Host,agent and environment interactions affecting Nervous necrosis virus infection in Australian bass Macquaria novemaculeata

Australian bass Macquaria novemaculeata were challenged by immersion with nervous necrosis virus (NNV) at different ages and under controlled conditions to investigate factors affecting disease expression. Fish challenged at 3 weeks of age with 10³ TCID₅₀/ml and higher doses developed clinical disease; a lower dose of 10² TCID₅₀/ml resulted in incidence below 100% and 10¹ TCID₅₀/ml was insufficient to cause infection. Additionally, fish were challenged at 5, 6 and 13 weeks of age at 17 and 21°C to assess the role of the age of the host and water temperature on disease expression. Although Australian bass challenged at all ages had evidence of replication of NNV, only those challenged at 3 weeks of age (20 and 24 days post?hatch [dph]) developed clinical disease. Higher water temperature had an additive effect on disease expression in larvae challenged at 24 dph, but it did not affect the disease outcome in older fish. Finally, isolates of NNV derived from fish with clinical or subclinical disease presentations caused similar cumulative mortality and clinical signs when larvae at 24 dph were challenged, suggesting that agent variation was not responsible for variation in clinical presentation in these field outbreaks of NNV infection.     

逆转录聚合酶链式反应(RT-PCR)检测5种养殖石斑鱼的神经坏死病毒

神经坏死病毒(Nervous necrosis virus)是导致多种海水鱼类神经性病害的致病原.发病及死亡的石斑鱼除了表现神经异常症状外,无明显的临床病症,体表及内脏组织也未发现明显病变及寄生虫感染.2003年4～8月,应用逆转录聚合酶链式反应(RT-PCR)技术从福建南部人工养殖的5种石斑鱼即紫石斑鱼(Epinephelus lanceolatus)、马拉巴石斑鱼(E. malabaricus)、青石斑鱼(E. awoara)、赤点石斑鱼(E. akaara)和云纹石斑鱼(E. moara)中检出5个神经坏死病毒分离株.检测了76份石斑鱼样品,这些石斑鱼NNV病毒的平均感染率约为90%.对这些病毒的RT-PCR产物421 bp核酸进行了测序和序列分析,其相同的序列超过99%.将这些序列与GenBank的石斑鱼(Epinephelus spp.)神经坏死病毒相关基因序列作比较,同源性在97%以上.对神经坏死病毒在石斑鱼体内的分布也进行了分析,在脑和眼组织的检出率最高,部分病鱼的肝、脾和肾组织也能检出病毒.结合流行病学特征,可确认神经坏死病毒为该传染病的主要致病原.RT-PCR方法是检测NNV等病原的一种理想的诊断方法.     

引起半滑舌鳎(Cynoglossus semilaevis Günther)鱼苗大规模死亡的神经坏死病毒病

2012和2013年,山东某育苗场15–20日龄的半滑舌鳎(Cynoglossus semilaevis Günther)鱼苗出现暴发性大规模死亡,7 d内死亡率高达90%–100%。本研究调查了疾病的发生情况和临床特征,采集病鱼样品进行了组织病理学检查,并运用RT-PCR方法进行了病原的检测和基因序列分析。结果发现,半滑舌鳎鱼苗一般在7月和8月发病,发病时养殖水温为22–24℃。病鱼游泳行为异常,表现为上下翻游、螺旋性游动、全身大幅度波浪状浮动症状,但病鱼体表无出血和溃疡症状。组织病理检查发现,病鱼脑和视网膜组织出现严重的空泡化及坏死。病鱼样品的RT-PCR检测结果全部呈鱼类神经坏死病毒阳性。对得到的RT-PCR产物测序,进行BLAST比对,发现该病毒与鱼类神经坏死病毒的赤点石斑鱼神经坏死病毒(Red-spotted grouper nervous necrosis virus, RGNNV)基因型的相似性达98%以上,而与鱼类神经坏死病毒的其他3个基因型：黄带拟鲹神经坏死病毒(Striped jack nervous necrosis virus,SJNNV)、红鳍东方鲀神经坏死病毒(Tiger puffer nervous necrosis virus, TPNNV)和条斑星鲽神经坏死病毒(Barfin flounder nervous necrosis virus,BFNNV)的相似性仅为71%–78%。由此可以判定,本研究发现的引起半滑舌鳎鱼苗大规模死亡的神经坏死病毒为RGNNV基因型,半滑舌鳎也是鱼类神经坏死病毒的天然宿主。该发现在半滑舌鳎疾病防治和鱼类神经坏死病毒的流行机制研究方面都具有重要意义。     

Resistance of pearlspot larvae,Etroplus suratensis,to redspotted grouper nervous necrosis virus by immersion challenge

Viral nervous necrosis (VNN) affects more than 120 species mostly belonging to the order Perciformes. However, none of the brackishwater species belonging to the family Cichlidae under the order Perciformes are reported to be susceptible. Hence, the present experiment was undertaken to study the susceptibility of the brackishwater cichlid, pearlspot, Etroplus suratensis to NNV. Thirty‐day‐old pearlspot larvae were infected with NNV by immersion. Mortality was recorded till 14 days post‐infection, and the infected larvae were subjected to nested RT‐PCR and histology. The virus was isolated from infected larvae using SSN‐1 cells. To study the replication of the virus in vitro, primary cultured brain cells of E. suratensis and IEK cells were infected with NNV. No mortality was observed in any of the control or experimentally infected larvae. However, the experimentally infected larvae were positive for NNV by nested RT‐PCR and the virus was isolated using SSN‐1 cells. Further, the infected pearlspot brain cells and IEK cells showed cytopathic effect at second and third passage of the virus and they were positive for NNV by nested RT‐PCR. Pearlspot is relatively resistant to VNN although the virus could replicate in the larvae and in cell culture.     

Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus)

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South‐East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV‐infected fish sharing a common water source.     

Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

Atlantic cod, Gadus morhua , averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2^−ΔΔCt method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.     

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.     

Immune Response and Disease Resistance of Barramundi,Lates calcarifer (Bloch), Offered Diets Supplemented with Replete Levels of Tryptophan or Whey

A basal diet containing 45% protein, 10% lipid, and 1.95% tryptophan (Trp) was formulated, and then supplemented with crystalline Trp (0, 0.1, 0.5, and 1% of the diet) or whey (16.9%) to evaluate the physiological effects of juvenile (initial weight: 0.57 ± 0.15 g) barramundi over a 58‐d rearing period. Significant reductions in growth and feed intake were observed in fish offered diets supplemented with Trp as compared to those offered the basal diet or the whey diet. Brain serotonin increased in fish fed the 1% Trp and whey diets albeit a decrease in cannibalism was not observed. Phagocytic activity, and Mx gene expression in spleens were not significantly changed among all groups. There were significant increases in lysozyme activity in the 1% Trp and whey groups, respiratory burst in the whey group, and Mx gene expression in head kidneys in the 1% Trp group 12 h after nerve necrosis virus (NNV) injection and in the whey 12 group and 24 h after NNV injection. Cumulative mortalities of fish fed the whey diet were significantly lower than those of fish fed the basal diet after Aeromonas hydrophila infection, but not Trp groups. Based on these results, diets supplemented with whey had reduced mortality when challenged with A. hydrophila and consequently function as an immunostimulant.     

鱼类神经坏死病毒研究进展与发展趋势

神经坏死病毒(nervous necrosis virus,NNV)是一种世界范围内流行、严重危害多种海水和淡水鱼类的传染性病原。NNV为单一正链、2节段RNA病毒,基因组由RNA1(3.1 kb)和RNA2(1.4 kb)组成。在病毒复制过程中,会合成亚基因组RNA3。RNA1编码RNA聚合酶。RNA2编码衣壳蛋白,为病毒的唯一结构蛋白。RNA3编码B1和B2两种非结构蛋白。根据病毒衣壳蛋白的基因序列,神经坏死病毒可以分成4种基因型,分别为拟鲹、红鳍东方鲀、条斑星鲽和赤点石斑神经坏死病毒基因型。但是,目前只发现A、B、C三种病毒血清型,A对应拟鲹神经坏死病毒基因型,B对应红鳍东方鲀神经坏死病毒基因型、C对应条斑星鲽神经坏死病毒和赤点石斑神经坏死病毒基因型。病毒存在垂直和水平两种传播途径,而且广泛分布于养殖和野生鱼类中。阻断病毒在野生与养殖鱼类之间的传播和开展新型鱼类疫苗研发是将来研究趋势。     

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.     

Modelling the effect of vaccination on transmission dynamics of nervous necrosis virus in grouper larvae Epinephelus coioides

Nervous necrosis virus (NNV) infection in susceptible grouper larvae has been reported to cause high mortalities, leading to great economic losses in aquaculture industry. Although the effects of NNV vaccines on grouper have been broadly investigated, vaccination strategies have not been fully established. To this end, we introduced the parsimonious epidemiological models that explored the assessment of key epidemiological parameters and how they changed when vaccinations showed the effects. We showed that the models capture the published cumulative mortality data accurately. We estimated a basic reproduction number R₀ = 2.44 for NNV transmission in grouper larvae without vaccination. To effectively control NNV transmission by vaccination, a model for disease control was also generalized to attain the goals of controlled reproduction number less than 1. Our results indicated that at least 60% of grouper population needed to be immunized for ~75 min. Our data-driven modelling approach that links the transmission dynamics of NNV and vaccination strategies for grouper has the potential to support evidence-based planning and adaptation of integrated control measures. We encourage that the epidemiology-based framework introduced here can be further implemented for establishing effective vaccination and mitigation actions aimed at controlling diseases in fish farming practices.     

Comparative pathogenicity study of ten different betanodavirus strains in experimentally infected European sea bass,Dicentrarchus labrax (L.)

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a severe pathological condition caused by RNA viruses belonging to the Nodaviridae family, genus Betanodavirus. The disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the Mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. To date, four different genotypes have been identified, namely red‐spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus and barfin flounder nervous necrosis virus, with the RGNNV genotype appearing as the most widespread in the Mediterranean region, although SJNNV‐type strains and reassortant viruses have also been reported. The existence of these genetically different strains could be the reason for the differences in mortality observed in the field. However, very little experimental data are available on the pathogenicity of these viruses in farmed fish. Therefore, in this study, the pathogenicity of 10 isolates has been assessed with an in vivo trial. The investigation was conducted using the European sea bass, the first target fish species for the disease in the Mediterranean basin. Naive fish were challenged by immersion and clinical signs and mortality were recorded for 68 days; furthermore, samples collected at selected time points were analysed to evaluate the development of the infection. Finally, survivors were weighed to estimate the growth reduction. The statistically supported results obtained in this study demonstrated different pathogenicity patterns, underlined the potential risk represented by different strains in the transmission of the infection to highly susceptible species and highlighted the indirect damage caused by a clinical outbreak of VER/VNN.     

Susceptibility of isogeneic ginbuna Carassius auratus langsdorfii Temminck et Schlegel to cyprinid herpesvirus‐2 (CyHV‐2) as a model species

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus‐2 (CyHV‐2), has affected the commercial production of the goldfish Carassius auratus and gibelio carp Carassius auratus gibelio. High water temperature treatments are reported to reduce the mortality rate of infected goldfish and elicit immunity in the survivors. To define the mechanism by which this intervention induces resistance, clonal ginbuna Carassius auratus langsdorfii, which is closely related to both species and has been used in fish immunology, may represent a promising model species. In this study, we investigated the susceptibility of clonal ginbuna strains to CyHV‐2 and the effect of high water temperature treatment on infected ginbuna and goldfish. Experimental intraperitoneal infection with CyHV‐2 at 25 °C caused 100% mortality in ginbuna strains, which was accompanied by histopathological changes typical of HVHN. Both infected ginbuna S3n strain and goldfish, exposed to high temperature for 6 days [shifting from 25 °C (permissive) to 34 °C (non‐permissive)], showed reduced mortalities after the 1st inoculation, and subsequent 2nd virus challenge to 0%, indicating induction of immunity. It was concluded that ginbuna showed a similar susceptibility and disease development in CyHV‐2 infection compared to goldfish, suggesting that ginbuna can be a useful fish model for the study of CyHV‐2 infection and immunity.     

Infections of nervous necrosis virus in wild and cage‐reared marine fish from South China Sea with unexpected wide host ranges

The concerns about the impact of the nervous necrosis virus (NNV) infections in wild fish have been raised. This paper presents the results of quarterly surveys of NNV in wild and cage‐reared marine fish from South China Sea. Samples of 892 wild fish belonging to 69 species and 381 cage‐reared fish belonging to 11 species were collected and were detected by seminested PCR and nested PCR. In the case of seminested PCR, the positive signal was detected in 3.0% and 3.1% samples of wild and cage‐reared fish, respectively. However, by nested RT‐PCR, the positive signal was observed in 42.3% and 63.0% samples of wild and cage‐reared fish, respectively. If the fish species were considered, the positive signal was detected in 21.7% and 72.7% species of wild and cage‐reared fish by seminested PCR assay, respectively. However, by nested RT‐PCR, the positive signal was observed in 65.2% and 100% species of wild and cage‐reared fish, respectively. The nucleotide sequences of the nested PCR products were determined. Phylogenetic tree showed that all the obtained viral isolates belonged to the red‐spotted grouper nervous necrosis virus (RGNNV) genotype. Thirty‐five species of the marine fish were the new hosts of NNV.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.