Location: Infectious Bacterial Diseases Research2008 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 research project include the identification of specific genes for Micobacterium avium subspecies paratuberculosis (MAP) 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 M. avium subsp. paratuberculosis proteins for study as potential diagnostic reagents. The proteins have been arrayed in a 96-spot blotting format for analysis. This work has identified unique M. avium subsp. paratuberculosis antigens that can be used for the development of new diagnostic tests for the management and control of paratuberculosis. Work was initiated to determine the impact of M. avium subsp. paratuberculosis infection on the microbial ecology of the gut in cattle. This information may be useful in distinguishing elements of pathogenesis for M. avium subsp. paratuberculosis through indirect effects on other bacterial populations. Additionally, new therapeutic strategies could be designed to replace or supplement bacterial populations to possibly mitigate the clinical symptoms of Johne’s Disease. The use of early detection methods such as the skin test and interferon-gamma tests to control and manage paratuberculosis in the field was performed in sheep flocks. Both the skin test and the interferon-gamma tests are proving efficacious in reducing the prevalence of paratuberculosis in sheep flocks using a test and cull protocol. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis. Work on M. avium subsp. paratuberculosis infections in cattle continued with an emphasis on the host response during the periparturient period, a very stressful period for cows. Results have 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 was conducted in collaboration on an acute experimental infection model for M. avium subsp. paratuberculosis in neonatal calves. 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 M. avium subsp. paratuberculosis. Modified-live strains of bacteria which express a subset of MAP immunogens were developed and will be evaluated as potential vaccine candidates. Vaccination is a recognized tool for the management of paratuberculosis in the field but vaccines with greater efficacy, reduced cross-reactivity with bovine tuberculosis, and fewer adverse side-effects are essential before expanded use of vaccination is recommended. This project addresses NP 103, component 6, problem statement 6A.
1. Impact of Mycobacterium avium subsp. paratuberculosis infection status on the bovine microbiome. A hallmark of cattle infected with M. avium subsp. paratuberculosis is the culture of viable organisms from feces. However, little is known about the impact of M. avium subsp. paratuberculosis infection on the bovine microbiome, which is defined as the community of microorganisms that normally reside in the host. The ribosomal DNA within fecal samples from infected and uninfected animals was analyzed in order to characterize the bacterial populations present. Sorting samples by bacterial populations present in feces allowed grouping of animals based on MAP infection status. This suggests that MAP infection status could be determined by identifying indirect effects on other bacterial populations. Additionally, new therapeutic strategies could be designed to replace or supplement bacterial populations to possibly mitigate the clinical symptoms of Johne’s Disease. This accomplishment addresses NP 103, component 6, problem statement 6A.
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. The proposed research should reveal potential mechanisms for the immunosuppression during the calving period 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. Results from this study demonstrate significant changes in immune function and immune cell populations due to the combined effects of calving and M. avium subsp. paratuberculosis infection. 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. This accomplishment addresses NP 103, component 6, problem statement 6A.
3. Antigen discovery in Johne’s disease. Within the past year, we capitalized on our long term project to develop a 96-spot protein array. It has taken 3 years to obtain the number of M. avium subsp paratuberculosis purified recombinant proteins necessary to construct this array. We used this protein array to profile the antibody response in cattle with Johne’s disease. Through this approach we have identified 6 novel antigens that may be used to develop an improved antigen-based test for Johne’s disease. Furthermore, protein array-based studies have identified M. avium subsp paratuberculosis proteins that appear to be detected very early following infection of cattle (within 70 days). Three manuscripts describing these studies were published in 2008. This accomplishment addresses NP 103, component 6, problem statement 6A.
5. Significant Activities that Support Special Target Populations
Kimura, K., Goff, J.P., Schmerr, M.J., Stabel, J.R., Inumaru, S., Yokomizo, Y. 2008. Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum. Journal of Dairy Science. 91(5):1852-1864.