The interaction of infectious salmon anaemia virus (ISAV) with the blue mussel,Mytilus edulis

Integrated multi‐trophic aquaculture (IMTA) is an alternative approach to mono‐culture aquaculture that reduces environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter‐feeding particulate‐bound organic nutrients. They may also increase or decrease disease risk on farms by serving as reservoirs or barriers for important finfish pathogens such as infectious salmon anaemia virus (ISAV). This study aimed to optimize culture and molecular assays in shellfish tissues and to determine the fate of ISAV in mussels, Mytilus edulis. To determine detection limits, qRT‐PCR and culture assays in both CHSE‐ and ASK cells were optimized in ISAV‐inoculated mussel tissue homogenates. Both qRT‐PCR and culture assays performed in ASK cells had comparable detection limits of 10^2.8 TCID₅₀ mL^?1. The ISAV RNA genome was consistently detected in digestive gland tissue of ISAV‐exposed mussels. Viable ISAV was not detected in mussel tissues by culture analysis in CHSE‐ and ASK cells. The fact that qRT‐PCR analysis resulted in positive cycle threshold (CT) values that corresponded to the detectable range of ISAV in ASK culture assays suggests that little to no viable ISAV particles are present in the mussel tissues.     

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.     

Estimation of infectious dose and viral shedding rates for infectious pancreatic necrosis virus in Atlantic salmon,Salmo salar L., post‐smolts

Infectious dose and shedding rates are important parameters to estimate in order to understand the transmission of infectious pancreatic necrosis virus (IPNV). Bath challenge of Atlantic salmon post‐smolts was selected as the route of experimental infection as this mimics a major natural route of exposure to IPNV infection. Doses ranging from 10² to 10^?4 50% end‐point tissue culture infectious dose (TCID₅₀) mL^?1 sea water were used to estimate the minimum infectious dose for a Scottish isolate of IPNV. The minimum dose required to induce infection in Atlantic salmon post‐smolts was <10^?1 TCID₅₀ mL^?1 by bath immersion (4 h at 10 °C). The peak shedding rate for IPNV following intraperitoneal challenge using post‐smolts was estimated to be 6.8 × 10³ TCID₅₀ h^?1 kg^?1 and occurred 11 days post‐challenge. This information may be incorporated into mathematical models to increase the understanding of the dispersal of IPNV from marine salmon sites.     

An alkaline phosphatase-based method for the detection of infectious salmon anaemia virus (ISAV) in tissue culture and tissue imprints

Currently, the presence of infectious salmon anaemia virus (ISAV) is often detected in Atlantic salmon by the use of an indirect fluorescent antibody test. This test is limited by the poor stability of fluorescein isothiocyanate which fades after about a week in storage, preventing the development of stained archive material as a reference source. One possible alternative would be the use of immunohistochemical staining methods to detect ISAV. An immunohistochemical method is presented that uses alkaline phosphatase‐conjugated antibodies and Vector^® Red as a substrate, to detect ISAV in kidney imprints. This paper also describes a procedure where Bouin's fluid is used to successfully inhibit endogenous alkaline phosphatase in tissue samples, prior to immunohistochemical processing. This method provides a stable stain that can be read for many weeks after staining or archived for future reference.     

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.     

Studies on the effect of temperature and pH on the inactivation of fish viral and bacterial pathogens

Disposal of fish by‐products in the European Community must comply with Regulation (EC) No 1069/2009 which categorizes animal by‐products according to risk, and specifies methods of disposal of by‐products according to that risk. There is provision under the regulation for composting or ensiling to be used for by‐products from aquatic animals. Biosecurity considerations require knowledge of the parameters of time and temperature, or time and pH, required to inactivate any fish pathogens that may be present. To provide those data, we undertook laboratory studies on the inactivation of a number of fish pathogenic viruses and bacteria at 60 °C, pH 4.0 and pH 12.0 as a preliminary to conducting subsequent trials with the most resistant viruses and bacteria in fish tissues. The most resistant bacterium to 60 °C, pH 4.0 as well as pH 12.0 was Lactococcus garvieae. Its concentration was reduced to the level of sensitivity of the test after 24–48 h exposure to 60 °C, but it survived for at least 7 days at pH 4.0 and 14 days at pH 12.0. The most resistant virus to 60 °C was infectious pancreatic necrosis virus, and to pH 12.0 was infectious salmon anaemia virus. The majority of the viruses tested survived exposure to pH 4.0 for up to 28 days. The results suggest that the process of acid ensiling alone is not an effective method for the inactivation of many viral and bacterial pathogens, and fish by‐products would need further treatment by a method approved under the regulation following ensiling, whereas alkaline or heat treatment are likely to provide an increased degree of biosecurity for on‐farm processing of mortalities.     

Development of a reproducible infectious pancreatic necrosis virus challenge model for Atlantic salmon, Salmo salar L.

Atlantic salmon smolts, previously unexposed to infectious pancreatic necrosis virus (IPNV), were placed into tanks of sea water at 10 °C. After 4 weeks, 40 fish were injected intraperitoneally (i.p.) with homogenized and filter‐sterilized kidney material obtained from salmon with clinical IPN in a marine farm in Shetland. The injected fish were cohabited with 40 untreated fish. Mortalities began in the injected fish on day 7 and reached a peak of 48% on day 14. In the cohabitation group, mortalities began on day 14 and reached a peak of 70% on day 27. The IPNV in the Shetland kidney homogenate was cultured in Chinook salmon embryo (CHSE) cells and passed twice. This cultured virus was injected i.p. into fish at various doses ranging from 10 to 10⁷ TCID₅₀ fish^?1 4 weeks after seawater transfer. Challenge tanks contained 30 injected fish and 30 cohabitees. Mortality rates and levels were dose‐dependent. The highest dose used resulted in a similar mortality pattern as obtained with a similar dose of the Shetland kidney homogenate, indicating that virulence was retained after two passes in tissue culture. Even with the lowest dose, mortality reached 12% in the injected group and 23% in the cohabitees. The IPNV titres were high (10⁶?10⁹ i.u. g^?1 kidney) in fish which died during the experiment and low (<10⁵ i.u. g^?1 kidney) or undetectable in surviving fish. The cultured virus (pass 3) was used in a challenge model where the population density of fish in the tanks was high (50 injected and 50 cohabitees) or low (15 injected and 15 cohabitees). In the high stocking density tank, mortalities peaked at about 35% in the injected group and at 52% in the cohabitees. In the low stocking density tank, mortalities peaked at about 40% in the injected fish but no mortality occurred in the cohabitees. However, IPNV was detected (up to 10⁴ i.u. g^?1 kidney) in 82% of cohabitees sampled on day 30. These data suggest that lethal lateral transmission of the virus is dependent on the infectious pressure from the injected group. A further trial was conducted to investigate the effect of time post‐seawater transfer on the susceptibility of post‐smolts to IPN. Groups of fish were challenged every 2 weeks from week 0–10. Few mortalities occurred at week 0 and virus titres were high in these fish. Most survivors became carriers, some with titres >10⁶ i.u. IPNV g^?1 kidney. From 2 to 10 weeks after seawater transfer, mortalities in both injected and cohabitees were substantial with viral titres >10⁷ i.u. g^?1 kidney. Survivors had lower titres and in many virus was undetectable. Throughout the experiments, moribund fish were sampled for histology and all showed typical IPN histopathology.     

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10⁵ TCID₅₀ mL⁻¹ for SAV1 and 3 × 10⁴ for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10² to 10⁷ TCID₅₀ mL⁻¹. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10² TCID₅₀ mL⁻¹. Clearly, salmon fry are susceptible to SAV infection and pancreas disease.     

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).     

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.     

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.     

Neutralizing antibody levels for protection against betanodavirus infection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), immunized with an inactivated virus vaccine

An inactivated betanodavirus, red‐spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 °C water temperature by a single intraperitoneal injection of formalin‐inactivated RGNNV. Fish immunized at vaccine doses of 10^8.5, 10^8.0, 10^7.5, 10^7.0 and 10^6.5 TCID₅₀ per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post‐immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre <1:80). When fish were challenged with RGNNV (10^5.0 and 10^4.0 TCID₅₀/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 10^8.5, 10^8.0, 10^7.5 and 10^7.0 TCID₅₀ per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 10^7.5 TCID₅₀ per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 10^6.5 TCID₅₀ per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 10^7.0 TCID₅₀ per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.     

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.     

Evaluation of the therapeutic effect of potassium permanganate at early stages of an experimental acute infection of Flavobacterium columnare in channel catfish,Ictalurus punctatus (Rafinesque)

The efficacy of potassium permanganate (KMnO₄) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus, was evaluated. Fish were experimentally challenged by waterborne exposure for 2 h to F. columnare after cutaneous abrasion, and treated with KMnO₄ at 2.0 mg L^?1 above the KMnO₄ demand at 0, 1, 2 or 4 h postchallenge for 24 h. Challenged non‐treated fish acted as a positive control and non‐challenged non‐treated fish acted as a negative control. Fish challenged and treated with KMnO₄ at 0, 1, 2 or 4 h postchallenge had mortalities of 26%, 63%, 64% and 83% respectively. The mortality of challenged fish treated with KMnO₄ at 0 h postchallenge (26%) was significantly less than the positive control (77%). The mortalities of challenged fish treated at 1, 2 or 4 h postchallenge were not significantly different from the positive control fish. The results suggest that KMnO₄ has a clear therapeutic value in early stages of columnaris infection but limited therapeutic value once the infection has progressed.     

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.