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Title: Interferon gamma responses to proteome-determined specific recombinant proteins: Potential as diagnostic markers for ovine Johne's disease

item HUGHES, VALERIE - Moredon Research Institute
item DENHAM, SUSAN - Moredon Research Institute
item Bannantine, John
item CHIANINI, FRANCESCA - Moredon Research Institute
item KERR, KAREN - Moredon Research Institute
item MAY, LINDA - Moredon Research Institute
item MCLUCKIE, JOYCE - Moredon Research Institute
item NATH, MINTU - Biomathematics And Statistics Scotland (BIOSS)
item STEVENSON, KAREN - Moredon Research Institute

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2013
Publication Date: 9/15/2013
Publication URL:
Citation: Hughes, V., Denham, S., Bannantine, J.P., Chianini, F., Kerr, K., May, L., McLuckie, J., Nath, M., Stevenson, K. 2013. Interferon gamma responses to proteome-determined specific recombinant proteins: Potential as diagnostic markers for ovine Johne's disease. Veterinary Immunology and Immunopathology. 155(3):197-204.

Interpretive Summary: With Johne’s disease in cattle and sheep, there are a few obstacles in developing a diagnostic test that will identify infected animals before they begin shedding the bacterial pathogen to their uninfected herd mates. One is that the test must detect infection early before clinical signs, such as weight loss and diarrhea, appear. Two is that the test must be sensitive enough to pick up the fairly weak immune response against the pathogen that occurs shortly after infection. The gamma interferon (IFN-gamma) test is one assay that fulfills both criteria. As the name indicates this test measures the host cytokine IFN-gamma which is actually present at elevated levels during the early stage of infection. In this communication, we evaluated several recombinant proteins derived from Mycobacterium avium subspecies paratuberculosis, the agent that causes Johne’s disease, for their ability to stimulate IFN-gamma production. It was found that four out of 30 recombinant proteins stimulated IFN-gamma production in infected sheep, but not healthy controls. Further testing in a sheep flock suggested that these proteins may serve a good antigens for building an improved IFN-gamma test for Johne’s disease.

Technical Abstract: Johne’s disease (JD), or paratuberculosis is a fatal chronic granulomatous enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). A long subclinical phase may ensue during which time the animal shows no signs of clinical disease. Diagnosis of JD is problematic and no single test can reliably detect sub-clinical disease. It is thought that Th1 responses to Map are the first to be activated with a later switch to Th2 responses and concomitant progression to clinical disease. Thus detection of a cell-mediated response, indicated by measuring interferon gamma (IFN-gamma) produced in response to mycobacterial antigens, may give an early indication of subclinical infection. Until fairly recently crude extracts of Map (PPDj) have been used to detect the cell-mediated response. However, more specific, quantifiable antigens used as reagents would improve the specificity and reproducibility of the test. A number of Map-specific proteins (determined by proteome analysis) were screened for their ability to raise a cell-mediated immune response in subclinically infected sheep. Thirty Map proteins were expressed and used in the IFN-gamma release assay on peripheral blood mononuclear cells (PBMCs) isolated from subclinically infected animals. From this initial screen four proteins were selected and tested using PBMCs from sheep, six subclinical animals and four from a JD-free flock. Three proteins elicited IFN-gamma levels, which were higher in the subclinical animal group than in the JD-free control group, two were statistically significant at the 95% confidence level. Thus these proteins have the ability to discriminate groups of infected and uninfected animals and may have use in diagnosis of JD.