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

Abstract

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.

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.

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).

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).

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).

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.

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.