Brucellosis is a zoonotic disease with the potential to be exploited as a bioterrorism disease. The agents of brucellosis, Brucella spp., have been classified as Category B pathogens because these bacteria are stable in the environment, easily aerosolized, highly infectious, difficult to treat, and have prolonged debilitating illness. We propose to develop and deliver novel diagnostics for brucellosis caused by Brucella melitensis. Tests to diagnose acute and chronic brucellosis are far from ideal, including issues of specificity and point- care-ruggedness and ease of use. Sensitive testing to identify pre-symptomatic patients at risk for brucellosis has never been tested. Our access to populations in Peru with a high incidence of brucellosis, both in the endemic and outbreak settings, will allow us to address these major issues of brucellosis diagnosis. Specific Aim 1 is to develop a well-defined clinical specimen bank for validating new assays to diagnose acute brucellosis by studying human populations in a Brucella me//tens/s-endemic country. This specimen bank will be comprised of clinical specimens from acute febrile patients at high risk for brucellosis. Gold standard culture (lysis centrifugation, automated blood culture), recently developed real time PCR assays, and standard serology (Rose Bengal, slide agglutination test, tube agglutination test) will be used to diagnose brucellosis. The clinical samples (isolates, sera, buffy coats) will be aliquoted and stored under Good Clinical Practice conditions as a resource for testing the new lateral flow assays described under Specific Aim 2. The specimen bank will also be available to NIH-funded biodefense investigators for testing other modalities for brucellosis detection. Specific aim 2 is to validate new lateral flow assays to optimize the rapidity and accuracy of brucellosis. Lateral flow assays have the advantage of being rapid, rugged, easy-to- use at point-of-care, and requiring small amounts of easily obtainable clinical sample to perform. We have already successfully used a lateral flow assay, based on Brucella LPS, to detect anti-Sruce//a antibodies in Peru and other brucellosis-endemic countries. This project proposes to transfer the technology needed to fabricate this assay to the PanBio Company (Maryland, USA and Brisbane, Australia), who will manufacture the lateral flow assay for testing on the clinical specimen bank described in Specific Aim 1 under Good Manufacturing Practice conditions. In addition, we propose to test the use of a newly characterized Brucella protein, VirB12, a protein invariably detected in small animal models of infection, in the early diagnosis of brucellosis. Finally, as an alternative to nucleic acid detection, we propose to develop a lateral flow-based antigen-detection method for the identification of Brucella infection in diverse clinical scenarios.