臺灣博碩士論文加值系統

家禽里奧病毒感染症在家禽中造成病毒性關節炎 (viral arthritis, VA)及營養不良症候群 (malabsorption syndrome, MAS) 等慢性消耗性的疾病，常造成家禽經濟產業的重大損失。本實驗以家禽里奧病毒 2408株 (營養不良症候群型) 足底接種 2 日齡 SPF 雛雞，可於接種後 3 至 4 日開始出現雛雞大量死亡情形。於肝臟及脾臟可見多發針尖大小黃白色壞死灶。組織病變上可見肝臟及脾臟有多發急性凝固性壞死灶，及脾臟有淋巴球大量流失。在免疫化學 (IHC) 染色下，可見 ARV σNS protein陽性訊號出現在接種足底、肝臟及脾臟的巨噬細胞中。而以往文獻僅進行胸腺及華氏囊摘除手術，並觀察家禽里奧病毒接種後之變化。並未有人提出摘除脾臟手術在家禽里奧病毒感染後所扮演的角色，為了研究肝臟與脾臟在家禽里奧病毒接種後病理變化的關聯性，故此次實驗將脾臟摘除後，再進行家禽里奧病毒 2408 足底接種後，觀察 SPF 雛雞的臨床症狀、肝臟之肉眼與組織病變、IHC 下陽性訊號分布及利用即時定量 PCR測定肝臟中之病毒核酸量。結果顯示，不同雞種下，僅有 SPF雛雞出現病變，市售蛋用、肉用雛雞則不受影響。在 SPF雛雞組，肝臟之 IHC 下病毒陽性訊號於高劑量組中的病毒陽性訊號則與低劑量組有顯著性差異出現，可知家禽里奧病毒接種有劑量效應存在，但於即時定量 PCR 中並無此結果出現。在手術控制組與脾臟摘除組方面，於各種比較上並無顯著性差異，僅在肝臟組織病變出現先後有不同，可見脾臟摘除組的肝臟急性凝固性壞死灶於接種後第二天出現，較手術控制組早。比較四種評估方法，包含肉眼與組織病變、IHC及即時定量 PCR，在脾臟方面因接種日程增加而有相同之趨勢；但肝臟卻無一致性，推測因肝臟中有多種酵素，導致在測定肝臟中之病毒核酸量受到干擾。由此推論，脾臟在家禽里奧病毒 2408 以足底接種感染時，以此四種方式評估並非重要的標的器官，卻可以使肝臟病變提早出現，顯示肝臟及脾臟間仍有某種作用，而何種臟器或是何種細胞為家禽里奧病毒首要感染器官，則有待後續研究繼續進行。

It was known that Avian reovirus (ARV) infection in poultry causes chronic wasting diseases, such as viral arthritis (VA) and malabsorption syndrome (MAS), often leads to a significant economic loss in poultry industry. In this investigation, we use ARV 2408 to inoculate 2-day-old SPF chicks via footpad route, which leads to a high mortality 3-4 days later. Multiple hepatic and splenic yellow-white pinpoint necrotic foci were demonstrated under necropsy, parallel with acute multifocal coagulative necrosis revealed in histopathological findings. Confirming by immunohistochemistry (IHC) stain, positive signals of ARV σNS protein were demonstrated in macrophages of inoculated footpad, liver, and spleen. Previous studies were limited in the descriptions of ARV inoculation after thymectomy and bursectomy, while no further information were available for effects caused by splenectomy in ARV infections. In order to study the correlation between ARV infection and consequential pathological changes in liver and spleen, we inoculated ARV 2408 in chicks by footpad route after splenectomy. Clinical traces of symptoms, gross lesions, hepatic histopathological changes, and viral distribution under IHC were demonstrated, parallel with viral nucleic acid quantification performed by qPCR. Significant differences were demonstrated between SPF chicks and commercial broilers and layers. In contrast to their counterparts, it appears that lesions only shown in SPF chicks. Dose dependent variations in SPF chicks were demonstrated by IHC, but no significant differences were revealed by qPCR. On the other hand, the only difference between operational control and splenectomized group was the timing that histopathological lesions appeared. Specifically, acute coagulative necrosis of splenectomized group appeared one day earlier than operational control group.
Summarizing the approaches of gross, histopathological lesions, IHC and qPCR, temporal dependent positive correlation was implied in spleen. However, no direct evidence suggests consequential outcomes in liver, but it was suspected that the accuracy of qPCR may be interfered by hepatic enzymes. Accordingly, our data suggested that spleen is not the primary target organ in ARV 2408 footpad inoculation, but splenectomy may advances the appearance of hepatic lesions. It is certain that there must have some crosstalk between liver and spleen, while the probing of definite ARV target organ will rely on further investigations.

目錄
中文摘要 I
Abstract II
目錄 III
圖次 VII
第一章 前言 1
第二章 文獻探討 2
第一節 家禽里奧病毒感染症簡介 2
1.1 疾病歷史與簡介 2
1.2 臨床症狀及其分類與病毒株 2
1.3 病毒特性及血清型 3
第二節 家禽里奧病毒感染症之病變 3
2.1 發病年齡層及主要病變 3
2.2 肝臟病變 3
2.3 脾臟病變 4
第三節 家禽里奧病毒感染之影響因子介紹 4
3.1 不同感染路徑引致之不同病變 4
3.2 不同病毒株引致之不同病變 5
3.3 接種之年齡效應 5
第四節 家禽里奧病毒感染症之偵測技術介紹 5
第五節 病毒性肝炎簡介 6
5.1 簡介 6
5.2 傳染途徑與主要病變 7
5.3 致病機轉：以人類病毒性肝炎為例 9
第三章 實驗材料與方法 11
第一節 病毒來源與增殖法介紹 11
1.1 攻毒病毒之來源 11
1.2 病毒增殖法 11
1.3 病毒力價測試 11
第二節 實驗動物 12
2.1 雞隻實驗分組 12
2.2 實驗動物來源 12
2.3 雞隻去脾手術 12
2.4 雞隻攻毒試驗 13
2.5 雞隻觀察與組織採樣及固定 13
2.6 組織切片製作 13
2.7 免疫組織化學染色切片製作 14
2.8 組織 RNA 萃取及 cDNA 製備 14
2.9 即時定量聚合酶鏈反應 (Real-time Quantitative Polymerase Chain Reaction, qPCR) 15
第三節 病理學檢查及組織病變指數介紹 15
第四節 影像分析系統介紹 15
第四章 實驗結果 17
第一節 病毒攻毒試驗 17
1.1 不同雞種腳底接種二日齡雛雞死亡率與臨床症狀之觀察 17
1.2 不同病毒力價腳底接種二日齡 SPF 雛雞死亡率與臨床症狀之觀察 17
1.3 雞隻去脾手術後以足底接種組之死亡率與臨床症狀之觀察 18
第二節 病毒接種後雞隻之病理變化 18
2.1 雛雞肉眼病理學與組織病理學之觀察結果 18
2.3 切片免疫組織化學染色之結果 20
2.4 qPCR 之結果 21
第三節 影像分析系統ImageJ之量化統計 21
3.1 免疫組織化學染色之病毒訊號分布區域統計 21
第五章 討論 22
參考文獻 29
附表 37
附圖 39

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