Pathological characteristics of olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> experimentally infected with <Emphasis Type="Italic">Streptococcus parauberis</Emphasis>

Pathological characteristics of olive flounder Paralichthys olivaceus experimentally infected with Streptococcus parauberis were studied. Various stressful conditions, aeration and netting stress in particular, led to induced mortality by S. parauberis. Netting stress-induced mortality was positively correlated to bacterial dose and stressful conditions. Inflammation of the heart and pericarditis was the major pathological change observed in olive flounder experimentally infected with S. parauberis. During the infected period, the number of bacteria in the infected olive flounder was recorded over time. S. parauberis remained in all fish organs tested, especially in the heart and brain.     

Effects of dietary fish oil replacement by microalgae raw materials on growth performance,body composition and fatty acid profile of juvenile olive flounder,Paralichthys olivaceus

Replacing dietary fish oil with DHA‐rich microalgae Schizochytrium sp. and EPA‐rich microalgae Nannochloropsis sp. for olive flounder (Paralichthys olivaceus) was examined. Three experimental isonitrogenous and isolipidic diets with lipid source provided by 50% fish oil (F50S50), 50% (M50F25S25) and 100% microalgae raw material (M100) respectively were compared with a soybean oil (S100) diet as control. Triplicate groups of olive flounder juveniles (16.5 ± 0.91 g) were fed the experimental diets, and a group was fed the control diets for 8 weeks in a recirculation system. Results showed feed efficiency and growth performance were not significantly changed when fish oil (FO) was totally substituted by soybean oil (SO) or microalgae raw material (MRM). The whole‐body composition, lipid content of liver and muscle, and lipid composition of plasma were not significantly influenced by the total substitution of FO by MRM. The polyunsaturated fatty acids (PUFA) content of muscle and liver declined in fish fed S100 diet, whereas it was not significantly reduced in fish fed M50F25S25 and M100 diets. The total substitution of FO by MRM not only maintained the levels of arachidonic acid, EPA or DHA but also increased n‐3/n‐6 ratio. In conclusion, MRM as the sole lipid source is sufficient to obtain good feed efficiency, growth performance and human health value in olive flounder juveniles.     

Dietary choline requirement of juvenile olive flounder (Paralichthys olivaceus)

An eight‐week study was conducted to determine the optimum dietary choline level in juvenile olive flounder, Paralichthys olivaceus. Seven diets were prepared to contain 0, 250, 500, 750, 1,000, 2,000 and 3,000 mg/kg diet. Juveniles (5.9 ± 0.03 g; 5.5 ± 0.4 cm; mean ± SD) were randomly distributed into 21 tanks (25 fish/tank) and fed one of the diets in triplicates. Survival rate of fish fed the diet containing the lowest choline level was significantly lower than those of fish fed the other diets (p < 0.05). Final body weight, weight gain, specific growth rate, feed efficiency and protein efficiency ratio significantly increased with increasing choline levels up to 1,000 mg/kg diet. Whole‐body protein and lipid contents increased in accordance with choline levels up to 750 mg/kg diet, beyond which they plateaued. Liver and muscle lipid contents elevated with increasing choline levels up to 2,000 mg/kg diet. Plasma cholesterol esters, triglycerides, cholesterol and total lipids were significantly influenced by the graded choline levels; however, responses of those indices were not identical. Broken‐line analyses of weight gain and liver choline concentrations responding to the graded choline levels revealed that choline requirements of the juvenile flounder could be between 847 and 1,047 mg/kg diet.     

<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

Efficacy and toxicity of praziquantel in helminth‐infected barbel (Barbus barbus L.)

This study evaluated efficacy and toxicity of the pyrazinoisoquinoline anthelmintic praziquantel (PZQ) in barbel infected with metacercariae of Diplostomum spathaceum and adult Pomphorhynchus laevis, and assessed antioxidant biomarkers and the lipid peroxidation response in juvenile barbel post‐treatment. The estimated 96‐hr LC50 of PZQ was 28.6 mg/L. For evaluation of efficacy, barbel naturally infected with D. spathaceum were exposed to a 10 and 20 mg/L PZQ 4‐day bath treatment. Both concentrations were 100% effective against D. spathaceum and significantly (p < .01) affected the activity of catalase, superoxide dismutase, glutathione reductase and glutathione‐S‐transferase as well as levels of reduced glutathione in liver and muscle. The efficacy of orally administered PZQ was assessed in adult barbel naturally infected with P. laevis. Fish were administered 10, 30 and 50 mg/kg of body weight and examined via gut dissection after 6 days. The 50 mg/kg dose significantly decreased the intensity of infection. Praziquantel is a feasible bath treatment for barbel infected with D. spathaceum and has potential for oral treatment of broodfish infected with P. laevis.     

Effects of dietary supplementation of citrus by‐products fermented with a probiotic microbe on growth performance,innate immunity and disease resistance against Edwardsiella tarda in juvenile olive flounder,Paralichthys olivaceus (Temminck & Schlegel)

Two consecutive studies were conducted to evaluate the dietary supplementation of citrus by‐products (CB) fermented with probiotic bacteria on growth performance, feed utilization, innate immune responses and disease resistance of juvenile olive flounder. In Experiment I, five diets were formulated to contain 0% (control) or 3% four different CB fermented with Bacillus subtilis (BS), Enterococcus faecium (EF), Lactobacillus rhamnosus (LR) and L. plantarum (LP) (designated as CON, CBF‐BS, CBF‐EF, CBF‐LR and CBF‐LP, respectively). During 10 weeks of a feeding trial, growth performance and feed efficiency were not significantly different among all the fish groups. However, fish fed CBF containing diets had significantly higher survivals than the CON group. Disease resistance of fish against Edwardsiella tarda was increased by the fermentation of CB. In Experiment II, we chose the BS as a promising probiotic and formulated five diets to contain 0%, 2%, 4%, 6% and 8% CBF‐BS. Growth performance was not significantly affected by the CBF‐BS supplementation during 6 weeks of a feeding trial. Innate immunity of fish was significantly enhanced by CBF‐BS supplementation. Myeloperoxidase and lysozyme activities were increased in a dose‐dependent manner by dietary CBF‐BS inclusions. In a consecutive challenge test against E. tarda, an increased disease resistance was found by CBF‐BS supplementation. These studies indicate that the fermentation process of CB with probiotic has beneficial effects on innate immunity and thereby increases disease resistance of olive flounder against E. tarda. Bacillus subtilis can be used as a promising probiotic microbe for by‐product fermentation in fish feeds.     

Genetic variants of Kudoa septempunctata (Myxozoa: Multivalvulida), a flounder parasite causing foodborne disease

Foodborne disease outbreaks caused by raw olive flounders (Paralichthys olivaceus) parasitized with Kudoa septempunctata have been reported in Japan. Origins of olive flounders consumed in Japan vary, being either domestic or imported, and aquaculture‐raised or natural. Although it is unknown whether different sources are associated with different outcomes, it is desirable to identify whether this is the case by determining whether unique K. septempunctata strains occur and if so, whether some are associated with foodborne illness. We here developed an intraspecific genotyping method, using the sequence variation of mitochondrial genes. We collected olive flounder samples from foodborne disease outbreaks, domestic fish farms or quarantine offices and investigated whether K. septempunctata genotype is associated with pathogenicity or geographic origin. The 104 samples were classified into three genotypes, ST1, ST2 and ST3. Frequency of symptomatic cases differed by genotypes, but the association was not statistically significant. Whereas K. septempunctata detected from aquaculture‐raised and natural fish from Japan were either ST1 or ST2, those from fish inspected at quarantine from Korea to Japan were ST3. Our method can be applied to phylogeographic analysis of K. septempunctata and contribute to containing the foodborne disease. The genotype database is hosted in the PubMLST website ( http://pubmlst.org/kseptempunctata/ ).     

Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP‐infected whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), in India

Stunted growth in pond‐reared Litopenaeus vannamei was observed in different farms located in Tamil Nadu and Andhra Pradesh, India. No mortality was associated with stunted growth. PCR assay on these samples revealed the presence of Enterocytozoon hepatopenaei (EHP) in stunted shrimp. Tissue distribution of EHP in naturally and experimentally infected shrimp was studied by PCR and histology. Histological examination revealed the presence of EHP in hepatopancreas and gut, but not in other organs. The PCR assay revealed the presence of EHP in all the organs tested in both naturally and experimentally infected shrimp. Healthy shrimp were challenged with E. hepatopenaei by intramuscular injection and oral route, and no mortality was observed in both routes after 30 days post‐challenge. Different developmental stages of the microsporidian parasite were observed in the hepatopancreatic epithelial cells. Biochemical parameters such as total protein, albumin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase were measured in the haemolymph of naturally and experimentally EHP‐infected shrimp. All biochemical parameters mentioned were found to be significantly higher in EHP‐infected shrimp when compared to normal shrimp. This is the first report relating AST and ALT levels to EHP infection in naturally and experimentally infected shrimp.     

Determination of hormones inducing oocyte maturation in <Emphasis Type="Italic">Chalcalburnus tarichi</Emphasis> (Pallas, 1811)

Chalcalburnus tarichi is an endemic cyprinid species living in the Lake Van basin, in eastern Anatolia, Turkey. The present study was undertaken to determine which hormones induce oocyte maturation in C. tarichi. The levels of 17α,20β,21-trihydroxyprogesterone (20β-S), progesterone (P), 17α-hydroxyprogesterone (17α-HOP), 11-deoxycortisol (11-DOC), and 17α-hydroxy-20β-dihydroprogesterone (17,20β-P) were measured in fish caught from Lake Van and the Karasu River, and injected with human chorionic hormone (hCG) (1,000 and 1,500 IU/kg). Oocytes of fish caught from the lake were also incubated in vitro with different doses (50, 200, and 1,000 ng/ml) of 20β-S, 17α-HOP, 11-DOC, and 17,20β-P. 11-DOC was found to be the most effective hormone among those measured for inducing oocyte maturation in vivo and in vitro. 17,20β-P could not be determined in the plasma of any fish in vivo (P < 0.05). 1,000 IU/kg dose of hCG given by injection caused a statistically significant increase in all plasma hormone levels (P < 0.05). It was found that there was a significant decrease in the P level only at 1,500 IU/kg dose of hCG injected (P < 0.05), while the level of other hormones increased at this dose (P < 0.05). It was also determined that all the hormones were effective in germinal vesicle breakdown (GVBD) in in vitro oocyte culture (P < 0.05). However, 11-DOC was found to be the most effective hormone in GVBD at a dose of 200 ng/ml (70% GVBD). In conclusion, 11-DOC synthesized during final oocyte maturation in C. tarichi was found to be a potent inducer of GVBD, which shows that 11-DOC may be described as an oocyte maturation steroid in this species.     

Dietary copper requirement of fingerling Heteropneustes fossilis for formulating copper‐balanced commercial feeds

Dietary copper requirement of Heteropneustes fossilis (6.74 ± 0.03 g) was determined by feeding purified diets containing same protein (400 g/kg) and gross energy (17.89 kJ/g) but different levels of copper for 12 weeks. Graded amount of CuSO₄.5H₂O (0, 1.96, 3.93, 5.89, 7.86, 9.82, 11.79 mg/kg) was supplemented to basal diet to attain desired dietary copper levels (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/kg). Analysed dietary copper concentrations were 4.28, 4.63, 5.28, 5.70, 6.19 and 6.69 mg/kg. Absolute weight gain, feed conversion ratio and protein gain improved with the increasing levels of dietary copper up to 5.28 mg/kg. Further inclusion of copper at a level of 5.70 mg/kg did not improve the above parameters. Significantly higher (p < .05) plasma ceruloplasmin, liver copper‐zinc superoxide dismutase, catalase activities and lower thiobarbituric acid reactive substances were evident in fish receiving diets with 5.28 and 5.70 mg/kg copper compared to other groups. Whole body and liver copper concentrations increased significantly (p < .05) with increasing dietary copper levels. Quadratic regression analysis of absolute weight gain, feed conversion ratio, protein gain and broken‐line regression analysis of plasma ceruloplasmin activity and liver TBARS value against the variable dietary copper levels depicted the dietary copper requirements for fingerling H. fossilis in the range of 5.24–5.68 mg/kg.     

Disseminated infection due to Exophiala pisciphila in Cardinal tetra,Paracheirodon axelrodi

Cardinal tetra, Paracheirodon axelrodi (Schultz, 1956), kept in an ornamental tank, was found to be affected by severe invasive mycosis. Externally, the disease manifested as abdominal swelling, and internally, the anterior part of the intestine was extremely bloated with abundant dematiaceous septate hyphae and an accumulation of fluid. Histopathologically, a granulomatous inflammatory response was observed in the intestine wall, kidney and spleen. We assume that the mycotic agent was primarily deposited in the intestine and was then gradually disseminated to the other organs. DNA sequencing of ITS and LSU rDNA regions and phenotypic characterization were used for identification of the isolated fungus. The obtained data confirmed that the infection was caused by Exophiala pisciphila. The disease was subsequently reproduced in the carp fingerling using intramuscular and intraperitoneal injection of a spore suspension. The 13th day after intramuscular infection, a marked elevation of neutrophils was recorded in the peripheral blood; this involved a proliferation of band forms and segmented forms. As far as we know, this is the first report of infection due to E. pisciphila in Cardinal tetra.     

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir,a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real‐time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue‐specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.     

Organic acids blend as dietary antibiotic replacer in marine fish olive flounder,Paralichthys olivaceus

The present study was conducted to evaluate the efficacy of organic acid blends as dietary antibiotic replacer in marine fish olive flounder, Paralichthys olivaceus. Fish averaging 3.5 ± 0.05 g (mean ± SD) were fed one of the four experimental diets: (1) without antibiotic or organic acid (Control/CON); (2) with antibiotic—50 mg oxytetracycline per kg body weight per day (OTC); (3) with organic acid blend A—4 g/kg diet (OA_A); and (4) with organic acid blend B—4 g/kg diet (OA_B), for 10 weeks. At the end of the experiment, total intestinal bacterial counts in fish‐fed OA_A, OA_B and OTC were significantly lower than that of fish‐fed CON diet (p < 0.05). Further, the group of fish‐fed organic acid blends (OA_A, OA_B) or antibiotic (OTC)‐supplemented diets exhibited lower intestinal Vibrio sp. counts compared with fish‐fed CON diet. Disease challenge test with bacteria Edwardsiella tarda showed significantly lower cumulative mortality rates for the group of fish‐fed OA_A, OA_B or OTC than that of fish‐fed CON diet (p < 0.05). There were no negative effects on the growth, serological characteristics and proximate composition among the group of fish‐fed different experimental diets. Therefore, the present experiment demonstrates that blends of organic acid could be a promising alternative to dietary antibiotics for the preventive and/or curative health management in marine fish olive flounder aquaculture.     

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.     

Effects of dietary soy isoflavones on growth,antioxidant status,immune response and resistance of juvenile grass carp (Ctenopharyngodon idella) to Aeromonas hydrophila challenge

The current study was conducted to evaluate the effect of dietary soy isoflavones (SI) on growth performance, antioxidant status, immune response and resistance to Aeromonas hydrophila in juvenile grass carp (Ctenopharyngodon idella). Six diets were formulated to contain 0 (control), 10, 50, 100, 500 or 1,000 mg SI per kg feed. Each diet was randomly allotted to triplicate net cages, and each net cage was stocked with 30 fish. The fish were fed one of the experimental diets to satiation twice per day for 60 days. The results showed that the WGR and DGC of the 500 mg/kg SI‐supplemented group were significantly higher than those of the non‐SI‐supplemented group (p < .05). Serum LZM and IgM activities in the SI‐supplemented groups were improved compared to the control group. SOD and GSH‐Px levels of fish fed the diet containing 500 mg/kg SI were significantly enhanced compared to those of fish fed the control diet (p < .05). Additionally, serum CAT, GSH‐Px and AKP activities in 50, 100 and 500 mg/kg SI‐supplemented groups were significantly higher than that in the control group (p < .05). The expression of most immune‐related genes (including IFN‐γ2, TNF‐α, M‐CSF2, IL‐6, IL‐12p40 and IL‐4) was significantly affected by dietary supplementation of SI. The group fed with 500 mg/kg SI had the highest 7‐day cumulative survival rate after challenge test (p < .05). The current results revealed that dietary inclusion of SI could improve the immune response and resistance against A. hydrophila and the supplementation level is suggested to be 500 mg/kg diet.     

The in vitro efficacy of oxytetracycline against re‐isolated pathogenic Aeromonas hydrophila carrying the cytolytic enterotoxin gene through hybrid catfish,Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) in Thailand

Aeromonas hydrophila is a pathogen infecting farmed hybrid catfish, Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) which incurs substantial economic losses in Thailand. The study aimed at a genetic tracking of A. hydrophila infection and the in vitro assessment of the efficacy of antibiotics against its virulent strains. Five clinical strains from catfishes and Nile tilapia were employed. They were 3‐passage re‐isolated through healthy hybrid catfish and the cytolytic enterotoxin gene (AHCYTOEN) of individuals was traced. Each of the re‐isolates at a dose of ~6.67 × 10⁵ CFU/g was intraperitoneally injected into ~15 g‐healthy hybrid catfish and their pathogenicity were observed for 7 days. It was found that AHCYTOEN was carried over whereas typical signs of motile aeromonas septicaemia were found in the specimens. The bacterial strains of Nile tilapia origin did not induce mortality but those of catfish origins (80%–100% rate of mortality). The strains were susceptible to the tetracycline antibiotics, and oxytetracycline produced MIC₅₀ and MBC as low as 0.007–0.031 μg/ml and 1–8 μg/ml respectively. As oxytetracycline specifically inhibited pathogenic A. hydrophila in vitro, it is recommended that an appropriate dosage regimen of the drug should be established.     

Immobilization antigen-independent protection of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

To determine whether olive flounder (Paralichthys olivaceus) immunized with lysate of Philasterides dicentrarchi could be protected immobilization antigen (i-antigen) type independently, fish were immunized with lysate of ciliates obtained from in vitro cultures or ciliates obtained from infected fish. Fish immunized with ciliates obtained from infected fish were completely protected against artificial infection with in vitro cultured P. dicentrarchi, in spite of a weak or no serum agglutination activity against in vitro cultured ciliates. The present results indicate that i-antigen-independent protection was elicited by immunization of fish with the ciliates lysate, and the cultured ciliates would provide a good source for preparation of vaccines, which are cross-protective against various i-antigen types of P. dicentrarchi.     

Identification and Characterization of Megalocytivirus Type 3 Infection with Low Mortality in Starry Flounder,Platichthys stellatus,in Korea

MVSF‐12 belonging to megalocytivirus type 3 was isolated from cultured starry flounder; Platichthys stellatus, at the moribund or subclinical stage with low mortalities in Korea. Of 20 apparently healthy fish in the farms, 17 were also confirmed in nested polymerase chain reaction to be infected by megalocytivirus. When starry flounder; rock bream, Oplegnathus fasciatus; and olive flounder, Paralichthys olivaceus, were artificially infected by MVSF‐12 or iridovirus sachun‐1 (IVS‐1, megalocytivirus type 1), starry flounder and olive flounder showed no mortality until Day 24, without any clinical signs including enlarged spleen, while rock bream showed 100% mortality by IVS‐1 infection within 11 d but no mortality by MVSF‐12. Although it was not pathogenic, MVSF‐12 in infected fish at Day 24 was viable when successfully cultured in vitro using primary rock bream embryo cells and produced a cytopathic effect (CPE) with the viral copy numbers between 1.76 × 10⁷ and 5.23 × 10⁷/mL of culture supernatant. In conclusion, this study demonstrates the low pathogenicity of MVSF‐12 and low susceptibility of starry flounder and olive flounder to both MVSF‐12 and IVS‐1. Indeed, MVSF‐12 at the subclinical stage could be replicated with CPE in vitro, indicating a possibility to induce pathogenic effects and mortality under adverse environment or physiologic conditions.