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Detection of OIE Notifibale Fish Virus Diseases and Comparison of Fish Immunoglobulins
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近年來國際間活魚及魚卵之貿易極為頻繁，因此實施嚴格之檢疫工作，方可避免國際間魚類重大傳染病之傳播。而依據OIE水生動物傳染性疾病列表中，高經濟價值的鮭鱒魚類疾病如Viral Hemorrhagic septicaemia (VHS)、Infectious hematopoietic necrosis virus (IHNV)及Infectious pancreatic necrosis virus (IPNV)等皆被各國列為主要監測的疾病。本研究目的旨在建立水生動物疾病監測系統並構築所檢測病原之陽性對照組，以達到精確快速之檢測依據。試驗首先為構築包含各檢測病原特異性基因片段之陽性質體，並應用試管內轉錄(in vitro transcription)之技術轉錄成RNA作為內部控制組(internal control)，再應用反轉錄聚合酵素連鎖反應(RT-PCR)同步來檢測檢體內之病毒核酸，以增加診斷作業之精確度。除測試反應最佳化之條件及敏感性外，並實際應用於進口鮭鱒魚、魚卵與國內養殖魚類之監測，以期達到快速精確之監測目的。而自95年7月至96年3月共篩檢台中地區160個進口樣品後，雖然尚未檢出IHNV及VHSV之陽性檢體，但於國內養殖之鱒魚及紅魽均可檢出IPNV陽性病例。此外，除了分子生物學診斷外尚可應用免疫血清學作為疾病監測，但由於水生動物的物種繁多，且跨種別間免疫球蛋白之差異性及對相同抗原之相互辨認度仍尚未明瞭，因此本研究亦針對不同魚種間之免疫球蛋白進行研究，嘗試評估不同魚種包括吳郭魚、淡水石斑、紅魽及虹鱒免疫球蛋白相互間之辨識能力，探討魚種間免疫球蛋白之差異性，以作為日後進一步研究魚類免疫機制之基礎。
Viral hemorrhagic septicaemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) are major pathogens listed on OIE notifiable fish diseases among several species of salmonid fish. As the international trade of egg, juvenile, and aquatic animal products between countries become more frequent, it is important to establish more precise and strict inspections under OIE regulation to exclude these pathogens from Taiwan. The aim of this study was to establish several internal positive controls for monitoring sensitivity and specificity during RT-PCR diagnosis. Several plasmids containing the conserved sequence of partial viral genome were constructed and RNAs were synthesized in vitro as internal positive controls for epidemiological surveillance. During July 2006 to March 2007, 160 collected samples including salmons (Salmo salar), rainbow trouts (Oncorhynchus mykiss), sweet fishes (Plecoglossus altivelis), and greater amberjacks (Seriola dumerili) from markets in Taichung area were screened. The results showed IPNV could be detected in rainbow trout and greater amberjacks, but neither IHNV nor VHSV could be found in all tested samples at this moment. Except for molecular biological diagnosis, the immunoserological assay is another alternative method for monitoring infectious fish diseases. However, the variation of antibodies among different fish species may dramatically affect the cross-species recognition of immunoglobulin. Therefore, a rabbit-anti-tilapia immunoglobulin antibody was produced and used to compare the difference of recognition between families of teleost using Western blotting method. These results may provide valuable information for understanding the immune system in different teleost fish.
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