Research Project: Development of Improved Diagnostic and Control Strategies for Brucellosis in Livestock and Wildlife

Location: Infectious Bacterial Diseases Research

2025 Annual Report

Objectives
Objective 1: Develop rationally-designed vaccines, including recombinants, that prevent disease or mitigate disease spread with an emphasis on platforms that allow for extended antigen release and remote delivery for cattle and elk. Subobjective 1A: Assess the potential of polyanhydride polymers as extended release vaccine platform using killed bacterial preparations to generate cell-mediated immune responses against Brucella in cattle and elk. Subobjective 1B: Evaluate immunogenicity/efficacy of a new recombinant vaccine in elk. Subobjective 1C: Develop new DIVA vaccine strains with targeted mutations using CRISPR. Objective 2: Improve current diagnostic tests for brucellosis that differentiate between Brucella species and can be used in multiple livestock and wildlife species. Subobjective 2A: Characterize sensitivity and specificity of diagnostic tests using synthetic Brucella antigens. Subobjective 2B: Identify new Brucella epitopes recognized by humoral responses of B. suis infected animals using phage library expression. Objective 3: Using transcriptomic approaches, define the immunopathogenesis of brucellosis at the tissue and cellular level by analyzing gene expression of peripheral immune responses and local immune responses to inform vaccine and therapeutic development. Subobjective 3A: Using a transposon generated mutant library of Brucella abortus, characterize how bacterial populations are limited during in vivo infection and identify bacterial genes critical for establishing infection in cattle. Subobjective 3B: Using a transposon-generated mutant library of Brucella abortus, characterize how pregnancy influences diversity of bacterial populations during in vivo infection in cattle. Subobjective 3C: Characterize the gene expression profiles of the peripheral immune response to Brucella in cattle, bison, and elk to determine species-specific differences in response to vaccination.

Approach
The long-term goals of this project are to facilitate the completion of brucellosis eradication programs in domestic livestock, and prevent reintroduction of brucellosis into livestock from wildlife reservoirs. Specifically, fundamental knowledge on Brucella pathogenesis will be gained, efficacious vaccination systems will be identified, and sensitive and specific diagnostic tools will be developed to aid eradication programs. Immunogenicity of vaccination strategies in targeted hosts (cattle, bison, elk, and swine), including novel vaccine platforms, will be evaluated in targeted species and efficacy characterized by experimental challenge. In addition, the project will try to improve the standard experimental challenge model for elk to better replicate the clinical effects of brucellosis under field conditions. By simultaneously characterizing the in vivo transcriptome of B. abortus and natural host during infection, we will develop knowledge of molecular mechanisms involved in regulation of host responses to infection, and genes expressed by the pathogen under in vivo conditions. This basic knowledge will identify future targets for development of new vaccines, diagnostics, immunomodulation, and possibly therapeutics. New diagnostics will be developed and analyzed for their ability to detect brucellosis in swine and cattle, and may allow differentiation of which Brucella spp. is associated with infection. The research will help resolve the risk of re-infection of domestic livestock from wildlife reservoirs of brucellosis, protect the financial investment that has been made in the U.S. brucellosis eradication program, and provide public health benefits by reducing the risk of zoonotic infection.

Progress Report
In Objective 1, immune responses of elk booster vaccinated with killed or live Brucella vaccines were characterized. Additional characterization of immunologic responses of bison to booster vaccination was conducted to identify protective immune mechanisms associated with this efficacious intervention strategy. New vaccine strains of Brucella were prepared and characterized under in vitro conditions to ensure correct genetic mutations and desired phenotypic properties. In studies under Objective 2, cattle were infected with Brucella abortus isolates genetically modified with multiple mutations throughout their genome to help identify Brucella genes essential for colonization and infection. The study demonstrated that pregnancy status did not influence pathogenesis of brucellosis in cattle. Objective 3, a gene expression approach was used to identify antigens of Brucella that are recognized by infected cattle for improving diagnostics. Differences in immunologic gene expression in bison, elk, and cattle were characterized in an effort to identify immune responses specifically associated with protection.

Accomplishments
1. Brucellosis vaccination of elk alters disease pathogenesis after infection. Brucella abortus is a zoonotic disease (can be transmitted between animals and humans) that infects bison, elk and cattle and causes economic losses to livestock producers from disease transmission from wildlife reservoirs. ARS scientists in Ames, Iowa, studied the long-term immune responses of elk to Brucella infection. Although vaccination did not reduce abortion rates in vaccinated elk when compared to non-vaccinated elk, inoculation did reduce the concentration of antibodies against Brucella as measured on serologic tests between 3 months and 8 months after infection. Reductions in antibody concentrations did not correlate with reduced infection in vaccinated elk. This data indicates that elk with prior exposure to Brucella abortus may have reduced antibody concentrations on diagnostic tests over time despite remaining infected with brucellosis. Therefore, serologic testing may not be accurate for identifying elk infected with Brucella, or for assessing disease prevalence in elk populations. This work will be of importance for protecting cattle herds from economic losses caused by brucellosis and be of interest to livestock producers, state and federal regulatory agencies, and researchers with interests in epidemiology of brucellosis in elk.

2. Phenotypic differences in susceptibility to brucellosis and efficacy of vaccination in wildlife reservoirs. Brucella abortus is a zoonotic disease (can be transmitted between animals and humans) that causes economic losses in cattle. Elk and bison in the areas around Yellowstone Park are reservoirs of brucellosis and can transmit brucellosis to cattle herds causing economic losses. ARS scientists in Ames, Iowa, did a metanalysis of data from multiple studies to compare differences in abortion and infection rates in both vaccinated and non-vaccinated bison and elk. Vaccination was efficacious in preventing abortion and infection in bison but was not effective in elk. Elk had lower rates of abortion and bacterial colonization in uterine and mammary tissues as compared to bison. This demonstrates that the current brucellosis vaccine is an effective intervention strategy for bison, but not for elk. This work provides information to guide intervention strategies that prevent economic losses to cattle herds and will be of interest to livestock producers, state and federal regulatory agencies, and researchers with interests in brucellosis in elk, bison, and cattle.

3. Genetic differences in immunologic genes. Bison within and in the areas surrounding Yellowstone National Park are infected with brucellosis, a zoonotic disease (can be transmitted between animals and humans) that causes reproductive losses in cattle. Cattle herds are being infected from this wildlife reservoir of disease. To understand the genetic basis that underlies their high susceptibility to clinical effects of brucellosis, ARS scientists in Ames, Iowa, compared genetic differences in immune function between the reservoirs of brucellosis in the United States (bison, elk and cattle). When compared to cattle, the overall genomic structure of the bison genome was largely conserved but specific regions had undergone significant evolutionary divergence. There was a substantial divergence of genomic structure, including numerous inversions, when the bison and elk genomes were compared. Specifically, differences were identified between bison, elk, and cattle in 11 genes associated with immune function. This work will guide the development of intervention strategies to prevent the economic losses caused by brucellosis in cattle herds and be of interest to livestock producers, state and federal regulatory agencies, and researchers with interests in brucellosis in elk, bison, and cattle.

Review Publications
Wiarda, J.E., Sarlo Davila, K.M., Trachsel, J.M., Loving, C.L., Boggiatto, P.M., Lippolis, J.D., Putz, E.J. 2025. Single-cell RNA sequencing characterization of Holstein cattle blood and milk immune cells during a chronic Staphylococcus aureus mastitis infection. Scientific Reports. 15. Article 12689. https://doi.org/10.1038/s41598-025-96657-5.
Boggiatto, P.M., Greiman, H., Falkenberg, S., Sarlo Davila, K.M., Putz, E.J., Olsen, S.C. 2024. Characterization of the adaptive cellular and humoral immune responses to persistent colonization of Brucella abortus strain RB51 in a Jersey cow. Frontiers in Veterinary Science. 11. Article 1367498. https://doi.org/10.3389/fvets.2024.1367498.
Menghwar, H., Boggiatto, P.M., Olsen, S.C., Slate, J., Goldkamp, A.K., Kanipe, C.R., Kaplan, B.S., Nielsen, D.W., Tatum, F.M., Casas, E., Dassanayake, R.P. 2025. Comparative innate immune responses of bison and cattle to Mannheimia haemolytica wildtype and LPS sialylation-deficient mutant strain. Research in Veterinary Science. 193. Article 105764. https://doi.org/10.1016/j.rvsc.2025.105764.
Kaplan, B.S., Dassanayake, R.P., Briggs, R.E., Kanipe, C.R., Boggiatto, P.M., Crawford, L., Olsen, S.C., Menghwar, H., Casas, E., Tatum, F.M. 2024. An injectable subunit vaccine containing Elongation Factor Tu and Heat Shock Protein 70 protects North American bison from Mycoplasma bovis infection. Frontiers in Veterinary Science. 11. Article 1408861. https://doi.org/10.3389/fvets.2024.1408861.
Sterle, H.M., Putz, E.J., Olsen, S.C., Boggiatto, P.M. 2024. Induction of CD4 T cell memory responses following BCG vaccination in cattle. Frontiers in Veterinary Science. 11. Article 1491424. https://doi.org/10.3389/fvets.2024.1491424.
Boggiatto, P.M., Buckley, A.C., Cassmann, E.D., Seger, H., Olsen, S.C., Palmer, M.V. 2024. Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Scientific Reports. 14. Article 11171. https://doi.org/10.1038/s41598-024-61414-7.
Putz, E.J., Andreasen, C., Stasko, J.A., Hamond, C., Olsen, S.C., Nally, J.E., Palmer, M.V. 2025. Circulating foamy macrophages and other features of bacillus Calmette-Guérin challenge in Golden Syrian hamsters. Vaccine. 55. Article 127037. https://doi.org/10.1016/j.vaccine.2025.127037.
Palmer, M.V., Jones, D.E., Bockenstedt, N.J., Boggiatto, P.M. 2025. Tertiary lymphoid structures in pulmonary granulomas of cattle experimentally infected with aerosolized Mycobacterium bovis. BMC Veterinary Research. 21. Article 403. https://doi.org/10.1186/s12917-025-04804-x.
Olsen, S.C., Boggiatto, P.M., Putz, E.J. 2025. Comparison of bison and elk susceptibility to experimental challenge with Brucella abortus strain 2308. Frontiers in Veterinary Science. 11. Article 1519453. https://doi.org/10.3389/fvets.2024.1519453.
Boggiatto, P.M., Palmer, M.V., Olsen, S.C., Falkenberg, S.M. 2025. Characterization of cellular and humoral immunity to commercial cattle BVDV vaccines in white-tailed deer. Vaccines. 13(4). Article 427. https://doi.org/10.3390/vaccines13040427.