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.     

Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines

The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by b-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 degrees C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 degrees C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

First detection and isolation of infectious haematopoietic necrosis virus from farmed rainbow trout in Nyeri County,Kenya

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis, a disease of salmonid responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in Kenya. In this study, rainbow trout fry and growers from two farms in Nyeri County were screened for IHNV. Whole fry (n = 4 from each farm) and kidney samples from growers (n = 15 and n = 6 from the two farms, respectively) were collected and preserved for cell culture examination or PCR analysis. Screening of samples was done by PCR followed by sequencing of the glycoprotein gene of the virus. Demonstration of the virus was done by propagation in EPC cells followed by the indirect fluorescence antibody test (IFAT). The results revealed the presence of IHNV at low prevalence of 0.1 and 0.4 for the two farms. The virus was confirmed both by IFAT and by partial sequencing of the G gene. Phylogenetic analysis revealed that the Kenyan isolates were identical to those of the J genogroup found mostly in Asia. The findings have implications for biosecurity measures and import regulations for the Kenyan rainbow trout industry.     

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.     

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).     

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.     

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.     

A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp

White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years.     

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.     

Atlantic salmon,Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U‐group strains ranged from 20 to 100%, four M‐group strains ranged 30‐63% and two L‐group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.     

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.     

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.     

Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full‐length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G‐gene to make the rIHNV‐Gsvcv. The chimeric rIHNV‐Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild‐type SVCV infection was tested. The rIHNV‐Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV‐Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV‐Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV‐Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.     

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.