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.     

Plasma protein changes in Atlantic salmon, Salmo salar L., with infectious salmon anaemia

Moderate to severe anaemia and hypoproteinaemia were reported in a Canadian outbreak of 'haemorrhagic kidney syndrome' in Atlantic salmon, later shown to be caused by a variant of infectious salmon anaemia virus (ISAV). The progressive anaemia associated with ISA has been previously reported, but hypoproteinaemia in salmon infected with European isolates of ISA virus has not been well documented. The present study showed a very significant positive correlation between decreasing haematocrit values and total plasma protein concentrations in Atlantic salmon infected with two Canadian and two Norwegian ISA viral isolates. However, variations in the concentration of individual plasma proteins, typical of acute phase responses in higher vertebrates, were not observed.     

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.     

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.     

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.     

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.     

Haemorrhagic kidney syndrome of Atlantic salmon, Salmo salar L.

This report describes a new syndrome affecting farmed Atlantic salmon on the Canadian east coast that has resulted in increased morbidity and mortality in affected stocks. The major pathological findings are apparent only microscopically and include renal interstitial haemorrhage and acute tubular necrosis and tubular casting. As a result, the disease has become known as haemorrhagic kidney syndrome (HKS). Affected fish are lethargic and anorectic, and lack external lesions. Clinically, HKS fish are anaemic, hypoproteinaemic and hyperosmolalic, with increased serum concentrations of sodium and chloride. At necropsy, internal changes ranged from apparently normal to include one or several of the following: swelling and/or patchy reddening of the kidney, pale gills, exophthalmos, serosanguinous ascites, darkening of the posterior intestine and splenomegaly. Ultrastructurally, viral inclusions were found in the cytoplasm of erythrocytes of HKS fish, and there were unusual electron‐dense inclusions within the tips of renal tubular microvilli of HKS fish. The significance and relevance of the ultrastructural findings to HKS are unknown. Virus isolation was attempted using CHSE, RTG‐2, FH‐10, BB and EPC cell lines; no virus was isolated. Bacteriological analysis failed to reveal significant pathogens. Analysis of tissues for heavy metals and pesticides was negative. Assays for clostridial toxins, lipopolysaccharide and verotoxins were negative. The aetiology of HKS remains unresolved.     

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.     

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).     

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.     

传染性鲑鱼贫血症病毒实时荧光环介导等温扩增检测方法的建立

根据ISAV的基因保守序列,利用LAMP Designer软件设计了6条引物,采用新型的环介导等温扩增设备进行扩增和检测,优化了反应条件,分析了所建立方法的特异性和灵敏度,并与RT-PCR和实时荧光RT-PCR进行比较。研究表明,该方法最适反应温度为64℃,反应10 min就可以观察到明显的扩增。该方法灵敏度高,检测限为78.4 fg RNA,比常规RT-PCR灵敏度高100倍,与实时荧光定量RT-PCR灵敏度相当;特异性好,与传染性胰腺坏死病毒(IPNV)、鲤春病毒血症病毒(SVCV)、出血性败血症病毒(VHSV)、鱼类病毒性神经坏死病病毒(VNNV)、鱼腹水病毒(YAV)等14种主要鱼类病毒没有交叉反应。结果表明,本研究建立了ISAV的实时荧光环介导等温扩增检测方法,实验能对整个扩增过程进行实时监测,提高检测灵敏度的同时,防止由于开盖跑电泳或加染料而导致的污染。     

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.     

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.     

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.     

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.     

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.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

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.