龐飛臺灣大學：獸醫學研究所賴佳琳Lai, Chia-LinChia-LinLai2007-11-282018-07-092007-11-282018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/59929豬隻離乳後多系統消耗性症候群 (PMWS)是一種新興的豬隻疾病，並證實PCV2為其主要的病原。此病毒廣泛的分佈於世界各地的養豬場，然而在流行病學上卻呈現高感染率但低發病率的情形。在一些研究中發現，當豬隻受到免疫活化亦或有其他病原共同感染時，如細菌、病毒，則PMWS的發病率會大為增加。截至目前為止，PCV2的標的細胞大多被認為主要是以單核球與吞噬球系列的細胞為主，另有少數人提出淋巴球亦具有感受性。本實驗室之前的研究指出，豬隻肺泡巨噬細胞經革蘭氏陰性菌脂多醣 (LPS) 刺激之後，PCV2會進入到核內複製，此外周邊血液淋巴球經致裂原concanavalin A (Con A)刺激後，其PCV2的抗原含有率亦呈顯著的增加。為探討免疫活化在PCV2的致病機制上所扮演的角色，因此擬檢測單核球與淋巴球分別及同時受到活化時，PCV2的抗原及核酸含有率是否會有更顯著的差異，以期模擬豬隻同時受到免疫活化以及細菌共同感染時，PCV2病毒含量的變化。 本實驗分別使用革蘭氏陰性菌脂多醣 (LPS) 和致裂原 (Con A) 刺激周邊血液單核細胞 (PBMCs) (其中含有淋巴球以及單核球)，以間接免疫螢光染色的方式偵測抗原含有率，並利用細胞表面抗原的染色來分辨PCV2陽性的主要細胞群；另外，亦使用及時定量聚合酶鏈鎖反應和原位雜合聚合酶鏈鎖反應，來觀察病毒核酸含有率的變化，並以豬腎-15細胞株測定病毒力價。結果顯示，以革蘭氏陰性菌脂多醣 (LPS) 及/或致裂原 (Con A) 同時刺激豬之PBMCs，不論是在抗原及核酸含有率均較不處理組或LPS處理組來的高，且隨著培養時間增加而有上升的趨勢，在病毒力價方面LPS與Con A共同處理組於第三天時亦較其他處理組高，此外有三分之二的陽性細胞為SWC3－的細胞。根據目前結果，可以推測單核球與淋巴球之間有正向的交互作用存在，以促使PCV2抗原及核酸的增加，這些證據顯示當豬隻受到免疫活化時，或許因PCV2病毒量的增加，導致受感染豬隻發病率的增加。Porcine circovirus type Ⅱ (PCV2) infection has been demonstrated to be an essential factor in the induction of the newly emerged disease, postweaning multisystemic wasting syndrome (PMWS), in pigs. PCV2 antibodies have been found in pigs worldwidely, usually with high seroprevalence. Although monocyte/macrophage lineage cells are considered as the major target cells, the role of lymphocytes on the disease development is still uncertain. Immune activation and co-factors such as bacteria or viruses have been suggested to be important factors in the induction of PMWS. Our previous studies have demonstrated that the PCV2 nucleic acid and antigens could be detected intranuclearly in bacterial lipopolysaccharide (LPS)-treated PCV2-inoculated swine alveolar macrophages (AMs) and in concanavalin A (Con A)-stimulated swine peripheral blood lymphocytes (PBLs). The objective of the present study was to further evaluate whether there is an enhancement effect on the PCV2-positive rate in either monocytes or lymphocytes of peripheral blood following monophasic or biphasic stimulation with LPS and/or Con A in healthy PCV2-carrier pigs. After stimulation with LPS and/or Con A, both PCV2 antigen- and nucleic acid-containing rates of the peripheral blood mononuclear cells (PBMCs) of healthy PCV2-carrier pigs measured by immunofluorescent assay (IFA), surface marker IFA, in situ hybridization-polymerase chain reaction (ISH-PCR), and real time PCR increased with time. The levels of the PCV2 antigen-containing rate in Con A-treated group and the group treated simultaneously with LPS and Con A ( (LPS + Con A)-treated groups) were significantly greater than those of the NT and LPS-treated groups. Significant difference was also seen among the LPS-, Con A, and (LPS + Con A)-treated groups. The viral titer of the (LPS + Con A)-treated group increased at 3 days post-incubation (DPI) as did antigen- and nucleic acid-containing rates. Two third of the PCV2-positive cells belonged to SWC3－ population; this implies that lymphocytes may also play an important role on PCV2 replication. The results indicate that interaction between PBMs and PBLs may exist and simultaneous activation of PBMs and PBLs may result in increased PCV2 load in both cells. The results further support that immune activation may increase the morbidity of PMWS in PCV2-infected pigs via the increase in viral load.Table of Contents 口試委員審定書 致謝 中文摘要 ...……………………………………………………….................. Ⅰ Abstract ………………………………………………………………............ Ⅲ Table of Contents ……………………………………………………............... Ⅴ Figures ………………………………………………………………........... Ⅷ 1 Introduction 1 2 Review of Literature 3 2.1 Porcine Circovirus 3 2.1.1 Taxonomy 3 2.1.2 Structure 3 2.1.3 Replication and Infection 5 2.2 Postweaning Multisystemic Wasting Syndrome (PMWS) 6 2.2.1 Introduction 6 2.2.2 Clinical Signs and Pathology 7 2.2.3 Target Cells and Tissue Distribution 8 2.2.4 Pathogenesis of PMWS 10 2.2.4.1 Mechanism of PCV2 Entering Host Cells 10 2.2.4.2 Roles of Immunoactivation and Co-infection on PMWS Development 11 2.2.4.3 Alterations in Immune System of PMWS-Affected Pigs 12 2.3 Introduction to Lipopolysaccharide (LPS) 14 2.4 Introduction to Mitogens 16 3 Materials and Methods 19 3.1 Experimental Design 19 3.1.1 Experimental Flow Chart 20 3.2 Experimental Animals 21 3.3 Materials 21 3.3.1 Preparation of Peripheral Blood Mononuclear Cells (PBMCs) 21 3.3.1.1 Method of Isolation 21 3.3.1.2 Culture Medium 22 3.3.1.3 Concentration of Lipopolysaccharide (LPS) 24 3.3.1.4 Concentration of Concanavalin A (Con A) 25 3.3.1.5 Antibodies 25 3.4 Methods 25 3.4.1 Cell Viability Detection 25 3.4.1.1 Trypan Blue Dye Exclusion Assay 25 3.4.1.2 Annexin V/ Propidium Iodide (AV/PI) 26 3.4.2 Indirect Immunofluorescent Assay (IFA) 27 3.4.2.1 Fluorescent Microscopy (Fixed Cells and IFA) 27 3.4.2.2 Flow Cytometry (Suspended Cell Fixation and IFA) 28 3.4.2.3 Cell Surface Marker and PCV2 Antigen (Suspended Cell Fixation and IFA) 29 3.4.3 Quantitative Real-Time Polymerase Chain Reaction (Real-time PCR) 29 3.4.4 In Situ Hybridization Polymerase Chain Reaction (ISH-PCR) 31 3.4.5 Viral Titration 32 3.5 Statistical Analysis 33 4 Results 34 4.1 Cell Viability 34 4.1.1 Trypan Blue Dye Exclusion Test 34 4.1.2 Annexin V(AV)/Propidium Iodide (PI) 34 4.2 PCV2 Antigen-Containing Rate (ACR) 35 4.2.1 Fluorescent Microscopy 35 4.2.1.1 Polyclonal Antibody 35 4.2.1.2 Monoclonal Antibody (mAb) 36 4.2.2 Flow Cytometry 37 4.3 PCV2 and Surface Marker 37 4.4 PCV2 Nucleic Acid-Containing Rate 38 4.4.1 Quantitative Real-Time Polymerase Chain Reaction 38 4.4.2 In Situ Hybridization Polymerase Chain Reaction 39 4.5 Viral Titration 39 5 Discussion 54 6 References 66613511 bytesapplication/pdfen-US豬隻離乳後多系統性消耗性症候群第二型環狀病毒單核球淋巴球交互作用PCV2Peripheral Blood Monocytes (PBM)Peripheral Blood lymphocyte (PBL)interactionLPSCon APMWSthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59929/1/ntu-96-R94629002-1.pdf