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

Location: Infectious Bacterial Diseases Research

2018 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 US 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 1.1, the in silico protein interaction network for MAP membrane proteins has been developed and cataloged. These results will be compared to protein interactions identified by Far Western blotting experiments. In support of Subobjective 2.1, immune responses to MAP infection in asymptomatic subclinical and cows demonstrating clinical disease were compared to responses of non-infected cattle (control). Infected cattle had increased cytokine secretion in peripheral blood mononuclear cells regardless of stage of disease, and higher levels of cytokine secretion as compared to responses of non-infected cattle. Infection resulted in increased secretion of pro-inflammatory cytokines, regardless of stage of disease. In contrast, IL-10 secretion was increased in clinical cattle compared to controls. Our data suggest that cows in the clinical stage of disease maintain robust immune responses to MAP infection but regulatory mechanisms prevent a pro-inflammatory immune response. Frozen tissues from infected and non-infected cattle were used to correlate stage of disease with macrophage phenotypes using immunofluorescent staining and confocal microscopy. Cattle with clinical disease had increased tissue macrophage numbers and greater MAP colonization. Macrophage populations in tissue were highly correlated with serum antibody titers, fecal shedding, and numbers of bacteria within tissue. A bioinformatic approach (STRING analysis) and a bovine macrophage cell line were used to evaluate interactions between MAP surface proteins and host cells. Improving understanding of host immune responses and identifying antigens contributing to disease progression will facilitate development of more sensitive and specific diagnostic tests for MAP, and assist in vaccine development. Improved management tools for MAP will benefit livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies. In support of Subobjective 2.3, cellular markers to differentiate B cell subsets have been identified, purchased, and are being evaluated in flow cytometric studies. Upon completion of antibody evaluation, the impact of disease status on B cell subsets will be assessed.

Accomplishments
1. Intestinal macrophage accumulation correlates with Johne’s disease severity. 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, correlated stage of clinical disease to macrophage populations and MAP bacteria in bovine ileal tissues using immunofluorescent (IF) and confocal microscopy. Macrophage populations and intracellular MAP bacteria were highest in cattle with clinical disease, followed by subclinical cows, and non-infected cattle. In clinical cows, macrophages were present throughout all layers of the ileal epithelium. Data indicates that macrophage populations correlate with disease progression. As many macrophages were not associated with MAP bacteria or antigens, our data suggests that bovine immune responses recruit macrophages into tissue in response to MAP invasion. In cows with clinical disease, macrophages are ultimately unable to clear MAP bacteria resulting in development of signs of disease. Fecal culture and antibody responses correlated to macrophage numbers and numbers of macrophages with intracellular MAP within ileal tissue. This research provides data on the host-pathogen interactions, disease pathogenesis, and demonstrates the relationships between ante-mortem tests and stage of disease. The research results provide critical information on the biology of MAP infection within the target tissue and are useful for producers, clinicians and researchers to gauge the involvement of tissue during the different stages of disease.

2. Circulating number and functional capacity of gamma delta T cells decline with Johne’s disease progression. The role of gamma delta T cells in the pathogenesis of mycobacterial infections is of increasing interest due to their diverse functions spanning both innate to adaptive immunity. Bovine gamma delta T cells differentiate into two subsets based on the surface expression of the workshop cluster 1 molecule (WC1). Lack of WC1 expression in gamma delta T cells is most common in organs such as spleen and intestine, whereas expression of WC1 is common in gamma delta T cells circulating in peripheral blood. In collaboration with scientists at Iowa State University, ARS scientists at Ames, Iowa, characterized gamma delta T cells in peripheral blood of cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP). Cattle infected with MAP had fewer gamma delta T cells in peripheral blood when compared to controls (non-infected) and the decrease circulation in blood was not due to migration to sites of MAP infection. Gamma delta T cells from infected animals without clinical signs (subclinical) had greater proliferation and production of interferon-gamma in response to MAP antigens when compared to cattle with clinical disease. The data indicates differences in gamma delta T cell function in cattle with subclinical or clinical form of Johne’s disease and provides insights into host-pathogen interactions during MAP infection. This information will be beneficial to scientists working to develop intervention strategies to reduce Johne’s disease in cattle.

Review Publications
Li, L., Wagner, B., Freer, H., Schilling, M., Bannantine, J.P., Campo, J.J., Katani, R., Grohn, Y.T., Radzio-Basu, J., Kapur, V. 2017. Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immuonassays. PLoS One. 12(12). https://doi.org/10.1371/journal.pone.0189783.
Samba-Louaka, A., Robino, E., Cochard, T., Branger, M., Delafont, V., Aucher, W., Wambeke, W., Bannantine, J.P., Biet, F., Hechard, Y. 2018. Environmental Mycobacterium avium subsp. paratuberculosis hosted by free-living amoebae. Frontiers in Cellular and Infection Microbiology. 8:28. https://doi.org/10.3389/fcimb.2018.00028.
Magombedze, G., Shiri, T., Eda, S., Stabel, J.R. 2017. Inferring biomarkers for Mycobacterium avium subsp. paratuberculosis infection and disease progression using experimental data. Scientific Reports. 7:44765. https://doi.org/10.1038/srep44765.
Bannantine, J.P., Campo, J., Randall, A., Pablo, J., Praul, C.A., Raygoza Garay, J.A., Li, L., Stabel, J.R., Kapur, V. 2017. Identification of novel seroreactive antigens in Johne’s disease cattle using the Mycobacterium tuberculosis protein array. Clinical and Vaccine Immunology. 24(7). https://doi.org/10.1128.CVI.00081-17.
Bannantine, J.P., Etienne, G., Laval, F., Lemassu, A., Daffe, M., Bayles, D.O., Ganneau, C., Bonhomme, F., Branger, M., Cochard, T., Bay, S., Biet, F., Stabel, J.R. 2017. Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide. Molecular Microbiology. 105(4):525-539. https://doi.org/10.1111/mmi.13717.
Albarrak, S., Hostetter, J., Waters, W.R., Stabel, J.R. 2017. WC1+ gamma delta T cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis respond differentially to stimulation with PPD-J. Veterinary Immunology and Immunopathology. 190:57-64. https://doi.org/10.1016/j.vetimm.2017.07.007.
Souza, C.D., Bannantine, J.P., Brown, W., Hwang, J.K., Laws, E., Ziaei, P., Norton, M.G., Abdellrezeq, G.S., Davis, W.C., Eren, M., Cardieri, M.D. 2017. A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages. Journal of Applied Microbiology. 123(1):54-65. https://doi.org/10.1111/jam.13491.
Li, L., Bannantine, J.P., Campo, J.J., Randall, A., Grohn, Y.T., Katani, R., Schilling, M., Radzio-Basu, J., Kapur, V. 2017. Identification of sero-reactive antigens for the early diagnosis of Johne's disease in cattle. PLoS One. 12(9). https://doi.org/10.1371/journal.pone.0184373.
Stabel, J.R., Jenvey, C.J. 2017. Sudan Black B masks Mycobacterium avium subspecies paratuberculosis immunofluorescent antibody labeling. Histology and Histopathy. 4(11). doi:10.7243/2055-091X-4-11.
Stabel, J.R., Jenvey, C.J. 2017. Autofluorescence and non-specific immunofluorescent labeling in frozen bovine intestinal tissue sections: Solutions for multi-color immunofluorescence experiments. Journal of Histochemistry and Cytochemistry. 65(9):531-541. https://doi.org/10.1369/0022155417724425.