Submitted to: Immunology Research Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 9/23/2003
Publication Date: 12/1/2003
Citation: Tuo, W., Fetterer, R.H., Boyd, P., Gasbarre, L.C., Dubey, J.P. 2003. Bovine T-cell response to antigens of neospora caninum. 2003. ARS Immunology Research Workshop. December 1-4, 2003, Bethesda, MD. p. 9.
Technical Abstract: Neospora caninum is an apicomplexan parasite that infects a number of cell types, causing neosporosis in several animal species including cattle. In the past decade, neosporosis has been recognized as a primary cause of abortion in cattle worldwide. It was estimated that approximately 20% of all bovine abortions are attributed to neosporosis; Neospora-related abortion costs the cattle industry in excess of $35 million a year in the state of California alone. In infected cows, N. caninum elicits a detectable antibody (Ab) response as shown by extensive studies, whereas N. caninum-induced cell-mediated immunity (CMI) is poorly understood. Previous exposure to, or infection by, the parasite does not provide protection against abortion or vertical transmission, in spite of the presence of Neospora-specific Ab in maternal circulation. The objective of this study was to define Neospora-specific CMI in cows infected with N. caninum and to identify immunodominant T helper cell Ag that may be used as vaccine candidates. Total Neospora Ag was prepared by freezing and thawing or sonication of N. caninum tachyzoites grown in a monkey kidney or a bovine macrophage cell line. Peripheral blood lymphocytes (PBL) from Neospora infected cows were stimulated with total soluble Neospora Ag weekly in the presence of irradiated PBL as a source of Ag presenting cells. Flow cytometry analysis showed 90-98% of these Neospora Ag specific T cells (week 3-8) were CD2+CD3+CD4+. A T cell proliferation assay was established and used to determine Ag specificity of Ag-specific T helper cells and antigenic activity of Neospora Ag. A bovine specific IFN-gamma ELISA was used to determine IFN-gamma content in supernatants of T cells stimulated with Neospora Ag. The total Neospora tachyzoite Ag was separated into 5 fractions by anion exchange using HPLC. Results indicate that high levels of IFN-gamma were produced by both PBL and T cell lines specific to Neospora Ag. Using the T cell proliferation assay, we showed that proteins from HPLC Fractions 1 and 2 had low or undetectable T cell-stimulating activity, whereas proteins from Fractions 4-6 had higher stimulatory activity. However, Fraction 5 contained the highest antigenic activity in all T cell lines assayed. Western blot analysis showed that Fraction 5 contained 8-10 distinctive Neospora protein bands that were defined by a rabbit antiserum raised against to Neospora tachyzoites (NC-1 strain). These studies suggest that Ag-specific T helper cells (CD4+CD3+) may be used to screen for immunodominant Neospora Ag. Neospora proteins that specifically stimulate T helper cell proliferation and cytokine production identified using our T cell proliferation and cytokine production assays may be used as vaccine candidates. This research will enhance our understanding of bovine CMI to Neospora infection and may facilitate the development of a vaccine against bovine neosporosis.