Detection of SJNNV and RGNNV genotypes using a relative quantification RT‐PCR assay

Viral Encephalopathy and Retinopathy (VER), is caused by a nodavirus included within the Betanodavirus genus of the Nodaviridae family. This disease affects more than 30 marine fish species worldwide and has been a major obstacle in the aquaculture industry; control of the disease is based on virus detection, essentially in carrier specimens. This study describes a real time PCR procedure for viral nervous necrosis virus detection from several organs of sea bass, Senegalese sole, and gilt‐head sea bream, from fish displaying either clinical symptoms or asymptomatic cases. The sensitivity of this technique was about 10⁶‐fold higher than that of the conventional RT‐PCR. The newly designed primers detected nodavirus isolates belonging to the RGNNV and SJNNV genotypes.     

Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production

The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using real-time PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV.     

Husbandry stress during early life stages affects the stress response and health status of juvenile sea bass, Dicentrarchus labrax

In aquaculture management it is important to establish objective criteria to assess health and welfare of the fish. Here we show that European sea bass (Dicentrarchus labrax) confronted with husbandry-associated stress (tank cleaning, i.e. scrubbing, and water temperature variation) during early life stages show poorer survival and disease resistance as juveniles. We evaluated several parameters for stress (plasma cortisol, glucose and lactate, hydromineral status), growth performance, the immune response (plasma IgM levels) and the effects of a nodavirus challenge. Principal component analysis allowed the establishment of a stress panel including plasma cortisol, osmolality, IgM levels and weight. Sea bass juveniles reared during early life in high and constant temperature perform best in terms of stress-related parameters assessed by principle component analysis. Variable water temperature triggers dramatic changes in plasma cortisol, osmolality, IgM levels, body weight and susceptibility to nodavirus that suggest a strong and prolonged activation of the HPI axis. Scrubbing induces some disturbances typical for mild short-term, acute stress, viz. increased plasma osmolality and decreased IgM levels, but does not affect plasma cortisol, growth or susceptibility to nodavirus of sea bass. Our data fit well with the concept of allostasis. We discuss the relevance of our work for sea bass aquaculture.     

Sea lice as a density-dependent constraint to salmonid farming

Fisheries catches worldwide have shown no increase over the last two decades, while aquaculture has been booming. To cover the demand for fish in the growing human population, continued high growth rates in aquaculture are needed. A potential constraint to such growth is infectious diseases, as disease transmission rates are expected to increase with increasing densities of farmed fish. Using an extensive dataset from all farms growing salmonids along the Norwegian coast, we document that densities of farmed salmonids surrounding individual farms have a strong effect on farm levels of parasitic sea lice and efforts to control sea lice infections. Furthermore, increased intervention efforts have been unsuccessful in controlling elevated infection levels in high salmonid density areas in 2009-2010. Our results emphasize host density effects of farmed salmonids on the population dynamics of sea lice and suggest that parasitic sea lice represent a potent negative feedback mechanism that may limit sustainable spatial densities of farmed salmonids.     

Susceptibility of spotted wolffish Anarhichas minor to experimental infection with nodavirus and infectious pancreatic necrosis virus

The spotted wolffish Anarhichas minor is a promising new species for cold-water aquaculture. The broad host-range of piscine nodavirus (NV) and infectious pancreatic necrosis virus (IPNV) makes them potentially pathogenic to new fish species in aquaculture. IPNV and NV strains highly pathogenic in farmed Atlantic salmon Salmo salar and halibut Hippoglossus hippoglassus, respectively, in Norway were used for the challenge of spotted wolffish. In general, water-borne infection with IPNV and NV resulted in significant mortality among juveniles <1 g. Cumulative mortality after bath-challenge and cohabitation was 60 to 75% in the smallest juveniles (0.3 g). Intramuscular and intraperitoneal injection of NV was 100% lethal to wolffish of 10 g, and the groups at 12 degrees C died before those at 7 degrees C. No cohabitants of this size died, but NV was still detectable in these individuals after 10 wk. A persistent IPNV infection with low mortality developed in bath-challenged juveniles of 0.7 g, in which IPNV was still detectable 4 mo later. This study comprises a demonstration of experimental viral infections in cultured spotted wolffish, although to date no natural outbreaks of viral diseases have been reported in this species.     

Environmental monitoring through protist next‐generation sequencing metabarcoding: assessing the impact of fish farming on benthic foraminifera communities

The measurement of species diversity represents a powerful tool for assessing the impacts of human activities on marine ecosystems. Traditionally, the impact of fish farming on the coastal environment is evaluated by monitoring the dynamics of macrobenthic infaunal populations. However, taxonomic sorting and morphology‐based identification of the macrobenthos demand highly trained specialists and are extremely time‐consuming and costly, making it unsuitable for large‐scale biomonitoring efforts involving numerous samples. Here, we propose to alleviate this laborious task by developing protist metabarcoding tools based on next‐generation sequencing (NGS) of environmental DNA and RNA extracted from sediment samples. In this study, we analysed the response of benthic foraminiferal communities to the variation of environmental gradients associated with salmon farms in Scotland. We investigated the foraminiferal diversity based on ribosomal minibarcode sequences generated by the Illumina NGS technology. We compared the molecular data with morphospecies counts and with environmental gradients, including distance to cages and redox used as a proxy for sediment oxygenation. Our study revealed high variations between foraminiferal communities collected in the vicinity of fish farms and at distant locations. We found evidence for species richness decrease in impacted sites, especially visible in the RNA data. We also detected some candidate bioindicator foraminiferal species. Based on this proof‐of‐concept study, we conclude that NGS metabarcoding using foraminifera and other protists has potential to become a new tool for surveying the impact of aquaculture and other industrial activities in the marine environment.     

Occurrence of Tetracycline Resistance Genes in Aquaculture Facilities with Varying Use of Oxytetracycline

The contribution of human activities to environmental reservoirs of antibiotic resistance is poorly understood. The purpose of this study was to determine if oxytetracycline (OTC) use in aquaculture facilities increased the detection frequency (i.e., prevalence) of tetracycline resistance (tet^R) genes relative to facilities with no recent OTC treatment. We used polymerase chain reaction to screen water and sediment from four noncommercial fish farms in northwestern Wisconsin for the presence of ten tet^R determinants: tet(A), tet(B), tet(D), tet(E), tet(G), tet(M), tet(O), tet(Q), tet(S), and tet(W). Water from farms with recent OTC use had significantly higher tet^R detection frequencies than did water from farms without recent OTC use, with prevalence in raceways and rearing ponds of farms with recent OTC use exceeding by more than twofold that of farms not using OTC. Effluent from all farms, regardless of treatment regime, had higher tet^R detection frequencies than their corresponding influent for all genes, but the specific combinations of tet^R genes detected in a sample were not different from their corresponding influent. Although OTC use was associated with the increased occurrence and diversity of tet^R genes in water samples, it was not found to relate to tet^R gene occurrence in sediment samples. Sediment samples from facilities with no recent OTC use had significantly higher frequencies of tet^R gene detection than did samples from facilities with recent OTC use. All of the tet^R genes were detected in both the medicated and nonmedicated feed samples analyzed in this study. These findings suggest that both OTC treatment in aquaculture facilities and the farms themselves may be sources of tet^R gene introduction to the environment. To our knowledge, this is the first study to use genotypic and cultivation-independent methods to examine tet^R gene occurrence associated with OTC use in aquaculture.     

Genetic screening of farmed Atlantic salmon escapees demonstrates that triploid fish display reduced migration to freshwater

Each year, hundreds of thousands of farmed Atlantic salmon escape from fish farms into the wild. Some of these escapees enter freshwater, and manage to interbreed with native populations. To hinder further genetic introgression in native populations, the use of sterile triploid salmon within commercial aquaculture is being examined. However, if triploid escapees migrate into freshwater, they may still have ecological impacts on local populations. In the present study, we used microsatellite DNA genotyping to determine the ploidy of 3794 farmed escapees captured in 17 Norwegian rivers in the period 2007–2014. Although a previous study has reported an average of 2 % triploids in Norwegian fish farms during this exact period, here, we only observed 7 (0.18 %) triploids among the escapees captured in freshwater. In addition, we identified three trisomic escapees. For the triploids where the within-river capture location was determined, they were only observed in the lower reaches and not on the spawning grounds. It is concluded that propensity for triploid Atlantic salmon to migrate into freshwater following escape from a fish farm is significantly lower than for normal diploid salmon escapees. Therefore, commercial production of triploids should not only be seen as an effective way of stopping genetic introgression, it will also significantly reduce the numbers of escapees entering rivers, which in turn limits ecological interactions and potential disease transmission.     

Molecular epidemiology of infectious hematopoietic necrosis virus reveals complex virus traffic and evolution within southern Idaho aquaculture

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which infects salmon and trout and may cause disease with up to 90% mortality. In the Hagerman Valley of Idaho, IHNV is endemic or epidemic among numerous fish farms and resource mitigation hatcheries. A previous study characterizing the genetic diversity among 84 IHNV isolates at 4 virus-endemic rainbow trout farms indicated that multiple lineages of relatively high diversity co-circulated at these facilities (Troyer et al. 2000 J Gen Virol. 81:2823-2832). We tested the hypothesis that high IHNV genetic diversity and co-circulating lineages are present in aquaculture facilities throughout this region. In this study, 73 virus isolates from 14 rainbow trout farms and 3 state hatcheries in the Hagerman Valley, isolated between 1978 and 1999, were genetically characterized by sequence analysis of a 303 nucleotide region of the glycoprotein gene. Phylogenetic and epidemiological analyses showed that multiple IHNV lineages co-circulate in a complex pattern throughout private trout farms and state hatcheries in the valley. IHNV maintained within the valley appears to have evolved significantly over the 22 yr study period.     

Jellyfish Stings Trigger Gill Disorders and Increased Mortality in Farmed Sparus aurata (Linnaeus, 1758) in the Mediterranean Sea

Jellyfish are of particular concern for marine finfish aquaculture. In recent years repeated mass mortality episodes of farmed fish were caused by blooms of gelatinous cnidarian stingers, as a consequence of a wide range of hemolytic, cytotoxic, and neurotoxic properties of associated cnidocytes venoms. The mauve stinger jellyfish Pelagia noctiluca (Scyphozoa) has been identified as direct causative agent for several documented fish mortality events both in Northern Europe and the Mediterranean Sea aquaculture farms. We investigated the effects of P. noctiluca envenomations on the gilthead sea bream Sparus aurata by in vivo laboratory assays. Fish were incubated for 8 hours with jellyfish at 3 different densities in 300 l experimental tanks. Gill disorders were assessed by histological analyses and histopathological scoring of samples collected at time intervals from 3 hours to 4 weeks after initial exposure. Fish gills showed different extent and severity of gill lesions according to jellyfish density and incubation time, and long after the removal of jellyfish from tanks. Jellyfish envenomation elicits local and systemic inflammation reactions, histopathology and gill cell toxicity, with severe impacts on fish health. Altogether, these results shows P. noctiluca swarms may represent a high risk for Mediterranean finfish aquaculture farms, generating significant gill damage after only a few hours of contact with farmed S. aurata. Due to the growth of the aquaculture sector and the increased frequency of jellyfish blooms in the coastal waters, negative interactions between stinging jellyfish and farmed fish are likely to increase with the potential for significant economic losses.     

Molecular Genetic Analysis of Stomach Contents Reveals Wild Atlantic Cod Feeding on Piscine Reovirus (PRV) Infected Atlantic Salmon Originating from a Commercial Fish Farm

In March 2012, fishermen operating in a fjord in Northern Norway reported catching Atlantic cod, a native fish forming an economically important marine fishery in this region, with unusual prey in their stomachs. It was speculated that these could be Atlantic salmon, which is not typical prey for cod at this time of the year in the coastal zone. These observations were therefore reported to the Norwegian Directorate of Fisheries as a suspected interaction between a local fish farm and this commercial fishery. Statistical analyses of genetic data from 17 microsatellite markers genotyped on 36 partially-degraded prey, samples of salmon from a local fish farm, and samples from the nearest wild population permitted the following conclusions: 1. The prey were Atlantic salmon, 2. These salmon did not originate from the local wild population, and 3. The local farm was the most probable source of these prey. Additional tests demonstrated that 21 of the 36 prey were infected with piscine reovirus. While the potential link between piscine reovirus and the disease heart and skeletal muscle inflammation is still under scientific debate, this disease had caused mortality of large numbers of salmon in the farm in the month prior to the fishermen''s observations. These analyses provide new insights into interactions between domesticated and wild fish.     

<Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>: an integrated view of a bacterial fish pathogen

Pasteurellosis, or pseudotuberculosis, is a bacterial septicaemia caused by the halophilic bacterium Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida). Although this disease was first described in wild populations of white perch and striped bass, currently the natural hosts of the pathogen are a wide variety of marine fish. The disease has great economic impact both in Japan, where it affects mainly yellowtail cultures, and in the Mediterranean area, due to the losses it causes in seabream and seabass farms. This microorganism serves as a perfect model to study a bacterial fish pathogen, either at an applied level, to resolve or to mitigate the high economic losses of fish farmers, or at a basic level, for a better understanding of P. damselae subsp. piscicida biology. This article discusses the methods employed in our laboratory to study the causative agent of pasteurellosis. It reviews important aspects, from the diverse procedures for the detection and isolation of the pathogen to the latest molecular studies that have allowed its correct taxonomic allocation. Characterization of some virulence mechanisms and the available methods to prevent the disease are also presented. Electronic Publication     

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.     

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.     

Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India

Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase. Darkened and moribund larvae showed no evidence of bacterial or parasitic infections. Tissue sections of brain and spinal cord showed clear necrotic vacuolation. Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells. The viral particles measured 28 to 30 nm in diameter. Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp. The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae. This is the first report of piscine nodavirus infection from the Indian sub-continent.