酒石酸钾-甘油密度梯度离心法纯化对虾肝胰腺细小病毒的方法学研究

阐述一种从对虾肝胰腺组织中纯化肝腺细小病毒的方法，将对虾肝胰腺组织样品研磨并进行适当脱脂处理后，进行一系列的浓缩和过３５％蔗糖垫离心，然后通过一个不连续的酒石酸钾－甘油密度梯度离心纯化。结果证实，该方法对样品中的病毒颗粒的分离和纯化效果均较理想，可以满足有关研究工作的需要。经与ＣｓＣｌ密度梯度离心法比较，提示两者效果相似，但本方法更加经济实用。     

对虾肝胰腺细小病毒滚环扩增检测方法的建立

根据对虾肝胰腺细小病毒(HPV)保守基因序列,设计特异性的锁式探针及其扩增引物,优化反应条件,建立了肝胰腺细小病毒超分支滚环扩增检测方法。实验中采用一步法连接,探针在Taq DNA连接酶作用下,58℃连接40 min、62℃扩增30 min便可以扩增出明显条带。反应特异性验证实验表明,该体系能够特异性地检测出HPV,而不与供试的其他对虾病原发生交叉反应;灵敏度分析结果显示该方法的检测极限为105 copies/μl,与PCR检测方法相比,一步法连接的滚环扩增的灵敏度低两个数量级。该方法反应过程中温度变化次数少,基本都在等温条件下进行,不需要PCR仪,可发展成为在简便实验条件下使用的简易检测方法。     

肝胰腺细小病毒和斑节对虾杆状病毒的PCR复合检测

肝胰腺细小病毒和斑节对虾杆状病毒是常见的2种DNA对虾病毒,在虾类养殖业中经常会出现混合感染。因此,建立这2种病毒的复合检测方法显得尤为重要。根据基因库中肝胰腺细小病毒和斑节对虾杆状病毒的保守序列,设计了肝胰腺细小病毒和斑节对虾杆状病毒的特异性引物,对这2种病毒PCR复合检测的反应条件和试剂浓度进行优化,退火温度55℃,主要试剂Mg2+终浓度为3mmol/L,dNTP终浓度为0.4 mmol/L,引物终浓度为0.8μmol/L。进一步比较了在优化后的PCR反应体系下,检测单种病毒和同时检测这2种病毒的灵敏度差异。     

中国明对虾肝胰腺细小病毒全基因组的克隆及序列分析

中国明对虾肝胰腺细小病毒是一种单链、线性DNA病毒.本研究利用DNA末端加尾和嵌套PCR首次完成了对虾肝胰腺细小病毒(hepatopancreatic parvovirus,HPV)中国分离株(HPV-Pc)全基因组序列的测定,研究结果表明,HPV-Pc株(GenBank登录号GU371276)的全基因组由6085个核...     

肝胰腺细小病毒(HPV) PCR检测及流行情况调查

采用水产行业标准《对虾肝胰腺细小病毒病诊断规程第1部分:PCR检测法》(SC/T7203.1-2007)的方法,对2011-2013年期间我国沿海7个省市主要养殖对虾品种不同生长阶段的对虾样品进行该病毒携带情况的筛查。该方法的检测灵敏度为0.07 fg,相当于大约20个病毒拷贝。结果显示,639份样品的HPV阳性检出率为18.47%。其中,在中国对虾(Fenneropenaeus chinensis)、凡纳滨对虾(Litopenaeus vannamei)和日本囊对虾(Marsupenaeus japonicus)中均有阳性检出,且仔虾、幼虾、成虾各个阶段均可检出HPV,表明HPV已在我国养殖对虾中存在并流行。本研究结果为对虾养殖生产提供了疫病的科学数据,为我国养殖对虾中该病的流行情况提供了参考依据。     

快速诊断中国对虾肝胰腺细小病毒病的冰冻切片方法

采用组织切片染色,冰冻发片染色,组织涂片染色和粪便涂片染色这四种观察包涵体的方法,对中国对虾肝胰腺细小病毒病进行诊断。四种诊断方法结果的比较表明,冰冻切片诊断方法具有快速,简便,不易漏检的优点,可弥被组织切片诊断法时间长,组织涂片诊断法不可靠和粪便涂片诊断法易漏检的缺点。     

犬细小病毒最佳血凝条件初探

应用不同离子强度、不同pH的磷酸盐缓冲液穴PBS雪。在不同温度、不同浓度的牛血清白蛋白穴BSA雪等条件下,对犬细小病毒穴CPV雪血凝穴HA雪试验的最佳条件进行研究。0郾5%醛化猪红细胞在0郾01M,pH6郾8含有0郾1%BSA的PBS缓冲液中,敏感性明显提高,且试验结果重复性好,反应时间为1h。采用这种改进的条件,在4℃进行,HA特异性强,敏感性高。     

一种可用于现场观察对虾病毒包涵体的染色法

筛选出两种既可混合在一起，又能将对虾肝胰腺涂片的细胞核和细胞质分别着色的染色剂：台盼蓝和伊红Ｙ（Ｔ—Ｅ）。讨论了它们混合染色的条件与方法。用含有０．６％台盼蓝和０．２％伊红的混合染色液涂染肝胰腺细小病毒样病毒感染的对虾肝胰腺，观察到了核内的病毒包涵体。观察结果与病理切片苏木精－伊红染色结果吻合。这一方法用于野外现场进行对虾病毒感染的观察诊断，方法简便迅速，诊断结果与经典的苏木精－伊红染色法基本一致。     

中国对虾溞状幼体的细小病毒(Parvovius)类疾病初检

通过对病变的中国对虾溞状幼体组织学切片观察发现,病虾的肠粘膜、肌肉、神经索、心脏等组织器官普遍坏死。明显的特征是,角质层皮下组织的细胞核内有巨大嗜伊红染料的包涵体。故推论此病是由细小病毒感染所致。     

对虾肝胰腺的微细结构

用光镜和电镜观察了对虾(Penaeus orientalis Kishnouye)肝胰腺的结构。结果为:对虾肝胰腺的结构与螫虾等甲壳类基本相同。肝胰腺由两侧对称的分枝上皮小管组成。上皮细胞分为:胚胎性、吸收、原纤维和分泌性4种。上皮的表面有整齐而紧密排列的微绒毛。上皮的基底面有电子致密度低的基板,其上有肌上皮细胞附着。     

A real‐time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Hepatopancreatic parvovirus (HPV) causes a common shrimp disease that occurs in many shrimp farming regions, especially in the Indo Pacific, and infects most of the cultured penaeid species. There are seven geographic HPV isolates known, so a method to detect different HPV types is needed. We developed a sensitive and generic real‐time PCR assay for the detection of HPV. A pair of primers and TaqMan probe based on an HPV sequence obtained from samples of Fenneropenaeus chinensis from Korea were selected, and they were used to amplify a 92 bp DNA fragment. This real‐time PCR was found to be specific to HPV and did not react with other shrimp viruses. A plasmid (pHPV‐2) containing the target HPV sequence was constructed and used for determination of the sensitivity of this assay. The assay could detect a single copy of plasmid DNA, and it was used successfully in finding HPV in shrimp samples from the China‐Yellow Sea region, Taiwan, Korea, Thailand, Madagascar, New Caledonia and Tanzania.     

Methodology for the identification and quantitative measurement of chemical stimulants for penaeid shrimp

In a recently developed paradigm for the feeding behaviour of Crustacea, chemical stimuli are postulated to play a key role in mediating the various stages of feeding from initial excitation to sustained feeding. A hierarchical testing protocol for the screening of compounds or mixtures as chemical stimuli for Penaeus spp. was designed, utilizing: (a) a static chamber for an initial rapid assay for behavioural excitation; (b) a novel flow-through chamber for demonstrating chemotaxis; (c) a modified Y-maze choice chamber for more detailed studies on chemotaxis and feeding stimulation; and (d) aquarium feeding trials in which the most potent chemical attractants and/or feeding stimulants are incorporated into experimental feeds. In shrimp aquaculture, efficient feed utilization is of paramount importance to the economics of production and water quality. Identification and incorporation into the feed of ingredients that act as chemoattrac-tants and/or chemical feeding stimulants should lead to better food utilization, e.g. lower feed conversion ratios (FCRs), thereby producing quicker growth rates and lowering the incidence of water pollution and disease.     

白斑综合征病毒感染与对虾的免疫防御反应

对虾白斑综合征病毒（WSSV）感染对虾后，最典型的免疫反应是对虾开放循环系统的血淋巴细胞数量急剧下降，血淋巴凝结功能下降，感染部位聚集了大量的血淋巴细胞，且以颗粒细胞为主。WSSV可感染对虾颗粒细胞和小颗粒细胞，其中小颗粒细胞感染率高、感染速率快，感染后大颗粒细胞占血细胞总数的比例可增加到50％；血淋巴中的总糖、总碳水化合物、总蛋白和游离氨基酸显著提高，超氧化物歧化酶、过氧化氢酶和诱导性一氧化氮合成酶的活性显著降低。自然状态下广泛存在WSSV潜伏感染，潜伏感染的存在会导致存在免疫反应情况下的感染复发，并且有助于病毒的传播；不同WSSV感染状态下过氧化物（POD）差异显著，其平均值由大到小依次为：潜伏感染虾样、中度感染虾样、严重感染虾样。而其抗菌活性（UA）、溶菌活性（UL）、酶氧化酶活性（PO）、碱性磷酸酶（ALP）和凝集效价（HAT）差异不显著；潜伏感染个体对再次接种WSSV有“类免疫应答”的抗性，这种抗性不是来源于发病期对虾的天然抗性，而是WSSV感染后的一种免疫系统增强。宿主细胞凋亡可能是感染对虾在高温时反而维持较高成活率的主要机制。免疫增强剂可对对虾防御WSSV感染产生影响，脂多糖、葡聚糖、肽聚糖、岩藻依聚糖和双链核糖核酸都已被证实可提高对虾抗病毒感染的免疫保护。WSSV主要囊膜蛋白VP28可诱导对虾对WSSV感染产生抗性降低累积死亡率，高效价的病毒抗血清具有良好的保护作用；对虾抗菌肽也可通过抑制病毒的复制而起保护作用。     

Isolation of a novel strain of Bacillus pumilus from penaeid shrimp that is inhibitory against marine pathogens.

A bacterium was isolated from the mid-gut of healthy black tiger shrimp, Penaeus monodon , based on a large zone of inhibition in mixed culture on solid medium. The isolate was a Gram-positive, motile spore former, with an optimum pH range for growth in tryptone soya broth containing 2% NaCl of between pH 6 and 9. The bacterium was highly salt tolerant with concentrations between 0% and 8% having no detrimental effect on growth. The isolate was identified as Bacillus pumilus based on physiological capabilities using the API50CHB and Biolog systems. Amplification and sequencing of the 16S rRNA gene followed by phylogenetic analysis confirmed its identity. The Bacillus pumilus isolate was strongly inhibitory against the marine bacterial pathogens Vibrio alginolyticus , V. mimicus and V. harveyi , and weakly inhibitory against V. parahaemolyticus in cross-streaking assays on solid medium. The organism was marginally self-inhibitory, and inhibited B. licheniformis and B. subtilis . The suitability of the B. pumilus isolate for use as a probiotic in farmed shrimp was further supported by the absence of any of the known B. cereus enterotoxin genes. Based on these in vitro results, in vivo safety and efficacy trials are underway to determine suitability of the novel strain as a commercial probiotic.     

Maturation and spawning of the small penaeid shrimp Metapenaeopsis dalei in Tosa Bay, Pacific coast of southern Japan

SUMMARY: Seasonal changes in the percentage of mature female Metapenaeopsis dalei were studied histologically, and the annual cycle of maturation and spawning of this species was discussed. Specimens were collected at night by a beam trawler in Tosa Bay (33°N, 133°E) during the period from April 1995 to March 1996. All individuals had a gonado somatic index [ GSI = 100 (gonado weight/body weight)] of higher than 8, and 66.7% of those individuals with a GSI of 6–8 had oocytes in the late phase of the prematuration stage after germinal vesicle shrinking and migrating to the margin of the oocyte or had oocytes in the maturation stage. Such individuals with developed oocytes appeared in April, June, July, August, October, November and March. Individuals with a GSI above 6 appeared throughout the year, except during September and February. Individuals with ovulated oocytes were sampled in March when the water temperature near the bottom depths was lowest (15.4°C) during the year, being similar to February (15.1°C). These facts indicate that maturation occurs all year-round and suggests that spawning in the Metapenaeopsis dalei population in Tosa Bay occurs all year-round. This is the first report of year-round maturation in penaeid shrimps in Japanese waters.     

Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody‐based farmer‐friendly flow‐through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect‐related (Pir) A toxin‐like protein of AHPND pathogen was used to immunize rabbits at 21‐day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross‐reactivity with AHPND‐free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer‐friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirA^VP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost‐effective. Collectively, sensitive FTA test would help shrimp farmers to take real‐time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.