台灣養殖及野生蟹類感染白點症病毒之研究：感染率及組織感染狀況之分析

Abstract

白點症病毒(White spot syndrome WSSV)是目前嚴重危害台灣以及其他亞洲地區養殖蝦類的重要病原體。罹病蝦之甲殼上普遍出現有白點，故名白點症(White spot syndrome)。目前針對對蝦類白點症病毒之檢測可利用二次聚合酵素鏈反應(2-step WSSV diagnostic polymerase chain reaction,PCR)來完成。近一年來，作者觀察到養殖鋸緣青蟳(Scylla serrata)第四步足(Ambulatory leg)及野生?斑蟳(Charybdis feriatus)頭胸甲(Carapace)出現白點症狀。利用WSSV DNA專一性之PCR引子對，針對各來源之養殖與野生蟹種，進行PCR檢測，發現養殖之鋸緣青蟳 ，野生蟹類(共計三屬五種)以及小型之三齒厚蟹皆受白點症病毒之感染。其中以紅星梭子蟹(Portunus sanguinolentus)之感染率最高，達88%，感染程度最嚴重。以HaeIII,HpaII, RsaI and Sau3AI等限制酵素對上述各蟹類之PCR正反應產物進行切割，其切割圖譜與白點症病蝦之PCR產物之切割圖譜相同。 進一步以WSSV PCR分析各種程度之感染蟹體各器官或組織之感染狀況，結果顯示，在嚴重感染之蟹體內，病毒呈系統性分佈，幾乎全身各組織與器官中均可偵測到其基因體之存在，呈一次PCR正反應之嚴重感染狀態。在輕微感染之蟹體，檢出率最高之器官為鰓、步足與血淋巴液，其次為胃、眼柄與顎腳，且皆於二次PCR始呈正反應。利用一般光學染色觀察受WSSV嚴重感染之組織與器官，發現都有細胞核染色深且脹大均質化的病變細胞存在，並以中胚層與外胚層演化之組織為主要侵犯器官。為了進一步證實細胞病變係由於WSSV感染所造成，以WSSV專一性探針進行原位雜合反應(in situ hybridization)，結果發現各細胞核脹大之病變細胞確實出現有藍紫色或棕黑色之正反應訊號。依照正反應訊號數目可確認各檢測器官之感染程度。分析結果顯示，鰓、胃與心臟為感染程度最嚴重之器官，而人為飼養的過程與輕微感染蟹體內病毒之增殖以及病程之發展相關。 穿透式電子顯微鏡觀察結果，發現許多具有封套(envelope)之桿狀病毒顆粒存在於嚴重感染之病變細胞中；利用氯化銫梯度離心法自嚴重感染之蟹體(Portunus sanguinolentus)組織純化所得之病毒顆粒，於電顯下負染觀察 ，病毒核蛋白鞘(nucleocapsid)呈桿形，長約325-350 nm，直徑約為60-70 nm，由核蛋白鞘次單元環繞相疊而呈特殊橫紋構造，橫紋寬度約為20nm，與病毒顆粒之縱軸垂直。所觀察到之病毒無論是大小或是核蛋白鞘之型態皆與純化自罹患白點症草蝦者極為相似。 除此之外，由養殖鋸緣青蟳苗之人工感染試驗結果，可證實對蝦類白點症病毒對於養殖蟹類具有病原性與致病力。即，蟳苗在感染後48 hr之鰓、胃及頭胸甲下表皮出現原位雜合之正反應訊號，感染後6天，全身各器官皆出現正反應訊號，且以全身之表皮為主要侵犯部位，與草蝦苗人工感染情況非常相似。 綜合以上之結果，顯示感染蟹類之白點症病毒與感染對蝦類之白點症病毒極有可能是相同或是親緣關係極為相近之病原體。

White spot syndrome virus (WSSV) is the causative agent of a disease which has been causing mass mortalities of cultured shrimps. The principle clinical sign of this disease is the presence of white spots on the exoskeleton, especially on the carapace of moribund shrimp. A two-step WSSV diagnostic polymerase chain reaction (PCR), based upon the specific primer sets, was routinely used to detect WSSV in shrimps. Recently, white spots had also been observed on the 4th pleopod (ambulatory leg) of cultured crabs (Scylla serrata) and the carapace of wild-caught portunid crabs (Charybdis feriatus). When DNA was extracted from crabs collected from three sources: cultured crabs, wild-caught portunid crabs and a non-cultured pest crab (Helice tridens), the PCR products showed the expected mobility, that is, they were coincident with the products amplified from the DNA prepared from WSSV-infected Penaeus monodon. The restriction profiles of these PCR products cleaved with HaeIII, HpaII, RsaI and Sau3AI were also the same. This is already strong evidence for the presence of WSSV in these various population. WSSV diagnostic PCR was also used to investigate the infection rate and the infected tissues of WSSV in wild-caught crabs. Almost all the tissues or organs of the seriously infected crabs showed WSSV positive after only one-step of amplification while tissues or organs of the lightly infected crabs were only positive in two-step WSSV PCR. The infection rate was particularly high in the gill, ambulatory leg and hemolymph, and only slightly less high in the stomach, eyestalk and maxilliped. The histopathological changes in the seriously infected tissues or organs were characterized by degenerated cells with hypertrophied nuclei. These nuclei were stained homogeneously by H & E. WSSV was confirmed as the causative agent by using in situ hybridization with a WSSV-specific probe. Various tissues from the mesoderm and ectoderm, such as connective tissue, epithelium, nervous tissue and muscle, could be infected by WSSV. Based on the number of the positive signals, the grades of infection could be distinguished. The gill, stomach and heart were the most seriously infected organs in crabs. Again, as with wild-caught shrimps which sometimes developed a patent infection after capture,the stress inherent in the cultured environment probably contributed to the wild-caught crabs, which were originally only lightly infected becoming seriously infected. In transmission electron micrographs of ultra-thin sections of infected tissues taken from infected wild-caught crabs (Portunus sanguinolentus), enveloped viral particles were readily observed in the nucleus of the infected cells. The viral agent was purified by CsCl gradient centrifugation. Negatively stained preparations showed the virus to be rod-shaped. The nucleocapsid measured 60-70 nm at its broadest point and was 325-350 nm long. The capsid was apparently composed of rings of subunits in a stacked series. The rings were aligned perpendicular to the longitudinal axis of the capsid. The thickness of the rings was very constant, usually being 20 nm. The morphological characteristics of this virus were thus very the similar to the viral agent purified from WSSV infected Penaeus monodon. Finally, healthy juvenile cultured crab, Scylla serrata, were exposed by immersion to epidermal filtrate from diseased P. monodon. Cumulative mortalities reached 40% within 2 weeks and WSSV was detected to be present in those experimentally infected crabs as early as 2 days post-infection. In situ hybridization with a WSSV-specific probe gave positive signals at 48 hr post-infection in the stomach, gill, cuticular epidermis and hepatopancreas. By 6 days post infection, almost all organs were heavily infected with WSSV. All of these results indicated that the causative agents of white spot syndrome of shrimps and crabs were in fact the same or closely related.