Abstract
Canine brucellosis is an emerging disease and Brucella canis can be a potential health threat for dog holders. Diagnosing canine brucellosis has pitfalls since serological methods that recognize B. abortus and B. melitensis infections fail to detect anti-B. canis antibodies. Therefore, we used an immunoproteomics approach to identify immunodominant proteins as antigen candidates to improve serological testing. We performed two-dimensional gel electrophoresis with B. canis cell extracts followed by MALDI-ToF MS analysis and could correlate 182 protein spots with 82 B. canis proteins. Western Blot analysis of 2D SDS-PAGE gels using the sera from B. canis-infected dogs, verified by culture, PCR or serological tests, and sera from uninfected dogs, detected 50 immunoreactive B. canis proteins. Hence, 32 out of 82 identified proteins did not react with any sera, although seven of these proteins have been identified in previous studies as immunodominant in B. abortus and/or B. melitensis. A total of 14 proteins, like GroEL, DnaK or KatA, were false-positive hits since they reacted with sera of B. canis - free dogs. In contrast, 36 immunogenic proteins were detected by serum from bacteremic dogs and 16 out of these proteins also reacted with serum from infected but non-bacteremic dogs. Some of these immunoreactive proteins, e.g. three periplasmic substrate-binding proteins, seem to be specific for B. canis and have not been described before as immunodominant in Brucella. A subset of immunodominant proteins were also found in B. canis outer membrane vesicles that might serve as vaccination platform. In summary, we identified several immunoreactive proteins for the detection of acute and chronic canine brucellosis.
