The pathogenesis of PMWS by evaluating the effect andinteraction of PCV2 and/or other possible co-factors,PRRSV or immune stimulator, on porcine immune cells in vitro

• Main Authors: Hui-Wen Chang, 張惠雯
• Other Authors: 龐飛
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/32507081595602371826

博士 === 國立臺灣大學 === 獸醫學研究所 === 94 === Postweaning multisystemic wasting syndrome (PMWS) has been recognized globally and become a major economic concern in many pig-producing countries, including Taiwan. To date, since the pathogenesis of the emerging syndrome is not yet completely understood, there is no effective method to control this disease. It is believed that porcine circovirus type 2 (PCV2) is the primary aetiological agent of PMWS, however, the existence of PCV2 does not definitively lead to PMWS in pigs. The pathogenesis of PMWS is considered multi-factorial. Bacteria or porcine reproductive and respiratory syndrome virus (PRRSV) is a common complicating factor of PMWS and is clinically important based on the observation of increases in postweaning mortality when co-infection is present. The objectives of the thesis were to evaluate the effect and interaction of PCV2 and/or other possible co-factors such as PRRSV and immune stimulator, including bacterial lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA), on porcine alveolar macrophages (AMs) and splenic macrophages (SMs) and the subsequent effect on co-cultured lymphocytes by in vitro inoculation and tried to correlate the findings to the pathogenesis of PMWS. It was demonstrated that PCV2 could be easily detected in the cytoplasm of AMs and SMs but no cell death occurred in these cells in vitro following the inoculation of PCV2 (Chapters II, III, IV, V). Thus, somewhat similar to the “Trojan horse” the monocyte/macrophage lineage cells (MLCs) may function as an important reservoir not only to help PCV2 to evade the immunosurveillance but also to assist PCV2 to spread. Moreover, PCV2 alone may impair AMs’ microbicidal capability and disturb various cytokine expressions in AMs and SMs (Chapters II, III, IV, V). The alterations may be potentially harmful to the local pulmonary defense system leading to secondary or opportunistic pathogen infection with subsequent development of mild to moderate pneumonia. When PRRSV was inoculated alone, a low but constant infectious rate accompanied with markedly reduced cell viability and significantly increased apoptosis was revealed in AMs (Chapters III, IV). The result suggests the presence of a remained clarified non-viral indirect mechanism in PRRSV-induced cytopathic effect on AMs. It was revealed that PRRSV also might impair phagocytosis and microbial killing ability of AMs and induce IL-8 and TNF-a production in AMs (Chapters III, IV). Significant up-regulation of Fas/FasL-mediated cell death and apoptosis of co-cultured lymphocytes and increase in IL-1b, IL-8, and TNF-a production were also demonstrated in PRRSV alone-inoculated SMs (Chapter V). It is suggested that PRRSV might trigger lymphocyte activation and subsequent apoptosis by the enhancement of Fas/FasL interaction. The finding may explain the pathogenesis of immunosuppression, interstitial pneumonia, and lymphoid depletion in PRRS-affected pigs. In PCV2 and PRRSV co-inoculated AMs (Chapters III, IV), it was observed that PCV2 inoculation occurring before or simultaneously with PRRSV inoculation could cause variable reductions in the PRRSV-associated infectious rate, cell death, and apoptosis when compared with PRRSV inoculation alone. The reduction in PRRSV infection in AMs was further proven being mediated by IFN-a generated by PCV2 inoculation (Chapter III). However, in AMs inoculated with PRRSV prior to the addition of PCV2, despite being inoculated with both PRRSV and PCV2 and also releasing a significant amount of IFN-a once PCV2 was added, PCV2 couldn’t reduce PRRSV infection in AMs once PRRSV has established its infection (Chapter IV). The dissimilar outcomes of the interaction of PCV2 and PRRSV in different inoculation orders in swine AMs may explain in part the variable morbidity of PMWS among pigs in the field, or even in the same pig herd. When PCV2 and PRRSV were co-inoculated, up-regulation of IL-8, TNF-a, and IFN-a, and significant increase in the level of FasL expression were seen in AMs as compared with mock- or those single virus-inoculated AMs, regardless of their inoculation orders (Chapter IV). There was a significant increase in Fas/FasL-mediated cell death of the co-cultured lymphocytes with the increase in IL-1b, IL-8, IL-10, and/or TNF-a expressions in PCV2 and PRRSV co-inoculated groups (Chapter V) when compared with mock- or those single virus-inoculated groups. The results imply that PCV2 and PRRSV may work synergistically on MLCs in the induction of overwhelming production of inflammatory mediators and intensification of Fas/FasL-mediated activation-induced cell death (AICD) in pigs with PCV2 and PRRSV dual infection. The observations may contribute, at least partially, to the more apparent interstitial pneumonia and lymphoid depletion seen in PMWS-affected pigs with PCV2 and PRRSV dual infection. Furthermore, it was found that the bacterial endotoxin, LPS, but not PMA, can induce translocation and transcription of PCV2 in AMs in vitro (Chapter VI). The result implies that co-infection with Gram negative bacteria as well as stimulation of macrophages may have the potential to promote PCV2 replication and increase PCV2 load in pigs. Thus, it is speculated that secondary Gram negative bacterial infection may induce PCV2 replication in PCV2-infected pigs and also be one of the important co-factors contributing to the full development of PMWS in the field. In conclusion, we have demonstrated that PCV2 alone and PRRSV alone have the ability to impair pulmonary defense mechanism and induce inflammatory mediators in the lungs resulting in the development of pneumonia. Although PCV2 can interfere with PRRSV infection by PCV2-induced IFN-a before PRRSV has established its infection, dual infection with PCV2 and PRRSV, regardless of their inoculation orders, will drastically enhance the production of inflammatory mediators and cause damage to AMs, including cell viability and anti-microbial activity. The alterations certainly will result in more apparent pneumonic lesion and more frequent secondary or opportunistic pathogen infection in PMWS-affected pigs with PCV2 and PRRSV dual-infection. Lymphoid depletion is a common pathological change seen in PMWS-affected pigs. Our results indicate that PRRSV alone may promote Fas/FasL and/or cytokine-mediated AICD in lymphocytes via infected MLCs. Although PCV2 itself was unable to induce Fas/FasL and/or cytokine-mediated AICD in lymphocytes, it could intensify the PRRSV-induced AICD in lymphocytes. This may explain, at least partially, why lymphoid depletion is more apparent in PMWS-affected pigs with dual infection of PCV2 and PRRSV. Based on the findings, it is assumed that the effect of PCV2 alone or PRRSV alone seems to be mild and restricted; however, both viruses can impair the pulmonary defense mechanism leading to higher susceptible to secondary infection. The secondary bacterial infection, especially Gram negative bacteria, may subsequently induce the replication of PCV2 via stimulation and activation of macrophages by the release of LPS. When PCV2 co-infected with PRRSV, PCV2 can work synergistically to intensify PRRSV-induced AICD in lymphoid organs and to significantly enhance cytokine production in lungs and lymphoid organs. It is suggested that although PCV2 is essential for the development of PMWS, it mainly acts as an immunosuppressor resulting in secondary infection and an enhancer in intensifying PRRSV- or immune activation-induced AICD rather than directly leads to the disease in pigs. The result may explain, at least partially, the pathogenesis and immunosuppression noted in PMWS-affected pigs. It also supports the general point of view that the mechanism for the development of full-spectrum PMWS is multi-factorial.