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-gamma) 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-gamma using a mouse anti-bovine IFN-gamma monoclonal antibody (clone CC302; shown to cross-react with recombinant equine IFN-gamma) and a rabbit anti-canine IFN-gamma polyclonal antibody. The percentage of PBMC synthesising IFN-gamma after in vitro stimulation with EHV-1 increased with age. In yearlings infected experimentally with EHV-1, PBMC showed two peaks of IFN-gamma synthesis, 11 and 56 days after infection. The IFN-gamma synthesis was principally associated with CD8(+) cells. The patterns of IFN-gamma synthesis detected by intracellular IFN-gamma 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-gamma using these simple techniques can now be applied to future studies on protective cellular immune responses following virus infection and/or vaccination of horses.
Paillot
R
R
Animal Health Trust, Centre for Preventive Medicine, Lanwades Park, Newmarket, Suffolk CB8 7UU, UK. romain.paillot@aht.org.uk
Daly
J M
JM
Juillard
V
V
Minke
J M
JM
Hannant
D
D
Kydd
J H
JH
D000367:Age Factors; D000818:Animals; D015925:Cryopreservation; D006566:Herpesviridae Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D004861:Herpesvirus 1, Equid / Q000276:immunology*; D006734:Horse Diseases / Q000276:immunology / Q000517:prevention & control*; D006736:Horses; D007371:Interferon-gamma / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology*; D008297:Male; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D007371:Interferon-gamma