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.

Diagnosis of bovine brucellosis by skin test: conditions for the test and evaluation of its performance.

Brucellergene OCB (Rhône-Mérieux) was used as an allergen to define the intrinsic parameters of a skin test and to compare its properties with serology for the diagnosis of bovine brucellosis. The skin test was also evaluated for its capacity to solve problems associated with false positive reactions in serological tests. The optimal reading delay for the skin test was 72 hours. The brucellosis allergic reaction was two to three times less intense than the tuberculosis allergic reaction. An increase of 1.1 mm or more in the skin thickness was therefore considered to be an adequate cut-off. The specificity calculated for 1192 brucellosis-free animals (including animals from brucellosis-free herds in which false positive serological reactions had been reported) was 99-83 per cent (95 per cent confidence interval [CI] 99-40 to 99-98 per cent). The sensitivity determined from 27 experimentally infected heifers ranged from 93 per cent (95 per cent CI 76 to 100 per cent) to 78 per cent (95 per cent CI 58 to 91 per cent) when measured respectively one and six months after the infection. Allergic reactions could be detected in vaccinated animals up to four-and-a-half years after the vaccination. On the other hand, no sensitisation was recorded in naïve animals after up to eight monthly injections of the allergen. The skin test gave valuable information, in combination with the serological tests, in both acute and chronic brucellosis. The skin test discriminated brucellosis clearly from false positive serological reactions due to infections with Yersinia enterocolitica O9.

Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/c mice.

BALB/c mice were immunized with recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis, a gene coding for a major outer membrane protein. Immunization resulted in the production of specific antibodies to B. melitensis in the serum, the production of which was considerably increased after boosting with a dose ten times lower than the first. A significant specific proliferative response of immune spleen cells to B. melitensis was observed 5 weeks after the first immunization but this response did not persist. Despite the induction of systemic humoral and cellular immune responses by recombinant E. coli expressing the B. melitensis omp31 gene, no significant protection against a challenge with smooth B. melitensis H38S was observed in immunized mice. These results demonstrate that despite the strong antibody response induced in mice, immunization with the recombinant Omp31 of B. melitensis does not confer any protective effect against a virulent smooth B. melitensis. However, its potential protective effect for protection against rough Brucella would be worth testing.

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.

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.

Efficacy of ELISA compared to conventional tests (RBPT and CFT) for the diagnosis of Brucella melitensis infection in sheep.

An indirect ELISA (iELISA) using a B. abortus smooth lipopolysaccharide (S-LPS) as coating antigen and a polyclonal anti-sheep IgG peroxydase-labeled serum as conjugate was developed. The iELISA was assessed in comparison to the Food and Agriculture Organisation/International Atomic Energy Agency (FAO/IAEA) standard bovine ELISA kit (IAEA kit) using an anti-bovine IgG conjugate that cross-reacts with sheep antibodies, and with complement fixation test (CFT) and Rose Bengal Plate Test (RBPT). The evaluation was performed on sera from ewes experimentally infected with Brucella melitensis 53H38 (H38), using negative and positive sheep reference sera. No significant difference was found as regards to the specificity, the lower limit of detection and the estimated sensitivity of the two iELISAs. This suggests that an anti-bovine conjugate could allow the development of only one ELISA protocol for all ruminants. The iELISA, if well standardized, proved to be a good screening test able to be used either alone or in addition to RBPT.

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.

Characterization, occurrence, and molecular cloning of a 39-kilodalton Brucella abortus cytoplasmic protein immunodominant in cattle.

Monoclonal antibodies and polyclonal antisera recognizing a 39-kDa protein (P39) of brucellin, a cytoplasmic extract from Brucella melitensis rough strain B115, were produced. The P39 was purified by anion-exchange chromatography. Eleven of fourteen Brucella-infected cows whose infections had been detected by the delayed-type hypersensitivity (DTH) test with brucellergen also developed a DTH reaction when purified P39 was used as the trigger. The T-cell proliferative responses to P39 of peripheral blood lymphocytes from Brucella-infected cows were also positive. None of the animals infected with other bacterial species that are presumed to induce immunological cross-reactions with Brucella spp. reacted to P39, either in DTH tests or in lymphocyte proliferation assays. A lambda gt11 genomic library of Brucella abortus was screened with a monoclonal antibody specific for P39, and the gene coding for this protein was subsequently isolated. The nucleotide sequence of the P39 gene was determined, and the deduced amino acid sequence is in accordance with the sequence of an internal peptide isolated from P39.

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.

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.

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.

Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification.

DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non-Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after EcoRI digestion.

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.

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.

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.

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.

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.

Outer-membrane protein- and rough lipopolysaccharide-specific monoclonal antibodies protect mice against Brucella ovis.

Brucella ovis, a naturally virulent rough Brucella species, is the aetiological agent of ram epididymitis. The identification of protective antigens is necessary to obtain a safe, specific subcellular vaccine. Monoclonal antibodies (MAbs) directed at both brucella outer-membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS) in a mouse protection test were used to identify potential targets for humoral immunity. Mixtures of MAbs directed at the 16.5-, 25-27-, 31-34- and 36-38-kDa OMPs conferred significant protection 7 days after challenge with reference strain B. ovis 63/290 compared with controls receiving either saline or an anti-brucella O-polysaccharide MAb. Furthermore, an anti-R-LPS MAb tested alone conferred protection at a level comparable with that obtained with the mixture of anti-OMP MAbs. The combination of protective OMP MAbs with the anti-R-LPS MAb was also strongly protective. One combination of OMP MAbs, which bound intensely to B. ovis in vitro, was ineffective. These results indicate that B. ovis OMPs and R-LPS are targets for protective antibodies and that they can be regarded as candidates for ram epididymitis subcellular vaccines.

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.

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.