玉米赤黴烯酮對豬免疫細胞及豬第二型環狀病毒複製的影響

Abstract

豬第二型環狀病毒 (PCV2)所引起的相關疾病稱為豬環狀病毒相關疾病(PCVAD)，PCVAD的形成需有許多共同調控因子，包含病毒、其他病原的共同感染、外來免疫刺激原如疫苗的給予、宿主及環境因素如飼料品質等。黴菌毒素是個重要的飼料品質因子，長期食用黴菌毒素汙染的飼料會造成免疫功能的影響，全球暖化及氣候異常使黴菌毒素的致害越趨重要。玉米赤黴烯酮(ZEN)為具有雌激素結構之非固醇類黴菌毒素，對肝細胞、血液、細胞基因及免疫系統均有毒性。本研究係探討ZEN對存於豬周邊血液單核細胞(PBMCs)及周邊血液淋巴細胞(PBLs)中的PCV2複製是否有直接影響，或是否會透過影響豬隻樹突細胞(DCs)或PBLs功能而間接影響到病毒的複製，以了解ZEN是否具有成為引發PCVAD的環境共同因子的潛力。第一階段實驗選擇臨床上健康的PCV2感染豬隻，以流式細胞儀檢測其PBMCs與不同濃度的ZEN共同培養24及72小時後細胞的存活率，依檢測結果選擇對於細胞存活率無影響的0.01-10 μg/ml低濃度ZEN，在有或無Con A刺激免疫活化的條件下，與PBMCs及PBLs共同培養24-120小時，發現在有Con A刺激條件下，1-10 μg/ml ZEN會使PBMCs中的PCV2核酸量顯著提升，但同濃度之ZEN對PBLs中的PCV2則無顯著影響。第二階段實驗則由PBMCs先分離出血液單核球(Mos)，於體外培養使其分化為DCs後，將DCs與10 μg/ml ZEN共同培養24小時，再於有或無Con A活化的條件下，加入同源的PBLs共同培養24-120小時，發現10 μg/ml ZEN 對於DCs在促使PBLs的增殖與提升PCV2核酸含量上無顯著影響。第三階段實驗將0.1-10 μg/ml ZEN在有或無Con A活化的條件下與PBMCs，以及在無Con A刺激的條件下與DCs，共同培養6-24小時，發現不論有無Con A刺激，10 μg/ml ZEN會顯著減少PBMCs之IL-2及IL-10 mRNA表現量，但同濃度之ZEN對DCs IL-10及IL-12 mRNA表現則無顯著影響。研究結果顯示，非細胞致死劑量之ZEN主要對於Con A活化的PBMCs中PCV2的複製及不論有無Con A刺激下的IL-2及IL-10 mRNA表現量的影響較大，對於DCs則無顯著影響。但由於PCVAD的發生與PCV2的病毒含量有顯著相關性，因此環境中之ZEN在豬隻有其他免疫刺激原存在時，是具有成為促使PCVAD產生的環境因子的潛力。

Porcine circovirus type 2 (PCV2), a single-stranded circular DNA virus, is the primary causative agent of PCV-associated diseases (PCVAD). Aside from PCV2, other factors such as co-infection with other viruses or bacteria and environmental factors also play an important role on PCVAD development. Immunosuppression caused by stress, drug or other environmental factors are suggested to promote PCV2 replication. Mycotoxins are climate dependent, plant- and storage-associated problems. Climate change like global warming represents a key agro-ecosystem driving force of fungal colonization and mycotoxin production. Prolonged low-dose exposure to mycotoxin may lead to the impairment of immunity. Zearalenone (ZEN) and its derivatives, produced by Fusarium spp. and known as non-steroidal, estrogenic mycotoxins, are known to be hepatotoxic, haematotoxic, genotoxic, and immunotoxic. The present study attempted to clarify the possible effects of ZEN on PCV2 replication and functional alterations in peripheral blood mononuclear cells (PBMCs), and the subsets monocyte-derived dendritic cells (DCs) and peripheral blood lymphocytes (PBLs); and to determine whether ZEN is a potential environmental co-factor for PCV2 in PCVAD development. The first part of the study was to incubate PCV2-containing PBMCs and PBLs obtained from healthy, sub-clinically PCV2-infected pigs with ZEN at different concentrations for 24 and 72 h, with or without concanavalin A (Con A) stimulation, to quantify PBMCs viability by flow cytometry. To incubate PBMCs and PBLs with non-lethal 0.01-10 μg/ml ZEN for 24-72 h and 24-120 h, respectively, with or without Con A, to quantify the viral load by real-time PCR. The results showed that there were significant differences in PCV2 load between 1-10 μg/ml ZEN-treated and control group under Con A stimulation, but not in PBLs. The second part of the study was to incubate porcine DCs with 10 μg/ml ZEN for 24 h prior to its co-culture with PBLs, with or without Con A stimulation, for 24, 72 and 120 h, to evaluate the differences in cell proliferation and PCV2 load. The result showed that there was no significant difference between ZEN-treated and control DCs, with or without Con A stimulation, in promoting PBLs proliferation and PCV2 load. The third part of the study was to incubate porcine PBMCs with 0.1-10 μg/ml ZEN, with or without Con A stimulation, for 6-24 h and incubate DCs with different concentrations of ZEN without Con A stimulation for 6-24 h, to evaluate the differences in the expression levels of IL-2 and IL-10 mRNA for PBMCs and IL-12 and IL-10 mRNA for DCs by using real-time PCR. The result showed a significant reduction in IL-2 and IL-10 mRNA expression in ZEN-treated than in control PBMCs at 10 μg/ml ZEN, with or without Con A stimulation; but there was no significant difference in IL-12 and IL-10 mRNA expression between ZEN-treated and control DCs at 0.1-10 μg/ml ZEN without Con A stimulation. In conclusion, the major effect of ZEN was in PBMCs, including significantly elevated PCV2 load and reduced IL-2 and IL-10 mRNA expression at higher but sub-lethal concentrations of ZEN under Con A stimulation. However, no significant effects were revealed in PBLs and DCs. Because PCV2 viral load is important in PCVAD development and under the stimulation of Con A ZEN could increase the PBMCs’ PCV2 viral load, it is thous suggested that ZEN may have the potential to be an environmental co-factor for PCV2 in the induction of PCVAD.