Abstract
Melanized focal changes in white skeletal muscle of farmed Atlantic salmon, “black spots”, is a quality problem affecting on average 20% of slaughtered fish. The spots appear initially as “red spots” characterized by hemorrhages and acute inflammation and progress into black spots characterized by chronic inflammation and abundant pigmented cells. Piscine orthoreovirus 1 (PRV-1) was previously found to be associated with macrophages and melano-macrophages in red and black spots. Here we have addressed the inflammatory microenvironment of red and black spots by studying the polarization status of the macrophages and cell mediated immune responses in spots, in both PRV-1 infected and non-infected fish. Samples that had been collected at regular intervals through the seawater production phase in a commercial farm were analyzed by multiplex fluorescent in situ hybridization (FISH) and RT-qPCR methods. Detection of abundant inducible nitric oxide synthase (iNOS2) expressing M1-polarized macrophages in red spots demonstrated a pro-inflammatory microenvironment. There was an almost perfect co-localization with the iNOS2 expression and PRV-1 infection. Black spots, on the other side, had few iNOS2 expressing cells, but a relatively high number of arginase-2 expressing anti-inflammatory M2-polarized macrophages containing melanin. The numerous M2-polarized melano-macrophages in black spots indicate an ongoing healing phase. Co-localization of PRV-1 and cells expressing CD8+ and MHC-I suggests a targeted immune response taking place in the spots. Altogether, this study indicates that PRV-1 induces a pro-inflammatory environment that is important for the pathogenesis of the spots. We do not have indication that infection of PRV-1 is the initial causative agent of this condition.
Highlights
This study aimed to clarify the role of Piscine orthoreovirus 1 (PRV-1) infection and the immune mechanisms involved in the development of melanized foci in white muscle of Atlantic salmon, using immune cell gene markers representing the macrophage polarization pattern, and the cytotoxic immune response
The myocyte degeneration in red muscle caused by PRV-1 [6], could be an additional driver for influx of macrophages, but is probably not the initial cause of red spots, as these are found at similar prevalence prior to PRV infection [2]
The iNOS2 expressing M1-polarized nonmelanized macrophages are mainly present in the period of the red focal changes, i.e. the time of inflammation, which suggests local production of nitric oxide (NO) and other oxygen radicals by the M1 macrophages
Summary
Melanized focal changes in the white skeletal muscle of farmed Atlantic salmon (Salmo salar), “black spots”, has emerged as a phenomenon that is found on average in 20% of the Atlantic salmon slaughtered at Norwegian processing plants [1]. The black spots are primarily observed at slaughter of seawater farmed Atlantic salmon [3], and there are no reports that such spots are common in wild fish. In a longitudinal study where the presence of spots was followed through the seawater production phase in a commercial farmed salmon population, it was concluded that red spots preceded the formation of black spots [2]. The red spots were found to have a stable low prevalence in the production period, while the black spots accumulated over time in the fish population in seawater [2]. Histopathological classification of the melanized spots show that they develop over the time the fish population has spent in sea water, and the most serious granulomatous inflammatory changes appear a few months before slaughter and are associated with Piscine orthoreovirus 1 (PRV-1) [2]. Aggregation of macrophages and other immune cells forming granulomatous structures in the black spots indicate a longterm activation of the immune response [4]
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