Notes Dermocystidium sp. in Scottish Atlantic salmon, Salmo salar: Evidence for impact on fish in marine fish farms

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1 Bull. Eur. Ass. Fish Pathol., 21(5) 2001, 209 Notes Dermocystidium sp. in Scottish Atlantic salmon, Salmo salar: Evidence for impact on fish in marine fish farms Bruno, D.W. FRS Marine Laboratory, Victoria Road, Aberdeen AB11 9DB Scotland Abstract Sea water mortality in farmed Atlantic salmon, Salmo salar was attributed to a Dermocystidium infection acquired in freshwater. Each of the four independent outbreaks recorded in Scottish waters were linked directly to a common source of juvenile fish. Renal enlargement with whitish nodules in the kidney with a progressing systemic granulomatous response occurred in internal organs. In addition, infection by Dermocystidium in the gill lamellae showed hyperplasia and oedema. Dermocystidium spp., infections occur in farmed salmonids and are reported from juvenile Atlantic salmon, Salmo salar, sockeye salmon, Oncorhynchus nerka, pink salmon, O. gorbuscha, rainbow trout, O. mykiss and brown trout, S. trutta in freshwater (McVicar & Wootten, 1980, Hedrick et al., 1989; Olson et al., 1991). Infections in fish are characterised by a chronic localised infection of the gills and skin (Allen et al; 1968; Olson et al., 1991; Landsberg & Paperna, 1992; Höglund et al., 1997), and occasionally as a systemic nodular infection involving visceral organs in salmon all in freshwater (Allen et al., 1968; McVicar & Wootten, 1980) and goldfish, Carassius auratus (Landsberg & Paperna, 1992). Different species of Dermocystidium or Dermocystidium-like organisms may be responsible for the localised and systemic pathology and this aspect is discussed by Höglund et al. (1997). Transmission and infectivity occurs in freshwater when the flagellated, free-swimming zoospores penetrate the gill epithelium (Olson et al., 1991). However, the freshwater host and therefore the full life cycle for this pathogen has not been determined. Taxonomically, Dermocystidium spp. that infect fish are poorly understood and the systematic position of the organism still remains unsettled, in contrast to Dermocystidium marinum in molluscs which have been placed in a new class Perkinsea with the sole member, Perkinsus marinus (Levine, 1978). Significant losses attributed to Dermocystidium spp. infection in fry are reported (Pauley 1967; Hoskins et al., 1976, McVicar & Wootten, 1980). This study examines increasing mortality in sea reared Atlantic salmon that was attributed to infection by Dermocystidium spp. acquired during their freshwater phase of growth. Fish Moribund Atlantic salmon were removed from sea cages of farms owned by four inde-

2 Bull. Eur. Ass. Fish Pathol., 21(5) 2001, 210 pendent companies in Scottish waters (both mainland and Shetland farms) during site visits by Fisheries Research Services fish health inspectorate. At all farms the fish had been transferred from a single freshwater source between April and May as S1 smolts in the same year. Necropsy and sampling of these fish was carried out between August and October when the mean weight was g. In addition one farm was sampled in September and again in October. Water temperatures in mid August were 13 0 C, and 15 0 C in mid September. Bacteriology and virology An inoculum from the kidney was streaked on tryptic soy agar (TSA) with 2.5% NaCl and Mueller-Hinton medium with added L- cysteine and selected antibiotics (MH). Bacteriology plates were incubated at 15 0 C and examined after 3 days for TSA and up to 10 weeks for the MH plates. Kidney, liver and heart were taken for infectious pancreatic necrosis virus (IPNV) testing and kidney for infectious salmon anaemia virus (ISAV) testing using RT-PCR (Mjaaland et al., 1997). ELISA testing was carried out on kidney tissue for Renibacterium salmoninarum, agent for bacterial kidney disease (BKD). Bacteriology testing was negative. Virus testing for IPNV was confirmed at one site, but absent from the remaining three farms. ISAV was not recorded by RT-PCR at any farm. Histological examination Spleen, kidney, heart, liver, pyloric caeca and gills were fixed in buffered formal saline. Sections were stained by haematoxylin and eosin (H&E), Gram, periodic acid Schiffs (PAS) and examined by light microscopy. Between August and October a systemic granulomatous pathology was noted in moribund fish from four sites in Scottish waters. Fish were lethargic, showed renal enlargement with scattered circular, whitish nodules visible in the kidney. The pyloric caeca were mauve, and the liver and gills pale, suggesting evidence of anaemia in the latter organs. The histological lesions, size of parasitic cells and spores were identical to reference material for Dermocystidium infection from salmon in freshwater as held by FRS Marine Laboratory and that previously described by other workers (McVicar & Wootten, 1980; Hedrick et al., 1989 and Olson & Holt, 1995). Most Dermocystidium from stained sections were spherical, 4-5µm in diameter, with some to 5-10µm in diameter. The outer spore wall stained strongly PAS positive and the large central vacuoplast of the spore refractile and PAS negative. Each cyst was surrounded by a thin homogeneous wall of parasite origin with no encapsulation of the cysts by host tissue. Extracellular spores were dominant, while others were located within hypertropic macrophages. Spores were characterised by a marked intracellular parasitism and macrophages with cell necrosis recorded. An extensive progressing granulomatous response with little fibrosis was noted in the liver, kidney, heart and spleen (Fig. 1). Within the spleen and liver loosely-defined granulomas containing uninucleate spores were observed with associated compression of adjacent tissue, reduced staining and a marked host response. Necrotic areas were identified centrally in the granulomas and in the kidney some free blood recorded, along

3 Bull. Eur. Ass. Fish Pathol., 21(5) 2001, 211 Figure 1. Dermocystidium infection in spleen tissue from Atlantic salmon. Numerous spores (arrowed)are associated with a loosely-defined granulomas response and compression of adjacent tissue (H&E, x 230). with an increase in melanomacrophages. Cysts were located in the glomerulus but not in the tubule endothelium. In the heart scattered cysts were primarily located in the spongiosum. Dermocystidium infection was recorded in the primary and secondary gill lamellae with hyperplasia and oedema of the secondary lamellae. Mortality and movement records Mortalities (expressed as weekly losses) were recorded and the stock movement records of fish onto and from the farms examined. The examination of fish stock movement records revealed that all four marine sites where moribund fish were removed purchased their juvenile stock from a common source. It was concluded that the Dermocystidium infection seen in the marinefarmed stock had originated from a single farm source in freshwater. Other stock with a different freshwater origin were not infected. There was no movement of stock between the marine sites. In addition, no mortality records were available to suggest infection had caused losses during the period of juvenile growth of the salmon. Mortality at one marine farm, and considered typical of farms with this infection, recorded a weekly loss of 1.1% of the year class (end August), which increased to 3.1% per week during September. A primary, systemic, visceral granulomatous response was reported in moribund Atlantic

4 Bull. Eur. Ass. Fish Pathol., 21(5) 2001, 212 salmon at four independently owned marine farms between August and October. A diagnosis of Dermocystidium was concluded following comparison with stained histological sections and available literature. The availability of a stock movement record book on each farm enabled the source of the original fish and hence the flagellated motile cells (Dermocystidium) to be traced to a single source of juvenile freshwater stock that had been supplied to each marine site. In freshwater, water borne transmission of Dermocystidium occurs and the organism infects salmonid gill epithelial cells (Olson et al., 1991). Dermocystidium infection in the cases reported in this study were postulated to have occurred in the stock prior to transfer to the sea water site. There is no evidence of infection in the marine environment. Mortality data is not widely reported for Dermocystidium infection, although Allen et al. (1968) described pre-spawning losses of 22% in female chinook salmon, O. tshawytscha. Mortality in the current study was moderate, attributed to the systemic Dermocystidium infection reaching a maximum of 3.1% per week over several weeks. Histological lesions were characterised by a marked parasitism with some macrophage involvement, fibrosis and a diffusing cell necrosis pushing aside normal tissue. The outbreak in post-smolts and lesion development was chronic in nature and represented the visceral and nodular form of this disease as described by Hedrick et al. (1989) and Nash et al. (1989) in freshwater. Although IPNV was found in fish from one site, in this instance this virus did not appear to contribute to fish losses, as there were no major differences in mortality between the IPNV positive site and the non-ipnv sites. Bacteriology culture and ELISA testing for R. salmoninarum were negative and concurrent infection and mortality at the four farms was attributed to the systemic Dermocystidium spp. infection. There was some evidence from farm records that the increase in water temperature of 2 0 C between August and September resulted in an increased mortality but the data here is incomplete, however, from another study, warm freshwater is reported to accelerate cyst and spore development (Olson and Holt, 1995). This study highlights infection by Dermocystidium spp., a freshwater parasite of salmonids, and its an impact on mortality following transfer of a single stock to four independent farms rearing Atlantic salmon in sea water Acknowledgement. The assistance of the FRS inspectorate, Aberdeen in collecting the original material is acknowledged. References Allen, R.L., Meekin, T.K. Pauley, G.B. and Fujihara, M.P. (1968) Mortality among chinook salmon associated with the fungus Dermocystidium. J. Fish.Res. Bd Can. 25, Cawthorn, R., Backman, S., Groman, D., O Halloran, J., and Johnson, G. (1990) Dermocystidium-like parasite in farmed Atlantic salmon. Can.Vet. J. 31, 591.

5 Bull. Eur. Ass. Fish Pathol., 21(5) 2001, 213 Hedrick, R.P., Friedman, C.S. and Modin, J. (1989) Systemic infection in Atlantic salmon Salmo salar with a Dermocystidium-like species. Dis. Aquat. Org. 7, Höglund, J., Alfjorden, A. and Nikkilä, T. (1997) Infection of juvenile salmon Salmo salar with a Dermocystidium-like organism in Sweden. Dis. Aquat.Org. 30, Hoskins, G.E., Bell, G.R., and Evelyn, T.P.T. (1976) The occurrence, distribution and significance of infectious diseases and of neoplasms observed in fish in the Pacific region up to the end of Can. Fish. Mar. Tech. Rep Landsberg, J.H. and Paperna, I. (1992) Systemic granuloma in goldfish caused by a Dermocysytidium-like aetiological agent. Dis. Aquat. Org. 13, Levine, N.D. (1978) Perkinsus gen. n. and other new taxa in the protozoan phylum Apicomplexa. J. Parasitology 64, 549. Mjaaland, S., Rimstad, E., Falk, K. and Dannevig, B.H. (1997) Genomic characterization of the virus causing infectious salmon anaemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost. J.Virol. 71, McVicar, A.H. and Wootten, R. (1980) Disease in farmed juvenile Atlantic salmon caused by Dermocystidium sp. In: Ahne, W. (ed) Fish diseases. Third COPRAQ-session, Springer- Verleg, Berlin, pp Nash, G., Southgate, P. and Richards, R.H. (1989) A systemic protozoal disease of cultured salmonids. J. Fish Dis. 12, Olsen, R.E., Dungan, C.F. and Holt, R.A. (1991) Water-borne transmission of Dermocysytidium salmonis in the laboratory. Dis. Aquat. Org. 12, Olson, R.E. and Holt, R.A. (1995) The gill pathogen Dermocystidium salmonis in Oregon salmonids. J.Aquat. Animal Hlth 7, Pauley, G.B. (1967) Prespawning adult salmon mortality associated with a fungus of the genus Dermocystidium. J. Fish. Res. Bd. Can. 24, Wootten, R. and McVicar, A.H. (1982) Dermocystidium from cultured eels, Anguilla anguilla L., in Scotland. J. Fish Dis. 5,