太平洋白蝦感染桃拉症病毒後血球之分析

Abstract

以桃拉症病毒（Taura syndrome virus, TSV）人工感染白蝦（Litopenaeus vannamei）後，分析對照組與感染組的全血球計數（Total hemocyte counts, THC），各類型血球計數（Differential hemocyte counts, DHC），酚氧化酵素活性（Phenoloxidase activity, PO activity），轉醯胺酵素活性（Transglutaminase activity, TGase）及超氧化陰離子的產量（Superoxide anion production）來評估病毒性疾病對白蝦細胞性免疫系統的影響。研究結果顯示受人工感染之白蝦於感染後第二天出現體色、泳足等附肢變紅、活動力差、軟殼、部分抽出之血液呈現紅色及血液較不易凝固的現象。受桃拉症病毒人工感染之白蝦於感染後第三天死亡率已達83.3％，至第四天全數死亡。桃拉症病毒感染後第二天的感染組白蝦較之未處理組白蝦或假感染組白蝦其全血球計數由1.6至2.2×107 cells／ml下降至0.3×107 cells／ml，明顯的（P＜0.001）減少79至84％；雖然桃拉症病毒感染並未改變各類型血球之比例，但若以全血球計數濃度乘以各類型血球比例而換算成各類型血球數，則透明血球由對照組的1.3至1.9×107 cells／ml劇降為0.3×107 cells／ml（減少76至84％），顆粒血球則由0.13至0.31×107 cells／ml劇降為0.022×107 cells／ml（減少83至93％），顯示桃拉症病毒急性感染會造成各類型血球的全面崩解。血球內超氧化陰離子的產生經由NBT stain定量，結果發現因病毒感染的影響，產量顯著地提升（P＜0.01）；而再受免疫刺激物β-1，3-1，6-glucan刺激血球免疫反應的能力卻明顯地衰退（P＜0.05）。這些結果表示桃拉症病毒急性感染刺激了血球使其吞噬能力上升，但由於血球已處於高度活化的狀態，對免疫刺激物β-1，3-1，6-glucan的刺激變得較不敏感，有所謂免疫疲乏的現象。血球內轉醯胺酵素活性也受到桃拉症病毒感染的嚴重影響，其活性從未處理組及假感染組的21至32 Unit／mg降低至感染組的12 Unit／mg，顯示白蝦一旦受到桃拉症病毒急性感染後其凝血作用受到嚴重的破壞。酚氧化酵素活性由未處理組及假感染組的0.04至0.14 unit提高至感染組的0.29 unit（P＜0.05），顯示酚氧化酵素系統也因病毒感染而活化，被活化之酵素可從血球中被釋放至血漿中。 經由本研究之結果我們推論病毒降低TGase之活性（質的減少），導致宿主未能有效進行凝血作用，這將有利於病毒的擴散；另一方面宿主受到病毒感染之刺激會增強其抗病活性，這可由①O2-產量增加（暗示吞噬活性增強）；②酚氧化酵素活性之增強，而得以證明。在病毒一宿主攻防的過程中，各類型血球幾乎全面崩解，這可能由於①TSV感染造成的溶血或血球再生能力之失常；也可能是②宿主血球離開體腔浸潤至病毒感染的組織中，或③血球進行去顆粒、細胞溶解等作用來增強其抗病能力。 由於病毒感染後這些血淋巴指數會劇烈變化，如果蝦體採樣的數目夠多，或許這些血淋巴指數可以作為白蝦是否受到桃拉症病毒急性感染的指標。

To evaluate the effect of viral infection on the cellular immune responses in the Pacific white shrimp (Litopenaeus vannamei), we intramuscularly injected shrimp with Taura syndrome virus (TSV). Hemolymph obtained from TSV-infected, mock-infected and untreated shrimps were measured for the total and differential hemocyte counts (THC and DHC). The separated hemocytes were further analyzed quantitatively for the intrahemocytic superoxide anion and the enzyme activities of transglutaminase (TGase) and phenoloxidase (PO). High mortality was observed in the TSV-infected shrimps. It reached up to 83.3% on the third day and 100% on the fourth day post infection. Shrimps showed a pale reddish coloration on the out-looking, lethargic and soft shell on the second day post infection. Withdrawn hemolymph showed a red coloration and coagulated poorly in some infected shrimps. Hematological analysis showed that the THC decreased significantly (p<0.001) from 1.6?2.2×107 cells/ml in the untreated or mock-infected shrimps to 0.3×107 cells/ml in TSV-infected shrimps. In other words a 79 to 84% decrease. Although there is no difference observed in the ratios of differential hemocytes among the untreated, mock-infected and TSV-infected shrimps, the hyalinocytes decreased from 1.3?1.9 to 0.3×107 cells/ml (a 76 to 84% decrease) and granulocytes decreased from 0.13?0.31 to 0.022×107 cells/ml (a 83 to 93% decrease). These indicates an almost completely breakdown of hemocytes in an acute phase infection. The TGase activity in hemocyte lysate was found to decrease significantly from 21?32 to 12 units/mg. It suggests that an acute infection hampered the blood coagulation dramatically. A significant increase of intrahemocytic superoxide anion was generated in TSV-infected shrimps using a nitroblue tetrazolium staining. However, the ratio of superoxide anion generation decreased in these hemocytes further stimulated with β-glucan, which is well-known an shrimp immunostimulant. This result suggests that a viral infection did enhance the phagocytic activity of hemocytes. Nevertheless, a poor response would occur in hemocytes on a further stimulation since they had been in an exhaused state. Plasma PO activity was found to increase significantly (p<0.05) from 0.04?0.14 to 0.29 units. This suggests that intrahemocytic PO was activated by a TSV-infection and then released into plasma. Since TSV infection hampered blood coagulation, it would benefit the virus to disseminate through the shrimp body. Invaded viral particles may induce hemolysis or decrease the proliferation activity of hematopoietic tissue. In the other hands, the circulating hemocytes may infiltrate into and play their functions in the infected foci. Increased intrahemocytic superoxide generation and PO activity via hemocyte degranulation or cytolysis, shrimps would strengthen their disease resistant and struggle against the viral infection. During the process of host-virus interacton some hemolymph parameters were found to vary significantly. We, therefore, suggest they might be useful in the disease diagnostics.