| Porcine circovirus type 2 (PCV2) is a pathogen cause post-weaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, porcine circovirus disease (PCVD). Porcine parvovirus (PPV) is an important collaborative pathogenic factor of PCVD. Porcine alveolar macrophages (PAM) were important immune cells and target cells of PCV2 and PPV. In this study, survival rate and phagocytosis of PAM and cytokine, antigen presenting relevant molecules in PAM were was systematically analyzed so that explore the cellular and molecular mechanisms of PCV2 and co-infection on immune functions in conventional piglets.The porcine antigen presenting function of related molecules CD80, CD86 and pig cytokines LI-1β, IFN-α1, TNF-α, etc and the housekeeping geneβ-actin part gene were amplified from PAM by RT-PCR, respectively, and construced relative recombined plasmids. restriction enzyme digestion and sequencing, were the corresponding elements of the positive recombinant plasmid. A standard curve of the above elements and linear regression equation were established of Real-time fluorescence quantitative PCR method (Real-time FQ-PCR) and determined its optimal reaction conditions.48 35-day-old healthy piglets were divided into 4 groups which were PCV2, PPV single-infected group, PCV2/PPV co-infected group and control group and included 12 piglets each group. The piglets were sacrificed on 3, 7, 14 and 35 day post infection(DPI) and PAM were collected. The survival rate and phagocytosis of PAM were measured at different times after infection by trypan blue staining and chicken red blood cell phagocytosis test. The results showed that the significant difference did not appear between the survival rates of PAM from the piglets infected by PCV2 alone and that of PCV2/PPV co-infection in whole. But the phagocytic activity of PAM from piglets infected by PCV2/PPV group significantly decreased when compared with that of piglets infected by PCV2.The expresstion levels of PAM proinflammatory cytokines, chemokines and regulation of cytokine mRNA were measured after infection by Real-time FQ-PCR. The results showed that the mRNA transcription levels of pro-inflammatory cytokines TNF-αand IL-1βof PAM from piglets of PCV2/PPV group increased significantly (P<0.01) in the early stages after infection; the mRNA level of IFN-α1 decreased and that of IL-6 increased persistently; the mRNA level of IL-8 were restrained persistently, as well as that of MCP-1 and MIP-1βwere significantly higher in early stage of infection but weakend significantly in later period (P<0.05); regulatory cytokine IL-10 and IL-12p40 was expressed strongly in early period and on 14DPI separately (P<0.01); however, the expressions of IFN-γ, IL-18 and GM-CSF were weakened persistently. Together, It is suggested that, PCV2/PPV of infection aggravated the local inflammatory response, caused the fuctions of early anti-virus and chemotactic reduced and induced functions of immune modulation decreased and appeared disorder.After infection, the expression levers of SLA-1, SLA-DR, mRNA of CD80 and CD86 molecules were detected by flow cytometry (FCM) and real-time FQ-PCR, respectively. The results indicated that the expression lever of SLA-1 was significantly lower (P <0.01) on 35 DPI and SLA-DR significantly lower on 7 DPI(P <0.05). Both mRNA transcription levels of CD80 and that of CD86 decreased strongly on 35 DPI. These results showed that the co-infections of PCV2 and PPV induced exogenous antigen presenting function being inhibitted gravely in early period while endogenous antigen presenting function significant reduction in later period.Taken together, the survival rate of PAM were not obviously affected by PCV2 and PPV co-infection, at the same time decreased strongly phagocytic activity of PAM; increased inflammation responses; resulted in the fuctions of early anti-virus and chemotactic decline, as well as functions of immune modulation and antigen presenting inhibition. PPV acted as a important role in the immunopathological process induced by PCV2. |
PDF Full Text Request