2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Characterize unique MAP proteins using previously cloned and expressed proteins for their effect on immune cells and to determine potential antigenicity by evaluating humoral and cellular immune responses and evaluate their use as new diagnostic tools. Subobjective 1.1: Construct 96-spot protein arrays and use them to probe sera from healthy and infected cattle. Subobjective 1.2: Screen recombinant proteins in a gamma-interferon (IFN-') microassay. Subobjective 1.3: Characterize a new protein that is expressed uniquely in MAP as well as other promising proteins from our initial screen. Subobjective 1.4: Develop a Luminex bead assay incorporating the most promising 10 MAP antigens and validate this test using 35 well-characterized cattle at different stages of Johne’s disease.
Objective 2: Develop an infection model that allows the evaluation of the host immune response to MAP in early and late infection by defining the factors that induce the shift of a T helper 1 to a T helper 2 response resulting in clinical disease. Subobjective 2.1: Compare methods of MAP inocula preparation on tissue infectivity in a calf model. Subobjective 2.2: Compare neonatal calves, sheep and goats as host animals for MAP challenge model. Subobjective 2.3: Evaluate host immunity in naturally infected dairy cows to identify markers associated with subclinical and clinical infection.
Objective 3: Develop new vaccines using novel vaccine platforms, adjuvants and strategies to control MAP based on the antigenic and genetic findings using various animal models. Subobjective 3.1: Determine immune responses elicited by vaccination with a commercial vaccine for MAP as well as effects of vaccination on the interpretation of M. bovis diagnostic tests. Subobjective 3.2: Evaluate cloned MAP proteins as potential vaccine candidates. Subobjective 3.3: Evaluate genomic DNA clones as potential vaccine candidates.
1b.Approach (from AD-416):
Within Objective 1 unique antigens of MAP will be evaluated as immunogens with particular emphasis on their utility as diagnostic reagents or vaccine candidates. In Objective 2, ruminant models of infection will be developed and compared for efficacy in establishing infection in the host with the goal of characterizing a model that will progress from asymptomatic subclinical infection to a more clinical state within a 12-month period. These infection challenge models will be useful for evaluating potential vaccine candidates characterized in Objective 3. Cloned MAP proteins that are antigenic in the ruminant host and genomic DNA clones arrayed in pools will be evaluated as potential vaccines to protect against infection under Objective 3. The 3 major objectives outlined within this project plan will work in an interactive manner to provide us with tools to control this disease.
Paratuberculosis (Johne’s Disease - JD) is a chronic progressive enteric disease characterized clinically by chronic or intermittent diarrhea, emaciation, and death. JD has worldwide distribution and economic impact on ruminant livestock production. Recent reports by the National Animal Health Monitoring System (NAHMS) demonstrated that over 70% of U.S. dairy herds had some incidence of Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in significant annual economic losses to the dairy industry, primarily due to reduced milk production and premature culling. Because host immune responses to MAP infection are complex, the primary objectives of this project include further understanding of host-pathogen interactions so that improved tools for diagnosis and prevention of disease can be developed. In support of this, recombinant MAP proteins were evaluated for reactivity to sera from infected cattle to assess humoral immunity, as well as in an IFN-gamma assay to assess cell-mediated immune responses. In addition, a unique monoclonal antibody that is highly specific to MAP has been identified and is currently being evaluated as a potential diagnostic tool. Experimental MAP infection in a neonatal calf model was initiated, as well as a comparative study to assess calves, sheep, and goats as ruminant models of infection. These studies will provide information on how methods of bacterial isolation affect strain virulence and infectivity in experimental models. The multi-species study will provide key information on the pathogenesis of MAP in different ruminant species, including species in which infection develops more rapidly or demonstrate more severe clinical disease. A third approach may yield new vaccine candidates for the control of paratuberculosis in the field. Cocktails of MAP recombinant proteins were evaluated in a mouse model for protection against MAP colonization in the tissues. One cocktail of 3 MAP proteins yielded significant protection and is currently being evaluated in a calf challenge model. Characterization of immune responses may result in more sensitive and specific diagnostic tests for the detection of paratuberculosis in the field. Understanding the pathogenesis of disease and developing more effective infection models will aid in the evaluation of diagnostic tools, therapeutics, and vaccines. New vaccine candidates would be beneficial in controlling the escalating spread of paratuberculosis in U.S. dairy herds. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis.
Completed whole genome sequencing of three U.S. sheep isolates of Mycobacterium avium subspecies paratuberculosis (MAP). Early data from our laboratory showed significant genomic differences between cattle and sheep isolates of this pathogen. Therefore, we sought to comprehensively define the genomic differences between cattle and sheep isolates of MAP. The genomes were compared to a bovine MAP strain previously sequenced in our lab. Comparative genomic analyses revealed large regions of the genome present in the sheep MAP isolates were absent in the bovine MAP isolates. ARS researchers in Ames, Iowa, performed a comprehensive study using next-generation sequencing technology combined with optimal mapping to demonstrate additional novel regions of difference between sheep and cattle strains of MAP. We now have a catalog of genetic differences between cattle and sheep strains of MAP. Tracking these differences at the genome level allows understanding of differences in pathogenesis of paratuberculosis between the two host species. This work will facilitate development of improved diagnostic assays more aligned with a specific ruminant species that could have worldwide relevance in identifying infected animals and preventing production losses.
Established infection models in calves to evaluate host immunity. False positive reactions in diagnostic tests can occur because of other mycobacteria that are present in the environment. This can lead to inaccurate diagnosis of an animal, creating production losses to producers due to culling because of erroneous diagnosis. ARS researchers in Ames, Iowa, conducted a study to evaluate the use of recombinant proteins from Mycobacterium avium subspecies paratuberculosis (MAP) to diagnose paratuberculosis in calves that were experimentally infected with different mycobacteria. The proteins were not able to differentiate between paratuberculosis and the other mycobacterial infections. However, information gained from this study about host immune responses to infection with other mycobacteria and how this compares to infection with MAP was informative. Results demonstrated that immune responses to MAP and Mycobacterium (M.) avium infections were similar to each other but very different from the immune response to M. bovis infection. These observations provide key information for development of new diagnostic tools that may be able to differentiate between animals that are either infected with, or vaccinated against MAP or M. bovis. Accurate detection of infection will be more efficient and cost-effective for producers, reducing the number of animals that are culled due to false positive diagnosis of infection and facilitates removal of truly diseased animals.
Allen, A.J., Stabel, J.R., Robbe-Austerman, S., Park, K.T., Palmer, M.V., Barrington, G.M., Lahmers, K.K., Hamilton, M.J., Davis, W.C. 2012. Depletion of CD4 T lymphocytes at the time of infection with M. avium subsp. paratuberculosis does not accelerate disease progression. Veterinary Immunology and Immunopathology. 149(3-4):286-291.
Stabel, J.R., Barnhill, A., Bannantine, J.P., Chang, Y.F., Osman, M.A. 2012. Evaluation of protection in a mouse model after vaccination with Mycobacterium avium subsp. paratuberculosis protein cocktails. Vaccine. 31(1):127-134.
Leite, F.L., Stokes, K.D., Robbe-Austerman, S., Stabel, J.R. 2013. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. Journal of Veterinary Diagnostic Investigation. 25(1):27-34.
Bradner, L., Robbe-Austerman, S., Beitz, D.C., Stabel, J.R. 2013. Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk. Journal of Clinical Microbiology. 51(5):1575-1577.
Bannantine, J.P., Wu, C., Hsu, C., Zhou, S., Schwartz, D.C., Bayles, D.O., Paustian, M.L., Alt, D.P., Sreevatsan, S., Kapur, V., Talaat, A.M. 2012. Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association. Biomed Central (BMC) Genomics. 13:89. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337245/pdf/1471-2164-13-89.pdf.
Bannantine, J.P., Lingle, C.K., Stabel, J.R., Ramyar, K.X., Garcia, B.L., Raeber, A., Schacher, P., Kapur, V., Geisbrecht, B.V. 2012. MAP1272c encodes a NlpC/P60 protein, an antigen detected in cattle with Johne's Disease. Clinical and Vaccine Immunology. 19(7):1083-1092.
Wadhwa, A., Bannantine, J.P., Byrem, T., Stein, T.L., Saxton, A.M., Speer, C.A., Eda, S. 2012. Optimization of serum EVELISA for milk testing of Johne's disease. Foodborne Pathogens and Disease. 9(8):749-754.