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.     

Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.     

Evidence of potential vertical transmission of tilapia lake virus

Tilapia lake virus disease (TiLVD) is an emerging viral disease in tilapia with worldwide distribution. Although the horizontal transmission of TiLV has been demonstrated through the cohabitation of infected fish with susceptible fish, no direct experiment showed the potential of vertical transmission from broodstock to progeny. In this study, natural outbreaks of TiLV in broodstock and fry in two tilapia hatcheries were confirmed. The TiLV genomic RNA was detected in liver and reproductive organs of infected broodstock, while infective virus was isolated in susceptible cell line. In situ hybridization assay confirmed the presence of TiLV in the ovary and testis of naturally infected fish and experimentally challenged fish. Moreover, early detection of TiLV in 2‐day‐old fry and the presence of TiLV genomic RNA and viable virus in the testis and ovary suggested the possible transfer of this virus from infected broodstock to progenies. As infective virus was present in gonads and fry in natural outbreak and experimental fish, the importance of biosecurity and prevention of the virus to establish in the hatchery should be emphasized. Hence, the development of TiLV‐free broodstock and the maintenance of high biosecurity standards in the hatcheries are essential for any attempt of virus eradication.     

Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease—Neutrase^®—was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.     

基于图像处理和线性拟合的鱼体尾柄测量方法研究

水产养殖场内的鱼体尾柄测量在养殖管理中有着重要意义。目前的尾柄测量方法主要依靠人工,操作复杂、效率低,且结果主观性强。研究提出一种基于图像处理和线性拟合的鱼体尾柄测量方法。首先在养殖场通过装置采集图像,通过边缘检测和轮廓提取得到鱼体的外部轮廓,基于鱼的形态特征这一先验知识,提取到尾柄感兴趣区域,最后对尾柄区域进行角点检测和最小二乘直线拟合,得到尾柄线长度。结果显示,该方法平均测量耗时1.01 s,平均测量偏差0.34 mm,测量精度达到95%以上。采用该方法不仅可以解决人工测量操作繁琐、主观性强等问题,而且能够避免测量过程耗时过长,为鱼体尾柄的大批量非接触式测量提供有效新途径。     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

Detection of channel catfish virus DNA in acutely infected channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction

Channel catfish virus (CCV) causes an acute haemorrhagic disease in channel catfish, Ictalurus punctatus (Rafinesque), fry and fingerlings. The present study describes a polymerase chain reaction (PCR)-based assay for detection of CCV DNA in the tissues of acutely infected juvenile catfish. The assay is rapid, sensitive and specifically detects CCV DNA derived from epidemiologically distinct viral isolates. The use of two independent PCR primers sets, each specific for particular CCV genes (open reading frames 8 and 59), provides a means to confirm the results and minimize false-positive results. The method identifies CCV DNA in several tissues of acutely infected fish, including the brain, blood, intestine, kidney and liver. The CCV PCR assay is useful for the diagnosis of acute CCV disease and for studies to investigate the molecular basis of CCV pathogenesis.     

传染性鲑鱼贫血症病毒实时荧光环介导等温扩增检测方法的建立

根据ISAV的基因保守序列,利用LAMP Designer软件设计了6条引物,采用新型的环介导等温扩增设备进行扩增和检测,优化了反应条件,分析了所建立方法的特异性和灵敏度,并与RT-PCR和实时荧光RT-PCR进行比较。研究表明,该方法最适反应温度为64℃,反应10 min就可以观察到明显的扩增。该方法灵敏度高,检测限为78.4 fg RNA,比常规RT-PCR灵敏度高100倍,与实时荧光定量RT-PCR灵敏度相当;特异性好,与传染性胰腺坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、出血性败血症病毒(VHSV)、鱼类病毒性神经坏死病病毒(VNNV)、鱼腹水病毒(YAV)等14种主要鱼类病毒没有交叉反应。结果表明,本研究建立了ISAV的实时荧光环介导等温扩增检测方法,实验能对整个扩增过程进行实时监测,提高检测灵敏度的同时,防止由于开盖跑电泳或加染料而导致的污染。     

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.     

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.     

Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel)

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.     

An improved in situ hybridization method for the detection of fish pathogens

A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.     

Development of a multiplex RT-PCR assay for simultaneous detection of the major viruses that affect rainbow trout (Oncorhynchus mykiss)

The major viral diseases that affect rainbow trout (Oncorhynchus mykiss) are viral haemorrhagic septicaemia, infectious haematopoietic necrosis, infectious pancreatic necrosis and sleeping disease. In the presented study, we developed a multiplex RT-PCR (mRT-PCR) assay for the simultaneous detection of these four rainbow trout viruses in a single assay. The choice of primers was carried out based on the expected size of the fragments, the temperature and time required for the amplification, and the specificity for the target sequence. Firstly, the method was optimised using reference strains of viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and sleeping disease virus (SDV) cultivated with permissive cell culture lines; subsequently, the method was used for the identification of these viral infections in rainbow trout samples. Twenty-two samples of rainbow trout, clinically suspected of having viruses, were analysed by the developed method to detect the presence of the four viruses, by directly analysing the animal tissues. The mRT-PCR method was able to efficiently detect the viral RNA in infected cell culture supernatants and in tissue samples, highlighting the presence of single infections as well as co-infections in rainbow trout samples. VHSV/SDV and IHNV/SDV co-infections were demonstrated for the first time in rainbow trout. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.