分子生物学技术在水产动物疾病诊断中的研究进展

近年来，分子生物技术在水产养殖疾病诊断上得以广泛应用。根据目前国内外研究的动向，对免疫检测技术、核酸检测技术、核酸技术与免疫学相结合方法等进行了综述，洋细阐明了各个技术的原理、特点及应用。病原体的提纯是应用这些方法的主要困难。     

观赏鱼体色的研究现状

体色是观赏鱼观赏价值的重要组成部分,如何改善观赏鱼的体色以提高其观赏价值,已成为提高我国观赏鱼档次的一个重要任务.对观赏鱼体色的研究进展进行了综述与分析.国内外对观赏鱼的体色研究较多,包括鱼类体色改变的原理以及利用饵料增色剂、雌核发育、性别控制、转基因等生物工程技术方法改善观赏鱼体色等.     

观赏鱼体色的研究现状

体色是观赏鱼观赏价值的重要组成部分,如何改善观赏鱼的体色以提高其观赏价值,已成为提高我国观赏鱼档次的一个重要任务.对观赏鱼体色的研究进展进行了综述与分析.国内外对观赏鱼的体色研究较多,包括鱼类体色改变的原理以及利用饵料增色剂、雌核发育、性别控制、转基因等生物工程技术方法改善观赏鱼体色等.     

快速检测水产动物疫病免疫技术研究

水产动物疾病的危害出现了日益严重的趋势,迫切需要各种灵敏、准确、快速的检测技术,其中免疫学方法以高特异性、高灵敏度在近20年里已广泛应用于水产动物疾病诊断,包括单克隆抗体技术、免疫荧光技术、免疫酶技术、免疫印迹等。免疫学方法比传统方法在许多方面有了较大进步,但也存在一些问题,随着现代分子生物学技术的高速发展,聚合酶链式反应(PCR)和核酸杂交等手段使免疫诊断显示了更加巨大的应用前景。一、单克隆抗体技术单克隆抗体与常规血清抗体相比,特异性强,能识别单一抗原决定簇,且容易制备,在对抗原,尤其是细胞表面抗原的特异性诊…     

小型热带观赏鱼与南美白对虾混养试验

正天津市是中国观赏鱼的主产地和主要集散地之一,在观赏鱼养殖品种数量及养殖规模、养殖水平、销售量及国内外影响力等方面已达到国内一流的水平。观赏渔业已成为天津市都市农业发展的重要组成部分。为了探索小型热带观赏鱼绿色高效养殖技术模式,天津市观赏鱼技术工程中心近几年开展了红剑鱼、燕尾黑玛丽、米奇鱼等小型观赏鱼与南美白对虾养殖试验,并取得了较好的经济效益、社会效益和生态效益。现将技术要点总结如下。     

征稿启示

为了进一步丰富本栏目内容,从2008年第1期开始,本栏目将进行改版,栏目内容包括观赏鱼养殖技术,观赏鱼产业发展方向,休闲垂钓技术、鱼类漫画、渔事渔趣、鱼类烹饪技巧、名人吃鱼的典故等内容,欢迎     

观赏鱼大赛获奖鱼只排行榜

2004年中国国际水族、观赏鱼大会与展览会的另一个这点是由国际知名专家裁判的高水平的观赏鱼比赛。比赛的参赛鱼包括龙鱼、金鱼、罗汉鱼、七彩神仙鱼、孔雀鱼、斗鱼等。这项国际级、高层次，奖品丰厚的赛事共有超过700尾观赏鱼参赛，是广州历来规模最大的观赏鱼比赛。     

应用生物技术防治水产养殖生物病害

本文主要介绍海洋生物技术在水产养殖生物病害防治中的研究与应用进展,内容包括应用海洋生物技术修复养殖生态环境;以单克隆抗体、核酸探针、PCR等海洋生物技术来诊断养殖生物的疾病;通过防止病毒的垂直与水平传播、免疫预防、转基因技术及破译病毒基因密码与开发基因工程药物等防治水产养殖生物疾病的海洋生物技术。     

观赏鱼类活体镜检病原的方法

我国目前饲养的观赏鱼类包括金鱼、热带鱼、红鲤和日本锦鲤。观赏鱼类通常饲养在鱼缸、水族箱和室外水泥池、人工喷水池等小型水体内。和其它饲养鱼类一样,观赏鱼是会患各种疾病,其预防和治疗的方法大致相同。由于观赏鱼类饲养在小水体中,一旦发病,容易被发现并捕捉转池进行治疗,因     

互联网上的观赏鱼网站

有关观赏鱼的网站成百上千 ,许多著名的观赏鱼网站均提供观赏鱼快讯、观赏鱼饲养和繁殖技术、疾病防治、品种介绍、观赏鱼图谱、电子公告板、观赏鱼书店、供求信息、市场行情、相关法规以及观赏鱼文化史等等资料。我们可以通过远程登录 (Ｔｅｌｎｅｔ)、文件传输 (ＦＴＰ)、电子邮件 (Ｅ ｍａｉｌ)、万维网 (ＷＷＷ )、网络新闻组 (ＵＳＥＮＥＴ)、电子公告板 (ＢＢＳ)等方法从互联网上了解国内外观赏鱼的动态 ,获取相关信息 ,交流经验 ,提高养殖、繁殖水平以及对观赏鱼的欣赏水平。观赏鱼的网站非常多 ,但水平不一 ,参差不齐。为了便于水产…     

Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay

Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study.     

Detection of channel catfish virus in channel catfish,Ictalurus punctatus (Rafinesque): use of a nucleic acid probe*

A nucleic acid probe for channel catfish virus (CCV) was constructed using recombinant DNA techniques. This probe consisted of a specific viral DNA fragment generated by digestion of CCV DNA with the restriction enzyme EcoRI. The probe was used to examine DNA isolated from tissues of fish that had been injected with CCV. Viral DNA was detected in some tissues of various injected fish. The sensitivity limit of detection was determined to be one viral DNA per cell.     

Detection of channel catfish virus in asymptomatic adult channel catfish,Ictalurus punctatus (Rafinesque)*

Abstract. Two populations of channel catfish were examined for the presence of channel catfish virus (CCV) by use of a nucleic acid probe. In one population of 22 fish with no history of CCV, viral DNA was found in every liver. These fish had previously been examined by a technique involving co-cultivation of their leucocytes with catfish tissue culture cells. The co-cultivation method had identified virus in 10 of these fish. The second fish population consisted of 14 adults that had survived a CCV outbreak in 1980. Of the 14 fish, 11 showed positive indication of CCV DNA. The tissue distribution of the CCV differed from fish to fish. All fish from the first group and one fish from the second group showed some alterations in the DNA banding patterns expected from pure CCV DNA. This might be indicative of modifications in the genomic structure of the CCV DNA when the virus is latent in a fish.     

Investigation of ornamental fish entering the EU for the presence of ranaviruses

A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.     

Detection methods of Cyprinid herpesvirus 2 infection in silver crucian carp (Carassius auratus gibelio) via a pORF72 monoclonal antibody

Cyprinid herpesvirus 2 (CyHV‐2) is the main pathogen responsible for causing haematopoietic necrosis disease in Carassius auratus gibelio. Although many nucleic acid‐based diagnostic methods have been applied, no stable and sensitive immunological diagnostic approaches have been reported. In this study, to detect CyHV‐2 in clinical samples using immunological methods, recombinant ORF72 protein (pORF72), encoded by the CyHV‐2 ORF72 gene, was used as a capture antigen to identify blood and tissues infected with CyHV‐2. First, ORF72 gene was amplified from the CyHV‐2 genome and cloned into a PGEX‐4t‐3 expression vector to produce pORF72 in Escherichia coli. The purified pORF72 was used as an immunogen to prepare monoclonal antibodies. The Western blotting assays revealed that the monoclonal antibody could specifically identify the pORF72. Furthermore, an immunohistochemical protocol and a blood smear method were established to detect CyHV‐2 in carps. The results indicate that the monoclonal antibody against pORF72 could be utilized as an effective detection tool for haematopoietic necrosis disease in Carassius auratus gibelio.     

水产养殖病原微生物的分子检测

随着水产养殖集约化程度的提高,虾类、贝类和鱼类养殖业受到病原微生物的严重影响。因此如何快速准确预测和诊断水产动植物疾病,就成为当前水产养殖业十分重要而突出的问题。随着分子生物学技术的发展,以核酸杂交、PCR、DNA指纹和16SrRNA检测等为代表的分子生物学技术在水产养殖病原体检测中已有大量应用并显示出巨大的应用前景。     

CRISPR-based platform for rapid,sensitive and field-deployable detection of scale drop disease virus in Asian sea bass (Lates calcarifer)

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource-limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR-Cas12a-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA-Cas12a). Using the viral adenosine triphosphatase (SDDV-ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked-eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi-nested PCR in field sample evaluation, our RPA-Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR-based methods. Therefore, owing to its high accuracy, rapidity and user-friendliness, the developed RPA-Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large-scale epidemics.     

Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real‐time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real‐time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real‐time PCR assays determined using genomic DNA templates from three target viruses, 12 non‐target viruses and 25 aquatic bacterial species were 100%. The intra‐run and inter‐run coefficients of variation of both assays were <5%. The real‐time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.     

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.     

Antimicrobial susceptibility pattern of Flavobacterium columnare isolates collected worldwide from 17 fish species

Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim‐sulfadimethoxine and trimethoprim‐sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards – amongst others – ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.