海水养殖鱼类病毒性神经坏死病防控技术研究进展

神经坏死病毒(Nervous necrosis virus,NNV)是一种能导致海水鱼脑、中枢神经及视网膜等神经系统坏死的病毒,该病毒引发的鱼类病毒性神经坏死病主要发生在稚鱼和幼鱼期,较强的致病力及高致死率给海水鱼养殖业带来了沉重的打击,成为海水鱼类养殖产业可持续发展最重要的限制因素之一。文章主要综述了NNV的基本特征、诊断技术、病毒传播、疫苗免疫学等研究进展,介绍了NNV的防控技术及策略,为海水养殖鱼类NNV的防控工作提供参考。     

石斑鱼病害防治

本文结合国内外资料介绍了近年来石斑鱼养殖过程中发现的主要疾病及其防治方法,供广大业者参考。 1石斑鱼苗培育过程常见病害 1．1病毒 石斑鱼苗培育过程,极易遭受病毒性病原的侵害,而简称为NNV的病毒性神经坏死症（Nervous Necrosis Virus）,是造成石斑鱼苗培育中经常发生的主要病毒性疾病。神经坏死病毒（NNV）主要感染寸苗前期的多种类石斑鱼苗,感染后的鱼体出现异常泳姿,     

斜带石斑鱼(Epinephelus coioides)病毒性神经坏死病毒的纯化分析及检测方法建立

根据GenBank中已有的鱼类病毒性神经坏死病毒(NNV)RNA2基因序列,设计引物,从福建厦门具有典型NNV发病症状的斜带石斑鱼中克隆了RNA2基因的全长序列,并将序列提交到GenBank获得登录号为MF510920,命名为XMNNV。系统进化树分析结果表明XMNNV与RGNNV聚类在一起,与SJNNV、BFNNV和TPNNV等其他鱼类神经坏死病毒亲缘关系较远,说明本研究分离得到的XMNNV属于RGNNV基因型。通过超速离心方法对病毒进行提纯,得到了纯化的NNV病毒。电镜观察结果表明,病毒粒子直径20～25 nm,结构为正二十面体,与已经报道的NNV结构一致。通过对XMNNV与其它RGNNV RNA2基因进行序列比对,在保守区设计引物,运用RT-PCR方法建立了RGNNV的PCR检测方法,该方法灵敏度高达67 copies/μL。纯化的病毒对E11(条纹月鳢细胞系)和大黄鱼肌肉细胞进行感染,结果表明该病毒可以感染这两种鱼类细胞。E11细胞被感染病毒后,细胞出现空泡化,并最终导致细胞分解死亡;大黄鱼肌肉细胞感染后,细胞变圆,慢慢从培养皿壁脱落,最终解体死亡。另外,对感染后细胞进行PCR检测,结果显示为阳性,进一步确定了分离的NNV具有感染这两种细胞的能力。本研究通过电镜观察和PCR检测两种方法确定了患病石斑鱼携带NNV,通过对两种鱼类细胞的感染实验,确定了该病毒具有一定的感染能力。综上,本研究为石斑鱼NNV疾病的诊断提供了有效的方法,对石斑鱼NNV疾病的预防具有一定的指导意义。     

逆转录聚合酶链式反应(RT-PCR)检测5种养殖石斑鱼的神经坏死病毒

神经坏死病毒(Nervous necrosis virus)是导致多种海水鱼类神经性病害的致病原.发病及死亡的石斑鱼除了表现神经异常症状外,无明显的临床病症,体表及内脏组织也未发现明显病变及寄生虫感染.2003年4～8月,应用逆转录聚合酶链式反应(RT-PCR)技术从福建南部人工养殖的5种石斑鱼即紫石斑鱼(Epinephelus lanceolatus)、马拉巴石斑鱼(E. malabaricus)、青石斑鱼(E. awoara)、赤点石斑鱼(E. akaara)和云纹石斑鱼(E. moara)中检出5个神经坏死病毒分离株.检测了76份石斑鱼样品,这些石斑鱼NNV病毒的平均感染率约为90%.对这些病毒的RT-PCR产物421 bp核酸进行了测序和序列分析,其相同的序列超过99%.将这些序列与GenBank的石斑鱼(Epinephelus spp.)神经坏死病毒相关基因序列作比较,同源性在97%以上.对神经坏死病毒在石斑鱼体内的分布也进行了分析,在脑和眼组织的检出率最高,部分病鱼的肝、脾和肾组织也能检出病毒.结合流行病学特征,可确认神经坏死病毒为该传染病的主要致病原.RT-PCR方法是检测NNV等病原的一种理想的诊断方法.     

海水鱼神经坏死病毒致病机理研究进展

神经坏死病毒(nervous necrosis virus,NNV)主要分布在地中海和太平洋西海岸,已在120多种鱼中被检测到,能引起鱼类病毒性神经坏死病(viral nervous necrosis,VNN),是对海水鱼类危害重大的病原之一.目前,随着神经坏死病毒的基因序列、类型、诊断和预防技术等方面研究的不断深入,...     

鱼类神经坏死病病毒衣壳蛋白在昆虫细胞中的表达及其特性

鱼类病毒性神经坏死病毒能引起多种海水鱼类中枢神经组织病变,给各国海水养殖业造成了巨大的损失。本研究利用RT-PCR技术获得病毒性神经坏死病毒0603株的衣壳蛋白基因,将其插入到杆状病毒Bac-To-Bac表达系统的pFastBacⅠ质粒中,构建了pFastBac-cp质粒。转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-cp,脂质体介导将其转染Sf9细胞产生有感染性的重组杆状病毒AcNPV-cp。利用AcNPV-cp感染Sf9细胞后,SDS-PAGE分析可见大小约为37kD的特异性蛋白带, Western-blotting分析发现,其可以与病毒性神经坏死病毒阳性血清反应出现特异性的杂交带,试验结果表明AcNPV-cp在Sf9细胞中成功地表达了病毒性神经坏死病毒的衣壳蛋白,其具有良好的免疫学活性。负染电镜观察发现,CP蛋白可自行装配成病毒样颗粒,其大小形态类似于病毒性神经坏死病毒。制备超薄切片后电镜观察发现,CP蛋白自行装配成的病毒样颗粒呈晶格状排列在细胞质中。 本研究为研制有效防控鱼类病毒性神经坏死病的新型颗粒性疫苗奠定了基础。     

谷氨酰胺对斜带石斑鱼GF-1细胞中C-Myc蛋白的表达与神经坏死病毒复制的影响

为探讨C-Myc表达、谷氨酰胺代谢和神经坏死病毒复制三者之间的关系,本研究首先克隆了斜带石斑鱼鳍条细胞(GF-1)中的C-Myc基因(GF-1-C-Myc),结果显示GF-1-CMyc基因cDNA全长814 bp,开放阅读框(ORF)为285 bp,编码95个氨基酸(aa),有亮氨酸拉链结构域与螺旋-环-螺旋(HLH)结构域。实验表达和纯化了GF-1-C-Myc蛋白,并制备其多克隆抗体。采用实时定量PCR技术(qRT-PCR)与免疫印迹法(WB)检测了GF-1-C-Myc基因的表达和神经坏死病毒的复制。结果显示,缺乏谷氨酰胺会同时抑制GF-1-C-Myc基因的表达和神经坏死病毒(NNV)的复制,添加谷氨酰胺可同时促进GF-1-C-Myc的表达和NNV的复制;此外,NNV感染可上调GF-1-C-Myc基因的表达,并显著消耗GF-1细胞培养液中的谷氨酰胺。研究表明,GF-1-C-Myc基因可调控宿主谷氨酰胺代谢,从而有利于神经坏死病毒的复制。本结果为防控NNV的感染提供了参考。     

鱼类病毒性神经坏死病毒衣壳蛋白在昆虫细胞中的表达及其特性

利用RT-PCR技术获得病毒性神经坏死病毒0603株的衣壳蛋白基因,将其插入到杆状病毒Bac-To-Bac表达系统的pFastBacI质粒中,构建了pFastBac-cp质粒.转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-cp,脂质体介导将其转染Sf9细胞产生有感染性的重组杆状病毒AcNPV-cp.利用AcNPV-cp感染Sf9细胞后,SDS-PAGE分析可见大小约为37 ku的特异性蛋白带,Western-blotting分析发现,其可以与病毒性神经坏死病毒阳性血清反应出现特异性的杂交带.试验结果表明,AcNPV-cp在Sf9细胞中成功地表达了病毒性神经坏死病毒的衣壳蛋白,其具有良好的免疫学活性.负染电镜观察发现,CP蛋白可自行装配成病毒样颗粒,其大小形态类似于病毒性神经坏死病毒.制备超薄切片后电镜观察发现,CP蛋白自行装配成的病毒样颗粒呈晶格状排列在细胞质中.为研制有效防控鱼类病毒性神经坏死病的新型颗粒性疫苗奠定了基础.     

鱼类神经坏死病毒研究进展与发展趋势

神经坏死病毒(nervous necrosis virus,NNV)是一种世界范围内流行、严重危害多种海水和淡水鱼类的传染性病原。NNV为单一正链、2节段RNA病毒,基因组由RNA1(3.1 kb)和RNA2(1.4 kb)组成。在病毒复制过程中,会合成亚基因组RNA3。RNA1编码RNA聚合酶。RNA2编码衣壳蛋白,为病毒的唯一结构蛋白。RNA3编码B1和B2两种非结构蛋白。根据病毒衣壳蛋白的基因序列,神经坏死病毒可以分成4种基因型,分别为拟鲹、红鳍东方鲀、条斑星鲽和赤点石斑神经坏死病毒基因型。但是,目前只发现A、B、C三种病毒血清型,A对应拟鲹神经坏死病毒基因型,B对应红鳍东方鲀神经坏死病毒基因型、C对应条斑星鲽神经坏死病毒和赤点石斑神经坏死病毒基因型。病毒存在垂直和水平两种传播途径,而且广泛分布于养殖和野生鱼类中。阻断病毒在野生与养殖鱼类之间的传播和开展新型鱼类疫苗研发是将来研究趋势。     

鱼类神经坏死病毒的检测与细胞培养技术研究进展

鱼类病毒性神经坏死病(Viral nervous necrosis,VNN)又称病毒性脑病和视网膜病(Viral encephlopathy and retinopathy,VER),在全世界多种鱼类中流行,对仔鱼和幼鱼危害很大,死亡率可达100%,对成鱼也有很高的致死率.由于其极高的传染性和危害性,被国际兽疫组织(OIE)列为重要的鱼类病害.该传染病的病原为神经坏死病毒(Nervous necrosis virus,NNV),是罗达病毒科(Nodaviridae)的一种小RNA病毒.罗达病毒科包括α-罗达病毒属(Alphanodavirus)和β-罗达病毒属(Betanodavirus).     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Status of aquatic and riparian biodiversity in artificial lake ecosystems with and without management for recreational fisheries: Implications for conservation

1. Humanity is facing a biodiversity crisis, with freshwater-associated biodiversity in a particularly dire state. Novel ecosystems created through human use of mineral resources, such as gravel pit lakes, can provide substitute habitats for the conservation of freshwater and riparian biodiversity. Many of these artificial ecosystems are subject to a high intensity of recreational use, however, which may limit their biodiversity potential.
2. The species richness of several taxa (plants, amphibians, dragonflies, damselflies, waterfowl, and songbirds) was assessed and a range of taxonomic biodiversity metrics were compared between gravel pit lakes managed for recreational fisheries (n = 16) and unmanaged reference lakes (n = 10), controlling for non-fishing-related environmental variation.
3. The average species richness of all the taxa examined was similar among lakes in both lake types and no substantial differences in species composition were found when examining the pooled species inventory. Similarly, there were no differences between lake types in the presence of rare species and in the Simpson diversity index across all of the taxa assessed.
4. Variation in species richness among lakes was correlated with woody habitat, lake morphology (surface area and steepness), and land use, but was not correlated with the presence of recreational fisheries. Thus, non-fishing-related environmental variables had stronger effects on local species presence than recreational fisheries management or the presence of recreational anglers.
5. Collectively, no evidence was found that anglers and recreational fisheries management constrain the development of aquatic and riparian biodiversity in gravel pit lakes in the study region; however, the conservation of species diversity in gravel pit lakes could benefit from an increasing reliance on habitat enhancement activities.