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

Location: Infectious Bacterial Diseases Research

2024 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
During FY24 progress was made in meeting the objectives of the brucellosis research project despite significant financial challenges. In Objective 1, elk were vaccinated with a killed Brucella strain alone, or in combination with a live Brucella vaccine strain to determine if an anamnestic response could be induced. New vaccine candidates have been developed using CRISPR techniques but have not yet progressed to in vivo evaluation. In studies under Objective 2, evaluation of swine responses to a synthetic diagnostic antigen for B. suis were completed demonstrating similar sensitivity and specificity to serologic responses on current diagnostic tests. For Objective 3, using previously acquired data, a transposon Brucella abortus library was modified and used to infect pregnant cattle to determine Brucella genes required for infection and colonization. Differences in innate gene expression and microbiome changes were characterized after Brucella vaccination. In addition, single cell genomic data was used to understand innate immune responses of cattle, bison and elk. In addition, studies were conducted on immune gene expression in Brucella-infected elk to help understand the molecular basis for differences in immune function between species. Comparative bioinformatic analysis of the bison, cattle, and elk genomes identified major changes in immunologic genes that may be the basis for the differences in susceptibility to Brucella infection.

Accomplishments
1. Genetic selection of cattle for increased milk production reduces immune responses. Selective breeding to increase production traits has provided economic benefits to livestock producers, but the effects of selection on immune function are not clear. ARS scientists in Ames, Iowa demonstrated that selective breeding for increased milk production in cattle reduced immune responses associated with defense against bacterial pathogens. This has resulted in phenotypes of increased pathogenicity and/or susceptibility to infection by pathogens that cause mastitis and reduction in expression of immune genes associated with protection. Identification of genes associated with increased susceptibility could allow use of selective breeding to improve immune function. This work will be of interest to livestock producers, veterinary practitioners, and researchers with interests in immune function.

2. Differences in Immune Cell Function in response to brucellosis among cattle, elk and bison. Brucella abortus is a zoonotic disease that causes abortions in cattle, elk, and bison. The three species differ in their susceptibility to brucellosis with bison being most susceptible to infection. ARS scientists in Ames, Iowa used genomic techniques to evaluate gene expression and cell types circulating in the bloodstream of the three species. Elk had more B lymphocyte cells, which are responsible for generating antibodies, circulating in comparison to the other three species. Differences in gene expression in immune cells were detected between the three species with bison and elk having the greatest number of differentially expressed immune genes compared to cattle and elk. Gene expression differences between the three species may influence immune cell function and contribute to phenotypic differences. This work will be of interest to livestock producers, veterinary practitioners, and researchers with interests in immune function.

3. Immune Responses Causing Chronic Brucella Infection. Brucella abortus is a zoonotic disease that can be shed to humans through milk. ARS scientists in Ames, Iowa characterized immune function in a Jersey cow persistently shedding Brucella abortus strain RB51 (RB51) in milk after vaccination. This phenomena has been documented in Jersey cattle and been associated with human infection after consumption of unpasteurized milk products. Data demonstrated a lack of peripheral CD4+ responses to strain RB51 and a concurrent high anti-RB51 antibody response. Cells from the cow could be stimulated to produce interferon-gamma by mitogens but did not produce this cytokine in response to RB51 indicating that they were not responding like most cattle. The cow did not demonstrate cellular immune responses to re-vaccination and data suggests the lack of immune responses were not due to T cell exhaustion. The cow failed to develop cellular immunity which is required for protection against intracellular pathogens. The lack of responses may provide information on protective immune responses in cattle that could allow development of more immunogenic vaccines.

Review Publications
Olsen, S.C., Boggiatto, P.M., Nol, P., Mccollum, M., Rhyan, J. 2021. Immune responses and efficacy of Brucella abortus strain RB51 in bison after delivery in a dry dart formulation or by parenteral inoculation. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2021.706160.
Sarlo Davila, K.M., Boggiatto, P.M., Olsen, S.C., Lippolis, J.D., Crooker, B., Putz, E.J. 2023. Effect of selection genotype on immune response to Brucella abortus RB51 in holstein cattle. Animal Genetics. 55(1):47-54. https://doi.org/10.1111/age.13372.
Palmer, M.V., Thacker, T.C., Kanipe, C.R., Boggiatto, P.M. 2021. Heterogeneity of pulmonary granulomas in cattle experimentally infected with Mycobacterium bovis. Frontiers in Veterinary Science. 8. https://doi.org/10.3389/fvets.2021.671460.
Tibbs-Cortes, B.W., Rahic-Seggerman, F.M., Schmitz-Esser, S., Boggiatto, P.M., Olsen, S.C., Putz, E.J. 2024. Fecal and vaginal microbiota of vaccinated and non-vaccinated pregnant elk challenged with Brucella abortus. Frontiers in Veterinary Science. 11. https://doi.org/10.3389/fvets.2024.1334858.
Bowden, C.F., Kiser, J.N., Miller, R.S., Buckley, A.C., Boggiatto, P.M., Giglio, R.M., Brown, V.R., Garrick, D., Neibergs, H.L., Piaggio, A.J., Speidel, S.E., Smyser, T.J. 2023. Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa). Frontiers in Genetics. https://doi.org/10.3389/fgene.2023.1292671.
Putz, E.J., Andreasen, C.B., Stasko, J.A., Fernandes, L.G., Palmer, M.V., Rauh, M.J., Nally, J.E. 2021. Circulating foamy macrophages in the golden syrian hamster (mesocricetus auratus) model of Leptospirosis. Journal of Comparative Pathology. 189(10):98-109. https://doi.org/10.1016/j.jcpa.2021.10.004.
Fernandes, L.G., Putz, E.J., Stasko, J.A., Lippolis, J.D., Nascimento, A.L., Nally, J.E. 2022. Evaluation of LipL32 and LigA/LigB knockdown mutants in Leptospira interrogans Serovar Copenhageni: impacts to proteome and virulence. Frontiers in Microbiology. 12. Article 799012. https://doi.org/10.3389/fmicb.2021.799012.
Putz, E.J., Bayles, D.O., Alt, D.P., Nally, J.E. 2022. Complete genome sequence of four strains of Leptospira borgpetersenii serovar Hardjo isolated from cattle in the Central United States. Journal of Genomics. 10. Pages 45-48. https://doi.org/10.7150/jgen.69822.
Putz, E.J., Fernandes, L., Sivasankaran, S., Bayles, D.O., Alt, D.P., Lippolis, J.D., Nally, J.E. 2022. Some like it hot, some like it cold; Proteome comparison of Leptospira borgpetersenii serovar Hardjo strains propagated at different temperatures. Journal of Proteomics. 262(2022). Article 104602. https://doi.org/10.1016/j.jprot.2022.104602.
Putz, E.J., Fernandes, L.G., Bayles, D.O., Lippolis, J.D., Nally, J.E. 2022. Proteomic dataset comparing strains of Leptospira borgpetersenii serovar Hardjo cultured at different temperatures. Data in Brief. 45. https://doi.org/10.1016/j.dib.2022.108713.
Putz, E.J., Fernandes, L.G., Sarlo Davila, K.M., Whitelegge, J., Lippolis, J.D., Nally, J.E. 2024. Proteomic profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures. Journal of Proteomics. 295. https://doi.org/10.1016/j.jprot.2024.105106.
Hamond, C., LeCount, K., Putz, E.J., Bayles, D.O., Camp, P., Goris, M.G., Van Der Linden, H., Stone, N.E., Schlater, L., Sahl, J.W., Wagner, D.M., Nally, J.E. 2022. Bovine leptospirosis due to persistent renal carriage of Leptospira borgpetersenii serovar Tarassovi. Frontiers in Veterinary Science. 9. Article 848664. https://doi.org/10.3389/fvets.2022.848664.
Hamond, C., LeCount, K., Anderson, T., Putz, E.J., Stuber, T., Hicks, J., Camp, P., Van Der Linden, H., Bayles, D.O., Schlater, L.K., Nally, J.E. 2024. Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States. Frontiers in Water. 6. https://doi.org/10.3389/frwa.2024.1278088.
Hamond, C., Adam, E., Stone, N.E., LeCount, K., Anderson, T., Putz, E.J., Camp, P., Hicks, J., Stuber, T., Van Der Linden, H., Bayles, D.O., Sahl, J.W., Schlater, L.K., Wagner, D.M., Nally, J.E. 2024. Identification of equine mares as reservoir hosts for pathogenic species of Leptospira. Frontiers in Veterinary Science. 11. https://doi.org/10.3389/fvets.2024.1346713.
Boggiatto, P.M., Kanipe, C.R., Putz, E.J., Olsen, S.C., Palmer, M.V. 2023. Wildlife immune responses to Mycobacterium bovis and to bacille of calmette-guerin. Journal of Immunology. https://doi.org/10.4049/jimmunol.2300323.
Falkenberg, S.M., Buckley, A.C., Boggiatto, P.M. 2023. Evaluation of the primeflow RNA assay as a method of detection of SARS-CoV-2 single and dual infections. Cytotechnology. https://doi.org/10.1007/s10616-023-00608-9.
Palmer, M.V., Kanipe, C.R., Lehman, K.A., Thacker, T.C., Putz, E.J., Boggiatto, P.M. 2023. Vaccination of white-tailed deer with Mycobacterium bovis bacillus calmette-guérin (BCG): effect of Mycobacterium avium ssp. paratuberculosis infection. Microorganisms. https://doi.org/10.3390/microorganisms11102488.