南台灣養殖石斑魚病毒性神經壞死症之研究

Abstract

病毒性神經壞死症（Viral Nervous Necrosis disease , VNN disease ) ，是一種新發現的魚類傳染性疾病，在世界各地造成多種海水魚的魚苗及幼魚大量死亡。近年來，在台灣具有高經濟價值的養殖石斑魚，也首次出現了類似病毒性神經壞死症的病例。本實驗即針對台灣南部養殖的石斑魚苗及幼魚，進行 VNN 疾病的診斷與病毒種類的鑑定，並探討病毒感染石斑魚之可能途徑，以及病毒在寄主體內對各組織的趨性。 光學顯微鏡的病理切片顯示，嚴重罹病之石斑魚苗其視網膜及腦部組織有明顯的空泡化現象；電子顯微鏡的觀察發現，在神經細胞的細胞質中有許多直徑約 20 nm 、不具外套膜的病毒顆粒。自病魚組織中抽取病毒核酸 RNA ，進行反轉錄反應成為 cDNA ，並以日本條紋?神經壞死症病毒（Striped jack nervous necrosis virus , SJNNV）的核酸引子，進行一次及二次 PCR 檢測, 確定罹病石斑魚成染了 NNV 。收集不同體長之魚苗及幼魚進行 PCR 檢測，發現依魚齡之不同，感染 NNV 的程度也有差異。用 PCR 檢測法篩檢不同來源之石斑魚卵，結果皆測到 NNV 病毒的核酸，顯示有垂直感染的可能性存在。檢測石斑養殖池中的混養小蝦，一次及二次 PCR 皆出現正反應，所以這些蝦子也可能是 NNV 的帶原者。檢測曾罹息 VNN 症之石斑魚苗而殘存下來、並漸長成無病癥的幼魚，發現這些幼魚帶有 NNV 病毒的基因。除了石斑魚之外，台灣養殖的鯇魚魚苗也曾測到 NNV 的核酸。 利用 DIG 標識之 NNV 核酸探針，對病魚組織切片進行原位雜交反應，結果顯示 NNV 在嚴重感染的魚苗中，可感染神經組織（視網膜、腦與脊髓）、內部器官（心臟、肝臟與消化道）、鰓以及骨骼肌。在較輕微感染的魚苗與幼魚體內，正反應出現的組織在不同魚體間有些差異，但主要是出現在視網膜、肝臟、鰓等部位。由此可知，石斑魚神經壞死症病毒，除了會破壞神經組織，還會侵犯其他組織。

Viral nervous necrosis (VNN) disease is a new worldwide disease that has caused high death rates of cultured marine fishes at larval and juvenile stage. From 1994, mass mortalities occurred repeatly among hatchery-reared larvae and juvenile of groupers in southern Taiwan. In this study, we examined the NNV infection of groupers by light microscopy, electron microscopy, polymerase chain reaction (PCR) and In Situ hybridization. The major clinical sign of diseased groupers is the abnormal swimming behavior. Light microscopy revealed vacuolation in brain and retina tissues, and numerous non-enveloped viral particles (20 nm in diameter) were found in the cytoplasm of nerve cells by electron microscopic observation. Two primer sets, (Fl, R3) and (F2, R3) designed for the stripped jack nervous necrosis virus (SJNNV) capsid protein gene, were used for the detection of NNV in groupers. Target fragment T2 of primer set (Fl, R3) and T4 of primer set (F2,R3) were separately derived after the PCR amplification of nucleic acids extracted from eggs, diseased larvae and juveniles, and the survival juveniles from the VNN diseased larvae. Dig-labeled T4 probe was used to study the tissue tropism of NNV by in situ hybridization. In heavily infected larvae, the positively reacted cells were not only shown in the nerve tissue (brain, retina, spinal cord), but also in the heart, liver, digestive tract, gill and muscle. In light infected groupers, the positively reacted tissues were different from fish to fish, but NNV were usually detected in the nerve tissue, gill and liver.