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United States Department of Agriculture

Agricultural Research Service

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Location: Infectious Bacterial Diseases Research

2012 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.

3. Progress Report:
The primary objectives of this project include the application of Mycobacterium avium subspecies paratuberculosis (MAP) recombinant proteins in platforms for development of new tools. Characterization of these 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 MAP proteins as well as application in 96-spot protein array and micro-IFN-g assays. This work has identified a MAP protein antigen that may be used for the development of new diagnostic tests for the management and control of paratuberculosis. This antigen, termed MAP1272c, has been evaluated as potential sero-diagnostic tool in an ELISA-based format and has demonstrated utility in the detection of infected cattle, although it did not outperform a commercially available ELISA test. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis. Studies evaluating experimental MAP infection in a neonatal calf model were initiated. Calves were inoculated with the same isolate of MAP from a cow with clinical disease but the isolate was in three different formats. The three formats consisted of pure bacteria from culture; raw feces; and the mucosal scrapings from the intestine. Each of these forms of the same isolate (pure bacteria; raw feces; mucosa) was fed to calves in milk replacer at 1-2 weeks of age. This study will provide valuable information on how methods of bacterial isolation can affect the virulence and, therefore, infectivity in an experimental model. We expect to find that the level of MAP infection in the tissues of calves fed the inoculum consisting of the cultured bacteria will be lower. Sample collection is underway on this study. Another study has been initiated to compare a multi-species infection of neonatal calves, sheep, and goats to determine which ruminant model will be present more advanced signs of infection and/or disease, such as increased infection and lesion formation in the tissues, diarrhea, and weight loss. Understanding the pathogenesis of disease and developing more effective infection models will aid in the evaluation of diagnostic tools, therapeutics, and vaccines. MAP proteins were evaluated as potential vaccine candidates in a mouse model. Recombinant MAP proteins were pooled (4 pools of 3 proteins each) and administered to mice. Mice were then infected with live MAP for a 3-month period. Reduced tissue infection was observed in mice vaccinated with 3 of the 4 pools of proteins, indicating some level of protection was attained by the immunization. Further studies are being initiated in a calf model to ascertain the true protective effects of the pooled proteins as a vaccine. Vaccines are beneficial management tools for producers to reduce infection within the herd, as they are relatively inexpensive and easy to administer.

4. Accomplishments
1. Novel antibody for Mycobacterium avium subspecies paratuberculosis (MAP). ARS researchers at Ames, Iowa, identified a new monoclonal antibody that selectively detects MAP and not other closely related bacterial strains. This antibody is the only one in the world that has this capability. Further research identified the protein, termed MAP1025, that this specific antibody binds. This antibody is the subject of a recently issued US patent. The identification of unique proteins or antibodies for MAP will be helpful in developing new diagnostic tools to detect infected animals.

2. Cloned Mycobacterium avium subspecies paratuberculosis (MAP) proteins for novel vaccines. Pools of recombinant MAP proteins, consisting of an overlapping array of 3 proteins in 4 pools, were used to vaccinate mice. Mice were then challenged with live MAP and tissue infectivity and immune responses were measured in cultured cells. ARS researchers at Ames, Iowa, identified that three of the 4 protein pools showed some level of protection as determined by lower levels of bacteria found in the tissues of vaccinated mice. In addition, immune responses in mice given the pooled proteins showed a shift in immune system responses (T and B cell populations and serum IgG2a) that would correlate with greater protection. The lack of vaccines for paratuberculosis, particularly vaccines that can discriminate between paratuberculosis and M. bovis infections in the field, makes this a compelling avenue of research. Vaccines can be used successfully to reduce the spread of infection and level of disease within a herd.

3. Characterize vaccine immunity and effects of vaccination on tuberculosis diagnostics. The immune responses of cattle to inoculation with a commercial vaccine for Mycobacterium avium subspecies paratuberculosis (MAP) were determined. In addition, these studies evaluated if the MAP vaccine would cross react with Mycobacterium bovis (M. bovis) diagnostic tests as a major drawback of current whole-cell vaccines for MAP is that they can produce false positive disease diagnosis with standard tests for bovine tuberculosis and paratuberculosis. A study conducted by ARS researchers in Ames, Iowa, to examine effects of immunization with a heat-killed whole cell vaccine on diagnostic test performance demonstrated that the whole-cell commercial vaccine interferes with diagnostic tools for paratuberculosis but results in low interference with the comparative cervical skin test and new serologic tests for M. bovis. This suggests that new vaccine preparations based upon defined antigens must be developed to discriminate between infected and vaccinated animals with paratuberculosis. A faster and more accurate way to identify infected animals will lead to timely herd management decisions which will save farmers thousands of dollars per herd.

Review Publications
Chen, J., Faisal, S.M., Chandra, S., Mcdonough, S.P., Moreira, M.A., Scaria, J., Chang, C., Bannantine, J.P., Akey, B., Chang, Y. 2012. Immunogenicity and protective efficacy of the Mycobacterium avium subsp. paratuberculosis attenuated mutants against challenge in a mouse model. Vaccine. 30(19):3015-3025.

Stabel, J.R., Bannantine, J.P., Eda, S., Robbe-Austerman, S. 2011. Induction of B Cell responses upon experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis. Clinical and Vaccine Immunology. 18(7):1139-1149.

Bannantine, J.P., Stabel, J.R., Lamont, E.A., Briggs, R.E., Sreevatsan, S. 2011. Monoclonal antibodies bind a SNP-sensitive epitope that is present uniquely in Mycobacterium avium subspecies paratuberculosis. Frontiers in Microbiology. 2(163):1-13.

Park, K.T., Allen, A.J., Bannantine, J.P., Seo, K.S., Hamilton, M.J., Abdellrazeq, G.S., Rihan, H.M., Grimm, A., Davis, W.C. 2011. Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease. Vaccine. 29(29-30):4709-4719.

Pithua, P., Godden, S.M., Wells, S.J., Stabel, J.R. 2011. Evaluation of the risk of paratuberculosis in adult cows fed Mycobacterium avium subsp paratuberculosis DNA-positive or -negative colostrum as calves. American Journal of Veterinary Research. 72(11):1456-1464.

Stabel, J.R., Waters, W.R., Bannantine, J.P., Lyashchenko, K. 2011. Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine. Clinical and Vaccine Immunology. 18(12):2079-2089.

Lamont, E.A., Bannantine, J.P., Armien, A., Ariyakumar, D.S., Sreevatsan, S. 2012. Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures. PLoS One [serial online]. 7(1):e30648. Available:

Last Modified: 06/26/2017
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