2007 Annual Report 1a.Objectives (from AD-416) 1)Develop effective strategies to prevent B. abortus or B. suis infections in free-ranging wildlife (bison, cervidae, feral swine) by characterizing immune responses, and determining safety and efficacy of available brucellosis vaccines. (2) Enhance immunologic responses of domestic livestock or free-ranging wildlife to brucellosis vaccines by use of adjuvants, vaccine engineering, or alternative delivery methods. (3) Develop and evaluate molecular markers to assist in diagnosis and epidemiology of brucellosis. (4) Characterize the genomic determinants of virulence and pathogenicity in wild-type Brucella species and vaccine strains. 1b.Approach (from AD-416) The project will use standardized methods to evaluate protection of brucellosis vaccines in domestic livestock or wildlife reservoirs of brucellosis. In an effort to develop improved brucellosis vaccines, immunologic methods will be used to identify antigens involved in protection, and immunomodulators which enhance vaccine efficacy. Molecular procedures will be used to develop assays which allow epidemiologic traceback of brucella strains, and characterize genes involved in virulence and pathogenicity. 4.Accomplishments Evaluation of a new brucellosis vaccine for bison. Tools are needed to help resolve the problem caused by the high prevalence of brucellosis in bison in Yellowstone National Park. We evaluated a new recombinant brucellosis vaccine in bison to determine if it might be better than currently available brucellosis vaccines. We found that the new vaccine was safe in bison calves, and induced immune responses that were comparable to its parent RB51 vaccine strain. If the new vaccine proves to be more protective, it could be the preferred choice for brucellosis vaccination of bison in Yellowstone National Park. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Brucellosis. Evaluation of a new brucellosis vaccine for swine. As feral swine expand into new areas of the United States, they pose a threat for spreading Brucella suis to cattle and people. In an effort to develop a vaccine that can be used to address the swine brucellosis problem, we evaluated a new vaccine strain for safety and efficacy in swine after delivery orally or hand vaccination. The vaccine strain was clinically safe in swine and induced some protection against infection with a virulent strain of B. suis. This vaccine may be beneficial under field conditions for reducing the prevalence of brucellosis in swine populations. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Brucellosis. Molecular tools for epidemiologic traceback of brucellosis. When a new case of brucellosis is identified, state and federal regulatory personnel need tools to identify the source of infection and to determine if the infection has spread to other herds. In a continuation of previous work, a DNA fingerprinting diagnostic test developed at NADC, was utilized to evaluate isolates from an area experiencing recurrent outbreaks of brucellosis. Samples collected from the area over the previous 11 years were compared to recent isolates. Data indicated a strong genetic similarity between recent and historical isolates indicating that the infection was an enduring local problem, not new infections from outside sources. This was the first retrospective use of molecular tools for brucellosis epidemiology in the U.S. and not only confirmed the likely source of recent infections in the area, but also demonstrated the value of the assay for use in brucellosis regulatory efforts. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Brucellosis. Effect of the innate immune system antimicrobial compounds on Brucella. The bacterial pathogen Brucella, causative agent of brucellosis, can survive in cells of the immune system that normally purge invading bacteria. The effect on Brucella of antimicrobial polycationic compounds and compounds with similar chemical characteristics that are secreted by immune cells was determined. Only one strain of 13 strains of Brucella spp. tested, the vaccine strain S19, was susceptible to any of the nine compounds tested, establishing that genetic resistance to these innate immune system antimicrobials likely aids in the persistence of Brucella. This study adds to our understanding of the biology of host-pathogen interactions and identifies a virulence mechanism of Brucella for further study. Knowledge of host pathogen interactions are critical to possible development of chemotherapeutic agents and construction of vaccine strains that do not persist in animals. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Brucellosis.  5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 1 Number of active CRADAs and MTAs 1 Number of non-peer reviewed presentations and proceedings 9 Review Publications Stoffregen, W.C., Olsen, S.C., Wheeler, C.J., Bricker, B.J., Palmer, M.V., Jensen, A.E., Halling, S.M., Alt, D.P. 2007. Diagnostic Characterization of a Feral Swine Herd Enzootically Infected with Brucella. Journal of Veterinary Diagnostic Investigation. 19(3):227-237. Halling, S.M., Jensen, A.E. 2006. Intrinsic and selected resistance to antibiotics binding the ribosome: analyses of Brucella 23S rrn, L4, L22, EF Tu1, EF Tu2, efflux and phylogenetic implications. BMC Microbiology. 6:84. Olsen, S.C., Fach, S.J., Palmer, M.V., Sacco, R.E., Stoffregen, W.C., Waters, W.R. 2006. Immune Responses of Elk to Initital and Booster Vaccinations with Brucella abortus Strain RB51 or 19. Clinical and Vaccine Immunology. 13(10):1098-1103.