Research Project: Identification of Antigens and Host Innate Immune Responses for Control of Johne's Disease

Location: Infectious Bacterial Diseases Research

2024 Annual Report

Objectives
Objective 1: Through genomic–scale approaches, identify MAP antigens for improved detection of Johne’s disease in cattle, including early stage infection. Building on this approach, identify and test selected antigens that could be used as vaccine candidates. Sub-objective 1.A: Design a synthetic antigen comprising strong epitopes from MAP antigens detected early in MAP infection of cattle. Sub-objective 1.B: Use genomic approaches to discover new vaccine and diagnostic targets in MAP and improve on current diagnostic tests. Sub-objective 1.C: Establish a CRISPR/Cas9 gene editing system in MAP for fast and targeted gene disruption to create live attenuated vaccine strains. Objective 2: Characterize the host’s innate-immune response to onset of MAP infection in order to identify and develop novel intervention strategies to control infection with MAP. Sub-objective 2.A: Characterize patterns of innate immune responses to natural infection in cattle in asymptomatic and clinical stages. Sub-objective 2.B: Characterize the presence of innate immune cells in tissues at the site of infection from naturally infected cattle.

Approach
First identified over a century ago, Johne’s disease (a.k.a. Paratuberculosis) has long been recognized as a serious economic and animal health problem throughout the world in domesticated ruminants such as dairy and beef cattle, sheep, and goats. Paratuberculosis results in more than $200 million in annual losses to the U.S. dairy industry each year with additional losses incurred by the other species. The agent that causes this disease is the slow growing bacterium Mycobacterium avium subspecies paratuberculosis (MAP). A growing recognition of MAP infection in wildlife species is also of considerable concern, as is the recent implication of the presence of MAP in retail milk sources that may make this pathogen a risk from a milk quality and food safety standpoint. The overall goal of the work described in this project is to reduce the impact of Johne’s disease on the livestock producer and their corresponding industries. This goal is best accomplished by understanding the bovine immune response and what role specific immune cells play in order to inform next generation vaccine development and to identify antigens and assay platforms with the ability to detect the disease earlier, before transmission and disease spread occurs. With this goal and approaches in mind, we propose two primary objectives: (1) Through genomic–scale approaches, identify MAP antigens for improved detection of Johne’s disease in cattle, including early stage infection. Building on this approach, identify and test selected antigens that could be used as vaccine candidates; (2) Characterize the host’s innate-immune response to onset of MAP infection in order to identify and develop novel intervention strategies to control infection with MAP. This research will not only expand the basic knowledge of the disease, but it will lead to countermeasures to combat the disease, providing significant economic benefits to livestock producers.

Progress Report
In support of Objective 1.C, two independent mutants (gene knockouts) of the Mycobacterium avium subsp paratuberculosis were prepared. These mutants were tested and differences in bacterial shape and metabolism, but not growth rate, were identified. The effects of the mutations on bacteria survival were also determined. In support of Objective 2, the effect of chemical treatments that are designed to enhance bacterial phagocytosis and destruction (autophagy) on functions of bovine macrophages were characterized. Prolonged treatment (24 hrs) did result in significant toxicity for three of the chemicals tested. However, reactive oxygen species (ROS) production, important for bacterial pathogen killing, was detected in bovine macrophages after chemical treatments. Differentiation of bovine macrophages into M1 and M2 phenotypes is associated with different functions by these two macrophage phenotypes. Use of immunofluorescence microscopy and antibodies that recognize external cellular markers demonstrated that M2 macrophages, which function in resolution and repair of tissue damage, were greater at sites of Johne’s disease infection. As M2 macrophages are less capable of killing the Johne’s pathogen, our data indicates a potential strategy of increasing M1 macrophages at sites of infection with Johne’s bacteria should benefit disease control.

Accomplishments
1. Bacterial fatty acids benefit biolubricant production. ARS scientists in Ames, Iowa and Peoria, Illinois discovered methods to control expression of a specific fatty acid, tuberculostearic acid, in Mycobacterium avium subspecies paratuberculosis (MAP) that is an important biolubricant. Biolubricants are prized for their high flash points, constant viscosity, and increased safety. Tuberculostearic acid from MAP is desirable in biolubricant production as it doesn’t break down at room temperature. Control of expression was achieved using genetic disruption (gene knockout) and through a chemical process. Analysis demonstrated that sheep strains of MAP do not express this fatty acid due to a genetic mutation. An engineered mutation in a bovine MAP strain confirmed the gene is associated with tuberculostearic acid production. These results highlight differences between sheep and cattle strains of Map and how differences may have implications for other applications, such as biolubricants. This work will be of interest to livestock owners, biolubricant producers, and researchers with an interest in Johne’s disease.

Review Publications
Bannantine, J.P., Stabel, J.R., Bayles, D.O., Biet, F. 2023. Improved DNA amplification of the hallmark IS900 element in Mycobacterium avium subspecies paratuberculosis: a re-examination based on whole genome sequence analysis. Applied and Environmental Microbiology. 89(2). https://doi.org/10.1128/aem.01682-22.
Bannantine, J.P., Duffy, S.C., Colombatti Olivieri, M.A., Behr, M.A., Biet, F., Price, N.P. 2024. Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis. Microbiology Spectrum. https://doi.org/10.1128/spectrum.00508-24.
Barletta, R.G., Bannantine, J.P., Stabel, J.R., Muthukrishnan, E., Anderson, D., Dutta, E., Manthena, V., Hanafy, M., Zinniel, D.K. 2023. Mycobacterium avium subsp. paratuberculosis candidate vaccine strains are pro-apoptotic in RAW 264.7 murine macrophages. Vaccines. 11(6). https://doi.org/10.3390/vaccines11061085.
Price, N.P., Jackson, M.A., Hartman, T.M., Bannantine, J.P., Naumann, T.A., Vermillion, K., Koch, A.A., Kennedy, P.D. 2023. Precursor-directed biosynthesis and biological testing of omega-alicyclic- and neo-branched Tunicamycin N-acyl variants. ACS Chemical Biology. https://doi.org/10.1021/acschembio.3c00324.
Thapa, S., Rathnaiah, G., Zinniel, D.K., Barletta, R.G., Bannantine, J.P., Huebner, M., Sreevatsan, S. 2024. The Fur-like regulatory protein MAP3773c modulates key metabolic pathways in Mycobacterium avium subsp. paratuberculosis under in-vitro iron starvation. Scientific Reports. https://doi.org/10.1038/s41598-024-59691-3.
Conde, C., Price-Carter, M.L., Cochard, T., Branger, M., Stevenson, K., Whittington, R., Bannantine, J.P., Biet, F. 2021. Whole-genome analysis of Mycobacterium avium subsp. paratuberculosis IS900 insertions reveals strain type-specific modalities. Frontiers in Microbiology. Vol. 12, Article 660002. https://doi.org/10.3389/fmicb.2021.660002.
Biet, F., Conde, C., Cochard, T., McIntosh, F.A., Behr, M.A., Bannantine, J.P. 2024. Complete genome sequence of an ovine ancestral strain of Mycobacterium avium subspecies paratuberculosis 6756. Microbiology Resource Announcements. 13(4). https://doi.org/10.1128/mra.01207-23.
Stabel, J.R., Bannantine, J.P., Humphrey, S.B. 2022. B cell phenotypes and maturation states in cows naturally infected with Mycobacterium avium subsp. Paratuberculosis. PLOS ONE. https://doi.org/10.1371/journal.pone.0278313.
Wherry, T., Dassanayake, R.P., Casas, E., Mooyottu, S., Bannantine, J.P., Stabel, J.R. 2022. Exogenous vitamin D3 modulates response of bovine macrophages to Mycobacterium avium subsp. paratuberculosis infection and is dependent upon stage of Johne's disease. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2021.773938.
Conde, C., Theze, J., Cochard, T., Rossignol, M., Fourichon, C., Delafosse, A., Joly, A., Guatteo, R., Schibler, L., Bannantine, J.P., Biet, F. 2022. Genetic features of Mycobacterium avium subsp. paratuberculosis strains circulating in the west of France deciphered by whole-genome sequencing. Microbiology Spectrum. https://doi.org/10.1128/spectrum.03392-22.