栉孔扇贝对急性病毒性坏死症病毒的生理和免疫应答

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	栉孔扇贝对急性病毒性坏死症病毒的生理和免疫应答
其他题名	The Physiological and Immune Response of Zhikong Scallop (Chlamys farreri) to
	唐保军
学位类型	博士
	2008-06-06
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	栉孔扇贝急性病毒性坏死症病毒生理免疫应答
摘要	栉孔扇贝（Chlamys farreri）是我国北方重要养殖扇贝种类，在海湾扇贝和虾夷扇贝引进以前，其年产量占我国扇贝总产量的80%。但自1997 年以来，我国北方大部分养殖区连续发生养殖栉孔扇贝大批死亡事件，严重影响和损害了栉孔扇贝养殖业的发展。我国栉孔扇贝大规模死亡是多种因素综合作用的结果。大致可分为生物因素与非生物因素：非生物的因素有夏季水温过高、养殖密度过高、养殖环境退化等。生物因素有流行性病原生物的侵害和扇贝种质的退化等。其中，急性病毒性坏死症（Acute Viral Necrobiotic Disease, AVND）病毒造成栉孔扇贝大规模死亡现象的研究已经开展。本研究通过生理学、免疫学技术和手段研究栉孔扇贝对急性病毒性坏死症病毒的生理和免疫应答，以期更好的了解栉孔扇贝对这一病毒的防御机制，为扇贝病害防治提供资料。本研究对不同温度下栉孔扇贝感染AVND 病毒后的耗氧率和排氨率进行了测定。结果显示，在17℃下，病毒组和注射生理盐水组栉孔扇贝的耗氧率逐渐升高，但两者无显著差异；方差分析显示，各组间排氨率的变化无显著差异。在25℃下，栉孔扇贝闭壳肌注射AVND 病毒和注射生理盐水组栉孔扇贝的耗氧率逐渐升高，在12 小时取得最大值，对照组则变化不大。方差分析显示，注射病毒组与注射生理盐水组和对照组之间有显著差异（P<0.05）。同时，对栉孔扇贝感染AVND 病毒的致病剂量进行了研究，在25℃水温下，栉孔扇贝肌肉注射感染AVND 病毒后，只有150 μl 组表现出明显患病症状并在第三天开始出现死亡现象，注射50、100 μl 组则无明显症状；17℃下栉孔扇贝感染AVND 病毒后无明显患病症状，表明AVND 病毒对栉孔扇贝的感染致病具有剂量和温度依赖性。对于25℃水温下栉孔扇贝感染AVND 病毒后血清中相关免疫酶类活力变化进行了测定。栉孔扇贝感染AVND 病毒后血清中SOD 的活性逐渐升高，在48小时达到最大值，方差分析显示不同时间点之间的SOD 活性有显著差异（P<0.05），在48 小时，病毒组和生理盐水组间的SOD 活性有显著差异（P<0.05），在其它时间点无显著差异。酸性磷酸酶（ACP）的活力在感染病毒2 小时后升高，在12 小时下降，然后又升高，在48 小时达到最大值。方差分析显示不同时间点之间的ACP 活性有显著差异（P<0.05），在2 小时和48 小时，病毒组和生理盐水组间的ACP 活性有显著差异（P<0.05）。碱性磷酸酶（AKP）的活力变化趋势与ACP 相同，在24 小时达到最大值。方差分析显示不同时间点之间的AKP 活性有显著差异（P<0.05）。在2 小时、12 小时、24 小时病毒组和生理盐水组间有显著差异（P<0.05）。栉孔扇贝酚氧化酶的活性最大值在48 小时，但与生理盐水对照组之间无显著差异。溶菌酶（Lysozyme）活性在病毒组和生理盐水对照组间无显著差异，病毒组最大值在2 小时取得，对照组在24 小时取得。结果表明栉孔扇贝通过升高或调节自身免疫相关蛋白酶类合成应对AVND 病毒侵染。采用荧光实时定量PCR 技术，对栉孔扇贝感染AVND 病毒后免疫相关基因的时空表达规律进行了研究。水温17℃下，栉孔扇贝肌肉注射感染AVND 病毒后超氧化物歧化酶（SOD）基因mRNA 的表达量逐渐上升，在注射后24 小时达到最大值，约为空白对照组的1.8 倍，方差分析显示，病毒组SOD 的表达量不同时间点之间有显著变化（P<0.05）；但与生理盐水对照组相比较，病毒组SOD表达量无显著差异。在水温25℃下SOD 基因mRNA 的表达量逐渐上升，在注射后6 小时达到最大值，约为对照组的1.5 倍，空白对照组的2.2 倍。病毒组SOD的表达量不同时间点之间有显著差异（P<0.05）；在感染后2、6、12、24 小时病毒组SOD 表达量比对照组有显著升高（P<0.05）。溶菌酶基因在肝胰脏中的表达升高，在6 小时达到最大值，约为生理盐水对照组的1.5 倍，空白对照组的2.7倍，在48 小时取得最小值（低于空白对照组）。病毒感染后不同时间之间溶菌酶基因表达有显著差异（P<0.05），感染后6、24 和48 小时病毒组溶菌酶基因表达比对照组有显著升高（P<0.05）。在AVND 病毒感染后6 小时，在肝胰脏、性腺、肌肉、鳃中溶菌酶mRNA 量急剧增加，分别达到了空白对照组的4.7 倍、3.8 倍、13.43 倍和25.15 倍。方差分析显示在不同组织部位的表达有显著差异（P<0.05）。表明AVND 病毒感染后栉孔扇贝免疫相关基因的表达具有时序性和组织部位特异性。
其他摘要	The Zhikong scallop (Chlamys farreri) is the important farming scallop specie in northern China. Before the bay scallop (Argopecten irradians) and Japanese scallop (Patinopecten yessoensis) were introduced into China, Zhikong scallop accounted for 80% of annual scallop output. However, Successive heavy mortalities in most farming areas in northern China since 1997 have seriously affected and damaged the aquaculture development of C. farreri. The massive mortality of C. farreri is caused by a variety of combined factors, including non-biological factors and biological factors. The non-biological factors include too high summer temperature, high stocking density, environmental degradation, and so on. Biological factors includes the pandemic biological pathogens and scallop germplasm degradation. The studies about C. farreri massive mortality caused by Acute Viral Necrobiotic Disease (AVND) virus have been carried out. This research studied the physiological and immune response of C. farreri to AVND virus by physiological and immunological technologies and means, try to help better understand the defensive mechanism of C. scallop to AVND virus and provide information for disease prevention and treatment. The oxygen consumption rate and ammonia excretion rate of Zhikong scallop (C. farreri) infected by AVND virus under different temperatures were measured. The results showed that at 17℃ the oxygen consumption rate and ammonia excretion rate of the groups of C. farreri with virus and saline injection gradually increased, but there was no significant difference between both groups. The control group had no significant change; variance analysis showed that there was no significant difference among three groups. At 25 ℃, the oxygen consumption rate of C. farreri with virus and saline injection gradually increased, and acquired maximum at 12 hours after injection. The control group had no significant change. Variance analysis showed that there was significant difference among the virus injection group, saline injection group and control (P <0.05). The results showed that the infection of AVND virus on C. farreri is dose-and temperature-dependant. The effects of AVND virus on the activity of immune-related enzymes in the serum of Zhikong scallop (C. farreri) under 25℃ were studied. The SOD activity in serum of C. farreri rose gradually in serum and reached the maximum at 48 hours after injection of AVND virus. The results of analysis of variance showed that there were significant differences among the time points (P<0.05). At 48 hours after infection, there is significant difference between the virus group and saline control group (P<0.05) and no significant difference at other time points. Acid phosphatase (ACP) activity rises in 2 hours after injection and declined in 12 hours, then rose and reached the maximum in 48 hours. The analysis of variance showed that there were significant differences among different time points. The virus group and saline control group has significant difference in 2 hours and 48hours (P<0.05). The change of alkaline phosphatase (AKP) had same trend with ACP. In 24 hours after infection, the AKP activity reached maximum, and there were significant differences among time points. The analysis of variance showed that there were significant differences between virus group and saline control group in 2, 12 and 48 hours (P<0.05). The phenoloxidase activity reached maximum in 48 hours after infection and there was no significant difference with saline control group. There was no significant difference of lysozyme activity between virus group and saline control group. The virus group scallop reached maximum in 2 hours and saline control group in 24 hours.The results indicated that C. farreri could increase or regulate protein synthesis immune-related enzymes to deal with the AVND virus infection. The expression of immune-related genes of Zhikong scallop (C. farreri) infected AVND virus was studied using fluorescent real-time PCR technology. The mRNA expression of SOD gene in serum of C. farreri gradually increased after injection of AVND virus at 17℃ and reached maximum in 24 hours after injection, about 1.8 times of blank control. There were significant differences among different time points (P<0.05), but no between the virus group and saline control group. The mRNA expression of SOD gene rose gradually in 25℃ water temperature and reached maximum in 6 hours after infection, about 1.5 times of blank control and 2.2 times of saline control group. There were significant differences among the time points and between the virus group and saline control group in 2, 6, 12, 24 hours after infection (P<0.05). After AVND virus infection, the Lysozyme gene expression in hepatopancreas of C. farreri increased, and reached maximum in 6 hours, about 1.5 times of saline control and 2.7 times of blank control group, and acquired minimum value in 48 hours after infection (lower than blank control group). There were significant differences of lysozyme gene expression among the times points after infection (P<0.05). The gene expression significantly increased in 6, 24, 48 hours after infection compared with saline control group (P<0.05). In 6 hours after AVND virus infection, the mRNA expression of lysozyme gene in hepatopancreas, gonad, muscle and gill sharply increased, and reached 4.7 times, 3.8 times, 13.43 times and 25.15 times of blank control group respectively. The analysis of variance showed that there were significant differences among different tissues of C. farreri (P<0.05). The results indicated that immune-related gene expression of C. farreri after AVND virus infection has time course dependence and tissue specificity.
页数	96
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/399
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	唐保军. 栉孔扇贝对急性病毒性坏死症病毒的生理和免疫应答[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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