Takafumi Ito

Severe oxidative stress is thought to be a principal cause of jaundice of yellowtail Seriola quinqueradiata

Abstract The jaundiced yellowtail Seriola quinqueradiata had yellow discolored skin and hyperbilirubinemia. Histology revealed that hemosiderin and ceroid occurred in the spleen. In the vascular system, there were vasculitis and sinusoidal dilatation. Hepatic cells showed degenerative figures with pyknotic or karyolytic nuclei, basophilic cytoplasm, cytoplasmic vacuoles in various sizes, and ceroidosis. In the bile duct system, however, there was no noticeable change. Hemoglobin contents of jaundiced fish were similar to those of control fish. Plasma taurine levels of jaundiced fish were significantly lower than those of control ( p 〈0.05). Judging from 2-thiobarbituric acid reactive substance values, in vivo lipid peroxidation progressed in the liver of jaundiced fish. In the plasma of jaundiced fish, contents of unconjugated bilirubin were significantly higher than those of conjugated bilirubin. A large amount of substance Xs, breakdown products of bilirubin scavenging active oxygen species, existed in the plasma of jaundiced fish. Therefore, jaundiced fish may suffer from severe oxidative stress, which might be a principal cause of jaundice. Liver superoxide dismutase and glutathione peroxidase activities of jaundiced yellowtail were significantly lower than those of controls.

Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus.

Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (μ1), μ2, μNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus ‘piscine orthoreovirus 2’ (PRV-2). Reverse transcription–quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.

Growth of cyprinid herpesvirus 2 (CyHV-2) in cell culture and experimental infection of goldfish Carassius auratus.

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20°C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25°C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.

Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-1O1H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 microgram/ml.

Susceptibility of Japanese Cyprininae fish species to cyprinid herpesvirus 2 (CyHV-2)

Cyprinid herpesvirus 2 (CyHV-2) is known as the causative agent of herpesviral haematopoietic necrosis (HVHN) of goldfish (Carassius auratus). Recently, the virus has also been detected from Prussian carp (C. gibelio) and crucian carp (C. carassius) from European and Asian countries. To analyze the risk of spreading to new host species, the susceptibility of other fish species to the virus is essential. In this study experimental infections of indigenous Cyprininae species in Japan were performed by immersion in and intraperitoneal injection of a CyHV-2 isolate. Although Edonishiki, a variety of goldfish, immersed with the virus showed a cumulative mortality of 90%, no mortality was observed in ginbuna C. auratus langsdorfii, nagabuna C. auratus buergeri, nigorobuna C. auratus grandoculis and common carp Cyprinus carpio. Cumulative mortality was 100, 20 and 10% in intraperitoneally injected Edonishiki, ginbuna and nagabuna, respectively. Furthermore all Edonishiki immersed with the virus died. However, even after stimuli of sudden temperature changes, the immersed ginbuna and nagabuna did not die. Moreover no mortality was observed in co-reared Ranchu, another variety of goldfish, with immersed ginbuna and nagabuna although all three Ranchu co-reared with immersed Edonishiki died. CyHV-2 DNA was detected and the virus was re-isolated from all dead fish. Moreover CyHV-2 DNA was detected from some of the surviving Carassius spp. These results revealed that susceptibility of Japanese indigenous Cyprininae fish species to CyHV-2 is much lower than for goldfish. In addition, ability of replication of CyHV-2 might be different among Carassius fish species.

Typing of viral hemorrhagic septicemia virus by monoclonal antibodies

Seven mAbs with specific reaction patterns against each of the four genotypes and eight subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24 reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57 reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of mAb VHS-1.24 to be aa 32-34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57 and VHS-3.75 were found to be aa 43 and 45-48, aa 117 and 121, and aa 103, 118 and 121 of the N-protein, respectively.

Stress response to daily temperature fluctuations in common carp, Cyprinus carpio L.

The littoral zone of lakes and lagoons is often used by fish for feeding or reproduction. However, the large changes in temperature that are typical of natural environments, including the littoral zone, represent a potential stressor for fish. Despite the importance of this habitat, little is known about the effect of daily temperature fluctuations on the stress responses of fish. We monitored daily temperature changes in the near-shore and offshore regions of a natural lagoon between May and July 2008–2010. We observed large temperature fluctuations more frequently in the near-shore zone than the offshore zone. We then exposed common carp (Cyprinus carpio) to a temperature regime similar to that observed in the near-shore zone and measured the levels of cortisol released into the water. The rate of cortisol release increased when carp were exposed to an increase in temperature of ~0.6°C/h over a 5-h period. Conversely, there was no change in the rate of release when temperatures decreased. Our results highlight the importance of maintaining high temporal resolution when evaluating the stress response to daily fluctuations temperature.

Effects of experimentally induced infections of goldfish Carassius auratus with cyprinid herpesvirus 2 (CyHV-2) at various water temperatures

In this study, we examined the influence of water temperature on the development of herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus after experimentally induced infection with cyprinid herpesvirus 2 (CyHV-2). In Expt 1, Ryukin goldfish were infected with CyHV-2 by intraperitoneal injection and maintained at 4 different water temperatures. Cumulative mortalities of the 15, 20, 25 and 30°C groups were 10, 90, 90 and 60%, respectively. Therefore, the temperature range of 20-25°C is considered highly permissive for HVHN. One of 6 surviving fish of the 15°C group died after a rapid temperature increase to 25°C at 30 d post infection. All 3 Edonishiki goldfish, co-reared with the surviving Ryukin in tanks where the water temperature was increased from 15 to 25°C, died. In Expt 2, Edonishiki goldfish were exposed to CyHV-2 by bath immersion at 13 or 24°C, resulting in cumulative mortalities of 0 and 87%, respectively, at 28 d post-exposure. No mortality of the surviving Edonishiki in the 13°C treatment was observed when the water temperature was increased to 24°C. In addition, in Expt 2, no mortality was observed in any Ranchu co-reared with CyHV-2-immersed Edonishiki in the group where water temperature was increased from 13 to 24°C, even after re-immersion challenge with CyHV-2. It is interesting to note that CyHV-2 DNA was detected in the kidneys of 4 of the 5 surviving Ranchu co-reared with the CyHV-2-immersed Edonishiki group where the water temperature was increased from 13 to 24°C. Therefore, it is likely that the surviving Edonishiki of the 13°C group were virus carriers. This study indicates that most fish infected with CyHV-2 at 13-15°C acquire resistance to HVHN, but as carriers they are able to infect naïve fish.

Differences in the susceptibility of Japanese indigenous and domesticated Eurasian common carp (Cyprinus carpio), identified by mitochondrial DNA typing, to cyprinid herpesvirus 3 (CyHV-3).

In 2004, a massive mortality of wild common carp (Cyprinus carpio) due to CyHV-3 infection occurred in Lake Biwa. Although common carp of two different mitochondrial types (Japanese indigenous and domesticated Eurasian) occur in the lake, the majority of the dead fish seemed to be the indigenous type. The apparent high mortality in the indigenous type implies a higher susceptibility of this type to CyHV-3. To test the hypothesis that the susceptibility of indigenous and Eurasian types differ, we performed experimental infections with CyHV-3 among 2 groups of the indigenous type, and for the Eurasian type 4 groups of domesticated common carp and 4 groups of koi carp. Fish were immersed in CyHV-3 isolate and kept at 24°C. Both groups of the indigenous type died more rapidly compared with the 8 groups of the Eurasian type. Cumulative mortality in both indigenous groups reached 95-100%, whereas the cumulative mortalities of domesticated common carp (30-95%) and koi carp (35-100%) were more varied. CyHV-3 genome in the organs of the indigenous type increased more rapidly after the viral exposure and reached higher peak levels than those of the domesticated strain. These findings revealed that susceptibility of the indigenous type of carp to CyHV-3 can be considered especially high.

Prevalence of red sea bream iridovirus among organs of Japanese amberjack (Seriola quinqueradiata) exposed to cultured red sea bream iridovirus

Red sea bream iridovirus (RSIV) is a representative of the genus Megalocytivirus which causes severe disease to aquaculture fish, mainly in Japan and South-east Asia. However, information to assess the viral kinetics of RSIV in fish is limited since reports on experimental infection by the immersion route, which is the natural infection route, are scarce. In this study, a method to evaluate the titre of RSIV was first developed. Experimental infections were continuously performed using RSIV cell culture as the inoculum to juvenile Japanese amberjack (Seriola quinqueradiata) (initial body weight 12.2 g) by immersion at three different concentrations. In addition, to investigate the prevalence of the virus among the organs of experimentally infected fish, viral DNA was measured at selected times by the real-time PCR method following viral inoculation by immersion. The developed titration method showed a 10(2) increase in sensitivity compared with the conventional method. We demonstrated that grunt fin cells can be used for continuous passage of RSIV. In the experimental infection, fish which were intraperitoneally injected with the RSIV cell culture or immersed with RSIV cell culture at 10(-2) and 10(-3) dilutions showed cumulative mortalities of 100 %. The results of measurements of the viral DNA of several organs from infected fish strongly suggest that the spleen is the target organ of RSIV in Japanese amberjack. Since the viral genome was detected from all the tested organs of two of five surviving fish which appeared to completely recover from the disease, it is suggested that these fish may become carriers.