Infectious gastroenteritis caused by Vibrio harveyi (V. carchariae) in cultured red drum, Sciaenops ocellatus

Summary An outbreak of serious mortality among the cultured red drum Sciaenops ocellatus (L.) characterized by a swollen intestine containing transparent yellow fluid (ascites and gastroenteritis) occurred in July 2000 in Taiwan. A motile strain Rd 0700 was isolated from head kidney and/or the intestinal yellow fluid on tryptone soya agar (TSA) supplemented with 2% (w/v) NaCl and/or thiosulfate citrate bile salt (TCBS) sucrose agar plates. Applying biochemical characteristics, this strain was characterized and identified as Vibrio harveyi (V. carchariae). The bacteria could be re‐isolated from kidney, liver, and the transparent yellow fluid of swollen intestine of fish after bacterial challenge. The LD₅₀ values of the organism and its extracellular products (ECP) were 2.9×10⁷ colony forming units (CFU) and 3.85 μg protein g^?1 fish body weight, respectively. All moribund/dead fish exhibited gastroenteritis except those killed within 12 h. This is a first report showing that intraperitoneal (i.p.) injection of the ECP from V. carchariae is lethal to red drum and can reproduce gastroenteritis in the fish.     

Isolation and Characterization of Vibrio carchariae,a Causative Agent of Gastroenteritis in the Groupers,Epinephelus coioides

An outbreak of serious mortality among the cultured groupers Epinephelus coioides, characterized by a swollen intestine containing yellow fluid, occurred in the summer of 1993 in Taiwan. A motile strain EmI82KL was isolated from the intestinal yellow fluid of the moribund groupers with tryptic soy agar supplemented with 2% NaCl and/or thiosulfate citrate bile salt sucrose agar. This strain was characterized and identified as Vibrio carchariae and was susceptible to chloramphenicol, doxycycline-HCl, nalidixic acid, oxolinic acid, oxytetracycline, and sulfonamide while resistant to ampicillin and penicillin G. In addition, the strain was neither auto-agglutinating nor hemagglutinating, but it was hemolytic against erythrocytes from sheep, rabbit, tilapia, and grouper. The bacteria could be reisolated from kidney, liver, and the transparent yellow fluid of swollen intestine of moribund groupers after bacterial challenge and re-identified as the same species. The LD₅₀ value was 2.53 × 10⁷ colony forming units/g grouper body weight. Received: 26 December 1996 / Accepted: 20 February 1997     

Infectious necrotizing enteritis and mortality caused by Vibrio carchariae in summer flounder Paralichthys dentatus during intensive culture

An epizootic causing mortality among cultured summer flounder Paralichthys dentatus occurred in summer of 1998 at a land-based facility on Narragansett Bay, Rhode Island, USA. The disease, flounder infectious necrotizing enteritis (FINE), was characterized by reddening around the anal area, distended abdomens filled with opaque serosanguineous fluid, enteritis and necrosis of the posterior intestine. In extreme cases of the disease, the posterior intestine was detached from the anus and was observed coming out the vent. The intestine of individuals that recovered from the disease ended in a blind-sac; the abdomens of these fish were distended, due to food and water inside the intestinal blind-sac. A bacterium was isolated from ascites fluid and kidney of moribund flounder and identified as the causative agent in challenge experiments. The pathogen was identified as Vibrio carchariae by morphological and biochemical characteristics and sequence of the 16S rRNA. The LD50 estimate was 5 x 10(5) colony-forming units injected intraperitoneally into 100 to 200 g summer flounder.     

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.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

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.     

三种免疫制剂对真鲷弧菌病的免疫保护性

菌体疫苗按不同的方式对真鲷进行免疫2周后,对实验鱼均具有免疫保护性,免疫保护性最好的免疫组,免疫保护率在初次免疫后高达60%,强化免疫后免疫保护率可提高到80%;粗制LPS经去毒处理后初次免疫真鲷,不同浓度的LPS对实验鱼具有不同程度的免疫保护性,强化免疫后,免疫保护率均有明显的提高,浓度越高,免疫保护性越强,对真鲷的免疫保护率最高可达90%,最小弧菌产生的外毒素经福尔马林灭活后制成毒素苗,这种毒素苗能产生较好的免疫保护性,其免疫保护率可达80%,这表明外毒素不仅是最小弧菌产生的毒力因子,同时也是菌体产生的有效保护性抗原。     

Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus

Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 microg protein/g prawn) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 microg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furthermore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by V. alginolyticus.     

Diet enriched with mushroom Phellinus linteus extract enhances the growth, innate immune response, and disease resistance of kelp grouper, Epinephelus bruneus against vibriosis

The effect of diet supplemented with Phellinus linteus fed for 30 days was investigated in grouper Epinephelus bruneus challenged with Vibrio anguillarum, Vibrio harveyi, Vibrio alginolyticus, and Vibrio carchariae; infected and treated fish had a significantly higher percent weight gain and feed efficiency. In groups fed with enriched diet and challenged with V. anguillarum and V. harveyi the mortality rate declined with a consequent rise in survival rate than with other pathogens. On the other hand, in groups fed with P. linteus enriched diet and challenged with V. anguillarum, V. harveyi, and V. alginolyticus the cellular and humoral immune responses, such as the alternative complement activity (ACH(50)), serum lysozyme activity, phagocytic activity (PA), phagocytic index (PI) significantly higher than in the control group. The respiratory bursts (RB), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were found significantly enhanced when the groups fed with enriched diet against V. anguillarum and V. harveyi. The results reveal that kelp grouper fed for 30 days with P. linteus enriched diet had higher cellular and humoral immune response and disease protection from vibriosis than the group fed on basal diet with the protection linked to stimulation of immune system.     

Pathogenicity of Vibrio alginolyticus for Cultured Gilt-Head Sea Bream (Sparus aurata L.)

The in vivo and in vitro pathogenic activities of whole cells and extracellular products of Vibrio alginolyticus for cultured gilt-head sea bream were evaluated. The 50% lethal doses ranged from 5.4 × 10⁴ to 1.0 × 10⁶ CFU/g of body weight. The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line. In addition, the in vitro ability of V. alginolyticus to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy. The biological activities of extracellular products of V. alginolyticus were hydrolytic activities; the products were able to degrade sea bream mucus. V. alginolyticus was cytotoxic for fish cell lines and lethal for sea bream. Moreover, the extracellular products could degrade sea bream tissues. However, experiments performed with the bath immersion inoculation technique demonstrated that V. alginolyticus should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.     

Attachment-inducing capacities of fish skin epithelial extracts on oncomiracidia of Benedenia seriolae (Monogenea: Capsalidae)

Attachment-inducing capacities of skin epithelial extracts of yellowtail, Japanese flounder and red sea bream on oncomiracidia of the monogenean Benedenia seriolae were examined. Clear differences were not detected in the capacity among the fish species, although B. seriolae infects only yellowtail and its congeners in Seriola. This suggests that either the capacity is not host specific or host-specific attachment-inducing capacity cannot be detected by the assay method. Further, the attachment-inducing capacities were suppressed by wheat-germ lectin and concanavalin A in skin epithelial extracts of Japanese flounder and yellowtail, respectively. This suggests that some sugar-related chemical substances existing in fish epithelia induce the attachment of B. seriolae oncomiracidia.     

Enhancement of Eriobotrya japonica extracts on non-specific immune response and disease resistance in kelp grouper Epinephelus bruneus against Vibrio carchariae

The present study investigated the effect of Eriobotrya japonica extracts at 0%, 0.1%, 1.0%, and 2.0% doses supplementation with feed on non-specific immune response, hematological and biochemical profile, and disease resistance against Vibrio carchariae in kelp grouper Epinephelus bruneus at weeks 1, 2, and 4. The white blood cell (WBC) significantly increased in fish fed with 0.1%, 1.0%, and 2.0% diets on weeks 1 and 2 when compared to the control. However, the glucose always decreased from the control except on week 2 against pathogen. The serum total protein, albumin, and globulin significantly increased at week 2 but they did not changed significantly at weeks 1 and 4. The superoxide anion, lymphokines production index, and phogocytosis did not significantly increased in any diet on the first week whereas it was significantly enhanced in 1.0% and 2.0% supplementation diets on weeks 2 and 4 against V. carchariae when compared to control. All diets significantly enhanced the serum lysozyme activity, bactericidal activity, and haemolytic complement activity from weeks 1-4 as compared to control. The serum agglutinating antibody titre did not significantly enhance on the first week whereas it was significantly enhanced on weeks 2 and 4. Fish fed with 1.0% and 2.0% doses diets was found lower mortality than 0.1% diet. Thus, this study suggested that 1.0% and 2.0% doses supplementation diets could be advocated to enhance the immune response and production disease from V. carchariae in E. bruneus.     

海水养殖真鲷最小弧菌外毒素的免疫学特性初步研究

按常规方法免疫健康家兔进行外毒素抗体的制备。将制备的兔抗血清与等体积的外毒素混合,37℃下作用1h后,观察毒素的溶血性及细胞毒性,取免疫前家兔血清(零号血清)作阴性对照,用毒素作阳性对照。结果表明,兔抗毒素血清能中和毒素,抑制毒素对人血细胞的溶血性及对Vero细胞的细胞毒性,中和毒素的兔抗毒素血清最高稀释度为1∶256,毒素对照显示出溶血性及细胞毒性。毒素与一定浓度的嗜水气单胞菌HEC毒素抗血清、霍乱肠毒素抗血清在37℃下分别作用1 h后,观察其溶血性及细胞毒性。结果表明,这2种抗血清都不能中和毒素,毒素与这2种抗血清作用后,仍然保持其溶血性及细胞毒性。用浓度为0.2%的福尔马林对浓度为5mg/mL的外毒素进行灭活处理,得到的灭活疫苗为毒素苗。一定稀释度的毒素苗,在初次免疫真鲷2周后利用病原菌对免疫组及对照组的实验鱼进行人工攻毒感染,毒素苗的免疫保护率可达80%,强化免疫后,免疫保护率有所提高。注射免疫的免疫保护率比浸泡免疫的高。     

Biochemical responses of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides

To elucidate the ichthyotoxic mechanisms of a harmful dinoflagellate Cochlodinium polykrikoides, biochemical responses of fish exposed to blooms were investigated. Particularly, based on our finding that oxidative damages of gill were associated with fish mortality (J. Plankton Res. 21 (1999) 2105-2115), dysfunction of ion-transporting enzymes and secretion of gill mucus of fish exposed to this bloom species were examined. The susceptibilities of several fishes to C. polykrikoides were different; the active pelagic fishes such as black scraper Thamnaconus septentrionalis, red sea bream Pagrus major, beakperch Oplegnathus fasciatus and seaperch Malakichthys wakiyae, were more vulnerable than the benthic fishes, flounder Paralichthys olivaceus and rockfish Sebastes inermis. In addition, the higher the algal cell density, the higher the fish mortality. When the test fishes were exposed to C. polykrikoides of 5000 cells ml(-1), the transport-related enzymes, carbonic anhydrase and Na(+)/K(+)-ATPase activities were significantly decreased. The activity of carbonic anhydrase was decreased with increasing algal cell density and exposure time. The quantity of total polysaccharide in gill mucus is higher in the fish exposed to C. polykrikoides than in the control fish; the magnitudes were higher in the pelagic fishes than that of benthic fishes. Moreover, a drop of blood pH and oxygen partial pressure (pO(2)) was also observed in red sea bream and flounder subjected to C. polykrikoides. These results suggest that the inactivation of gill transport-related enzymes activities, the fall in blood pO(2) and abnormal secretion of gill mucus by the C. polykrikoides may be one of the principal causes of fish kill.     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.     

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.     

Derivation of a pluripotent embryonic cell line from red sea bream blastulas

A pluripotent cell line, sea bream embryonic stem‐like cells (SBES1), was developed from blastula‐stage embryos of the cultured red sea bream, Chrysophrys major . The SBES1 cells were cultivated in Dulbecco's modified eagles medium (DMEM) medium supplemented with foetal bovine serum, marine fish serum, fish embryo extract, selenium, basic fibroblast growth factor and leukemia inhibitory factor. They were small and round or polygonal, and grew actively and stabely in culture. The cells exhibited a positive alkaline phosphatase activity upon histochemical staining. When the cells were treated with all‐ trans retinoic acid, they differentiated into various types including neuron‐like, neuroglia‐like and muscle‐like cells, suggesting that the SBES1 cells remained pluripotent in culture. Chromosome analysis revealed that SBES1 cells had a normal diploid karyotype with 2 n  = 2 st  + 46 t . At present, SBES1 cells have been cultured for >180 days with more than 60 passages. High survival rate has been obtained after cryopreservation of cell cultures. This embryonic cell line may potentially be used for the production of transgenic red sea bream.     

Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilt-head sea bream (Sparus aurata L.).

The kinetics of adhesion of Vibrio strains isolated from diseased fish to skin mucus of gilt-head sea bream was studied. A modified Langmuir adsorption isotherm was calculated, and the results obtained indicate that the strains tested (Vibrio alginolyticus DP1HE4 and Vibrio anguillarum-like DC12R8 and DC12R9) showed a saturation kinetics except for V. alginolyticus (CAN), which showed a proportional adsorption kinetics. The adhesive capability for skin mucus does not seem to be an essential virulence factor of pathogenic strains of Vibrio, since this specific interaction depended on several environmental factors, temperature and salinity being the most important. However, the absence of an inhibitory effect of mucus on the pathogenic microorganisms, and the capability of the Vibrio strains to utilize mucus as a carbon source, could favor their settlement on the skin with a potential for infection of cultured, stressed fish.