Development of a widely applicable positive control strategy to support detection of infectious salmon anaemia virus (ISAV) using Taqman real-time PCR

Real-time PCR assays are being increasingly applied to the detection of fish pathogens due to their sensitivity, specificity and potential for high throughput sample processing. Such assays allow for the ready and efficient inclusion of appropriate quality controls which are fundamental to scientific integrity and to satisfying the demands of diagnostic test accreditation. In this article, we report development of a universal positive control strategy for real-time PCR assays, which has been used to support and improve a previously published method for detection of infectious salmon anaemia virus (ISAV). The strategy employed uses an RNA mimic template, which is based on the ISAV segment 8 target sequence but includes an artificial universal positive control sequence. Inclusion of this sequence, which is targeted by a second specific probe carrying a different fluorophore to the primary assay, allows for convenient screening of all real-time PCR reactions for the presence of contaminating positive control material. The development of readily distinguishable artificial positive control material offers distinct advantages to real-time PCR assays over using control material derived from clinical material.     

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.     

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).     

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.     

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.     

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.     

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.     

Comparative virulence of Infectious salmon anaemia virus isolates in Atlantic salmon, Salmo salar L.

Infectious salmon anaemia virus (ISAV) surveillance in the Bay of Fundy has identified the existence of a large number of genetically distinct ISAV isolates which appear to be of variable virulence. Genetically distinct isolates are currently being designated based on sequencing of the hyper polymorphic region (HPR) of genomic segment 6, which encodes the haemagglutinin–esterase protein, but it has been difficult to elucidate a clear association between these molecular variations and variations in virulence. This has hampered the establishment of proactive management decisions regarding infected fish, and ISAV infections, regardless of type, must be treated as one. Field data of ISAV infections is difficult to collect and to compare between infections because of a wide range of confounding factors including time of year, fish stock, cage site location, mitigating factors and stressors. An important tool in determining the relationship between molecular differences and virulence comes from analysis of quarantine studies. The goal of this study was to compare the virulence, by co-habitation and intraperitoneal injection, of four regionally common and recent ISAV isolates in a controlled environment. We found significant differences in mortality between ISAV molecular isolates, and present data showing that survival of ISAV infection confers significant resistance to re-infection with a different ISAV isolate. These findings, if borne out in field studies, will significantly alter the way ISAV infections are managed in the Bay of Fundy and elsewhere.     

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.     

Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool

Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.