Effectiveness of a vaccine against red sea bream iridoviral disease in a field trial test.

Since 1990, red sea bream iridovirus (RSIV) has caused high mortalities in the summertime in cultured red sea bream Pagrus major in southwest Japan. To establish control measures for red sea bream iridoviral disease (RSIVD), the effectiveness of a formalin-killed viral vaccine was evaluated in a field trial. Two groups each consisting of 1000 juvenile red sea bream were either intraperitoneally inoculated with vaccine (vaccinated group) or were not vaccinated (non-vaccinated group). After vaccination, the fish were held for 1 wk, then transferred to a marine net pen and observed for 12 wk. The cumulative mortalities caused by RSIVD in the vaccinated group or control group were 19.2 and 68.5%, respectively. Additionally, the presence of virus antigen in the spleen was investigated and body weight was measured 6 and 12 wk post vaccination. In the vaccinated group, viral antigen was not detected. The increase in body weight of vaccinated fish was significantly (p < 0.05) greater than that of control fish. These results suggest that the vaccine against RSIVD was effective in 1 field trial.     

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.     

Histopathological and ultrastructural features of enlarged cells of humpback grouper Cromileptes altivelis challenged with Megalocytivirus (family Iridoviridae) after vaccination

The genus Megalocytivirus in the family Iridoviridae encompasses isolates of red sea bream iridovirus (RSIV) and grouper sleepy disease iridovirus (GSDIV). In the present study, humpback grouper Cromileptes altivelis juveniles were challenged with GSDIV after vaccination with a commercial RSIV vaccine. The unvaccinated group (in duplicate) showed higher mortalities (59.3 to 66.7%) than the vaccination group (0% mortalitiy, in duplicate). Surviving fish in the vaccinated group displayed masses of enlarged cells in the spleen. Electron microscopy revealed that they contained hemosiderin granules within the cytoplasm. In contrast, moribund fish from the unvaccinated group exhibited large numbers of inclusion body-bearing cells (IBCs) in the spleen, while surviving fish displayed masses of enlarged cells in which a small number of GSDIV virions were assembled.     

鳜鱼传染性脾肾坏死病毒(ISKNV)PCR检测方法的建立及虹彩病毒新证据

利用真鲷虹彩病毒（ＲＳＩＶ）核苷酸还原酶小亚单位（ＲＮＲＳ）基因保守区设计的一对引物,建立了鳜鱼传染性脾肾坏死病毒（ＩＳＫＮＶ）特异的ＰＣＲ检测体系。运用该体系检测ＩＳＫＮＶ,具有简便、快速、敏感、特异等特点,为诊断与预防ＩＳＫＮＶ提供了一个重要的手段。通过对ＰＣＲ产物的克隆与序列分析,发现ＩＳＫＮＶ ＰＣＲ扩增产物与ＲＳＩＶ ＲＮＲＳ基因相应序列的同源性很高,达到９２．５％,进一步证明ＩＳＫＮＶ     

Phenotypic Diversity of Infectious Red Sea Bream Iridovirus Isolates from Cultured Fish in Japan

Megalocytivirus is causing economically serious mass mortality by infecting fish in and around the Pacific region of Asia. The recent emergence of many new iridoviruses has drawn attention to the marked taxonomic variation within this virus family. Most studies of these viruses have not included extensive study of these emergent species. We explored the emergence of red sea bream iridovirus (RSIV) on a fish farm in Japan, and we specifically endeavored to quantify genetic and phenotypic differences between RSIV isolates using in vitro and in vivo methods. The three isolates had identical major capsid protein sequences, and they were closely related to Korean RSIV isolates. In vitro studies revealed that the isolates differed in replication rate, which was determined by real-time quantitative PCR of viral genomes in infected cells and cell culture supernatant, and in cell viability, estimated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay for infected cells. In vivo studies showed that the isolates exhibit different virulence characteristics: infected red sea bream showed either acute death or subacute death according to infection with different isolates. Significant differences were seen in the antigenicity of isolates by a formalin-inactivated vaccine test. These results revealed that variant characteristics exist in the same phylogenetic location in emergent iridoviruses. We suggest that this strain variation would expand the host range in iridoviral epidemics.Iridoviruses are designated as icosahedral cytoplasmic DNA viruses; this group of viruses has different hosts, including fish, amphibians, and insects, causing economic and environmental problems. The family Iridoviridae includes five genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and Megalocytivirus (45). Piscine iridoviruses belong to the genera Ranavirus, Lymphocystivirus, and Megalocytivirus. In recent years, the genotypical variation between newly found iridovirus strains included in the genus Ranavirus has been studied in this viral family (5). However, the properties of and variation between iridovirus species have not been well characterized except for a few iridoviruses isolated from amphibians (11, 46), fish (8, 18, 19), and insects (23, 42, 44). Most studies attempting to differentiate variants have relied on genotypic rather than phenotypic properties.Members of the Megalocytivirus genus produce characteristic basophilic inclusion bodies in the enlarged cells of host fish organs, which have been collected from mass mortalities occurring in wild and cultured fish species (14, 37). Red sea bream (Pagrus major) aquaculture has suffered great losses from the prevalence of red sea bream iridovirus (RSIV) infection in Japan. RSIV has been assigned to the Megalocytivirus genus. The virus was first isolated from cultured red sea bream in western Japan in 1990 (22).Many other piscine iridoviruses have been reported in Asian countries (7, 24, 25) from more than 100 different species (41), including freshwater and marine fish (19). RSIV is closely related genetically to viruses isolated from ornamental fish in Southeast Asia, based on nucleotide sequence studies (21, 39). A formalin-inactivated RSIV vaccine has been used in juvenile marine fish against this disease (31, 32).The incidence of iridovirus infection has been increasing among cultured fish in Japan (29, 30). In addition, genetic and phenotypic iridovirus variants suggest the presence of diverse variants in this virus group (16). In the present study, we examined the detailed properties of three RSIV isolates from two fish species collected from fish farms in western Japan. Using in vitro and in vivo processes, we focused close examination specifically on quantitative genetic and phenotypic differences between the three isolates. The aim of this study was to increase understanding of the epidemiology of RSIV.     

Application of the arming system for the expression of the 380R antigen from red sea bream iridovirus (RSIV) on the surface of yeast cells: a first step for the development of an oral vaccine

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell-surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell-surface display technology in yeast.     

Protection of red sea bream Pagrus major against red sea bream iridovirus infection by vaccination with a recombinant viral protein

Megalocytivirus infections cause serious mass mortality in marine fish in East and Southeast Asian countries. In this study the immunogenicity of crude subunit vaccines against infection by the Megalocytivirus RSIV was investigated. Three capsid proteins, 18R, 351R and a major capsid protein, were selected for use as crude subunit vaccines. High homology among Megalocytivirus types was found in the initial sequence examined, the 351R region. Red sea bream (Pagrus major) juveniles were vaccinated by intraperitoneal injection of recombinant formalin‐killed Escherichia coli cells expressing these three capsid proteins. After challenge infection with RSIV, fish vaccinated with the 351R‐recombinant bacteria showed significantly greater survival than those vaccinated with control bacteria. The 351R protein was co‐expressed with GAPDH from the bacterium Edwardsiella tarda in E. coli; this also protected against viral challenge. A remarkable accumulation of RSIV was observed in the blood of vaccinated fish, with less accumulation in the gills and spleen tissues. Thus, the 351R‐GAPDH fusion protein is a potential vaccine against Megalocytivirus infection in red sea bream.     

Characterization of an iridovirus detected from cultured turbot Scophthalmus maximus in Korea

Juvenile turbot Scophthalmus maximus that became sick during an outbreak of disease at mariculture facilities at Go-Chang, Korea, in 2003, were examined to identify the cause of the disease. The fish had pale body color, an enlarged abdomen, protruding eyes, an enlarged spleen and kidney, and pale gills and/or liver. Histopathogical examination revealed basophilic enlarged cells in the kidney, spleen, gills, heart, stomach, intestine, liver, pancreas and skin. Hexagonal viral particles with a diameter of 136 to 159 nm were observed in the enlarged cells. A specific 1299 bp fragment of the major capsid protein (MCP) gene of the turbot iridovirus (TBIV) was amplified by PCR. Sequence homology was greater than 93.76% between the MCP gene in TBIV and the same gene in 5 viruses in the tentatively proposed genus Tropivirus (family Iridoviridae): red sea bream iridovirus, sea bass iridovirus, grouper sleepy disease iridovirus, African lampeye iridovirus and dwarf gourami iridovirus. These results suggest that the virus detected from turbot is similar to the proposed genus Tropivirus.     

Identification of Quantitative Trait Loci for Resistance to RSIVD in Red Sea Bream (<Emphasis Type="Italic">Pagrus major</Emphasis>)

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIβ, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIβ. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIβ gene and could be useful for MAS of red sea bream.     

Molecular cloning and expression analysis of two lipopolysaccharide-induced TNF-α factors (LITAFs) from rock bream,Oplegnathus fasciatus

Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α factor (LITAF) plays an important role controlling the expression of TNF-α and the other cytokine genes in the presence of LPS. However, two LITAF homologues have not been characterized in fish. In this study, we cloned two distinct LITAF (RbLITAF1 and RbLITAF2) cDNAs from rock bream (Oplegnathus fasciatus) and characterized their expression profiles after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding regions of RbLITAF1 and RbLITAF2 cDNAs were 492 bp and 417 bp, encoding 153 and 138 amino acid residues, respectively. The genes consisted of a LITAF domain. RbLITAF1 was highly expressed in the spleen and heart of healthy rock bream, whereas RbLITAF2 was highly expressed in the gill, intestine and stomach. In spleen, the gene expression of RbLITAF1 and RbLITAF2 were increased until 5 days post-infection (dpi), and then decreased at 7 dpi. In kidney, E. tarda and RSIV infection led to induction of the RbLITAF1 gene at 1 dpi, RbLITAF2 gene was down-regulated after pathogen infection. These results suggest that RbLITAFs may be involved in the LITAF-mediated immune response and regulate systemic immune responses against pathogen infection.     

Molecular characterization of peroxisome proliferator-activated receptors (PPARs) and their gene expression in the differentiating adipocytes of red sea bream Pagrus major

To investigate the molecular mechanism of fish adipocyte differentiation, the three subtypes of PPAR genes (alpha, beta and gamma) were characterized in a marine teleost red sea bream (Pagrus major). The primary structures of red sea bream PPARs exhibited high degrees of similarities to their mammalian counterparts, and their gene expression was detected in various tissues including adipose tissue, heart and hepatopancreas. During the differentiation of primary cultured red sea bream adipocytes, three PPARs showed distinct expression patterns: The alpha subtype showed a transient increase and the beta gene expression tended to increase during adipocyte differentiation whereas the gene expression level of PPARgamma did not change. These results suggest that they play distinct roles in adipocyte differentiation in red sea bream. In the differentiating red sea bream adipocytes, mammalian PPAR agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2), ciglitazone and fenofibrate did not show clear effects on the adipogenic gene expression. However, 2-bromopalmitate increased the PPARgamma and related adipogenic gene expression levels, suggesting the gamma subtype plays a central role in red sea bream adipocyte differentiation and in addition, fatty acid metabolites can be used as modulators of adipocyte function. Thus our study highlighted the roles of PPARs in fish adipocyte differentiation and provided information on the molecular mechanisms of fish adipocyte development.     

Pathogenesis of Gastroenteritis Caused by Vibrio carchariae in Cultured Marine Fish

Serious mortality among the cultured grouper Epinephelus coioides, characterized by a swollen intestine containing yellow fluid (gastroenteritis), occurred in 1993 in Taiwan. A bacterium isolated from the intestinal fluid and head kidney of moribund groupers was identified as Vibrio carchariae. Since then, the same Vibrio species has also been isolated from moribund black sea bream Acanthopagrus schlegeli, yellowfin sea bream A. latus, Japanese sea bass Lateolabrax japonicus, and red drum Sciaenops ocellatus suffering from the same syndrome. Each isolate was virulent to the respective fish. Recently, a similar syndrome, flounder infectious necrotizing enteritis, also caused by V. carchariae in summer flounder Paralichthys dentatus, was reported in Rhode Island. The extracellular products (ECPs) of V. carchariae strains EmI82KL (from grouper), Rd (from red drum), and SfUSA (from summer flounder, U.S.A.) were virulent to the grouper or red drum. A 33-kDa serine protease partially purified from the ECP of strain EmI82KL was lethal to the fish. All the moribund or killed fish exhibited gastroenteritis except those killed within 12 hours. This report is the first to show that intraperitoneal injection of the ECP or protease in the fish is virulent and can reproduce gastroenteritis. The serine protease was suggested as a major toxin in the grouper or red drum secreted by V. carchariae.     

Histopathology of cultured sea bream Sparus aurata infected with sanguinicolid trematodes

The present study is the first report of a sanguinicolid infection affecting sea bream Sparus aurata cultured in net cages in the NE of Spain. The disease was associated with trickling mortalities during the cold season (1999 and 2000). Examination of gill wet mounts of the affected population revealed that sanguinicolid infection was present in 82.6 and 100% of the fish sampled in 1999 and 2000, respectively. Adult flukes, which were located in the kidney, were tentatively identified as members of the family Sanguinicolidae, subfamily Cardicolinae. Eggs and miracidia were found in the gill vascular structures. The inflammatory response triggered by the parasites was moderate and the lesions caused by either eggs and miracidia in the gills or adult flukes in the kidney were not extremely severe, possibly because of the moderate intensity of the parasitosis. Histological observations of sanguinicolid infected sea bream presented here are compared with those reported in other fish species. The role played on sea bream morbility and mortality by other factors (occurrence of a simultaneous moderate monogenean infection, immunological impairement related to low water temperatures) is discussed.     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

Mineral composition in fillets of sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata): a comparison of cultured and wild fish

The objective of this study was to determine some of the trace mineral elements in four commercial feeds commonly available in Turkey for marine culture and in fillets of cultured and wild sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The feeds and cultured fish were from four different fish farms operating in the same region but with four slightly different feeds. Concentrations of Iron (Fe), Zinc (Zn), Manganese (Mn), Copper (Cu), Lead (Pb), Cobalt (Co), Nickel (Ni), Chromium (Cr), and Cadmium (Cd) were analyzed in the feeds and fillets of cultured as well as wild fish. Significant differences in the mineral concentrations existed within feed groups, cultured fish groups as well as between cultured and wild fish. Fe, Zn, and Mn in the feeds, Fe, Co, and Zn in sea bass and Fe, Zn and Co in sea bream were predominant among the nine analyzed minerals. Fe, Zn, Mn, Cr, and Ni contents in the fillets of wild sea bass were significantly, (P < 0.05) lower than those of the cultured sea bass groups. Co, Cr, Pb, and Ni in the fillets of wild sea bream were also significantly (P < 0.05) lower than those of cultured sea bream groups. These differences in trace elements in the cultured and wild fish were probably related to differences in their dietary mineral concentrations.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.     

斑点叉尾鮰病毒实时荧光LAMP检测方法的建立

斑点叉尾鮰病毒(Channel catfish virus,CCV)属于大DNA病毒,是一种能引起斑点叉尾鮰(Letalurus punetaus)病毒性疾病的重要病原.研究以编码产物为磷酸激酶的CCV ORF77基因为靶标,设计了能识别靶基因上的8个独立区域的6条特异引物,利用基于环介导等温扩增技术(Loop-med...