豬第二型環狀病毒 (Porcine circovirus type 2；PCV2) 被認為於養豬產業中引起呼吸系統之疾病中重要的病毒性疾病之一。PCV2一旦與其他病毒性病原混合感染，特別是與豬生殖和呼吸症候群病毒 (Porcine reproductive and respiratory syndrome virus；PRRSV) 混合感染造成養豬產業重大經濟上的損害。因而本試驗之目的為評估商品化豬第二型環狀病毒疫苗於田間使用之保護效益與病毒在組織中分佈之研究。本試驗以 300 頭離乳豬分成施打疫苗組和對照組各 150 頭，於疫苗施打前後第 0、7、21、35 和 49 天，分別於兩組中隨機選取4頭豬隻進行剖檢及採集樣品，利用 Annexin V/PI 及Tunel assay 共同分析組織中細胞凋亡之情形、以 ELISA 的方法檢測血清中 PCV2 及PRRSV特異性抗體、評估肺臟肉眼病變、以 Real-time PCR 檢測各臟器之病毒含量以及生長性能表現之評估。實驗結果發現，疫苗組豬隻在細胞凋亡作用及組織中 PCV2 病毒量與對照組相較之下，兩組間具有顯著性差異 (P<0.05)。PCV2 及 PRRSV 抗體方面，疫苗組豬隻可顯著增加 PCV2 特異性抗體以及同時減低組織中 PCV2 抗原量，實驗後期疫苗組豬隻於生長體重和日增重之表現均較對照組佳。綜合以上試驗結果顯示，PCV2疫苗使用於田間豬隻具有一定的保護效益。
關鍵字：豬第二型環狀病毒、細胞凋亡

Porcine circovirus type 2 (PCV2) is considered to be one of important viral pathogens causing respiratory disease in swine industry worldwide. Once co-infected with other viral pathogens, especially the porcine reproductive and respiratory syndrome virus (PRRSV), PCV2 causes a significant economic loss in the swine industry. The purpose of this study is to evaluate the protective efficacy and distribution of PCV2 in tissues after vaccination in the pigs. Three hundreds 4-week-old pigs were divided into vaccinated and Non-vaccine groups, with 150 pigs each in three pens respectively in the same house. The pigs in the vaccinated group were intramuscularly injected with one dose of Ingelvac CircoFLEX® vaccine and the pigs in the Non-vaccine group with one ml of saline. Among them, four pigs in each group were humanely euthanized at days 0, 7, 21, 35 and 49 after vaccination. The lung lesions were macroscopically examined and scored, and spleens were collected to examine splenocytic apoptosis using Annexin V/PI and Tunel assay. The loads of PCV2, and PRRSV were also examined by real-time PCR, and PCV2-specific antibody, PRRSV-specific antibody by ELISA. ANOVA analysis was performed to compare significant difference between groups. Comparison of PCV2 viral load in tissues, PCV2-specific antibodies and splenocytic apoptosis showed statistical significant difference at various days post-vaccination between these two groups (P<0.05). In conclusion, the commercial PCV2 vaccine provides a certain level of protection for pigs in the field conditions.

中文摘要……………………………………………………………………….…….……...i
英文摘要……………………………………………………………………………….…...ii
目次…………………………………………………………………..…………….............iii
表次…………………………………………………………………………………….…...v
圖次…………………………………………………………………………………….…..vi
前言……………………………………………………………….....……..…….1
文獻探討………………………………………………………………...……….2
豬環狀病毒之分類及結構………………………………………………...3
1.1豬環狀病毒臨床症狀與病理變化…………………………………….3
1.2豬環狀病毒與其他病毒的感染以及流行病學……………………….5
1.3豬環狀病毒感染組織內分布及其標的細胞………………………….6
1.4豬隻環狀病毒之複製模式…………………………………………….7
1.5豬環狀病毒之致病機制…………………………………………..….10
病毒誘導細胞凋亡與持續感染之機轉……………………………….…11
2.1豬環狀病毒與細胞凋亡之相關性…………………………………...11
目前商品化疫苗發展及趨勢………………………………………….…12
3.1市售商品化疫苗評估效益…………………………………………...13
探討豬第二型環狀病毒疫苗在田間使用之效益……………………………..15
實驗設計………………………………………………………….………15
材料及方法
分離豬隻脾臟淋巴細胞………………………………...………………….16
細胞凋亡之偵測
2.1 TUNEL assay & quantitation…………………………………………...17
2.2 Annexin V與Propidium iodide 雙重染色………………………….…17
肺臟肉眼病變評估分數………………………………………………...….18
淋巴系統及肺臟病變嚴重度之判別方法…………………………………18
組織研磨製備同步萃取病毒核酸
5.1萃取豬第二型環狀病毒核酸………………..........................................19
5.2萃取豬生殖和呼吸症候群病毒核酸………………………….……….19
質體之純化…………………………………………………………………20
6.1質體DNA轉殖與培養…………………………………………………21
6.2質體DNA之萃取……………………………………………….……..21
即時定量PCR
7.1標準曲線……………………………………. …………………………22
7.2檢測各組織中豬第二型環狀病毒量…………………………………..22
7.3檢測肺臟、淋巴結及脾臟中豬生殖和呼吸症候群病毒量……..........23
特異性抗體檢測…………………………………………………………....23
8.1豬第二型環狀病毒特異性抗體ELISA檢測……………………….…23
8.2豬生殖和呼吸症候群病毒特異性抗體ELISA檢測…………….……24
生長性能評估……………………………………………………………....24
數據統計分析……………………………………………………………....25
結果………………………………………………………………......................26
細胞凋亡之偵測
1.1 TUNEL assay & quantitation……………………………………...……26
1.2 Annexin V與Propidium iodide 雙重染色…………………………….26
肺臟肉眼病變評估分數…………………………………………………..27
顯微病變，淋巴系統及肺臟病變嚴重度之判別結果……………….......27
Real-time PCR 各臟器PCV2病毒量……………………….....................28
Real-time PCR 脾臟、肺臟及淋巴結PRRSV病毒量………………......28
特異性抗體檢測
6.1豬第二型環狀病毒特異性抗體ELISA檢測……………………….…28
6.2豬生殖和呼吸症候群病毒特異性抗體ELISA檢測..………….……29
生長性能評估…………………………………………….………………29
討論………………………………………………………………....................30
結論………………………………………………………………....................36
參考文獻………………………………………………………………………………….37

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