Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (SJNNV, RGNNV, TPNNV and BFNNV) and it is suggested that host specificity is different among these genotypes. In the present study, a betanodavirus [sevenband grouper nervous necrosis virus (SGNNV)] belonging to the redspotted grouper nervous necrosis virus (RGNNV) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of several marine fish species. When challenged with the virus by a bath method (10(5.1) TCID50 mL(-1)), sevenband grouper, Epinephelus septemfasciatus, Japanese flounder, Paralichthys olivaceus, and tiger puffer, Takifugu rubripes, displayed behavioural abnormalities and mortalities with distinct histopathological signs of viral nervous necrosis and heavily immunostained cells were observed in the central nervous tissues and retina. Bath-challenged rock fish, Sebastiscus marmoratus, and a hybrid of sevenband grouper and kelp grouper, E. moara, did not display any behavioural abnormality or mortality during the experimental period, although many fish showed slight signs of viral infection in nerve cells. Kelp grouper and red sea bream, Pagrus major, showed no behavioural abnormality, mortality or immunohistopathological changes after the virus challenge. These results are, in part, consistent with the natural host range of RGNNV, indicating the complexity in the host specificity of betanodaviruses.     

赤点石斑鱼病毒性神经坏死症的组织病理和电镜观察

用逆转录-聚合酶链式反应（RT-PCR）检测患病赤点石斑鱼苗，呈Beta诺达病毒阳性。光学显微镜下观察到病鱼的脑、视网膜、脊髓有空泡．在脑部，空泡主要分布在端脑、间脑和小脑。受感染的细胞明显收缩、致密变化和嗜碱性。包涵体常为圆形，大小不一。透射电镜下，在感染细胞的细胞质可观察到含有病毒粒子的致密体。病毒粒子呈等面体，无外膜，直径为25～28nm，随机分布在细胞质或在致密体内排列成品格状。致密体大小不一。偶尔观察到较大致密体的外膜已破裂，病毒粒子被释放到细胞质。     

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli – expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.     

Lumpfish (Cyclopterus lumpus,Linnaeus) is susceptible to viral nervous necrosis: Result of an experimental infection with different genotypes of Betanodavirus

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail‐biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV‐infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.     

The effects of dietary lipid levels on performance and heat‐shock protein response of juvenile white seabass,Atractoscion nobilis

A feeding trial was conducted to study the effect of dietary lipid on growth performance and heat‐shock protein (HSP70 and HSP60) response of white seabass (WSB), Atractoscion nobilis. Five diets were formulated to contain 440 g kg^?1 protein from 300 g kg^?1 fish meal, 240 g kg^?1 soybean meal and 100 g kg^?1 soy protein concentrate with different levels of lipid: 100, 120, 140, 160 or 180 g kg^?1. At the end of the trial, heat shock response based on HSP70 and HSP60 was measured in liver and white muscle from fish at ambient temperature and temperature shock conditions. Final weight and percent gain were significantly higher for fish fed the 100 g kg^?1 lipid diet than for fish fed the rest of the diets (P ≤ 0.05). Feed conversion ratio was lowest for fish fed the 100 g kg^?1 lipid diet. The HSP70 and HSP60 responses were positively correlated to dietary lipid levels following temperature shock. At ambient temperature, HSP60 and HSP70 responses in muscle and HSP60 response in liver increased with dietary lipid level. Temperature shock significantly increased the HSP response of fish in all treatments. Results of this study demonstrated that a moderate (110–120 g kg^?1) level of dietary lipids would be recommended for production diets but a higher dietary lipid level may be required for optimal stress tolerance.     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.     

Evaluation of carbohydrate‐to‐lipid ratio in diets supplemented with Bacillus subtilis probiotic strain on growth performance,body composition and digestibility in juvenile white seabass (Atractoscion nobilis,Ayres 1860)

This study aimed to evaluate the effect of increasing the carbohydrate to lipid ratio in diets supplemented with Bacillus subtilis in white seabass (Atractoscion nobilis) juveniles on digestibility, growth performance, feed utilization efficiency and tissue composition. Four experimental isoenergetic and isoproteic diets supplemented with B. subtilis were formulated with increasing starch to lipid ratios (g/g) of 10:19; 14:17; 18:15 and 22:13 (diets PD10, PD14, PD18 and PD22 respectively). A negative control diet for B. subtilis supplementation was also formulated to be similar to diet PD10 (diet CD10). Each experimental diet was fed to triplicate groups of 20 fish per tank (IBW: 9.5 g), three times a day, to apparent satiation, for 60 days. Supplementation with B. subtilis, of the low starch‐high lipid diet (10:19), significantly enhanced the overall growth performance, digestibility and feed efficiency. Moreover, the increase in starch to lipid ratio, in supplemented probiotic diets, significantly increased growth performance, feed intake, feed utilization efficiency and protein and energy digestibility, being maximum for the highest dietary starch to lipid level. Lipid content of whole body, muscle and liver and hepatosomatic index significantly increased with the increase in starch to lipid ratio. The results of this study indicate that B. subtilis supplementation significantly enhanced growth performance, digestibility and feed utilization of low starch‐high lipid diet. However, further improvement of growth performance and feed utilization efficiency may be achieved combining B. subtilis supplementation with increased dietary starch to lipid ratio to 22:13.     

鱼病毒性神经坏死病病毒（VNNV）不同基因型鉴别方法的建立及在VNN检疫和监测中的应用

从GenBank中查找出乙型野田村病毒组中海水鱼类各病毒的序列,并用Sequencher多重序列比较软件将其分到条纹踢神经坏死病毒(striped jack nervous necrosis virus,SJNNV)组、条纹星鲽神经坏死病毒(barfin flounder nervous necrosis virus,BFNNV)组、红点石斑鱼神经坏死病毒(redspotted grouper nervous necrosis virus,RGNNV)组和虎斑东方纯神经坏死病毒(tiger puffer nervous necrosis virus,TPNNV)组4个基因型的组别中。用DNAsis序列比较软件比较同一基因型各基因序列之间的同源性,均在815％以上;不同基因型之间序列的同源性,均在66％以下。结合Premier引物设计软件和Sequencher序列多重比较软件,设计了4对引物,采用逆转录聚合酶链式反应(RT—PCR)来鉴别这4个不同的基因型。对从深圳口岸进境的产地为台湾的海水鱼苗和广东、福建两省养殖的主要海水鱼类进行检疫和监测,结果在进境的海水鱼苗中检出有RGNNV基因型的VNNV,在福建和广东省养殖的石斑鱼成鱼和鱼苗的病鱼体内均检测到RGNNV基因型的VNNV,对上述扩增产物基因序列进行比较,相似性均在96．5％以上,推导出的氨基酸序列与玛拉巴石斑鱼神经坏死病毒(MNNV)序列相似性均为100％。     

Field tests of Poly(I:C) immunization with nervous necrosis virus (NNV) in sevenband grouper, Epinephelus septemfasciatus (Thunberg)

It was recently reported that Poly(I:C) immunization with live nervous necrosis virus (NNV) confers protection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), from NNV infection. In the present study, we conducted field tests with sevenband grouper for the evaluation of Poly(I:C) immunization efficacy. In the first experiment, sevenband grouper were immunized with NNV followed by Poly(I:C) administration 7 weeks before natural occurrence of viral nervous necrosis (VNN). Survival rate of the naïve fish was 71.0%, whereas that of the immunized fish was 99.8%. In the second experiment, sevenband grouper were immunized 10 months before VNN occurrence and survival rate of the non‐treated and vaccinated fish was 79.5% and 97.5%, respectively. In the third experiment, we administered Poly(I:C) to sevenband grouper at 20 days after natural occurrence of VNN. The survival rate of the non‐treated fish was 9.8%, whereas that of fish administered Poly(I:C) was 93.7%. Based on these results, it was concluded that Poly(I:C) immunization conferred protection in fish against NNV infection in field tests and the protection lasted more than 10 months. Furthermore, even after occurrence of VNN, fish mortality could be reduced by Poly(I:C) administration and there was an unexpected curative effect on VNN‐affected fish.     

Fisheries independent assessment of a returning fishery: Abundance of juvenile white seabass (Atractoscion nobilis) in the shallow nearshore waters of the Southern California Bight, 1995–2005

Nearshore, coastal and embayment areas off southern California were sampled to determine the spatial and temporal patterns abundance and size distributions of young white seabass in the shallow (5–14 m) waters from Santa Barbara south to Imperial Beach off San Diego. A total of 19 stations, 13 in nearshore coastal waters and 6 in embayments, dispersed along the Southern California Bight were surveyed in each sampling month using 45.7 m variable mesh, monofilament gill nets. In the 11-year period of sampling (April 1995–June 2005), a total of 8075 juvenile white seabass was captured in 42 sampling months. The mean catch-per-unit-effort (CPUE: 2.0 fish/net ± 0.2) for juvenile white seabass varied significantly among stations during the 10-year period (1996–2005) of the full station sampling. Stations located near large rocky headlands, such as Palos Verdes, Santa Barbara, Newport, and La Jolla yielded the highest catches. Although CPUE peaked in August 1999 as a result of strong year classes in 1996–1998, overall, catches tripled over the 11-year sampling period at seven coastal sites increasing significantly at a rate equivalent to 0.22 fish/(net year). These relatively high catches of wild, juvenile fish over the last decade, along with significant increases in commercial CPUE and increased recreational catches overall, indicate that the natural population of white seabass is in the process of recovery. Commercial catches are again comparable to levels attained prior to the fishery collapse in the 1970s and 1980s. Therefore, we propose that the white seabass now represents one of the first documented cases of a recovering, demersal species of commercial importance. The ban of nearshore commercial gill net fishing by Proposition 132 probably contributed greatly to the increase in the population size that led to this recovery. In addition, the succession of warm water years that occurred from 1983 to the strong El Niño event of 1997–1998 also played an important role in the successful recruitment of white seabass.