Genetic diversity of piscine myocarditis virus in Atlantic salmon Salmo salar L. in Ireland

Piscine myocarditis virus (PMCV) is a double‐stranded RNA virus which has been linked to cardiomyopathy syndrome (CMS) in Atlantic salmon (Salmo salar L.). The first recorded outbreak of CMS in Ireland occurred in 2012. Heart tissue samples were collected in the current study from farmed Atlantic salmon from various marine sites around Ireland, and the open reading frames (ORFs) 1 and 3 were amplified and sequenced in order to examine the genetic diversity of PMCV. Results showed PMCV to be largely homogenous in Irish samples, showing little genetic diversity. However, several amino acid positions within both ORF1 and ORF3 showed consistent variations unique to the Irish PMCV strains when compared with previously published Norwegian strains. The phylogeny generated in the present study suggests that PMCV may have been introduced into Ireland in two waves, both coming from the southern part of PMCV's range in Norway. In addition, over three‐quarters of the PMCV strains which were sequenced came from fish not exhibiting any clinical signs of CMS, suggesting that either PMCV is evolving to become less virulent in Ireland or Irish Atlantic salmon are developing immunity to the disease.     

Indications for a vertical transmission pathway of piscine myocarditis virus in Atlantic salmon (Salmo salar L.)

Losses due to cardiomyopathy syndrome (CMS) keep increasing in salmon‐producing countries in the North‐Atlantic. Recently, Piscine myocarditis virus (PMCV) has been detected in post‐smolts shortly after sea‐transfer, indicating a possible carry‐over from the hatcheries. In addition, there are reports of prevalences of PMCV as high as 70%–90% in certain groups of broodfish, and a recent outbreak of CMS in the Faroe Islands has been linked to the importation of eggs from a CMS‐endemic area. Thus, there is a need to investigate whether PMCV can be transmitted vertically from infected broodstock to their progeny. In the present study, samples from eggs, larvae, fingerlings and presmolt originating from PMCV‐positive broodstock from two commercial Atlantic salmon producers were tested for PMCV. The prevalence of PMCV in the broodstock was 98% in the hearts, 69% in the roe and 59% in the milt. Piscine myocarditis virus was detected in all stages of the progeny until and including the 40 g stage. Piscine myocarditis virus was also detected in presmolt sampled for tissue tropism. This provides farmers with several options for minimizing the risk of transfer of PMCV from broodstock to progeny, including screening of broodstock and aiming to use only those that are negative for PMCV or have low levels of virus.     

Monitoring infection with Piscine myocarditis virus and development of cardiomyopathy syndrome in farmed Atlantic salmon (Salmo salar L.) in Norway

An epidemiological study was carried out in Norway in 2015–2018, investigating the development of infection with Piscine myocarditis virus (PMCV) and development of cardiomyopathy syndrome (CMS) in farmed Atlantic salmon. Cohorts from 12 sites were followed and sampled every month or every other month from sea transfer to slaughter. PMCV was detected at all sites and in all sampled cages, and fish in six sites developed clinical CMS. The initial infection happened between 1 and 7 months post‐sea transfer, and the median time from infection with PMCV until outbreak of CMS was 6.5 months. Generally, fish from sites with CMS had higher viral titre and a higher prevalence of PMCV, compared to sites that did not develop clinical CMS. The virus persisted until the point of slaughter at most (11 out of 12) of the sites. The detection of PMCV in all sites suggests that PMCV is more widespread than previously known. Screening for PMCV as a tool to monitor impending outbreaks of CMS must be supported by observations of the health status of the fish and risk factors for development of disease.     

Modelling salmon lice,Lepeophtheirus salmonis,reproduction on farmed Atlantic salmon,Salmo salar L

The aim of this study was to model sea lice levels and the effect on reproduction by a stochastic simulation model and to evaluate the uncertainty of lice estimates based upon counts. Two empirical data sets were examined to parameterize the models. An overall fit of the data to the Poisson distribution was found and thus was used as the base of the stochastic models. In the model, salmon lice reproduction is not linear with the number of adult females and at low lice loads a smaller proportion of the adult female lice will reproduce. Depending on the variance structure, it was estimated that between 40% and 60% of the adult female lice will reproduce at an abundance of 0.5 adult females per fish. Lice counts, especially when examining few fish at low lice loads, are uncertain and at a true abundance of 0.1 one may count between 0 and 5 lice when examining 10 fish. Understanding the dynamics of sea lice reproduction is a key factor in the development of sustainable control strategies.     

Viral co‐infections in farmed Atlantic salmon,Salmo salar L., displaying myocarditis

Several different viruses have been associated with myocarditis‐related diseases in the Atlantic salmon aquaculture industry. In this study, we investigated the presence of PMCV, SAV, PRV and the recently identified Atlantic salmon calicivirus (ASCV), alone and as co‐infections in farmed Atlantic salmon displaying myocarditis. The analyses were performed at the individual level and comprised qPCR and histopathological examination of 397 salmon from 25 farms along the Norwegian coast. The samples were collected in 2009 and 2010, 5–22 months post‐sea transfer. The study documented multiple causes of myocarditis and revealed co‐infections including individual fish infected with all four viruses. There was an overall correlation between lesions characteristic of CMS and PD and the presence of PMCV and SAV, respectively. Although PRV was ubiquitously present, high viral loads were with a few exceptions, correlated with lesions characteristic of HSMI. ASCV did not seem to have any impact on myocardial infection by PMCV, SAV or PRV. qPCR indicated a negative correlation between PMCV and SAV viral loads. Co‐infections result in mixed and atypical pathological changes which pose a challenge for disease diagnostic work.     

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes

Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A. salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A. salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A. salmonicida have an intraerythrocytic phase. Here, we evaluate whether A. salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A. salmonicida did not kill primary S. salar erythrocytes, even in the presence of high bacterial loads, but A. salmonicida invaded the S. salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A. salmonicida showed bacteraemia 5 days post‐infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.     

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.     

Cardiomyopathy syndrome in Atlantic salmon Salmo salar L.: A review of the current state of knowledge

Cardiomyopathy syndrome (CMS) is a severe cardiac disease affecting Atlantic salmon Salmo salar L. The disease was first recognized in farmed Atlantic salmon in Norway in 1985 and subsequently in farmed salmon in the Faroe Islands, Scotland and Ireland. CMS has also been described in wild Atlantic salmon in Norway. The demonstration of CMS as a transmissible disease in 2009, and the subsequent detection and initial characterization of piscine myocarditis virus (PMCV) in 2010 and 2011 were significant discoveries that gave new impetus to the CMS research. In Norway, CMS usually causes mortality in large salmon in ongrowing and broodfish farms, resulting in reduced fish welfare, significant management‐related challenges and substantial economic losses. The disease thus has a significant impact on the Atlantic salmon farming industry. There is a need to gain further basic knowledge about the virus, the disease and its epidemiology, but also applied knowledge from the industry to enable the generation and implementation of effective prevention and control measures. This review summarizes the currently available, scientific information on CMS and PMCV with special focus on epidemiology and factors influencing the development of CMS.     

Erythrocytic inclusion body syndrome virus in wild Atlantic salmon, Salmo salar L

An investigation was undertaken to establish aspects of the epizootiology of erythrocytic inclusion bodies in wild Atlantic salmon, Salmo salar L., in Scotland. From 1992 to 1995, adult and juvenile salmon, from Scottish rivers, were screened for the presence of erythrocytic inclusion bodies and haematological parameters measured. The nature of the inclusion bodies was assessed through transmission electron microscopy, negative-staining and blood smear-staining techniques and was demonstrated to be viral in origin with characteristics similar to a member of the family Togaviridae. Specifically, these were a viral genome of single-stranded RNA, spherical virion morphology with an icosahedral core, average size of 70 nm and a buoyant density of 1.15-1.20 g cm(-3). The cytoplasmic inclusions were either large, single inclusions (1-2 microm) or smaller multiple inclusions (0.5-1 microm). A total of 4.2% (n=48) and 27.7% (n=213) of the parr and adult salmon, respectively, were positive for the presence of the inclusions. The intensity of the inclusions, when present, varied from light in parr to moderate and heavy in adults, when graded according to the number of inclusions per field of view. Neither haematological variations nor clinical disease was associated with the presence or absence of viral inclusions.     

Strong genetic influence on IPN vaccination-and-challenge trials in Atlantic salmon, Salmo salar L

Two series of experimental challenge trials were performed for evaluation of multivalent oil‐adjuvanted vaccines with and without an infectious pancreatic necrosis virus (IPNV) antigen component. In both the trial series, Atlantic salmon were hatched, reared, vaccinated and subjected to temperature and light manipulation to induce smoltification. When ready for sea the fish were transported to the VESO Vikan experimental laboratory for bath or cohabitant challenge with IPNV. In the first series, four vaccination and bath challenge trials involving 2‐year classes of experimental fish were conducted. In the second series, three groups of eyed eggs of Atlantic salmon allegedly differing in their innate resistance to IPNV were used (Storset, Strand, Wetten, Kjøglum & Ramstad 2007). Hatching, rearing and smoltification were synchronized for each group, and fish from each genetic group were randomly allocated IPN vaccine, reference vaccine or saline before being placed into parallel tanks for bath or cohabitant challenge. In the first series of trials, IPN‐specific mortality commenced on day 10–12 after bath challenge. Replicates showed similar results. In trials 1 and 2 belonging to the same experimental fish year class, the average cumulative control mortality reached 60.6% and 79.5%, respectively, whereas in trials 3 and 4 belonging to the following year class the control mortality was consistently below 50%. In the second series of trials, the experimental fish originating from allegedly IPN susceptible parents consistently showed the highest cumulative mortality among the unvaccinated controls (>75%) whereas smolts derived from allegedly IPNV resistant parents showed only 26–35% control mortality. The IPN‐vaccinated fish experienced significantly improved survival vs. the fish immunized with reference vaccine, with RPS values above 75% in the IPN susceptible strain. In the IPN resistant strain, the protection outcomes were variable and in part non‐significant. The outcome of both the trial series suggests that control mortalities above 50% are necessary to reliably demonstrate specific protection with IPN vaccines.     

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon,Salmo salar L. parr

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re‐isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated ‘AP’ (‘activated through passage’). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose–yeast extract.     

Gill pathology in Scottish farmed Atlantic salmon,Salmo salar L., associated with the microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009

Gill disorders have emerged in recent years as a significant problem in the production of marine‐stage Atlantic salmon Salmo salar L. The multi‐aetiological condition ‘proliferative gill inflammation’ (PGI) has been reported to cause heavy losses in western Norway, yet reports of Scottish cases of the disease have remained anecdotal. In the present study, histopathological material from a marine production site in the Scottish Highlands experiencing mortalities due to a seasonal gill disease with proliferative‐type pathology was examined using light microscopy, special staining techniques and transmission electron microscopy (TEM). The microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009 (syn. Paranucleospora theridion) was identified by staining using a Gram Twort method and TEM associated with distinctive proliferative and necrotic pathology confined to the interlamellar Malpighian cell areas of the primary filaments. Epitheliocystis was not a feature of the gill pathology observed. It is believed this is the first report of D. lepeophtherii being identified associated with pathology in a Scottish gill disease case, and supports anecdotal reports that a disease at least partly synonymous with PGI as described by Norwegian researchers is present in Scottish aquaculture.     

Colonization of Aeromonas salmonicida subsp. masoucida strains in Atlantic salmon (Salmo salar L.) during infection

Aeromonas salmonicida subsp. masoucida (ASM) is classified as atypical A. salmonicida and brought huge economic damages to the local salmonid aquaculture in China. An ASM strain named AS‐C4 was used to investigate the colonization of ASM in Atlantic salmon (Salmo salar L.) by an immersion challenge with the control group (T0, no AS‐C4), group T1 (2.67 × 10⁴ CFU/ml AS‐C4) and group T2 (2.67 × 10⁷ CFU/ml AS‐C4). The numbers of AS‐C4 copies in different fish tissues (gill, intestine, skin, blood, muscle, spleen, liver and kidney) were determined at different time points post challenge using the quantitative real‐time PCR (qRT‐PCR). AS‐C4 were detected in the gill and intestine as early as 0 hr after the challenge both in T1 and T2 groups, suggesting that the gill and intestine were probably the portals of entry of AS‐C4 into salmon. Although AS‐C4 could not be detected in the skin until 24 hr after the challenge in T1 group, it could be detected in the skin as early as 0 hr after the challenge in T2 group, indicating that the skin may also be a portal of entry of AS‐C4 into salmon. AS‐C4 was immediately detected in the blood within 3 hr after it entered the host, suggesting that AS‐C4 successfully invaded the bloodstream of fish. After AS‐C4 colonized the host, it colonized the internal tissues, such as the spleen, liver, kidney and muscle. The results of this study will contribute to the understanding of the pathogenesis of the ASM strains and give a broader understanding of the infection route of ASM in it's host, providing more information for the development of new therapeutic strategies to protect against this pathogen in aquaculture.     

Apoptosis inhibition of Atlantic salmon (Salmo salar) peritoneal macrophages by Piscirickettsia salmonis

To improve the understanding of the piscirickettsiosis pathogenesis, the in vivo apoptosis modulation of peritoneal macrophages and lymphocytes was studied in juvenile Salmo salar intraperitoneally injected with Piscirickettsia salmonis. Five fish were sampled at post‐exposure days 1, 5, 8 (preclinical), 20 (clinical) and 40 (post‐clinical period of the disease), and the leucocytes of their coelomic washings were analysed by flow cytometry (using the JC‐1 cationic dye), TUNEL and cytology to detect apoptotic cells. A selective and temporal pattern of apoptosis modulation by P. salmonis infection was observed. Apoptosis in lymphocytes was not affected, whereas it was inhibited in macrophages but only during the preclinical stage of the induced piscirickettsiosis. Hence, it is postulated that P. salmonis inhibits macrophage apoptosis at the beginning of the disease development to survive, multiply and probably be transported inside these phagocytes; once this process is complete, macrophage apoptosis is no longer inhibited, thus facilitating the exit of the bacteria from the infected cells for continuing their life cycle.