Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 3/24/2008
Publication Date: 4/23/2008
Citation: Feng, X., Fetterer, R.H., Tuo, W. 2008. Interferon-gamma stimulating activities of the fractionated neospora caninum tachyzoite lysate. BARC Poster Day.
Technical Abstract: Neospora caninum is an obligate intracellular protozoan parasite, causing bovine abortion worldwide. Our recent research showed that N. caninum tachyzoite lysate elicits production of the T cell cytokine interferon-gamma (IFN-g) by both bovine and murine T cells, which may be critical to host protection. The purpose of the present study was to identify Neospora proteins that are responsible for the induction of IFN-g in T cells of the mouse model. Neospora lysate (NcAg) was prepared and separated by high pressure liquid chromatography using a size exclusion column eluted at 1 ml per min with phosphate buffer (pH 7.2). Fractions (200 ul) were then subjected to analysis by gel electrophoresis followed by silver staining. The results showed that fractions 28-59 had visible protein bands by silver stain. Spleen cells of female adult BALB/c mice were prepared and used to detect IFN-g inducing activities. One million spleen cells were incubated in 48-well plates with complete medium (RPMI-1640 supplemented with 10% FBS, 25 mM glutamine and 10ug/ml gentamicin, culture medium alone control), 20 ul phosphate buffer (negative control), 0.5ug/ml Con A (positive control), or 20 ul from each of the fractions for 48 hr at 37C with 5% CO2 and 95% air atmosphere. Culture supernatants were collected and assayed by enzyme-linked immunosorbent assay for IFN-g concentrations. High levels of IFN-g were stimulated by Fractions 28-30, 41, 43-44, 47-52, and 56-57. These preliminary results indicate that the IFN-g inducing activity in NcAg was separated by size-exclusion chromatography and present in different fractions representing Neospora proteins with variable molecular weights. Further research has been planned to identify and characterize the proteins responsible for the induction of IFN-g.