亞臨床第二型豬環狀病毒感染豬的免疫狀態-血液病毒量對周邊血液單核細胞對致裂原及第二型豬環狀病毒刺激反應的影響

Abstract

由第二型豬環狀病毒(PCV2)所引起的豬環狀病毒相關疾病中，以離乳後多系統性消耗症候群(PMWS)所引起的致害最為嚴重，造成離乳豬嚴重生長遲緩、體重下降，甚至死亡，此病在養豬產業造成重大的經濟損失，根據文獻可知，病毒量對於PCV2感染的嚴重度有重要的影響。由於大部分PCV2的感染豬隻為亞臨床感染，而有關於亞臨床PCV2感染免疫狀態的研究仍極為有限，因此，本研究以周邊血液單核細胞為檢測對象，以活體外給予致裂原刺激及PCV2攻毒，模擬田間免疫活化及PCV2再暴露等狀況，探討含不同病毒量的亞臨床染豬隻的細胞存活率、增殖能力、PCV2含量及細胞激素變化，以進一步了解在亞臨床感染的條件下PCV2可能的免疫致病機制。此外，豬場的PCV2疫苗使用，亦可能會影響亞臨床PCV2感染豬隻的免疫狀態，故也將其列入討論。實驗結果顯示，若依病毒量將豬隻分為大於103 copies/μL高病毒量豬隻及小於103 copies/μL低病毒量豬隻兩群，可見在給予Con A或PCV2處理後，含高病毒量豬隻的細胞存活率皆較含低病毒量豬隻為低；但就細胞增殖能力而言，在所有處理組中皆可發現含低病毒量豬隻會高於含高病毒量豬隻，此外，亦可見無疫苗注射且含低病毒量豬隻以及有疫苗注射但含高病毒豬隻的細胞增殖指數有較高的表現。在病毒量方面，含高病毒量豬隻的數量在有給予疫苗注射豬群中較無疫苗注射豬群為低，且平均PCV2病毒量也較低；而有疫苗注射且含低病毒量豬隻，亦能夠將病毒量維持在相對低值；此外，亦發現PCV2病毒的增加與細胞增殖能力上升有正相關。在細胞激素方面，含高病毒量豬隻的IL-1β、IL-8及IL-10 mRNA表現量較含低病毒量豬隻有較高的趨勢，而且在PCV2處理組的表現量上會明顯上升，尤其是在含高病毒量豬隻；IL-4 mRNA表現量，同樣在高病毒量豬隻有較高的表現趨勢；含低及高病毒量豬隻的IL-2、IL-4及IFN-ϒ mRNA表現量，在給予Con A後均有增加的趨勢，但是PCV2處理組則與對照組無明顯差異；而有給予疫苗但含高病毒量豬隻的IL-1β、IL-8及IL-10 mRNA表現量均會增加，同時，其細胞增殖能力也會明顯上升。根據實驗結果推測，從亞臨床PCV2感染到出現PCVAD臨床症狀可能為一漸進性病程，在亞臨床感染豬隻的免疫系統雖尚能維持正常調節，但病毒量的增加可能造成較嚴重的炎症並進而引發組織病變以及免疫抑制。此外，疫苗的注射有助於減低病毒量，但是在給予疫苗注射後所出現的含高病毒量豬隻，則可能有較嚴重的組織發炎病變及免疫抑制的傾向，而較有利於PCV2的複製。

Porcine circovirus type 2 (PCV2) infection induces porcine circovirus associated diseases (PCVAD) among which postweaning multisystemic wasting syndrome (PMWS) is the most serious, which causes severe growth retardation, weight loss, and death in weaned pigs leading to considerable economic loss in pig industry. It is known that PCV2 load determines the outcome of PCV2 infection. However, the majority of PCV2-infected pigs in the field are subclinical and information regarding the immune status of the subclinically PCV2-infected pigs is rather limited. Therefore, using peripheral blood mononuclear cells (PBMC) treated with mitogen and recall antigen in vitro to mimic the immune system activation by vaccination, infection with other pathogens or PCV2 re-exposure in the field situation and to evaluate the survival rate, proliferation ability, PCV2 load, and cytokine expression profiles in subclinically PCV2-infected pigs with different PCV2 loads to determine the immune status of the asymptomatic pigs may provide a more clear picture in understanding the immunopathogenesis of PCV2 infection in pigs. Due to the fact that PCV2 vaccine is widely used in the field, the effects of vaccination on the immune status of subclinically PCV2-infected pigs are discussed as well. According to the PCV2 loads, we subdivided the pigs into two groups, including the high viral load group (HVL) which had PCV2 genome number more than 103 copies/μL and the low viral load group (LVL) which had PCV2 genome number less than 103 copies/μL. In Con A or PCV2 treated groups, the result of cell viability of PBMC displayed that pigs with HVL had lower cell viability than those with LVL. In general, the proliferation abilities of PBMC in pigs with LVL were higher than pigs with HVL. Furthermore, non-vaccinated pigs with LVL and vaccinated pigs with HVL had higher cell proliferation abilities. In the result of PCV2 load, it showed that the vaccinated pigs had lower viral loads and only a relatively small number of pigs had high PCV2 loads. Besides, vaccinated pigs with LVL could maintain a low level of PCV2 load. Pigs with HVL were prone to have higher mRNA expression levels of IL-1β, IL-8, and IL-10, which were more prominent in PCV2-treated group, especially in the HVL group. The level of IL-4 mRNA expression in the HVL pigs was higher than those in pigs with LVL. The expression levels of IL-2, IL-4, and IFN-ϒ became higher when treated with Con A but they were not responsive to PCV2 re-exposure. Besides, the mRNA expression levels of IL-1β, IL-8, and IL-10 increased in vaccinated pigs with HVL and had increasing cell proliferation ability in PBMC. According to the results, it is considered that pigs transform from subclinical PCV2 infection to PCVAD might be a progressing course of disease development. In subclinically infected pigs, the immune system might regulate normally but inflammation and immunosuppression could be more severe since the increasing PCV2 viral loads. Although PCV2 vaccination would reduce the viral loads quite successfully, vaccinated pigs with high PCV2 loads might be prone to have more severe inflammation and immunosuppression and more conductive to PCV2 replication than non-vaccinated ones.