對蝦類白點症病毒自然感染之研究-天然雌種草蝦感染狀況之分析

Abstract

白點症病毒（white spots syndrome associated virus, WSSV）是近年來造成養殖蝦大量死亡的重要病原體。目前白點症病毒的檢測可利用聚合酵素鏈反應（polymerase chain reaction, PCR）完成，為提昇檢測靈敏度，我們評估不同的檢測方法，結果發現以內部引子對進行二次PCR檢測反應的靈敏度較一次PCR檢測反應及一次PCR檢測反應加上南氏雜合反應或點墨雜合反應均要高，因此後續檢測均用二次PCR檢測反應進行。 依照二次 PCR 檢測 WSSV 於天然雌種草蝦體內分佈情況之結果，可將種蝦區分成三級，嚴重感染之種蝦除了肝胰腺及腹神經，幾乎全身各器官均遭受 WSSV 嚴重攻擊；輕微感染之種蝦則在第二次 PCR 檢測反應中才有器官出現陽性反應，其檢出率以血淋巴液為最高，其次為步足、泳足、鰓、卵巢及腹節肌肉，而以心臟及頭胸甲下表皮為最低；不帶有 WSSV 的種蝦在二次 PCR 檢測反應中均呈陰性反應。 利用一般光學染色觀察受 WSSV 嚴重感染種蝦之病理切片，可見許多器官中有脹大、染色深且均質化細胞核之病變細胞，其中以表皮細胞最為明顯，但有些器官則因組織之特性，無法明顯區別病變與正常細胞，為了進一步證實病變細胞是由 WSSV 造成，故以原位雜合反應進行檢測，各細胞核脹大的病變細胞，確實出現藍紫色之陽性反應訊號，而不易區別之病變細胞亦在原位雜合反應中呈陽性反應，可明顯區別於周圍之正常細胞，利用此法，可明顯發現卵巢組織中，卵泡細胞、卵原細胞及染色絲核仁期之卵細胞均遭受 WSSV 的感染，但以 H & E 染色觀察，則僅能見到卵泡細胞中具有脹大、染色深且均質化之細胞核。以穿透式電子顯微鏡觀察 WSSV 嚴重感染種蝦之卵巢，在被感染的卵泡細胞中， WSSV 出現在細胞核及細胞質中，而在被感染的卵原細胞中 WSSV 則常出現在細胞核中。 1995 年 7 月－1996 年 2 月間，引進天然雌種草蝦共 48 隻，其中 6 隻在第一次 WSSV PCR 檢測反應中即出現陽性反應，而在第二次 WSSV PCR 檢測反應中共有 30 隻出現陽性反應，總感染率高達62.5 %，然而各季節捕獲之種蝦之 WSSV 感染率依季節之不同而有所差異。 在實驗觀察期間，引進之種蝦並非全都產卵，成功產卵之種蝦均非嚴重感染者，一次 WSSV PCR 出現陽性反應之種蝦則在產卵之前均已死亡，然而在產卵後，幾乎所有的種蝦均在一次 WSSV PCR 檢測反應中出現陽性反應，白點則明顯可見，推測產卵為逆境因數（stress）導致白點病毒暴發。 分析種蝦親代與子代之間感染 WSSV 的情況，發現雖然 WSSV 輕微感染的種蝦其子代可能不帶 WSSV，然而未感染之種蝦其子代卻曾有 WSSV PCR 陽性反應的結果出現，關於 WSSV 是否會垂直感染，或者子代被外界環境病毒所感染，或因病毒沾於體表造成污染仍是有待研究之項目。

White spot syndrome associated virus (WSSV) is the causative agent of a disease which has recently been causing mass mortalities of cultured shrimps. The detection of WSSV in shrimps has been performed by polymerase chain reaction (PCR). In an attempt to establish a highly sensitive assay, we evaluated different methods for the detection of the presence of WSSV. The sensitivity of two-step PCR with nested primers was found to be higher than that of one-step PCR alone, one-step PCR with southern blot, and one-step PCR with dot blot, and so two-step PCR was used in the subsequent parts of this study. Three grades of infection were detected in wild-caught Penaeus monodon spawners: Except for the hepatopancreas and nerve tissues, WSSV was present in the other organs of the seriously infected spawners; in the lightly spawners only of the organs were positive in two-step WSSV PCR, in particular the hemolymph, followed by the pereiopods, pleopods, gills, overies and muscle, and finally the heart and integument; all the organs of the uninfected spawners were WSSV negative in two-step WSSV PCR. Using a light microscope to observe histopathological changes in seriously infected spawners, degenerated cells were found to be characterized by hypertrophied nuclei being stained homogeneously in some organs. This phenomenon was particularly distinct in the cuticular epidermis, while in other tissues, the degenerated cells were not so readily distinguishable because the nuclei were not so obviously hypertrophied and in fact remained close to their normal size. We used in situ hybridization to confirm that the cell degeneration was caused by WSSV. Each cell with a clearly hypertrophied nuclei gave a positive signal (i.e. a blue-purple precipitate) and degenerated cells with non-obviously hypertrophied nuclei were also positive , and were thus rendered distinguishable from normal uninfected cells. Using in situ hybridization, some of the follicle cells, oogonia and chromatin nucleolus oocytes in the ovary of the seriously infected spawners were found to be infected by WSSV. This compare with histological observation using H&E stain, in which only the follicle cells were characterized by hypertrophied nuclei. With TEM observation of WSSV seriously infected spawners, viral particles were readily observed in the nucleus and cytoplasm of the infected follicle cells, but while they were often seen in the nucleus of the infected oogonia , they were only seldom seen in the cytoplasm. From July 1995 to February 1996, 48 broodstock specimens of P. monodon were caught from the wild. Six of these were positive in one-step WSSV PCR and 30 were positive in two-step WSSV PCR, giving a rate of infection of 62.5%. This rate of WSSV infection was different in different seasons and different locations. Not all of the broodstock specimens spawned during the observation period (several days to several months). Of those that did spawn successfully, none were found to have been seriously infected; Conversely, specimens that were one-step WSSV PCR positive invariably died before spawning. After spawning, however, almost all of the specimens become one-step PCR positive with tiny white spots becoming visible on the carapace. This was probably due to the stress inherent in spawning. Conversely, although the offspring of lightly infected (i.e. two-step PCR positive) spawners were sometimes found to be two-step WSSV PCR negative. On the other hand, offspring of uninfected spawners were occasionally found to be two-step WSSV PCR positive. These contrary phenomenon occurred quite rarely and inconsistently but nonetheless will require consideration in further studies of WSSV in vertical transmission and environment contamination.