Identification Of Apoptotic Cells And Analysis Of Apoptosis-associate Factors In Thymus Of Piglets Infected With HP-PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease of pigs,which is characterized by severe respiratory distress and poor growth in piglets, and severe reproductivefailures as well as abortion in sows. PRRS was first recognized in the1990s in Europe, the UnitedStates and then worldwide. At present, PRRS has become an endemic disease in the swine industry.PRRS virus (PRRSV) is an enveloped positive single-stranded RNA virus, which belongs to the familyArteriviridae. In2006, the causative agent of "swine high fever" disease had been identified as highlypathogenic PRRSV (HP-PRRSV), which caused enormous economic losses in swine industry. Ourprevious studies had demonstrated that significant thymus atrophy could be observed in piglets infectedwith HP-PRRSV (HuN4strain), which related to the numerous apoptotic cells in thymocytes. Theobjective of the present study was to further identify the apoptotic cells in thymus, and detect mRNAexpression levels of apoptosis-associate factor in the thymus of piglets infected with HP-PRRSV.In order to demonstrate the type of apoptotic cells and infected cells, and their relationship in thethymus of piglets infected with HP-PRRSV. The thymus of piglets infected with HP-PRRSV andcontrol piglets were obtained, subsequently frozen sections were made, then performedimmunofluorescence staining and observed by Laser scanning confocal microscope (LSCM) to identifythymus apoptosis in HP-PRRSV infected piglets. The results demonstrated that the apoptotic cells werenot infected by HP-PRRSV, and most of them were CD3+T cells, and HP-PRRSV infected cells wereonly found in CD14+cells.To further analyze the alterations of apoptosis-associate factors, a series real time SYBR Green IRT-PCR methods were developed for TNF-α, TNFR1, FasL, Fas, Bax, Bcl-2, Caspase-8, and Caspase-3,respectively. And the mRNA expression in thymus of weaned piglets infected with HP-PRRSV andcontrol piglets were detected. Compared with the control piglets, the mRNA levels of TNF-α, TNFR1,Caspase-8, and Caspase-3increased significantly (p<0.05) in the thymus of piglets infected withHP-PRRSV, which mediated exogenous apoptotic pathway. These results demonstrated that theapoptosis of thymocytes induced by HP-PRRSV was mainly mediated by the exogenous pathway.