Knowledge of the construction of immune organs of different species is important to understand the evolution of immunity. In birds, the bursa of Fabricius is a cloaca-based lymphoid organ that has been vital for our current understanding of the adaptive immune system of vertebrates. So far, this structure has not been described in other species. Here, we show a substantial lymphoepithelial tissue in a previously undescribed bursa in the Atlantic salmon, situated analogous to the placement of the avian bursa of Fabricius, also developing from the ectoderm. Investigations of three different age groups of Atlantic salmon revealed a dynamic lymphoepithelium that regressed following sexual maturation. This is similar to the situation in birds. Morphological investigations including dissection, radiography, in situ hybridization and immunohistochemistry revealed presence of T and B cells embedded within a CCL19-positive lymphoepithelium. CCL19 expression is indicative for lymphoid organs, and thus we suggest that the salmonid bursa should be regarded as such. We found however no evidence for primary lymphoid organ functions including recombination events in the lymphocytes, this contrasting the situation in birds. Our results demonstrate the presence of a salmonid lymphoepithelial compartment with anatomical and developmental similarities to early stages in the organogenesis of the avian bursa of Fabricius. Such information provides novel and exciting possibilities both for our understanding of the function of the immune system in salmon and for our understanding of the development of lymphoid structures and niches in vertebrates.

Melanised skeletal muscle changes (black spots) are a serious problem for the salmon industry world-wide, causing extensive production losses. Piscine orthoreovirus (PRV) is associated with the condition. The initial cause for the focal changes remains obscure. Early changes start as muscle bleedings and extensive muscle necrosis, and so far, these changes cannot be explained, However, from this acute phase, the changes may progress into melanised focal changes appearing as chronic granulomatous reactions. Granulomatous melanised changes have been consistently observed in PRV infected fish, and in serious changes, the tissue persistence of virus dominate the changes despite the inflammatory changes. Here, we address the pathogenesis of PRV infection and phases of melanised focal changes development. Fluorescent in situ hybridisation (FISH) assay confirmed the colocalisation of iNOS2 transcripts (M1 activated macrophages) with PRV in red spot whereas both M1 and M2 (Arg2 transcripts) macrophages localized at PRV infected clusters in black spots. Melano-macrophages were found positive with Arg2 transcripts demonstrating their role in tissue repair mechanism. In PRV negative fish low expression of M1 and M2 specific transcripts suggest PRV act as an accelerator for the melanisation in these focal changes. Transcripts specific for melanin production were observed in non-melanised cells in early stages of the condition and in melano-macrophages in late stages. The nature of the melano-macrophages is disputed and the rationale for their melanin production is not understood. Our investigations reveal new characteristics both of the pathogenesis of focal melanised changes as well as of the melano-macrophages involved.

Research on the immune system of salmonid fish is attracting increased attention, however, progress within this field has been hampered by the general lack of cell markers targeting different immune molecules. Here, we circumvent these challenges by applying in situ hybridization (ISH) to reveal basic cellular responses in salmon immune reactions. Using RNAscope® (ACD, Newark, CA), we have established ISH targeting immunoglobulin (Ig) M and T RNA. Further, we also target other immune genes where we previously had no markers, and tyrosinase, thought to be important for the function of the melano-macrophages which are special, melanin-producing immune cells found in ectothermic vertebrates. We provided target gene sequences to the manufacturer, which supplied us with probes, and we performed ISH according to the manufacturer’s instructions. Different inflammatory conditions in farmed salmon, including cancerous changes from the gut and muscle tissue with chronic myositis and melanization due to the presence of melano-macrophages, were screened by standard histology and subsequently subjected to ISH. For the IgM results, we compared IHC and ISH results, as we have antisera targeting IgM. The results were highly similar. ISH thus seems as a good method that may be used in salmon when applicable and especially to target transcripts where no antibody recognizing the resulting protein is available. Further, we reveal hitherto non-described cellular compositions of inflammatory changes in salmon using probes detecting several different immune cell transcripts. Our results show that ISH using RNAscope® works in Atlantic salmon and is highly reproducible.