Diffuse melanization of the red skeletal musculature in farmed Atlantic salmon (Salmo salar L.).

Farmed Atlantic salmon (Salmo salar L.) are susceptible to melanization of the white muscle (‘the filet’), where melanized spots have a prevalence of 20%–30% at slaughter (Mørkøre et al., 2015). More than 90% of melanized changes in the filet manifest as focal discoloration in the cranio-ventral part (Bjørgen et al., 2019). These changes develop from focal haemorrhages, that is, red focal changes, which are the initial and acute stage of the condition (Bjørgen et al., 2015, 2019). The haemorrhages are believed to progress into a chronic inflammatory condition with melanization (Koppang et al., 2005; Larsen et al., 2012). Although the development of severe melanized focal changes has been associated with persistent PRV-1 infection (Bjørgen et al., 2019), the initial causes of red focal changes are unknown (Bjørgen et al., 2020). The focal appearance and restricted localization argue for underlying anatomical predispositions, and hypotheses such as fat accumulation and fat-induced inflammation, local ischaemia and costal abnormalities have all been proposed as hypotheses (Bjørgen et al., 2019; Jiménez-Guerrero et al., 2022). Still, no single cause has been pinpointed and the aetiology of the condition is probably of multi-factorial origin. Melano-macrophages are common immune cells in fish and are normally found in abundance in lymphoid organs such as kidney, spleen and liver (Bjørgen & Koppang, 2021; Thorsen et al., 2006). They are especially active in chronic, granulomatous inflammation and can thus be found in a range of different inflammatory conditions (Koppang et al., 2005; Poppe & Breck, 1997). Although focal melanization is the most prevalent manifestation in white muscle, other forms have been reported, including melanization near the vertebrae (Trangerud et al., 2020), in the dorsal part of the fillet and diffuse melanization of the dorsal and ventral white muscle (Ugrovatov, 2016). In summary, the white muscle could seem more disposed for inflammatory conditions attracting melano-macrophages. On the contrary, melanization is rarely reported to occur in the red skeletal muscle but has occasionally been observed by producers or customers (Personal observation, Line Rønning, Lerøy Seafood Group). As the red skeletal muscle is found on the lateral side of the trunk (reviewed by Kiessling et al., 2006), beneath the skin, melanization is often left undiscovered due to the fileting methods. Although conditions such as wounds and foreign bodies can induce the reaction of melano-macrophages (Roberts et al., 1973), melanization of the red skeletal muscle has not been reported as a quality concern. Here, we report the appearance of melanization in farmed Atlantic salmon affecting solely the red skeletal muscle. We provide histopathological description of the condition and discuss possible causes of this presumed uncommon condition. In November 2016, approximately 52,000 harvest-ready Atlantic salmon from Lerøy's location Kjørvikgrunn were slaughtered according to production standards. The fish was of the AquaGen QTL IPN/PD breed and had an average weight of 4613 g. All fish had been vaccinated with Alpha Ject micro 6 (Pharmaq, part of Zoetis). Heart and skeletal muscle inflammation (HSMI) caused by PRV-1 infection was confirmed by histology in November 2015. Salmonid alphavirus 2 (SAV2) was detected in the population by RT-qPCR in July 2016 and Pancreas disease (PD) caused by SAV2 infection was confirmed by RT-qPCR and histological investigations in August 2016. Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) was suspected in August 2016. This was confirmed by histological investigations in December 2016. Increased mortality was registered in relation to all three disease outbreaks. At slaughter, a high prevalence of melanization in the red skeletal muscle was detected. Affected (n = 6) and unaffected fillets (n = 6) were collected at the production line and shipped on ice to the School of Veterinary Medicine, Adamstuen, Oslo, Norway. The changes were photographed, and samples were collected from three different areas of the filet (cranial, mid, and caudal parts, see Figure 1) and put in buffered formalin. Both cross-sectional and longitudinal samples were obtained. From each fish, a sample collected from the red muscle (mid part) was put in RNAlater and sent to PatoGen AS, Ålesund, Norway, for RT-qPCR analysis for common viral infections affecting the skeletal muscle: Piscine orthoreovirus 1 (PRV-1) and salmonid alphavirus 2 (SAV2). The samples were also analysed for infectious pancreas necrosis virus (IPNV). The RT-qPCR analyses are accredited and validated to ISO7025 standards. Details of purification method and PCR conditions could not be disclosed by PatoGen due to issues related to competing patents. Samples were defined as positive when having a PRV-1, SAV2 or IPNV Ct lower than 37.0. Statistical analysis of PRV-1 data was performed using Mann–Whitney U-test in STATA (StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC). The samples were embedded in paraffin and stained according to standard procedure with haematoxylin and eosin. In addition, all samples were stained for detection of fibrin according to Lendrums Maritus Scarlet Blue (MSB) staining protocol, Giemsa staining to differentiate nucleus morphology and cytoplasm of blood cells, Van Gieson (VG) for detection of collagen and Gram staining for detection of Gram-positive/negative bacteria. Severe, diffuse melanization of the red skeletal muscle was observed as superficial, black discoloration in the fillet of the affected fish (Figure 1). The extent of the discoloration was identical to the anatomical placement of the red skeletal muscle. In transverse sections of the fillet, a clear demarcation in melanization confirmed that solely the red, and not the white skeletal muscle was affected (Figure 1c). All the control individuals were devoid of discoloration. In H&E-stained sections, the macroscopically observed demarcation of melanization between white and red muscle was confirmed, as the white muscle had normal appearance while the red muscle was severely affected (Figure 2a). The changes in the red muscle were dominated by muscle fibre degeneration and fibrosis. Fibrosis was confirmed by VG stain (Figure 2b). Scattered immune cells, predominantly macrophage-like cells were observed and melano-macrophages and what appeared as either large accumulations of melanin or collections of melano-macrophages were detected (Figure 2c). Some muscle fibres showed signs of regeneration, including cytoplasmic basophilia and rowing of nuclei (Figure 2d). The histological features were similar in all affected fish and for the different areas sampled. Giemsa and Gram staining did not show presence of bacteria. No pathological changes were detected in the control fish (Figure 3a,b); however, scattered melano-macrophages were occasionally detected in the endomysium of red muscle. (Figure 3c,d). Tissue from the mid part of the filet was analysed for the presence of PRV-1 and SAV2 (Table 1) but also for IPNV, by RT-qPCR, as IPN is a differential diagnosis to PD. All affected samples were positive for PRV-1 with a median Ct value of 30.2, for the control fish, four samples were PRV-1 positive, and two were negative. When the negative value is set to 37, that is, cut-off for the RT-qPCR, there were statistically significant difference between the Ct values for PRV-1 for affected versus non-affected groups (p = .0022). SAV2 was detected in three affected samples, of which one had a Ct 23.2, and in two non-affected samples. No samples were positive for IPNV. In this case report, we present melanization affecting the whole red skeletal muscle in farmed Atlantic salmon. The condition, that in general is considered as a rather rare quality and possibly welfare problem, was detected during quality control after filleting, as a black longitudinal sheath directly underneath the skin. Histological analysis revealed diffuse inflammation with infiltrates of inflammatory cells where melano-macrophages were abundant. There was extensive fibrosis. In general, there were several histological similarities with the more common melanized focal changes; however, no granulomas were detected in the red skeletal muscle. Granulomas are hallmark traits for severe melanized focal changes in the white muscle and typical for some chronic inflammatory conditions (Koppang et al., 2005; Shah et al., 2017). The absence of organized granulomas may imply that the underlying cause differs from that of the melanized focal changes in white muscle. Alternatively, anatomical and physiological conditions in red muscle could contribute to the development of the changes seen in our study. Nevertheless, granulomatous inflammation in red muscle has been reported in rainbow trout affected by proliferative kidney disease (PKD) (Fernandez-De-Luco et al., 1997); thus such an immune reaction can also occur in red muscle. Signs of regeneration were seen in most changes in our work, indicating that the condition is temporary and heals over time. Macrophage-like cells and myophagocytosis were observed in the affected red muscle, but scattered melano-macrophages were also detected in the endomysium between intact muscle fibres from non-affected fish. Melanin is rarely observed in the red skeletal muscle in routine diagnostics (personal communication, Dr. Hege Hellberg, Pharmaq Analytic, Norway); however, this could be a normal trait of slaughter-ready farmed salmon, or alternatively the scattered melano-macrophages could be remnants from a preceding regeneration and clearing of the condition. Red muscle is anatomically different from white muscle in several important aspects. Although the red muscle makes up <10% of the total muscle mass in Atlantic salmon, it is important in enduring and slow movement as it is rich in mitochondria and heavily vascularized, and thus adapted towards aerobic exercise (Anttila & Manttari, 2009; Kryvi & Poppe, 2016). White muscle, on the other hand, generates speed and extreme anaerobic movements, but has a relatively poor blood supply and only scattered mitochondria (Hudson, 1973; Nag, 1972). These differences could have an impact on the process of regeneration and healing in red and white muscle which subsequently can lead to different forms of melanization. Of the viruses commonly affecting the skeletal muscle in salmon, some are prone to cause red muscle damage (Bruno et al., 2013). PRV-1, which is the cause of HSMI may specifically affect the red muscle and induce myositis, and although it is not a common reaction, melano-macrophages have been described in the red skeletal muscle of HSMI diseased fish (Wessel et al., 2017). SAV2 and 3, which are the causes of PD in farmed salmon in Norway, can affect both red and white muscle, typically late in the course of the disease development (Bruno et al., 2013). The endomysial inflammation and fibrosis observed in our material could result from both SAV2 and PRV-1 infection. One could speculate that SAV2 was the key contributor due to the ongoing outbreak concurrent with slaughter; however, SAV2 was only detected in three of the six affected samples. The Ct values for PRV-1 of affected fish were statistically significantly lower (p = .0022) than from non-affected fish. Although the RT-qPCRs were run without a normalizing RNA reference, and the amount of RNA thus cannot be quantified, the Ct values of PRV-1 reflects in general that the load of PRV-1 was significantly higher in melanized red muscle than in normal red muscle tissue. This indicates a correlation of the amount of PRV-1 and the condition, but it does not necessarily specify causation. All affected fish were infected with PRV-1, including the three SAV2 infected. The two SAV2 infected non-affected fish were negative for PRV-1 by RT-qPCR. The number of samples here is very small, but it could be speculated that the findings indicate that SAV2 infection alone does not induce red muscle melanization. The co-infection with PRV-1 and SAV2 found in three of six affected samples is difficult to speculate upon. The fish came from a population where HSMI and PD had been confirmed prior to slaughter, that is, the population had probably gone through serious infections with both PRV-1 and SAV2. In addition, CMS caused by PMCV was suspected and later detected at the location; however, PMCV does not infect skeletal muscle. Melanization is a general inflammatory response where one important function is to protect the surrounding tissue against oxidative damage (Larsen et al., 2012). Melanization has been detected not only in association with intraperitoneal injections (Koppang et al., 2005; Poppe & Breck, 1997) but also in several viral infections, for example, PRV-1 has been associated with severe melanized focal changes in the white skeletal muscle (Bjørgen et al., 2015, 2019) and melanization of the heart atrium has been reported in fish with CMS (Fagerland et al., 2013). In an epidemiological study, PD diagnosis was correlated with increased prevalence of melanized focal changes in the filet (Lund et al., 2018). Although statistical analysis of our material could indicate that PRV-1 infection is associated with red muscle melanization, other infections or even co-infections with SAV2 or PMCV may have contributed to the development of the condition. In situ detection of viruses in the red muscle could have added information about the presence of PRV-1 and SAV2 in relation to the histopathological changes, but this was not possible to perform due to sub-optimal fixation of the field material. There is no experimental model that are known to induce the condition, and one is dependent on samples collected at the slaughter line in field. In conclusion, we have reported a case of melanization solely affecting the red skeletal muscle of farmed Atlantic salmon. The condition has unknown aetiology and histologically appears diffuse and not granulomatous as is the dominant form in melanized focal changes in the white muscle. The involvement of SAV2 and PRV-1 infection might participate in the development of the changes and should be a target in future research if the condition develops into a more generalized problem for the industry. MB: carried out histological analysis and wrote the manuscript. LR: sampled field material and commented on the manuscript. ER: commented on the manuscript. EOK: conducted sample preparations and histological experiments, commented on the manuscript. HB: conducted sample preparations and histological experiments, commented on the manuscript. Dr. Hege Hellberg, Pharmaq Analytic, is acknowledged for fruitful scientific discussions. The authors declare no conflicts of interest. The data that support the findings of this study (histological slides) are available from the corresponding author, HB, upon reasonable request.