Histopathological description of an emerging disease in Norwegian salmonid aquaculture caused by an x‐cell parasite

In the autumn 2002 a disease outbreak in farmed rainbow trout (Oncorhynchus mykiss (Walbaum)) was reported on the Western coast of Norway, involving a parasitic protist. This parasite occurred as a plasmodia that upon partial small subunit ribosomal DNA sequencing was found to be related to the Perkinsea among the alveolates (Karlsbakk et al., 2003; Nylund et al., 2006). In the autumn 2017, cases with parasitic disease with similar findings both clinical and histologically re-appeared, and since then an increasing number of cases has been reported annually for rainbow trout, and in the autumn 2020 a similar disease condition appeared in farmed Atlantic salmon (Salmo salar L.). The observed increase in number of cases has raised concern that this might be an emerging disease in salmonid aquaculture in Norway. However, as this disease is not notifiable, and as there are several providers of diagnostic services in Norway, the exact number of cases are not known. All cases have been located to the Western coast of Norway and observed in fish in sea-water phase, both from on-growing sites from conventional net-pens as well as from broodfish. So far, a histopathological description of lesions caused by this parasite is lacking. In this short communication, typical histopathological lesions observed in field cases of infected rainbow trout and Atlantic salmon are presented in order to increase the awareness of this parasite. Typical reported clinical observations in diseased fish include decreased or ceased feeding, lethargy and increased mortality. Macroscopically, typical lesions included multifocal, white to yellow nodules or cauliflower-like lesions in internal organs, ranging in size from pinpoint to several millimetres (Figure 1A-C). In advanced cases, lesions aggregated to a thick, confluent whitish covering could be seen (Figure 1d). Changes were most often observed in liver, heart and red skeletal muscle, but parasite-containing nodules have also been seen in other tissues like spleen, kidney, intestine, gills, gonads, peri-pancreatic adipose tissue and white skeletal muscle in fish with protracted course of disease. Other less specific internal changes included ascites, haemorrhages in the viscera and the abdominal wall, and yellowish discoloration of the liver. Usual anamnestic information from fish health personnel submitting the samples included suspicion of granuloma formation and/or neoplasia. Histological examination was performed on formalin-fixed, paraffin-embedded sections (2–3 μm thickness) prepared after standard procedures (Suvarna et al., 2019) and stained with haematoxylin and eosin. In this material, a histological investigation of a total of 72 individuals of rainbow trout and three individuals of Atlantic salmon sampled in the period autumn 2017 until spring 2021 are included (Table 1). In addition, selected sections were stained with Gram, Ziehl Neelsen, Giemsa and Luna to assess the staining properties of the parasite. Both the appearance and distribution of the tissue changes were similar for rainbow trout (Figure 2a-O) and Atlantic salmon (Figure 3a-f). The tissue changes typically consisted of focal or multifocal to coalescing areas of coagulative necrosis and haemorrhage with various numbers of parasites. These changes corresponded with the white nodules as observed macroscopically. Surprisingly, given the severity of the tissue lesions, only sparse infiltration of inflammatory cells was observed. The structure of the parasites also appeared similar in lesions from both rainbow trout and Atlantic salmon. The parasite appeared as circular to slightly elongated, basophilic structures, with an outer membrane surrounding several nuclei and resembled the x-cell plasmodia observed by Dyková et al. (1993). Also, nuclei lying free in necrotic tissue were seen, presumably representing ruptured plasmodia. The size of the x cells in histological sections varied from 5 µm to 48 µm; however, the majority measured 10–30 µm in diameter. At most, 22 nuclei were counted in a single x-cell plasmodium. In stained smears from diseased rainbow trout, up to 47 nuclei within a single plasmodium has been reported (Karlsbakk et al., 2003). In Giemsa-stained sections, the parasites appeared light basophilic with darker blue (basophilic) nuclei (Figure 4). The parasite did not stain distinctly with Gram, Ziehl Neelsen or Luna stain. Both macroscopically and as inferred from histology, the highest occurrence of lesions was found in the liver, followed by the heart. Subsequently, lesions could appear in most well-vascularized tissues like the red part of the skeletal muscle, spleen, kidney, intestine and gills. During severe perkinsus infection in anurians, a similar pattern of prevalence was seen, with the most frequently affected organs being the liver, spleen, kidney and pancreas (Isidoro-Ayza et al., 2019). It therefore seems likely that the parasite is spread systemically via the blood and that it might have preferences for highly vascularized tissues. The observed high prevalence in the liver could imply infection through the intestine followed by a primary infestation of the liver, although this suggestion requires investigation. Also, in cases with infection in both the liver and the heart (and possibly other organs), the lesions appeared more chronic in the liver than in the heart, suggesting a protracted course of disease in the former. Further, the presence of multinucleated x cells in both lamellar vascular structures of the gill and the epithelium of various parts of the intestine, in fish with advanced disease, could indicate that the parasite exits the fish from these sites. Importantly, this condition has only been diagnosed in the period between mid-October and mid-February. In health check follow-ups in sites where this parasite was diagnosed earlier, only scar tissue was seen from mid-February onwards. The observed lesions and the intralesional parasites show similarities with the disease multinucleate sphere X (MSX) in the eastern oyster (Crassostrea virginica) caused by Haplosporidium nelsoni (Carnegia & Burreson, 2012; Lynch et al., 2013). The parasites also show similarities with X cells, associated with tumour-like formations in marine fish (Feist & Bass, 2017; Freeman et al., 2017). These x cells have been difficult to affiliate to a phylum; however, Freeman et al. showed that they are related to Perkinsus spp. among the Alveolata (Freeman, 2009; Freeman et al., 2017). Infection with a protist parasite referred to as Perkinsus sp. has been reported as common in farmed Atlantic salmon in the Bay of Fundy at the border between Canada and North America (Cawthorn et al., 1991). That parasite has not been described, but was considered similar to Dermocystidium sp. and the rosette agent, a parasite later named Sphaerothecum destruens (Harrell et al., 1986; Hedrick et al., 1989), both now ichthyosporeans (Adl et al., 2012). So far, the mode of transmission of the xcelliids to different hosts is unknown (Bower, 2006). In shellfish, variation in pathogenicity between different Perkinsus species is thought to be caused by the difference in the production of extracellular proteins (McLaughlin et al., 2000). Based on the extensive necrotic lesions observed in salmonids, toxin or enzyme production by the parasite may be suspected. Further work should focus on genetic studies to reveal the phylogenetic placement of the parasites and to determine whether the infection seen in rainbow trout and Atlantic salmon represents the same parasite as suggested from the morphology. Very recently, the name Salmoxcellia vastator was proposed for this salmonid xcelliid parasite (Karlsbakk et al., 2021). Further, modes of transmission and whether this parasite has a direct or indirect life cycle should be investigated. Of particular importance is whether this parasite can be vertically transmitted. The authors would like to express gratitude to Professor Trygve Poppe for the valuable discussion regarding the histopathological manifestations covered in this short communication. The authors declare that there are no conflicts of interest associated with this publication. No shared data are available.

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