Buzzy
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OLD MOOS - NEW MOOS - 2010/01/20 03:42
Hi Mamas & George,
I know this is OLD news, but it is still interesting.
MOOOOOOOOOOO.
Buzzy
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ource: IOWA STATE UNIVERSITY submitted to
BOVINE MACROPHAGE AND T CELL RESPONSES TO MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS INFECTION
PROJECT DIRECTOR: Jones, D. E. Steadham, E. Hostetter, J. Waters, R.
PERFORMING ORGANIZATION
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
AMES,IA 50011
NON TECHNICAL SUMMARY: Currently there is no vaccine available to prevent or treat the cause of Johne's disease. Development of a vaccine, or treatment, is likely to be impeded without an understanding of the mechanisms that allow Map to survive in vivo. The purpose of this project is to understand those mechanisms.
OBJECTIVES:Johne's disease (Mycobacterium avium subspecies paratuberculosis or Map) has a significant impact on the dairy industry nationwide. Milk production can be reduced from 2 to 19 percent per cow when compared to uninfected herdmates. The impact of Map on beef cattle is not as readily apparent, but loss of body weight and inefficient feed conversion, as well as premature culling, are consequences of Map infection that can adversely effect cattle revenues. Currently there is no vaccine available to prevent or treat the cause of Johne's disease. The ability of Map to produce disease is related to its ability to survive and replicate within the host macrophage. Paradoxically, macrophages typically play an essential role in host defense of Map by ingesting and killing invading bacilli as part of a cell-mediated immune response that is driven by IFN-g from antigen-specific CD4+ T cells (Th1 cells). The mechanisms by which Map survives within macrophages are not known, nor are the mechanisms that subvert the Th1 cell response and the induction of protective immunity. Development of a vaccine, or treatment, is likely to be impeded without an understanding of the mechanisms that allow Map to survive in vivo. The objectives of this proposal are based on our understanding that persistent infection develops because bovine macrophages infected with Map fail to kill the bacteria. We hypothesize that there is a failure in the ability of Map-specific CD4+ a/b T cells to maintain Th1 phenotype and effectively activate macrophages to kill bacteria. This project has two objectives towards understanding the immunopathology associated with Johne's disease in cattle and will critically examine one of the four scenarios, outlined above, that may impact the expression of the Th1 CD4+ T cell phenotype during this chronic disease. Objective 1: Determine the effects of CD4+ T cell secreted or membrane associated products on macrophage activation in vitro and their influence on Map killing and intracellular trafficking. Objective 2: Test the hypothesis that there is a failure of CD4+ a/b T cells to remain committed to a Th1 cell phenotype.
APPROACH:We will test the hypothesis by assaying bacterial killing by macrophages when stimulated with committed Th1 cells or cell products. Th1 cells will be produced in vitro by culturing CD4+ T cells from naive animals with anti-CD3 and IL-12. Infected macrophages will be cultured with the Th1 cells and/or their supernatants. At various time points after infection the bacteria will be recovered from the macrophages and their viability will be determined by staining and CFU assay. We will also evaluate several other aspects of macrophage activation under these conditions, specifically, the production of reactive nitrogen and oxygen intermediates and phagosome maturation. We will also determine the commitment of CD4+ cells to the Th1 phenotype by repeatedly stimulating cells in vitro and assaying for proliferation and the production of IFN-g. IL-12 receptor b1 and b2 levels will be assayed as an additional indicator of Th1 phenotype. Receptor expression will be determined by amplifying the mRNA from Th1 cells by PCR. We will also determine the Th1 cell phenotype of Map-specific CD4+ T cells obtained from in vivo Map infections. Their commitment to a Th1 phenotype will be tested by repeated stimulation in vitro. These Th1 cells from Map infected animals will be compared to the equivalent cell type from M. bovis-infected animals. M. bovis infection of cattle results in a persistent Th1 type immune response and are used as a positive control. Evaluation of these aspects of the immune response to Map will facilitate our understanding of the immune response of cattle to Map infection and help in the evaluation of vaccine candidates
CRIS NUMBER: 0192185 SUBFILE: CRIS
PROJECT NUMBER: IOWV-411-23-0021 SPONSOR AGENCY: NIFA
PROJECT TYPE: ANIMAL HEALTH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2001 TERMINATION DATE: Sep 30, 2004
GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)
CLASSIFICATION
Knowledge Area (KA) Subject (S) Science (F) Objective (G) Percent
311 3310 1090 4.2 50%
311 3410 1100 4.2 50%
CLASSIFICATION HEADINGS
KA311 - Animal Diseases
S3310 - Beef cattle, live animal
S3410 - Dairy cattle, live animal
F1090 - Immunology
F1100 - Bacteriology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks
RESEARCH EFFORT CATEGORIES
BASIC (N/A)%
APPLIED 100%
DEVELOPMENTAL (N/A)%
KEYWORDS: mycobacterium paratuberculosis; johnes disease; mycobacterium bovis; macrophages; t cells; immune response; infection; beef cattle; dairy cattle; animal diseases; bacterial diseases (animals); immunology; cell mediated immunity; interferon; defense mechanisms; host pathogen relations; phenotypes; animal pathology; secretion; cell membrane; cell culture; interleukin; intermediates; nitrogen; oxygen; cell proliferation; comparative analysis; vaccines
PROGRESS: Jan 1, 2002 TO Dec 31, 2002
A method of experimentally infecting calves with Mycobacterium avium subspecies paratuberculosis (Map) was developed. This has allowed us to investigate the development of the infection through the first eight months of exposure. Five months after intra-tonsilar inoculation with the bacteria, calves developed an antibody and lymphocyte response to Map antigens. The cells proliferating upon exposure to Map antigens, in vitro, were evaluated by flow cytometry and found to be CD4+ T lymphocytes. In addition, these cells were found to be the primary cell type producing IFN-gamma. We have also titrated naive primary bovine monocyte-derived macrophages for their response to IFN-gamma in vitro. We have developed assays for evaluating reactive nitrogen intermediates, reactive oxygen intermediates, and changes in the cellular morphology of macrophages in response to IFN-gamma. This was a necessary first step in evaluating the ability of Map antigen-specific T cells to activate Map-infected bovine macrophages by the IFN-gamma secreted into their medium in vitro.
IMPACT: 2002-01-01 TO 2002-12-31 The inability to experimentally infect calves with Map has retarded progress in studying Johne's disease. For the first time we are able to describe the length of time required for an antibody response, as well as a cell-mediated response, to develop. In addition, we will have a reproducible and consistent source of antigen responsive T cells to use in determining their ability to activate Map infected macrophages.
PUBLICATION INFORMATION: 2002-01-01 TO 2002-12-31
No publications reported this period
PROJECT CONTACT INFORMATION
NAME: Jones, D. E.
PHONE: 515-294-4682
FAX: 515-294-5423
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