豬第二型環狀病毒感染對 Concanavalin A 刺激周邊血 液單核細胞增殖的影響

Abstract

豬第二型環狀病毒（porcine circovirus type 2; PCV2），為全球豬隻新興疾 病-離乳後多系統消耗性症候群（postweaning multisystemic wasting syndrome; PMWS）的主要病原。本病為多因子影響的疾病，而血清中含高量 PCV2 核酸 （3.9×105 至 1.8×1012 copies/mL) 與臨床發生 PMWS 的嚴重程度有顯著相關性。 近年報告指出 Con A 具有促進淋巴球內 PCV2複製且增加淋巴組織病毒力價的現 象，而 PCV2 於血清或淋巴組織中的含量或力價與臨床症狀顯著相關。因此本實 驗的目的以 Con A 模擬免疫刺激探討豬第二型環狀病毒如何影響刺激下的細胞增 殖反應。實驗設計包括：一、以自然感染帶原豬，分析不同 PCV2 的病毒含量， 與 Con A 刺激 PBMC 增殖反應之相關性。二、於實驗感染 SPF 豬，探討含高病 毒豬群的 PBMC 及脾臟細胞，PCV2 superinfected 是否影響 Con A 刺激細胞增 殖。實驗中細胞增殖反應以流式細胞儀檢測，IFN-γ 及 IL-10 以 ELISA 檢測，病 毒量以 TagMan® Real-time PCR 測定。細胞的處理分四組包括不處理組，PCV2 組，Con A 刺激組及 Con A/PCV2 處理組。實驗第一部分結果僅 PCV2 superinfected Con A 刺激之細胞增殖反應與血清及病毒量呈負相關，單獨以 PCV2 superinfected 或 Con A 刺激 PBMC 均無顯著差異，而 IL-10 濃度與病毒量 成正相關。實驗第二部分結果，於周邊血液細胞，控制組 PCV2 superinfection 有 促進 Con A 刺激 PBMC 之細胞增殖反應，但相同處理在攻毒組有抑制細胞增殖 的現象，此細胞增殖的抑制與細胞內含高病毒量有關但與細胞之凋亡及壞死無 關，此外也可能與培養上清液 IL-10 濃度有關。在脾臟細胞，控制組中細胞增殖 反應、病毒含量及 IL-10 分泌無顯著變化，而 PCV2 superinfection 可促進 Con A 刺激脾臟分泌 IFN-γ。本研究以 Con A 刺激模擬免疫活化模式，觀察高病毒量或 於 PCV2 superinfection 可能藉由 IL-10 分泌影響免疫細胞增殖。推論感染豬若經 過免疫刺激，則增加病毒複製及 IL-10 分泌，可能使免疫細胞功能受損，而易有 臨床症狀的產生。

Porcine circovirus type 2 (PCV2) is associated with a new emerging disease, postweaning multisystemic wasting syndrome (PMWS) which causes severe economic losses in swine industry worldwild. PMWS-affected pig is characterized by high PCV2 load in serum, ranging from 3.9×105 to 1.8×1012 PCV2 copies/mL, which has significantly correlated with severe clinical signs of PMWS. Several reports reveal immune stimulation is an important factor in the induction of PMWS. Con A enhances PCV2 replication and high viral titer in PBMC and in lymphoid tissues, respectively, that has correlated with PMWS as well. The aim of this study is to investigate the impact of PCV2 infection on Con A stimulated cell proliferative response. The experimental designs include: 1. To evaluate the correlation between viral load in serum and Con A stimulated cell proliferation as well as IFN-γ and IL-10 secretions in PBMC from PCV2-carrier pigs. 2. To evaluate whether PCV2 superinfection affect Con A stimulated cell proliferative response in experimental PCV2 infected SPF pigs. Flow cytometry was used to detect cell proliferation response and ELISA was used to evaluate the level of IL-10 and IFN-γ. Viral load was measured by TagMan® Real-time PCR. Culture of PBMC or spleen cells were separated into 4 group of treatment as mock, PCV2 superinfection, Con A treated and Con A/PCV2. In the first part of the study, the tendency of inverse correlation between seral PCV2 load and proliferation response of PBMC at Con A/PCV2 group was observed. The postitive correlation between IL-10 level and viral load was also noted. In the second part of the study, PBMC proliferation after Con A stimulation could be enhanced by PCV2 superinfection in the control group. However, this enhancement was not observed in the experimental PCV2 infected SPF pigs. In this group, the inhibition of enhanced cell proliferation was correlated to the cellular viral load but was not associated with the process of necrosis or apoptosis and might be associated with high IL-10 level. In spleen, viral load in spleen cells and IL-10 secretions were insignificant but the level of IFN-γ was enhanced at Con A/ PCV2 group. Our data suggested that high viral load could inibit Con A stimulated cell proliferation and the inhibition maybe associated with induction of IL-10.