Location: Infectious Bacterial Diseases Research
2020 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
Johne’s Disease is a chronic progressive intestinal disease caused by the bacterium Mycobacterium paratuberculosis (MAP) and is characterized clinically by chronic or intermittent diarrhea, emaciation, and death. The disease has a worldwide distribution, and over 70% 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 Objective 1, immunogenic antigens were identified using a protein microarray and characterized in antibody-based detection assays using synthetic peptides to define specific epitopes on the proteins. Using improved annotation software, the project identified 227 previously unknown genes in the genome of MAP. The majority of these new genes (67%) were identified by analysis as hypothetical proteins, suggesting they are unique and not comparable to genes previously reported in other bacteria. Transcriptomic analysis studies demonstrated that these genes are expressed (not pseudogenes) and the new genes are being evaluated to see if infected animals develop immune responses to them. Genes unique to MAP were also identified by comparing the genomes of MAP with other closely related Mycobacterium species to identify new targets for diagnostic evaluation. In support of Objective 2, immunofluorescent staining and confocal microscopy were used to characterize intestinal macrophage populations and correlate differences in populations with clinical severity of disease. The project demonstrated that a novel subunit vaccine protects calves and reduces colonization of tissues with MAP. Vaccinated calves had increased IL-17 and IFN-gamma gene expression, indicating that pro-inflammatory cytokines expression correlates with observed reductions in MAP colonization. Vaccinated calves had serologic responses to MAP, but not to M. avium or M. bovis, suggesting the vaccine will not cause cross-reactivity on diagnostic tests.
Accomplishments
1. Host immune response to MAP infection. Johne’s Disease is a chronic progressive intestinal disease caused by the bacterium Mycobacterium paratuberculosis (MAP) and is characterized clinically by chronic or intermittent diarrhea, emaciation, and death. Dairy herd infected with Johne’s disease have significant economic losses due to reduced milk production and premature culling. Understanding the host immune response to MAP infection is critical to the optimization of diagnostic tools and vaccine development. ARS scientists in Ames, Iowa, compared immune responses of naturally-infected dairy cattle that were either asymptomatic (subclinical) or clinical, to non-infected cattle. Our data suggest a complex coordination of immune responses during MAP infection, with a shift to a less pro-inflammatory response as the host transitions from subclinical to clinical. Basic understanding of immunologic changes during infection will be of interest to producers, veterinarians, and academic scientists interested in development of intervention strategies and understanding the physiologic mechanisms related to disease progression.
2. Development of MAP infection models. Johne’s Disease is a chronic progressive intestinal disease caused by the bacterium Mycobacterium paratuberculosis (MAP) and is characterized clinically by chronic or intermittent diarrhea, emaciation, and death. Dairy herd infected with Johne’s disease have significant economic losses due to reduced milk production and premature culling. Animal models of MAP are critical for evaluating vaccine efficacy and developing therapeutics for treatment. ARS scientists in Ames, Iowa, compared immune responses and infection in neonatal sheep, goats, and calf models of MAP infection. Fecal shedding was higher for calves throughout the study. Colonization of tissues was highly variable but tended to be higher in calves and sheep. Calves developed more robust immune responses than sheep and goats. Expression of cytokine genes typically associated with inflammation was higher in goats and correlated with lower colonization of MAP in tissues. Although the data does suggest similarities in sheep, goats, and calves in susceptibility, functional differences in host immune responses appear to correlate with differences in MAP infection between species. This information on infection models will be of interest to producers, veterinarians, and academic scientists interested in identifying intervention strategies to reduce MAP infection.
3. Completed genome of the bacterium causing Johne’s Disease. Johne’s Disease is a chronic progressive intestinal disease caused by the bacterium Mycobacterium paratuberculosis (MAP) and is characterized clinically by chronic or intermittent diarrhea, emaciation, and death. Dairy herd infected with Johne’s disease have significant economic losses due to reduced milk production and premature culling. The type III strain of MAP is found in sheep and goats and has characteristics of being more fastidious and difficult to culture than other MAP strains. It is also phylogenetically different. ARS scientists in Ames, Iowa, completed the genomic sequence of a MAP type III strain and published it for public use. The genomes of type I and type II strains of MAP were previously published. These data advance our understanding of genetic diversity among all MAP subtypes and contribute to an understanding of how genetic differences influence virulence. This information on MAP genetics will be of interest to producers, veterinarians, and academic scientists interested in understanding MAP virulence and identifying intervention strategies to reduce infection.
Review Publications
Abdellrazeq, G.S., Fry, L.M., Elnaggar, M.M., Bannantine, J.P., Schneider, D.A., Chamberlain, W.M., Mahmoud, A.H.A., Park, K.T., Hulubei, V., Davis, W.C. 2020. Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium supsp. paratubeculosis peptide vaccine. Vaccine. 38(8):2016-2025. https://doi.org/10.1016/j.vaccine.2019.12.052.
Stabel, J.R., Reinhardt, T.A., Hempel, R.J. 2018. Vitamin D status and responses in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis. Journal of Dairy Science. 102(2):1594-1600. https://doi.org/10.3168/jds.2018-15241.
Jenvey, C.J., Shircliff, A.L., Bannantine, J.P., Stabel, J.R. 2019. Phenotypes of macrophages present in the intestine are impacted by stage of disease in cattle naturally infected with Mycobacterium avium subsp. paratuberculosis. PLoS One. 14(5). https://doi.org/10.1371/journal.pone.0217649.
Bannantine, J.P., Zinniel, D., Barletta, R.G. 2019. Transposon mutagenesis in Mycobacterium avium subspecies paratuberculosis. Methods in Molecular Biology. 2016/117-125. https://doi.org/10.1007/978-1-4939-9570-7_11.
Li, L., Bannantine, J.P., Campo, J.J., Randall, A., Grohn, Y.T., Schilling, M., Katani, R., Radzio-Basu, J., Easterling, L., Kapur, V. 2019. Identification of sero-diagnostic antigens for the early diagnosis of Johne's disease using MAP protein microarrays. Scientific Reports. 9:17573. https://doi.org/10.1038/s41598-019-53973-x.
Stabel, J.R., Bannantine, J.P. 2019. Divergent antigen-specific cellular immune responses during asymptomatic subclinical and clinical states of disease in cows naturally infected with Mycobacterium avium subsp. paratuberculosis. Infection and Immunity. 88(1):e00650-19. https://doi.org/10.1128/IAI.00650-19.
Stabel, J.R., Bannantine, J.P., Hoestetter, J.M. 2020. Comparison of sheep, goats, and calves as infection models for Mycobacterium avium subsp. paratuberculosis. Veterinary Immunology and Immunopathology. 225(110060). https://doi.org/10.1016/j.vetimm.2020.110060.
Bannantine, J.P., Wadhwa, A., Stabel, J.R., Eda, S. 2019. Characterization of ethanol extracted cell wall components of Mycobacterium avium subsp. paratuberculosis. Veterinary Sciences. 6(4):88. https://doi.org/10.3390/vetsci6040088.
Shoyama, F.M., Janetanakit, T., Bannantine, J.P., Barletta, R., Sreevatsan, S. 2020. Elucidating the regulon of a fur-like protein in Mycobacterium avium subsp. paratuberculosis (MAP). Frontiers in Microbiology. 11:598. https://doi.org/10.3389/fmicb.2020.00598.
