Salmonid alphavirus (SAV) and pancreas disease (PD) in Atlantic salmon,Salmo salar L., in freshwater and seawater sites in Norway from 2006 to 2008

A cohort study was initiated in the spring of 2006 to investigate epidemiological aspects and pathogenesis of salmonid alphavirus (SAV) subtype 3 infections and pancreas disease (PD). The aims were to assess involvement of the freshwater production phase, the extent and frequency of subclinical infections and to follow PD‐affected populations throughout the entire seawater production cycle, as well as investigate possible risk factors for PD outbreaks. Fish groups from 46 different Atlantic salmon freshwater sites in six counties were sampled once prior to seawater transfer and followed onto their seawater sites. A total of 51 Atlantic salmon seawater sites were included, and fish groups were sampled three times during the seawater production phase. SAV subtype 3 was not identified by real‐time RT‐PCR from samples collected in the freshwater phase, nor were any SAV‐neutralizing antibodies or histopathological changes consistent with PD. In the seawater phase, SAV was detected in samples from 23 of 36 (63.9%) studied sites located within the endemic region. No SAV subtype 3 was detected in samples from seawater sites located outside the endemic region. The cumulative incidence of PD during the production cycle amongst sites with SAV detected was 87% (20 of 23 sites). Average fish weight at time of PD diagnosis ranged from 461 to 5978 g, because of a wide variation in the timing of disease occurrence throughout the production cycle. Mortality levels following a PD diagnosis varied greatly between populations. The mean percentage mortality was 6.9% (±7.06) (range 0.7–26.9), while the mean duration of increased mortality following PD diagnosis was 2.8 months (±1.11) (range 1–6).     

鲑鱼甲病毒E1基因高保守区融合表达及免疫特性的分析

根据Gen Bank中公布的鲑鱼甲病毒(salmonid alphavirus,SAV)SAV 1、SAV 2和SAV3三个基因型中E1基因,选择高保守序列702 bp(436-1137)合成基因,命名为SAV E1,将其克隆到原核表达载体p Cold TF中构建重组质粒。然后将重组质粒转化到大肠杆菌感受态细胞BL21中,经终浓度为1.0 mmol/L的IPTG诱导表达,SDSPAGE和Western blot鉴定,重组蛋白均获得了表达,表达E1重组蛋白约95 k D。用镍离子亲和层析柱纯化重组蛋白,制备抗血清。间接ELISA结果显示,鼠抗重组蛋白E1血清效价为1∶25 600;间接免疫荧光结果显示,鼠抗重组E1蛋白血清可与SAV发生特异反应,由此表明表达的E1重组蛋白具有良好的免疫原性和免疫反应性,为SAV检测方法的建立提供理论依据。     

Effect of pancreas disease caused by SAV 2 on protein and fat digestion in Atlantic salmon

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co‐varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies.     

Mortality and weight loss of Atlantic salmon,Salmon salar L., experimentally infected with salmonid alphavirus subtype 2 and subtype 3 isolates from Norway

Pancreas disease (PD) caused by salmonid alphavirus (SAV) has a significant negative economic impact in the salmonid fish farming industry in northern Europe. Until recently, only SAV subtype 3 was present in Norwegian fish farms. However, in 2011, a marine SAV 2 subtype was detected in a fish farm outside the PD‐endemic zone. This subtype has spread rapidly among fish farms in mid‐Norway. The PD mortality in several farms has been lower than expected, although high mortality has also been reported. In this situation, the industry and the authorities needed scientific‐based information about the virulence of the marine SAV 2 strain in Norway to decide how to handle this new situation. Atlantic salmon post‐smolts were experimentally infected with SAV 2 and SAV 3 strains from six different PD cases in Norway. SAV 3‐infected fish showed higher mortality than SAV 2‐infected fish. Among the SAV 3 isolates, two isolates gave higher mortality than the third one. At the end of the experiment, fish in all SAV‐infected groups had significantly lower weight than the uninfected control fish. This is the first published paper on PD to document that waterborne infection produced significantly higher mortality than intraperitoneal injection.     

Molecular cloning of MDA5, phylogenetic analysis of RIG‐I‐like receptors (RLRs) and differential gene expression of RLRs,interferons and proinflammatory cytokines after in vitro challenge with IPNV,ISAV and SAV in the salmonid cell line TO

The RIG‐I receptors RIG‐I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG‐I‐like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full‐length MDA5 sequence in Atlantic salmon, and compared it with RIG‐I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa‐d) and proinflammatory cytokines (TNF‐α1, TNF‐α2, IL‐1β, IL‐6, IL‐12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV‐infected cells. In the two aforementioned, TNF‐α1 and TNF‐α2 were highly upregulated, while in SAV‐infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.     

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C‐E3‐E2‐6K‐E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post‐vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus‐neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus‐neutralizing antibodies.     

对虾白斑综合症（WSS）的分子流行病学研究进展

本文主要依据1993年以来关于白斑综合症(WSS)的研究结果,对白斑综合症的致病因子与暴露、生物标志与生物学效应、易感性、预防策略进行综合评述,以期为有效控制WSS疫情、重振对虾养殖业提供参考。     

基于线粒体COI基因的部分鲇形目鱼类系统发育研究

本文选择COI基因片段作为分子标记,对部分鲇形目鱼类(Siluriformes)进行系统发育研究。应用通用引物PCR扩增得到13种鲇形目鱼类的134条COI基因,并与Gen Bank中获得15种鲇形目鱼类的51条COI基因进行比对分析。结果显示:鲇形目鱼类COΙ基因存在碱基插入缺失现象较少,为越南隐鳍鲇(Pterocryptis cochinchinensis)、丝尾鳠(Hemibagrus wyckioides)和黄颡鱼(Pelteobagrus fulvidraco)3种共计5个位点;平均碱基含量A+T(55.5%)显著高于G+C(44.5%)。利用Kimura’s 2-parameter计算28个物种的种间平均遗传距离为0.195,23个物种的种内平均遗传距离为0.006。系统发育树的分析结果表明,Neighbour-Joining(NJ)树较Maximum Likelihood(ML)树更为适合鲇形目鱼类的遗传发育分析;COI基因在科及其以下水平的系统进化关系与传统分类方法所认同的结果一致性较高,达到82.9%以上;在目水平的一致性的可信度较低,仅为71%;半鲿属聚为单系类群,黄颡鱼属、属和拟鲿属三者聚为一支,黄颡鱼属与拟鲿属的亲缘关系较近。     

Polymorphisms of the growth hormone gene in domesticated red sea bream populations (Pagrus major) based on minisatellite genotypes and nucleotide sequences

The genetic diversity of the growth hormone gene in domesticated red sea bream (pmaGH) was evaluated using a minisatellite DNA marker located in intron 3 (pmaGH22) and nucleotide sequences. The number of alleles of pmaGH22 was largely decreased in domesticated strains of red sea bream, and the possibility of selection pressures was also detected based on the analysis of the Hardy–Weinberg equilibrium in some strains. However, each strain inherited a small number of alleles of pmaGH22, and the entire domesticated population (combining all strains) showed a large number of alleles (n = 17), similar to the allelic richness of the wild population (n = 18.5). Based on nucleotide sequencing analysis, three synonym mutations were found in the coding regions, and also several SNPs and indels were found in the noncoding regions. In addition, four genealogies of growth hormone haplotypes were identified based on principal coordinate analysis, and these genealogies of pmaGH partly reflected allele size ranges of pmaGH22. Several haplotypes shared alleles of pmaGH22, and also fragment size homoplasy in pmaGH22 was suspected. These alleles of pmaGH22 and the haplotypes will be a useful indicator for divergence of pmaGH and for broodstock individual selection with minimum inbreeding effect.     

禽源致病性沙门氏菌四环素耐药性基因tetC的PCR扩增及序列分析

通过对临床分离并经生化试验和血清学试验鉴定的20株禽源沙门氏菌和1株标准株C79-13进行药敏试验,结果证明:有19株有高耐药性,另外2株有低耐药性,耐药率达100%;对其四环素耐药基因tetC进行了PCR扩增,结果获得以质粒为模板的特异性产物,与药敏试验符合率为100%;利用PCR检测四环素耐药基因tetC具有很高的敏感性和特异性,从而为禽类致病性沙门氏菌的耐药性检测提供了高效敏感的手段.     

基于RAG2基因序列分析仿石鲈科鱼类及其相关种类系统进化关系

为探讨仿石鲈科鱼类目前存在的分类争议问题,测定了仿石鲈科(Haemulidae),金线鱼科(Nemipteridae),眶棘鲈属(Scolopsis)等相关科属共33种鱼类RAG2基因部分序列,利用最大简约法(MP法)和贝叶斯分析法(BI法),并以黄鹦嘴鱼作为外类群构建分子系统进化树.结果显示,33种鱼类共形成三大类群,其中仿石鲈科8个属聚成一类群,金线鱼属和眶棘鲈属的种类聚成另一类群,眶棘鲈属未能与仿石鲈科形成单系.同时,基于属间遗传距离比较,眶棘鲈属与金线鱼科的遗传距离比其与仿石鲈科各属的遗传距离要小得多,表明眶棘鲈属与金线鱼科有更近的亲缘关系,支持眶棘鲈属隶属于金线鱼科的观点.此外,传统分类资料中归类于仿石鲈科的髭鲷属(Hapalogenys)也没有与仿石鲈科聚成单系,而是独自形成一支,位于进化树基部,显示出髭鲷属与仿石鲈科关系较远,与近年来认为髭鲷属应该从仿石鲈科中划分出去的结果一致.在仿石鲈科内部,少棘胡椒鲷属(Diagramma)中的少棘胡椒鲷与胡椒鲷属(Plectorhinchus)的种类关系很近.在进化树上,少棘胡椒鲷位于胡椒鲷属的内部,花尾胡椒鲷最先分化出来,位于该分支基部,支持少棘胡椒鲷归为胡椒鲷属,名称沿用原来的学名胡椒鲷的观点.     

Phylogenetic analysis and molecular methods for the detection of lymphocystis disease virus from yellow perch, Perca flavescens (Mitchell)

Lymphocystis disease is a prevalent, non-fatal disease that affects many teleost fish and is caused by the DNA virus lymphocystis disease virus (LCDV). Lymphocystis-like lesions have been observed in yellow perch, Perca flavescens (Mitchell), in lakes in northern Alberta, Canada. In an effort to confirm the identity of the virus causing these lesions, DNA was extracted from these lesions and PCR with genotype generic LCDV primers specific to the major capsid protein (MCP) gene was performed. A 1357-base pair nucleotide sequence corresponding to a peptide length of 452 amino acids of the MCP gene was sequenced, confirming the lesions as being lymphocystis disease lesions. Phylogenetic analysis of the generated amino acid sequence revealed the perch LCDV isolate to be a distinct and novel genotype. From the obtained sequence, a real-time PCR identification method was developed using fluorgenic LUX primers. The identification method was used to detect the presence/absence of LCDV in yellow perch from two lakes, one where lymphocystis disease was observed to occur and the other where the disease had not been observed. All samples of fin, spleen and liver tested negative for LCDV in the lake where lymphocystis disease had not been observed. The second lake had a 2.6% incidence of LCD, and virus was detected in tissue samples from all individuals tested regardless of whether they were expressing the disease or not. However, estimated viral copy number in spleen and liver of symptomatic perch was four orders of magnitude higher than that in asymptomatic perch.     

九种鲱科鱼类线粒体DNA细胞色素b基因相对速率测试及分歧时间估计

对鲱科9种鱼类的线粒体细胞色素b(cytb)基因序列进行综合分析。结果表明:(1)用Ne ighbour-Join ing(NJ)法构建的分子系统树显示盖纹沙丁鱼属的3种鱼类形成一个单系类群,鲱属2种鱼类形成一个单系类群,它们的bootstrap支持率均为100%。金色小沙丁鱼和短体小沙丁鱼形成一个单系类群,在NJ树中的支持率达到或超过50%。欧洲黍鲱与太平洋鲱和大西洋鲱是单系起源,其bootstrap支持率高达100%。系统发育结果还支持沙丁鱼属鱼类与盖纹沙丁鱼属鱼类是单系类群,支持率分别为87%和95%。但单系类群I(包括大西洋鲱、太平洋鲱、欧洲黍鲱)和单系类群Ⅱ(包括欧洲沙丁鱼、加州沙丁鱼、南美拟沙丁鱼、斑点盖纹沙丁鱼)之间的系统发育关系尚不明确,因为它们之间的支持率很低。(2)dN/dS的比值显著的小于1(Z-test),提示由于功能限制,cytb基因受到强烈的负选择作用。(3)基于Tajim a的1D和2D相对速率测试表明,分子钟假说在鲱科鱼类中是成立的;鲱科鱼类的分歧时间是0.12至13.45百万年间。     

Genetic structure of the whitefish (Coregonus lavaretus) population inhabiting the Miedwie Lake, Poland, based on partial ND-1 and ITS-1 gene sequences

The aim of this study was to characterize whitefish and peled populations in Miedwie Lake by means of the genetic analysis of ND-1 (NADH dehydrogenase 1) gene and the ITS-1 (internal transcribed spacer 1) region in order to distinguish native forms from whitefish/peled hybrids. In the analysis, archival specimens of Coregonus lavaretus maraena from the Berlin Museum für Naturkunde were used. Genetic analysis performed with the aid of MEGA 4.0 software explicitly indicated that samples from Miedwie Lake belonged entirely to a native (rapidly growing) form of whitefish. Furthermore, the conducted research has also provided crucial information for a C. lavaretus management program for Miedwie Lake.     

基于12SrRNA和ND3基因序列分析巨[鱼丕]遗传多样性及系统进化

为从分子水平研究巨[鱼丕](Bagarius yarrelli Sykes)的遗传多样性和系统进化关系,本实验对采集于怒江、澜沧江、元江3河流5种群中的60个个体进行12S rRNA和ND3基因序列的PCR扩增,同时与红河河口群体12个个体的12S rRNA和ND3基因序列进行比对分析,采用Mega5.02软件对碱基组成、Kimura-2 parameter遗传距离、可变位点等进行分析。应用DnaSP软件进行遗传多样性相关参数的计算。结果显示:12S rRNA和ND3基因序列长度分别为954 bp和346 bp(349 bp),分别定义了51个单倍型和42个单倍型,12S rRNA和ND3序列中的碱基平均含量分别为:T为20.8%、C为25.7%、A为32.1%、G为21.4%和T为29.5%、C为28.2%、A为28.1%、G为14.1%,表现出明显的A+T偏倚性;6个群体的群体内和群体间的遗传距离分别为0.003~0.284(12S rRNA),0.009~0.059(ND3)和0.004~1.062(12S rRNA),0.008~0.143(ND3);12S rRNA基因的51个单倍型和ND3基因的42个单倍型序列总的单倍型多样性(Hd)、核苷酸多样性(Pi)及核苷酸平均差异数(K)分别为0.972和0.937、0.035和0.079、31.414和27.176,均显示出丰富的遗传多样性。结合从GenBank中下载的12S rRNA和ND3基因同源序列的[鱼丕]科9属10种鱼类进行系统发育树的构建,结果表明巨[鱼丕]独自汇聚成一大单系群,怒江潞江坝群体、怒江三江口群体及澜沧江临沧群体间关系较近,澜沧江景洪群体可单独构成一个单系种群,红河河口群体、红河曼耗群体与其它巨[鱼丕]构成一单系种群后再同澜沧江景洪群体汇聚成一支。     

Analysis of bacterial diversity in the intestine of grass carp (Ctenopharyngodon idellus) based on 16S rDNA gene sequences

In the current study, we assessed bacterial diversity in the gut content of pond-reared grass carp (Ctenopharyngodon idellus), in the associated habitat environments (pond water and sediment) and in the ingested food (commercial feed and the reed Phragmites australis) by analysing 16S rDNA sequences from clone libraries. The highest bacterial diversity was observed in the gut content and was determined by the total number of operational taxonomic units, Shannon diversity index (H), Shannon equitability index (E_H), Coverage (C_good) and rarefaction curves calculated from the 16S rDNA gene libraries. Our data indicated that allochthonous gut microbes of grass carp were distinctively different from the corresponding environmental microbes. The pairwise similarity coefficient (C_s) for microbe communities between gut content and ingested food was higher than for those between the gut content and habitats, indicating that the allochthonous microbiota identified in the intestines of grass carp were phylogenetically closer to those in the ingested food than to those in the habitat. Based on our study and previous research, we suggest that the digesta of grass carp harbours a microbiota phylogenetic core of Proteobacteria and Firmicutes and this observation deserves further investigations with respect to a potential pool of probiotics to grass carp.     

珠带鱼和带鱼未定种Trichiurus sp.2的分子系统进化关系

针对南海大型带鱼新种——珠带鱼(Trichiurus margarites Li,1992)与日本产带鱼未定种T.sp.2(sensu Nakabo,2002)之间的系统进化关系及分类地位不明确问题,本研究通过测定采自海南岛西岸珠带鱼的线粒体16S rRNA基因部分序列,结合已报道的T.sp.2及其近缘种的同源序列,对其16S rRNA基因序列变异进行分析。基于Kimura双参数模型计算,海南岛西岸珠带鱼与日本九州、海南岛东岸及印度洋T.sp.2间的净遗传距离(0.000～0.006)远低于属内其他种间的净遗传距离(0.037～0.061),表明二者的遗传分化程度处于种内水平。邻接法(NJ)和最大似然法(ML)构建的分子系统树显示,珠带鱼与T.sp.2以极高的置信度(NJ 100%,ML 96%)聚为一支单系,并与其他带鱼形成姐妹群。根据所得分子数据并结合形态学研究资料,可以确定日本产带鱼未定种即是珠带鱼,它广泛分布于印度西太平洋。综合3种带鱼属鱼类在西北太平洋的种间分化事件,对珠带鱼的起源及演化进行了初步推测。