Skip to main content
ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Research Project #432135

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

Location: Infectious Bacterial Diseases Research

2021 Annual Report

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.

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
This will be the final report for project 5030-32000-221-00D terminating October 5, 2021. Research will continue under a bridging (or new) project. Overall, solid progress was made on research objectives despite intractable problems with the two-hybrid assays used in Subobjective 1.1 for defining bacterial-host protein interactions. New detection reagents for Johne’s disease were developed in support of Subobjective 1.2. Specifically, over 80 novel DNA targets from a whole genome analysis were identified for detecting Mycobacterium avium subspecies paratuberculosis (MAP) bacteria. A synthetic lipid representing a component of the MAP bacterial cell wall was constructed and shown to be sensitive as an antigen in an enzyme linked immunosorbent assay (ELISA)-based test. A diagnostic phage assay to detect MAP was tested using samples from infected dairy cattle. In work addressing Objective 2, host immunity was evaluated and correlated to the pathogenesis of Johne’s disease. T cell populations in the intestinal tissues of cows were characterized and data suggest that specific T cell types could be predictive of early stages of Johne’s disease. This observation was used to demonstrate similarities and differences in the T cell responses of calves infected with tuberculosis or Johne’s strains of mycobacteria. Data suggests that diagnostics can be developed to distinguish M. bovis infection from MAP infection. During the five-year project plan, advances in knowledge were made that will be beneficial for controlling Johne’s disease in domestic livestock. Improvements were made in diagnostics including antigen-based and DNA-based diagnostic platforms for detecting MAP infection in animals. A protein array was used to identify peptides that demonstrate reactivity to serum from cows with Johne’s disease. Some antigens induced antibody responses throughout disease stages, whereas other antigens were detected by humoral responses at different stages of infection (i.e. early or later in infection). Immunoreactive proteins identified by the MAP protein array were further tested in ELISA and multiplex bead assay formats and responses of infected and non-infected cattle compared. Assays using some of the proteins had better sensitivity in detecting infected animals when compared to detection by commercially available Johne’s diagnostic tests. New targets were also developed for use in DNA-based diagnostics. Knowledge on disease pathogenesis was developed including the observation that low levels of vitamin D correlate with a higher incidence of Johne’s disease in cattle. A variety of approaches, including functional genomics and characterization of host responses were used to understand immunologic responses. This work characterized the T cell response during infection, correlated cytokine gene expression to disease progression, and defined the composition of cell wall lipids and gene expression in MAP during infection. New live attenuated vaccine strains and recombinant proteins were evaluated for their immunogenicity and efficacy in cattle. A commercially available disinfectant that is environmentally friendly was demonstrated to be effective at killing MAP. Overall, this work advanced scientific knowledge of Johne’s disease and identified new vaccine and diagnostic strategies that can be used to control this disease in domestic livestock.

1. New Johne’s vaccine for cattle. ARS researchers in Ames, Iowa, tested a new subunit vaccine containing a cocktail of recombinant proteins in trials in dairy calves. In two trials, a strong reduction in MAP colonization of intestinal tissues was observed with highest dosage of the vaccine demonstrating the greatest reduction in infection. The vaccine also reduced fecal shedding of the pathogen which is important for stopping on-farm transmission. Data has been used to patent this vaccine. These data will be of interest to producers, regulatory personnel, and researchers interested in intervention strategies for preventing Johne’s disease in domestic livestock.

2. New diagnostic strategies for MAP. ARS researchers in Ames, Iowa, improved diagnostics of Johne’s disease during the past year by development of antibody and DNA detection assays that have improved performance in detecting a lipid-based enzyme linked immunosorbent assay (ELISA) test, an electrochemical based antibody test, identification of new targets for DNA-based detection assays, and diagnostics for detecting early stages of Johne’s disease. A synthetic lipid that has water soluble properties was developed for a lipid-based enzyme-linked immunosorbent assay test that demonstrated sensitivity and specificity equivalent to the native lipid in MAP. For development of a fast, cow-side, diagnostic test results, an electrochemical test was constructed that has improved sensitivity for detecting infected cattle as compared to a standard diagnostic platform. Sophisticated genome comparisons were used to identify 80 new DNA-based diagnostic targets. These new targets were validated for sensitivity in detecting MAP in polymerase chain reaction tests. Predictive modelling of T cell populations during infection suggests that characteristics of these populations could be used to develop diagnostics for early detection of Johne’s infections. These data will be of interest to researchers, producers and regulatory personnel with interests in improved diagnostics for Johne’s disease.

3. Inactivation of Johne’s disease. Two improved methods for effectively killing Mycobacterium avium subspecies paratuberculosis (MAP) were developed. ARS scientists in Ames, Iowa, and in Peoria, Illinois, collaborated to identify and evaluate peptides produced by natural killer immune cells that are toxic to MAP. The work identified two peptides that were most potent in killing MAP were shown to act by destroying the bacterial cell membrane. These peptides could be used to develop intervention strategies to promote natural killer cell activity at the site of infection or recombinantly express the peptides. An environmentally friendly disinfectant was identified that kills MAP on laboratory and field conditions and compared to traditional disinfectants. The new disinfectant was as effective at killing MAP as the harsher phenolic compounds that are typically used. Development of validated inactivation methods for MAP are needed for improving biosafety and biosecurity. These data will be of interest to researchers, regulatory personnel, and producers with interests in Johne’s disease.

Review Publications
Stabel, J.R., Bannantine, J.P. 2021. Reduced tissue colonization of Mycobacterium avium subsp. paratuberculosis in neonatal calves vaccinated with a cocktail of recombinant proteins. Vaccine.
Greenstein, R.J., Su, L., Grant, I.R., Foddai, A.C., Turner, A.M., Nagati, J.S., Brown, S.T., Stabel, J.R. 2021. Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease. Gut Pathogens.
Jenvey, C.J., Shircliff, A.L., Obando Marrero, E.E., Stabel, J.R. 2021. Prediction of Johne's disease state based upon quantification of T cell markers and their interaction with macrophages in the bovine intestine. Veterinary Research.
Dassanayake, R.P., Wherry, T.L., Falkenberg, S.M., Reinhardt, T.A., Casas, E., Stabel, J.R. 2021. Bovine NK-lysin-derived peptides are bactericidal against Mycobacterium avium subspecies paratuberculosis. Veterinary Research. 52. Article 11.
Bannantine, J.P., Conde, C., Bayles, D.O., Branger, M., Biet, F. 2020. Genetic diversity among Mycobacterium avium subspecies revealed by analysis of complete genome sequences. Frontiers in Microbiology. 11.
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.
Hatate, K., Rice, J., Parker, K., Wu, J., Turner, A.M., Stabel, J.R., Eda, S. 2021. Electrochemical detection of serum antibodies against Mycobacterium avium subspecies paratuberculosis. Frontiers in Veterinary Science. 8.
Bay, S., Begg, D., Ganneau, C., Branger, M., Cochard, T., Bannantine, J.P., Kohler, H., Moyen, J., Whittington, R.J., Biet, F. 2021. Engineering synthetic lipopeptide antigen for specific detection of Mycobacterium avium subsp. paratuberculosis infection. Frontiers in Veterinary Science. 8.
Bannantine, J.P., Bayles, D.O., Biet, F. 2021. Complete genome sequence of a type III ovine strain of Mycobacterium avium subspecies paratuberculosis. Microbiology Resource Announcements. 10.
Bannantine, J.P., Stabel, J.R., Bayles, D.O., Condes, C., Biet, F. 2021. Diagnostic sequences that distinguish M. avium subspecies strains. Frontiers in Veterinary Science. 7.
Stabel, J.R., Turner, A.M., Walker, M.E. 2020. An eco-friendly decontaminant to kill Mycobacterium avium subsp. paratuberculosis. Journal of Microbiological Methods. 176.