卤虫携带白斑综合征病毒的研究

卤虫是是,蟹苗种培育的关键动物性饵料,查清其能否携带白斑综合征病毒,对培育健康苗种至关重要。2002年7月至10月,自盐田中取野生卤虫,经暂养后选20尾抱卵雌虫,每尾置1个烧杯中,投喂单胞藻,产卵后取出亲体;20d后,每个烧杯中取5尾子一代个体,利用巢式体外DNA扩增技术对亲体和子一代成体进行WSSV病毒检测,检测结果显示：卤虫中可扩增出特异性的,与引物设计吻合的DNA片段;DNA测序分析表明,扩增片段的DNA序列与白斑综合征病毒序列一致;野生卤虫携带白斑综合征病毒的阳性率为58%,实验条件下野生卤虫后代的阳性率为43%,同一家系中,亲代与子代携带病毒的情况不完全一致,不能确实白斑综合征病毒可否通过繁殖,在卤虫世代间进行垂直传播,研究结果表明：卤虫很可能是白斑综合征病毒的携带者,苗种培育和养殖期间投喂卤虫可能造成虾,蟹感染白斑综合征病毒,卤虫检疫是培育健康苗种的关键措施。     

对虾6种病毒多重PCR检测方法的建立

本研究针对养殖对虾6种病毒,包括白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、肝胰腺细小病毒(HPV)、桃拉综合征病毒(TSV)、对虾杆状病毒(BP)和传染性肌肉坏死病毒(IMNV),选择各自的基因分别设计特异性引物和探针,首先进行了单一病毒的PCR验证,在此基础上建立了同时特异性检测6种对虾病毒的多重PCR检测体系。对反应条件进行优化并进行特异性和灵敏度的验证。50μl反应体系,Mg2+的最佳浓度为5mmol/L,ExTaq酶最佳用量为3.75U,反应程序中最佳退火温度为55.5℃。6种病毒之间以及与对虾基因组都存在很好的特异性。最终经试验验证,该系统的检测灵敏度对WSSV可达104拷贝,IHHNV可达102拷贝,HPV可达104拷贝,TSV可达103拷贝,BP可达105拷贝,IMNV可达105拷贝。虽然该多重PCR方法灵敏度不如单一的PCR检测高,但是通过实际样品检测验证了该方法省时、消耗较少,又不失准确性,在实际应用中具有可靠性和应用价值。     

WSSV口服卵黄抗体的制备及在螯虾中的应用

将提纯的WSSV接种于蛋鸡后获得了高效价的IgY(卵黄抗体),与一定比例的保护剂混合后,瞬间喷雾干燥制成干粉其效价基本维持不变。在以螯虾为实验模型的攻毒及治疗试验结果表明:饲料中0.1%的IgY含量能在一定程度上延缓螯虾死亡时间,而0.2%的IgY含量则能够使螯虾的最终死亡率从100%降至30%左右。这表明WSSV特异性IgY制剂是安全有效的抗WSSV感染的的新型生物制剂。     

对虾白斑综合症病毒PCR检测体系的优化研究

通过改进WSSVPCR模板制备方法、引物的筛选、PCR反应的优化,建立稳定的WSSVPCR检测体系。结果表明,该体系具有稳定、快速、灵敏等特点,可用于生产中对WSSV的检测。     

Experimental white spot syndrome virus challenge of juvenile Litopenaeus vannamei (Boone) at different salinities

In recent years, the shrimp industry has turned to inland freshwater culture as one method to avoid problems such as the introduction of possible vectors of viral pathogens into seawater ponds. Our experiments evaluated susceptibility to white spot syndrome virus (WSSV) in Litopenaeus vannamei held under different salinity regimens. Juvenile L. vannamei that were conditioned at salinities of 35, 25, 15, 5 and 2 g L⁻¹ were challenged with WSSV. In order to assess the severity of white spot disease, histological analysis and nested polymerase chain reaction (PCR) tests were carried out on the challenged shrimp every 4 h after 48 h post challenge. The results indicated that significantly more severe infections resulted at 15‰ than at other salinities. Mortality could not be compared due to the sampling design and because severe WSSV infections occurred in all test groups such that few shrimp remained alive in each challenged group at the end of the test. Despite this, the results suggest that salinity may affect the course and outcome of WSSV infections.     

用动物模型检验消毒剂对对虾白斑综合征病毒的灭活效果

以克氏原螯虾为动物模型，检验消毒剂对虾白斑综合征病毒（WSSV）的灭活效果。将病毒液分别与不同浓度的消毒剂37℃作用2h，再感染螯虾，结果显示，40μg/ml和60μg/ml的特种双链季铵盐络合碘能有效灭活病毒，显著降低接种螯虾的死亡率，80μg/ml的该药能完全灭活病毒。相同浓度的该药与二溴海因，溴氯海因，有机氯类相比，所接种螯虾的平均死亡时间前者较后者长0．9－1．2d，显示了较好的消毒效果。     

对虾白斑综合征病毒在螯虾动物模型的感染特性

应用克氏原螯虾作为动物模型研究对虾白斑综合征病毒（ＷＳＳＶ）的感染增殖特性，涉及感染温度，感染途径，继发感染，半数致死量（ＬＤ５０），免疫保护及保存期等，结果显示，２２－２５℃时，接种ＷＳＳＶ的螯虾一般于２－７d内死亡，温度升高对病毒增殖影响不显著，３０－３２℃时，平均死亡时间２－６d，温度降低对病毒增殖影响较显著，１５－１９℃时，接种螯虾平均２－１０d内死亡，８－１０℃时，平均死亡时间３－６d，感染途径分别用腹节肌肉，腹节皮下洲射及口服，均能使螯虾感染发病，而浸泡方式不能使螯虾发病，细菌分离和细菌定量结果表明，寄考于螯虾心脏，肝胰腺内的阴沟肠杆菌在感染后期大量增殖，菌量分别是正常螯虾的２５和３０倍，形成继发感染，用螯虾心脏，肝胰腺内的阴肠杆菌在感染后期大量增殖，菌量分别是政党螯虾的２５和３０倍，形成继发梁。用螯虾测定ＷＳＳＶ的ＬＤ５０为１０^-6.5.mL^-1种毒液，将病毒５６℃，３０min灭活后免疫螯虾，不能使螯虾形成免疫保护，ＷＳＳＶ匀浆液－３０℃冻存１年后失活，而－３０℃冻存于螯虾体内ＷＳＳＶ保存１年后仍有活力。     

白斑综合征病毒感染梭子蟹试验及组织病理学观察

利用对虾白斑综合征病毒WSSV，采用注射和投喂方式，对养殖三疣梭子蟹（Portunus trituberculatus）进行人工感染，以确定WSSV对养殖梭子蟹的易感性和可能存在的潜在危害。结果发现，在水温24—25℃条件下，病毒注射感染组全部发病，10-16d全部死亡；投喂组死亡率33％；对照组未出现死亡。采用荧光定量PCR对各试验组梭子蟹的肌肉和肝胰腺混合物进行WSSV检测，发现注射感染组阳性检出率为100％，均为强阳性；投喂组阳性检出率为50％；对照组的阳性检出率均为O。光镜下可观察到感染梭子蟹的肝胰腺、肌肉组织、鳃组织的靶细胞核肿大、变形，并出现以坏死为主的变质性病变；电镜下病变组织细胞中可见形态、大小与WSSV一致的病毒粒子。由此表明：对虾WSSV可以感染三疣梭子蟹，并表现出明显的病理症状，感染与病毒的数量以及梭子蟹自身的体质、环境条件密切相关。     

病原感染条件下中国对虾二倍体和三倍体血液学变化

以热休克诱导获得中国对虾三倍体，通过口服或注射的方式对二倍体和三倍体中国对虾进行了白斑综合征病毒(WSSV)和鳗弧菌感染实验，研究了病原感染前后二倍体和三倍体中国对虾血液学的变化并首次报道了三倍体中国对虾血细胞的组成。实验结果表明：三倍体和二倍体中国对虾均对WSSV敏感，三倍体未显示出明显的抗性。WSSV感染4d后，对虾外周循环系统的血细胞数量明显减少，三倍体和二倍体对虾血细胞数量均降为对照组的10％左右。对虾血细胞组成发生变化，大颗粒细胞明显减少，小颗粒细胞相应增加，而透明细胞所占比例基本不变；大颗粒细胞和小颗粒细胞内颗粒物质均有所减少，出现空泡或细胞外突起等异常结构。注射鳗弧菌感染后两种对虾的血细胞数量均有所减少，血细胞数量的减少与弧菌的注射量呈正相关关系：三倍体中国对虾血细胞组成与二倍体相似，但其密度仅为二倍体的40％左右：本研究结果为三倍体对虾抗逆特性的研究提供了一定依据和参考。     

Exposure to probiotics and β-1,3/1,6-glucans in larviculture modifies the immune response of Penaeus vannamei juveniles and both the survival to White Spot Syndrome Virus challenge and pond culture

The effect of the inclusion of probiotics (Vibrio alginolyticus) and β-1,3/1,6-glucans in Penaeus vannamei larviculture was evaluated by measuring the immune response and survival of shrimp juveniles subjected to white spot syndrome virus (WSSV) challenge tests (per os) and pond culture. Treatments were designed to contrast the probiotic factor (inclusion vs non-inclusion) and β-1,3/1,6-glucans supplementation in various larvae stages; starting from early stage (Zoea II), to middle stage (PL 12), late stage (15 days pre-challenge) or non-supplemented. In larviculture, the highest survival was obtained in animals treated with β-1,3/1,6-glucans from Zoea II. The use of probiotics enhanced survival during the first 0-52 h post-WSSV challenge period. During 56-156 h post-WSSV challenge period, interactions were observed between β-1,3/1,6-glucans × time and β-1,3/1,6-glucans × time × probiotics. Significant differences in final survival rates between treatments were not observed. In the second WSSV challenge, immune parameters were analysed. Significant interaction between probiotics and β-1,3/1,6-glucans was observed for plasmatic protein (PP) concentration, super oxide anion (O₂⁻) generation, antibacterial activity (AA), and total haemocyte count (THC). The use of probiotics in larviculture had a negative effect on the PP, but increased the AA and THC, while β-1,3/1,6-glucans had a negative effect on the O₂⁻ generation. The most relevant results were obtained from treatments T2 (probiotics in larviculture, β-1,3/1,6-glucans from Z II) and T4 (probiotics in larviculture, β-1,3/1,6-glucans 15 days before challenge). Treatment T2 presented the highest survival rate in larviculture. After WSSV infection, the animals of this treatment displayed resistance to the virus, a strong AA and increase of THC. Treatment T4 increased the amount of PP, increased the O₂⁻ generation and THC. Histological analysis showed that the animals of treatment T2 and T4 were able to limit the spread of the virus during the first hour after challenge with WSSV. The survivors from treatments T2 and T4 had a high THC, accompanied by a lack of white spot disease (WSD) injuries. A bioassay was carried out under farm conditions during the warm-rainy season using larvae from treatments T2 and T4. The animals were stocked at 18 animals/m² in earth ponds of 0.20 ha (three ponds/larvae kind). WSD outbreak was not detected, and the survival was significantly higher in ponds stocked with larvae from treatment T4 (70 ± 3%) than in ponds stocked with larvae from treatment T2 (49 ± 9%).