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.     

七带石斑鱼染色体核型研究

采用PHA体内直接注射法制备了七带石斑鱼(Epinephelus septemfasciatus)头肾组织染色体标本并分析其核型。结果表明,七带石斑鱼二倍体染色体数为48,24对染色体均为端部着丝粒染色体,其染色体臂数(NF)为48,核型公式为:2n=48t,NF=48。未发现与性别相关的异型染色体,第24对染色体长度明显小于其他染色体。通过与其他22种石斑鱼染色体核型进行比较,发现七带石斑鱼具有石斑鱼属鱼类的原始核型特征,属于石斑鱼属鱼类的原始类群。     

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.     

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.     

Histopathological studies on viral nervous necrosis of sevenband grouper, Epinephelus septemfasciatus Thunberg, at the grow-out stage

Abstract Viral nervous necrosis caused by sevenband grouper nervous necrosis virus (SGNNV) has occurred in grow-out stages (0-3 years old) of sevenband grouper, Epinephelus septemfasciatus, since the 1980s. In the present study, based on histopathological features of the central nervous system (CNS) in naturally diseased fish, pernasal infection experiments using grow-out fish were performed and pernasal infection was established as a putative invasion route of SGNNV. The definite SGNNV-targeted cells were determined by histopathological studies including indirect fluorescent antibody test and electron microscopy. Nerve cells in the olfactory lobe were most extensively necrotized with vacuolation followed by infiltration of microglia and macrophages. Purkinje cells and Golgi cells were extensively infected in the cerebellum. Megalocells and small nerve cell nuclei were also infected in the preoptic area, thalamus, medulla oblongata and spinal cord. Only a few small nerve cells were infected in the olfactory bulb and optic tectum. The retina of some diseased fish displayed vacuolated bipolar cells of the inner nuclear layer and in the ganglion cell layer. These SGNNV-infected nerve cells displayed viroplasmic inclusions containing virions, vacuoles and myelin-like structures. Based on observed histopathological changes, the lesion of the CNS was characterized by encephalitis but not encephalopathy.     

Immune responses of Asian sea bass, Lates calcarifer Bloch, against an inactivated betanodavirus vaccine

Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.     

Skeletal development and deformities in cultured larval and juvenile seven-band grouper, Epinephelus septemfasciatus (Thunberg)

The seven‐band grouper, Epinephelus septemfasciatus (Thunberg), is currently recognized as a potential new species for aquaculture in Japan. This study describes normal and abnormal skeletal development of the jaw and vertebrae in cultured larvae and early juveniles of E. septemfasciatus. The ontogenetic stages at which skeletal deformities of jaw and vertebra developed were also described for this species. Osteological observations were made using a clearing and staining method for larvae and soft X‐ray photographs for juveniles. A high incidence of skeletal deformities was observed in the jaws and vertebral column during the larval and juvenile stages. Most of the jaw deformities were explained by an abnormal maxilla curvature. Jaw deformities were visually evident from flexion stage after ossification of the deformed elements. Deformities in the vertebral column (mostly lordosis) were observed from the post‐larval stage and became more evident as growth proceeded. The lordosis generally occurred on the positions of the 8–11th vertebra. These types of deformities are compared with those of other species, and possible causative factors of the skeletal deformities are discussed.
     

Characterization and viral susceptibility of a brain cell line from brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål) with persistent betanodavirus infection

A continuous cell line designated BMGB (brown‐marbled grouper brain) was established from the brain tissues of the brown‐marbled grouper Epinephelus fuscoguttatus and characterized. BMGB cells were identified as astroglial progenitor cells because they expressed glial fibrillary acidic protein and keratin and were persistently infected by betanodavirus, as confirmed through immunocytochemistry, polymerase chain reaction and immunoblot analyses. Because few intact virions were present in the BMGB cell culture fluid, the cytopathic effect (CPE) was not observed when the culture fluid was inoculated with GBC1 cells. However, BMGB cells displayed typical CPE after infection with additional betanodavirus, megalocytivirus and chum salmon reovirus. BMGB cells showed low myxovirus resistance (Mx) protein expression, which increased following betanodavirus and reovirus infection. Because the cells contained several unusual or degraded viral proteins, the persistent infection of betanodavirus in the BMGB cells may have resulted from a mechanism that destroys the viral proteins rather than the result of Mx protein expression. Despite the persistent betanodavirus infection, BMGB cells proliferated in a manner similar to other normal tropic fish cells and supported the propagation of several piscine viruses; however, the yield was lower than that of normal cells. The BMGB cells will be useful for investigating virus and host cell interaction.     

In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Mouse monoclonal antibodies (MAbs) were produced by using yellow grouper nervous necrosis virus (YGNNV) as an immunogen, isolated from infected yellow grouper, Epinephelus awoara (Temminck & Schlegel), and propagated in GB cells. In enzyme linked immunosorbent assay (ELISA), 43 hybridoma clones secreting MAbs strongly reacted with the purified virus. Ten of them showed a higher neutralization index (NI) value between 6.5 and 4.5 (log₁₀ NI) than the other 33 MAbs against YGNNV infection in cell culture. All 10 MAbs belonged to the IgG isotype with a κ light chain and recognized the 42 kDa coat protein of YGNNV by Western blot analysis. Immunohistochemical results demonstrated that the viral signals co-located with pathological lesions observed in retina, brain and spinal cord. These results indicate that the MAbs are useful for confirmative diagnosis of YGNNV infection.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

Detection of betanodavirus in juvenile barramundi, Lates calcarifer (Bloch), by antigen capture ELISA

Betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. Betanodaviruses possess a bipartite single-stranded RNA genome consisting of the 3.1 kb RNA1 encoding an RNA-dependent RNA polymerase and the B2 protein, while the 1.4 kb RNA2 encodes the viral nucleocapsid protein, alpha. A panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (GGNNV) was developed for use in diagnostics. All antibodies reacted with native and recombinant alpha in immunoblot and indirect immunofluorescence assays. Each of the monoclonal antibodies reacted against discrete regions of the alpha protein, though none reacted with the extreme C-terminal region of the protein. One of the monoclonal antibodies, specific for the K151-T246 region of alpha, was used for the development of an antigen capture ELISA. In this assay we could detect 10(3)-10(4) TCID(50) units of virus derived from infected tissue culture supernatants. Head tissue extracts prepared from experimentally infected barramundi, Lates calcarifer, juveniles were assayed for GGNNV using the antigen capture assay and a clear increase in alpha antigen was detected from 5 to 15 days post-challenge. The assay thus represents a useful method for field-based detection of betanodavirus in fish hatcheries.     

Development,distribution and expression of a DNA vaccine against nodavirus in Asian Seabass,Lates calcarifier (Bloch, 1790)

Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.     

Establishment of a new cell line from the heart of giant grouper,Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility

A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC₅₀) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL⁻¹, respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.     

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety‐three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.     

Apparent digestibility of selected ingredients in diets for juvenile grouper, Epinephelus coioides (Hamilton)

Apparent digestibility coefficients (ADCs) for dry matter (ADC_dm) and crude protein (ADC_cp) of selected feed ingredients were determined in vivo for grouper using passive faeces collection (Guelph System). A reference diet (RF) and test diets (consisted of 70% RF and 30% test ingredient) with 1% Cr₂O₃ as an inert indicator were used. An RF contained 45% protein, 10% fat and 15.7 kJ g^?1 metabolizable energy. Three isonitrogenous and isocaloric diets, each contained a test ingredient (white fish meal, white cowpea meal and ipil‐ipil leaf meal), were used in a growth study based on ADC_cp of feed ingredients. An RF without Cr₂O₃ was a control. The ADC values of experimental diets were also determined. In grouper, the ADC_dm of white cowpea meal, defatted soybean meal, wheat flour and shrimp meal (74–76%) were significantly lower than that of squid meal (99%), but comparable with those of the fish meals (84–89%). No significant difference was observed between the ADC_dm of ipil‐ipil leaf meal, rice bran and wheat flour (56–73%). The ADC_cp of white cowpea meal and defatted soybean meal were similar to those of the fish meals, squid meal and shrimp meal (94–99%). The ADC_cp of wheat flour was comparable with that of ipil‐ipil leaf meal (79–83%). Rice bran had the lowest ADC_cp value of 43%. Based on specific growth rate (SGR), the growth of fish fed white cowpea meal‐based diets was similar to that of the control fish (3.2–3.3% day^?1). Also, no significant difference was observed between the ADC_dm (68–72%) and ADC_cp (88–91%) of white cowpea meal‐based diet and the control diet. The results suggest that ADC values can be used as indicators to determine the nutritional value of feed ingredients. White cowpea meal can be incorporated as a protein source in practical diet for grouper at 20.5% of the diet with no adverse effect on growth.     

Effect of temperature,hypoxia and disinfection with ozonated seawater during somitogenesis on muscular development of the trunk in larval seven‐band grouper,Epinephelus septemfasciatus (Thunberg)

The cause of abnormal musculature on both sides of the same myotome in artificially cultured seven‐band grouper Epinephelus septemfasciatus (Thunberg) larvae remains unclear. We evaluated the effect of three factors: changes in temperature (between 22 and 24°C), disinfection with ozonated seawater (0.3 mg TROs L^?1, 1 min), and hypoxia (22 or 14% saturation) during the early (20 h post‐fertilization, hpf) and mid‐ (24 hpf) somite stages on trunk muscular development in larval seven‐band grouper. The thermal regimes and disinfection with ozonated seawater had no effect on muscle development (P > 0.05, G‐test). In contrast, exposure to hypoxia during both the early and mid‐somite stages increased the incidence of abnormal musculature in the trunk (P < 0.01, G‐test). These larvae had abnormal musculature on both sides of the trunk. In addition, the number of abnormal myotomes differed among these larvae. Exposure in the early and mid‐somite stages resulted in the highest frequency of abnormalities in the 11th and 13th myotome respectively. Our results suggest that hypoxia during somitogenesis can lead to abnormal muscle development in the trunk of artificially cultured seven‐band grouper larva.     

Systemic and mucosal antibody response in tilapia, Oreochromis niloticus (L.), following immunization with Flavobacterium columnare

Specific antibody responses to Flavobacterium columnare (isolate ATCC 23463T) were characterized in plasma and mucus of tilapia following intraperitoneal (i.p.) injection or immersion immunization with formalin-killed sonicated or whole cell preparations. Fish (30 per treatment) received a primary immunization and were booster immunized 4 weeks later. An enzyme-linked immunosorbent assay was developed for detection and quantification of specific anti-F. columnare antibody, and it was found that formalin-killed sonicated cells in Freund's complete adjuvant (FCA) injected i.p. stimulated a significant systemic antibody response within 2 weeks (mean titre 11,200) which increased to 30,600 following secondary immunization. At 10 weeks post-immunization, the mean titre remained significantly elevated above the controls. Antibodies were also observed in cutaneous mucus of fish immunized i.p. with formalin-killed sonicated cells in FCA at 6 and 8 weeks post-immunization (mean titres 67 and 33, respectively). Although some individual fish responded, mean plasma and cutaneous mucus antibody titres were not significantly greater than controls in any of the other treatment groups. The results of this study demonstrate that tilapia can mount a significant humoral response in plasma and cutaneous mucus to F. columnare, but i.p. immunization with FCA is required to elicit this response.