Biomedical subjects

A Cloeckaert

Publications and source records attributed to A Cloeckaert.

At least 55 records · Page 3Linked to original sources

PubMed1997

Rapid identification of rough Brucella isolates by a latex coagglutination assay with the 25-kilodalton outer membrane protein and rough-lipopolysaccharide-specific monoclonal antibodies.

A latex coagglutination assay was developed to identify rough (R) isolates of Brucella. Latex beads were coated, via protein A, with either an anti-Brucella rough-lipopolysaccharide (R-LPS) monoclonal antibody (MAb) or an anti-Brucella 25-kDa outer membrane protein (Omp25) MAb. Slide agglutination tests were done for 68 strains of Brucella spp., including type strains of all biovars as well as field isolates. Latex beads coated with MAb to R-LPS coagglutinated only R strains, whereas latex beads coated with MAb to Omp25 coagglutinated all the R Brucella isolates except Brucella ovis. Coagglutination was easier to read than agglutination with rabbit R-Brucella-specific antiserum. Thus, this assay accurately differentiates B. ovis from other R Brucella isolates. The latex coagglutination assay can substitute, to advantage, for the current anti-Brucella (R) rabbit monospecific serum.

Antibodies, Monoclonal↗

PubMed1997

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.

Antibodies, Monoclonal↗

PubMed1996

Molecular and immunological characterization of the major outer membrane proteins of Brucella.

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s by selective extraction techniques and classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 kDa and 25-27 kDa OMPs which belong to the group 3 proteins. Variation in apparent molecular mass is essentially due to association with peptidoglycan subunits of different sizes. Two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of homology (> 85%), encode the 36 kDa porin proteins, now named Omp2a and Omp2b proteins respectively. Two genes code for the group 3 OMPs and are named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. Furthermore, all Brucella major OMPs share amino acid sequence homology with the major OMPs RopA or RopB of Rhizobium leguminosarum, which supports the close genetic relationship of brucellae with members of the alpha-2 subdivision of the class Proteobacteria. Another characteristic common to the major OMPs of R. leguminosarum and Brucella is that they are tightly, probably covalently, associated with the peptidoglycan. The major OMP genes display diversity among Brucella species, biovars and strains allowing their differentiation, and the polymorphic markers identified have brought new insights into the evolutionary development of the genus Brucella, antigenic variability of brucellae, and future prospects in the field of vaccine development.

Antigenic Variation↗

PubMed1996

Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep.

We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev. 1 vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.

Amino Acid Sequence↗

PubMed1996

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection.

Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.

Amino Acid Sequence↗

PubMed1996

Purification and antigenic analysis of the major 25-kilodalton outer membrane protein of Brucella abortus.

The major 25-kDa outer membrane protein (Omp25) of Brucella abortus was purified and antigenically characterized by use of monoclonal antibodies (mAbs). Purification was achieved from the sodium dodecyl sulphate-insoluble (SDS-I) cell wall (CW) fraction of vaccine strain B. abortus B19 which was shown by use of mAbs to contain the two major outer membrane proteins of 25 and 36 kDa linked to peptidoglycan, smooth lipopolysaccharide (S-LPS), and rough LPS (R-LPS). Purity of Omp25 was checked with a number of mAbs directed to the different components of the SDS-I fraction. In ELISA, five anti-Omp25 mAbs, which showed significant binding to B. abortus whole cells and which are probably directed to conformational epitopes well-exposed on the bacterial surface, reacted poorly or not at all with the purified Omp25. Addition of R-LPS to purified Omp25 restored the binding capacity of these mAbs, which suggested that R-LPS may play an important role in reconstitution and exposure of conformational epitopes of Omp25. Immunoelectron microscopy showed that Omp25 was inserted into the R-LPS vesicles. Four of these anti-Omp25 mAbs probably recognize the same or closely located epitopes on Omp25, since one of the mAbs conjugated to peroxidase was inhibited in its binding in ELISA by the three others. Other anti-Omp25 mAbs showed strong binding to purified denatured Omp25 and their binding capacity was not affected by the addition of R-LPS to the purified Omp25. Thus, these results confirmed, as defined by the mAbs, the presence of both sequential and at least one conformational epitope on Omp25.

Antibodies, Monoclonal↗

PubMed1996

Competitive enzyme-linked immunosorbent assay using monoclonal antibodies to the Brucella melitensis BP26 protein to evaluate antibody responses in infected and B. melitensis Rev.1 vaccinated sheep.

Competitive enzyme-linked immunosorbent assay (C-ELISA) was performed using 15 monoclonal antibodies (MAbs), specific for Brucella BP26 (previously also called CP28), a periplasmic protein antigen, to investigate antibody responses in naturally and B. melitensis H38 experimentally infected and B. melitensis Rev.1 vaccinated sheep. The antigen preparation consisted of cytosoluble protein extract (CPE) of B. melitensis B115. By combining the C-ELISA results of several MAbs, a high percentage of naturally infected animals were detected which showed different status in the current conventional diagnostic tests. Indeed, 90% of sheep which were positive in the conventional bacteriological and serological tests were positive in C-ELISA. 72% of the bacteriologically negative but serologically and delayed type hypersensitivity positive sheep were also positive in the C-ELISA. Moreover, 79% of the bacteriologically and serologically negative sheep but delayed type hypersensitivity positive were also detected by C-ELISA. Thus, these results confirmed the importance of BP26 as a frequently recognized target of the humoral immune response of infected sheep. The 8 B. melitensis H38 experimentally infected sheep showed various degrees of antibody responses at the 90th day after infection, which was delayed in comparison to that against O-polysaccharide (O-PS). Of the 15 MAbs tested, only one MAb was weakly inhibited (20 to 35% inhibition) by 56% of negative control sera. Furthermore, no antibody response against BP26 was detected in B. melitensis Rev.1 vaccinated sheep. Results of the C-ELISA with the 15 MAbs showed individual variability of the antibody responses against BP26. Thus, it is suggested that several epitopes of BP26 are of interest for diagnosis of B. melitensis infection in sheep.

Animals↗

PubMed1996

Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker.

The dnaK gene and surrounding sequences from reference strains of the six Brucella species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the B. ovis dnaK gene and studied for polymorphism with nine restriction endonucleases. The restriction patterns were identical for all species with all restriction endonucleases tested except for B. melitensis strain 16M that showed a different pattern with EcoRV, consistent with the presence of a single site instead of two for the other Brucella species. The absence of the second EcoRV site for B. melitensis 16M was confirmed by DNA sequence analysis. The second EcoRV site in other Brucella species was located in a 12-bp segment, which was missing from the published dnaK sequence of B. ovis, between the stop codon of the dnaK gene and its putative transcription terminator sequence. The difference between B. ovis strain 63/290 and B. melitensis 16M was due to an additional base-pair in B. melitensis 16M. Subsequently, 71 other field, vaccinal and reference strains of the six Brucella species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the dnaK locus with EcoRV. The presence of a unique EcoRV site was specific to B. melitensis strains. Southern blot analysis of whole genomic DNA digested with EcoRV and with the dnaK gene used as probe also detected a distinct pattern for B. melitensis. These results indicate that both PCR-RFLP and Southern blot analysis of the dnaK locus can be used to distinguish B. melitensis strains from the other Brucella species and may be useful for typing and diagnostic purposes as well.

Base Sequence↗

PubMed1996

Production and characterisation of monoclonal antibodies to Brucella melitensis cytosoluble proteins that are able to differentiate antibody responses of infected sheep from Rev. 1 vaccinated sheep.

Monoclonal antibodies (MAbs) were produced to Brucella melitensis cytosoluble proteins (CP) with apparent molecular masses of 12, 24 and 28 kDa (CP12, CP24 and CP28) which were previously shown by immunoblotting to differentiate antibody responses of infected sheep from those of B. melitensis strain Rev. 1 vaccinated sheep. These MAbs were derived from mice infected with virulent smooth (S) B. melitensis strain H38. Most MAbs obtained were directed to CP28, which indicated (as was shown in infected sheep) that this protein was also highly immunogenic in mice. A large number of MAbs that showed reactivity to CP in ELISA but did not show reactivity in immunoblotting of CP were also obtained and might recognise conformational epitopes of these proteins. MAbs were used to localise CP12, CP24 and CP28. None of the MAbs reacted with whole B. melitensis cells in ELISA but showed reactivity with sonicated bacteria in ELISA, which indicated an internal localisation of these proteins. Among several B. melitensis B115 subcellular fractions tested, the anti-CP12 and anti-CP28 MAbs reacted essentially with the CP extract (CPE) in both ELISA and immunoblotting, whereas the anti-CP24 MAbs reacted with both CPE and cell envelope fraction (CEF)-although with lower intensity to the latter fraction. The internal localisation of these proteins was confirmed by immuno-electron microscopy of thin-sectioned B. melitensis B115 or B. melitensis 16M cells. Immunogold labelling was mainly observed in the cytoplasm and, consequently, CP12, CP24 and CP28 are probably cytoplasmic proteins. Immunoblotting of whole cell lysates with the MAbs also showed the presence of these proteins in all Brucella species and biovars, including the vaccine strains B. melitensis Rev. 1 and B. abortus B19. The use of these MAbs should help further study of antibody responses in sheep and other hosts and may be of considerable value for developing new diagnostic tests for ovine brucellosis.

Animals↗

PubMed1996

Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937-derived phagocytes.

In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat-shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37 degrees C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937-derived phagocytes resulted in long-lasting DnaK induction in the wild-type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30 degrees C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild-type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37 degrees C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature-independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.

Bacterial Proteins↗

PubMed1996

Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev.1-vaccinated sheep.

The problem of differentiating sheep infected with Brucella melitensis from those vaccinated or exposed to cross-reaching organisms has not been resolved by conventional serological tests or through the use of the smooth lipopolysaccharide in primary binding assays. We therefore analyzed sera from ewes experimentally infected with B. melitensis H38, from ewes naturally infected with B. melitensis, and from B. melitensis Rev.1-vaccinated ewes by enzyme-linked immunosorbent assay with three antigenic fractions: O polysaccharide, a cytosoluble protein extract (CPE) from the rough strain B. melitensis B115, and a partially purified cytosoluble protein of 28 kDa (CP28) from the CPE. Immunoglobulin G anti-O polysaccharide and anti-CPE responses were detected in all groups of animals tested (Rev.1 vaccinated and B. melitensis infected). However, false-positive reactions with CPE occurred with sera from Brucella-free ewes. The use of partially purified CP28 abolished these false-positive reactions. Furthermore, no immunoglobulin G antibodies against CP28 were detected in sera from vaccinated ewes, whereas 80% (8 of 10) of ewes experimentally infected with B. melitensis H38 and 89% (25 of 28) of naturally infected ewes showed various degrees of anti-CP28 reactivity (absorbance values of between 0.5 and 2.5). The results obtained with CP28 showed the potential usefulness of this antigen to permit the detection of B. melitensis-infected ewes and their differentiation from B. melitensis Rev.1-vaccinated ones.

Animals↗

PubMed1996

Characterization of a monoclonal antibody specific for Brucella smooth lipopolysaccharide and development of a competitive enzyme-linked immunosorbent assay to improve the serological diagnosis of brucellosis.

The reactivity of monoclonal antibody (MAb) 12G12 was analyzed in regard to the main biovars of Brucella species and some members of the families Enterobacteriaceae and Vibrionaceae which present serological cross-reactions with the smooth lipopolysaccharide (S-LPS) of Brucella species. This MAb was strictly directed against the common specific epitope of the Brucella S-LPS. It recognized all of the smooth Brucella strains and biovars except B. suis biovar 2. In order to improve the specificity of the serological diagnosis of brucellosis, a competitive enzyme-linked immunosorbent assay (cELISA) was developed with the horseradish peroxidase-conjugated MAbs 12G12 and S-LPS of B. melitensis Rev1. The specificity of the cELISA was analyzed with 936 serum samples from healthy cattle. The assay was evaluated with sera from heifers (n = 18) experimentally infected with B. abortus 544. After infection, the performance of the cELISA was in agreement with those of the complement fixation test and the rose Bengal plate test. Finally, the specificity of the assay was also evaluated in regard to false-positive serological reactions by using sera from heifers experimentally infected with Yersinia enterocolitica 0:9 (n = 4) and with field sera presenting false-positive reactions (n = 74). The specificity of the cELISA was greater than the specificities of the complement fixation test and the rose Bengal plate test. Indeed, the new assay detected only 31 of the 101 false-positive serum samples detected by at least one serological test.

Animals↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Cloning, nucleotide sequence, and expression of the gene coding for a ribosome releasing factor-homologous protein of Brucella melitensis.

The gene coding for a Brucella melitensis cytosoluble protein (CP24) that is immunogenic in infected sheep and a major component of brucellin INRA was cloned and sequenced. As in Brucella cells, CP24 was located in the cytoplasm of recombinant Escherichia coli. The amino acid sequence predicted from the cloned gene revealed 48 and 46% identity with the ribosome releasing factor, a protein factor required for release of the 70S ribosome from the mRNA, of E. coli and Haemophilus influenzae Rd, respectively. Sera from naturally infected sheep and sheep experimentally infected with B. melitensis H38 showed antibody reactivity against recombinant CP24.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Cloning, nucleotide sequence, and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein.

The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced. A B. melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10. Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter. omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb. Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da. Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da. The predicted amino acid sequence of B. melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv. viciae 248 and 34.3% identity with Omp25 of B. abortus 544. As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E. coli. The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B. melitensis and recombinant E. coli. The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein. The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective activity and their potential for the development of subcellular vaccines against brucellosis.

Amino Acid Sequence↗

PubMed1995

Identification of sero-reactive Brucella melitensis cytosoluble proteins which discriminate between antibodies elicited by infection and Rev.1 vaccination in sheep.

Distinction between Brucella melitensis infected and vaccinated sheep is needed to fully achieve ovine brucellosis eradication in several countries. For this purpose, we probed immunoblots of cytosoluble protein extract (CPE) of the rough (R) B. melitensis strain B115 with sera of Brucella-free, naturally infected, B. melitensis H38 experimentally infected and B. melitensis Rev.1 vaccinated sheep to identify immunogenic Brucella cytosoluble proteins which may lead to the development of more useful diagnostic tests and which may eventually differentiate vaccinated sheep from infected sheep. Brucella-free sheep sera showed IgM antibody reactivity to protein bands between 19 and 92 kDa. The use of conjugate specific for sheep IgG avoided non specific reactivity to all these bands except to the 39 and 50 kDa bands which were still detected in some Brucella-free sheep sera. In sera of B. melitensis H38 experimentally infected sheep, a specific IgG antibody response was observed against proteins of molecular masses of 19, 24, 28, 32, 39, 50 and 54 kDa. These proteins were also variably detected by IgG of sera from naturally infected sheep. Other proteins of 10, 12, 23, 36, 38, 42, 46, 68, 80 and 92 kDa were also detected by the latter sera. In sera from B. melitensis Rev.1 vaccinated sheep, an IgG antibody response was only observed against proteins with molecular masses of 39 and 50 kDa. These results suggest that the 19, 24, 28, 32 and 54 kDa proteins (the first recognized after experimental infection and that provoked a consistent humoral response during natural infection) could be interesting to develop serological tests for differentiating B. melitensis infection from B. melitensis Rev.1 vaccination.

Animals↗

PubMed1995

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 I immunoblot analysis of the antibody response to Brucella protein antigens in bovine brucellosis.

Sera from three groups of Brucella abortus infected cattle were examined in immunoblots with the following antigens: sodium dodecyl sulfate/mercapto ethanol (SDS/ME) extracts of two rought B. abortus strains (45/20 and RB51) and rough B. ovis, smooth lipopolysaccharides (SLPS) from B. abortus strain 99 and Y. enterocolitica 0:9, and a cytoplasmic extract from smooth B. abortus strain 19-S. The sera groups were: (1) 26 sera from animals, experimentally infected with B. abortus strain 544, which were all positive in the conventional brucellosis serological tests; (2) 152 sera from naturally infected cattle herds with varying titres in the conventional brucellosis tests, and (3) 30 sera from naturally infected cattle with varying titres in the conventional brucellosis tests and from which B. abortus was cultured. B. abortus strain 99 and Y. enterocolitica serotype 0:9 SLPS staining showed up frequently in all sera groups and correlated well with the strength in the conventional brucellosis tests, confirming the immunodominance of SLPS in B. abortus infections. Another immunodominant component of 50-80 kDa was found in the rough B. abortus 45/20 antigen preparation but not in the B. abortus RB51 and in the B. ovis cell extracts. This component was also recognised by sera from Y. enterocolitica 0:9 infected cattle and is probably a protein-lipopolysaccharide complex. Although many of the sera from B. abortus infected cattle with high titres in the conventional brucellosis tests showed complex protein staining patterns in blots, no protein bands other than the 50-80 kDa bands were found to be immunodominant.

Animals↗