Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis

Abstract

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.

FIG. 1.

Alignment of the BP26 amino acid sequences from Brucella spp. Amino acid differences for each strain in comparison to the published B. melitensis 16M BP26 are highlighted in black. Abbreviations: Ba1, B. abortus 544 (biovar 1); Ba3, B. abortus Tulya (biovar 3); S19, B. abortus S19 vaccine strain; RB51, B. abortus RB51 vaccine strain. The BP26 amino acid sequence for the five remaining B. abortus biovar reference strains and for B. suis and B. ovis reference strains was identical to B. abortus 544 and B. melitensis 16M BP26.

FIG. 2.

Reactivity in Western blotting of BP26-specific MAbs against recombinant E. coli/pCP2801. Positions of protein molecular mass markers are shown on the left. MAbs: V78/09B12/B02 (lane 1), V78/11A07/G06 (lane 2), V78/06E10/H10 (lane 3), V78/04D01/A10 (lane 4), V78/10F02/E10 (lane 5), V78/10A07/H09 (lane 6), V78/03G01/F01 (lane 7), V78/02D06/C08 (lane 8), V78/05B10/F07 (lane 9), V78/02E08/F03 (lane 10), V78/05G03/H03 (lane 11), V78/09C08/A01 (lane 12), V78/05G06/C01 (lane 13), V78/04G07/H05 (lane 14), V78/07G06/A09 (lane 15), V78/10A03/A02 (lane 16), V78/06D11/G06 (lane 17), V78/04D06/B04 (lane 18).

FIG. 3.

Fragments of BP26, synthesized as fusion proteins with LacZ in E. coli, reacting in colony blotting with the BP26-specific MAb V78/10A07/H09.

FIG. 4.

BP26-specific MAbs reacting in colony blotting with the BP26 fragments synthesized as fusion proteins with LacZ in E. coli/pCP28112 and E. coli/pCP28124.

FIG. 5.

Reactivity in Western blotting of sera from sheep naturally infected by B. melitensis or from Brucella-free sheep with E. coli/pCP2801 synthesizing the entire recombinant BP26 (A) or E. coli/pCP28124 synthesizing amino acids 55 to 152 of BP26 (B). Reactivity with the BP26-specific MAb V78/04D01/A10 is shown in lanes 1. The same lane number corresponds to the same serum in both panels. O.D., optical density provided by sera in indirect ELISA with purified recombinant BP26 (2).

FIG. 6.

Reactivity in Western blotting of sera from rams naturally infected by B. ovis with E. coli/pCP2801 synthesizing the entire recombinant BP26 (A) or E. coli/pCP28124 synthesizing amino acids 55 to 152 of BP26 (B). Reactivity with the BP26-specific MAb V78/04D01/A10 is shown in lane 1 in panels A and B. The same lane number on panels A and B corresponds to the same serum.