南美白对虾的病害及其控制

对虾是一种生长快、生长周期短、对环境适应能力强、养殖效益高的理想海水养殖对象。目前我国对虾养殖的品种主要有中国对虾 (Ｐｅｎａｅｕｓｃｈｉｎｅｎｓｉｓ)、斑节对虾 (Ｐ .ｍｏｎｏｄｏｍ )、长毛对虾(Ｐ .ｐｅｎｉｃｉｌｌａｔｕｓ)、日本对虾 (Ｐ .ｊａｐｏｎｉｃｕｓ)、墨吉对虾 (Ｐ .ｍｅｒｇｕｉｅｎｓｉｓ)、刀额新对虾 (Ｍｅｔａｐｅ ｎａｅｕｓｅｎｓｉｓ)、独角新对虾 (Ｍ .ｍｏｎｏｃｅｒｏｓ)、周氏新对虾 (Ｍ .ｊｏｙｎｅｒｉ)、脊尾白虾 (Ｐａｌａｅｍｏｎｃａｒｉｎｉ ｃａｕｄａ)以及南美白对虾 (Ｐｅｎａｅｕｓｖａ…     

中国对虾淋巴器的酚氧化酶

酚氧化酶 (ｐｈｅｎｄｏｘｉｄａｓｅ ,ＰＯ)作为一个类补体系统[1] 在甲壳动物中的作用日益受到人们的重视 ,国内外学者在酚氧化酶与免疫的关系方面作了许多工作。一致的看法是 ,微生物细胞壁中的多糖成份可激活酚氧化酶原系统[2 ] 。甲壳动物血细胞中的酚氧分酶原系统被激活后可促进血细胞的吞噬与包襄 ,介导凝集和凝固 ,产生杀菌物质[1-4 ] 。但对于ＰＯ与病毒的关系则研究较少 ,在被病毒感染的对虾组织中直观地显示ＰＯ活性及ＰＯ对病毒的作用目前尚未见报道。对虾淋巴器被认为是对虾的免疫器官[5,6] 。本工作以健康和患病中国对虾的淋…     

长毛对虾幼体荧光病的防治

在长毛对虾育苗过程中 ,常常会发生对虾幼体在夜间发光的现象。研究发现 ,这是一种由细菌引起的疾病 ,不是发光原生动物 -夜光虫造成的。本文综合多年生产实践及有关资料将该病的防治方法介绍如下。一、病原体病原体为假单胞菌科 (Ｐｓｅｕｄｏｍｏｎａｄａｃｅａｅ)的荧光假单胞菌 (Ｐｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｎｓｃｅｎｓ ) 〔1〕和弧菌科 (Ｖｉｂｒｉｏｎａｃｅａｅ )的哈维氏弧菌 (Ｖｉｂｒｏｈａｒｖｅｙｉ ) 〔2〕。假单胞菌为革兰氏阴性短杆菌 ,两端圆形 ,大小为 0 .3～ 1 .0 μｍ× 1 .0～ 4.4μｍ ,在极端有 1～ 6根鞭毛 …     

对虾皮下及造血组织坏死杆状病毒单克隆抗体的ELISA快速检测

对虾病毒性暴发病对我国对虾养殖业的危害十分严重,其病原为皮下及造血组织坏死杆状病毒(ｈｙｐｏｄｅｒｍａｌａｎｄｈｅｍａｔｏｐｏｉｅｔｉｃｎｅｃｒｏｓｉｓｂａｃｕｌｏｖｉｒｕｓＨＨＮＢＶ)[1]或称为对虾白斑综合症病毒(ｗｈｉｔｅｓｐｏｔｓｙｎｄｒｏｍｅｂａ?..     

鳖白底板病、腐皮病并发症病原菌及药物治疗

中华鳖(Ｔｒｉｏｎｙｘｓｉｎｅｎｓｉｓ)的白底板、腐皮病是两种较严重的传染病,两者并发症的感染率可达80%～90%,死亡率100%。陈晓凤等[1995]研究认为白底板病的病原是嗜水气单胞菌(Ａ.ｈｙｄｒｏｐｈｉｌａ)、迟缓爱德华氏菌(Ｅ.ｔａｒｄａ)和变形杆菌(Ｐ.ｖｕｌｇａｒｉｓ)。而谢军[1996]认为该病的病原可能是病毒。腐皮病的病原也众说不一,孙佩芳等[1998]多次从患腐皮病的中华鳖血液和肝脏中分离到温和气单胞菌,而其他人也从患腐皮病鳖体内分离到气单胞菌、假单胞菌和无色杆菌等菌种,其中认为气单胞菌为主要致病菌[中国科学院水生生物…     

用于PCR检测对虾皮下及造血组织坏死杆状病毒(HHNBV)的一种快速提取DNA方法

使用采样液ＳＥＭＰ－Ｔｒｉｓ保存人工感染ＨＨＮＢＶ（ＷＳＳＶ的一个分离株）的中国对虾组织，然后分别用酚抽提法、玻璃乳（Ｇｌａｓｓ Ｍｉｌｋ）回收法、硝酸纤维素膜结合法和煮沸－乙醇沉淀法提取ＤＮＡ。应用ＨＨＮＢＶ引物对提取的ＤＮＡ进行ＰＣＲ扩增，使用琼脂糖凝胶电泳对扩增产物进行鉴定和检测。通过比较表明，煮沸－乙醇沉淀法是一种最快速、简便的从采样液ＳＥＭＰ－Ｔｒｉｓ保存的病虾组织中制备ＰＣＲ模板的方法     

中华绒螫蟹幼体日粮和生长效率的初步研究

目前国内对物质和能量的主要研究对象是鱼类、对虾类和龟鳖类[1～ 7] 。何林岗[8,9] 曾测定了河蟹和青虾各期幼体的摄食量 ;周鑫[4 ] 研究了河蟹Ⅰ期蚤状幼体对钝顶螺旋藻的摄食和消化率。周名江[10 ] 对单胞藻 -卤虫的能量流动进行了研究。至今未见中华绒螯蟹 (Ｅｒｉｏｓｈｅｉｒｓｉｎｅｎｓｉｓ)幼体物质和能量转换效率方面的报道。研究物质和能量的转换效率不仅在生态学上具有重要意义 ,而且能指导蟹类的养殖生长及科学管理1　材料与方法1 .1　实验场地和用水实验于 1 997年在海南万宁市万州罗氏沼虾孵化场进行 ,海水为天然海水 (盐度 …     

微生态制剂在对虾病害防控中的应用开发进展

正1对虾养殖病害现状及措施1.1对虾养殖的病害威胁我国养殖产量较多、经济价值较高的对虾有凡纳滨对虾(Litopenaeus vannamei)、斑节对虾(Penaeus monodon)、日本囊对虾(Marsupenaeus japonicas)、中国明对虾(Fenneropenaeus chinensis)、长毛明对虾(F.penicillatus)、墨吉明对虾(F.merguienrsis)等[1]。但是目前养殖病害已经成为制约对虾产业发展的重要障碍,其中细菌性疾病是最主要的对虾疾病之一,尤其弧菌病是当前造成虾     

微藻EPA和DHA的研究现状及前景

高度不饱和脂肪酸 (ＰＵＦＡ)在营养和医学上的重要作用 ,已引起人们广泛的兴趣 ,特别是二十碳五烯酸 (ＥＰＡ)和二十二碳六烯酸 (ＤＨＡ)两者对防治心脏疾病、动脉硬化、癌症、风湿关节炎、气喘和糖尿病等人类疾病有明显效果[1～ 3 ] ,已成为研究开发的热点。在水产上 ,ＥＰＡ和ＤＨＡ是许多鱼类幼体、对虾幼体、双壳类幼虫的必需脂肪酸 ,关系到幼虫和幼体的生长发育和存活。ＥＰＡ和ＤＨＡ虽然对有些动物不是必需的 ,但在饵料中适当添加这些物质 ,可提高投喂动物的生长速度和存活率[4～ 6] 。目前国内外对微藻中的ＥＰＡ和ＤＨＡ已进行了…     

稀土元素对中国对虾卵子孵化和幼体变态的影响↑（*）

稀土元素对淡水生物藻类培养[1]、鱼类养殖[2]影响的研究表明,微量稀土元素对淡水生物有促进生长,改善品质的作用。但稀土元素对海洋生物的影响迄今未见报道。天然海水中稀土元素水平属超痕量,仅为0.01μｇ/Ｌ[3]。本文首次研究稀土元素对中国对虾(Ｐｅｎａｅｕｓｃｈｉｎ...     

多重PCR检测对虾白斑综合症病毒和传染性皮下组织坏死病毒

采用对虾白斑综合症病毒和传染性皮下组织坏死病毒的特异引物,在一个PCR反应中同时扩增到大小分别为271 bp和356 bp的病毒特异片段,多重PCR条件优化后可从最少100 fg级病毒DNA模板中定性检测出此两种病毒。     

凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测

2013年,河北、天津等地区养殖的凡纳滨对虾(Litopenaeus vannamei)育苗期出现死苗、出苗率低的情况,生产上,仔虾个体大小差异较大,造成了严重损失.本研究采用荧光定量PCR方法(Real-time PCR)对天津大港地区采集的108尾凡纳滨对虾仔虾样品进行单尾病原检测.结果显示,传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)和虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)均有检出.IHHNV阳性检出率100％,每微克对虾组织DNA的病毒拷贝数为103-107,且个体较大的样品(1.2-2.0 cm)携带病毒拷贝数偏高;EHP阳性检出率为49.1％,每微克对虾组织DNA的拷贝数为103-105,且集中于个体较小样品(0.7-1.1 cm).对IHHNV和EHP阳性凡纳滨对虾样品进行生物学体长与病毒载量指数相关性分析,显示IHHNV载量指数与对虾生长速率呈正相关,虾组织IHHNV平均载量达8.51×104 copies/μg DNA,为较高的感染水平;EHP的载量与对虾生长速率呈负相关关系,与较大个体阳性检出率较低相对应,虾组织EHP平均载量达到2.19× 104 copies/μg DNA,为较高的感染水平.由此,该批凡纳滨对虾仔虾患病为IHHNV和EHP的混合感染所致,本研究数据为IHHNV和EHP病原混合感染流行情况及其对养殖育苗期仔虾生长的影响提供科学依据.     

常规PCR、实时定量PCR检测传染性皮下及造血器官坏死病毒的效果分析

对虾传染性皮下及造血器官坏死病毒(IHHNV)可感染世界各地养殖对虾,给对虾养殖业造成严重经济损失。本实验首次采用实时定量PCR法对广西地区的84份凡纳滨对虾样品进行检测,同时以常规PCR检测作对照。实时定量PCR检测阳性率为79·8%,常规PCR检测阳性率为40·5%,表明广西地区养殖的凡纳滨对虾IHHNV的感染率较高。将二者检测均呈阳性的30份样品扩增产物进行序列分析测序,测序结果通过DNA STAR软件包进行分析,并通过NCBI Blast与GenBank中的序列进行比对。结果证明,测定的是IHHNV序列。30份样品的IHHNV序列很保守,可以分为4种类型,仅有两个碱基的位置发生变异。实时定量PCR检测IHHNV,快速、灵敏、准确,特异性好,可以作为检测对虾感染病毒的有效方法。     

多重RT-PCR体系检测4种虾病毒的方法

根据多重RT-PCR的技术原理,利用对虾传染性表皮与造血组织坏死症病毒、白斑综合征病毒、黄头病毒和桃拉综合征病毒的基因序列分别设计了4对特异引物,建立多重RT-PCR体系用于虾4种病毒的检测。多重RT-PCR体系能特异地扩增出IHHNV、WSSV、YHV和TSV的目的片段:TSV特异性扩增片段508 bp,WSSV 特异性扩增片段435 bp,IHHNV 特异性扩增片段301 bp 和YHV。特异性扩增片段614 bp。结果表明,多重PCR虾病毒检测系统具有较高的特异性和敏感性,并对其它对虾病原呈阴性。IHHNV、TSV、WSSV和YHV模板在多重PCR虾病毒检测体系中的检测下限分别为0.1,1,0.02和0.2 pg。病毒感染病料检测试验中,该检测体系的检测结果与单纯PCR的检测结果呈现出较好的吻合度。     

双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)

根据Genbank中对虾白斑病由白斑综合征病毒（WSSV）和传染性皮下及造血器官坏死病毒（IHHNV）的基因序列，设计了两对能分别检测WSSV和IHHNV保守片段基因的特异性引物，而且这两对引物在同一反应体系中可以同时对WSSV和IHHNV的DNA模板进行多重PCR扩增，得到大小分别为110bp（WSSV）和356bp（IHHNV）的扩增条带。对影响PCR反应的主要因素Mg“浓度和退火温度进行了优化，证明当Mg^2＋浓度为2．0～4．0mmd，退火温度为57～59℃时可获得最佳的扩增和检测效果。特异性试验结果表明，这两对引物检测WSSV和IHHNV具有很好的特异性，对其它对虾常见病原的PCR扩增结果均为阴性。敏感性测定结果表明，该反应体系最低能检测100pg的WSSV和IHHNV的DNA模板。临床检测表明，所建立的双重PCR方法可以适用于WSSV和IHHNV的同时检测和鉴别。     

Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

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。     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

Simultaneous presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Type A virus-related sequence in Penaeus monodon from India

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp is complicated by the fact that certain virus-related sequences are integrated into the genome of Penaeus monodon in some parts of the world, which has been reported so far from Africa and Australia. In this study, we evaluated the highly specific and sensitive diagnostic primer sets for detection of infectious IHHNV and integrated virus-related sequence in 177 samples of P. monodon from India. A nested primer set, IHHNV648F/R and IHHNV309F/R was used to specifically detect infectious IHHNV and not the virus-related sequences. IHHNV was detected in 67.4% postlarvae (PL) and 34% adult samples using this primer set. The OIE recommended primers IHHNV392F/R and IHHNV389F/R gave positive reaction for 86.7% PL and 67% adult samples, while the primer pair 77012F and 77353R gave positive reaction with 46.7% PL and 20% adult samples. These primers were found to detect virus-related sequence integrated into the shrimp genome. The analysis of virus-related sequence by MG831F/R primers showed that 33.7% PL and 31.7% adult shrimp possessed Type A virus-related sequence. 22.8% PL and 10.5% adults had both IHHNV and Type A virus-related sequence. Cloning and sequencing 832 bp virus-related sequence from P. monodon from India revealed presence of five shrimp DNA markers between 439 and 825 bp. This study is the first conclusive report on the presence of Type A virus-related sequence in P. monodon from India.     

Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection

A susceptibility study of postlarvae (PL) and juvenile Super Shrimp®, a selected line of Penaeus stylirostris, was conducted to compare their resistance to infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection to that of a specific pathogen free (SPF) population of P. vannamei. Super Shrimp® PLs were fed with IHHNV-infected shrimp tissue for 2 days and then maintained on a pelletized ration for an additional 28 days. PLs were sampled at days 0, 1, 2, 3, 4, 6, 10, 15, 20, 25 and 30. There was no apparent mortality during the experimental period. Tissue DNA extracted from the PLs was analyzed for the presence of IHHNV by PCR. Low levels of IHHNV were detected only in DNA extracts from samples at days 1, 2, and 3. No IHHNV DNA was detected from days 4 to 30. The days that the PLs were weakly IHHNV-PCR positive were during the period that they were being fed with IHHNV-tissue, and thus, the IHHNV DNA signal was suspected to be from the infected tissue used as a feed. Through both histology and in situ hybridization, we confirmed that tissues of Super Shrimp® PLs were not infected with IHHNV. PCR results of another IHHNV challenge study with juveniles of Super Shrimp® were similar to those with PLs. These results indicate that IHHNV did not replicate in the PL and juvenile Super Shrimp®. In contrast, P. vannamei juveniles, which were used as a positive control, showed a more intense IHHNV infection, as determined by PCR detection, beginning at 6 days postchallenge and increasing throughout the remainder of the study. In addition, the IHHNV-infected P. vannamei at 30 days postchallenge showed histological changes characteristic of IHHNV infection and had a positive reaction for IHHNV with in situ hybridization. Our studies show that Super Shrimp® are resistant to IHHNV infection. This is the first unequivocal demonstration of resistance to viral infection in shrimp.