不同饵料对中国对虾幼虾生长及感染WSSV存活率的影响

采用4种饵料投喂中国对虾（Fenneropenaeus chinensis）幼虾，用投喂感染的方法人工感染WSSV。测定体长、体重以及各组的攻毒存活率，实验周期15d。ANOVA分析结果表明，投喂鲜活卤虫组体长、体重的增长明显优于其他各组，差异达极显著水平（P〈0．01）；投喂人工配合饵料实验组的体长、体重增长量最小；投喂鱼肉组体长增长慢于投喂蛤蜊肉组，而体重的增长快于投喂蛤蜊肉组，差异均不显著（P〉0．05）。投喂卤虫成体和投喂鱼肉两组的攻毒存活率最高，明显高于投喂配合饵料和蛤蜊肉两实验组，差异达极显著水平（P〈0．01）；投喂卤虫组和投喂鱼肉组之间存活率无显著差异（P〉0．05），投喂人工配合饵料组和蛤蜊肉组差异不显著（P〉0．05）。巢式PCR检测表明，人工感染前的中国对虾幼虾少数携带WSSV，人工感染后全部个体检测到病毒特征片段。     

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。     

WSSV人工感染量和饵料对中国明对虾存活时间的影响

为揭示不同白斑综合征病毒(white spot syndrome virus,WSSV)病毒量对于中国明对虾存活时间和存活率的影响,实验设计了逐尾、定量人工感染实验,在确保每尾中国明对虾进食特定量WSSV毒饵后进行观察、分析.结果显示,分别喂食含5.2×108 copies、1.0×109copies、2.1×109 copies WSSV的毒饵,对虾平均存活时间分别是389.3、323.3和187.3 h,差异极显著(P＜0.01);对虾最终累计死亡率都为100％.研究表明,致死量范围内,WSSV的感染量越低,对虾的平均存活时间越长.为了揭示饵料对中国明对虾抗病性能的影响,对已感染WSSV的中国明对虾投喂不同饵料.结果显示,分别喂食活卤虫、鲜蛤肉和配合饲料,对虾平均存活时间分别是281.7、173.9和164.9 h;喂食活卤虫的实验组平均存活时间显著高于喂食配合饲料和鲜蛤肉的实验组(P＜0.01);喂食配合饲料和鲜蛤肉的对虾平均存活时间无显著差异(P＞0.05);3组累计死亡率都为100％,结果表明,与配合饲料和鲜蛤肉相比,喂食活卤虫更能增强对虾抗WSSV的能力.     

感染白斑综合症病毒中国对虾雄性生殖系统的PCR检测与电镜观察

通过人工投喂携带WSSV的毒饵，对性腺发育成熟的中国对虾（Fenneropenaeus chinensis）雄虾（♂）进行感染实验。采用nest-PCR（巢式PCR）技术，检测感染后的中国对虾雄性生殖系统受WSSV感染情况，同时选取感染严重的虾样进行电镜观察。巢式PCR检测结果表明，感染组中国对虾的精巢、输精管和精囊均被WSSV感染，其中精囊呈阳性的最多，输精管次之，精巢最少。通过电镜进一步观察发现，WSSV粒子只存在于精巢、输精管和精囊的结缔组织中，而在其他组织和生殖细胞中均未发现病毒粒子。其中，精巢中WSSV粒子存在于精巢内两个生精小管之间的结缔组织；输精管中WSSV粒子存在于管壁的结缔组织；精囊中WSSV粒子也只存在于精囊内膜的结缔组织和精荚膜的结缔组织中。PCR检测和电镜观察结果均表明，WSSV粒子能感染中国对虾的雄性生殖系统且对性腺感染存在着一定的组织特异性。     

不同饵料组合对中国对虾幼体WSSV携带量及存活率的影响

采用单胞藻（SA）、配合饵料（AF）、轮虫（BP）和卤虫幼体（BS）4种饵料,设计了SA+AF、SA+AF+BP、SA+AF+BS和SA+AF+BP+BS 4种饵料组合并用于中国对虾苗种培育,投喂SA+AF饵料的受精卵分设“碘伏”消毒及未消毒组,投喂其他饵料的受精卵均为“碘伏”消毒组。分析各期幼体成活率、P10体重及WSSV携带量,结果表明,N-Z期间,受精卵消毒与否及投喂不同饵料对中国对虾的成活率差异均不显著（P>0.05）;Z-P期间,投喂BP的成活率明显高于未投喂BP组（P<0.01）;投喂SA+AF+BS的幼体在M-P期间成活率与其他饵料组差异极显著（P<0.01）;投喂SA+AF+BP 饵料组P10幼体的平均体重小于投喂SA+AF+BP+BS组的幼体（P<0.01）;SA+AF消毒组P10仔虾WSSV携带量为10.52±3.3 copies/ng DNA,低于其他饵料组仔虾的WSSV携带量（P<0.05）。在P11-P60培育期间,分别投喂菲律宾蛤仔足肌（CF）+配饵（AF）、60Co γ辐照菲律宾蛤仔足肌(RCF)+配饵（AF）、高锰酸钾消毒菲律宾蛤仔足肌（DCF）+配饵（AF）和配饵（AF）4种饵料组合。结果表明,CF+AF组对虾体重和体长增长最大,但与AF组差异不显著,而CF+AF组和AF组与其余两组差异显著（P<0.05）;4组对虾存活率差异不显著（P>0.05）。WSSV人工感染实验结果表明,4种饵料投喂的对虾累积死亡率都在90%以上,差异均不显著(P>0.05)。     

二种对虾白斑综合征组织与细胞病理研究

对自然发病、投喂感染和注射感染的中国对虾、凡纳滨对虾白斑综合征进行组织与细胞病理研究，结果发现：中国对虾与凡纳滨对虾的病理变化相似，但不同感染方式的患病对虾有差异。临床病理：相同点表现为游动无力，反应迟钝，胃中无食，体色变暗，肌肉浑浊，肝胰腺肿大；不同点为自然发病和投喂感染的对虾临床病理变化比注射人工感染明显，病程长。显微病理：相同点表现为细胞核肿大，细胞变性、坏死等；不同点为自然发病和投喂感染的病虾胃、肝胰腺的病理变化比注射感染严重，坏死细胞数量多，组织大面积坏死溶解；注射感染病虾的肌肉组织显微病理变化比自然发病、投喂感染严重。超微病理：相同点表现为细胞肿大、变性、坏死、溶解，线粒体、内质网等细胞器形态变异，肿大，膜溶解或整体崩解；细胞核肿大或固缩或溶解．部分细胞核中可见WSSV；不同点为注射感染的患病对虾肌肉组织的超微病理变化比自然发病、投喂感染严重，肌细胞核中观察到WSSV的几率较高；投喂感染、自然发病对虾的胃上皮细胞、肝胰腺细胞的超微病理变化比注射感染严重。另外，不同类型的细胞对WSSV的易感性不相同，血细胞为最常见的被病毒感染的靶细胞，血细胞通过细胞变形或形成伪足，贴附于其它组织细胞膜上。     

应用PCR和RT-PCR技术对4种对虾病毒的检测

探讨了应用PCR和RT－PCR技术对4种主要的对虾病毒进行检测的方法。同时使用该方法检测了对虾天然饵料－－卤虫中的4种对虾病毒。结果显示，含病毒核酸的阳性对照样品分别扩增出了大小为824bp,705bp,260bp和216bp的预期产物，但未能从卤虫样品中检测到此4种病毒的存在。本文报道的病毒检测方法具有快速、灵敏、准确的特点，可以用于进出口贸易中对活体或冰冻对虾，虾苗，对虾饵料等进行相关对虾病毒的检疫，也为制定我国对虾病毒检疫检验规范提供了技术参考。     

中国对虾雄性生殖系统感染WSSV在其垂直传播中的作用

通过人工感染实验,对中国对虾(Fenneropenaeus chinensis)雄性亲虾进行投喂感染。在确定其携带WSSV粒子后,将被WSSV感染的精荚人工移植到健康的雌虾纳精囊内。在无其他病源的情况下,促其产卵繁殖,统计各组子代的受精率、孵化率及无节幼体至溞状幼体的变态率贸?式PCR技术对亲虾及子代进行WSSV检测。结果表明,受WSSV感染的精荚能够把病毒传播给健康雌虾,雌虾能产出携带WSSV的卵子,培育出带毒幼体。各组子代的受精率、孵化率及变态率的统计结果表明,感染组和对照组在受精率上没有明显区别,受WSSV感染的精卵细胞可以正常结合。对照组受精卵的孵化率明显高于感染组,差异显著(P=0.045<0.05)。对照组无节幼体的变态率也高于感染组。说明WSSV的入侵对受精卵及幼体的发育有影响,WSSV感染导致部分受精卵及幼体不能正常发育或死亡。     

对虾传染性皮下及造血组织坏死病毒(IHHNV) PCR检测方法的建立

对虾传染性皮下及造血组织坏死病毒(IHHNV)病是国际兽疫局(OIE)划定的甲壳类其他重要疾病之一,它分布较广,危害严重,对世界对虾养殖业发展影响重大.本文根据Genbank登录的IHHNV基因序列(AF218266),设计了1对特异性引物,从纯化的IHHNV DNA和感染IHHNV凡纳滨对虾(Litopenaeus vannamei)组织DNA中成功地扩增出产物大小为703bp的DNA片段,该对引物对IHHNV DNA的检测灵敏度为19.85fg(8.83×103病毒拷贝),与健康对虾组织DNA、对虾白斑综合征病毒(WSSV) DNA及对虾肝胰腺细小病毒(HPV) DNA无交叉反应.本方法可快速、灵敏、特异地检测出对虾感染和携带IHHNV状况,为对虾健康养殖、无特定病原(SPF)种群选育及流行病调查提供了有效的检测手段.     

热灭活WSSV诱导的中国明对虾(Fenneropenaeus chinensis)在不同温度下的免疫致敏反应

本研究拟探究经热灭活的白斑综合征病毒(White spot syndrome virus,WSSV)能否诱导中国明对虾(Fenneropenaeus chinensis)产生免疫致敏反应.将具有典型白斑综合征症状的对虾肌肉剁碎后经60℃灭活lh,采用单尾定量口饲的方法,连续6d投喂不同水温条件(15℃、23℃、28℃、32℃)下的实验组对虾(E15℃、E23℃、E28℃、E32℃);同时设置常温(23℃)条件下阳性对照组C23℃(投喂未经灭活的WSSV毒饵)、对照组CF23℃(只投喂商业配合饲料).在实验第13天对存活个体进行第二次人工WSSV感染,结果显示,WSSV经60℃处理lh可彻底失活,表现为连续投喂6d至第二次人工感染期间无对虾死亡,而阳性对照组C23℃死亡率为100％.截止实验第19天,E15℃、E23℃、E28℃、E32℃各组存活率分别为80.41％、33.29％、8.47％、16.43％,CF23℃组的存活率为8.89％,E15℃组与其他各实验组差异极显著(P＜0.01),E230℃与CF23℃、E28℃、E32℃组差异显著(P＜0.05),E28℃和E32℃组差异不显著(P＞0.05);对各组实验材料进行WSSV绝对荧光定量检测,结果显示,经热灭活WSSV诱导的中国明对虾二次感染WSSV后,28℃环境下病毒增殖速度最快,高温(32℃)和低温(15℃)都会不同程度抑制WSSV的增殖速度.实验表明,热灭活WSSV可以诱导中国明对虾产生免疫致敏反应,对受WSSV感染的中国明对虾具有一定的保护作用;温度与WSSV的增殖速度密切相关.     

Bioencapsulation of Therapeutic Quantities of the Antibacterial Romet-30 in Nauplii of the Brine Shrimp Artemia and in the Nematode Panagrellus redivivus

Nauplii of the brine shrimp, Artemia , and cultures of the soil nematode, Panagrellus redivivus , were fed a suspension of the water-insoluble antibacterial Romet-30 (sulfadimethoxine plus ormetoprim) to determine if they could be used as a mechanism for delivery of the drug to larval penaeid shrimp.
Romet-30 was added to groups of brine shrimp nauplii and nematodes in 1 L beakers to achieve a concentration of 3 mg of drug per ml of seawater. After a 4 h uptake period, predetermined numbers of organisms were assayed for antibiotic content using the micro-plate diffusion method and a Romet-30 sensitive strain of Vibrio alginolyticus as the indicator. The resulting zones of inhibition were compared to that of a standard Kirby-Bauer sensitivity disc containing 25 μg of the drug that was placed on each plate as standard control. Negative controls consisted of equal numbers of nauplii and nematodes that were not exposed to the antibiotic. Artemia nauplii and nematodes accumulated approximately 0.1 μg of the drug per nauplius and approximately 0.25 μg per nematode. They exhibited no signs of toxicity from exposure to the drug and contained the antibiotic in sufficient quantity to provide a theoretical therapeutic dose of the drug when fed to penaeid larvae.
In subsequent tests, normal and diseased larval Penaeus stylirostris readily accepted Romet-30 enriched Artemia . Diseased larvae fed the drug in this manner exhibited a statistically greater overall survival and a greater survival to the post-larval stage by Student's t -test at the 5% significance level.     

浙江省和广东省部分地区2005年虾病发生的病原学调查研究

对2005年浙江省和广东省部分养殖场南美白对虾的急性传染病病原进行了研究。临床症状发现大部分病虾带有白斑综合症的明显症状,后通过组织病理学观察,PCR检测及测序分析,证实本次病害的主要病原是白斑综合症病毒,说明白斑综合症在对虾养殖中仍存在着流行趋势。     

Performance of Larval and Postlarval Pacific White Shrimp,Litopenaeus vannamei (Boone), Fed Two Commercial Liquid Diet Supplements

Abstract

Two commercial liquid diet supplements were evaluated as a partial replacement for live foods fed to larval and postlarval Pacific white shrimp, Litopenaeus vannamei. Larvae were stocked in 20-L buckets at a density of 113 shrimp/L and fed one of four diets (5 replicate buckets per diet): (1) a control diet consisting of the diatom, Chaetoceros neogracile, and live Artemia; (2) the control diet plus Treflan (a fungicide); (3) LiquaLife liquid larval diet supplements with reduced concentrations of C. neogracile and Artemia; and (4) Epifeed liquid larval diet supplements with reduced concentrations of Artemia. Diets were evaluated by comparing shrimp rostro-caudal length and survival to PL-8, survival to osmotic and pH stress, as well as shrimp gut fullness, gut lipid content, and fouling. Rostro-caudal length and survival to PL-8 were not significantly different among the four treatments. However, mean survival to PL-8 was 12-34% greater in the Epifeed treatment than in the other three treatments. Shrimp fed Epifeed exhibited greater survival (P < 0.05) when exposed to 7 ppt water for two hours than shrimp in the control treatments. In contrast, there was no significant difference in survival (P > 0.05) among the four treatments when shrimp were exposed to apH of 3.35 for two hours. Although shrimp fed liquid diet supplements had more fouling (P < 0.05) than shrimp in either of the control treatments, commercial liquid diet supplements can be used in penaeid shrimp hatcheries to partially replace Artemia without compromising shrimp growth or survival.     

自然养殖水体投喂CpG ODN和表面展示VP28的解脂耶罗维亚酵母对凡纳滨对虾(Litopenaeus vannamei)抗WSSV的免疫保护作用

用添加CpG寡聚核苷酸(CpG ODN)和表面展示VP28的解脂耶罗维亚酵母(VP28-yl)的饵料投喂凡纳滨对虾,进行田间中试实验。投喂30 d后进行WSSV感染实验,评估其对凡纳滨对虾的免疫保护作用。投喂实验结束后,CpG ODN投喂组对虾的相对增重率达到(65.8±7.8)% (P<0.05),这暗示CpG ODN可能具有促生长作用。WSSV攻毒后,CpG ODN和VP28-yl投喂组对虾中WSSV拷贝数与对照组相比均显著降低(P<0.05),相对免疫保护率分别可达到26.7%和36.7%。在投喂结束和WSSV刺激后,CpG ODN组对虾中的呼吸爆发水平均显著升高(P<0.05)。而在VP28-yl投喂组,WSSV引起的细胞凋亡则显著受到抑制(P<0.05)。此外,WSSV刺激后,STAT基因在CpG ODN组和VP28-yl组对虾中的表达水平均显著上调(P<0.05),分别在第5天和第3天达到最大值,而对照组中则显著下调。研究结果表明,CpG ODN和VP28-yl增强了凡纳滨对虾抗病毒免疫力,对养殖对虾病毒性疫病的防控具有显著作用,可以作为免疫增强剂添加在饵料中,具有在养殖生产中推广使用的前景。     

卤虫在不同饵料培养介质中的生长规律

以啤酒酵母和单胞藻为投喂饵料，新疆艾比湖卤虫（Artemia parthenogenetica)为投喂对象，建立三个实验组分别进行研究，观察在不同饵料条件下艾比湖卤虫的生长，探讨不同饵料对卤虫的生长、成活的影响。通过对卤虫体长和存活率的测量，建立了卤虫在不同饵料情况下的生长方程、生长曲线和存活率曲线，并通过对方程和曲线的分析获得以下结论：投喂单胞藻组卤虫的生长最慢，存活率最低25表面以后全部死亡；投喂酵母组卤虫生长最快，在0≤t≤42.6时体长值最高，存活率较低，投喂单胞藻和酵母的混合饲料组卤虫的前期生长次于酵母组，后期生长超过之，存活率较高且成熟个体产虫量和产卵量也多于酵母组。     

A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp

White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years.     

Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus

White spot syndrome virus (WSSV) is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimp are cultured, as well as in the Gulf of Mexico and SE USA. The virulence of six geographic isolates of WSSV was compared using Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. For challenge studies, virus infected tissues were given per os to L. vannamei postlarvae and Fa. duorarum juveniles. Resultant WSSV infections were confirmed by histological examination. The cumulative mortality of L. vannamei postlarvae reached 100% after challenge with each of the six geographic isolates of WSSV. However, the Texas isolate caused mortalities more rapidly than did the other shrimp isolates; the crayfish WSSV isolate was the slowest. In marked contrast, cumulative mortalities of juvenile Fa. duorarum reached only 35–60%, and varied among the geographic isolates of WSSV. Interestingly, in Fa. duorarum, the Texas WSSV isolate was also the most virulent, while the crayfish WSSV was the least virulent. The findings suggest that slight differences in virulence exist among geographic isolates of WSSV, and that susceptibility may vary with species and lifestages of the host.     

Passive immunity in tiger shrimp (Penaeus monodon) fed with monoclonal antibody to white spot syndrome virus

A study was conducted on the stability of monoclonal antibody (MAb) in the hepatopancreas and hemolymph of Penaeus monodon and its effect on protection against white spot syndrome virus (WSSV) upon challenge. MAb C-5 raised against WSSV was purified and coated onto a commercial shrimp feed at dosages of 5, 10 and 15 mg/kg feed. The feed was fed to P. monodon and stability of the MAb in hepatopancreas and hemolymph was determined by immunodot and Western blot. Immunodot results indicated the presence of MAb for 2 h post-feeding in hepatopancreas and hemolymph which was dose-dependent. MAb was also detected in hemolymph by Western blot up to 1 h post-feeding. Shrimp fed with MAb were challenged with WSSV by oral and injection methods. In shrimp fed with 15 mg antibody/kg feed (0.45 μg MAb/g shrimp/day) WSSV infection significantly delayed both in oral and injection challenges with a survival of 65 and 70 % (p < 0.05), respectively, during 15 days post-challenge. MAb was stable in shrimp for passive immunization against WSSV and could be a potential tool for prophylaxis against the virus.     

蚯蚓与蝇蛆对中国对虾生长及抗白斑综合征病毒感染的研究

以中国对虾（Fenneropenaeus chinensis）为实验材料，分别投喂配合饲料、蚯蚓、蛤蜊、蝇蛆等四种饵料，利用生长和抗病性指标综合评价蚯蚓和蝇蛆作为中国对虾饵料的可行性。生长实验前测定每个实验组的初始体长和体重，养殖40d后再次测定生长指标，之后，分别投喂不同毒饵量进行人工感染实验。方差分析表明：投喂四种饵料后，蛤蜊组生长最快，其次是蚯蚓，再次是蝇蛆，最后是配合饲料，各组之间对虾体长增长差异显著（P<0.05），体重增长差异极显著(P<0.01) 。利用线性固定模型分析不同饵料及不同病毒量感染下对中国对虾存活率的影响，其中蚯蚓组存活率最高，配合饲料组存活率最低，并且蚯蚓组存活率显著高于蛤蜊组和配合饲料组(P<0.05)，蝇蛆组显著高于配合饲料组（P<0.05）。研究结果表明：蚯蚓和蝇蛆可显著提高中国对虾的生长速度，明显提高中国对虾的抗病性，是很好的对虾饵料。