Equine interferon gamma synthesis in lymphocytes after in vivo infection and in vitro stimulation with EHV-1

Equine cytotoxic T lymphocyte (CTL) responses to equine herpesvirus-1 (EHV-1) are well characterised but little is known about the cytokine response after infection or vaccination. EHV-1 is common in horses and infects lymphocytes in vivo. This virus was used as a model to measure the synthesis of interferon gamma (IFN-γ) by equine peripheral blood mononuclear cells (PBMC) after in vivo infection and/or in vitro stimulation with EHV-1. Both flow cytometry and ELISPOT assays were used to quantify equine IFN-γ using a mouse anti-bovine IFN-γ monoclonal antibody (clone CC302; shown to cross-react with recombinant equine IFN-γ) and a rabbit anti-canine IFN-γ polyclonal antibody. The percentage of PBMC synthesising IFN-γ after in vitro stimulation with EHV-1 increased with age. In yearlings infected experimentally with EHV-1, PBMC showed two peaks of IFN-γ synthesis, 11 and 56 days after infection. The IFN-γ synthesis was principally associated with CD8+ cells. The patterns of IFN-γ synthesis detected by intracellular IFN-γ staining or ELISPOT were compared with CTL data and shown to be similar. These methods were also applied successfully to frozen samples of PBMC. Measurement of equine IFN-γ using these simple techniques can now be applied to future studies on protective cellular immune responses following virus infection and/or vaccination of horses.