Diseased Eels Research Articles

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and visual detection of Anguillid herpesvirus 1

China has the largest aquaculture eel production in the world. High-density cultivation pattern often results in an outbreak of epidemic diseases. Since the 1990s, eel “mucus sloughing and hemorrhagic septicemia disease” was often broke out in China, and brought huge economic losses to eel breeders. Anguillid herpesvirus 1 (AngHV) was detected and isolated from the diseased eel, and proved to be the pathogen of the disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive, and specific detection of AngHV. A set of six primers targeting the ORF51 gene of AngHV was designed, which could effectively detect purified AngHV virions, AngHV-infected cells, or eel tissue samples. The suitable reaction temperature is 63℃, and the reaction time is 40 min. There was no cross-reaction with eel and other fish viruses, including Infectious pancreatic necrosis virus (IPNV), Marine birnavirus (MABV), Rana grylio virus (RGV), Cyprinid herpesvirus 3 (CyHV-3), and Eel iridovirus (EIV). The lower detection limit of the AngHV LAMP assay is 10 copies of AngHV genome DNA, which is at least 100 times more sensitive than conventional PCR in detecting AngHV. The assay could effectively detect AngHV from collected samples with typical clinical symptoms of AngHV infection. It suggested that the LAMP assay could be used in specific detection of AngHV and has great potential for early diagnosis of AngHV infection in the farm.

Regulatory role of VvsB protein on serine protease activity of VvsA in Vibrio vulnificus.

Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB. This study examines the contribution of VvsB to the functionality of VvsA. In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA. The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA. The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.

Complete genome sequence of Edwardsiella sp. NBRC12716 isolated in 1962 from the liver of diseased eel.

We report the complete genome sequence of Edwardsiella sp. NBRC12716 isolated from a diseased eel in 1962. The genome consists of a single, circular chromosome 3,771,060 bp in length with 59.74% GC content and encodes 25 rRNA, 96 tRNA, and 3,182 protein-coding genes.

Mortality in farmed European eel (Anguilla anguilla) in Italy due to Streptococcus iniae

BackgroundStreptococcal infections are one of the main causes of fish disease. During the last decade, Streptococcus iniae has become one of the most important aquatic pathogens worldwide, causing high losses in marine and freshwater finfish. Clinical signs in farmed fish include loss of appetite, lethargy and grouping at the bottom of the tank. Gross changes comprise darkening of the skin and haemorrhage at the basis of fins and opercula. To date, S. iniae has been isolated from several wild and farmed fish species but never in the European eel (Anguilla anguilla). In Europe, eel production from aquaculture is around 4500 tonnes and Italy is the third largest producer. This communication represents the first report of an outbreak of S. iniae infection in European eels.Case presentationThe outbreak occurred at an eel farm in northern Italy between May 2021 and September 2021. The outbreak caused about 2% mortality per month, resulting in the loss of about 10% of the farmed fish. The diseased eels showed apathy, lethargy, inactivity and inappetence. In July 2021, three eels were necropsied. Necropsy revealed skin and branchial hyperaemia, a few skin ulcers, and diffuse peritoneal congestion with a few haemorrhagic-like spot lesions. Swab samples for bacteriology were taken from the kidneys, liver, spleen, and brain. Additionally, four eels were opened and swap samples as above were taken. All the investigated eels were found dead. Bacteriological examination revealed growth of Streptococcus spp. from all samples. Identification of S. iniae was done by biochemical characterization, the API20STREP microsystem, 16S rDNA sequencing, and MALDI-TOF. Antimicrobial therapy (oxytetracycline and erythromycin) was ineffective.ConclusionsThis is the first report of S. iniae infection in the European eel. Although this may be an isolated outbreak, it is of concern due to the losses associated with this pathogen in fish worldwide and because the European eel is an endangered species. Due to the difficulties of controlling the disease with antimicrobials, it is advisable to plan other effective control measures, such as improving water quality and the environmental conditions, reducing fish density, improving biosecurity, and by using immunostimulants and, when possible, vaccines.

Fast and accurate identification by MALDI-TOF of the zoonotic serovar E of Vibrio vulnificus linked to eel culture.

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Investigation of the Expression of Serine Protease in Vibrio vulnificus.

Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a metalloprotease-negative strain isolated from a diseased eel, produces a 45-kDa chymotrypsin-like alkaline serine protease known as VvsA. The gene encoding vvsA also includes another gene, vvsB with an unknown function; however, it is assumed to be an essential molecular chaperone for the maturation of VvsA. In the present study, we used an in vitro cell-free translation system to examine the maturation pathway of VvsA. We individually expressed the vvsA and vvsB genes and detected their mRNAs. However, the sample produced from vvsA did not exhibit protease activity. A sodium dodecyl sulfate (SDS) analysis detected the VvsB protein, but not the VvsA protein. A Western blotting analysis using a histidine (His)-tag at the amino terminus of proteins also showed no protein production by vvsA. These results suggested the translation, but not the transcription of vvsA. Factors derived from Escherichia coli were used in the in vitro cell-free translation system employed in the present study. The operon of the serine protease gene containing vvsA and vvsB was expressed in E. coli. Although serine proteases were produced, they were cleaved at different sites and no active mature forms were detected. These results indicate that the operon encoding vvsA and vvsB is a gene constructed to be specifically expressed in V. vulnificus.

Isolation of a novel adomavirus from cultured American eels, Anguilla rostrata, with haemorrhagic gill necrosis disease.

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29±0.23 at 6days post-infection. The virions were non-enveloped with a diameter of 75-85nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A.rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.

First isolation of Flavobacterium psychrophilum associated with reports of moribund wild European eel (Anguilla anguilla) in Scotland.

In late April 2015, the River Dee Trust informed Marine Scotland Science, Fish Health Inspectorate (FHI), that there had been observations of dead and moribund European eels on the River Dee. Later in May, the Spey Fishery Board also reported a number of moribund European eels in a rotary screw smolt trap on the River Spey. In total, 10 cases involving moribund eels were investigated in 2015 and one case in 2016. In addition, a health screen was conducted to investigate the potential presence of Flavobacterium psychrophilum in healthy eels and Atlantic salmon from the River Dee in 2015. Externally, the diseased eels demonstrated white patches in different locations of the body. In all cases, F.psychrophilum was detected by bacterial isolation and/or molecular methods. Three isolates were further characterized by whole-genome sequencing (WGS) as belonging to sequence type 15 (ST15). Histological examination of diseased European eels revealed lesions at the level of the integument. The pathogen screen for F.psychrophilum in wild healthy fish tested negative by PCR. Further investigation is required to understand the pathogenicity of this bacterium on the health of eels and the potential impact on the wild salmonid population.

Persistent development of adomavirus and aquareovirus in a novel cell line from marbled eel with petechial skin haemorrhage.

In Taiwan, a petechial haemorrhage disease associated with mortality has affected marbled eels (Anguilla marmorata). The eels were revealed to be infected with adomavirus (MEAdoV, previously recognized as a polyoma-like virus). In this study, cell line DMEPF-5 was established from the pectoral fin of a diseased eel. DMEPF-5 was passaged >70 times and thoroughly proliferated in L-15 medium containing 2%-15% foetal bovine serum at 20-30°C. Transcripts of neural cell adhesion molecule 1 and nestin genes, and nucleic acids of MEAdoV and a novel reovirus (MERV) in the cells were demonstrated by reverse transcription-polymerase chain reaction analysis. Phylogenetic analysis revealed that the AdoV LO8 proteins mostly relate to adenovirus adenain, whereas MERV is close to American grass carp reovirus in Aquareovirus G, based on a partial VP2 nucleotide sequence. DMEPF-5 cells are susceptible to additional viral infection. Taken together, the marbled eels with the haemorrhagic disease have coinfection with MEAdoV and MERV, and the pathogenic role of MEAdoV and MERV warrants research. DMEPF-5 has gene expression associated with mesenchymal stem and progenitor cells and is the first cell line persistently infected with adomavirus and aquareovirus. DMEPF-5 can facilitate studies of such viruses and haemorrhagic disease.

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum.The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA–DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.

Genome Sequence of a Marbled Eel Polyoma-Like Virus in Taiwan.

ABSTRACTWe report here the complete genome sequence of a virus isolated from a diseased marbled eel (Anguilla marmorata) in Taiwan. The virus has been characterized as being related to Japanese eel endothelial cell-infecting virus (JEECV), with a large T-antigen-like protein. The sequence of the marbled eel virus displays low homology to the JEECV.

Regulation systems of protease and hemolysin production in Vibrio vulnificus.

Vibrio vulnificus, a gram-negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema-forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone-like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.

Phylogenetic analysis of the pathogenic genus Aeromonas spp. isolated from diseased eels in China

Analyses of 16S rRNA and housekeeping genes (HKGs) were valuated as identification markers for pathogenic Aeromonas isolated from diseased eels. The interrelationships of 32 Aeromonas strains which had been verified as pathogens to eels were studied using phylogenetic analysis with 16S rRNA and HKG sequences (cpn60, gyrB, rpoB and dnaJ) and identified by Biolog automatic microbiology analysis system (gene III). From the analysis of 5 genes, the mean gene divergences of 16S rRNA, cpn60, gyrB, rpoB and dnaJ in 32 isolates were 1.4 ± 0.2%, 7.1 ± 0.7%, 5.2 ± 0.5%, 2.2 ± 0.4% and 6.8 ± 0.5%, respectively. The results of comparative phylogeny between nucleotide based analyses (excluding the third codon position) of four HKGs with the sequences from 55 strains of Aeromonas (including 23 referenced strains of Aeromonas) showed cpn60 and dnaJ have higher discriminate power than gyrB and rpoB comparing with the taxonomical identification by Biolog system. In addition, amino acid sequences of concatenated cpn60-rpoB-gyrB is a good method for Aeromonas pathogens identification. This study showed analysis of HKG sequences can be used as an alternative method for sound identification of bacterial pathogens isolated from diseased eels in China.

Isolation of a novel polyomavirus, related to Japanese eel endothelial cell-infecting virus, from marbled eels, Anguilla marmorata (Quoy & Gaimard).

Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T-antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell-infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK-1 and EO-2 cells at temperatures 25-30°C, and the progeny virus yields were 10(7.0) , 10(7.4) and 10(7.7) TCID50 mL(-1) , respectively. The purified virions were icosahedral particles, 70-80nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.

Outbreak of Edwardsiella tarda infection in farm-cultured giant mottled eel Anguilla marmorata in China

In 2012, a freshwater farm in south China experienced a severe disease outbreak in cultured giant mottled eel Anguilla marmorata, resulting in high mortality. The diseased eels showed extreme ulcerative injuries throughout their bodies and obvious swelling of the liver and body kidney. A pure culture of a bacterial strain, designated HZHM, was isolated and identified as Edwardsiella tarda based on its physiological and biochemical characteristics and on phylogenetic analysis of 16S rRNA and fimbrial genes. The HZHM strain was unable to produce hydrogen sulphide (H2S) from thiosulphate in biochemical testing. In addition, results of the challenge trial confirmed that the HZHM strain was virulent for A. marmorata, with an LD50 value of 1.3 × 106 colony-forming units (CFU). Histologically, severe hemorrhage occurred in most organs, increased melano-macrophage reaction was observed in the spleen and body kidney, and cytoplasmic vacuolation and liquefactive necrosis were found in the brain. To our knowledge, this is the first report of E. tarda-associated pathogenicity in A. marmorata.

Phylogenomics characterization of a highly virulent Edwardsiella strain ET080813T encoding two distinct T3SS and three T6SS gene clusters: Propose a novel species as Edwardsiella anguillarum sp. nov.

As important zoonotic organisms causing infections in humans, Edwardsiella bacteria are also notorious leading fish pathogens haunting worldwide aquaculture industries. However, the taxa are now widely recognized to be misclassified, which hurdled the understanding of the epidemiology and development of effective diagnostics and vaccines. Currently the genus Edwardsiella consists of three species Edwardsiella tarda, E. ictaluri, and E. hoshinae. Previous phylogenomic analysis revealed that E. tarda strains display two major highly divergent genomic types (genotypes), EdwGI and EdwGII, and the former represents a genotype of fish-pathogenic isolates and being recently proposed as a novel species E. piscicida, sp. nov. Here multiple phylogenetic analyses and the genome-level comparisons of EdwGI strains disclose that the phylogroup strains from diseased eel formed an obviously distinct cluster that could be equated with a new species status. The phylogenetic evidence for the new species assignment was also supported by corresponding DNA–DNA hybridization estimation values and by phenotypic characteristics. Interestingly, further comparative genomics reveals that these strains have acquired the locus of enterocyte effacement (LEE) genes and as a result these bacteria contain at least 2 sets of distinct T3SS and 3 sets of T6SS gene clusters, respectively. It is therefore proposed that the phylogroup strains from diseased eel should be classified as Edwardisella anguillarum sp. nov., and the type strain is ET080813T (=DSM27202T=CCUG 64215T=CCTCC AB2013118T=MCCC 1K00238T). These findings will contribute to development of species-specific control measures against Edwardsiella bacterium in aquatic animals, while also shedding light on the pathogenesis evolution in Edwardsiella bacterium.

Antimicrobial susceptibility and genetic characterisation of oxytetracycline-resistant Edwardsiella tarda isolated from diseased eels

Edwardsiellosis is one of the most important bacterial diseases in eels. Edwardsiella tarda (E. tarda) isolates (n=94) from diseased eels were screened against the eight most commonly used antimicrobial agents...

Vibrio vulnificus outbreaks in Dutch eel farms since 1996: strain diversity and impact

Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab®). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab® is recommended for epidemiological purposes during V. vulnificus outbreaks.

Regulation system of serine protease production in Vibrio vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel

Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in cultured eels or shrimps, as well as in immunocompromised humans. An extracellular metalloprotease has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA. In the present study, we first clarified the regulatory characteristics of the VvsA production in V. vulnificus strain NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel. V. vulnificus coordinates expression of virulence genes in response to the bacterial cell density, which is termed quorum sensing (QS) and is mediated by the small diffusible molecule called autoinducer 2 (AI-2). When cultivated at 26°C, the vvsA expression was closely related with expression of the luxS gene encoding the synthase of the AI-2 precursor LuxS. Both VvsA and AI-2 were sufficiently secreted at early stationary phase of the bacterial growth. In contrast, when cultivated at 37°C, far less amounts of the AI-2 and VvsA were produced even at the stationary phase. Disruption of the luxS gene was found to decrease significantly the vvsA expression and VvsA production. Disruption of luxO encoding the central response regulator of the QS circuit caused an increase in the vvsA expression and VvsA production at the logarithmic growth phase. These findings indicate that VvsA production is positively regulated by the V. vulnificus LuxS-dependent QS system, which operated more effectively at 26°C than at 37°C.

양식 동남아산 뱀장어, Anguilla bicolor의 Anguillid herpesvirus-1 (AngHV-1) 감염증

양식 동남아산 뱀장어 Anguilla bicolor에 아가미의 출혈 및 간장의 충혈을 특징으로 하는 질병의 원인을 조사한 결과 Anguillid herpesvirus (AngHV-1) 의 감염이 확인되었다. 병어는 병리조직학적으로 아가미의 상피세포의 증생과 모세혈관의 출혈, 간장의 공포변성 및 중심정맥의 충혈이 관찰되었다. 병어를 <TEX>$35^{\circ}C$</TEX>에 10일간 승온 사육한 결과 아가미의 형태가 건강어의 아가미와 유사한 형태로 회복되었으며, 폐사율도 10%로 승온 처리가 폐사율 감소에 효과적인 것으로 나타났다. Diseased eel (Anguilla bicolor) displayed severe hemorrhages in the gills, and congestion and swelling in the liver. During the epizootic, the water temperature was <TEX>$28^{\circ}C$</TEX> and the morality rates were about 5%. No parasites were found on the gills and skin. Bacteria were not cultured from any internal organs using TSA or SS agar at <TEX>$28^{\circ}C$</TEX> for 48 hrs. Histopathologically, the gills showed epithelial hyperplasia in the base of secondary gill lamellae and hemorrhages in the capillaries. Some cells in the proliferated interlamellar epithelia exhibited marginal hyperchromatosis. And severe vacuolated changes in the parenchymal cells and congestion in the central veins were observed in the liver. The specific amplicon (396 bp) was detected from gills and opercula of affected eel PCR using Anguillid herpesvirus-1 (AngHV-1) -specific primer sets HVAPOLVPSD (5-'GTG TCG GGC TTT GTG GTG C-3') and HVAPOLOOSN (5'-CAT GCC GGG AGT CTT TTT GAT-3'). Sequencing analysis of the amplicon demonstrated that this gene was 99% homologous to the AngHV-1 sequence deposited in GenBank. This is the first report of AngHV-1 outbreak in the farmed shortfin eels (A. bicolor) in Korea. When diseased fish were maintained for 10 days at water temperatures of <TEX>$32^{\circ}C$</TEX> and <TEX>$35^{\circ}C$</TEX>, the cumulative mortalities were 100% and 10%, respectively. Even though the AngHV-1 genome in the gills from the eel kept at <TEX>$35^{\circ}C$</TEX> was detected using PCR, the structure of gill filaments was similar with that of normal fish. Increasing the water temperature to <TEX>$35^{\circ}C$</TEX> was an effective way to diminish the mortality of AngHV-1 affected eel.