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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.  相似文献   

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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。  相似文献   

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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.  相似文献   

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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.  相似文献   

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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 102.8 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.  相似文献   

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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).  相似文献   

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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.  相似文献   

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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.  相似文献   

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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.  相似文献   

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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.  相似文献   

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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.  相似文献   

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The efficacy of emamectin benzoate (SLICE) against sea lice infestations of Atlantic salmon, Salmo salar L., is typically assessed using untreated fish, or fish treated with alternative therapeutants, as controls. The State of Maine, USA, is currently under active management for the OIE-notifiable pathogen, infectious salmon anaemia virus (ISAV); consequently, neither control group is feasible in this region. Untreated salmon risk extensive damage from the ectoparasites, and threaten to increase vector-borne exposure or susceptibility of farms to ISAV; and the only treatment presently available in Maine is SLICE. However, because sea lice infestations are unlikely to resolve spontaneously, and response to treatment occurs within weeks, use of a pretreatment baseline is a reasonable alternative for confirmatory studies. We evaluated SLICE efficacy on Atlantic salmon farms in Cobscook Bay 2002-2005, in the absence of untreated controls, using pretreatment lice loads as a reference for calculation. Maximum efficacy ranged from 68% to 100% reduction from initial levels. Time-to-maximum efficacy ranged from 1 to 8 weeks after treatment initiation. Efficacy duration, measured between first reduction and first progressive rise in counts, ranged from 4 to 16 weeks.  相似文献   

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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.  相似文献   

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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.  相似文献   

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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.  相似文献   

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