【作者】 朱斐；

【导师】 许梓荣；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 对虾白斑综合征病毒(WSSV)是对全球对虾养殖业危害最大,传播最广的病毒之一,尤其是对中国对虾养殖业带来了毁灭性的打击,至今已经造成了数十亿美元的损失。但目前尚无行之有效的防治手段,因此寻找预防和控制该病毒病一直是近年来的研究热点。许多研究结果表明WSSV囊膜蛋白可以诱导甲壳动物产生对WSSV的抵抗力,从而大幅提高WSSV攻毒后的成活率。多糖(如葡聚糖、壳聚糖、甲壳素)也被认为能提高甲壳动物的免疫力,并被广泛用于提高甲壳动物抗细菌性和病毒性疾病的研究。本研究从制作WSSV灭活制剂着手,经口服途径免疫克氏原螯虾,进行主动免疫保护克氏原螯虾抗WSSV感染的效果试验,并初步研究WSSV灭活制剂的保护作用机理。同时在螯虾饲料中添加不同比例(5mg/g、10mg/g、15mg/g)的三种多糖(壳聚糖、甲壳素、葡聚糖),研究多糖对克氏原螯虾抗WSSV感染的影响。主要研究内容和结果如下：1 BEI灭活WSSV制剂(BIW)的研制应用双乙烯亚胺(Binary Ethyleneimine, BEI)灭活WSSV,经螯虾肌肉注射试验证实白斑综合征病毒(WSSV)在2mM BEI,37℃下处理24h可以被完全灭活,注射试验表明死亡率为零,同时PCR检测也表明受试螯虾没有感染WSSV,结果表明BEI灭活WSSV制剂(BIW)对于螯虾是安全的。对BEI灭活WSSV的PCR检测证明了BEI的作用是破坏病毒的DNA分子,虽然扫描电镜观察发现WSSV粒子的囊膜表面有轻微的变化,但是SDS-PAGE分析说明结构蛋白的基本组成几乎没有改变,在灭活后WSSV的主要囊膜蛋白VP19. VP28. VP281、VP38A、VP51B和VP110等条带均清晰可辨,说明BEI的灭活作用对WSSV的囊膜蛋白基本无损坏影响,从而保持了其诱导甲壳动物抗白斑综合征的能力。2口服BIW诱导螯虾抗白斑综合征效果的研究结果表明口服BIW能显著提高克氏原螯虾攻毒后的相对存活率。不同次数口服免疫试验表明,随着口服BIW次数的增多,螯虾对WSSV的抵抗力也相应增强,但随着口服次数增多差异也越小,结果显示口服三次BIW就可以诱导螯虾对WSSV产生较强的抵抗力。口服三次BIW后,螯虾在第7、14、21、28天攻毒的相对存活率RPS分别为70%、53%、38%和20%,均显著高于对照组(P<0.05)。但是口服BIW螯虾的死亡率随着攻毒时间的推后也持续的升高,存活率随之下降,在14天之内口服BIW的螯虾可以保持50%以上的存活率。口服BIW的螯虾在口服结束后第一天血清酚氧化酶活力、超氧化物歧化酶活力和溶菌酶活力均有一定程度的提高,但差异不显著(P>0.05)；口服BIW结束后第七天四种酶活力水平有显著的提高(P<0.05),后逐步下降,至第4周基本与对照组无明显差异。PCR检测结果发现,病死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏六种组织的PCR结果均为WSSV阳性,而口服BIW螯虾的各组织检测结果均为阴性。光镜和电镜观察也证实了这一点,病死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏等组织均发生了不同程度的病变,而口服BIW螯虾的组织结构形态与健康螯虾无明显差异。3 BIW诱导螯虾抗白斑综合征机理的研究口服BIW的螯虾,在口服结束后第7天和第21天后攻毒存活率均显著高于对照组(P<0.05),相对存活率分别为60%和42%,而口服热灭活WSSV的相对存活率仅为10%和5%。注射BIW的螯虾在免疫结束第7天和21天后攻毒存活率均显著高于对照组,相对存活率分别为75%和60%。而注射热灭活WSSV的相对存活率仅为10%和5%。另外,电镜观察发现在BEI灭活WSSV的囊膜表面仅有轻微的变化,而热灭活WSSV的囊膜则几乎从核衣壳上脱落,说明BEI灭活对WSSV囊膜的影响要比热灭活小的多。SDS-PAGE也证明了这一点,BEI灭活WSSV可见VP19、VP28、VP281等囊膜蛋白条带都十分清晰,而热灭活WSSV则未见清晰的条带。对照攻毒试验说明WSSV灭活制剂所提供的保护作用是与灭活WSSV囊膜蛋白密切相关的。4多糖添加剂对克氏原螯虾抗WSSV影响的研究在虾饲料中添加不同比例(5mg/g、10mg/g、15 mg/g)的三种多糖(壳聚糖、甲壳素、葡聚糖),连续投喂20天以后口服攻毒。结果发现,与阳性对照相比,壳聚糖组5 mg/g、10 mg/g和15]mg/g三个浓度的相对存活率分别为25%、40%和25%；甲壳素组三个浓度的相对存活率分别为5%、15%和15%；葡聚糖组三个浓度的相对存活率分别为20%、25%和30%。其中以10mg／g壳聚糖组的保护作用最好,15mg／g葡聚糖组次之,相对存活率均显著高于对照组(P<0.05)。但同种多糖添加剂不同浓度的相对存活率之间却无显著差异(P>0.05)。酶活力测定发现,壳聚糖、甲壳素、葡聚糖对螯虾酶活力的提高作用以葡聚糖最好,壳聚糖次之,甲壳素则较差。而10 mg/g壳聚糖组的酶活力水平相对较高,与该组较高的存活率相一致。结果表明,上述四种酶活力水平可能与螯虾抗WSSV的能力有一定的相关性。研究发现,2mM双乙烯亚胺(BEI)对WSSV有确切的灭活效果,BEI灭活的WSSV经PCR检测和SDS-PAGE分析表明,BEI破坏了病毒的DNA,而对WSSV的囊膜蛋白无损坏影响,保持了其诱导甲壳动物抗白斑综合征的能力。三次可使螯虾对WSSV产生较强的抵抗力,在14天之内攻毒可以保持50%以上的存活率,且螯虾血清酚氧化酶活力、超氧化物歧化酶活力、溶菌酶活力均显著提高。通过光镜和电镜观察发现,口服BEI灭活WSSV攻毒后存活螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏六种组织均未感染WSSV,且PCR结果均为WSSV阴性。对照攻毒试验表明,WSSV灭活制剂所诱导的对白斑综合征的抵抗力是与灭活WSSV囊膜蛋白密切相关的。更多还原

【Abstract】 White spot syndrome virus (WSSV), which cause high mortality in many economic shrimp Aquaculture, and is widespread over the world, make the large economic losses to the shrimp farming industry in China. But no very effective treatment against WSSV was found, so more and more study focused on the adaptive immunity of crustacean. Polysaccharides like glucan, chitosan and chitin have been successfully used to enhance resistance of crustacean against bacterial and viral infections (Sung et al.1994; Itami et al.1998; Chang et al.1999; Chang et al.2003; Chotigeat et al.2004).This study was carried out to explore the possibility of protecting Procambarus clarkii from WSSV by oral vaccination with BEI-inactivated WSSV. And different concerntration of chitosan, chitin, glucan (5 mg/g、10 mg/g、15 mg/g) was tested to enhace the relative percent of survival in crayfish when they were challenged with WSSV.1 WSSV inactivation by BEIIM injection experiment showed that WSSV had been inactivated by 2mM BEI completely and the solution was safety for oral vaccination. The cumulative mortalities in the 37℃heated WSSV group indicated WSSV could keep the infectivity even exposed to 37℃over 24h. PCR detection and SDS-PAGE revealed that BEI only destroy the nucleic acid but not destroy envelope proteins in the process of inactivation. Intramuscular injection (IM) experiment showed that WSSV had been inactivated completely and the solution was safety for feeding.2 Protective effects of BEI-inactivated WSSV (BIW) in crayfish by oral deliveryThe potentiality of oral vaccination against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. Efficacy of BEI-inactivated WSSV was tested by oral vaccination followed by oral challenge of crayfish with WSSV. The crayfish fed with the food pelletes coated with BIW showed a resistance to WSSV on the 7th day post-vaccination (dpv). Calculated RPS values were 60%,70%and 75% for the vaccinated once, twice and thrice with BIW. Crayfish vaccinated thrice showed significantly higher RPS values compared to the control groups (P<0.05), the RPS values of 70%、53%、38%and 20%for crayfish challenged at 7,14,21 and 28 days after cessation of oral vaccination. The immunological parameters analyzed revealed that crayfish vaccinated with inactivated WSSV showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD) and lysozyme (LZM) compared to the control groups at 7 days post vaccination (dpv). But all of four enzyme activity decreased at 14,21 and 28 dpv. The result indicated that the resistance induced BEI-inactivated WSSV could persist for 20 days. Crayfish that survived from oral challenges were positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. This find provide a feasible way to control the prevalence of WSSV in prawn breeding through oral administration and perhaps has good effect to control other envelope virus.3 A pilot study on mechanism of protective effects conducted by BIWEfficacy of BIW was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish fed with BIW showed a better survival (P< 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared with the control. Calculated relative percent survival (RPS) values were 60%and 42%on the 7th and 21st dpv for BIW. The crayfish injected with BEI-inactivated WSSV showed a better survival (P< 0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared with the control. Calculated relative percent survival (RPS) values were 75%and 60% on the 7th and 21st dpv for BIW. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect crsyfish from WSSV. 4 Protective effects of Polysaccharide (chitosan, chitin andβ-glucan) in crayfishCrayfish, Procambarus clarkii, were fed with chitosan, chitin or p-glucan at 5,10 and 15mg/g for four weeks, and oral challenged with WSSV. The cumulative mortalities in the groups fed with chitosan at 10mg/g was significantly lower than the control (P<0.05) but the other groups was not. The RPS showed that 10mg/g chitosan and 15mg/gβ-glucan provided significantly better protection against WSSV compared to the control (P<0.05). PCR analysis demonstrated that the surviving crayfish were WSSV-negative. The immunological parameters analyzed revealed that the crayfish fed with chitosan and glucan showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD), peroxidase (POD)and lysozyme (LZM) compared to the control groups. The high levels of prophenoloxidase, superoxide dismutase, peroxidase and lysozyme may be responsible for enhancing resistance against WSSV in crayfish fed with chitosan or glucan.IM injection experiment had showed that WSSV had been inactivated by 2mM BEI completely and the solution was safety for oral vaccination. PCR detection and SDS-PAGE revealed that BEI only destroy the nucleic acid but not destroy envelope proteins in the process of inactivation. The immunological parameters analyzed revealed that crayfish vaccinated with inactivated WSSV showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD), and lysozyme (LZM) compared to the control groups at 7 days post vaccination (dpv). Crayfish that survived from oral challenges were positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. This study indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect crsyfish from WSSV.更多还原