利用WSSV (White Spot Syndrome Virus) PCR檢測技術探討影響池蝦養成之原因--分析種蝦與子代間帶原相關性

Abstract

蝦類白點症病毒檢測系統的建立，對於輕微感染及未出現明顯病徵之帶原者均能準確檢測出。於台南地區的田間試驗顯示，在受檢之25個草蝦養殖池中，成功養殖至收成有13池，但在監測過程中僅有2池（11.8％）未曾測得白點症病毒聚合?鏈反應正反應，其餘收成池均曾偵測到輕微的白點症病毒帶原，比較這些養殖池的收成狀況，發現白點症病毒帶原的收成池其收成量變動很大，同時發現一但蝦池中有白點症病毒聚合?鏈反應出現一階正反應檢體，則池蝦於2-3天內即大量死亡，並且若有養殖池發病，則相鄰的養殖池亦容易受到影響。針對放養密度進行分析的結果顯示放養密度大於50 pcs/m2的養殖池，無一池養殖成功，而放養密度介於20-40 pcs/m2的養殖池具有較高的養成機會。此外，蝦苗的品質也深深地影響到收成的結果。因此除了環境因素外，蝦苗帶原情形也是左右養殖成敗之另一重要因素。由實驗室初步調查結果可知目前養殖蝦普遍是白點症病毒帶原蝦，而環境變動所造成的緊迫，又往往促使白點症病毒爆發，因此本論文亦針對溫度變化探討其對白點症病毒帶原蝦體所造成的影響。利用不同稀釋度病毒液浸泡感染的方式，以得到輕微感染白點症病毒之草蝦與斑節蝦，再施以不同溫度的緊迫，發現水溫24℃提高到29℃，蝦體於一天內即迅速死亡；溫度變化由15℃提高至24℃，於72小時內輕微感染草蝦的死亡率達100％，而輕微感染斑節蝦的死亡率僅約40％；而當溫度由15℃提高至29℃時，在輕微感染下草蝦或斑節蝦之死亡率均高達100％，顯示高溫對帶原草蝦的影響比帶原斑節蝦為大。實驗室先前調查發現天然捕獲的草蝦及斑節蝦均有很高比率的蝦白點症病毒帶原，本論文分析比較天然草蝦種蝦產前與產後的帶原情形由約67％提高至80％，因此種蝦生產過程亦是一緊迫因素，會造成蝦體中蝦類白點症病毒的大量複製。持續探討白點症病毒對種蝦產卵的影響，可知產前為嚴重感染的種蝦（即一階聚合?鏈反應為正反應）於捕獲後大部分於1-4天內死亡，僅有20％能成功產卵，但所產的卵均無法成功孵化。輕微感染及無帶原種蝦成功產卵的機率均很高（78％及81％）；無帶原之種蝦產後本身及與所產的卵均無蝦白點症病毒正反應出現；而輕微感染種蝦中，則發現產後有47％已成為一階聚合?鏈反應正反應的種蝦，且其受精卵大部分為二階聚合?鏈反應正反應（89％）；產後仍為輕微感染種蝦，所產的卵僅31％帶原，顯示帶原情況越輕微的種蝦，其蝦卵的病毒帶原檢出率也越低。本研究結果除可確認產卵之緊迫對蝦體的影響外，並且藉由此研究試圖建立一個種蝦的檢測模式，期望對於優質蝦苗的取得有所助益。

White spot syndrome virus (WSSV) has been detected in cultured and captured shrimps, crabs, pest crabs and other arthopods. In this study, healthy Marsupenaeus japonicus and Penaeus monodon juveniles were immersed in a sub-lethal dose of filtrate prepared from diseased or healthy shrimp. After 10 days, the sub-lethally infected and the healthy control shrimp were both subjected to different temperature conditions. The experimental groups of both shrimp species began to die of high temperature (29℃) treatment, while at low temperatures (15℃) the mortalities were lower. With an initial exposure to low temperatures for 24 h, after which the water temperature was increased to 24 or 29℃, P. monodon mortality reached 90%-100% at either temperature, while at 29℃, M. japonicus mortality reached 100% within a further 48 h, but only reached 40% at 24℃. The moribund experimentally infected shrimp under temperature shifts were all one-step WSSV PCR positive. These results demonstrate that temperature shifts alone can induce an outbreak of WSS disease in populations in which viral infection is only two-step WSSV positive. In order to discover the pattern of growth and survival of WSSV-infected shrimp on typical shrimp farms, as well as the interaction between the cultured shrimp and endemic WSSV carrier, a field survey of WSSV infection in cultured shrimp in 25 ponds in southern Taiwan at two-week intervals was carried out. Out of 25 ponds, 13 were successfully harvested. Only 2 out of 25 ponds were found to be 2-step PCR negative during the growth period. Even though the ponds were harvested in which WSSV-infected poatlarvae during growth period was detected, the survival rate, the yield, and the average body weight were variable. Mass mortality will predictably occur for any pond stocked with WSSV-infected postlarvae. At high density (>50pcs/m2), no ponds were harvest in our survey. Because the host range of WSSV is wide and hard to eliminate completely from the open culture system, it is important to monitor both the post-larve before stocking and larvae carefully during the growth period by WSSV diagnostic PCR. We re-tested stored (frozen) DNA samples in five independent PCR replicates and confirmed that equivocal tests results from a previous study on WSSV in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. The prevalence of WSSV among spawners was 67% before spawning and 80% after. This could demonstrate that spawning stress may trigger WSSV replication. Only 60% of brooders spawned successfully, and those heavily infected when captured mostly died within 1 to 4 days, but only 20% managed to spawn. All their egg batch sub-samples were 1-step PCR positive, and many failed to hatch. The spawning rates for lightly infected spawners and negative spawners were high (81 and 78%, respectively). None of the negative spawners became WSSV positive after spawning, and none gave egg samples positive for WSSV. A full 47% of lightly infected brooders became heavily infected after spawning, and almost all egg sample replicates (89%) tested 2-step PCR positive. They remained lightly infected brooders after spawning, and only 31% of their egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests, screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV free or lightly infected PL. This may be used to stock shrimp ponds under low stress rearing conditions.