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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND IMMUNOLOGICAL CHARACTERISTICS OF JOHNE'S DISEASE
2009 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 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 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. Work was continued to determine the impact of MAP infection on the microbial ecology of the gut in cattle. Work during the past year has focused on examining the colonization of cattle by microorganisms immediately after birth and evaluating what genes are utilized by MAP when it is present in either the environment or infecting cattle. The analysis of cattle colonization by microorganisms has been completed and manuscripts describing the work are currently being prepared. Experiments evaluating the response of MAP to the environment have been completed and the samples are currently being examined. Preliminary studies designed to evaluate the response of MAP during an infection have been performed, but additional experiments are warranted in order to develop a reasonable model of infection in cattle that also allows us to retrieve enough bacteria to study. This information may be useful in distinguishing elements of pathogenesis for MAP through indirect effects on other bacterial populations. New therapeutic strategies could be designed to replace or supplement bacterial populations to possibly mitigate the clinical symptoms of Johne’s Disease. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis. Work on MAP 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. 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. A potential candidate for a vaccine was developed by insertion of genes from MAP into the coding region of Mannheimia haemolytica. This hybrid construct may be efficacious as a vaccine to prevent paratuberculosis. 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 recommeded.


4.Accomplishments
1. Impact of M. paratuberculosis infection status on the bovine microbiome. The normal microbial flora, or microbiome, residing in the digestive tract of animals is a source of important biological activities, including unique metabolic functions, immune system modulation, and energy utilization. The mechanisms by which microbial flora interact are poorly understood and only a small percentage of the individual microbes that comprise this microbiome have been identified due to the difficulties in culturing these organisms. The development of DNA-based culture-independent methods has opened deeper and broader avenues of inquiry into the identification of microorganisms present in a wide variety of environments. The bacterial DNA content of fecal samples and intestinal contents taken from calves at several time points beginning at birth was performed in order to identify the subpopulations of microorganisms present in the digestive tract. Populations within fecal samples did not readily cluster by individual or age, but samples taken from the same anatomical site tended to have similar types of microorganisms present. This study represents one of the first attempts to examine the initial colonization of neonates by environmental microbes. Understanding how the digestive tract microbiome develops after birth should allow us to determine how this process varies between individual animals, as well as how it might be manipulated to provide additional short-term and long-term health benefits.

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 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. Identification and characterization of a new antigen from M. paratuberculosis. There is a need for better diagnostics for identification of cattle infected with M. paratuberculosis. We identified a new M. paratuberculosis antigen that is strongly recognized by sera from infected cattle. The antigen, MAP 1272c, appears to be the best candidate antigen from a panel of 100 proteins for use in developing a new diagnostic test. In collaboration with investigators at the University of Missouri-Kansas City, we have now crystallized a section of the protein and evaluated it's function. These results further our understanding of the biology of this protein. We have also prepared two monoclonal antibodies against the protein that will be used for research and diagnostic development.


6.Technology Transfer

Number of the New/Active MTAs (providing only)5
Number of New Patent Applications Filed1
Number of Other Technology Transfer1

Review Publications
Stabel, J.R., Khalifeh, M.S. 2008. Differential Expression of CD5 on B Lymphocytes in Cattle Infected with Mycobacterium avium subsp. paratuberculosis. Veterinary Immunology and Immunopathology. 126(3-4):211-219.

Wu, C., Schmoller, S.K., Bannantine, J.P., Eckstein, T.M., Inamine, J.M., Livesey, M., Albrecht, R., Talaat, A. 2009. A Novel Cell Wall Lipopeptide Is Important for Biofilm Formation and Pathogenicity of Mycobacterium avium subspecies paratuberculosis. Microbial Pathogenesis. 46(4):222-230.

Olsen, I., Tollefsen, S., Aagaard, C., Reitan, L.J., Bannantine, J.P., Sollid, L.M., Lundin, K.E. 2009. Isolation of Mycobacterium avium Subspecies paratuberculosis Reactive CD4 T Cells from Intestinal Biopsies of Crohn's Disease Patients. PLoS One [serial online]. 4(5):e5641. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005641.

Hughes, V., Bannantine, J.P., Denham, S., Smith, S., Garcia-Sanchez, A., Sales, J., Paustian, M., Mclean, K., Stevenson, K. 2008. Immunogenicity of Proteome-determined Mycobacterium avium subsp. paratuberculosis-specific Proteins in Sheep with Paratuberculosis. Clinical and Vaccine Immunology. 15(12):1824-1833.

Li, L., Singh, S., Bannantine, J.P., Kanjilal, S., Kapur, V. 2009. Mycobacterium avium subspecies paratuberculosis. In: Nene, V., Kole, C., editors. Genome Mapping and Genomics in Animal-Associated Microbes. Berlin/Heidelberg, Germany: Springer-Verlag. p. 65-83.

Seth, M., Janagama, H.K., Lamont, E.A., Widdel, A., Vulchanova, L., Stabel, J.R., Waters, W.R., Palmer, M.V., Sreevatsan, S. 2009. Biomarker Discovery in Subclinical Mycobacterial Infections of Cattle. PLoS One [serial online]. 4(5):e5478. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005478.

Khalifeh, M., Al-Majali, A.M., Stabel, J.R. 2009. Role of Nitric Oxide Production in Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis. Veterinary Immunology and Immunopathology. 131(1-2):97-104.

Stabel, J.R. 2008. Pasteurization of Colostrum Reduces the Incidence of Paratuberculosis in Neonatal Dairy Calves. Journal of Dairy Science. 91(9):3600-3606.

Karcher, E.L., Bayles, D.O., Bannantine, J.P., Beitz, D.C., Stabel, J.R. 2008. Osteopontin: A Novel Cytokine Involved in the Regulation of Mycobacterium avium subsp. paratuberculosis Infection in Periparturient Dairy Cattle. Journal of Dairy Science. 91(8):3079-3091.

Stabel, J.R., Palmer, M.V., Harris, B.N., Plattner, B., Hostetter, J., Robbe-Austerman, S. 2009. Pathogenesis of Mycobacterium avium subsp. paratuberculosis in Neonatal Calves after Oral or Intraperitoneal Experimental Infection. Veterinary Microbiology. 136(3-4):306-313.

Karcher, E.L., Johnson, C.S., Beitz, D.C., Stabel, J.R. 2008. Osteopontin Immunoreactivity in the Ileum and Ileoceccal Lymph Node of Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis. Veterinary Immunology and Immunopathology. 126(1-2):142-148.

Last Modified: 10/25/2014
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