Systemic megalocytivirus infection in threespined stickleback Gasterosteus aculeatus

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1 Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 227 Systemic megalocytivirus infection in threespined stickleback Gasterosteus aculeatus M. Marcos-López 1 *, S. W. Feist 2, R. Hicks 2 and P. A. Noguera 1 1 Marine Scotland Science, Marine Laboratory, AB11 9DB, Aberdeen, UK; 2 Centre for Environment, Fisheries and Aquaculture Science, DT4 8UB, Weymouth, UK Abstract Iridoviruses have been reported from a wide range of freshwater and marine fish species worldwide and in recent years megalocytiviruses (family Iridoviridae) have emerged as important fish pathogens affecting commercially important species. The present case describes a systemic megalocytivirus infection in a three-spined stickleback Gasterosteus aculeatus L. Histology revealed the presence of numerous cytomegalic cells in several locations throughout the body. Transmission electron microscopy (TEM) revealed the presence of high numbers of viral particles, with a morphology compatible with an iridovirus, throughout the cytoplasm of the affected cells. The histopathological features and the TEM observations are consistent with a megalocytivirus infection. Introduction Iridoviruses are known to affect invertebrates, fish, amphibians and reptiles worldwide (Mao et al., 1999; Weber et al., 2009). The family Iridoviridae contains 5 genera, 3 of which are found in fish: Lymphocystivirus, Ranavirus, and Megalocytivirus (Chinchar et al., 2009; Weber et al., 2009; Whi ington et al., 2010). Lymphocystis was the first iridoviral infection reported in fish (Weissenberg, 1965) and is generally characterized by chronic superficial infections and absent or very low mortalities. Mortalities may occur on the rare occasions where internal organs are affected or due to debilitation and/or secondary infections (Williams et al., 2005). Megalocytiviruses and ranaviruses, however, have emerged in recent years as important fish pathogens, producing severe systemic infections and high mortalities (Williams et al., 2005; Whi ington et al., 2010). Ranaviruses in fish include, among others, epizootic haematopoietic necrosis virus (EHNV) (Reddacliff and Whi ington, 1996), European catfish virus (ECV) (Bigarre et al., 2008) and Santee-Cooper ranavirus or largemouth bass virus (Grizzle et al., 2002). Megalocytiviruses affect several fish species and some of the most studied are red sea bream iridovirus (RSIV) (Inouye et al., 1992), infectious spleen and kidney necrosis virus (ISKNV) (Wang et al., 2007) and turbot iridovirus (TBIV) (Oh et al., 2006). Other fish viruses such as erythrocytic necrosis virus (ENV) (Reno and Nicholson, 1981) and white sturgeon iridovirus (WSIV) (Hedrick et al., 1990) have been included in the iridovirus family on the basis of the morphologi- * Corresponding author s mar.marcos-lopez@scotland.gsi.gov.uk

2 Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 228 cal features of the viral particles but without further molecular or serological characterization, therefore they remain unassigned. The stickleback Gasterosteus aculeatus is widely used as an experimental model, particularly for carcinogenesis, endocrinology and chemical testing studies (Katsiadaki et al., 2006). However, there is a paucity of information on viral infections in this species (Mao et al., 1999). The present study reports a megalocytivirus infection in stickleback where histology revealed numerous hypertrophic cells distributed throughout the body, predominantly in the subcutis, musculature and kidney. The affected cells showed pronounced cytomegalia, a pathological change compatible with certain viral agents such as megalocytiviruses. Material was analysed by transmission electron microscopy (TEM), which confirmed the suspected megalocytivirus infection. Materials and methods Sample collection and histology Wild caught sticklebacks, 68.7 mm and 3.19 g on average, were reared in an experimental facility in British Columbia, Canada, in ambient sea water conditions (8 to 10.5ºC). A batch of 180 sticklebacks were sampled for histological analysis as part of a sea lice experimental challenge. Fish were fixed whole in 10% (w/v) neutral buffered formalin, and a transversal portion of the body at the dorsal fin level of each individual was routinely processed into paraffin wax blocks. Blocks were sent to Marine Scotland Science, Marine Laboratory, Aberdeen, UK, where 25 of the blocks were sectioned (3-5 μm), stained with haematoxylin and eosin (H&E) and analysed under light microscopy. Samples detected positive at the H&E screening were further stained with Feulgen stain for DNA. Transmission electron microscopy Blocks from samples detected positive by histological assessment were sent to the Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UK, for TEM analysis. A small portion of the wax embedded material was removed from the block, de-waxed in xylene and rehydrated through a series of alcohols to water. The samples were then fixed in 1% osmium tetroxide in 0.1M phosphate buffer (ph 7.4) for 1 hour and rinsed in the same buffer before dehydration through a graded acetone series. They were then embedded in Agar 100 epoxy resin (Agar Scientific, Agar 100 pre-mix kit, medium) and polymerised overnight at 60 C. Semi-thin sections (1-2 μm) were stained with Toluidine Blue for viewing by light microscopy and selected regions were identified for ultrastructural study. Ultrathin sections (70-90 nm) were obtained and stained with uranyl acetate and Reynold s lead citrate. Grids were examined on a JEOL JEM 1210 transmission electron microscope and digital images captured using a Gatan Erlangshen ES500W camera and Gatan Digital Micrograph TM so ware. Results Histopathology Examination revealed numerous cytomegalic cells in a single fish. These cells occupied the subcutis, especially around the lateral line, and invaded the adjacent dermis and skeletal muscle (Figure 1A). They were also observed throughout the main horizontal septum between the epaxial and hypaxial musculature, at the median septum surrounding the spinal cord, vertebral column and dorsal vessels (Figure 1B),

3 Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 229 Figure 1. A. Cytomegalic cells present in the subcutis (*) and adjacent dermis (D) and skeletal muscle (M). H&E. B. Cytomegalic cells occupying the median septum and surrounding the dorsal vessels (V). H&E. C. Cytomegalic cells in kidney parenchyma (*) and glomeruli (arrow). H&E. D. Cytomegalic cells in intestinal submucosa (*). H&E. and at the dorsal and pelvic fins. Identical cells Transmission electron microscopy were found in renal glomeruli, occupying the whole kidney parenchyma (Figure 1C) and the gastrointestinal submucosa (Figure 1D), and in Affected cells showed hypertrophy of the cytoplasm and contained large numbers of subspherical and hexagonal viral particles distrib- the peritoneum surrounding the gastrointestinal tract. Other internal organs were not included in the sample. The cytomegalic cells appeared enlarged, basophilic, with a pale granular ap- uted throughout the cytoplasm (Figure 3A). Viral particles appeared unenveloped, were pearance and frequently with an inconspicuous eccentric nucleus. The Feulgen stain revealed 3B). In a few cells, structured arrays of particles were present. In most cases, the host cell nucleus positive red-purple inclusions in the cytoplasm of the affected cells and in the nucleus of all was not apparent, although accumulations of coarse electron dense material, suggestive of cells (Figure 2). The cytoplasm of uninfected cells stained green. chromatin, were observed. between 120 and 145 nm in diameter, and comprised empty capsids and nucleocapsids (Figure

4 Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 230 Figure 2. Cytomegalic cells in kidney parenchyma (*) and inside vessel (V). Cytoplasm of affected cells stain red-purple, showing presence of DNA. Feulgen stain (x40). Figure 3. A. Infected host cell of indeterminate origin from the subcutis with organised clusters and individual virions distributed throughout the cytoplasm. Nuclei and cellular organelles are not discernable. B. Detail from Fig. 3A showing the stratified arrangement of intact iridovirus-like nucleocapsids.

5 Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 231 Discussion Histology and TEM were the approaches used to characterise the pathology and identify the aetiology of the megalocytivirus infection. Viruses are classified according to their virion architecture, morphology, the nature and structure of their genetic material, and the type of pathology they induce. Iridoviruses are large, nm in diameter, icosahedral, and double-stranded DNA viruses (Smail and Munro, 1989; Whittington et al., 2010) that assemble in the cytoplasm of the host cells, where they appear unenveloped. Unlike other virus families, the 3 genera of Iridoviridae that affect fish can be distinguished by histology according to the type of pathology they induce. Lymphocystis viruses produce marked cytomegaly of dermal fibroblasts of skin and gills (Smail and Munro, 1989), ranaviruses are generally associated with systemic necrotizing infections (Whi ington et al., 2010), and megalocytiviruses cause characteristic cytomegalia of cells in multiple organs (Weber et al., 2009). Given the observed cellular pathology and virion location and morphology in the present study, a megalocytivirus was determined as the aetiological agent. The associated pathology resembles those reported in other fish species. Turbot Pse a maxima suffering from reddish body syndrome, caused by the turbot iridovirus ( nm in diameter), show enlarged cells in spleen, kidney and gills, the spleen being the main target organ (Shi et al., 2004). Red sea bream iridovirus was first detected in red sea bream Pagrus major, but has now been identified in more than 30 fish species (Lua et al., 2005). Infected fish show enlarged cells in spleen, kidney, liver and gills (Inouye et al., 1992). Some of the reports described haemorrhage, necrosis and inflammation associated with the viral infection in the affected organs (Rodger et al., 1997; Gibson-Kueh et al., 2003). In this case, mild haemorrhage was observed in the kidney, but this is thought to be induced by damage of the vascular endothelium due to pressure from the enlarged cells. No necrosis or inflammation was noticed. In the current report, although detected only in a single individual, the pathological changes, the abundance of virions in affected cells and their widespread distribution make this case significant at the individual level. The severity of the infection is likely to have compromised the host, as the infected cells would have been functionally impaired. However, in the original batch of fish there was no mortality, abnormal behaviour or significant macroscopic lesions recorded during sampling. Information on viral diseases of stickleback is scarce. Mao et al. (1999) isolated a ranavirus from a single stickleback, stickleback virus (SBV). Characterization showed that SBV was very closely related with frog virus 3 (FV3). In the same study, an identical virus was isolated from the red-legged frog, Rana aurora. Recently, Waltzek et al. (2010) also reported a low level mortality in stickleback and a ributed this to a megalocytivirus. Viral haemorrhagic septicaemia (VHS), an important disease of rainbow trout Oncorhynchus mykiss and turbot caused by a rhabdovirus, was isolated from stickleback during the investigation of mortalities in a wild stickleback population (Gagne et al., 2007). Gibson-Kueh et al. (2003) reviewed and studied the pathology of systemic megalocytivirus infections in several marine and freshwater fish species. Cytomegalic cells were found in multiple organs; mainly in spleen (parenchyma and

6 Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 232 capsule), kidney (glomeruli and interstitium), intestine (lamina propia), and connective tissue through the body, but also in eye, pancreas, liver, heart, gills, and brain. The specific type of cells affected in systemic megalocytivirus infections is however unclear from published work, although epithelial and endothelial cells have been shown to be not affected (Gibson- Kueh et al., 2003). The type of cells involved in the present case is difficult to identify due to the presence of massive numbers of viral particles distorting their normal structure, however, their distribution may suggest an inflammatory origin, as they were found in highly vascularised locations and in organs where, in normal conditions, high numbers of inflammatory cells may be present, such as renal interstitium and intestinal lamina propia. Other organs likely to have been affected are gills and spleen, but unfortunately these were not included in the sample (Armstrong and Ferguson 1989) and 1989 (Anderson et al., 1993), when megalocytiviruses infections were detected in tropical fishes imported from Singapore into Canada and Australia respectively. In addition, iridoviruses are very stable in aquatic environments, which may help their transmission by water borne routes (Go et al., 2006). The current case together with the finding reported by Waltzek et al., (2010) are the first reports of this infection in a member of the Order Gasterosteiformes. Acknowledgments The authors would like to thank Nichola Mc- Mannus for her technical assistance in processing samples for light microscopy, to Dr. Campbell Pert and Dr. Simon Jones who provided the samples, and to Dr Hugh Ferguson for advice in histopathology interpretation. The support of Defra under contract FB002 (to SWF) is gratefully acknowledged. Megalocytiviruses have emerged in recent years as important fish pathogens. Megalocytiviruses have their origin in Southeast Asia, where outbreaks were firstly recorded in red sea bream (Pagrus major) from Japan in 1990 (Inouye et al., 1992), in brown spo ed groupers (Epinephelus tauvina) from Singapore in 1992 (Chua et al., 1994) and in Malabar groupers (Epinephelus malabaricus) and sea bass (Lateolabrax spp.) from Thailand in 1993 (Danayadol et al., 1996). Anthropogenic factors such as increased global trade in ornamental fish may have facilitated the spread of megalocytiviruses to distant geographic regions such as Australia, North America and Europe, and enabled the emergence of disease in new host species (Go et al., 2006; Go and Whi ington, 2006; Dong et al., 2010). First evidences date back as far as References Anderson IG, Prior HC, Rodwell BJ and Harris GO (1993). Iridovirus-like virions in imported dwarf gourami (Colisa lalia) with systemic amoebiasis. Australian Veterinary Journal 70, Armstrong RD and Ferguson HW (1989). Systemic viral disease of the chromid cichlid Etroplus maculatus. Diseases of Aquatic Organisms 7, Bigarre L, Cabon J, Baud M, Pozet F and Castric J (2008). Ranaviruses associated with high mortalities in catfish in France. Bulletin European Association of Fish Pathologists 28, Chinchar VG, Hya A, Miyazaki T and Williams T (2009). Iridoviridae: poor viral relations no longer. Current Topics in Microbiology and Immunology 328, Chua HC, Ng ML, Woo JJ and Wee JY (1994).

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