【作者】 闫冬春；

【导师】 董双林； 黄倢；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2004， 博士

【摘要】 白斑综合症病毒(white spot syndrome virus, WSSV)是迄今为止危害最为严重的一种对虾病毒。自1992年暴发以来，已在世界范围内传播开来，每年给对虾养殖业造成巨大经济损失，至今仍未得到完全控制，成为目前对虾养殖业可持续发展的主要障碍之一。十年来，人们对WSSV的传播途径进行了大量研究，确认了幼苗带病毒和水中浮游动物、池塘堤坡动物(如蟹类)和鸟粪携带病毒等的主要传播途径。近些年来，人们在生产上除采用养殖抗病力强的种类(如凡纳滨对虾)外，还采用培育健康虾苗、限制一些鲜活饵料的使用、养殖用水消毒等措施，以切断WSSV的传播途径，使得养殖生产得到了一些恢复。但是，每年全国发病的面积比例仍然很大，特别是北方地区的损失仍然相当严重。其基本原因之一就是人们对WSSV传播的某些途径仍不十分清楚，致使某个(些)传播途径仍未被有效地切断。 本研究采用灵敏、可靠的检测技术，结合具体的养殖实践，调查了WSSV在池塘养殖系统中的分布情况，并对池塘底泥中的轮虫休眠卵在传播WSSV中的作用和机制进行了初步研究，以期为今后有效杀灭WSSV提供理论依据。本文所得主要结果如下： 设计了2对引物用于检测对虾白斑综合症病毒(WSSV)，外引物用于PCR扩增，内引物用于合成探针进行斑点杂交。结果表明，外引物PCR-电泳的检出极限为1pg WSSV DNA；PCR产物经内探针斑点杂交，可检出10fg的WSSV DNA；单纯的内探针斑点杂交只能检测出1ng以上的WSSV DNA。PCR-斑点杂交的检测灵敏度比PCR-电泳高2个数量级，比单纯斑点杂交高5个数量级。Southern杂交表明，PCR-斑点杂交检测WSSV特异、可靠，该方法可用于痕量WSSV的检测。白斑综合症病毒(WSSV)的PCR检测条件优化及浮游动物中间宿主的调查研究 PCR检测对虾白斑综合症病毒过程中用尿嗯睫一DNA糖基酶(UNG)防遗留污染。使用UNG时，该对引物的适宜dUTP和M扩浓度分别为0.组耐和2.OmM;UNG存在时，PCR检测WSSV DNA的最低量为1 pg，UNG的使用使PCR的检测灵敏度降低了一个数量级;进行正常PCR扩增前，0.SU UNG可消除至少IOng含dU的wSSV DNA的PCR扩增产物。 PCR检测攻毒鳌虾鳃样品时出现阴性，而经10一106倍稀释后的样品却呈现阳性。结合核酸探针斑点杂交及组织切片结果，判断PCR出现了假阴性，并推算出PCR能够成功扩增的引物与模板的比例范围为2 .43 Xl扩一2.43 X10，。。探讨了PCR出现假阴性的原因及防范措施。 于2002年采用PCR一核酸探针斑点杂交法检测了乳山对虾养殖场1000余份样品携带白斑综合症病毒(WSSV)的情况，结果显示:639例对虾样品阳性检出率为26.6%;77例蟹类样品阳性检出率为18.2%;266例浮游动物样品阳性检出率为38.3%，3月份至9月份浮游动物阳性率呈下降趋势，消毒后水体中浮游动物的阳性率仍很高;30例螺、贝样品及22例抽滤海水样品检测均为阴性;204例底泥样品中，阳性检出率为17.6%。 2002年在乳山养殖场采用两种围栏养殖模式养殖了中国明对虾和凡纳滨对虾，并对其防病效果进行了比较。结果表明，中国明对虾在养殖过程中WSSv阳性率逐渐增高，浮游动物WSSV阳性率一直很高;凡纳滨对虾在养殖过程中wSSV阳性率有下降趋势，浮游动物wSSV阳性率较低;布围隔的防病效果好于网围隔。 采用PCR一核酸探针斑点杂交法检测了对虾养殖池塘底泥中轮虫休眠卵及孵出轮虫携带WSSV的情况。表面消毒后的检测结果表明，WSSV极有可能存在于休眠卵的内部，轮虫休眠卵在对虾养殖池塘隔冬传播WSSv中可能起着重要作用。 对采于海水养殖池塘的壶状臂尾轮虫休眠卵及孵出轮虫进行了电镜观察。结果表明，轮虫休眠卵表面有明显的、不规则的褶皱突起，月_附着一些污染物。轮虫细胞内线粒体、内质网密集。wSSv浸浴攻毒后轮虫出现细胞核及核仁膨大。 初步研究了池塘轮虫休眠卵及其孵出轮虫传播白斑综合症病毒(WSSV)的白斑综合症病毒(Wssv)的PCR检测条件优化及浮游动物中间宿主的调查研究可能性。以轮虫及其休眠卵粗提液注射淡水克氏原鳌虾，结果注射轮虫休眠卵粗提液组未出现阳性，注射轮虫粗提液组出现1例阳性，表明轮虫有可能传播WSSV。更多还原

【Abstract】 White spot syndrome virus (WSSV) was the most destructive shrimp virus so far. It had spread all over the world since occurred in 1992. This virus brought great economic losses in world shrimp farming every year and has not been controlled completely till today, it was currently the main disease threat to sustainable shrimp farming. In past 10 years, much work has been done about WSSV transmission modes and several routes of infection have been found. These include not only carrier shrimp larvae, other crustaceans (e.g., crabs) and plankton in and around culture ponds, but also bird feces. Shrimp farmers have taken care to culture WSSV-resistant species (e.g., Litopenaeus vannamei), to breed WSSV-free post larvae, to restrict live food inputs, to disinfect inlet water in order to limit horizontal and vertical transmission of WSSV. Despite implementation of measures to reduce transmission, the occurrence of WSSV outbreaks was still very high in China’s mainland, especially in more northernly regions. This may be due to the fact that some major transmission routes have not yet been discovered.WSSV distribution in shrimp pond system was studied combined with shrimp farming practice by sensitive, crediable detection techniques. Effect and mechanism of rotifer resting eggs in shrimp pond sediments in transmitting WSSV were studied primarily. These study could offer theory information to prevent WSSV transmission effectively. The primary results of the study were as follows:Two pairs of primer were designed for white spot syndrome virus detection. The outer one was for polymerase chain reaction (PCR), the inner one was for producing probe. The results showed that the detection limit of outer primers PCR-electrophorisis was Ipg WSSV DNA, which was lOfg WSSV DNA in PCR-dot blot hybridization; Single inner primers dot blot hybridization could only detect more than Ing WSSV DNA. The sensitivity of PCR-dot blot hybridization was 102 higher than PCR-electrophorisis, 105 higher than single dot blothybridization. The specificity of PCR- dot blot hybridization was confirmed by Southern hybridization. The results showed that PCR-dot blot hybridization was suit for trace WSSV detection.Uracil-DNA glycolsylase (UNO) was used to control carry-over contamination in WSSV DNA detection by PCR . Optimal dUTP and Mg2+ concentrations for this pair of primers were 0.4mM and 2.0mM respectively when UNG was present, the detection limit of WSSV DNA by PCR amplification was Ipg on this condition. UNG made this PCR sensitivity degrade one-tenth. At least lOng WSSV DNA containing dU could be abolished by 0.5U UNG prior to the normal PCR amplification .The negative result was ocurred in WSSV injected Cambarus proclarkii detection with PCR. But, positive results were occurred when the DNA sample was diluted to 10?106 times. Together with the results of DNA probe dot blot hybridization and histopathological section, PCR false negative result could be concluded. The ratio of primer to template for sceessful amplification was 2.43 x]05 ?2.43xl010; The reason and prevent methods for PCR false negative results werediscussed.In 2002, more than 1 000 samples were detected by PCR-DNA probe dot blot hybridization for white spot syndrome virus (WSSV) carrying status. The results showed that 26.6% was WSSV positive in 639 slirimp samples, 18.2% was positive in 77 crab samples, respectively. The WSSV positive ratio was 38.3% in 266 zooplankton samples, and the positive ratio was declined from March to September. Noticeable, the zooplankton positive ratio was still very high after water disinfection. All the 30 snail and shellfish sampbs and 22 filtered seawater samples were WSSV negative. The WSSV positive ratio was 17.6% in 204 mud samples.In 2002, two pen-closing culturing natterns were used to culture Fenneropenaeus chinesis and Litopenaeus vannamei in Rushan shrimp farm. The effects of white spot syndrome prevention were compared. The results showed that the WSSV positive ratio was increased in Fenneropenaeus chinesis culturing process, a更多还原