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.     

Spatial and non-spatial risk factors associated with cage-level distribution of infectious salmon anaemia at three Atlantic salmon, Salmo salar L., farms in Maine, USA

The distribution of infectious salmon anaemia (ISA) was examined among 80 cages from three Atlantic salmon grow-out farms in Maine, USA that were stocked with smolts from a single hatchery. Cage-level disease was broadly defined as one or more moribund fish testing positive for infectious salmon anaemia virus (ISAV) by RT-PCR and a second confirmatory test (IFAT, culture or genotype sequence). Spatio-temporal and cage-level risks were explored using logistic regression and survival analysis. Non-spatial risk factors associated with ISA, or shortened survival time to disease, included increased predation, trucking company choice for smolt transfers, a finely-sedimented benthic substrate, and smaller average size of smolts at stocking. Univariable analysis identified the time-dependent spatial factor 'adjacency to newly infected cages' to be predictive of new infection in neighbouring cages 11-12 weeks later. However, none of the spatial factors, or their lags retained relevance in multiple-variable models. The results suggest a diffuse distribution of virus exposure throughout infected sites, with host-susceptibility factors probably influencing disease manifestation in individual cages. The narrow focus of the current study may limit application of the findings to other sites and year-classes. However, these data support the relevance of husbandry efforts to optimize fish health in regions affected by ISAV.     

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.     

Mapping quantitative trait loci for infectious salmon anaemia resistance in a North American strain of Atlantic salmon

Infectious salmon anaemia (ISA) is a highly virulent viral disease of Atlantic salmon that causes massive economic losses to infected aquaculture operations. Our goal was to detect and map quantitative trait loci (QTL) that confer resistance to ISA in an admixed commercial strain of Atlantic salmon that was largely founded from the Saint John River (SJR) in North America. Full‐sibling families were challenged with a virulent strain of ISA virus. Mortality was tracked during two annual trials with individual fish that survived to the end of the trial being classified as ‘resistant’, and those that died were classified as ‘susceptible’. Ten families with intermediate levels of mortality and an average size of 54.2 individuals were chosen for genotyping with a 50K SNP array designed for the SJR strain. Single nucleotide polymorphisms that were segregating within families were first used to make a composite 11K female linkage map that was then used to find the positions of QTL for ISA resistance using a half‐sib model. The dam‐based HS model detected a total of three QTL for ISA resistance including an experiment‐wide significant QTL on Ssa25 that accounted for 8.3% of the phenotypic variance and chromosome‐wide significant QTL on Ssa03 and on Ssa04 that accounted for 6.0% and 6.6% respectively. We conclude that classic linkage mapping within families continues to be an important method of detecting QTL for oligogenic traits in strains founded from multiple populations. Single nucleotide polymorphisms with moderate trait effects are being used to select within families for more ISA‐resistant strains of Atlantic salmon.     

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.     

Risk factors associated with mortalities attributed to infectious salmon anaemia virus in New Brunswick, Canada

Outbreaks of unexplained mortalities attributed to infectious salmon anaemia (ISA) were examined in the 1996 year class of Atlantic salmon in three regions of New Brunswick, Canada. A total of 218 net pens at 14 sites deemed to have been exposed to ISA virus (ISAV) were surveyed for mortality records and management, environmental and host characteristics. Based on definitions of mortality patterns, clinical ISA disease outbreaks occurred in 106 net pens. There were eight sites in which >50% of net pens experienced ISA outbreaks during the study period. Factors related to their potential role in transmission of virus to new sites or new net pens at the same site were identified as sea lice vectors, divers visiting multiple sites, sites belonging to companies with more than one site, exposure to other year classes at the site, and proximity to other infected net pens. Host resistance factors associated with greater risk of outbreaks were identified as larger groupings, general health following smolt transfer, stressful husbandry procedures during growout, and health or productivity during colder water periods. Despite very close proximity between sites, modification of these management factors would probably influence the severity of mortalities caused by ISAV.     

Selective breeding can increase resistance of Atlantic salmon to furunculosis, infectious salmon anaemia and infectious pancreatic necrosis

We reasoned that by challenging large numbers of Atlantic salmon families with the causative agents of furunculosis, infectious salmon anaemia (ISA) and infectious pancreatic necrosis (IPN), we could show unequivocally that resistance to these diseases expresses moderate‐to‐high levels of additive genetic variation, and that the resistances are weakly correlated genetically. We tested this reasoning by challenging Atlantic salmon from 920 (approximately) full‐sib families with the causative agents of furunculosis and ISA, and fish from 265 of these families with the causative agent of IPN. Additive genetic variation and genetic correlations were estimated by fitting a threshold liability model to the resistances assessed as binary traits. Resistance to furunculosis, ISA and IPN was moderate –to highly heritable. The marginal posterior means for heritability on the underlying liability scale were 0.37 for resistance to ISA, and 0.55 and 0.62 for resistance to IPN and furunculosis. Genetic correlations between the resistances were weak (?0.11 to 0.07). These levels of additive genetic variation indicate that resistance to furunculosis, ISA and IPN will respond to selection. The weak genetic correlations indicate that it should be relatively easy to improve resistance to the diseases simultaneously. We believe that there is now strong evidence that selectively breeding Atlantic salmon for resistance can be highly successful.     

Assessment of infectious salmon anaemia virus prevalence for different groups of farmed Atlantic salmon, Salmo salar L., in New Brunswick

Abstract Infectious salmon anaemia (ISA) virus (ISAV) has been causing disease in New Brunswick since 1996. As a control measure, all fish in an outbreak cage are killed. The objective of this study was to compare ISAV prevalence in cages experiencing an outbreak with healthy cages from the same farm, neighbouring farms and distant farms. Atlantic salmon from five different groups were tested using an RT-PCR test. Groups included moribund fish from a cage experiencing an outbreak (A), healthy fish from an outbreak cage (B), healthy fish from a negative cage from a farm experiencing an outbreak in a different cage (C), healthy fish from a negative farm near an outbreak farm (D), and healthy fish sampled at a negative farm located in an area with only negative farms (E). Apparent prevalences (standard error) for the different groups (A-E) were 0.94 (+/-0.026), 0.41 (+/-0.062), 0.29 (+/-0.040), 0.08 (+/-0.037) and 0.08 (+/-0.037), respectively. All groups were significantly different (P < 0.002) from each other except for groups B and C and groups D and E. Because the prevalence of the virus was significantly higher in the outbreak cage (B) compared with other sites, early harvest of outbreak cages will remove one source of virus. However, ISA negative cages (C) that remain on the positive farm may potentially act as a viral reservoir.     

Multiple passage of infectious salmon anaemia virus in rainbow trout,Oncorhynchus mykiss (Walbaum), did not induce increased virus load

The infectious salmon anaemia virus (ISAV) has not been observed to cause natural disease in farmed rainbow trout, Onchorhynchus mykiss (Walbaum), but may cause high mortality in farmed Atlantic salmon, Salmo salar L. In this study, ISAV was passaged 10 times in succession by intraperitoneal injections of serum from previous passage into naïve rainbow trout. The serum viraemia was monitored by real‐time qPCR. The rainbow trout in this study became infected but did not develop ISA. No clinical signs were observed in the rainbow trout in any passage, but replication of ISAV was detected from Day 4 post‐infection (p.i.). Neither increased relative virus loads nor histopathological and immunohistochemical findings consistent with ISA were observed. However, the expression of interferon type I and Mx genes were slightly up‐regulated in the hearts of some individual fish at day 17 p.i. Sequencing of all open reading frames in the ISAV genome of the 10th passage revealed two nucleotide mutations, one in segment 6 coding for the haemagglutinin–esterase (HE) and one in segment 1 coding for the basic polymerase 2 (PB2). The mutation in HE resulted in an amino acid substitution T/K₃₁₂.     

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.     

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.     

Presence of infectious salmon anaemia virus (ISAV) in tissues of Atlantic salmon, Salmo salar L., collected during three separate outbreaks of the disease

Abstract. Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon, Salmo salar L., in Norway. The enveloped virus particles (100nm) believed to be the causative agent of the disease have been observed budding from endothelial cells in heart blood vessels. However, it is not known if the virus propagates in endothelial cells in all tissues/organs, if other target cells exist or if material collected from different salmon farms with natural outbreaks of ISA contain the same virus particles. Salmon smolts from three hatcheries with no history of disease were taken into the laboratory and experimentally challenged with ISA collected from Atlantic salmon during natural outbreaks of the disease in three different fish farms outside Bergen. Norway. Tissues for TEM studies were Collected from: (1) organs that showed clinical signs of ISA (i.e. used in the diagnosis of the disease); (2) tissues believed to be important in transmission of the virus (integument, kidney, urinary bladder, gut and somatic muscle); and (3) hormone-producing tissues (pituitary gland, saccus vasculosus, thymus, thyroid, ultimobranehial gland, gonad, head kidney, heart and ventral aorta). The same virus as that believed to be the causative agent of ISA was found in all tissues examined from the challenged fish, i.e. a multiorgan infection with the same virus present in salmon from all three fish farms. The virus particles are about 100 nm in diameter, consisting of a slightly pleomorphic unit membrane envelope within which are a number of granules about 10–12nm in diameter. The granules seemed to be arranged in two concentric circles (spheres). The virus was seen budding from the surface of endothelial cells in blood vessels/sinus only. However, the virus was found intracellularly in both endothelial cells and in leucocytes.     

Study of the interaction between a persistent infectious pancreatic necrosis virus (IPNV) infection and experimental infectious salmon anaemia (ISA) in Atlantic salmon, Salmo salar L.

Abstract. An infectious pancreatic necrosis virus (IPNV) carrier stock of Atlantic salmon parr (100 g) was divided between two tanks and inoculated experimentally with tissue homogenate containing the aetiologic agent of infectious salmon anaemia (ISA) and non-ISA tissue homogenate (control), respectively. Plasma and kidney samples from ISA-infected and control fish were taken twice weekly for 25 days. In the kidney samples, IPNV was quantified by a plaque assay. In plasma, anti-IPNV antibodies were measured using an indirect ELISA. The ISA-infection did not seem to activate the IPNV-infection. Neither the proportion of fish with IPNV or anti-IPNV antibodies, nor the IPNV titre or level of anti-IPNV antibodies showed any specific trend during the study. Independently of ISA, IPNV was detected in 54 out of 132 fish (41%), while 71 out of 195 fish (36%) had plasma antibodies against IPNV. No association was found between detection of IPNV, and presence or level of anti-IPNV antibodies in individual fish.     

In vivo virulence and genomic comparison of infectious Salmon Anaemia Virus isolates from Atlantic Canada

The infectious salmon anaemia virus (ISAV) is capable of causing a significant disease in Atlantic salmon, which has resulted in considerable financial losses for salmon farmers around the world. Since the first detection of ISAV in Canada in 1996, it has been a high priority for aquatic animal health management and surveillance programmes have led to the identification of many genetically distinct ISAV isolates of variable virulence. In this study, we evaluated the virulence of three ISAV isolates detected in Atlantic Canada in 2012 by doing in vivo‐controlled disease challenges with two sources of Atlantic salmon. We measured viral loads in fish tissues during the course of infection. Sequences of the full viral RNA genomes of these three ISAV isolates were obtained and compared to a high‐virulence and previously characterized isolate detected in the Bay of Fundy in 2004, as well as a newly identified ISAV NA‐HPR0 isolate. All three ISAV isolates studied were shown to be of low to mid‐virulence with fish from source A having a lower mortality rate than fish from source B. Viral load estimation using an RT‐qPCR assay targeting viral segment 8 showed a high degree of similarity between tissues. Through genomic comparison, we identified various amino acid substitutions unique to some isolates, including a stop codon in the segment 8 ORF2 not previously reported in ISAV, present in the isolate with the lowest observed virulence.     

Leucocytes from Atlantic salmon, Salmo salar L., experimentally infected with infectious salmon anaemia (ISA) exhibit an impaired response to mitogens

Abstract. The proliferative response to mitogens of head kidney leucocytes from Atlantic salmon, Salmo salar L., experimentally infected with infectious salmon anaemia (ISA) was examined. The mean haematocrits of ISA-inoculated fish were significantly lower than the mean haematocrits of control-inoculated fish at day 14. Mortality in ISA-inoculated fish appeared at day 16 after inoculation. Seven days after inoculation, leucocytes from ISA-inoculated fish showed an increased response to phytohaemagglutinin (PHA) compared to control-inoculated fish, while no change in the response to lipopolysaccharide from Escherichia coli (LPS) could be observed. At days 14 and 22 after inoculation, the responses to both LPS and PHA of leucocytes from ISA-inoculated fish were severely impaired. These suppressions of the immune response of leucocytes from ISA-inoculated fish were found in fish with low haematocrits (< 15) as well as in fish with haematocrits higher than 30, suggesting that suppression of the immune system and the development of anaemia are independent events in the pathogenesis of ISA.     

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.     

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.