Limiting dilution analyses for the quantification of cellular immune responses in bovine ostertagiasis

Abstract

A sensitive limiting dilution analysis (LDA) was used to quantitate the local and systemic cellular immune response of cattle after immunization with keyhole limpet hemocyanin (KLH) and infection with Ostertagia ostertagi. The assay measures the proliferative response of bovine T-cells after in vitro stimulation with antigen. Interleukin 2 activity was supplied by supernates from mitogen-stimulated bovine peripheral blood lymphocytes (PBL) and accessory cell function was in the form of irradiated autologous PBL. The assay measures the response of a single cell and was most easily demonstrated in the lymph nodes draining the site of antigen inoculation. Comparison of cell frequencies and maximal responses generated in conventional proliferative assays showed several differences between the two assays. First, after antigen injection, the highest cell frequencies were seen in the draining lymph nodes within 3 days, and decreased by 10 days post-immunization. In contrast, in mass cultures maximal stimulation was not seen until 7–10 days after injection, but remained high up to 4 weeks after immunization. Second, at 17 days post-infection, a time of eruption of the parasite from the gastric glands, high frequencies of inducible cells were demonstrated by LDA in all lymphoid populations tested. In contrast, low levels of proliferation were seen in mass cultures. The reasons for these differences may include different sensitivities to suppression or more stringent requirements for specificity between the two assays. Finally, it was found that immunologically naive calves have relatively high frequencies of Ostertagia-specific cells in PBL, and that after infection these frequencies decrease. These results indicate either active suppression of the potential anti- Ostertagia response or an extra-vascularization of these cells to the site of infection.