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研究生:鄭煜堯
研究生(外文):Cheng, Yu-Yao
論文名稱:飼料添加靈芝醱酵物和接種豬環狀病毒第二型疫苗對離乳仔豬生長性能和免疫反應之交互影響
論文名稱(外文):Effects Of A Culture Of Ganoderma lucidum Supplemented Diet And The Porcine Circovirus 2 Vaccine On The Growth Performance And Immune Response Of Weaned Pigs
指導教授:李德南 博士許惠貞 博士
指導教授(外文):Lee, Der-Nan, Ph. D.Hsu, Hui-Chen Ph. D.
口試委員:顏宏達楊天樹余祺
口試日期:2012-07-02
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物資源學院碩士在職專班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:88
中文關鍵詞:靈芝環狀病毒疫苗
外文關鍵詞:Ganoderma lucidumPigPorcine circovirus type 2Vaccine
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摘要
仔豬因離乳緊迫之影響,常降低免疫力與生產效率,若遭豬環狀病毒第二型(Porcine circovirus type 2)感染,則易出現臨床症狀並提高死亡率。離乳仔豬餵飼傳統天然藥用真菌之靈芝醱酵物(cultured of Ganoderma lucidum, CGL),經證實可改善生長性能與免疫能力,但對豬隻免疫細胞之作用與感受PCV2之效果仍未明瞭。故本研究區分為兩試驗進行探討,試驗一為探討飼料添加CGL和接種PCV2疫苗對離乳仔豬生產性能之影響,試驗二為利用體外試驗測定靈芝多醣對豬隻免疫細胞之影響。試驗一選取144頭4週齡雜交離乳仔豬,以2 × 2複因子逢機完全區集設計分配至添加CGL及接種PCV2疫苗與否組成之四種處理。試驗重複6次(欄),每欄飼養豬隻6頭,公母各半,基礎飼糧以玉米-大豆為主,添加先前證實之CGL最佳劑量;PCV2則採用商用疫苗。試驗豬隻飼料分成0-2週、3-6週和7-8週共三期,飼料與飲水任飼,試驗第2天依處理分別肌肉注射生理食鹽水或接種PCV2疫苗。豬隻隔週秤重,於試驗開始與結束每欄逢機採取4頭豬隻血液樣品。結果發現,飼料添加CGL可改善試驗7-8週之飼料效率與提高T細胞之CD4+CD8+亞群比例,亦降低嗜中性球比例、受PMA(phorbol 12-myristate 13-acetate)刺激之嗜中性球活性氧物質(reactive oxygen species, ROS)分泌量及經美洲商陸裂殖原(pokeweed mitogen, PWM)刺激之周邊血液單核細胞(peripheral blood mononuclear cell, PBMC)增生反應。豬隻接種PCV2疫苗可提高試驗7-8週之日增重、經PWM刺激之PBMC增生反應及PCV2疫苗與靈芝多醣之抗體力價,另外亦降低血液淋巴球和豬隻病毒血症比例。飼料添加CGL和接種PCV2疫苗對豬隻血液單核球比例、嗜中性球吞噬能力、T細胞之CD4-CD8-與CD4+亞群比例、血漿血紅素結合蛋白與血漿干擾素-γ濃度具交互作用。試驗二為分離離乳仔豬嗜中性球和PBMC於體外培養,經添加不同濃度液態醱酵靈芝水萃多醣。結果發現添加3.1與50 μg/mL之靈芝多醣,可分別較對照組增加嗜中性球吞噬作用和自發性ROS分泌量,而添加1.6 μg/mL以上之靈芝多醣可抑制經PMA刺激之嗜中性球ROS分泌量。添加1.9-7.8 μg/mL靈芝多醣可增加PBMC之自發性增生反應,而添加62.5-250 μg/mL靈芝多醣則有平復伴刀豆球蛋白(concanavalin A)誘增PBMC增生反應之能力。綜上所述,飼料添加CGL及接種PCV2疫苗可分別改善仔豬離乳後7-8週飼料效率和日增重,並具調節豬隻免疫功能,而在接種疫苗與否之情況中,飼料添加CGL對豬隻免疫狀態具不同作用,故靈芝醱酵物之多醣具有調節離乳仔豬免疫細胞之功能,雖未直接影響豬隻抵抗PCV2病毒之能力,但可藉調節免疫能力而增進豬隻生產效率。
關鍵字:靈芝、豬、環狀病毒、疫苗 
ABSTRACT
Weanling results in decreased of production efficiency and immunocompetence, which may be attributed to stress in piglets. Recently weaned piglets are susceptible to PCV2 and have high mortality rates.The culture of Ganoderma lucidum is a fungus used in traditional herbal medicine, and previous studies have demonstrated that CGL could improved the growth performance and immunity of weaned pigs. However, the effects of CGL polysaccharides on the immune cells and the ability of anti-PCV2 in weaned pigs have not been elucidated. Therefore, the aim of this study was to explore whether CGL influences the immune response and production performance of weaned pigs. The research involved two experiments. In Exp. 1, we investigated the effects of a CGL supplemented diet as well as PCV2 immunization on the production performance of weaned pigs. In this experiment, 144 pigs were randomly allotted into four treatments groups according to 2 × 2 factorial experiment design. Each group was divided into six pens containing six pigs each. The factor containing supplemented with CGL and PCV2 vaccination. Results revealed that CGL increased FE between 6-8 weeks, percentage of CD4+CD8+ T cells. However, by week 8 of the CGL treatment, the percentage of neutrophils as well as ROS production in PMA stimulated neutrophils had decreased. The PCV2 vaccine treatment increased ADG between 6-8 weeks, PBMC proliferation in response to PWM stimulation. Furthermore, antibody titers revealed that the PCV2 vaccine increased the production of G. lucidum polysaccharide(GLPS) antibodies and PCV2 vaccine antibodies. However, this treatment decreased the percentage of lymphocytes. When the diet of piglets was supplemented with CGL and the PCV2 vaccine have found the interactions on percentage of monocytes, neutrophils phagocytosis, percentages of CD4-CD8- and CD4+ T cells, and the concentrations of plasma haptoglobin and IFN-γ. In Exp. 2, GLPS was extracted by 121℃ water from liquid fermentation. The extracted GLPS were then added to peripheral blood mononuclear cells (PBMC) and neutrophils of weaned pigs, in vitro. The results reveal that the addition of GLPS increased ROS production in neutrophil and the spontaneity proliferation of PBMC, However, ROS production by PMA-stimulated neutrophils and the proliferation of con A stimulated PBMC both decreased. In conclusion, this study confirmed treating weaned piglets with a CGL supplemented diet and PCV2 immunizations improved FE and ADG, respectively. Furthermore, the modified immune response in weaned pigs may have resulted from the combined effects of the CGL supplemented diet and the PCV2 immunization
Key words: Ganoderma lucidum, Pig, Porcine circovirus type 2, Vaccine
目錄
誌謝........................................................................................................................I
中文摘要.............................................................................................................III
英文摘要..............................................................................................................V
目錄....................................................................................................................VII
表目錄.................................................................................................................XI
圖目錄..............................................................................................................XIII
壹、 前言...............................................................................................................1
貳、 文獻檢討.......................................................................................................4
一、 多醣結構種類與免疫特性...................................................................4
二、 靈芝多醣特性與免疫調節能力...........................................................6
三、 靈芝多醣抗病毒功效之研究...............................................................8
四、 PCV2 對豬隻的影響與致病機制.......................................................9
五、 PCV2 疫苗保護原理與刺激免疫作用.............................................11
參、 材料與方法.................................................................................................14
一、 試驗一:飼料添加靈芝醱酵物和接種豬環狀病毒第二型疫苗對豬隻生長性能和免疫反應之交互影響(田間試驗).............................14
(一) 靈芝固態醱酵物....................................................................14
1. 菌種...................................................................................14
2. 培養與活化.......................................................................14
3. 固態醱酵培養...................................................................15
(二) 靈芝多醣萃取與分析............................................................15
1. 粗多醣萃取.......................................................................15
2. 粗多醣含量測定...............................................................16
(三) 疫苗來源................................................................................16
(四) 試驗設計與飼料處理............................................................16
(五) 動物飼養與管理....................................................................17
(六) 血液樣品採集與周邊血液單核細胞純化...........................19
1. 樣品採集...........................................................................19
2. 周邊血液單核細胞純化...................................................19
(七) 先天性免疫活性測定............................................................20
1. 白血球計數.......................................................................20
2. 白血球組態...............................................................20
3. 嗜中性球活性氧物質分泌量...........................................20
4. 嗜中性球吞噬能力...........................................................21
(八) 細胞性免疫活性測定............................................................22
1. 周邊血液單核細胞增生反應...........................................22
2. T淋巴細胞亞群組成........................................................22
(九) 體液性免疫活性測定............................................................23
1. 血漿總IgG測定................................................................23
2. 血漿豬環狀病毒第二型病毒抗體力價測定...................23
3. 血漿靈芝多醣特異性IgG抗體力價測定........................23
(十) 血漿豬環狀病毒第二型病毒定量分析................................24
(十一) 免疫狀態...........................................................................25
1. 血紅素結合蛋白...............................................................25
2. 血漿干擾素-γ....................................................................27
(十二) 統計分析...........................................................................27
二、 試驗二:靈芝多醣對豬隻免疫細胞之影響(體外試驗)...................28
(一) 靈芝液態醱酵........................................................................28
1. 菌種、培養與活化...........................................................28
2. 液態醱酵培養...................................................................28
3. 靈芝粗多醣萃取、分離與測定.........................................28
(二) 免疫細胞樣品採集與純化....................................................30
(三) 免疫細胞活性測定................................................................30
1. 嗜中性球活性氧物質分泌...............................................30
2. 嗜中性球吞噬能力...........................................................30
3. 周邊血液單核細胞增生反應...........................................31
(四) 統計分析................................................................................31
肆、 結果.............................................................................................................32
一、 試驗一:飼料添加靈芝醱酵物和接種豬環狀病毒第二型疫苗對豬隻免疫反應之影響(田間試驗).........................................................32
(一) 靈芝固態醱酵物多醣含量....................................................32
(二) 豬隻生長性能........................................................................32
(三) 先天性免疫活性....................................................................34
(四) 細胞性免疫活性....................................................................39
(五) 體液性免疫活性....................................................................42
(六) 豬環狀病毒第二型病毒量....................................................44
(七) 免疫狀態................................................................................46
二、 試驗二:靈芝多醣對豬隻免疫細胞之影響(體外試驗)...................48
(一) 靈芝液態醱酵物成份分析....................................................48
(二) 先天性免疫調節....................................................................48
(三) 細胞性免疫調節....................................................................51
伍、 討論.............................................................................................................54
陸、 結論.............................................................................................................59
柒、 參考文獻.....................................................................................................60


表目錄
表 1-1 基礎飼糧組成和營養分含量..............................................................18
表 1-2 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬生長性能之
影響......................................................................................................33
表 1-3 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬血液白血球
組態之影響..........................................................................................35
表 1-4 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬嗜中性球活
性氧物質(ROS)分泌量之影響..........................................................36
表 1-5 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬嗜中性球吞
噬能力之影響......................................................................................38
表 1-6 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬周邊血液單
核細胞增生能力之影響......................................................................40
表 1-7 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬周邊血液單
核細胞T細胞亞群分佈之影響........................................................41
表 1-8 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬血漿豬PCV2
抗體力價、IgG濃度與靈芝多醣特異性IgG抗體力價之影響..........43
表 1-9 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬血漿PCV2病
毒量之影響..........................................................................................45
表 1-10 飼料添加靈芝醱酵物與接種PCV2疫苗對離乳仔豬血漿血紅素
結合蛋白與干擾素-γ濃度之影響.....................................................47
表 2-1 靈芝液態醱酵培養基配方..................................................................29
表 2-2 添加靈芝多醣對豬隻嗜中性球活性氧物質分泌量之影響............49
表 2-3 添加靈芝多醣對受PMA刺激的豬隻嗜中性球活性氧物質分泌量
之影響..............................................................................................50
表 2-4 添加靈芝多醣對豬隻嗜中性球吞噬作用之影響..............................52
表 2-5 添加靈芝多醣對豬隻周邊血液單核細胞增生反應之影響..............53


圖目錄
圖 1-1 PCV2標準質體選殖片段架構圖.......................................................26
圖 1-2 PCV2即時定量系統標準曲線...........................................................26
圖 1-3 接種PCV2疫苗對離乳仔豬血漿PCV2病毒量分佈比率之影
響..........................................................................................................45



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