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United States Department of Agriculture

Agricultural Research Service

2010 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.

3.Progress Report
The primary objectives of this project include the identification of specific genes for Mycobacterium avium subspecies paratuberculosis (MAP) with an emphasis on differentiation of MAP antigens from closely related Mycobacterium avium subspecies 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 MAP proteins for study as potential diagnostic reagents. This work has identified unique MAP antigens that can be used for the development of new diagnostic tests for the management and control of paratuberculosis. One unique protein in particular has demonstrated promise as a strong antigen. These antigens are being evaluated as potential diagnostic tools and vaccine candidates in our laboratory and in collaboration with other laboratories. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis. Research on MAP infections in cattle with an emphasis on the host response during the periparturient period, a very stressful period for cows, was completed and data summarized in manuscript form. Results have demonstrated significant changes in the expression and secretion of cytokines, major modulators of host immunity, and on immune cell phenotypes. A novel cytokine, osteopontin, was expressed differently in naturally infected cattle and healthy noninfected cattle. This work will help define which attributes of host immunity are initiated in the early stages of infection and help in the evaluation of vaccine candidates for MAP. Studies evaluating different modes of experimental MAP infection in a neonatal calf model were completed along with data analyses. This work suggested that the use of a clinical isolate of MAP was more effective in establishing infection in calves, although all experimental modes tested resulted in an asymptomatic calf model. Experimental infection resulted in the identification of key markers of early host immune responses to MAP, including T and B cell-mediated responses. These results will allow discernment of markers for early detection of MAP infection.

1. Identified a novel antigen expressed uniquely by Mycobacterium avium subspecies paratuberculosis. The gene encoding this unique protein, termed UP1, was not annotated because it was buried in the genome sequence on the opposite DNA strand of an annotated gene. There are no similarities to this protein in the protein databases. ARS researchers at Ames, Iowa, have shown by RT-PCR and antibody detection that this gene is expressed and protein produced from it only with Mycobacterium avium subspecies paratuberculosis strains tested thus far. This finding opens up an entirely new level of complexity for comparative genomics.

2. 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. Cows with paratuberculosis often break with clinical disease signs shortly after calving. Understanding potential mechanisms for the immunosuppression during the calving period was undertaken by characterizing cytokine expression and secretion and immune cell function in cows with subclinical and clinical infection as compared to healthy non-infected controls. Cows in different states of infection were sampled during the 4 weeks before calving and the 4 weeks following calving. These studies by ARS researchers at Ames, Iowa, demonstrated that parturition decreased host immunity in cows regardless of infection status. Furthermore, this research identified a novel protein, osteopontin, involved in host immunity was affected by parturition and infection status of the cow. Subsequent evaluation of tissues postmortem demonstrated higher levels of osteopontin in the intestinal tissue of clinically infected cows compared to healthy noninfected cows. These results suggest that the periparturient period is a highly significant period for the dairy cow and may result in increased susceptibility to infectious diseases due to changes in host immunity. This information can be used to improve management of dairy cows to alleviate the escalation of disease in this time period. These studies will alert cattle and dairy producers to potential periods of increased disease susceptibility.

3. Develop an experimental infection model in calves that will demonstrate the transition from subclinical to clinical infection. Work has been completed to evaluate different modes of experimental inoculation of neonatal calves to develop an infection model for paratuberculosis. The majority of experimental models for ruminants have utilized an oral inoculation of live Mycobacterium avium subspecies paratuberculosis (MAP) in order to establish infection, thereby mimicking the fecal-oral route of transmission generally observed in the field. The current studies by ARS researchers at Ames, Iowa, included comparison of oral inoculation with a laboratory-adapted strain and a clinical isolate of MAP, oral inoculation with MAP after immunosuppression with dexamethasone, and intraperitoneal inoculation with MAP. These studies suggested that oral inoculation with a clinical isolate of MAP yielded higher infection levels in the tissues of calves, however, all experimental methods resulted in asymptomatic subclinical infection. We were able to utilize these experimental methods to elucidate early markers of T cell and B cell-mediated immune responses of the host to MAP infection. Interferon-gamma was a very robust indicator of infection in all inoculated calves and appeared consistently as early as 90 days post-inoculation. In addition, our study is the first to demonstrate that B-cell responses are highly upregulated in early infection, even though this doesn’t readily link to antibody production. This information is useful in deciphering the host immune response to early exposure and infection with MAP and provides some information about the utility of various markers to diagnose infection.

Review Publications
Stabel, J.R. 2010. Immunology of Paratuberculosis Infection and Disease. In: Behr, M.A., Collins, D.M., editors. Paratuberculosis Organism, Disease, Control. Wallingford, Oxfordshire, UK: CABI. p. 230-243.

Janagama, H.K., Senthilkumar, T., Bannantine, J.P., Rodriquez, M.G., Smith, I., Paustian, M., Mcgarvey, J.A., Sreevatsan, S. 2009. Identification and Functional Characterization of the Iron-dependent Regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis. Microbiology. 155(Pt 11):3683-3690.

Bannantine, J.P., Paustian, M., Kapur, V., Eda, S. 2010. Proteome and Antigens of Mycobacterium avium subspecies Paratuberculosis. In: Behr, M.A., Collins, D.M., editors. Paratuberculosis Organism, Disease, Control. Wallingford, Oxfordshire, UK: CABI. p. 94-108.

Abomoelak, B., Chi, J., Hoye, E.A., Bannantine, J.P., Talaat, A.M. 2009. MosR, A Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis. Journal of Bacteriology. 191(19):5941-5952.

Paustian, M., Bannantine, J.P., Kapur, V. 2010. Mycobacterium avium subsp. paratuberculosis Genome. In: Behr, M.A., Collins, D.M., editors. Paratuberculosis Organism, Disease, Control. Wallingford, Oxfordshire, UK: CABI. p. 73-82.

Barnhill, A.E., Hecker, L.A., Kohutyuk, O., Buss, J.E., Honavar, V., West-Greenlee, H.M. 2010. Characterization of the Retinal Proteome During Rod Photoreceptor Genesis. BMC Research Notes 2010. 3:25. Available:

Bannantine, J.P., Stabel, J.R., Bayles, D.O., Geisbrecht, B. 2010. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set. Protein Expression and Purification. 72(2):223-233.

Pithua, P., Wells, S.J., Godden, S.M., Sreevatsan, S., Stabel, J.R. 2010. Experimental Validation of a Nested Polymerase Chain Reaction Targeting the Genetic Element ISMAP02 for Detection of Mycobacterium avium subspecies paratuberculosis in Bovine Colostrum. Journal of Veterinary Diagnostic Investigation. 22(2):253-256.

Last Modified: 4/20/2014
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