2008 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. 3.Progress Report During the past year, collaborative projects evaluating brucellosis vaccines in bison and elk were initiated. New vaccine strains were created that will be initially evaluated in laboratory animal models. Brucella species and biovars were evaluated in an effort to identify unique genetic fingerprints that can be used for identification. The project is working to develop brucellosis vaccines for domestic livestock and wildlife that will facilitate eradication of this disease in the United States. New diagnostics being developed in the project will be beneficial in conducting epidemiologic tracebacks of brucellosis infections. Both the vaccines and diagnostics developed in the project will be useful for management of brucellosis in the remaining reservoirs within the U.S. This project addresses NP 103, component 3, problem statement 3A. 4.Accomplishments 1. Characterized the efficacy of a new brucellosis vaccine in 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. Although the new vaccine was safe in bison calves, it did not enhance protection against brucellosis when compared to its parent strain. Our data suggests the new vaccine would not be the preferred choice for brucellosis vaccination of bison. This accomplishment addresses NP 103, component 3, problem statement 3A. 2. Development of more efficacious vaccination strategies for free-ranging wildlife. Booster vaccination may be a management tool that could be used to enhance vaccine protection and herd immunity. Bison were booster vaccinated and samples obtained to ascertain if repeat vaccination stimulated greater immunologic responses. Bison will be experimentally challenged to determine if booster vaccination enhanced vaccine efficacy. This accomplishment addresses NP 103, component 3, problem statement 3A. 3. Genomic characterization of a Brucella vaccine strain. Genomic characterization of Brucella vaccine strains may provide insight into genes which enhance protective immunity and attenuation. In collaboration with other scientists, the genome sequence for the Brucella vaccine strain, S19 was determined and several mutated genes were identified that may have relevance to attenuation in this strain. This information can be applied to designing new brucellosis vaccines. This accomplishment addresses NP 103, component 3, problem statement 3A. 4. Development of Molecular Epidemiological Tests. Despite near eradication of brucellosis in cattle, Brucella-infected herds continue to be found and epidemiological investigations are conducted to identify the source of infection. Previously we developed a powerful new technique called HOOF-Printing to genetically fingerprint the disease strain’s DNA, that assists in determining genetic relationships between Brucella strains. To advance the ability to determine relationships, we are currently evaluating the use of single nucleotide changes (SNPs). These point mutations will be used in conjunction with HOOF-Print data to increase confidence in interpreting genetic comparisons of Brucella strains. During 2008 unique single nucleotide polymorphisms (SNPs) were found that differentiate the vaccine strain RB51, from all other Brucella abortus strains. We continue to find new SNP markers and analyze them against a panel of Brucella abortus field strains for development of a more accurate diagnostic test. This accomplishment addresses NP 103, component 3, problem statement 3A. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 1 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Crasta, O.R., Folkerts, O., Fei, Z., Mane, S., Evans, C., Martino-Catt, S., Bricker, B.J., Yu, G., Du, L., Sobral, B.W. 2008. Genome Sequence of Brucella abortus Vaccine Strain S19 Compared to Virulent Strains Yields Candidate Virulence Genes. PLoS One. 3(5):e2193. Available: http:www.plosone.org/article/info:doi/10.1371/journal.pone.0002193. Olsen, S.C., Waters, W.R., Stoffregen, W.C. 2007. An Aerosolized Brucella spp. Challenge Model for Laboratory Animals. Zoonoses and Public Health. 54(8):281-285. Halling, S.M., Jensen, A.E., Olsen, S.C. 2008. Defensin Susceptibility and Colonization in the Mouse Model of AJ100, a Polymyxin B Resistant, Brucella abortus RB51 Isolate. Current Microbiology. 56(2008):274-278.