白蝦感染白斑病爆發臨界值之評估

Abstract

本實驗的目的，希望藉由Capture-ELISA的方式監控蝦類血淋巴中病毒的變化量。首先製備標準曲線，先利用純化後的WSSV免疫小鼠，製備單源抗體，當作Capture antibody。然後，以純化後的WSSV免疫大鼠，製備多源抗體，當作binding antibody。以純化過的WSSV為抗原製備標準曲線。得知WSSV蛋白質濃度變化和吸光值之間呈現線性關係，迴歸方程式為Y＝0.0001x＋0.0332 R2＝0.9106。 接著，進行感染實驗，分別以肌肉注射、餵食、浸泡三種方式感染健康白蝦。每日抽取其血淋巴進行Capture-ELISA分析。將得到的吸光值，利用標準曲線換算成病毒濃度。將每日的病毒濃度變化配合累積死亡率發現，以肌肉注射、餵食、浸泡三種方式感染健康白蝦，都可以在最大累積死亡率出現之前，觀察到病毒變化量的高峰。由這三種感染方式，也可以找到造成大量死亡的病毒濃度致死點（10.4ng WSSV／μl hemolymph－16ng WSSV／μl hemolymph）。也就是說，經由每日監測病毒的變化情形，可以推估其大量死亡的時間點。 將來期望將此Capture-ELISA分析系統推廣到養殖場作實際的應用。由蝦子的血淋巴監控其健康情形，推估其大量死亡的時間點。以方便養殖業者做出最好的經濟策略。

The goal of this study is to establish a Capture enzyme-linked immunosorbent assay (ELISA) system for the quantification assay of the whitespot syndrome virus (WSSV). This system is designed as that a monoclonal antibody recognizing a major viral envelope protein, VP 28, was used as the primary antibody and the polyclonal antibodies affinity-purified from WSSV-immunized rats sera as the secondary antibody. First, a standard curve was plotted from purified WSSV containing known amounts protein verse the optical density read from capture ELISA. This plot is linear relation between 0.33 and 42.40 ng WSSV/μl hemolymph. And a regression equation of Y=0.0001X + 0.0332, R2=0.9106 was thus induced. The capture ELISA was subsequently applied to monitor the concentration changes of WSSV proteins in infected shrimp hemolymph. Three infection trials were proceeded in healthy adult penaeus vannamei using muscle injection, feeding and immersion. The hemolymph was daily collected from individual shrimp and analyzed by the Capture-ELISA system. The concentrations of WSSV proteins within each sample were calculated from the standard curve. Additionally, the cumulative mortality was daily observed from the other replicate of shrimp. Combining the daily changes of WSSV concentration with the cumulative mortality, a common phenomenon was observed that the peak of WSSV concentration was reached 1 day before the appearance of the highest cumulative mortality amongst the three infection trials. The range of WSSV protein concentration (10.4 to 16 ng WSSV/μl hemolymph) was suggested to be a critical point that could indicate the outbreak of the serve disease and the massive mortality of infected shrimp. The Capture-ELISA system will be fourthly applied to field cultures. The condition of WSSV infection within culture shrimp populations will be monitored using this system. And a better culture strategy could be adopted by the farmers.