Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum)

A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.     

Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon,Salmo salar,infected with salmonid alphavirus (SAV3)

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on‐farm qualitative test and an in‐laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ₃₄₀). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ₃₄₀ in infected fish rose from undetectable to a maximum at 6 weeks post‐infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.     

Salmonid alphavirus (SAV) and pancreas disease (PD) in Atlantic salmon,Salmo salar L., in freshwater and seawater sites in Norway from 2006 to 2008

A cohort study was initiated in the spring of 2006 to investigate epidemiological aspects and pathogenesis of salmonid alphavirus (SAV) subtype 3 infections and pancreas disease (PD). The aims were to assess involvement of the freshwater production phase, the extent and frequency of subclinical infections and to follow PD‐affected populations throughout the entire seawater production cycle, as well as investigate possible risk factors for PD outbreaks. Fish groups from 46 different Atlantic salmon freshwater sites in six counties were sampled once prior to seawater transfer and followed onto their seawater sites. A total of 51 Atlantic salmon seawater sites were included, and fish groups were sampled three times during the seawater production phase. SAV subtype 3 was not identified by real‐time RT‐PCR from samples collected in the freshwater phase, nor were any SAV‐neutralizing antibodies or histopathological changes consistent with PD. In the seawater phase, SAV was detected in samples from 23 of 36 (63.9%) studied sites located within the endemic region. No SAV subtype 3 was detected in samples from seawater sites located outside the endemic region. The cumulative incidence of PD during the production cycle amongst sites with SAV detected was 87% (20 of 23 sites). Average fish weight at time of PD diagnosis ranged from 461 to 5978 g, because of a wide variation in the timing of disease occurrence throughout the production cycle. Mortality levels following a PD diagnosis varied greatly between populations. The mean percentage mortality was 6.9% (±7.06) (range 0.7–26.9), while the mean duration of increased mortality following PD diagnosis was 2.8 months (±1.11) (range 1–6).     

Mortality and weight loss of Atlantic salmon,Salmon salar L., experimentally infected with salmonid alphavirus subtype 2 and subtype 3 isolates from Norway

Pancreas disease (PD) caused by salmonid alphavirus (SAV) has a significant negative economic impact in the salmonid fish farming industry in northern Europe. Until recently, only SAV subtype 3 was present in Norwegian fish farms. However, in 2011, a marine SAV 2 subtype was detected in a fish farm outside the PD‐endemic zone. This subtype has spread rapidly among fish farms in mid‐Norway. The PD mortality in several farms has been lower than expected, although high mortality has also been reported. In this situation, the industry and the authorities needed scientific‐based information about the virulence of the marine SAV 2 strain in Norway to decide how to handle this new situation. Atlantic salmon post‐smolts were experimentally infected with SAV 2 and SAV 3 strains from six different PD cases in Norway. SAV 3‐infected fish showed higher mortality than SAV 2‐infected fish. Among the SAV 3 isolates, two isolates gave higher mortality than the third one. At the end of the experiment, fish in all SAV‐infected groups had significantly lower weight than the uninfected control fish. This is the first published paper on PD to document that waterborne infection produced significantly higher mortality than intraperitoneal injection.     

Longitudinal serological surveys of Atlantic salmon, Salmo salar L., using a rapid immunoperoxidase-based neutralization assay for salmonid alphavirus

Longitudinal serological surveys for salmon pancreas disease virus (SPDV), the causal agent of pancreas disease (PD), were conducted on multiple caged populations of Atlantic salmon, Salmo salar L., on two farms over a 77-week period (farm 1, freshwater and marine stages) and a 36-week period (farm 2, marine stage only), using a microtitre-based virus neutralization (VN) assay. Collected sera were also screened for viraemia with SPDV, and pancreas, heart and muscle tissues were examined for lesions consistent with PD. Outbreaks of PD occurred during the marine phase on both farms, as demonstrated by seroconversion, the isolation of virus and progressive histopathological changes consistent with a PD outbreak. All populations monitored showed a progressive increase in seroprevalence of 90-100%, typically accompanied by rises in geometric mean antibody titres. With the exception of one caged population, which showed a marked biphasic seroprevalence pattern, the seroprevalence figures in the remaining four monitored populations remained high (> or =70%) until the end of the study period. Peak VN titres of > or =1/1280 were detected on both farms. The results provide essential baseline information for the interpretation of SPDV VN serology results, and indicate that this methodology is suited to both the diagnosis and seroepidemiology of SPDV infections.     

Genetic characterization of salmonid alphavirus in Norway

Pancreas disease (PD), caused by salmonid alphavirus subtype 3 (SAV3), emerged in Norwegian aquaculture in the 1980s and is now endemic along the south‐western coast. In 2011, the first cases of PD caused by marine salmonid alphavirus subtype 2 (SAV2) were reported. This subtype has spread rapidly among the fish farms outside the PD‐endemic zone and is responsible for disease outbreaks at an increasing numbers of sites. To describe the geographical distribution of salmonid alphavirus (SAV), and to assess the time and site of introduction of marine SAV2 to Norway, an extensive genetic characterization including more than 200 SAV‐positive samples from 157 Norwegian marine production sites collected from May 2007 to December 2012 was executed. The first samples positive for marine SAV2 originated from Romsdal, in June 2010. Sequence analysis of the E2 gene revealed that all marine SAV2 included in this study were nearly identical, suggesting a single introduction into Norwegian aquaculture. Further, this study provides evidence of a separate geographical distribution of two subtypes in Norway. SAV3 is present in south‐western Norway, and marine SAV2 circulates in north‐western and Mid‐Norway, a geographical area which since 2010 constitutes the endemic zone for marine SAV2.     

Effect of pancreas disease caused by SAV 2 on protein and fat digestion in Atlantic salmon

Salmonid alphavirus (SAV) causes pancreas disease (PD) in farmed Atlantic salmon (Salmo salar L.), and exocrine pancreas tissue is a primary target of the virus. Digestive enzymes secreted by the exocrine pancreas break down macromolecules in feed into smaller molecules that can be absorbed. The effect of SAV infection on digestion has been poorly studied. In this study, longitudinal observations of PD outbreaks caused by SAV subtype 2 (SAV2) in Atlantic salmon at two commercial sea sites were performed. The development of PD was assessed by measurement of SAV2 RNA load and evaluation of histopathological lesions typical of PD. Reduced digestion of both protein and fat co‐varied with the severity of PD lesions and viral load. Also, the study found that during a PD outbreak, the pen population comprise several subpopulations, with different likelihoods of being sampled. The body length of sampled fish deviated from the expected increase or steady state over time, and the infection status in sampled fish deviated from the expected course of infection in the population. Both conditions indicate that disease status of the individual fish influenced the likelihood of being sampled, which may cause sampling bias in population studies.     

Lack of evidence for vertical transmission of SAV 3 using gametes of Atlantic salmon, Salmo salar L., exposed by natural and experimental routes

Pancreas disease (PD) is an important cause of losses in farmed salmonids in Norway, the United Kingdom and Ireland. As the spread of salmonid alphavirus (SAV), the causal agent, to naïve populations is of major concern to the farming industry, it is important to uncover the transmission routes of the virus. This study was conducted to investigate the potential for vertical transmission of SAV subtype 3. Progeny of broodstock with signs of late‐stage PD and persistent RT‐PCR signals for SAV were followed from fertilization to smoltification in an experimental facility. Fertilized ova were either not disinfected or taken through one of three different disinfection regimes. Also, ova and milt from uninfected broodfish from a different population were exposed to a cell‐cultured strain of SAV 3 immediately before fertilization to simulate a viraemic phase in parent fish. A group of uninfected controls were also included in the study. Fertilized ova from bath exposed and negative control groups were double disinfected. Following fertilization, experimental fish went through a normal freshwater phase. However, fry were stressed at first feeding to enhance replication of possibly latent virus. Smoltification was induced by an artificial light regime, and experimental fish were followed to the late smoltification phase. Selected samples were investigated by real‐time RT‐PCR for SAV, by histology for evidence of PD and by serology for neutralising antibodies against SAV. All analysed samples of progeny were negative. This result shows that SAV 3 is not readily transmitted vertically from parents to offspring. Additional negative PCR results from salmon sampled in commercial hatcheries support these findings. Also, recent studies have shown that risk factors for the horizontal transmission route explain the vast majority of PD outbreaks in Norway. It is concluded that if it happens at all, vertical transmission is of minor importance in the spread of SAV 3.     

Inactivated Piscine orthoreovirus vaccine protects against heart and skeletal muscle inflammation in Atlantic salmon

Heart‐ and skeletal muscle inflammation (HSMI) caused by infection with Piscine orthoreovirus (PRV) is one of the most common viral diseases in farmed Atlantic salmon (Salmo salar) in Norway, and disease outbreaks have been reported in most countries with large‐scale Atlantic salmon aquaculture. Currently there is no vaccine available for protection against HSMI, partly due to the lack of a cell line for efficient virus propagation. Erythrocytes are the primary target cells for PRV in vivo and a potential source for isolation of PRV particles. In this study, PRV was purified from infected erythrocytes, inactivated and used in a vaccination trial against HSMI. A single immunization with adjuvanted, inactivated PRV induced protection against HSMI in Atlantic salmon infected by virus injection 6 weeks later, while a moderate protection was obtained in fish infected through natural transmission, i.e. cohabitation. The PRV vaccine significantly reduced PRV loads and histopathological lesions typical for HSMI compared to the unvaccinated control group. This is the first demonstration of protective vaccination against PRV, and promising for future control of HSMI in Atlantic salmon aquaculture.     

病毒性出血性败血症病毒单链抗体的原核表达及其功能鉴定

为制备病毒性出血性败血症病毒(VHSV)单链抗体(single chain variable fragment antibody,ScFv)并鉴定其生物学功能,本研究提取抗VHSV单抗1G5的杂交瘤细胞株总RNA并反转录获得cDNA模板,通过PCR扩增VHSV抗体的轻链可变区(VL)和重链可变区(VH)编码序列,将其拼接成单链抗体Sc Fv基因后插入载体pET28a中,构建原核表达重组质粒并在大肠杆菌中诱导表达。结果表明,单链抗体主要以可溶性形式表达,分子量约28 ku,能特异性识别VHSV病毒的G蛋白并对VHSV病毒具有体外中和活性,其对VHSV病毒的G蛋白亲和力(KD)达到1.4×10~(–8) M。单链抗体ScFv的制备为进一步研究VHSV的治疗性抗体、快速诊断试剂奠定了基础。     

Effect of the arachidonic acid/vitamin E interaction on the immune response of juvenile Atlantic salmon (Salmo salar) challenged against Piscirickettsia salmonis

Atlantic salmon (Salmo salar) were fed 6 experimental diets containing three levels of arachidonic acid (ARA) (0.18 g kg^?1, 0.28 g kg^?1 and 0.63 g kg^?1 for low, medium and high levels, respectively) and two levels of vitamin E (150 and 730 mg kg^?1 for low and high levels, respectively). At the end of the experimental period, fatty acids in the liver and immunity markers (lysozyme activity, respiratory burst and phagocytic activity) were determined and fish subjected to a challenge test against the salmonid rickettsial syndrome (SRS) pathogen. ARA, vitamin E or their interaction did not exert an effect on fish performance, whereas ARA alone clearly increased the deposition of ARA. Dietary vitamin E only enhanced liver vitamin E deposition, while the interaction of ARA and vitamin E influenced lysozyme activity and EPA/ARA ratio pointing out the effect of both nutrients on the fish immune system and metabolism. Only the medium concentration contributed to reducing mortality when the fish were exposed to the SRS pathogen. In conclusion, different levels of supplementation with ARA and vitamin E in the diet had no effect on productivity, but did have effects on immune markers and cumulative mortality when fish were exposed to the SRS pathogen.     

Geographical distribution of salmonid alphavirus subtypes in marine farmed Atlantic salmon, Salmo salar L., in Scotland and Ireland

Sequence data from salmonid alphavirus (SAV) strains obtained from farmed marine Atlantic salmon, Salmo salar L. , over a 20-year period between 1991 and 2011 was reviewed to examine the geographical distribution of the genetically defined SAV subtypes in twelve regions across Ireland and Scotland. Of 160 different Atlantic salmon SAV strains examined, 62 belonged to subtype 1, 28 to subtype 2, 34 to subtype 4, 35 to subtype 5 and 1 to subtype 6. SAV subtypes 1, 4 and 6 were found in Ireland, while subtypes 1, 2, 4 and 5 were found in Scotland. In the majority of regions, there was a clear clustering of subtypes, with SAV subtype 1 being the dominant subtype in Ireland overall, as well as in Argyll and Bute in Scotland. SAV subtype 2 predominated in the Shetland and Orkney Islands. The emergence in Atlantic salmon of subtype 2 strains typically associated with sleeping disease in rainbow trout in Argyll and Bute, strongly suggesting transmission of infection between these species, was noted for the first time. SAV subtype 4 was the most common subtype found in the southern Western Isles, while SAV subtype 5 predominated in the northern Western Isles and north-west mainland Scotland. No single strain was dominant on sites in the western Highlands, with a number of sites in this region in particular having more than one subtype detected in different submissions. The significance of these results in relation to aspects of the epidemiology of infection, including transmission, biosecurity and wildlife reservoirs are discussed and knowledge gaps identified.     

Immune response of DNA vaccine against lymphocystis disease virus and expression analysis of immune‐related genes after vaccination

In this study, we found that an intramuscular injection of Japanese flounder (Paralichthys olivaceus, 60–80 g in weight and 15–20 mL in length) with 5 μg of a DNA vaccine (pEGFP‐N2‐LCDV‐cn‐MCP 0.6 kb, containing lymphocystis disease virus major capsid protein gene) induced a strong immune response. Subsequent real‐time polymerase chain reaction showed that the expression of immune‐related genes [e.g., major histocompatibility complex (MHC) class I α, MHC II α, T‐cell receptor (TCR), tumour necrosis factor (TNF), tumour necrosis factor receptor (TNFR), Mx, interleukin (IL)‐1β, CXC and IL‐8R] was significantly changed after DNA vaccination. The most remarkable alternation was the expression of MHC I α and MHC II α genes: MHC II α reached the maximum on day 8 in different tissues, and MHC I α on day 2 in the intestine and gills. The expression of TCR increased and reached a plateau in 2 days in the spleen, gills, kidney and liver after vaccination and then decreased after day 8. In contrast, the expression of TCR in the intestine increased and reached a plateau in 8 days. The expression of IL‐8R reached the maximum on day 2 in different tissues and then decreased on day 8. Mx increased in the gills, kidney, spleen and liver on days 2, 8, 2 and 2, but decreased in the intestine, gills, spleen and liver on days 2, 8, 8 and 8 respectively. The TNFR expression increased in the spleen, kidney and gills on days 2, 8 and 8, but decreased in intestine, liver and gills on days 2, 8 and 8 respectively. The expression of TNF, CXC and IL‐1β increased 2 and 8 days after the injection of DNA vaccine. However, the expression of TNF, CXC and IL‐1β altered on days 2 and 8 with different patterns in different tissues respectively. The fish responded to the DNA vaccine by yielding a specific immunoglobulin against lymphocystis disease virus (LCDV) as observed with indirect ELISA. The DNA vaccine induced a unique humoral response, suggesting that the DNA vaccine activated both cellular and humoral defences of the specific immune system of Japanese flounder.