臺灣博碩士論文加值系統

傳染性胰臟壞死病毒（Infectious pancreatic necrosis virus, IPNV）普遍存在本省多種養殖魚類，雖然本省所分離出之IPN病毒株病原性不高，但是以往的研究結果顯示，逆境因子(鹽度以及重金屬)會提高石班魚對IPN病毒之感受性，進而引發高死亡率。因此，本論文以分離自虹鱒之IPN病毒，T42G為病原體，探討IPN病毒和鋅鎘重金屬對石斑魚之致病機制。
首先觀察IPN病毒與鋅、鎘重金屬交互作用對石斑魚組織病理變化之影響，結果發現IPN病毒浸泡組石斑魚最明顯的組織病理變化出現在胰臟，感染第十二天檢體的胰臟腺泡細胞，呈現局部性壞死及炎症細胞的浸潤，在壞死區並可觀察到包涵體樣構造；在腎臟可觀察到腎小管的上皮細胞壞死；而鰓等其他臟器則無明顯病變發生。鋅、鎘重金屬處理所造成的病變情形相似，以鰓最為嚴重，鰓出現上皮肥大、增生、上提與黏液細胞增生、鰓絲融合及血竇形成等病變情形；重金屬浸泡也會造成石斑魚腎小管上皮細胞的壞死，肝細胞水腫、萎縮及壞死等傷害，但在胰臟卻未觀察到明顯的病變。病毒和重金屬交互作用時對石斑魚的影響，在於各臟器出現病變的時間較單一處理組魚提前，其中以胰臟病變提早在第六或第八天出現最為明顯，且胰臟病變的檢體數也有增加的現象。顯示，重金屬與IPN病毒共同作用確實會提高石班魚各臟器病變的發生率，進而引發魚體死亡。
以T42G特異性抗體利用DAB呈色之免疫染色結果顯示，IPN病毒感染之石斑魚，尤其是和重金屬交互處理後之石斑魚的鰓、肝、胰、腸、腎、脾等臟器中，皆有病毒的存在，顯示IPN病毒在石斑魚體內會造成全身性的感染。
以QAI金屬染色觀察，鋅在細胞內呈藍紫色，而鎘則是呈現紅色。進而以此染色法觀察鋅、鎘處理組之石斑魚體內重金屬離子分佈情形，結果發現對照組和鋅處理組魚的肝、腎、脾、鰓、肌肉等所有臟器均呈現藍紫色的反應，顯示一般魚體內原就有相當含量的鋅存在。而鎘處理後，除了在魚的一級鰓薄板的骨質部分和肝臟血管周圍有紅色的呈色反應，其他部分都呈現藍紫色。觀察IPN病毒與重金屬交互作用組石斑魚的重金屬分布情形，結果和單一重金屬處理組結果並無差別，顯示病毒並不影響重金屬在魚體內的蓄積。
以病毒在分離檢測IPN病毒在石斑魚體內各臟器的增殖情形，結果發現所有組別的病毒檢出率均以後腎最高，胰臟次之，而各組之間（單一病毒處理和病毒重金屬交互處理）在檢出率和病毒力價並無差異，顯示重金屬與病毒交互作用並不影響病毒在魚體內的增殖。
最後以NBT染色法測定頭腎細胞之胞內超氧離子的產生情形，藉以分析IPN病毒與重金屬對石斑魚頭腎細胞吞噬活性之影響，結果發現病毒處理組魚頭腎的吞噬活性相較於對照組魚，有提高的現象；而重金屬（尤其是鎘處理）與病毒交互作用明顯會抑制頭腎細胞超氧離子的產生。

摘要……………………………………………………………………………I
前言……………………………………………………………………………1
文獻整理………………………………………………………………………3
一、傳染性胰臟壞死病毒之簡介……………………………………………3
二、重金屬的污染……………………………………………………………6
三、重金屬染色………………………………………………………………10
四、魚類的免疫系統…………………………………………………………11
材料與方法……………………………………………………………………13
一、實驗材料…………………………………………………………………13
(一)實驗用魚…………………………………………………………………13
(二)細胞株繼代培養…………………………………………………………13
(三)IPN病毒…………………………………………………………………15
(四)重金屬貯備溶液…………………………………………………………15
(五)重金屬染劑OAI(2-(8-quinolylazo)-4,5-diphenylimidazole)之合成與配製…………………………………………………………………………15
二、實驗方法………………………………………………………………..16
(一)IPN病毒與鋅、鎘重金屬交互作用對石斑魚組織病理變化之影響………………………………………………………………………………18
(二)以免疫化學染色法觀察IPN病毒在石斑魚體內各臟器之感染情形………………………………………………………………………………18
1.免疫化學染色法特異性之確認……………………………………………18
2.IPN病毒在石斑魚各臟器之分佈情形……………………………………19
(三)以QAI金屬染色法觀察重金屬在石斑魚體內各臟器之分佈情
形………………………………………………………………………………19
1.鋅、鎘離子在QAI金屬染色後之呈色反應………………………………19
2.鋅、鎘離子在石斑魚各臟之分部情形……………………………………19
(四)重金屬對IPN病毒在石斑魚體內增殖情形之影響……………………20
1.實驗處理與採樣……………………………………………………………20
2.病毒再分離…………………………………………………………………20
3.病毒力價測定………………………………………………………………20
(五)IPN病毒與重金屬對石斑魚頭腎細胞吞噬活性之影響………………21
1.實驗處理與採樣……………………………………………………………21
2.頭腎細胞吞噬活性之分析…………………………………………………21
結果……………………………………………………………………………23
一、IPN病毒與鋅、鎘重金屬交互作用對石斑魚組織病理變化之影響……………………………………………………………………………23
二、以免疫化學染色法觀察IPN病毒在石斑魚體內各臟器之感染情形…24
三、以QAI金屬染色法觀察重金屬在石斑魚體內各臟器之分佈情形……25
四、重金屬對IPN病毒在石斑魚體內增殖情形之影響……………………26
五、IPN病毒與重金屬對石斑魚頭腎細胞吞噬活性之影響………………26
討論……………………………………………………………………………27
參考文獻………………………………………………………………………32
圖表…………………………………………………………………………44
附錄……………………………………………………………………………61

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