Research Project: Characterization of Antigens, Virulence Markers, and Host Immunity in the Pathogenesis of Johne’s Disease

Location: Infectious Bacterial Diseases Research

2019 Annual Report

Objectives
Objective 1: Identify MAP antigens including protein to protein interactions using proteomic and genomic tools to better understand their function in pathogenesis of Johne’s disease and develop improved diagnostic tools. Subobjective 1.1: Define pathogenic mechanisms of MAP through bacterial community interactions as well as protein interactions with the host. Subobjective 1.2: Detection reagents for Johne’s Disease. Objective 2: Characterize host immunity and pathogenesis of disease using immunophenotypic and cell signaling markers in response to asymptomatic and clinical MAP infection, as well as vaccination. Subobjective 2.1: Characterize patterns of Th17-mediated immune responses to natural infection in cattle in asymptomatic and clinical stages. Subobjective 2.2. Characterize key differences in host immunity upon vaccination compared to infection. Subobjective 2.3: Assess B cell mediated immunity to natural infection in cattle in asymptomatic and clinical stages using maturation and activation markers for B cell subsets. Subobjective 2.4: Characterize the impact of infection on the phenotypes of antigenpresenting cells in target tissues of infected cattle. Objective 3: Investigate genetic variability among MAP isolates of livestock using whole genome sequencing to develop improved epidemiological tools and evaluate the genetic basis of virulence. Subobjective 3.1: Identify the genotypes of MAP present in U.S. dairies using whole genome sequencing. Subobjective 3.2: Identify and characterize virulent strains of MAP.

Approach
Within Objective 1 the function of MAP proteins as antigens will be identified using genomic and proteomic tools to better understand their role(s) in pathogenesis of Johne’s Disease and to develop improved diagnostic tools. In Objective 2, tools such as cellular phenotype and secretion of cytokines involved in cell signaling will be measured to characterize host immune responses in asymptomatic and clinical stages of infection, as well after vaccination, to gain knowledge as to correlates involved in controlling the disease. Genetic variability of MAP isolates of livestock will be investigated using whole genome sequencing under Objective 3. This will lead to improved epidemiological tools in the field and understanding of MAP genes involved in virulence. The 3 major objectives outlined within this project plan will work in an interactive manner to provide us with tools to control this disease.

Progress Report
Mycobacterium paratuberulcosis (Johne’s Disease, MAP) is a chronic progressive enteric disease characterized clinically by chronic or intermittent diarrhea, emaciation, and death. The disease has a worldwide distribution and over 70 percent of U.S. dairy herds are infected. Dairies infected with Johne’s disease have significant economic losses due to reduced milk production and premature culling. Because the host immune responses to MAP is complex, the project is characterizing host-pathogen interactions to develop new diagnostic and vaccination tools. In support of Subobjective 1, defining pathogenic mechanisms of MAP, protein-protein interaction studies between MAP bacilli and MAP proteins, and MAP bacilli and bovine macrophages have been conducted. Clones simultaneously expressing multiple MAP antigens have been constructed and authenticated by sequencing and mass spectrometry analysis. These will be used in planned studies evaluating vaccine efficacy. In studies in support of Objective 2 host immune responses of asymptomatic subclinical, cows with clinical disease, and non-infected cattle were compared. Cows with clinical disease had reduced secretion of IL-17, a key pro-inflammatory cytokine. This correlates with a previously observed reduction in numbers of TauCR+ T cells in clinical cows, since TauCR+ T cells are an important source of IL-17. Our data suggest that IL-17 is an important mediator in MAP infection in cattle and decreased expression of IL-17 may disrupt protective immunity. The project also characterized B cell populations in subclinical or clinical cows and characterized differences in tissue macrophage populations in the ileum of cattle at different stages of disease. The project also completed a vaccination study in calves and evaluated efficacy after experimental infection. Improving our understanding of host immune responses and identifying pathogenic mechanisms related to MAP will allow development of better diagnostic tests and vaccines. Improved intervention strategies for MAP will be beneficial for livestock producers, veterinarians, diagnostic laboratories, and regulatory personnel.

Accomplishments
1. Intestinal macrophage phenotypes in Johne’s Disease. Intestinal macrophage phenotypes in Johne’s disease. Johne’s disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Following ingestion, the bacteria are translocated across the intestinal epithelium and taken up by intestinal macrophages. Once ingested by macrophages, MAP may be killed or may persist intracellularly. ARS scientists at Ames, Iowa, used immunofluorescent (IF) and confocal microscopy to correlate the stage of disease to presence of MAP bacteria and macrophage populations/phenotypes within bovine ileal tissues. Cows in the clinical stage of disease had higher numbers of tissue repair macrophages and lower numbers of host defense macrophages in ileal tissues. Asymptomatic, subclinical cows had a nearly equal ratio of host defense and repair macrophage phenotypes, and noninfected cows demonstrated only host defense macrophage populations in ileal tissues. The reduction in host defense macrophages in cows with clinical disease explains why infected cattle have high MAP tissue burdens and fail to control or clear infections. This data provides critical information on the pathophysiology of MAP infection within cattle and will be useful for producers, clinicians and researchers in understanding immune differences at different stages of disease.

2. Vitamin D and Johne’s Disease. Vitamin D plays a key role in both innate and adaptive immunity by influencing macrophage activation and T cell function. Reduced vitamin D concentrations have been associated with increased susceptibility to infection with mycobacterial pathogens. ARS scientists at Ames, Iowa, found that cows with clinical Johne’s disease had lower serum vitamin D levels. Expression of genes associated with vitamin D metabolism in the ileal tissue of cows were also reduced in cattle with clinical disease. A decrease in vitamin D concentrations may be associated with reduced macrophage activation and reduced ability to clear the MAP infection. Reductions in serum vitamin D could contribute to the pathogenesis of Johne’s disease. This information will be beneficial to livestock producers, veterinarians, and scientists working to develop intervention strategies to reduce Johne’s disease in cattle.

3. New diagnostics for Johne’s disease. Improved diagnostic tests are needed to aid producers in controlling this disease. Using a protein array, scientists in Ames, Iowa identified patterns of serological reactivity in cows with Johne’s disease. Some antigens induced antibody responses throughout disease stages, where responses to other antigens were detected late in the course of disease, or early in infection with a decline in responses at later stages of disease. The most promising proteins identified from the Map protein array were tested by ELISA and a multiplex bead assay and distinguished infected from non-infected healthy animals with sensitivities ranging from 65 percent to 93 percent. Some of the proteins had better sensitivity on clinical samples when compared to a commercially available Johne’s disease diagnostic test. These data will be of interest to livestock producers, veterinarians, and diagnostic laboratories who want more accurate tests identifying animals infected with Johne’s disease.

Review Publications
Abdellrazeq, G.S., Elnaggar, M.M., Bannantine, J.P., Park, K.T., Souza, C.D., Backer, B., Hulubei, V., Fry, L.M., Khaliel, S.A., Torky, H.A., Schneider, D.A., Davis, W.C. 2018. A Mycobacterium avium subsp. paratuberculosis relA deletion mutant and a 35 kDa major membrane protein elicit development of cytotoxic T lymphocytes with ability to kill intracellular bacteria. Veterinary Research. 49:53. https://doi.org/10.1186/s13567-018-0549-3.
Greenstein, R.J., Su, L., Fam, P.S., Stabel, J.R., Brown, S.T. 2018. Failure to detect M. avium subspecies paratuberculosis in Johne’s disease using a proprietary fluorescent in situ hybridization assay. BMC Research Notes. 11:498. https://doi.org/10.1186/s13104-018-3601-5.
Lyashchenko, K.P., Grandison, A., Keskinen, K., Sikar-Gang, A., Lambotte, P., Esfandiari, J., Ireton, G.C., Vallur, A., Reed, S.G., Jones, G., Vordermeier, H.M., Stabel, J.R., Thacker, T.C., Palmer, M.V., Waters, W.R. 2017. Identification of novel antigens recognized by serum antibodies in bovine tuberculosis. Clinical and Vaccine Immunology. 24(12):e00259-17. https://doi.org/10.1128/CVI.00259-17.
Rathnaiah, G., Zinniel, D., Bannantine, J.P., Stabel, J.R., Grohn, Y.T., Collins, M.T., Barletta, R.G. 2017. Pathogenesis, molecular genetics, and genomics of Mycobacterium avium subsp. paratuberculosis, the etiologic agent of Johne’s disease. Frontiers in Veterinary Science. 4:187. https://doi.org/10.3389/fvets.2017.00187.
Bannantine, J.P., Stabel, J.R., Lippolis, J.D., Reinhardt, T.A. 2018. Membrane and cytoplasmic proteins of Mycobacterium avium subspecies paratuberculosis that bind to novel monoclonal antibodies. Microorganisms. 6(4):127. https://doi.org/10.3390/microorganisms6040127.
Jenvey, C.J., Hostetter, J.M., Shircliff, A.L., Bannantine, J.P., Stabel, J.R. 2018. Relationship between the pathology of bovine intestinal tissue and current diagnostic tests for Johne's disease. Veterinary Immunology and Immunopathology. 202:93-101. https://doi.org/10.1016/j.vetimm.2018.06.012.
Jenvey, C.J., Hostetter, J.M., Shircliff, A.L., Bannantine, J.P., Stabel, J.R. 2019. Quantification of macrophages and Mycobacterium avium subsp. paratuberculosis in bovine intestinal tissue during different stages of Johne's disease. Veterinary Pathology. https://doi.org/10.1177/0300985819844823.
Albarrak, S.M., Waters, W.R., Stabel, J.R., Hostetter, J.M. 2018. Evaluating the cytokine profile of the WC1+ gamma delta T cell subset in the ileum of cattle with the subclinical and clinical forms of MAP infection. Veterinary Immunology and Immunopathology. 201:26-31. https://doi.org/10.1016/j.vetimm.2018.05.003.