Immune responses of orange-spotted grouper,Epinephelus coioides,against virus-like particles of betanodavirus produced in Escherichia coli

Betanodaviruses are the causative agents of viral nervous necrosis (VNN), a serious disease of cultured marine fish worldwide. Virus-like particles (VLPs) are one of the good novel vaccine candidates to control this disease. Until now, betanodavirus vaccine studies mainly focused on the humoral immune response and mortality after virus challenge. However, little is known about the activation of genes responsible for cellular and innate immunity by vaccines. In the present study, VLPs of orange-spotted grouper nervous necrosis virus (OGNNV) were produced in prokaryotes and their ability to enter Asian sea bass cells was the same as native virus, suggesting that they possess a similar structure to OGNNV. VLPs immunogenicity was then determined by intramuscularly vaccinating Epinephelus coioides at different concentrations (1.5 or 15 μg g^?1 fish body weight, FBW) and immunizing frequencies (administration once, twice and thrice). A single vaccination with the dosage of 1.5 μg g^?1 FBW is enough to provoke high titer antibodies (average 3 fold higher than that of negative control) with strong neutralizing antibody titer as early as 1 week post immunization. Furthermore, quantitative PCR analysis revealed that eleven genes associated with humoral, cellular and innate immunities were up-regulated in the liver, spleen and head kidney at 12 h post immunization, correlating with the early antibody response. In conclusion, we demonstrated that VLP vaccination induced humoral immune responses and activated genes associated with cellular and innate immunity against betanodavirus infection in orange-spotted grouper.     

Genome analysis of Betanodavirus from cultured marine fish species in Malaysia

Betanodavirus is the causative agent of the viral nervous necrosis (VNN) or viral encephalopathy and retinopathy disease in marine fish. This disease is responsible for most of the mass mortalities that occurred in marine fish hatcheries in Malaysia. The genome of this virus consists of two positive-sense RNA molecules which are the RNA1 and RNA2. The RNA1 molecule contains the RdRp gene which encodes for the RNA-dependent RNA polymerase and the RNA2 molecule contains the Cp gene which encodes for the viral coat protein. In this study, total RNAs were extracted from 32 fish specimens representing the four most cultured marine fish species in Malaysia. The fish specimens were collected from different hatcheries and aquaculture farms in Malaysia. The RNA1 was successfully amplified using three pairs of overlapping PCR primers whereas the RNA2 was amplified using a pair of primers. The nucleotide analysis of RdRp gene revealed that the Betanodavirus in Malaysia were 94.5-99.7% similar to the RGNNV genotype, 79.8-82.1% similar to SJNNV genotype, 81.5-82.4% similar to BFNNV genotype and 79.8-80.7% similar to TPNNV genotype. However, they showed lower similarities to FHV (9.4-14.2%) and BBV (7.2-15.7%), respectively. Similarly, the Cp gene revealed that the viruses showed high nucleotide similarity to RGNNV (95.9-99.8%), SJNNV (72.2-77.4%), BFNNV (80.9-83.5%), TPNNV (77.2-78.1%) and TNV (75.1-76.5%). However, as in the RdRp gene, the coat protein gene was highly dissimilar to FHV (3.0%) and BBV (2.6-4.1%), respectively. Based on the genome analysis, the Betanodavirus infecting cultured marine fish species in Malaysia belong to the RGNNV genotype. However, the phylogenetic analysis of the genes revealed that the viruses can be further divided into nine sub-groups. This has been expected since various marine fish species of different origins are cultured in Malaysia.     

Viral encephalopathy and retinopathy of Dicentrarchus labrax and Sparus aurata farmed in Tunisia

Viruses belonging to the Nodaviridae family cause disease worldwide among a large number of species of marine fish, and have been described in all continents. In the present study, a total of 69 farmed Tunisian sea bass (Dicentrarchus labrax) and 24 sea bream (Sparus aurata) samples were tested monthly for the detection of betanodavirus. The virus was identified in both species using indirect immunofluorescence assays (IFAT) and RT-PCR. In addition sequence analysis of part of the coat protein gene indicated that both species were infected by highly related, but distinct, strains belonging to the RGNNV genotype. The sequence of the coat protein gene of several strains was identical but up to 9 different sequences were detected in a single farm. In addition, viral sequences obtained from fish that were held at lower temperature (<20°C) were distinct from the rest of the sequences.     

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10¹ to 10⁸ copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (C_t) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of C_t values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R² > 0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID₅₀/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.     

鱼类病毒性神经坏死病研究现状

鱼类病毒性神经坏死病是近年来严重危害海水鱼类,引起暴发性流行的重大病害之一。目前,已知受神经坏死病毒感染的鱼类达40多种。该病毒包括4 种血清型,即红鳍东方鲀神经坏死病毒、拟鲹神经坏死病毒、条斑星鲽神经坏死病毒、赤点石斑鱼神经坏死病毒。患病鱼常表现出游泳异常,身体失去平衡等典型神经性疾病症状。病理组织学观察可见中枢神经系统特别是脑和视网膜出现严重的坏死、空泡化。病毒可通过垂直和水平两种途径传播。关于病毒的命名、感染机制及其防治还需深入研究。     

Caligus sclerotinosus (Copepoda: Caligidae), a serious pest of cultured red seabream Pagrus major (Sparidae) in Korea

Caligid copepods (Crustacea) known as sea lice are pests of cultured fish, causing serious diseases and economic losses in fish aquaculture worldwide. One species, Caligus sclerotinosus Roubal, Armitage & Rohde, 1983 (Caligidae), is considered a serious pest of the highly prized red seabream Pagrus major (Temminck and Schlegel, 1843) (Sparidae) cultured in Japan. Recently, in neighboring Korea, red seabream culture has intensified and almost replaced yellow tail culture. However, until now, there have been no reports on infection of this sea louse from red seabream in Korea. We surveyed 120 (20 fish per month) P. major from a sea ranched Tongyeong Marine Research Center aquaculture facility, Gyeongsangnamdo, Korea for six months in 2011 (June to November). We recorded severe infection by the sea louse C. sclerotinosus on the skin of P. major. Prevalence was 100%, mean intensity 7.06, maximum intensity 49, and minimum intensity 2. Adult females (624), males (219) and few chalimi (5) were observed and identified by their morphology. As an average of all our collections, less than 0.6% of individuals were chalimi. We suggest, therefore, that adults of C. sclerotinosus undergo ontogenetic host switching after their final moult. No infection of C. sclerotinosus was found on wild P. major collected from Tongyeong and Yeosu fish markets on the southern coast of Korea. Severe infection by this sea louse may cause secondary infections of the host. This copepod is already reported from Australia and Japan and hence, this is the first report from Korea. We expect this pest to have an impact on Korean red seabream fisheries equally serious to that being experienced in Japan.     

In vitro study of the replication capacity of the RGNNV and the SJNNV betanodavirus genotypes and their natural reassortants in response to temperature

Betanodaviruses are the causative agents of viral nervous necrosis and affect a broad range of fish species worldwide. Their bi-segmented genome is composed of the RNA1 and the RNA2 molecules encoding the viral polymerase and the coat protein, respectively. In southern Europe the presence of the RGNNV and the SJNNV genotypes, and the RGNNV/SJNNV and RGNNV/SJNNV reassortants has been documented. Several studies have reported a correlation between water temperature and disease onset. To explore the replication efficiency of betanodaviruses with different genomes in relation to temperature and to understand the role of genetic reassortment on viral phenotype, RGNNV, SJNNV, RGNNV/SJNNV and RGNNV/SJNNV field isolates were fully sequenced, and growth curves generated in vitro at four different temperatures (15, 20, 25, 30 °C) were developed for each isolate. The data obtained, corroborated by statistical analysis, demonstrated that viral titres of diverse betanodavirus genotypes varied significantly in relation to the incubation temperature of the culture. In particular, at 30 °C betanodaviruses under investigation presented different phenotypes, and viruses containing the RNA1 of the RGNNV genotype showed the best replication efficiency. Laboratory results demonstrated that viruses clustering within the same genotype based on the polymerase gene, possess similar growth kinetics in response to temperature, thus highlighting the key role of RNA1 in controlling viral replication at different environmental conditions. The results generated might have practical implications for the inference of viral phenotype according to genetic features and may contribute to a better understanding of betanodavirus ecology.     

Antigenic properties and experimental transmission to several fish species of a marine birnavirus isolated from sole (Solea senegalensis).

A cross-neutralization test was used to study the antigenic relationship of an aquabirnavirus isolated from sole (Solea senegalensis), named solevirus, and several infectious pancreatic necrosis virus (IPNV) strains. Solevirus was antigenically similar to IPNV strain Sp. Transmission of the solevirus to other fish species has been determined by inoculation to freshwater and marine fish species (two salmonids and gilt-head seabream). A higher pathogenicity was obtained for the marine fish species, although solevirus caused an asymptomatic infection in all species tested, as demonstrated by the detection of viral RNA and of viral antigens in fish leucocytes, respectively, using polymerase chain reaction (PCR) and flow cytometry (FC).     

Failure of the Polymerase Chain Reaction (PCR) Method to Detect Striped Jack Nervous Necrosis Virus (SJNNV) in Striped Jack Pseudocaranx dentex Selected as Spawners

Abstract

The causative agent of viral nervous necrosis (VNN) in striped jack Pseudocaranx dentex is transmitted from female and male spawners to larvae, and elimination of carrier spawners, determined by the detection of striped jack nervous necrosis virus (SJNNV) via polymerase chain reaction (PCR), has been used to prevent transmission of the disease in larval production facilities. However, some outbreaks of VNN occurred in larvae obtained from SJNNV-negative spawners. We compared the occurrence of infection between groups of larvae obtained from SJNNV-negative spawners and those from SJNNV-positive spawners. Viral nervous necrosis occurred in all seven groups of larvae obtained from the virus-positive spawners between the 3rd and 7th day of rearing. The virus was detected only occasionally after 2 weeks in four of six groups obtained from the virus-negative spawners. These results confirmed vertical transmission of the virus and revealed that a very small amount of SJNNV in spawners escaped PCR detection and could produce infection, although the frequency of infection occurring in the SJNNV-negative group was much lower than that among the larvae from SJNNV-positive spawners.     

Indications for both host-specific and introduced genotypes of Staphylococcus aureus in marine mammals

Staphylococcus aureus is present in the marine environment and causes disease in marine mammals. To determine whether marine mammals are colonized by host-specific strains or by strains originating from other species, we performed multi-locus sequence typing on ten S. aureus strains isolated from marine mammals in the U.K., the Netherlands, and the Antarctic. Four new sequence types of S. aureus were discovered. S. aureus strains from a southern elephant seal (n=1) and harbour porpoises (n=2) did not cluster with known S. aureus strains, suggesting that they may be host species-specific. In contrast, S. aureus strains from harbour seals (n=3), other harbour porpoises (n=3), and a grey seal (n=1) clustered with S. aureus strains previously isolated from domestic ruminants, humans, or birds, suggesting that these S. aureus strains in marine mammals were introduced from terrestrial species.     

Kinetics of viral load and erythrocytic inclusion body formation in Pacific herring artificially infected with erythrocytic necrosis virus

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.     

Salinity ranges of some southern African fish species occurring in estuaries

The recorded salinity ranges of 96 fish species occurring in southern African estuaries are documented. Factors influencing the tolerance of fishes to low and high salinity regimes are discussed, with most species tolerant of low rather than high salinity conditions. This is important since most systems are subject to periodic freshwater flooding, especially during summer. The penetration of freshwater teleosts and elasmobranchs into estuaries is examined and the occurrence of marine fishes in rivers documented. Mortalities arising from salinity extremes in southern African estuaries are discussed, with temperature a key factor initiating such fish kills. Only eight fish species have been recorded spawning in estuaries although the fry of many species are attracted to estuarine waters. It is concluded that a small percentage of southern African fishes have succeeded in utilizing estuaries and this may be attributed to the paucity of strong osmoregulators     

Kinetics of Viral Load and Erythrocytic Inclusion Body Formation in Pacific Herring Artificially Infected with Erythrocytic Necrosis Virus

Abstract

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic—a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0–4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

Received September 7, 2011; accepted March 8, 2012     

Measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion

Killer whales and sea otters maintained in captivity are the subjects of routine health monitoring programs, and interest in immunologic studies in sea otters has been rising recently in response to potential impacts from infectious disease and environmental pollution on the threatened southern sea otter population. Development of species-specific reagents for immunologic studies in these two marine mammals is currently in its infancy. In this study, killer whale and sea otter immunoglobulin-specific polyclonal antibodies were generated, and used to develop tests for serum Ig concentration in the killer whale (Orcinus orca) and the southern (Enhydra lutris nereis) and northern sea otter (Enhydra lutris lutris). Killer whale serum IgG was purified using caprylic acid/ammonium sulfate precipitation. Sea otter plasma IgG was purified using protein-A-agarose. Polyclonal anti-Ig antisera were produced in rabbits, and specificity confirmed by immunoelectrophoresis. Radial immunodiffusion was used to measure Ig concentration in serum or plasma samples derived from 21 captive killer whales, 18 wild and 4 captive southern sea otters and 15 wild and 4 captive northern sea otters grouped by age. Mean killer whale serum Ig concentration (+/-95% confidence interval) ranged from 15.04 +/- 3.97 g/l for animals aged 0-5 years to 26.65 +/- 9.8 g/l for animals aged >10 years. Mean sea otter serum Ig concentration (+/-95% confidence interval) ranged from 28.39 +/- 11.00 g/l for southern sub-adults to 32.76 +/- 11.58 g/l for southern adults. No significant difference in serum Ig concentration was found between southern and northern sea otters. Serum Ig concentrations in two northern sea otter pups were low compared to those of adult sea otters. The two serum Ig quantitation assays produced were highly specific and reproducible and will be useful additions to the limited number of tests available for immune function in these marine mammal species.     

Experimental Infection of Australian Freshwater Fish with Epizootic Haematopoietic Necrosis Virus (EHNV)

Abstract

The ranavirus, epizootic hematopoietic necrosis virus (EHNV), is endemic to southern Australia with natural outbreaks resulting in mass mortality events in wild Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and less severe disease in farmed Rainbow Trout Oncorhynchus mykiss. To further investigate the host range for EHNV, 12 ecologically or economically important freshwater fish species from southeastern Australia were exposed experimentally to the virus. A bath-challenge model at 18 ± 3°C was employed with limited use of intraperitoneal inoculation to determine if a species was likely to be susceptible to EHNV. Of the species tested, Murray–Darling Rainbowfish Melanotaenia fluviatilis and Dewfish Tandanus tandanus (also known as Freshwater Catfish) were considered to be potentially susceptible species. EHNV was isolated from approximately 7% of surviving Eastern Mosquitofish Gambusia holbrooki, indicating this widespread alien fish species is a potential carrier. The infection of Silver Perch Bidyanus bidyanus and Macquarie Perch Macquaria australasica and the lack of infection in Murray Cod Maccullochella peelii peelii and Golden Perch Macquaria ambigua ambigua after exposure to EHNV via water confirmed earlier data from Langdon (1989). Five other species of native fish were potentially not susceptible to the virus or the fish were able to recover during the standard 35-d postchallenge observation period. Overall, it appeared that EHNV was less virulent in the present experimental model than in previous studies, but the reasons for this were not identified.

Received May 21, 2012; accepted November 1, 2012     

Emerging pathologies in aquaculture: effects on production and food safety

Infectious diseases represent a limiting factor for the further development of Italian aquaculture. The recent introduction and spreading of new pathogens, along with the global climatic change, has contributed to a considerable decrease in trout production. Emerging pathologies in rainbow trout culture include viral diseases, e.g. infectious haematopoietic necrosis (IHN), bacterial diseases, such as lactococcosis and visceral flavobacteriosis, and parasitical diseases, e.g. proliferative kidney disease (PKD). Higher mortality rates in trout fry and fingerlings are generally induced by visceral flavobacteriosis and IHN, while the main losses in large trout during the warm season are due to lactococcosis and PKD. Mariculture has at present a better sanitary status compared to trout culture, but a rapid dissemination of pathogens, including zoonosis agents, is envisaged also for seabass and seabream. Emerging pathologies in sea bass include VNN, pseudotuberculosis, streptococcosis and tuberculosis. Seabream is much more resistant and is mainly affected by novel Vibrio infections and enteromyxidiosis. A good sanitary management of fish farms is essential for avoiding or limiting losses caused by emerging pathologies. Transmission of zoonosis agents to man, through the consumption of cultured fish, is very remote in Italy. On the contrary, transmission of Streptococcus iniae, Vibrio vulnificus and Mycobacterium marinum by means of improper manipulation of infected fish, could represent a potential hazard for fish farmers and fish processors, as well as for people preparing fish meals.     

Evidence of Brucella sp. infection in marine mammals stranded along the coast of southern New England.

After recent isolations of Brucella sp. from pinnipeds and cetaceans, a survey was initiated to investigate the prevalence of Brucella sp. infections and serologic evidence of exposure in marine mammals stranded along the coasts of Connecticut and Rhode Island. One hundred and nineteen serum samples from four species of cetaceans and four species of pinnipeds were collected from 1985 to 2000 and tested for antibodies to Brucella sp. using the brucellosis card test, buffered acidified plate antigen test, and rivanol test. In addition, 20 of these were necropsied between 1998 and 2000, with lymphoid and visceral tissues cultured for Brucella sp. Three of 21 (14%) harbor seals (Phoca vitulina) and four of 53 (8%) harp seals (Phoca groenlandica) were seropositive. Brucella sp. was isolated from two of four (50%) harbor seals and three of nine (33%) harp seals. Of the five animals with positive cultures, two were seropositive and three seronegative. Brucella sp. was most frequently cultured from the lung and axillary, inguinal, and prescapular lymph nodes. Tissues from which Brucella sp. was isolated showed no gross or histopathologic changes. These results indicate that marine mammals stranded along the coast of southern New England can be exposed to and infected with Brucella sp.     

Bacterial diseases of marine fish

The principal bacterial diseases found among wild and cultured marine fish are reviewed. The bacterial agents discussed include the Gram-negative pathogens in the Vibrio, Aeromonas, Pasteurella and Edwardsiella genera, Renibacterium salmoninarum and the myxobacteria, streptococci, mycobacteria, nocardias and anaerobic organisms which have been associated with fish diseases. Methods for the isolation and identification of these organisms are described.