Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S‐23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype‐specific biochemical characteristics and genetic characteristics of 16S‐23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.     

Clonal analysis of Yersinia ruckeri based on biotypes, serotypes and outer membrane protein-types

Abstract. The biotypes, serotypes and outer membrane protein-types (OMP-types) of 135 isolates of Yersinia ruckeri were analysed in an attempt to identify clonoal groups and to examine to further detail the relationships between islotes from wide geographic areas. Each isolate could be assigned to one of two biotypes (1 and 2). one of five 0-serotypes (01, 02, 05, 06 and 07), and one of five OMP-types (1–5). Outer membrane protein analysis was able to differentiate between isolates within a given serotype. Thus, serotype 01 isolates types 1 and 2. and serotype 07 isolates consisted of OMP-types 1 and 5. Whereas OMPwith serotype 07, OMP-types 1 and 2 were associated with serotypes 01, 02, 05, 06 and 07, for the majority of the disease outbreaks in Europe and represented 79% of the European isolates. A combination of biotype, serotype and OMP-type analysis was demonstrated to be useful in epidemiological studies of Y. ruckeri.     

Genetic and phenotypic comparison of Nocardia seriolae isolated from fish in Japan

The phenotypic and genetic characterizations of 58 isolates of the fish pathogen Nocardia seriolae , from amberjack, Seriolae dumerili , yellowtail, Seriola quinqueradiata , Japanese flounder, Paralichthys olivaceus , and chub mackerel, Scomber japonicus, in Japan from 1970–2005, were examined to investigate the epidemiological relationship between isolates. The phenotypic and genetic characterizations were determined by α-glucosidase activity and biased sinusoidal field gel electrophoresis (BSFGE) analysis, respectively. There was no α-glucosidase activity in strains isolated from 2000–05 ( n  = 50) with a few exceptions ( n  = 3), while all strains isolated from 1970–90 ( n  = 8) were positive. In BSFGE analysis, digestions with restriction enzymes Xba  I and Ase  I produced 15 and 16 restriction patterns, respectively. All restriction patterns obtained from 50 strains isolated during 2000–05 were unrelated to those obtained from eight strains isolated during 1970–90, with the exception of two strains isolated during recent outbreaks. Based on the phenotypic and genetic characterizations, recent outbreaks of nocardiosis in Japan are suggested to be epidemiologically unrelated to earlier outbreaks in Japan. Although a low genetic relationship was observed in the restriction pattern between recent and earlier isolates, identity was confirmed between these groups of isolates because five representative strains showed 99.9% homology with N. seriolae ATCC43993^T in the 16S rRNA sequence.     

Occurrence and distribution of settling and newly settled spotted halibut Verasper variegatus and Japanese flounder Paralichthys olivaceus in shallow nursery grounds around Shimabara Peninsula, western Japan

The occurrence and distribution of settling and settled spotted halibut Verasper variegatus were compared with those of Japanese flounder Paralichthys olivaceus in shallow habitats around Shimabara Peninsula, western Japan, to elucidate the settlement and recruitment characteristics of spotted halibut, a rare pleuronectid flatfish in Japan. Push-net sampling conducted at nine sites in March and April 2003 collected spotted halibut from four nurseries located at eastern areas of Shimabara Peninsula in Ariake Bay; Japanese flounder were sampled from eight nurseries. Comparison of microhabitat distributions in a tidal-flat nursery during February?CApril in 2003?C2007 revealed that metamorphosing larvae and juveniles of spotted halibut, which showed the drastic body coloration (black to brown) and behavioral changes (frequent swimming to settlement), were distributed mainly in the lower edge of the intertidal area, whereas Japanese flounder inhabited both intertidal and subtidal areas. Metamorphosing larvae of spotted halibut (12.6?C16.5?mm standard length; n?=?24) were collected in March when the highest densities were observed, whereas metamorphosing larvae of Japanese flounder (10.0?C15.9?mm, n?=?55) were observed during March?CApril. Our results suggest that spotted halibut has a narrower time/space window for recruitment to the specific nurseries compared with Japanese flounder.     

Tunic morphology and viral surveillance in diseased Korean ascidians: soft tunic syndrome in the edible ascidian,Halocynthia roretzi (Drasche), in aquaculture

‘Soft tunic syndrome’ causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non‐diseased specimens using chinook salmon embryo (CHSE‐214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.     

牙鲆雌核发育研究进展

牙鲆(Paralichthys olivaceus)是一种重要的海水经济鱼类,其雌性生长速度显著快于雄性。因此,生产全雌牙鲆对提高养殖产量和经济效益具有重要意义。雌核发育是一种特殊的有性生殖方式,在这种生殖方式中,灭活的精子进入卵子,激活胚胎发育,但精核不与卵核融合形成合子,所以雌核发育的个体只具有母本的遗传信息。目前,牙鲆已成为雌核发育技术应用于产业的代表性鱼类。本文对牙鲆人工诱导雌核发育的研究进展进行综述,介绍牙鲆减数分裂雌核发育和有丝分裂雌核发育的诱导方法,以及雌核发育技术在性别控制、克隆制备等育种研究中的应用。最后,作者提出"雌核发育育种体系"构想,并对其在鱼类育种和遗传研究中的应用前景进行展望。     

Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan

Abstract. During the period from 1965 to 1980, 263 Vibrio anguillarum strains from ayu, Plecoglossiis altivelis (Temminck & Schlegel), two from rainbow trout. Salmo gairdneri Richardson, and two from eel, Anguilla japonica (Temminck & Schlegel), were collected from fish suffering from vibriosis in various parts of Japan. On the basis of cross-agglutination and cross-absorption tests with thermostable (O) antigens, six distinct serotypes (A, B, C, D, E and F) were established among 12 selected strains of V. anguillarum . 241 strains isolated from ayu and two strains from rainbow trout belonged to serotype A, six strains from ayu and one strain from eel to serotype B, 12 strains from ayu to serotype C, three strains from ayu to serotype D, one strain from ayu to serotype E, and one strain from eel to serotype F. V. anguillarum strains belonging to serotypes D, E and F have not been detected from ayu, rainbow trout and eel since 1973; these serotypes appear to be minor types. V. anguillarum strain NCMB 6 and 1669 belong to our serotype A and V. anguillarum 813 to our serotype C.     

Influence of Hatchery Protocols on Mitochondrial DNA Variation in Japanese Flounder Juveniles

The Japanese flounder Paralichthys olivaceus is a commercially important fish that is stocked extensively from hatchery rearing programs in Japan. To examine the genetic variability of hatchery-raised juveniles of the Japanese flounder that are used for stocking into natural waters, we analyzed a portion of the mitochondrial genome. The mtDNA region extending from the 3'half of the cytochrome b gene to the central domain of the control region was PCR amplified and analyzed using 11 restriction endonucleases. We identified 34 polymorphic cleavage sites out of a total of 61 sites, which resulted in 67 different haplotypes in a total of 265 offspring, examined from eight hatchery stocks. Haplotype diversity of offspring at each of the eight hatcheries ranged from 0.49 ± 0.09 (SE) to 0.94 ± 0.03 (SE). Also, we observed 40 polymorphic sites out of a total of 59 sites, which resulted in a total of 50 haplotypes in 60 wild flounder. Haplotype diversity of the wild population was 0.98 ± 0.01 (SE). The use of subcultured fishes as broodstock appears to be one of the most important causes of reduced genetic diversity in hatchery-raised flounder juvenile. Our results suggest that the use of wild fish for broodstock is an effective way to maintain genetic variability in Japanese flounder offspring.     

Hg2+对克隆牙鲆和普通牙鲆的急性毒性

以相同规格的牙鲆Paralichthys olivaceus普通群体为对照,采用静水生物测试法开展了Hg2+对牙鲆克隆群体的急性毒性实验,实验所用幼鱼为130日龄,体长为(12.38±1.13)cm,体质量为(17.97±5.79)g。实验期间,水温为(21±1.2)℃,pH为8.1-8.3,盐度为31.0,硬度6200 mg/L,连续充气保证溶氧充足。研究表明,实验初始阶段,高浓度组克隆牙鲆在容器内躁动不安,上升到水面沿池壁缓慢转圈游动,一段时间后安静伏在池底;而普通牙鲆则仅个别鱼在水面游动,多数静止在池底不动。随中毒程度加深,用玻璃棒触碰普通牙鲆时,其反应剧烈,在水中侧翻、打转、急速游动或上下窜动,而克隆牙鲆则仅沿池底缓慢游动。最终克隆牙鲆和普通牙鲆均身体僵直,失去呼吸能力死亡。Hg2+对克隆牙鲆的24、48、96 h的半致死质量浓度分别为1.50、0.86、0.84 mg/L,而对普通牙鲆的半致死质量浓度则分别为1.93、1.04、0.90 mg/L。结果表明,克隆牙鲆对Hg2+的耐受性弱于普通牙鲆,对Hg2+更加敏感。从死亡过程看,各处理组的克隆牙鲆从出现死亡个体开始,死亡时间集中在8 h内,具有同步性;普通牙鲆则在实验期间陆续出现死亡,持续时间较长,同步性较差。     

Immunodiagnosis of virulent strains of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) using a monoclonal antibody

Abstract. A virulent strain of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) was used to produce monoclonal antibodies that identified virulent strains of A. hydrophila. Antibodies from a clone, designated as F26P5C8, were found to identify the A. hydrophila serotype I isolates associated with EUS fish, and which were found to be virulent after subsequent inoculation studies. Immunodiagnosis of a large number of A. hydrophila from Australia and Japan showed some additional isolates to be identified by F26P5C8, but the status of their virulence is presently unknown.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens

The present study was undertaken to examine the antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder Paralichthys olivaceus . Newly hatched larvae of flounder were held in a 100 L plastic circular tank and fed rotifiers, Artemia nauplii and commercial feeds, depending on the developmental stage of the fish. Genera Aeromonas , Moraxella and Vibrio were predominantly isolated from the intestinal tracts of Japanese flounder at larval and juvenile stages, whereas Aeromonas , Bacillus , coryneforms, Moraxella , Pseudomonas and Vibrio were detected at high densities in live diets and artificial feeds. Antibacterial bacteria accounted for 1.7–24.3% of the intestinal isolates against Lactococcus garvieae , Pasteurella piscicida , Vibrio anguillarum and V. vulnificus . In particular, as much as 53.3% of Vibrio spp. other than Vibrio -swarmer isolated from 197-day-old juveniles inhibited the growth of P. piscicida . These results suggest that intestinal bacteria having antibacterial activity play a role in the prevention of infectious diseases.     

牙鲆主要生长性状与体质量的灰色关联度分析

采用灰色关联分析方法对牙鲆(Paralichthys olivaceus)10个选育家系的9个生长性状与体质量的关联度进行分析和评价。首先对体质量与生长性况进行数列无量纲化处理,计算出每个生长性状与体质量的关联系数和关联度,根据灰色关联度构建两两比较判断矩阵定量分析各生长性状对体质量的影响度。结果显示,每个生长性状与体质量的关联度由大到小依次为:全长(0.875 8)、体长(0.859 3)、尾柄高(0.830 7)、躯干长(0.827 7)、头长(0.803 5)、尾长(0.781 2)、体高(0.740 2)、吻长(0.665 7)和尾柄长(0.654 0)。体质量的主控因素为:全长、体长、尾柄高和躯干长4个性状,影响度分别为:28.51%、21.62%、16.41%和11.69%,四者的影响度总和达到78.23%。分析表明,与体质量关联度最大的分别是全长、体长、尾柄高和躯干长,且它们对体质量的影响度最高,可以将其作为培育高产牙鲆的重要评价指标,以提高选择效率。本研究旨在为制定理想的牙鲆选育目标、提高育种效率提供理论依据,同时拓展灰色关联度方法在水产动物育种研究中的应用。     

Effects of clioquinol on the scuticociliatosis‐causing protozoan Miamiensis avidus in olive flounder Paralichthys olivaceus

Scuticociliatosis is a devastating and intractable protozoal disease in olive flounder, leading to a significant loss throughout the year. This study aimed to investigate a systemically effective antiscuticociliatosis agent for olive flounder for better absorption into the infected internal organs. The in vitro and in vivo antiscuticociliatosis effects of clioquinol (CQ) were examined after screening 30 biocidal agents against the highly pathogenic scuticociliate Miamiensis avidus. CQ was the most potent in vitro drug of those tested against cultured M. avidus. CQ was the least toxic in healthy olive flounder among the drugs that exhibit high potencies. In olive flounder, a single intramuscular injection of 40 mg/kg CQ significantly reduced mortality caused by artificial infection with M. avidus, and 10–20 mg/kg CQ increased fish survival times. CQ was also effective in naturally infected scuticociliatosis. Ciliate cell numbers were lower when CQ was injected in most organs, including the brain. CQ was well absorbed by the internal organs after intramuscular injection. This study suggests that CQ can be considered as a potential antiscuticociliatosis agent for systemic administration in olive flounder.     

Molecular characterization of Portuguese strains of Yersinia ruckeri isolated from fish culture systems

A total of 23 Portuguese strains of Yersinia ruckeri , the causative agent of enteric redmouth disease (ERM), were comparatively studied by means of lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, plasmid profiling and ribotyping in order to investigate the heterogeneity among isolates and the usefulness of these methods as epidemiological markers. Only two LPS profiles were observed among the isolates studied, corresponding with the serotypes O1 and O3 of Y. ruckeri . A higher heterogeneity was detected analysing the OMP, seven different patterns being observed. Although some isolates carried different small plasmids, all the serotype O1 isolates showed a plasmid band of 50 MDa and all the serotype O3 strains shared in common a extrachromosomic DNA band of 30 MDa. The analysis of the ribopatterns obtained using three different enzymes, separated the strains into 10 ribotypes, indicating genetic heterogeneity among the isolates. The heterogeneity was greater within serotype O1 isolates, six ribotypes being detected, than within serotype O3 in which only three ribotypes were found. Although OMP analysis and ribotyping can be useful for the differentiation of strains on the basis of farm and/or season of isolation, we consider that ribotyping is the best candidate for epidemiological studies because it is easier to perform and offers a slightly better discriminative power.     

Nutritional comparison in muscle of wild,pond and factory cultured Japanese flounder (Paralichthys olivaceus) adults

Muscle proximate composition, amino acid profiles, fatty acid compositions and selected minerals of wild, pond and factory cultured Japanese flounder adults were compared in this study to elucidate their nutritive values and dietary nutrition requirements. Wild and pond cultured Japanese flounder flesh had higher crude proteins, but crude lipid contents of factory cultured fish were 3.8~4.0 fold greater. Major amino acids in Japanese flounder were aspartic acid, glutamic acid and lysine. Wild fish had higher levels of total amino acids, essential amino acids, nonessential amino acids and half‐essential amino acids and the contents in pond cultured samples were similar. Arachidonic acids (ARA), eicosapentaenoic acids (EPA), docosahexaenoic acids (DHA), polyunsaturated fatty acids (PUFA), DHA + EPA and n–3 PUFA in wild Japanese flounder were significantly higher than that in factory cultured fish, whereas fatty acid levels were close between pond cultured and wild fish. Japanese flounder were rich in zinc, iron and selenium, but poor in copper, chromium and nickel. Results indicated wild Japanese flounder had higher nutritional value and better meat quality, but the nutritional compositions of pond cultured fish were close to wild Japanese flounder, which suggested that pond culture of Japanese flounder offers broad application prospects.     

Distribution of marine birnavirus in cultured marine fish species from Kagawa Prefecture, Japan

To determine the distribution of marine birnavirus (MABV) in cultured populations of different marine fish species, 1291 pooled tissue samples from 2672 fish belonging to 22 species and one hybrid were collected from Kagawa Prefecture, Japan, during 1999-2001. Using cell-culture MABV was isolated from three species: yellowtail, Seriola quinqueradiata Temminck & Schlegel (positive number/sample number, 10/419), amberjack, S. dumerili (Risso) (4/72), and Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel) (41/481). Using PCR on MABV-negative samples, the MABV genome was detected in the same three species [yellowtail (9/409), amberjack (4/68) and Japanese flounder (93/440)] and two additional species, spotted halibut, Verasper variegatus (Temminck & Schlegel) (5/11), and goldstriped amberjack, S. lalandi Valenciennes (1/5). These MABV-positive species can be taxonomically divided into two groups: the genus Seriola and flatfish. In Japanese flounder, MABV was detected during all seasons, and the infection rate was correlated with water temperature. Aquaculture sites with MABV-positive fish were evenly distributed over the surveyed area, suggesting that MABV is widely distributed at aquaculture sites in Kagawa Prefecture. The nucleotide sequence at the variable region, the VP2/NS junction, revealed that the 39th base mutation occurs host-specifically for flatfish. Flatfish are suspected to be the main reservoir of MABV and might be responsible for establishing the infection cycle in aquaculture environments.     

乳酸菌对牙鲆稚鱼养殖水体和肠道菌群的影响

在牙鲆稚鱼中投喂添加由单一鼠李糖乳杆菌P15制备的微生态制剂和黄霉素,在60d的投喂期内,采用平板计数法检测好氧性异养菌的总数、总弧菌数和乳酸菌的数量。结果表明,在投喂菌液和冻干菌粉后,养殖水体和牙鲆肠道的乳酸菌数均呈上升趋势,在30d后乳酸菌数量达到稳定并在肠道内定植。同时,由于乳酸菌的抑制作用,弧菌的数量下降,以肠道中的弧菌最明显。乳酸菌对养殖水体和牙鲆肠道的好氧性异养菌没任何影响。乳酸菌对养殖水体和肠道菌群的影响与抗生素具有相近的效果,表明乳酸菌作为饲料添加剂可以取代抗生素应用在牙鲆的养殖中。     

Effect of taurine supplemented practical diet on growth performance and taurine contents in whole body and tissues of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effect of dietary taurine on juvenile Japanese flounder was determined by feeding three taurine-supplemented experimental diets (TAU) and a commercial diet (CD) to evaluate a practical diet for juvenile Japanese flounder. Juvenile Japanese flounder were reared on the three experimental diets supplemented with taurine at 0, 0.5, 1.0% and CD. These diets were fed to juvenile Japanese flounder of an initial mean body weight of 0.2 g for 6 weeks at 20°C and the taurine contents of the whole body and tissues were analyzed. The final average body weight of juvenile Japanese flounder fed the 1.0% TAU was significantly higher than that of the other groups. Taurine contents in the whole body and tissues increased with the increase in dietary taurine level. These results indicate that juvenile Japanese flounder require at least 15 mg/g taurine in the diet, even though a combined mix of fish, krill and squid meal was the main protein source in the experimental diets.