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Infection and Immunity, November 2001, p. 7020-7028, Vol. 69, No. 11
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.11.7020-7028.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Minor Nucleotide Substitutions in the omp31 Gene of Brucella ovis Result in Antigenic Differences in the Major Outer Membrane Protein That It Encodes Compared to Those of the Other Brucella Species

Nieves Vizcaíno,1,* Reinhold Kittelberger,2 Axel Cloeckaert,3 Clara M. Marín,4 and Luis Fernández-Lago1

Departamento de Microbiología y Genética, Edificio Departmental, Universidad de Salamanca, 37007 Salamanca,1 and Unidad de Sanidad Animal, Servicio de Investigación Agroalimentaria, Diputación General de Aragón, 50080 Zaragoza,4 Spain; National Centre for Disease Investigation, Ministry of Agriculture and Forestry, Upper Hutt, New Zealand,2; and Station de Pathologie Aviaire et Parasitologie, Institut National de la Recherche Agronomique, Centre de Tours, 37380 Nouzilly, France3

Received 16 July 2001/Accepted 6 August 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

As a result of DNA-DNA hybridization studies, the genus Brucella has been proposed as monospecific (31, 32). However, the classical six-species organization of the genus is still maintained, as it is in accordance with the pathogenicity and host preference characteristics of each species. Moreover, DNA markers distinguishing the Brucella species and some of their biovars have been found (33).

Ovine brucellosis is mainly caused by Brucella melitensis (also responsible for caprine brucellosis) and Brucella ovis, the latter being the most frequent cause of contagious ram epididymitis. Infection by B. ovis reduces fertility in rams, and abortions in ewes have also been reported, constituting an important limiting factor for the development of sheep production in many countries.

Diagnosis of B. ovis infection is mainly performed with serological methods, of which the complement fixation test (CFT) is most extensively used (3). However, the CFT has several disadvantages, including its complexity, lack of sensitivity, prozone phenomena, and incompatibility with hemolyzed or anticomplementary sera (25, 26, 28). In order to find a serological test to substitute for the CFT, several enzyme-linked immunosorbent assays (ELISAs) have been developed (1, 22, 25, 26, 28). All of them use complex antigenic extracts that are not well characterized, and they have never replaced the CFT as the routine diagnostic test. ELISA with the B. ovis rough lipopolysaccharide (LPS) employs a more purified antigen, containing less than 0.8% protein that is not detectable in Western blotting (22), but it is less sensitive than ELISAs with antigenic mixtures, such as the hot saline extract (22). Therefore, the development of serological tests for the improved diagnosis of B. ovis infection requires the identification and characterization of new antigens.

Recently, it has been shown that a humoral immune response against a 29-kDa outer membrane protein (OMP) is detected by electrophoretic immunoblotting in 93 to 100% of sheep experimentally infected with B. ovis (20). The 29-kDa antigen was determined to be composed of two OMPs, Omp25 and Omp31 (19), Omp31 being the major immunodominant antigen (18). A panel of monoclonal antibodies (MAbs) specific for the B. ovis Omp31 protein and sera from B. ovis-infected rams reacted in Western blotting with the Omp31 protein extracted from B. ovis (18). However, the same sera and MAbs exhibited poor reactivity with recombinant B. melitensis Omp31, extracted from recombinant Escherichia coli by a temperature-dependent Triton X-114-based technique (18). It was suggested that differences in the amino acid sequence between the B. ovis and B. melitensis Omp31 proteins might explain this poor reactivity (18).

Interest in the Brucella Omp31 protein is determined not only by its usefulness for diagnostic purposes but also by its role in protective immunity against B. ovis infection. Passive protection experiments with a MAb specific for the B. melitensis Omp31 protein have shown that Omp31 is a promising candidate for the development of subcellular vaccines against infections caused by B. ovis (5, 6).

In the present work we have determined the nucleotide sequence of omp31 from all the Brucella species and biovar reference strains, with the exception of Brucella abortus, which lacks omp31 (36). We have shown that the nucleotide substitutions found in the B. ovis omp31 gene result in antigenic differences as measured by reactivity with MAbs specific for Omp31 and sera from B. ovis-infected rams. The epitope mapping of the B. ovis Omp31 protein, by using a panel of MAbs and recombinant DNA techniques, is also presented.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and plasmids. All Brucella strains (Table 1), except B. ovis 020, were obtained from the Institut National de la Recherche Agronomique Brucella Culture Collection, Nouzilly, France. B. ovis 020 was isolated in New Zealand. Bacteria were grown on tryptic soy agar (Difco Laboratories, Detroit, Mich.) supplemented with 0.1% yeast extract (Difco Laboratories). For B. ovis strains, 5% horse serum (GibcoBRL, Life Technologies, Barcelona, Spain) was also added to the culture medium. The strains were checked for purity and species and biovar characterization by standard procedures (2). E. coli JM109 (Promega, Madison, Wis.) bearing each recombinant plasmid was cultured on Luria-Bertani medium containing 50 µg of ampicillin ml-1, with isopropyl-1-thio-beta -D-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (X-Gal) when necessary. Recombinant E. coli used for Western blotting and ELISA was cultured in the presence of IPTG as described previously (15).

                              
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  		TABLE 1.   Brucella strains used in this study

Plasmid pNV3123 bears the B. melitensis 16M omp31 gene and was constructed as described previously (15). Briefly, omp31 was PCR amplified with primers 31sd and 31ter and cloned into plasmid pCRII (TA cloning kit; Invitrogen, San Diego, Calif.). The insert of the recombinant plasmid was excised by digestion with SacI and XbaI and cloned into pUC19 (Invitrogen) digested with the same enzymes. B. melitensis 16M omp31 is expressed in pNV3123 under the control of the lacZ promoter, and its expression is induced by IPTG. Plasmid pNV3147 is the result of cloning the PCR-amplified (primers 31sd and 31ter) B. ovis 63/290 omp31 gene into pGEM-T (Promega). B. ovis omp31 is also expressed in pNV3147 under the control of the lacZ promoter. To construct pNV31114 and pNV31115, a fragment of the B. melitensis 16M and B. ovis 63/290 omp31 genes, respectively, was PCR amplified with primers 3148 and 3183 and cloned into pGEM-T Easy (Promega). Plasmid pNV31118 is pGEM-T Easy into which a PCR-amplified fragment of B. ovis DNA containing omp31 and adjacent DNA at both sides of the gene has been cloned. This plasmid was used for epitope mapping of the B. ovis Omp31 protein (see below). The remaining plasmids were obtained during the epitope mapping of the B. ovis 63/290 Omp31 protein (see below) and contain different fragments of the B. ovis 63/290 omp31 gene cloned in pGEM-7Zf (Promega). All of them synthesize a fragment of B. ovis 63/290 Omp31 as a fusion protein with the lacZ-encoded protein.

MAbs and sera. Supernatants of the hybridoma cultures were used as sources of MAbs. MAb A59/10F09/G10 was raised against the B. melitensis Omp31 protein and was obtained as previously described (8). MAbs 8F2, 9C2, 11E7, 12G7, 4B2, 11E3, 14D5, 17E8, 12H9, and 18B7 were raised against the B. ovis Omp31 protein and obtained as described previously (18). MAb A01/08H06/G02 was raised against the B. ovis Omp31 protein and was obtained by infection of BALB/c mice with B. ovis Reo 198 following a protocol previously described (8).

Sera from Brucella-free sheep (n = 4), naturally B. melitensis-infected ewes (n = 11), and naturally B. ovis-infected rams (n = 12) were obtained at the Unidad de Sanidad Animal, Servicio de Investigación Agroalimentaria, Disputación Genêral de Aragón, Zaragoza, Spain. B. melitensis or B. ovis was isolated from all the infected ewes and rams, respectively. Sera from B. melitensis-infected ewes gave positive reactions in CFT and ELISA with a smooth Brucella extract and sera from B. ovis-infected rams were positive in CFT and ELISA with B. ovis hot saline extract. Sera from Brucella-free sheep were negative in CFT and ELISA.

Immunological techniques. Indirect ELISA (iELISA) was performed as described previously (37). Plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated with either nonsonicated or sonicated bacterial suspensions at an optical density (OD) of 1.0 at 600 nm. MAbs were tested at a 1/5 dilution, and the goat anti-mouse immunoglobulin G (IgG) (Fc specific)-peroxidase conjugate (Sigma, St. Louis, Mo.) was used at a 1:4,000 dilution. A solution of 2,2'-azino-di-(3-ethylbenzothiazoline-sulfonic acid) (ABTS; Boehringer Mannheim, Mannheim, Germany) was used as the substrate. OD values were recorded at 405 nm, against a 630-nm reference filter, in a microplate reader 550 (Bio-Rad, Hercules, Calif.). A sonicated E. coli (pGEM-7Zf) suspension was used as a negative control for reactivity of the Omp31-specific MAbs.

For colony blotting, plates were overlaid with a 0.45-µm-pore-size nitrocellulose membrane and kept for 2 h at 37°C. Next, the membranes were placed for 10 min over a filter paper soaked with 10% sodium dodecyl sulfate (SDS) and washed three times with Tris-buffered saline (TBS; 0.15% NaCl, 10 mM Tris-HCl [pH 7.5]). The membranes were saturated for 30 min with TBS-1% skim milk and incubated overnight with the Omp31-specific MAbs diluted 1/5 in TBS-0.33% skim milk. After three washing steps with TBS-0.05% Tween-20, the membranes were incubated for 1 h with a goat anti-mouse IgG (Fc specific)-peroxidase conjugate (Sigma) diluted 1:500 in TBS-0.33% skim milk and washed three times with TBS. The reaction was developed with TBS containing 0.06% 4-chloro-1-naphthol (Sigma) and 5 mM H2O2.

For Western blotting, SDS-polyacrylamide gel electrophoresis (PAGE) was performed as described previously (21), with IPTG-induced recombinant E. coli resuspended in Laemmli sample buffer at an OD (600 nm) of 3 and boiled for 10 min (20 µl per lane). After electrophoresis, proteins were transferred for 75 min, at 0.8 mA cm-2, to a nitrocellulose membrane. Detection of the protein bands reacting with the Omp31-specific MAbs was performed as described for colony blotting after the saturation step. Sera from Brucella-free, B. melitensis-infected, or B. ovis-infected sheep were adsorbed with E. coli(pGEM-7Zf) prior to Western blotting to remove cross-reactivity with E. coli proteins. Briefly, 60 µl of serum, diluted in 3 ml of TBS-0.33% skim milk (1:50 dilution), was incubated overnight with a nitrocellulose strip of E. coli(pGEM-7Zf) (SDS-PAGE was performed with 20 µl of an IPTG-induced culture adjusted at an OD at 600 nm of 20 in Laemmli sample buffer per lane, and proteins were transferred to nitrocellulose). Then, sera were incubated for 3 h with 200 µl of sonicated and boiled E. coli(pGEM-7Zf) (from an IPTG-induced culture adjusted in H2O to an OD value of 40 at 600 nm) and centrifuged at 10,000 × g for 5 min, and the supernatants were collected. Adsorbed sera were tested against recombinant E. coli in Western blotting that was performed as described for colony blotting after the saturation step, but using a donkey anti-sheep IgG (whole molecule)-peroxidase conjugate (Sigma).

DNA amplification and sequencing. PCR was performed with the Expand long-template PCR system (Boehringer Mannheim) according to the instructions of the manufacturer, using 100 ng of Brucella DNA template, extracted as previously described (35), and a 2 µM concentration of each primer. Cycling conditions were those described previously (35). All the primers were selected according to the published sequence for the B. melitensis 16M omp31 and adjacent DNA to both sides of the gene (35). DNA sequencing was performed by primer-directed dideoxy method (27) with an ABI Prism 37 DNA sequencer (Perkin-Elmer, Foster City, Calif.).

The omp31 gene was PCR amplified from the different Brucella species and biovars with primers 31sd (5'-TGACAGACTTTTTCGCCGAA-3') and 31R2 (5'-TATGGATTGCAGCACCGC-3'). The PCR products were electrophoresed through an agarose gel, purified from the gel with the Geneclean II kit (Bio 101, La Jolla, Calif.), and sequenced with primers 31ter (5'-CATTCAGGACAATTCCCGCC-3') and omp31-2 (5'-GCAGACTTGACCTTACCA-3') located in the reverse strand of omp31.

PCR-amplification of the omp31 DNA fragment from B. melitensis 16M and B. ovis 63/290, cloned in pNV31114 and pNV31115, respectively, was accomplished with primers 3148 (5'-TCAACGCCGGTTACGCAG-3') and 3183 (5'-CCGACGAAGCCGCCAGCT-3').

The B. ovis 63/290 omp31 gene cloned in pNV3147 was amplified with primers 31sd and 31ter, and the B. ovis 63/290 DNA fragment, containing omp31 and adjacent DNA on both sides of the gene, cloned in pNV31118 was amplified with primers 31D (5'-CGTACATATTGGCGAGGG-3') and 31R2.

The B. ovis 63/290 omp31 fragments cloned in pNV31120, pNV31121, pNV31131, pNV31132, pNV31135, pNV31136, pNV31138, pNV31141, pNV31144, and pNV31148 were sequenced with the universal and reverse pUC19 primers, using the plasmid DNA obtained with the Wizard Plus SV miniprep system (Promega) as specified by the manufacturer.

Epitope mapping. Recombinant plasmid pNV31118, containing B. ovis 63/290 omp31 and adjacent DNA to both sides of the gene, was digested with EcoRI and the DNA insert purified from an agarose gel after electrophoresis by using the Geneclean II kit. Approximately 6 µg of the insert was randomly digested with DNase I (Boehringer Mannheim), for 6 min at 24°C, as described previously (34). The resulting fragments were resolved by agarose gel electrophoresis. Fragments sizing between 100 and 500 bp were purified from the gel with the Geneclean II kit, end repaired by treatment with T4 DNA polymerase (Boehringer Mannheim) in the presence of deoxynucleotide triphosphates, and then ligated, in the SmaI site of lacZ, to pGEM-7Zf (Promega). E. coli JM109 was transformed with the ligation mixture and plated on Luria-Bertani agar containing ampicillin, IPTG, and X-Gal. Bacterial colonies were replated and screened by colony blotting with the Omp31-specific MAbs as described above. The positive colonies with one or more MAbs were selected, their plasmid DNA was extracted, and the insert DNA was sequenced and converted into amino acids. The shortest region of Omp31 common to all the plasmids giving reactivity with an individual MAb delimits its specific epitope.

DNA and protein analysis. Multiple DNA and amino acid alignments were performed with CLUSTAL W (29) (http://www2.ebi.ac.uk/clustalw/). Hydrophilicity, antigenic index, surface probability, and flexible regions were determined with the DNAStar Protean program (DNASTAR, Inc., Madison, Wis.).

Nucleotide sequence accession numbers. The omp31 nucleotide sequences of the 14 Brucella strains have been deposited in the GenBank/EMBL/DDBJ databases under accession numbers AF366061 to AF366074.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Sequencing of omp31 in the Brucella species and biovars. The omp31 gene was PCR amplified and sequenced from all recognized Brucella species and biovar reference strains. The seven biovars of B. abortus were not included in this study, as they lack omp31 (36). The alignment of the Omp31 amino acid sequences, deduced from the determined nucleotide sequence, is shown in Fig. 1. The Omp31 amino acid sequences of B. melitensis 63/9 (biovar 2), B. suis 1330 (biovar 1), B. suis 686 (biovar 3), B. suis 513 (biovar 5), and B. neotomae 5K33 were identical to that of the published B. melitensis 16M (biovar 1) Omp31 protein (34) and are not included in the figure. B. melitensis Ether (biovar 3), B. suis 40 (biovar 4), and B. canis RM6/66 displayed only one specific amino acid substitution compared to the B. melitensis 16M Omp31 protein. The B. suis Thomsen (biovar 2) Omp31 protein differed in two amino acids, while B. ovis 63/290 was revealed as the most differing strain, with seven amino acid differences compared to the B. melitensis 16M Omp31 protein. The Omp31 amino acid sequences of four other B. ovis strains (Table 1), from different geographical origins, were also analyzed and were identical to that obtained for the B. ovis 63/290 reference strain (data not shown).


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  		FIG. 1.   Alignment of the Omp31 amino acid sequences from the Brucella species and biovar reference strains. Amino acid differences for each strain in comparison with the B. melitensis 16M Omp31 protein are highlighted in black. The two immunodominant regions of the B. ovis protein identified in this work by mapping with MAbs are highlighted in grey. M1, B. melitensis 16M (biovar 1); M3, B. melitensis Ether (biovar 3); S2, B. suis Thomsen (biovar 2); S4, B. suis 40 (biovar 4); C, B. canis RM6/66; O, B. ovis 63/290.

At the DNA level, the three biovars of B. melitensis differed from the other Brucella species and biovars at nucleotide position 6 of omp31, but this difference did not lead to a change in the encoded amino acid. The Brucella species and biovars showing differences in the Omp31 amino acid sequence, compared to the B. melitensis 16M Omp31 sequence, displayed the same number of differences in the omp31 nucleotide sequence with the exception of B. ovis (data not shown). The B. ovis omp31 gene showed nine differences in the nucleotide sequence (data not shown) while only seven differences were detected at the amino acid level. One of the two additional nucleotide changes did not alter the encoded amino acid, and the second one was located in a codon showing two nucleotide substitutions compared to the B. melitensis 16M omp31 gene that altered the encoded amino acid.

Reactivity of a panel of MAbs specific for Omp31 against the recombinant Omp31 protein from B. melitensis 16M and B. ovis 63/290. Eleven MAbs raised against the B. ovis Omp31 protein and one raised against that of B. melitensis were used to analyze the antigenic properties of the Brucella Omp31 proteins.

The 11 MAbs raised against the B. ovis Omp31 protein reacted in iELISA, with variable intensities, with either sonicated B. ovis 020 or sonicated recombinant E. coli(pNV3147), synthesizing the B. ovis Omp31 (Table 2). In general, the OD values of the 11 MAbs were higher with B. ovis 020 cells than with recombinant E. coli(pNV3147) cells (Table 2). However, it is difficult to determine if this fact is due to a change in the antigenic properties of the recombinant B. ovis Omp31 protein synthesized in E. coli(pNV3147) or to a different relative amount of Omp31 at the surface of B. ovis and recombinant E. coli(pNV3147). The 11 MAbs also reacted with nonsonicated B. ovis 020 cells (Table 2), although the OD values were lower than those obtained with sonicated B. ovis 020, as has also been reported for other OMPs (10), showing that the epitopes recognized by the MAbs on the B. ovis Omp31 protein are exposed on the bacterial surface. In contrast, only 2 of the 11 MAbs (8F2 and A01/08H06/G02) gave a positive reaction, which was very weak, with sonicated recombinant E. coli(pNV3123) synthesizing the B. melitensis Omp31 protein (Table 2). MAb A59/10F09/G10, raised against B. melitensis Omp31, reacted in iELISA to a similar extent with either recombinant E. coli(pNV3123) or E. coli(pNV3147) (Table 2).

                              
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  		TABLE 2.   Reactivity in iELISA of the twelve Omp31-specific MAbs with B. ovis 020, recombinant E. coli (pNV3147), and recombinant E. coli (pNV3123)

The 11 MAbs raised against the B. ovis protein recognized in Western blotting the recombinant Omp31 protein from B. ovis 63/290 synthesized in E. coli (pNV3147) (Fig. 2A). However, only four of them (8F2, 9C2, 11E7 and 12G7) reacted, but very weakly, with the recombinant Omp31 protein from B. melitensis 16M synthesized in E. coli(pNV3123) (Fig. 2B). MAb A59/10F09/G10, raised against the B. melitensis Omp31 protein, strongly reacted with the recombinant B. melitensis Omp31 protein [E. coli(pNV3123)] (Fig. 2B) and to a lesser extent with the recombinant B. ovis Omp31 protein [E. coli(pNV3147)] (Fig. 2A).


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  		FIG. 2.   Reactivity in Western blotting of Omp31-specific MAbs with the recombinant B. ovis 63/290 Omp31 protein (A) and the recombinant B. melitensis 16M Omp31 protein (B) synthesized in E. coli (pNV3147) and E. coli (pNV3123), respectively. Positions of protein molecular mass markers are shown on the left. MAbs specific for B. ovis Omp31: 8F2 (lanes 1), 9C2 (lanes 2), 11E7 (lanes 3), 12G7 (lanes 4), 4B2 (lanes 5), 11E3 (lanes 6), 14D5 (lanes 7), 17E8 (lanes 8), 12H9 (lanes 9), 18B7 (lanes 10), and A01/08H06/G02 (lanes 11). MAb A59/10F09/G10 is specific for the B. melitensis Omp31 protein (lanes 12).

Reactivity of sera from naturally B. melitensis- and B. ovis-infected sheep with the recombinant B. melitensis and B. ovis Omp31 proteins. Twelve sera from naturally B. ovis-infected rams were tested in Western blotting against the recombinant B. ovis Omp31 protein synthesized in E. coli(pNV3147). All of them showed reactivity with the recombinant B. ovis Omp31 protein (Fig. 3A, lanes 2 to 13) as they developed a protein band with the same apparent molecular mass as that revealed with the Omp31-specific MAb A01/08H06/G02 (Fig. 3A, lane 1). Some other protein bands were observed in some strips and presumably correspond to E. coli proteins reacting with serum antibodies developed in response to common exposure of animals to this bacterium that were not completely removed after adsorption. Sera from Brucella-free ewes did not react with the Omp31 protein (Fig. 3A, lanes 14 to 17).


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  		FIG. 3.   Reactivity in Western blotting of sera from B. ovis-infected rams against recombinant E. coli (pNV3147), synthesizing the B. ovis 63/290 Omp31 protein (A), and recombinant E. coli (pNV3123), synthesizing the B. melitensis 16M Omp31 protein (B) (lanes 2 to 13). Positions of protein molecular mass markers are shown on the left. Lanes 1, MAb A01/08H06/G02 (A) and MAb A59/10F09/G10 (B). Sera from B. ovis-infected rams: 9163 (lanes 2), 13001 (lanes 3), 14001 (lanes 4), 76795 (lanes 5), 78872 (lanes 6), 9248 (lanes 7), 1EM (lanes 8), 2EM (lanes 9), 8EM (lanes 10), 9EM (lanes 11), 11EM (lanes 12), and 78889 (lanes 13). Sera from Brucella-free ewes: 6011 (lanes 14), 6008 (lanes 15), 5118 (lanes 16), and 5117 (lanes 17).

When the sera from naturally B. ovis-infected rams were tested against the recombinant B. melitensis Omp31 protein [E. coli(pNV3123)], only four of them reacted with the recombinant protein (Fig. 3B, lanes 2, 4, 12, and 13).

Eleven sera from naturally B. melitensis-infected ewes were tested in Western blotting against the recombinant B. melitensis Omp31 protein synthesized in E. coli(pNV3123). None of them reacted with the recombinant protein (Fig. 4), suggesting that Omp31 does not induce an important humoral immune response in sheep infected by B. melitensis.


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  		FIG. 4.   Reactivity in Western blotting of sera from B. melitensis-infected ewes against recombinant E. coli (pNV3123), synthesizing the B. melitensis 16M Omp31 protein (lanes 2 to 12). Positions of protein molecular mass markers are shown on the left. Lane 1, MAb A59/10F09/G10. Sera from B. melitensis-infected ewes: 14052 (lane 2), 2588 (lane 3), 2712 (lane 4), 518 (lane 5), 14307 (lane 6), 17590 (lane 7), 18179 (lane 8), 17165 (lane 9), 18209 (lane 10), 18011 (lane 11), and 17002 (lane 12).

Epitope mapping of the B. ovis Omp31 protein. As the Western blotting assays with sera from B. ovis-infected rams revealed the recombinant B. ovis Omp31 protein as a promising antigen for the diagnosis of B. ovis infection in rams, we searched for Omp31 immunodominant epitopes. The insert DNA of pNV31118, containing the B. ovis omp31 gene and adjacent DNA on both sides of the gene, was digested with DNase I, and the fragments ranging between 100 and 500 bp were cloned in plasmid pGEM-7Zf. Fragments of the B. ovis omp31 gene cloned in phase with lacZ will synthesize regions of the Omp31 protein as fusion proteins with the lacZ-encoded protein. Reactivity of each fusion protein with the MAbs specific for the B. ovis Omp31 protein was tested in colony blotting. The insert DNA contained in the plasmids of the bacterial colonies giving a positive reaction with the MAbs was sequenced and translated into amino acids according to the known B. ovis Omp31 sequence. The shortest region of Omp31 common to all the plasmids giving reactivity with an individual MAb delimits its specific epitope (Fig. 5). Plasmid pNV31115 was also included in the study. It contains, cloned in phase with lacZ of pGEM-T Easy, the B. ovis omp31 gene region corresponding to the epitope recognized by MAb A59/10F09/G10 on the B. melitensis Omp31 protein (amino acids 48 to 83) (34).


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  		FIG. 5.   (A) Reactivity in colony blotting of the MAbs raised against the B. ovis Omp31 protein with the fragments of Omp31 synthesized as fusion proteins with the lacZ-encoded protein in E. coli. *, MAb A01/08H06/G02. (B) Hydrophilicity plot, antigenic index, surface probability exposure, and flexible regions of the B. ovis Omp31 protein determined with the DNAStar Protean program. The two immunodominant regions identified by the epitope mapping of the protein are indicated with a grey shaded area.

MAb 14D5, a MAb showing a very weak reaction in Western blotting against the recombinant B. ovis Omp31 protein synthesized in E. coli(pNV3147) (Fig. 2A), did not show reactivity with the fusion proteins of any of the recombinant plasmids. This MAb also reacted weakly in iELISA with both sonicated B. ovis and E. coli(pNV3147) cells (Table 2), and its specific epitope might be poorly accessible on Omp31 and the fusion proteins. MAb 18B7 recognized only a fusion protein containing the amino acids 13 to 108, and it gave a weak reaction against Omp31 in Western blotting (Fig. 2A) and iELISA (Table 2), which might also be related to poor accessibility of its specific epitope. The specific epitope for MAbs 8F2, 9C2, 11E7, and 12G7 was delimited to a region of 45 amino acids located near the C end of the B. ovis Omp31 protein (amino acids 180 to 224) that is predicted to contain two fragments of high antigenic index (Fig. 5). Reactivity of these four MAbs against recombinant E. coli(pNV31138), synthesizing amino acids 180 to 240 of Omp31 (Fig. 5), was tested in iELISA and Western blotting. Surprisingly, while they clearly reacted in colony blotting with E. coli(pNV31138), they did not show reactivity in Western blotting or iELISA (data not shown). This suggests that binding of these MAbs to their specific epitope is highly dependent on the conformation of the protein, which would be different in the three immunological tests. Considering that the specific epitope for MAbs 8F2, 9C2, 11E7, and 12G7 is located in a region of Omp31 that has the same amino acid sequence in B. ovis and B. melitensis (Fig. 1), similar reactivities with the B. melitensis and B. ovis Omp31 proteins would be expected for these MAbs. However, they showed an intense reaction in iELISA and Western blotting with sonicated cells of recombinant E. coli(pNV3147) (B. ovis Omp31) (Fig. 2A; Table 2) but poor reactivity with sonicated cells of recombinant E. coli(pNV3123) (B. melitensis 16M Omp31) (Fig. 2B; Table 2). These results seem to confirm that binding of these four MAbs to their specific epitope is highly influenced by the conformation of the protein. The seven amino acid differences detected between the B. melitensis and B. ovis Omp31 proteins (Fig. 1) would modify the conformation of Omp31, either changing the shape of the epitope(s) or hindering the accessibility of the MAbs. The epitope for MAb 4B2 was located between amino acids 149 and 182, and four other MAbs (11E3, 17E8, 12H9, and A01/08H06/G02) recognized a region matching the epitope previously identified on the B. melitensis Omp31 protein for MAb A59/10F09/G10 (34) (Fig. 5). These four MAbs reacted strongly in Western blotting with the fusion protein synthesized in E. coli(pNV31115) that contains amino acids 48 to 83 of the B. ovis Omp31 protein (for example, see the reactivity of MAb A01/08H06/G02 in Fig. 6, lane 1), but they did not react with the same region of the B. melitensis 16M Omp31 protein synthesized in E. coli(pNV31114) (data not shown). MAbs 11E3, 17E8, 12H9, and A01/08H06/G02, mainly 12H9 and A01/08H06/G02, react better in Western blotting than in iELISA with the B. ovis Omp31 protein (Fig. 2A; Table 2), and they show a strong reactivity in Western blotting with the fusion protein of E. coli(pNV31115) (amino acids 48 to 83 of the B. ovis Omp31 protein) (Fig. 6). Their specific epitope(s) would be better exposed under the denaturing conditions of Western blotting.


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  		FIG. 6.   Reactivity in Western blotting of sera from B. ovis-infected rams (lanes 2 to 13) against amino acids 48 to 83 of the B. ovis Omp31 protein synthesized as a fusion protein with the lacZ-encoded protein in E. coli (pNV31115). Positions of protein molecular mass markers are shown on the left. Lane 1, MAb A01/08H06/G02. Sera in the individual lanes are as in Fig. 3.

Reactivity of sera from B. ovis-infected rams with pNV31115. The epitope recognized by MAb A59/10F09/G10 on the B. melitensis Omp31 protein was located in the most hydrophilic region of the protein (34), and four MAbs (11E3, 17E8, 12H9 and A01/08H06/G02) raised against the B. ovis Omp31 protein are specific for an epitope(s) located in the same region (Fig. 5). This region of the B. ovis Omp31 protein has been shown to be surface exposed (4, 8, 34) (Fig. 5) and is predicted to have a high antigenic index (Fig. 5). Accordingly, we analyzed the reactivity, in Western blotting, of sera from B. ovis-infected rams against this region of the B. ovis Omp31 protein synthesized as a fusion protein in E. coli(pNV31115).

MAb A01/08H06/G02 reacted with the fusion protein of E. coli(pNV31115) (Fig. 6, lane 1). The 12 sera from B. ovis-infected rams reacted in Western blotting with the recombinant B. ovis Omp31 protein of E. coli(pNV3147) (Fig. 3A), but only four of them gave reactivity with the fusion protein of E. coli(pNV31115) (Fig. 6, lanes 2, 3, 5, and 6). However, these four sera gave a very strong positive reaction.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The Brucella Omp31 protein has been identified as an interesting candidate for both the diagnosis of infections caused by B. ovis in rams (18, 19) and the development of a subcellular vaccine against ram epididymitis caused by B. ovis (5, 6). However, the use of the Brucella Omp31 protein for diagnostic and vaccination purposes requires the antigenic characterization of the protein for optimal results.

In a previous study, sera from B. ovis-infected rams and MAbs specific for the B. ovis Omp31 protein showed good reactivity with Omp31 extracted from B. ovis but poor reactivity with the recombinant B. melitensis Omp31 protein (18). The different behavior of the MAbs and sera against the B. melitensis and B. ovis Omp31 proteins might be explained if the two Brucella species display different amino acid sequences for Omp31. Although the six species of the genus Brucella display a high degree of DNA homology (16, 17, 31, 32), recent studies show that the six species and some of their biovars can be differentiated on the basis of DNA polymorphism (33). Additionally, it has been shown that in some genes minor differences in the nucleotide sequence between the Brucella species lead to changes in the antigenic behavior of the encoded proteins (10). Accordingly, we have determined the nucleotide sequence for omp31 in all Brucella species and biovar reference strains, searching for DNA variability that might be related to Omp31 antigenic differences between the Brucella species. The gene was quite conserved in the genus (Fig. 1), B. ovis being the most divergent species, which is in accordance with other studies regarding DNA polymorphism (9, 10, 11, 23). The five B. ovis strains from different geographical origins included in this study displayed exactly the same nucleotide sequence, showing that the omp31 gene has not evolved within the species B. ovis. The differences observed in omp31 between the Brucella species and biovars are in accordance with previous results obtained by PCR-restriction fragment length polymorphism about the polymorphism in the Brucella omp31 gene (36).

The nine nucleotide differences found between the B. melitensis and B. ovis omp31 genes (Fig. 1) strongly modify the antigenic properties of the encoded proteins (Fig. 2 and 3; Table 2). A modification of the antigenic behavior has also been demonstrated for another B. ovis OMP. A deletion of 36 bp in the B. ovis omp25 gene, compared to omp25 of the other Brucella species, resulted in an antigenic shift in the encoded protein, as shown by the binding properties of a panel of anti-Omp25 MAbs (10). It has been suggested that the Brucella species must be considered clonal populations that have evolved separately in order to survive in each animal host (24). This hypothesis was raised considering that each Brucella species preferentially infects an animal host and survives almost exclusively in infected hosts and that no mechanisms of genetic exchange have been observed in this genus (24). Thus, phenotypic and genotypic differences observed between the Brucella species might be related to a particular adaptation mechanism to survive in the host. B. ovis shows a restricted host preference, infecting sheep almost exclusively and having a preference for male reproductive organs, while most other Brucella species have a wider range of susceptible animal species and have a preference for placental tissues. The higher degree of divergence observed in B. ovis than in the other Brucella species might be a result of its adaptation to survive in the host. The three major OMPs sequenced in all the species of the genus Brucella (Omp2, Omp25, and Omp31) display in B. ovis the highest number of differences (9, 10, 11; this work), and even these differences modify the antigenic properties of the protein (10; this work). Protein polymorphism of bacterial surface antigens, as is the case for Omp2, Omp25, and Omp31, is considered an important mechanism for survival in the host, allowing either evasion of the immune system or modulation of the host range (7).

Reactivity of sera from B. ovis naturally infected rams against recombinant B. ovis Omp31 in Western blotting (Fig. 3) reveals recombinant B. ovis Omp31 as a promising candidate for the diagnosis of B. ovis infections in rams. These results are in accordance with previous observations where good reactivity of sera from infected rams was obtained with the Omp31 protein extracted from B. ovis (18). The antigenic differences detected between the B. melitensis and B. ovis Omp31 proteins (Fig. 2 and 3; Table 2) may explain why the same sera gave poor reactivity with the recombinant B. melitensis Omp31 protein (18). Accordingly, the best antigen for an Omp31-based diagnostic test for B. ovis infection would be the B. ovis Omp31 protein and not the B. melitensis protein, but further analysis should be performed with a larger number of sera to evaluate its real usefulness. Likewise, the B. ovis Omp31 protein should be more suitable for the evaluation of protective activity of Omp31 against B. ovis infection.

The antibody response against Omp31 was also tested in ewes naturally infected by B. melitensis. Eleven sera were tested in Western blotting against the recombinant B. melitensis Omp31 protein, but none of them gave a positive reaction (Fig. 4). Although the number of sera tested was limited, these results suggest that antibodies against Omp31 do not highly contribute to the overall humoral immune response induced in sheep infected by B. melitensis and, therefore, that Omp31 would not be a good diagnostic antigen for B. melitensis infections in sheep. Although it must be taken into account that B. melitensis and B. ovis display different pathogenic characteristics, the absence of a detectable humoral immune response against Omp31 in B. melitensis-infected ewes might be related to the presence of O polysaccharide chains in the B. melitensis LPS which might reduce the immunogenic properties of Omp31. A better reactivity with rough Brucella strains than with smooth strains has been observed for MAbs specific for Omp31 and other Brucella OMPs (4, 8), suggesting that the LPS O polysaccharide chains reduce the antigenic properties of these OMPs, probably masking the epitopes to the antibodies. Moreover, while the Omp31-specific MAb A59/10F09/G10 showed protective activity in mice against infection by naturally rough B. ovis (5, 6), immunization of mice with E. coli(pNV3123) induced good levels of anti-Omp31 antibodies, but they were not protective against infection by smooth B. melitensis (15). These results also suggest that the smooth LPS hinders the binding of the antibodies to Omp31. The presence of LPS O chains in the bacterial surface might modify not only the antigenic properties of Omp31 but also its immunogenic properties. This would not happen in B. ovis, as this bacterium lacks O chains.

Epitope mapping of the B. ovis Omp31 protein using MAbs identified two immunodominant regions on the protein. Four MAbs recognize a region of the protein located near the C end with the same amino acid sequence as the corresponding region of the B. melitensis Omp31 protein. Surprisingly, these four MAbs reacted weakly in Western blotting and only one of them reacted very weakly in iELISA with the recombinant B. melitensis Omp31 protein (Fig. 2B; Table 2). These results suggest that the reactivity of these MAbs would be highly dependent on the conformation of the protein that would be different between B. ovis and B. melitensis due to the seven amino acid differences observed between Omp31 of both species (Fig. 1). This C-terminal region of the B. ovis Omp31 protein is predicted to contain stretches of amino acids with high antigenic index, flexibility, and relative surface exposure probability (Fig. 5), three factors that usually characterize the regions containing the most defined epitopes (12, 13, 14, 30). Four other MAbs were specific for a hydrophilic region of the B. ovis Omp31 protein (amino acids 48 to 83) with a high antigenic index (Fig. 5) that seems to be well exposed on the bacterial surface (4, 8, 34) (Fig. 5) and that displays three of the seven amino acid differences found with the B. melitensis Omp31 protein (Fig. 1). The four MAbs reacted with the recombinant B. ovis Omp31 protein (Fig. 2A, lanes 6, 8, 9 and 11; Table 2), but they did not react with the recombinant B. melitensis Omp31 protein (Fig. 2B; Table 2), demonstrating that minor differences in the amino acid sequence of Omp31 strongly modify its antigenic properties. According to these results and to those obtained with sera from B. ovis-infected rams against the recombinant B. melitensis and B. ovis Omp31 proteins (Fig. 3), it was thought that this hydrophilic region of the B. ovis Omp31 could contribute to a great extent to the humoral immune response specific for Omp31 in rams infected by B. ovis. However, while all the sera reacted with the whole recombinant B. ovis Omp31 protein (Fig. 3A), only four of them reacted with the fusion protein containing amino acids 48 to 83 of the B. ovis Omp31 protein (Fig. 6). Thus, the humoral immune response specific against the B. ovis Omp31 might involve conformational epitopes or linear epitopes not identified in this study. The epitope mapping of the protein has been accomplished by using MAbs raised against Omp31 in mice, and the antibody response induced in mice does not necessarily involve the same epitopes as that in sheep. Moreover, to obtain the MAbs specific for the B. ovis Omp31 protein, mice were given boosters of a denatured B. ovis Omp31 (18, 19), and the hybridomas were selected with the same antigen, which probably underestimates the antibody response generated against Omp31 during the course of infection. The four sera reacting with the hydrophilic region of the B. ovis Omp31 protein gave a very strong signal, which contrasts with the absence of reactivity of the other sera (Fig. 6). Antibody response against this region of the protein might be associated with the stage of the B. ovis infection and might appear after the processing of Omp31 by antigen-presenting cells. The analysis of the antibody response, specific for Omp31 and the epitope synthesized in E. coli(pNV31115), during a B. ovis experimental infection in rams might help to clarify this point.

Further studies are necessary to determine the real usefulness of the recombinant B. ovis Omp31 protein for the diagnosis of B. ovis infections and to evaluate its contribution to protective immunity against B. ovis. However, special care must be taken when the antigen presentation for these assays is selected, as the antigenic and probably the immunogenic properties of the B. ovis Omp31 protein seem to be highly dependent on the conformation of the protein.


    ACKNOWLEDGMENTS

We thank Manuel Sánchez Hernández for his valuable help with DNA sequence determination and Jean Michel Verger and Maggy Grayon for providing the Brucella strains.

This work and Nieves Vizcaíno were financed by project FAIR5-CT97-3360 from the European Union.


    FOOTNOTES

* Corresponding author. Mailing address: Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain. Phone: 34-923-294532. Fax: 34-923-224876. E-mail: vizcaino{at}www-micro.usal.es.

Editor:   D. L. Burns


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, November 2001, p. 7020-7028, Vol. 69, No. 11
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.11.7020-7028.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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