanisms into play in response to stress conditions. For example, the ribosome releasing factor in B. melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B. melitensis in response to heat stress. Some of the identified proteins and their potential specific regulation could be required for the adaptation of B. melitensis to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence.

Acids↗
PubMed1999

Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae.

Brucellae have been reported to be phylogenetically related to bacteria of the family Rhizobiaceae. In the present study, we used a panel of monoclonal antibodies (MAbs) to Brucella outer membrane proteins (OMPs) to determine the presence of common OMP epitopes in some representative bacteria of this family, i.e., Ochrobactrum anthropi, Phyllobacterium rubiacearum, Rhizobium leguminosarum, and Agrobacterium tumefaciens, and also in bacteria reported to serologically cross-react with brucella, i.e., Yersinia enterocolitica O:9, Escherichia coli O:157, and Salmonella urbana. In particular, most MAbs to the Brucella outer membrane lipoproteins Omp10, Omp16, and Omp19 cross-reacted with O. anthropi and P. rubiacearum, which are actually the closest relatives of brucellae. Some of them also cross-reacted, but to a lower extent, with R. leguminosarum and A. tumefaciens. The putative Omp16 and Omp19 homologs in these bacteria showed the same apparent molecular masses as their Brucella counterparts. None of the antilipoprotein MAbs cross-reacted with Y. enterocolitica O:9, E. coli O:157, or S. urbana.

Animals↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

Serological cross-reactivity between Brucella abortus and yersinia enterocolitica 0:9: IV. Evaluation of the M- and C-epitope antibody response for the specific detection of B. abortus infections.

Smooth lipopolysaccharides (SLPS) from Brucella abortus contain A-epitopes against which the majority of serum antibodies are directed during infections. SLPS from Yersinia enterocolitica 0:9 possesses identical epitopes, which are the cause for serological cross-reactivity. All Brucella spp. possess M- and C-epitopes which are not present in Y. enterocolitica 0:9. In order to examine the usefulness of these M- and C-epitopes for discriminatory serological testing, a panel of sera were used in this study, comprising sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected cattle of comparable strength in the serological brucellosis tests to the sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected bovines with strong serological reactions and sera from animals free from B. abortus or Y. enterocolitica infections. These sera were tested in blocking ELISAs with seven M- and one C-epitope-specific monoclonal antibodies in combination with SLPS from B. melitensis M16 high in M-epitopes as antigen. Strong B. abortus sera inhibited most strongly, while negative sera showed no or little inhibition. Sera with weak or intermediate titres blocked to a lower extent. Unexpectedly, the sera from Y. enterocolitica 0:9-infected heifers showed inhibition behaviour virtually identical to the comparable sera from B. abortus infected animals. Absorbing out of the A-epitope specific serum antibodies with either Y. enterocolitica 0:9 SLPS or with Y. enterocolitica 0:9 bacteria, indicated the presence of M- or C-epitope-specific serum antibodies in some sera from B. abortus-infected cattle but not in the sera from Y. enterocolitica 0:9-infected animals. These results demonstrate that the M- or C-epitope-specific antibody response in sera from B. abortus infected cattle is only of limited value for the serological discrimination between B. abortus and Y. enterocolitica 0:9 infections.

Animals↗
PubMed1998

Characterization of an immuno-dominant antigen in Brucella ovis and evaluation of its use in an enzyme-linked immunosorbent assay.

A panel of 45 Brucella ovis serologically positive sera were tested in immunoblots against B. ovis outer membrane proteins Omp31 and Omp25, purified by preparative SDS-gel electrophoresis. Forty-three sera reacted with Omp31, while only 11 reacted with Omp25, suggesting that Omp31 is identical to the previously reported immuno-dominant 29-kDa protein. Attempts to purify Omp31 on a larger scale by using procedures such as ion exchange-, reversed phase-, affinity- and gel filtration chromatography suggested that the outer membrane proteins were aggregated with rough lipopolysaccharide. Only denaturing SDS-gel filtration chromatography was able to separate proteins of about 29 kDa from rough lipopolysaccharide but did not separate Omp31 from Omp25 in B. ovis preparations. When used in an enzyme-linked immunosorbent assay, this 29-kDa protein preparation was less sensitive and less specific than the routinely used heat-extracted B. ovis antigen. A readily available recombinant E. coli, expressing the gene for Omp31 from Brucella melitensis 16 M, was used to extract and enrich recombinant Omp31 by a temperature-dependent Triton X-114-based technique. When this material was used in immunoblots with the 45 sera from B. ovis-infected sheep and with 10 monoclonal antibodies, raised against B. ovis Omp31, major differences in the antibody reactivity between the recombinant B. melitensis Omp31 and the B. ovis Omp31 were found. Such differences were unexpected because of the known structural and immunological relatedness of outer membrane proteins from various Brucella species. These results indicated that the antibody-response in B. ovis naturally-infected sheep against the immuno-dominant Omp31 was directed against epitopes which were only accessible when the protein was aggregated with rough lipopolysaccharides, or which were formed after aggregation but were not present in the recombinant protein.

Animals↗
PubMed1998

Evaluation of immunogenicity and protective activity in BALB/c mice of the 25-kDa major outer-membrane protein of Brucella melitensis (Omp25) expressed in Escherichia coli.

The antibody response specific to the 25-kDa major outer-membrane protein (Omp25) of Brucella melitensis expressed in Escherichia coli was assessed in BALB/c mice. Groups of mice were immunised and boosted either with sonicated E. coli carrying plasmid pAC2533-E. coli (pAC2533)-expressing the gene coding for Omp25 (omp25 gene) of B. melitensis, or with E. coli carrying plasmid pUC19-E. coli (pUC19). One control group received saline. The evolution of antibody responses was investigated by indirect ELISA with whole rough (R) B. melitensis H38 cells as antigen. Serum antibody titres of mice immunised with E. coli (pAC2533) were appreciably higher than those of mice immunised with E. coli (pUC19). The specificity to Omp25 of murine antibodies induced by E. coli (pAC2533) was demonstrated by SDS-PAGE and immunoblotting of five B. melitensis strains. Binding of antibody in E. coli (pAC2533) immune sera to the surface of B. melitensis strains differing in their smooth lipopolysaccharide (S-LPS) expression was also studied by whole-cell ELISA and by flow cytometry. Antibody reactivity to R and smooth-rough (S-R) was much stronger than that to smooth (S) B. melitensis strains, indicating a much better accessibility of Omp25 to antibody on strains lacking or expressing less O-polysaccharide on their surface. The antibodies to Omp25 were predominantly of IgG2a isotype. The capacity of E. coli (pAC2533) to induce protective immune responses against four challenge strains of B. melitensis was further evaluated in mice. Significant reductions in splenic infections, in comparison with mice immunised with E. coli (pUC19) and unimmunised (saline injection) mice, were observed in R B. melitensis B115, S-R B. melitensis EP and S B. melitensis H38 infected mice. Protection against S B. melitensis 16M was not significant. The data from the present study, together with previous results, suggest that humoral immunity against probably conformational, well-exposed epitopes of the Omp25 could contribute to protective mechanisms against B. melitensis infection in mice.

Animals↗
PubMed1998

Evaluation of an enzyme-linked immunosorbent assay using recombinant major surface protein 5 for serological diagnosis of bovine anaplasmosis in Venezuela.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of bovine anaplasmosis with purified recombinant major surface protein 5 (MSP5) of Anaplasma marginale produced in Escherichia coli. Serum antibody responses against MSP5 were detected in calves experimentally infected with A. marginale as early as 21 days postinfection and reached maximum titers at 28 days postinfection. The MSP5 ELISA performed with serum samples taken from field cattle from different regions of Venezuela showed a seroprevalence of 47%, which seems to be in accordance with the reported epidemiological status of bovine anaplasmosis in Venezuela. Positive results obtained in the MSP5 ELISA were further confirmed by immunoblotting, with the recombinant MSP5 as the antigen. Thus, these results confirmed the importance of MSP5 as a suitable antigen for the serological diagnosis of bovine anaplasmosis.

Anaplasmosis↗
PubMed1998

O-Polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry.

Smooth Brucella strains are classified into three serotypes, i.e., A+M-, A-M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.

Animals↗
PubMed1998

Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages.

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages.

Amino Acid Sequence↗
PubMed1997

Localization and characterization of a specific linear epitope of the Brucella DnaK protein.

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the alpha-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the alpha-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.

Adenosine Triphosphatases↗
PubMed1997

Mapping and identification of Brucella melitensis proteins by two-dimensional electrophoresis and microsequencing.

Two-dimensional (2-D) gel electrophoresis was used to map Brucella melitensis proteins. The 2-D proteins map of B. melitensis B115 revealed 595 silver-stained protein spots separated by both isoelectric point and molecular mass. Twenty-five proteins were identified either by immunoblotting using monoclonal antibodies (MAbs) or by N-terminal microsequencing. The protein spots identified by MAbs were the 89 kDa outer membrane protein, DnaK, bacterioferritin, CP24, and BP26. Some spots were identified by N-terminal microsequencing as proteins whose sequences had been reported previously from Brucella, such as three heat-shock proteins, namely DnaK, GroEL and GroES; bacterioferritin; Cu-Zn superoxide dismutase; and the 50S ribosomal protein L7/L12. Other proteins had amino acid sequences homologous with those of various proteins from other bacteria found in protein databases: ClpP; the 10K-S protein; the ORFU phosphoprotein; succinyl-CoA synthetase alpha sub-unit; an inorganic pyrophosphatase; the Fe and/or Mn superoxide dismutase; the nucleoside diphosphate kinase, an amino acid ABC type transporter, and an electron transfer flavoprotein small subunit. Seven proteins were identified with N-terminal sequences not yet reported in databases. The 2-D map established in this study will be the basis for comparative studies of protein expression in Brucella.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of Brucella ovis immunogenic proteins using two-dimensional electrophoresis and immunoblotting.

In a previous report, proteins from Brucella melitensis were characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and N-terminal microsequencing. In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B. ovis, responsible for ram epididymitis and infertility. The combination of 2-D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B. ovis on the 2-D pattern. These proteins comprised cytoplasmic, periplasmic, and some membrane proteins except the major outer membrane proteins. By comparing 2-D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readily identified. Serum from a ram naturally infected with B. ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection. This serum showed antibody reactivity against approximately 82 protein spots. Twenty-one of these proteins were identified either by use of monoclonal antibodies or by N-terminal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu-Zn superoxide dismutase. Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases; NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl-CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/Ile/Val-binding protein precursor, and ClpP. The remaining eight proteins had N-terminal sequences lacking similarity to existing databases entries. Thus, the 2-D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.

Amino Acid Sequence↗