Infectious salmon anaemia – pathogenesis and tropism

Infectious salmon anaemia (ISA) is a serious disease of farmed Atlantic salmon caused by the aquatic orthomyxovirus infectious salmon anaemia virus (ISAV). ISA was first detected in Norway in 1984 and was characterized by severe anaemia and circulatory disturbances. This review elucidates factors related to the pathogenesis of ISA in Atlantic salmon, the dissemination of the virus in the host and the general distribution of the 4‐O‐acetylated sialic acids ISAV receptor. The knowledge contributes to the understanding of this disease, and why, almost 30 years after the first detection, it is still causing problems for the aquaculture industry.     

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV‐3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin‐4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.     

Enhanced detection of infectious salmon anaemia virus using a low‐speed centrifugation technique in three fish cell lines

Infectious salmon anaemia (ISA), caused by ISA virus (ISAV), is a serious disease of farmed Atlantic salmon, Salmo salar L. Recently, molecular‐ and immunofluorescent‐based techniques have become powerful diagnostic tools for ISAV detection, but culture‐based techniques remain the gold standard. A disadvantage of ISAV culture is that the incubation time required before cytopathic effect (CPE) is observed in cell monolayers. To decrease time until CPE is observed, a low‐speed centrifugation technique was applied to existing standard operating procedures for ISAV culture in three fish cell lines. Time until CPE observation was compared in CHSE, SHK and ASK cells, treated or not treated with low‐speed centrifugation after inoculation with ISAV. Low‐speed centrifugation treatment significantly enhanced observable cell infection. Compared to control cells, the length of time until ISAV CPE observation decreased in centrifuged ASK and CHSE cells. Low‐speed centrifugation was also incorporated into a modified clinical shell vial assay. At 48 h post‐inoculation with approximately 20 viral particles, ISAV was detected by an immunofluorescence antibody test in treated ASK and SHK1 cells but not in control cells. Finally, this enhanced viral adsorption assay performed in ASK cells demonstrated higher sensitivity than a real‐time RT‐PCR assay performed on RNA isolated from ISAV‐spiked salmon kidney homogenates.     

Host tropism of infectious salmon anaemia virus in marine and freshwater fish species

The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net‐pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod‐like and perch‐like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists.     

Replication of the infectious salmon anaemia virus (ISAV) in rainbow trout, Oncorhynchus mykiss (Walbaum)

Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon, Salmo salar L., in Norway. However, in laboratory experiments, the virus has also been found to propagate in trout, Salmo trutta L., where it is a persistent infection. It is crucial for the management of ISA in Norway, and for the prevention of spread to fish in other countries, that possible carriers of the virus are found. Another possible salmonid reservoir species is the rainbow trout, Oncorhynchus mykiss (Walbaum). After experimental infection, the ISA virus was found to be present in O. mykiss as late 28 days after infection, with a peak around day 20. The infection resulted in a significant drop in the haematocrit and haemorrhages on the liver in some specimens. The ISA virus was seen budding from endothelial cells lining blood vessels in the heart ventricle of challenged rainbow trout. It is concluded that the ISA virus is able to propagate in O. mykiss and that this species may function as a reservoir when cultured in areas where the virus is found. The virus does not seem to cause any significant mortalities of infected rainbow trout.     

Experimental infection of Atlantic salmon, Salmo salar L., with infectious salmon anaemia virus: a histopathological study

Recent reports of the isolation of infectious salmon anaemia virus (ISAV) from Atlantic salmon, Salmo salar L., affected by haemorrhagic kidney syndrome (HKS) suggest that ISAV can cause severe renal haemorrhage and necrosis in addition to well-known pathognomonic hepatocellular necrosis and haemorrhage. The prevalence of ISAV-induced pathognomonic renal HKS lesions and their correlation to pathognomonic hepatic lesions of infectious salmon anaemia (ISA) is not known. The present experimental infection of Atlantic salmon with a Canadian isolate of ISAV found that pathognomonic hepatic ISA lesions were present in 90.6% and pathognomonic renal HKS lesions in 78.1% of fish which died after the experimental challenge. Both pathognomonic hepatic ISA lesions and pathognomonic renal HKS lesions were found together in 65.6% of fish which died after ISAV challenge. The present study clearly demonstrates that ISAV can cause a very high prevalence of both HKS and ISA pathognomonic lesions.     

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon,Salmo salar L.

The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real‐time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post‐infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortality of 100% as compared to 20% in the LVI group by day 23 post‐infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).     

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.     

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.     

Relative resistance of Pacific salmon to infectious salmon anaemia virus

Infectious salmon anaemia (ISA) is a major disease of Atlantic salmon, Salmo salar, caused by an orthomyxovirus (ISAV). Increases in global aquaculture and the international movement of fish made it important to determine if Pacific salmon are at risk. Steelhead trout, Oncorhynchus mykiss, and chum, O. keta, Chinook, O. tshawytscha, coho, O. kisutch, and Atlantic salmon were injected intraperitoneally with a high, medium, or low dose of a Norwegian strain of ISAV. In a second challenge, the same species, except chum salmon, were injected with a high dose of either a Canadian or the Norwegian strain. Average cumulative mortality of Atlantic salmon in trial 1 was 12% in the high dose group, 20% in the medium dose group and 16% in the low dose group. The average cumulative mortality of Atlantic salmon in trial 2 was 98%. No signs typical of ISA and no ISAV-related mortality occurred among any of the groups of Oncorhynchus spp. in either experiment, although ISAV was reisolated from some fish sampled at intervals post-challenge. The results indicate that while Oncorhynchus spp. are quite resistant to ISAV relative to Atlantic salmon, the potential for ISAV to adapt to Oncorhynchus spp. should not be ignored.     

The effect of competition among three salmonids on dominance and growth during the juvenile life stage

Although non‐native species can sometimes threaten the value of ecosystem services, their presence can contribute to the benefits derived from the environment. In the Great Lakes, non‐native brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) support substantial recreational fisheries. With current efforts underway to restore once‐native Atlantic salmon (Salmo salar) to Lake Ontario, there is some concern that Atlantic salmon will impede non‐native contributions to the recreational fishery because Atlantic salmon exhibit niche overlap with brown trout and rainbow trout, particularly during the juvenile life stage. We therefore examined competition and growth of juvenile Atlantic salmon, brown trout and rainbow trout in semi‐natural streams. We found that brown trout were the most dominant and had the greatest growth rate regardless of what other species were present. Rainbow trout were more dominant than Atlantic salmon and consumed the most food of the three species. However, in the presence of brown trout, rainbow trout fed less frequently and exhibited negative growth as compared to when the rainbow trout were present with only Atlantic salmon. These data suggest that, outside of density‐dependent effects, Atlantic salmon will not impact stream production of brown trout and rainbow trout.     

Variation in semen production of farmed Atlantic salmon and rainbow trout

The variation in semen production among farmed Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) has been studied. Both species were stripped at weekly intervals, the Atlantic salmon four times and the rainbow trout three times.The individual variation in volume of semen was very high, particularly in rainbow trout. The total volume of semen obtained was 137 ml (20 ml/kg body weight) in Atlantic salmon and 23 ml (5 ml/kg body weight) in rainbow trout. The intraclass correlation for volume of semen was estimated at 0.73 in Atlantic salmon and at 0.59 in rainbow trout. The correlations between volume of semen and body size (weight and length) were all positive. They were all significant and medium in Atlantic salmon whereas in rainbow trout they were all low and significant only for volume of semen at first stripping.The number of males needed to supply the Norwegian fish farming industry with semen is discussed. It is concluded that the possibility of disseminating genetic improvement throughout the whole population of farmed Atlantic salmon and rainbow trout by transport of semen from selected males is considerable.     

High‐oil residue camelina meal,a viable source of protein at low levels in diets for juvenile salmonids

The oilseed Camelina sativa has been studied as a lipid source for farmed salmonids, but recommended inclusion as a protein source has not been determined. This study evaluated low inclusion of camelina high‐oil residue meal (HORM) at 20, 40 and 60 g/kg of the diet, to determine an adequate level for Atlantic salmon parr (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Salmon and trout were fed experimental diets containing up to 60 g/kg HORM for 16 weeks. At 40 g/kg HORM, trout and salmon growth performance were similar to those fed a control diet. However, at 60 g/kg HORM, trout showed lower final weight, weight gain and feed intake than those fed the control diet. Rainbow trout fed 40 and 60 g/kg HORM showed significantly lower whole body ash (p = .005), slightly lower whole body protein levels and higher fat than the control. In salmon fed 60 g/kg HORM diets, whole body ash (p = .024), and the submucosal layer of the intestine was thicker than the control (p = .007). Current results indicate that up to 40 g/kg HORM can be included in diets for rainbow trout and salmon juveniles.     

Distended, water-filled stomach in seawater farmed rainbow trout, Oncorhynchus mykiss (Walbaum), provoked experimentally by osmoregulatory stress

The present study evaluates if abdominal dis-tension caused by a water-filled stomach in seawater farmed rainbow trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon, Salmo salar L., could be provoked experimentally by live chilling in sea water (0.5 °C). Fifty rainbow trout and 50 salmon were visually classified, either as normal or suffering from the condition. Prior to chilling, no rainbow trout or salmon suffered from a water-filled stomach. During chilling, 25% of the rainbow trout and 2.5% of the salmon developed water-filled stomach. Affected rainbow trout had significantly higher blood plasma osmolality and significantly lower body weight than normal trout. The frequency of trout with a water-filled stomach increased significantly with increasing chilling time and increasing plasma osmolality. The regression coefficients revealed that the proportion of affected rainbow trout in-creased by 0.82% h⁻¹ of chilling and by 0.46% per unit increase in mosmol L^−1. The present study revealed that abdominal distension in seawater farmed rainbow trout was far more severe than in Atlantic salmon and could be provoked experimentally by osmoregulatory stress. This is consistent with the observation that water-filled stomach appear only occasionally in seawater farmed Atlantic salmon.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.