Location: Infectious Bacterial Diseases Research2011 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 MAP induces significant changes in the expression and secretion of cytokines and immune cell phenotypes. A novel cytokine, osteopontin (Opn), was found to be expressed differently in healthy cattle as compared to MAP-infected cattle. This work characterized host immunity in the early stages of infection and will help in the evaluation of vaccine candidates for MAP. Further work to understand the interaction of cytokines in different stages of infection was performed with cell culture systems. These studies demonstrated a unique interplay between interferon-g (IFN-g) and interleukin-10 (IL-10), two cytokines with conflicting roles in immunity to intracellular bacteria. Studies evaluating different modes of experimental MAP infection in a neonatal calf model were completed. This work suggested that the use of a clinical isolate of MAP was more effective in establishing infection in calves, although no calves demonstrated clinical symptoms typical of MAP. Experimental infection resulted in the identification of key markers of early host immune responses to MAP, including T and B cell-mediated responses, which will facilitate discernment of markers for early detection of MAP infection.
1. Identified a novel protein expressed uniquely by Mycobacterium avium subspecies paratuberculosis. The gene encoding this unique protein, termed UP1, was not identified (and thus annotated) because it was buried in the genome sequence on a DNA strand opposite of an identified gene. There are no similarities to this protein in sequence databases. ARS researchers at NADC, Ames, IA, have shown by real time polymerase chain reaction (RT-PCR) and antibody detection that this gene is expressed and a protein produced only by strains of Mycobacterium avium subspecies paratuberculosis. This finding opens up an entirely new level of complexity for comparative genomic research because it shows you must go deeper than simply comparing two DNA sequences. The identification of unique proteins or genes for MAP will be helpful in developing new diagnostic tools to detect infected animals.
2. Evaluate host immunity in naturally infected dairy cows. Characterizing immune function in cows with subclinical and clinical infection will help us determine what are the important mediators of host immunity during infection. ARS researchers at NADC, Ames, IA, isolated cells from cows in different stages of infection (clinical and subclinical) and stimulated them with live Mycobacterium avium subspecies Paratuberculosis (MAP). These studies demonstrated that interferon-g (IFN-g) and interleukin-10 (IL-10), have a unique interplay with each other during infection. The secretion of IFN-g is much higher for cells from subclinically infected cattle in the early stages of disease but decreases as cows progress to clinical stages of disease. Conversely, secretion of IL-10 is more prominent in clinical cows. These results indicate IFN-g is involved in controlling infection and protects against escalation to clinical disease. Addition of IFN-g to cell cultures resulted in lower secretion of IL-10 from cells isolated from clinical cows. Conversely, the addition of IL-10 resulted in the downregulation of IFN-g in MAP-infected cows. Furthermore, a novel secreted protein, osteopontin (Opn), was found to be affected by the clinical stage of infection. Upon the addition of Opn secretion of IFN-g by cells from subclinically infected cows was increased, indicating a mechanism for induction of host immunity. This information improves our understanding of host immune responses during stages of infection and may facilitate divelopment of new vaccines. It may be possible to select vaccine candidates that enhance secretion of IFN-g or Opn, thereby reducing the likelihood that cattle will progress to the more advanced stage of disease. Vaccine management of dairy cows is key to alleviating the escalation of disease within infected herds.
3. Evaluated experimental infection models in calves that would demonstrate the transition from subclinical to clinical infection. Previous work by ARS researchers at NADC, Ames, IA, evaluating different modes of experimental inoculation of neonatal calves in an effort to develop an infection model for paratuberculosis was extrapolated to study the induction of early immune responses to Mycobacterium avium subspecies Paratuberculosis (MAP). The studies included comparison of oral inoculation with MAP, oral inoculation with MAP after immunosuppression with a steroid (dexamethasone), and intraperitoneal inoculation with MAP. Oral inoculation yielded higher infection levels in the tissues of calves, however, all experimental methods resulted in subclinical infection and did not transition to a clinical state within the 12-month period. We were able to utilize these experimental methods to identify early markers of host immunity to MAP infection. Interferon-g (IFN-g), a secreted protein, was a very robust indicator of infection in all inoculated calves and appeared consistently as early as 90 days after infection. These results indicate IFN-g may be useful to identify infected animals in the very early stages of infection. These studies were successful in identifying unique markers for T cells (a subset of immune cells). In addition, this study was the first to demonstrate that B-cell (another subset of immune cells) responses are highly upregulated early in MAP infection and that B cells may be actively involved with T cells in coordinating a protective response. This information is useful in understanding the host immune response to early exposure and infection with MAP. In addition, we can use these markers to help identify animals in the very early stages of infection and manage those animals by culling or isolation before further spread of the disease within the herd.
Collins, M.T., Stabel, J.R. 2011. Diseases of dairy animals: infectious diseases: Johne's disease. In: Fuquay, J.W., Fox, P.F., McSweeney, P.L.H., editors. Encyclopedia of Dairy Sciences. 2nd edition. Oxford, UK: Academic Press (Elsevier). p.174-180.