Application of a Whole Genome Approach to the High Throughput Discovery of Novel Diagnostic Antigens for Brucella abortus

Abstract

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that can be transmitted to humans through direct contact with infected animals or consumption of contaminated food products. Current serological tests used to identify infected animals rely on the detection of antibodies to O-antigens of the smooth lipopolysaccharide (sLPS) of B. abortus. Due to the presence of structurally similar O-antigens of bacteria such as E. coli O:157 and Yersinia enterocolitica O:9, these tests can produce false-positive results, which requiring alternative protein target antigens. We hypothesize that through comparative genomics and bioinformatics analysis of all ORFs within the B. abortus genome, followed by profiling of the humoral immune responses to surface and extracellular proteins, novel protein antigens with diagnostic potential may be discovered. In this study, the genomes of thirteen strains were analyzed using a subcellular localization prediction database (PSORTb) to identify proteins in the outer membrane and extracellular space. A total of 100 ORFs coding for such proteins including known immunogenic proteins reported in literature were identified and selected for recombinant protein expression using high-throughput in vivo cloning and in vitro transcription/translation strategies. The in vitro expression of 67 of these candidates has been successfully demonstrated. These recombinant B. abortus proteins were subsequently probed with E. coli pre-adsorbed sera from infected animals for the identification of immunoreactive protein antigens. Ten unique candidates were demonstrated to be antigenic and have the potential for diagnostic applications. This study illustrates a unique, high throughput strategy to express and screen proteins of a bacterial pathogen for novel diagnostic antigen discovery.