Location: Infectious Bacterial Diseases Research
2022 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
This report is the first of the new project plan and the initial milestones were all met. In Subobjective 1.A, the eight proteins listed in Table 1 of the project plan were successfully expressed in E. coli and purified by affinity chromatography and overlapping peptide libraries were synthesized. In support of Subobjective 1.B, two distinct lipid antigens produced by the Mycobacterium avium subspecies paratuberculosis (MAP) bacterium were synthesized and used to test for the presence of bacteria in cow’s milk. Both antigens demonstrated sensitivity for detecting the Mycobacterium avium subspecies paratuberculosis (MAP) bacteria in milk samples. Genomic sequences of additional MAP strains were completed for use in this subobjective next FY. Genomic materials were prepared (plasmid DNAs and the single guide RNA) and were constructed for transformation of MAP in Subobjective 1.C.
In work addressing Objective 2, host cellular immunity was evaluated in goats and calves infected with MAP. Goats demonstrate increased interferon-gamma cytokine production and changes in T cell populations (CD4+ and gamma-delta) after vaccination against MAP. MAP infected calves also show changes in T cell populations (CD4+, CD8+ and gamma delta). These observations demonstrate similarities in T cell responses of calves and goats. Overall, these studies provide insight on how immune responses protect against MAP infection.
Accomplishments
1. The role of Vitmin D on immune cells of cattle. Vitamin D is depleted in animals with Johne's disease but is necessary for enhancing phagocytosis of the pathogen and is beneficial in controlling inflammation. ARS researchers in Ames, Iowa, examined the effects of two vitamin D analogs on peripheral blood mononuclear cells and macrophages of Mycobacterium avium subspecies paratuberculosis-infected cattle (Johne's disease). Our results show that vitamin D modulates cytokine signaling in cattle at different stages of MAP infection. This modulation impacts both pro- and anti-inflammatory immune responses, thus negatively impacting bacterial growth. This work has implications for potential approaches for treatment of Johne's disease in domestic livestock. These data are of interest to producers, veterinarians, and researchers interested in alternative intervention strategies for preventing Johne's disease.
Review Publications
Thomas, S., Abraham, A., Rodriguez-Mallon, A., Unajak, S., Bannantine, J.P. 2021. Challenges in veterinary vaccine development. In: Thomas, S., editor. Vaccine Design. NY, NY: Humana. 1124. p. 3-36. https://doi.org/10.1007/978-1-0716-1888-2_1.
Bannantine, J.P., Stabel, J.R., Kapur, V. 2022. Immunological evaluation of goats vaccinated with a commercial vaccine against Johne’s disease. Vaccines. 10(4). Article 518. https://doi.org/10.3390/vaccines10040518.
Stabel, J.R., Waters, W.R., Bannantine, J.P., Palmer, M.V. 2021. Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis. Veterinary Immunology and Immunopathology. 237. https://doi.org/10.1016/j.vetimm.2021.110268.
Bannantine, J.P., Bayles, D.O. 2021. Draft genome sequences of two Bison-type and two Sheep-type strains of Mycobacterium avium subspecies paratuberculosis. Microbiology Resource Announcements. 10(28). Page 21. https://doi.org/10.1128/MRA.00526-21.
Hosseiniporgham, S., Biet, F., Ganneau, C., Bannantine, J.P., Bay, S., Secche, L. 2021. A comparative study on the efficiency of two Mycobacterium avium subsp. paratuberculosis (MAP)-derived lipopeptides of L3P and L5P as capture antigens in an in-house milk ELISA test. Vaccines. 9(9). Article 997. https://doi.org/10.3390/vaccines9090997.
