2007 Annual Report
1a.Objectives (from AD-416)
Objective 1: Systematically identify and characterize novel and specific antigens from the M. paratuberculosis genome sequence project.
Objective 2: Determine the genetic variability among M. paratuberculosis isolates and examine the transcriptional profile of the M. paratuberculosis genome.
Objective 3: Develop and evaluate methods to evaluate the host immune responses to M. paratuberculosis in early and late infection to distinguish elements of protective immunity.
Objective 4: Evaluate the sensitivity and specificity of cell-mediated diagnostic tests in sheep and cattle for early detection of M. paratuberculosis infection.
1b.Approach (from AD-416)
Within Objective 1 unique antigens of M. paratuberculosis will be
evaluated as immunogens with particular emphasis on their utility as
diagnostic reagents or vaccine candidates. Objective 2 will compare
and contrast the genetic content of various strains of M.
paratuberculosis, both within and between species of animals to
provide information on the characteristics of infectivity and
pathogenicity for different strains. The host immune response to M.
paratuberculosis infection will be evaluated in Objective 3 in both
experimentally and naturally infected animals to gain an
understanding of how the disease progresses from a subclinical to a
more clinical state. Objective 4 will examine the efficacy of skin
testing and a blood assay for the early detection of disease in
naturally infected and noninfected cattle and sheep.
The Johne’s Disease Research Project is aligned with NP 103 Animal Health to conduct innovative cutting-edge research, which delivers effective and practical solutions to agricultural problems of high national priority. The primary objectives of this research project include the identification of specific genes for Mycobacterium avium subsp. paratuberculosis with an emphasis on differentiation of MAP antigens from closely related M. avium subsp. avium antigens. Characterization of these genes and their representative proteins will result in more sensitive and specific diagnostic tools for the detection of infection in the field. Progress in the past year has included the further cloning and expression of Mycobacterium avium subsp. paratuberculosis proteins for study as potential diagnostic reagents. The proteins are being evaluated by Western blotting techniques and in the secretion of cytokines such as interferon-gamma and IL-12 as potential markers of infection. Monoclonal antibodies have been generated to some of the cell surface proteins of Mycobacterium avium subsp. paratuberculosis, providing tools that can be used for understanding the disease process. Sequencing of the ovine strain of Mycobacterium avium subsp. paratuberculosis has been initiated and will provide information on key differences between cattle and sheep strains. This information may be useful in distinguishing elements of pathogenesis for Mycobacterium avium subsp. paratuberculosis. The development of early detection methods such as the skin test and interferon-gamma tests and evaluation of these tests in the field have been initiated in sheep and dairy calves. Early detection of Mycobacterium avium subsp. paratuberculosis infection will reduce the spread of disease in a flock or herd. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis. Further diagnostic tests that have been modified and improved in the past year include a quantitative real-time PCR test using a unique Mycobacterium avium subsp. paratuberculosis target gene that has been validated for milk, feces, and tissue. Research on the immunology and pathology of Mycobacterium avium subsp. paratuberculosis infections in cattle has continued with an emphasis on the host response during the periparturient period, a very stressful period for cows. Results from one periparturient study demonstrated significant changes in the expression and secretion of cytokines, major modulators of host immunity, and on immune cell phenotypes. Further work on early host responses to infection has been conducted through the development and evaluation of experimental infection models for Mycobacterium avium subsp. paratuberculosis in neonatal calves. This work will help define which attributes of host immunity are initiated in the early stages of infection. Experimental infection models will also be useful for future work evaluating vaccine candidates for Mycobacterium avium subsp. paratuberculosis.
Evaluation of experimental infection models for MAP in the neonatal calf.
Infection models are useful for studying host responses to infection to aid in the development of diagnostic tools and vaccines. The majority of experimental models for ruminants have utilized an oral inoculation of live Mycobacterium avium subsp. paratuberculosis in order to establish infection, thereby mimicking the fecal-oral route of transmission generally observed in the field. The current study was designed to compare the effectiveness of oral and intraperitoneal inoculation on the host immune response to Mycobacterium avium subsp. paratuberculosis infection. Calves were purchased and assigned to one of 5 experimental infection treatment groups, including control, oral, oral with dexamethasone (DXM) treatment, intraperitoneal, and oral/mucosal. Animals were monitored using a variety of assays throughout the course of the study. Results are pending the terminal endpoint of the study. A bovine infection model is critical to the success of evaluating diagnostic tools and vaccines for paratuberculosis and is helpful in evaluating dynamic host immune responses to infection. This will help cattle and dairy producers. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce and number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Johne's Disease.
Evaluate host immunity in naturally infected dairy cows in both subclinical and clinical stages of infection and noninfected control cows during the periparturient period.
Previous studies indicate that the periparturient period is a period of stress with concomitant immunosuppression. Dogmatic information indicates that cows with paratuberculosis often break with clinical disease signs shortly after calving. The proposed research should reveal potential mechanisms for the immunosuppression during the calving period by characterizing cytokine expression and secretion and immune cell function during the periparturient period in cows with subclinical and clinical infection as compared to healthy non-infected controls. Thus far, cows have been pre-screened by ELISA and fecal PCR for purchase, acclimated on-site, and sampling has begun for the immediate periparturient period from 4 weeks before calving through 4 weeks post-calving. If we can identify factors that contribute to the immunosuppression noted at calving, we can use this information to improve management of dairy cows that will alleviate the escalation of disease in this time period. These studies will alert cattle and dairy producers to potentially disease susceptible periods. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Johne's Disease.
Genome sequencing of an ovine Mycobacterium avium subsp. paratuberculosis isolate.
Previous work in our laboratory has demonstrated that Mycobacterium avium subsp. paratuberculosis isolates obtained from sheep and cattle can be distinguished from each other by the presence or absence of certain genetic material. The genomic DNA from a Mycobacterium avium subsp. paratuberculosis isolate obtained from a sheep was subjected to whole-genome sequencing in order to identify all of the genes present in the organism. The nearly complete genome sequence will provide a framework for future research into the host specificity of Mycobacterium avium subsp. paratuberculosis isolates and will enable a more detailed comparison with the previously sequenced cattle isolate. This work has provided a road map of a second strain of M. avium subsp. paratuberculosis to investigators in the field of Johne's Disease research. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Johne's Disease.
Antibody production in Johne's Disease.
Within the past year, we have produced several novel monoclonal antibodies against Mycobacterium avium subsp. paratuberculosis. These antibodies have opened up new areas of research in pathogenesis and diagnostic studies. In addition, they have gathered the interest of the private sector as indicated by the numerous MTAs with companies for transfer of these antibodies. Two manuscripts have just been published describing these antibodies. We have also identified antibodies, produced in sheep with Johne’s Disease, that react with BOTH a sheep protein AND a protein produced by Mycobacterium avium subsp. paratuberculosis. This new information provides the first clues to the immunopathology observed in Johne's Disease, and may suggest an autoimmune component to this disease. This study was also published in 2007. This accomplishment aligns with National Program 103, Animal Health, "Strategic Goal 4: Enhance protection and safety of the Nation's Agriculture and Food Supply", "Objective 4.2: Reduce the number, severity and distribution of agricultural pest and disease outbreaks", as it relates to diagnosis and control of Johne's Disease.
5.Significant Activities that Support Special Target Populations
Methods development, optimization, and evaluation for sheep diagnostic tests for MAP was conducted. The sheep producers in the US specifically requested that ARS perform work in this area.
|Number of new CRADAs and MTAs||7|
|Number of active CRADAs and MTAs||7|
|Number of invention disclosures submitted||1|
Bannantine, J.P., Radosevich, T.J., Stabel, J.R., Sreevatsan, S., Kapur, V., Paustian, M. 2007. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis. Clinical and Vaccine Immunology. 14(5):518-526.
Bannantine, J.P., Radosevich, T.J., Stabel, J.R., Berger, S., Griffin, J.F., Paustian, M. 2007. Production and Characterization of Monoclonal Antibodies Against a Major Membrane Protein of Mycobacterium avium subsp. paratuberculosis. Clinical and Vaccine Immunology. 14(3):312-317.
Talaat, A.M., Ward, S.K., Wu, C., Rondon, E., Tavano, C., Bannantine, J.P., Lyons, R., Johnston, S.A. 2007. Mycobacterial Bacilli are Metabolically Active during Chronic Tuberculosis in Murine Lungs: Insights from Genome-Wide Transcriptional Profiling. Journal of Bacteriology. 189(11):4265-4274.
Radosevich, T.J., Reinhardt, T.A., Lippolis, J.D., Bannantine, J.P., Stabel, J.R. 2007. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187. Journal of Bacteriology. 189(3):1109-1117.
Li, L., Munir, S., Bannantine, J.P., Sreevatsan, S., Kanjilal, S., Kapur, V. 2007. Rapid Expression of Mycobacterium avium subsp. paratuberculosis Recombinant Proteins for Antigen Discovery. Clinical and Vaccine Immunology. 14(1):102-105.
Stabel, J.R., Kimura, K., Robbe Austerman, S. 2007. Augmentation of Secreted and Intracellular Gamma Interferon Following Johnin Purified Protein Derivative Sensitization of Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis. Journal of Veterinary Diagnostic Investigation. 19:43-51.
Stabel, J.R. 2006. Host Responses to Mycobacterium avium subsp. paratuberculosis: A Complex Arsenal. Animal Health Research Reviews. 7(1):61-70.
Harrentien, L.A., Finnegan, M.V., Woodford, N.L., Mansfield, K.G., Waters, W.R., Bannantine, J.P., Paustian, M., Garner, M.M., Bakke, A.C., Peloquin, C.A. 2006. Mycobacterium avium in Pygmy Rabbits (Brachylagus Idahoensis): 28 Cases. Journal of Zoo and Wildlife Medicine. 37(4):498-512.