Novel and Unique Diagnostic Biomarkers forBacillus anthracisInfection

A search for bacterium-specific biomarkers in peripheral blood following infection with Bacillus anthracis was carried out with rabbits, using a battery of specific antibodies generated by DNA vaccination against 10 preselected highly immunogenic bacterial antigens which were identified previously by a genomic/proteomic/serologic screen of the B. anthracis secretome. Detection of infection biomarkers in the circulation of infected rabbits could be achieved only after removal of highly abundant serum proteins by chromatography using a random-ligand affinity column. Besides the toxin component protective antigen, the following three secreted proteins were detected in the circulation of infected animals: the chaperone and protease HtrA (BA3660), an NlpC/P60 endopeptidase (BA1952), and a protein of unknown function harboring two SH3 (Src homology 3) domains (BA0796). The three proteins could be detected in plasma samples from infected animals exhibiting 10(3) to 10(5) CFU/ml blood and also in standard blood cultures at 3 to 6 h post-bacterial inoculation at a bacteremic level as low as 10(3) CFU/ml. Furthermore, the three biomarkers appear to be present only in the secretome of B. anthracis, not in those of the related pathogens B. thuringiensis and B. cereus. To the best of our knowledge, this is the first report of direct detection of B. anthracis-specific proteins, other than the toxin components, in the circulation of infected animals.