高温应激对克氏原螯虾免疫酶活性及抗WSSV感染的影响
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摘要
为探明高温应激对克氏原螯虾(Procambarus clarkia)抗病力的影响,分别采用30℃和35℃水温进行高温应激试验,应激前和应激后3、6、12、24和48 h分别采集血淋巴样本,测定总超氧化物歧化酶(T-SOD)、多酚氧化酶(PPO)、溶菌酶(LYZ)、酸性磷酸酶(ACP)以及碱性磷酸酶(ALP)等免疫相关酶类指标。并在上述高温应激下开展白斑综合征病毒(WSSV)人工感染实验,同时设置25℃为阳性和阴性对照组,于感染后3、6、12、24、48、72 h取鳃组织定量测定WSSV在组织中的载量,同时记录克氏原螯虾的发病和死亡情况,统计累积死亡率。结果显示,克氏原螯虾血淋巴中的T-SOD与LYZ含量在高温应激后都出现了显著性降低,而PPO、ACP和ALP的变化差异不大。在35℃饲养条件下,WSSV在克氏原螯虾体内的增殖受到抑制,实验虾发病慢,累积死亡率相对较低,但在30℃饲养条件下,WSSV在克氏原螯虾体内的增殖较常温(25℃)下更活跃,发病和死亡更快。结果表明,高温应激能导致克氏原螯虾机体非特异性免疫力降低,30℃时能加快克氏原螯虾感染WSSV的发病进程,而35℃时因病毒复制被抑制而导致疾病的发展受阻。
The activity of some immune-related enzymes in hemolymph of Procambarus clarkia and its ability of anti-white spot syndrome virus(WSSV) infection were evaluated to reveal the effect of high temperature stress on the disease resistance of the crayfish. The high temperature stress(HTS) experiment was carried out at the temperature of 30 and 35 ℃, respectively. The hemolymph samples were sampled before stress and at 3, 6, 12, 24, 48 h posttemperature stress(PTS) which were used for T-SOD, PPO, LYZ, ACP, ALP testing. Meanwhile, WSSV artificial infection experiment was carried out under HTS, taking 25 ℃ as the positive and negative infection group, respectively. The gill tissue was sampled at 3, 6, 12, 24, 48 h and 72 h PTS for the quantitative analysis of the viral load, and the mortalities were recorded. The results showed that, T-SOD and LYZ decreased significantly after HTS, while the variety of PPO, ACP and ALP was not significantly changed. The propagation of WSSV in P. clarkia was inhibited at 35 ℃, the disease developed slowly and resulted a relevant low death rate. The propagation activity of WSSV at 30 ℃ was higher than that at 25 ℃, and the infected crayfish dead faster. It revealed that, HTS could decrease the nonspecific immunity of P. clarkia. The process of WSSV infection was accelerated at 30 ℃, while it was stayed because of the inhibition of viral replication at 35 ℃.
The activity of some immune-related enzymes in hemolymph of Procambarus clarkia and its ability of anti-white spot syndrome virus(WSSV) infection were evaluated to reveal the effect of high temperature stress on the disease resistance of the crayfish. The high temperature stress(HTS) experiment was carried out at the temperature of 30 and 35 ℃, respectively. The hemolymph samples were sampled before stress and at 3, 6, 12, 24, 48 h posttemperature stress(PTS) which were used for T-SOD, PPO, LYZ, ACP, ALP testing. Meanwhile, WSSV artificial infection experiment was carried out under HTS, taking 25 ℃ as the positive and negative infection group, respectively. The gill tissue was sampled at 3, 6, 12, 24, 48 h and 72 h PTS for the quantitative analysis of the viral load, and the mortalities were recorded. The results showed that, T-SOD and LYZ decreased significantly after HTS, while the variety of PPO, ACP and ALP was not significantly changed. The propagation of WSSV in P. clarkia was inhibited at 35 ℃, the disease developed slowly and resulted a relevant low death rate. The propagation activity of WSSV at 30 ℃ was higher than that at 25 ℃, and the infected crayfish dead faster. It revealed that, HTS could decrease the nonspecific immunity of P. clarkia. The process of WSSV infection was accelerated at 30 ℃, while it was stayed because of the inhibition of viral replication at 35 ℃.
引文
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[12] Hikima S,Hikima J,Rojtinnakorn J,et al.Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species [J].Gene,2003,316:187-195.
[13] Escobedo B C M,Alday-Sanz V,Wille M,et al.A review on the morphology,molecular characterization,morphogenesis and pathogenesis of white spot syndrome virus [J].J Fish Dis,2010,31(1):1-18.
[14]温周瑞.中国小龙虾白斑综合征的研究进展及防控对策[J].湖北农业科学,2017,(9):1601-1604.
[15]丁正峰,薛晖,夏爱军,等.白斑综合征病毒在养殖克氏原螯虾中感染流行研究[J].南京农业大学学报,2008,(4):129-133.
[16] Du H H,Li W F,Xu Z R,et al.Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii [J].Dis Aquat Org,2006,71(2):175-178.
[2]陈昌福,杨军,刘远高,等.克氏原螯虾暴发性疾病病原及其传播途径的初步研究[J].华中农业大学学报,2008,(6):763-767.
[3]徐进,魏开金,徐滨,等.克氏原螯虾对高温应激的生理学响应[J].淡水渔业,2017,47(6):9-13.
[4]黄倢,杨冰,陈爱平,等.GB/T 26831.2—2012 白斑综合征(WSD)诊断规程第2部分:套式PCR检测法[S].北京:国家质量监督检验检疫总局,2012.
[5]徐进,范玉顶,周勇,等.常规PCR与巢式PCR法快速检测克氏原螯虾白斑综合征病毒(WSSV)[J].淡水渔业,2008,38(6):52-54.
[6]张玲,唐小千,绳秀珍,等.实时荧光定量PCR检测克氏原螯虾体内白斑症病毒(WSSV)的动态变化[J].中国动物检疫,2015,(6):69-74.
[7]赵建华,杨德国,陈建武,等.鱼类应激生物学研究与应用[J].生命科学,2011,(4):394-401.
[8]刘波,谢骏,戈贤平,等.大黄蒽醌提取物对罗氏沼虾抗高温应激的影响[J].中山大学学报(自然科学版),2009,(6):109-114.
[9]董亮,何永志,王远亮,等.超氧化物歧化酶(SOD)的应用研究进展[J].中国农业科技导报,2013,(5):53-58.
[10] De L F M.Effects of antioxidants on immune system ageing [J].Eur J Clin Nutr,2002,56(3):5-8.
[11]朱毅菲.不同浓度氨氮、不同低pH突变对克氏原螯虾免疫功能的影响[D].武汉:华中农业大学,2006:65.
[12] Hikima S,Hikima J,Rojtinnakorn J,et al.Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species [J].Gene,2003,316:187-195.
[13] Escobedo B C M,Alday-Sanz V,Wille M,et al.A review on the morphology,molecular characterization,morphogenesis and pathogenesis of white spot syndrome virus [J].J Fish Dis,2010,31(1):1-18.
[14]温周瑞.中国小龙虾白斑综合征的研究进展及防控对策[J].湖北农业科学,2017,(9):1601-1604.
[15]丁正峰,薛晖,夏爱军,等.白斑综合征病毒在养殖克氏原螯虾中感染流行研究[J].南京农业大学学报,2008,(4):129-133.
[16] Du H H,Li W F,Xu Z R,et al.Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii [J].Dis Aquat Org,2006,71(2):175-178.