The Dynamic Of Cytokines Associated With Th17 Cells In Goats Infected With ORFV

Contagiouts Ecthyma(CE), ORF, is a viral disease of sheep and goats that continues to present problems to farms around the world. In our country, epidemic areas include Jilin, Ningxia, Yunnan, Henan, Hubei, Xinjiang, Shaanxi, et al. Goats are infected easier than sheep. In some of goat farms, the incident could reach 10 %~100 %. It is rarely fatal adult goats, but it seriously damage to newborn lambs. Because it often lead open ulcerations on limps to influence their eating behavior, and that could cause high mortality rate on newborn lambs. Besides, ORF could cause induce mastitis or other serious disease and injure adult female goats production performance. The characteristic of ORF includes papule, blister, pustule and scab on limps. The ORF process is a serious typical inflammatory response. In recently studying, the activating cells, related to inflammation, and the secreted cytokines, which participate this inflammation, are still unrevealed.In this study, we used variety methods, which include pathological slices, lymphocyte proliferation, real-time PCR, prokaryotic expression, et al. firstly, collecting tissue of limp lesions on lambs infected with ORFV in different periods, we studied the characteristic of epithelium tissue lesions in ORF process. Then, stimulating peripheral blood mononuclear cells(PBMCs) of ORF virus-negative lambs by wild or attenuated ORF virus, we detected PBMCs proliferation and mRNA level of cytokines. In addition, we detected mRNA level of cytokines protected by PBMCs in different period of ORF process. Finally, we expressed goats IL-1β and IL-6 proteins. The results of this study as following:1. Analysis of the pathological slices of variety periods of ORF lesions suggests that lots of inflammatory cells infiltration have appeared under matrix cells of skin lesions from pimple period. With growing of inflammation process, the number of infiltration cells, including neutrophil, lymphocyte and macrophage, has grown up.2. In this study, goats PBMCs grown effectively by ORF virus stimulated in vitro. And the proliferated effect of wild ORF virus was stronger than attenuated one. The result shows that the mRNA level of IL-17 raised obviously, and the mRNA levels of IL-1β, IL-6 and IL-23 grown significantly and TGF-β dropped down. In addition, analysis of cytokines mRNA levels in variety periods of ORF process PBMCs shows that IL-17 mRNA grown up significantly in pustule period, and the mRNA levels of IL-1β, IL-6 and IL-23 grown up significantly in blister period. Beside, the level of IFN-γ also was grown up significantly, in pustule period.3. We cloned gene of goats IL-1β and IL-6, and their sizes are 463 bp and 552 bp respectively. Through using prokaryotic expression system, we successfully gained goat cytokines protein, and amino acid sizes are 37.4 kDa and 40.6 kDa respectively. Then created polyclonal antibodies of goats IL-1β and IL-6 by injected rabbits, and the serum antibody titers are 1/16 and 1/8 respectively, which detected by the agar double diffusion method.In summary, the pathological process of ORF is immune pathological response. And, the cells, participating this response process, are neutrophil in early period and lymphocyte in later period. The major cell, meditating ORF immune pathological response, is IL-17. According to the result of mRNA level of other cytokines, it suggests that the IL-17, protecting in ORF process, were created by Th17 cell. In addition, Th1 also participates ORF inflammation response. Finally, we cloned gene of goats IL-1β and IL-6 and expressed them, and prepared polyclonal antibodies for future study of immune pathological mechanism of ORF.