Piscine orthoreovirus‐3 is prevalent in wild seatrout (Salmo trutta L.) in Norway

In 2017, a PCR‐based survey for Piscine orthoreovirus‐3 (PRV‐3) was conducted in wild anadromous and non‐anadromous salmonids in Norway. In seatrout (anadromous Salmo trutta L.), the virus was present in 16.6% of the fish and in 15 of 21 investigated rivers. Four of 221 (1.8%) Atlantic salmon (Salmo salar L.) from three of 15 rivers were also PCR‐positive, with Ct‐values indicating low amounts of viral RNA. All anadromous Arctic char (Salvelinus alpinus L.) were PCR‐negative. Neither non‐anadromous trout (brown trout) nor landlocked salmon were PRV‐3 positive. Altogether, these findings suggest that in Norway PRV‐3 is more prevalent in the marine environment. In contrast, PRV‐3 is present in areas with intensive inland farming in continental Europe. PRV‐3 genome sequences from Norwegian seatrout grouped together with sequences from rainbow trout (Oncorhynchus mykiss Walbaum) in Norway and Coho salmon (Oncorhynchus kisutch Walbaum) in Chile. At present, the origin of the virus remains unknown. Nevertheless, the study highlights the value of safeguarding native fish by upholding natural and artificial barriers that hinder introduction and spread, on a local or national scale, of alien fish species and their pathogens. Accordingly, further investigations of freshwater reservoirs and interactions with farmed salmonids are warranted.     

Real-time RT-qPCR assay to detect sequences in the Piscine orthoreovirus-1 genome segment S1 associated with heart and skeletal muscle inflammation in Atlantic salmon

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.     

Preservation Methods for Retaining N-3 Polyunsaturated Fatty Acids in Alaska Coho Salmon (Oncorhynchus kisutch) Products

Abstract

Coho salmon (Oncorhynchus kisutch) fillets were processed using five different methods (smoking, canning, freezing, acidifying, and salting) to evaluate the effect of preservation choice on the quality of polyunsaturated fatty acids (PUFA). Salmon preserved by smoking, canning, or freezing retained higher values of total fatty acids, including n-3 PUFAs such as eicosapentaenoic acid and docosahexaenoic acid. Salting and acidifying (pickling) treatments resulted in a significant decrease in PUFAs. The results of this study are intended to provide direction for handling and storage of salmon to retain the maximum levels of high-value n-3 PUFAs.     

Heavy Metal Content of Coho Oncorhynchus kisutch and Chinook Salmon O. tschawytscha Reared in Deep Upwelled Ocean Waters in Hawaii1

Coho (Oncorhynchus kisutch) and chinook salmon (O. tschawyrscha) were reared in seawater pumped from the 600 m depth at the Natural Energy Laboratory of Hawaii. Fish were reared in either 100% deep water at 11 C, or a mixture of 55% deep and 45% surface waters at 17 C. Salmon flesh was analyzed for mercury, zinc, cadmium and lead. Mercury concentration in coho salmon reared at 11 C was significantly lower than the concentration in coho reared at 17 C (P < 0.05), but other heavy metal concentrations in tissue did not differ between the two species or between the two rearing temperatures. Compared with wild salmon from the northwest Pacific Ocean, salmon reared in Hawaii had significantly lower concentrations of mercury and lead (P < 0.05), but significantly higher concentrations of zinc and cadmium. These differences in heavy metal concentration between wild and cultured salmon were most readily explained by differences in the heavy metal content of diets and the lack of heavy metal concentrations in rearing water. The concentration of mercury in salmon tissue was at least 20 times less than the 1.0 μ/g U.S. Food and Drug Administration action level for mercury. Action levels have not been established for zinc, cadmium, and lead.     

Cross-sectional study of histopathology and piscine orthoreovirus during a marine production cycle of farmed Atlantic salmon (Salmo salar L.) in British Columbia,Canada

Two cohorts of farmed Atlantic salmon, Salmo salar L., in British Columbia, Canada, were sampled for histopathology (nine organs) and piscine orthoreovirus (PRV-1) PCR after seawater entry at 2, 4, 6, 8, 10, 13, 16 and 19 months (20 fish per cohort per date). One cohort—from a PRV+ hatchery—remained PRV+ throughout the study (sample prevalence 80%–100%). In an adjacent pen, the other cohort—from a PRV− hatchery—was 0% PRV+ at 78 days, 30% PRV+ at 128 days and ≥95% PRV+ thereafter. Among sample cohorts that were ≥80% PRV+, median Ct values were nominally less among fish sourced from the PRV− hatchery (28.7–33.3) than the PRV+ hatchery (30.8–35.2). No microscopic lesions were associated with PRV Ct value (minimum = 25.6). About 3% of fish in both cohorts had moderate inflammatory heart lesions; among these fish, only one had skeletal muscle inflammation (mild), and PRV Ct values were similar to unaffected cohorts sampled the same day. Also, among 16 moribund or freshly dead fish sampled opportunistically during the study, 14 were PRV+, and none had significant inflammatory heart lesions. These data support the hypothesis that British Columbia PRV-1 does not contribute to mortality.     

Piscine reovirus (PRV) in wild Atlantic salmon,Salmo salar L., and sea‐trout,Salmo trutta L., in Norway

This is the first comprehensive study on the occurrence and distribution of piscine reovirus (PRV) in Atlantic salmon, Salmo salar L., caught in Norwegian rivers. PRV is a newly discovered reovirus associated with heart and skeletal muscle inflammation (HSMI), a serious and commercially important disease affecting farmed Atlantic salmon in Norway. A cross‐sectional survey based on real‐time RT‐PCR screening of head kidney samples from wild, cultivated and escaped farmed Atlantic salmon caught from 2007 to 2009 in Norwegian rivers has been conducted. In addition, anadromous trout (sea‐trout), Salmo trutta L., caught from 2007 to 2010, and anadromous Arctic char, Salvelinus alpinus (L.), caught from 2007 to 2009, were tested. PRV was detected in Atlantic salmon from all counties included in the study and in 31 of 36 examined rivers. PRV was also detected in sea‐trout but not in anadromous Arctic char. In this study, the mean proportion of PRV positives was 13.4% in wild Atlantic salmon, 24.0% in salmon released for stock enhancement purposes and 55.2% in escaped farmed salmon. Histopathological examination of hearts from 21 PRV‐positive wild and one cultivated salmon (Ct values ranging from 17.0 to 39.8) revealed no HSMI‐related lesions. Thus, it seems that PRV is widespread in Atlantic salmon returning to Norwegian rivers, and that the virus can be present in high titres without causing lesions traditionally associated with HSMI.     

Mortality outbreak by perch rhabdovirus in European perch (Perca fluviatilis) farmed in Italy: Clinical presentation and phylogenetic analysis

This work reports a mortality outbreak, occurred in 2015 and affecting juveniles of European perch (Perca fluviatilis L.) farmed in Italy. Perch rhabdovirus (PRV) was detected by viral isolation and biomolecular investigations. Phylogenetic analysis clustered our isolate into genogroup B, which also includes PRV isolates from Perca fluviatilis identified in France (2004–2009); diagnostic investigations also revealed opportunistic bacteria (Aeromonas hydrophila) and parasites (Chilodonella piscicola). Since, occasionally, PRV has been reported in the natural environment, which is often a source of eggs and broodstock for farms, it could be possible that both similar France and Italian isolate were imported from a same place elsewhere and have a common origin. Improving biosecurity measures (batch control) and disinfection of egg strings with an iodine‐based solution helps prevent apparent vertical transmission of PRV.     

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.‐approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA‐positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.     

Piscine reovirus in wild and farmed salmonids in British Columbia,Canada: 1974–2013

Piscine reovirus (PRV) was common among wild and farmed salmonids in British Columbia, western Canada, from 1987 to 2013. Salmonid tissues tested for PRV by real‐time rRT‐PCR included sections from archived paraffin blocks from 1974 to 2008 (n = 363) and fresh‐frozen hearts from 2013 (n = 916). The earliest PRV‐positive sample was from a wild‐source steelhead trout, Oncorhynchus mykiss (Walbaum), from 1977. By histopathology (n = 404), no fish had lesions diagnostic for heart and skeletal muscle inflammation (HSMI). In some groups, lymphohistiocytic endocarditis affected a greater proportion of fish with PRV than fish without PRV, but the range of Ct values among affected fish was within the range of Ct values among unaffected fish. Also, fish with the lowest PRV Ct values (18.4–21.7) lacked endocarditis or any other consistent lesion. From 1987 to 1994, the proportion of PRV positives was not significantly different between farmed Atlantic salmon, Salmo salar L. (44% of 48), and wild‐source salmonids (31% of 45). In 2013, the proportion of PRV positives was not significantly different between wild coho salmon, Oncorhynchus kisutch (Walbaum), sampled from British Columbia (5.0% of 60) or the reference region, Alaska, USA (10% of 58).     

Oxytetracycline depletion and withdrawal time estimation following intraperitoneal administration in three species from Chilean salmon farming

This study was conducted to evaluate the half‐time (T½) and withdrawal time (WT) of oxytetracycline (OTC) following intraperitoneal (i.p.) administration of OTC (24.8–34.7 mg/kg) in three farmed salmonid species, Coho salmon (Oncorhynchus kisutch), Rainbow trout (Oncorhynchus mikiss) and Atlantic salmon (Salmo salar). A detection technique in fish skin muscle through a high‐performance liquid chromatography (HPLC) with a diode array detector (DAD) was developed and validated. The depletion studies were carried out in controlled conditions (nine studies) and under field conditions (one study). The T½ and WT estimations from the skin muscle after the i.p. administration of OTC in salmonids appear to be longer than studies where the OTC was orally administrated. Furthermore, the OTC maximum concentration in muscle seems to be also higher in the i.p. treatment. Due to the prolonged WT following the i.p. OTC administration, cautions related to the salmon harvest time should be consider in order to prevent OTC traces in the final product.     

Inactivated Piscine orthoreovirus vaccine protects against heart and skeletal muscle inflammation in Atlantic salmon

Heart‐ and skeletal muscle inflammation (HSMI) caused by infection with Piscine orthoreovirus (PRV) is one of the most common viral diseases in farmed Atlantic salmon (Salmo salar) in Norway, and disease outbreaks have been reported in most countries with large‐scale Atlantic salmon aquaculture. Currently there is no vaccine available for protection against HSMI, partly due to the lack of a cell line for efficient virus propagation. Erythrocytes are the primary target cells for PRV in vivo and a potential source for isolation of PRV particles. In this study, PRV was purified from infected erythrocytes, inactivated and used in a vaccination trial against HSMI. A single immunization with adjuvanted, inactivated PRV induced protection against HSMI in Atlantic salmon infected by virus injection 6 weeks later, while a moderate protection was obtained in fish infected through natural transmission, i.e. cohabitation. The PRV vaccine significantly reduced PRV loads and histopathological lesions typical for HSMI compared to the unvaccinated control group. This is the first demonstration of protective vaccination against PRV, and promising for future control of HSMI in Atlantic salmon aquaculture.     

Piscine reovirus,but not Jaundice Syndrome,was transmissible to Chinook Salmon,Oncorhynchus tshawytscha (Walbaum), Sockeye Salmon,Oncorhynchus nerka (Walbaum), and Atlantic Salmon,Salmo salar L.

A Jaundice Syndrome occurs sporadically among sea‐pen‐farmed Chinook Salmon in British Columbia, the westernmost province of Canada. Affected salmon are easily identified by a distinctive yellow discolouration of the abdominal and periorbital regions. Through traditional diagnostics, no bacterial or viral agents were cultured from tissues of jaundiced Chinook Salmon; however, piscine reovirus (PRV) was identified via RT‐rPCR in all 10 affected fish sampled. By histopathology, Jaundice Syndrome is an acute to peracute systemic disease, and the time from first clinical signs to death is likely <48 h; renal tubular epithelial cell necrosis is the most consistent lesion. In an infectivity trial, Chinook Salmon, Sockeye Salmon and Atlantic Salmon, intraperitoneally inoculated with a PRV‐positive organ homogenate from jaundiced Chinook Salmon, developed no gross or microscopic evidence of jaundice despite persistence of PRV for the 5‐month holding period. The results from this study demonstrate that the Jaundice Syndrome was not transmissible by injection of material from infected fish and that PRV was not the sole aetiological factor for the condition. Additionally, these findings showed the Pacific coast strain of PRV, while transmissible, was of low pathogenicity for Atlantic Salmon, Chinook Salmon and Sockeye Salmon.     

Presence and genetic variability of Piscine orthoreovirus genotype 1 (PRV‐1) in wild salmonids in Northern Europe and North Atlantic Ocean

Piscine orthoreovirus genotype 1 (PRV‐1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV‐1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV‐1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV‐1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV‐1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV‐1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV‐1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free‐living fish and provides rationale for screening wild broodfish used in restocking programmes.     

Climate effects on size‐at‐age and growth rate of Chinook Salmon (Oncorhynchus tshawytscha) in the Fraser River,Canada

Decline in size‐at‐age of Chinook Salmon (Oncorhynchus tshawytscha) has been observed for many populations across the entire Northeast Pacific Ocean, and identifying external drivers of this decline is important for sustainable management of these ecologically, economically, and culturally valuable resources. We assessed size‐at‐age of 96,939 Chinook Salmon sampled in the Fraser River watershed (Canada) from 1969 to 2017. A broad decline in size‐at‐age was confirmed across all population aggregates of Fraser River Chinook Salmon, in particular since year 2000. By developing a novel probability‐based approach to calculate age‐ and year‐specific growth rates for Fraser River Chinook Salmon and relating growth rates to environmental conditions in specific years through a machine learning method (boosted regression trees), we were able to disentangle multi‐year effects on size‐at‐age and thus identify environmental factors that were most related to the observed size‐at‐age of Chinook Salmon. Among 10 selected environmental variables, ocean salinity at Entrance Island in spring, the Aleutian Low Pressure Index and the North Pacific Current Bifurcation Index were consistently identified as important contributors for four of the seven age and population aggregate combinations. These top environmental contributors could be incorporated into future stock assessment and forecast models to improve Chinook Salmon fisheries management under climate change.     

Molecular identification of iridoviruses infecting various sturgeon species in Europe

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low‐to‐high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74–88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus‐European (AcIV‐E). Moreover, three samples infected with AcIV‐E showed genetic heterogeneity, with the co‐existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV‐E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV‐E and an NV‐related virus.     

Differences in stability effects on the marine survival of hatchery pink salmon (Oncorhynchus gorbuscha) within the upwelling and downwelling domains of the northeast Pacific Ocean

Regional coastal conditions have a strong influence on juvenile salmon survival during their critical first months in the marine environment. Salmon (genus Oncorhynchus) survival has been thought to be favored within the high latitude downwelling domain if water column stabilities increase, whereas stability may have the opposite effect in upwelling‐dominated lower latitudes. In this study, the relationships between water column stabilities during early marine residence of pink salmon (Oncorhynchus gorbuscha) in both the upwelling and downwelling domains of the northeast Pacific Ocean and marine survival rates for hatchery stocks ranging from Vancouver Island, British Columbia, to Kodiak Island, Alaska, were explored. Contrary to expectation, there was no clear difference in the effect of stability on marine survival rates in the downwelling and upwelling domains. In both domains, marine survival rates increased for pink salmon stocks that experienced below‐average stability on the inner shelf during early marine residence. Stability effects from the outer shelf showed no consistent relationship to marine survival within the northeast Pacific.     

Health status of two salmonid aquaculture facilities in North Portugal: characterization of the bacterial and viral pathogens causing notifiable diseases

A comparative bacteriological and virological survey was conducted in two fish farms in the North of Portugal. The fish species examined included cultured rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., as well as wild fish captured near both facilities. The microbial load in the internal organs of apparently healthy fish was nonitored over a year, an all the disease problems occurring during this period were investigated. Although both farms presented intermediate levels of infection(30–40% infected fish), farm B showed the poorest microbiological quality since constant but low mortalities were observed throughout the year. Flavobacterium and Psedomonas-Xanthomonas were the predominant bacterial groups, comprising around 40–50% of the isolates from each farm. In farm B, members of the Enterobacteriaceae and mortile Aeromonas also showed significant prevalence (about 20%). The only outbreak of a notifiable disease was an occurrence of furunculosis, caused by Aeromonas salmonicida subsp. salmoncida, in farm A. However, Yersinia ruckeri was isolated not only from diseased fish, but also from asymptomatic fish, usually in mixed infections with motile Aeromonas or infections with motile Aeromonas or infectious pancreatic necrosis virus (IPNV). While Y. ruckeri isolates associated with mortalities belonged to the serotype O1 (subgroup a), those isolated from asymtomatic fish corresponded to serotype O3. Two strains of IPNV (serotype Ab) were isolated in farm B, which represents the first viral agent detected in Portuguese aquaculture. Qualitative and quantitative differences in microbial load were observed between cultured and wild fish. No notifiable bacterial or viral pathogens were detected in any of the feral species studied.     

Inactivation of Piscine orthoreovirus

Piscine orthoreovirus infects various salmonid fish species, and the infection is associated with diseases such as heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). There are no vaccines available or genetically selected resistant hosts that can efficiently control piscine orthoreovirus (PRV) infection. Currently, the only prophylactic measure against PRV is general biosecurity measures aiming to break the transmission cycle. Methods to eradicate infectious virus from contaminated facilities are desirable, but the knowledge on how to inactivate PRV is lacking. A major bottleneck for inactivation studies is the lack of ability to propagate PRV in cell culture. Therefore, in this study we developed an in vivo model for detection of infectious PRV particles after treatment of the virus with inactivation tools such as heat, pH, iodine, UV and commercially available disinfectants. The results show that standard iodine treatment is efficient in inactivation of the virus, and similarly are high and low pH extremes and treatment with Virocid, a commercially available disinfectant. A UV dose of at least 50 mJ/cm² is required for inactivation, and the virus has high resistance against heat treatment.     

Melanized focal changes in skeletal muscle in farmed Atlantic salmon after natural infection with Piscine orthoreovirus (PRV)

Melanized focal changes in skeletal muscle of farmed Atlantic salmon (Salmo salar) are a major quality problem. The aetiology is unknown, but infection with Piscine orthoreovirus (PRV) has been associated with the condition. Here, we addressed the pathogenesis of red and melanized focal changes and their association with PRV. First, a population of farmed fish (PRV‐negative prior to sea transfer) was sequentially investigated throughout the seawater period. The fish were autopsied and tested for PRV infection. Muscular changes were described by macroscopy and histology, and a classification system was established. Second, in an experimental infection trial, PRV was injected intramuscularly to induce changes. The farmed fish was gradually infected with PRV. Red focal changes occurred throughout the observation period with a low prevalence regardless of PRV status. Melanized changes were highly diverse and their prevalence increased during the trial. Changes of low macroscopic grade and histological category were more prevalent in PRV‐negative fish. Diffuse granulomatous melanized changes only occurred after PRV infection. No muscular changes were observed in the experimentally challenged fish. Our studies do not indicate that PRV infection causes red focal changes, but seems important in the development of granulomatous melanized changes.