Alteration Of Cellular Immunity Responses In Lambs During Infection Of Orf Virus

Contagious pustular dermatitis (CPD), also known as Orf, is an acute, highly contagious pustular skin lesions disease of sheep, goats, and humans. It is caused by contagious pustular dermatitis virus (CPDV), also called Orf virus (ORFV). There are different versions of sheep's immunologic response caused by ORFV, and most esearch Result tend to cellular immunity. In this study, Real-Time PCR and MTT were used to determine the Thl/Th2/Toll-like recetor mRNA expression and the lymphopoiesis of sheep to study the general and tissue immunity of sheep caused by ORFV.Three major factors which affected the peripheral blood lymphocyte proliferatation of lamb including concertration of stimulus, cell, lymphocyte and time had been studied. The test showed the best condition of reaction was 10μ.g/mL PHA, 1×107/mLcell concentration; or 40μg/mL ConA,2×10'/mL cell concentration, reaction condition is 37℃incubated 48 hours. Real-time PCR assays based on SYBR Green I for detection of Thl/Th2/Toll-like receptor and GAPDH gene were established using primers derived from sheep Thl/Th2/Toll-like receptor and GAPDH gene. The assays were highly sensitive and had a detection limit of 1×102 copies/μL of initial templates. These assays were highly specific and there was single specific melting peak for each cytokine. It was highly reproducible and had a coefficient of variation less than 3 percent for intra-and inter-assay. The high sensitivity, specificity and reproducibility of the assays indicated that the SYBR Green I real-time PCR could be used as an effective tool for detection and quantification of sheep Thl/Th2/Toll-like receptor cytokines mRNA level.In this study, we have demonstrated using MTT assay to determine the immunity state that there are intense cellular immune responses of sheep cause by Orf virus; we have detected the expression of some important mRNA during the infection period by analyzed PWBC. We have found IL-2 and IFN-y have the same change, increased at 5 dpi and there were linear correlation between them; the level of IL-1βand TNF-a mRNA was obviously increasing from 3 dpi; TLR-4 and TLR9 mRNA was increasing: but the level of IL-4 mRNA was not regular. It showed type 1 immune responses of sheep with Orf virus infection in general immunity. At the same time, we detected Th1/Th2/Toll-like receptor mRNA expression in the lip, tounge, spleen, lymph node, heart, liver, lunge, kidney and intestine. We have found there were very intense the immunologic response in the lip and tongue; a high-level cell immunity in kidney and intestine should be connected with the high expression ofγδ+T cell and macrophage which resided in intestinal mucosal and deep cell, separately. The spleen lymph nodes a major immune organ also have a intensive cell immunity response. these results indicate that orf virus predominantly induces strong Thl cellular immune responses rather than humoral immune responses, which has important implications in understanding the host immune responses initiated against orf virus. It is a very useful basis for further studying the ORFV.