Spined Stickleback Gasterosteus Aculeatus

Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 227 Systemic megalocytivirus infection in three- spined stickleback Gasterosteus aculeatus M. Marcos-López1*, S. W. Feist2, R. Hicks2 and P. A. Noguera1 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, mortalities (Williams et al., 2005; Whiington fish, amphibians and reptiles worldwide (Mao et al., 2010). Ranaviruses in fish include, among et al., 1999; Weber et al., 2009). The family Irido- others, epizootic haematopoietic necrosis virus viridae contains 5 genera, 3 of which are found in (EHNV) (Reddacliff and Whiington, 1996), Eu- fish: Lymphocystivirus, Ranavirus, and Megalocy- ropean catfish virus (ECV) (Bigarre et al., 2008) tivirus (Chinchar et al., 2009; Weber et al., 2009; and Santee-Cooper ranavirus or largemouth Whiington et al., 2010). Lymphocystis was bass virus (Grizzle et al., 2002). Megalocyti- the first iridoviral infection reported in fish viruses affect several fish species and some of (Weissenberg, 1965) and is generally character- the most studied are red sea bream iridovirus ized by chronic superficial infections and absent (RSIV) (Inouye et al., 1992), infectious spleen or very low mortalities. Mortalities may occur and kidney necrosis virus (ISKNV) (Wang et on the rare occasions where internal organs are al., 2007) and turbot iridovirus (TBIV) (Oh et affected or due to debilitation and/or secondary al., 2006). Other fish viruses such as erythrocytic infections (Williams et al., 2005). Megalocytivi- necrosis virus (ENV) (Reno and Nicholson, ruses and ranaviruses, however, have emerged 1981) and white sturgeon iridovirus (WSIV) in recent years as important fish pathogens, (Hedrick et al., 1990) have been included in the producing severe systemic infections and high iridovirus family on the basis of the morphologi- * Corresponding author’s e-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 228 cal features of the viral particles but without detected positive at the H&E screening were further molecular or serological characteriza- further stained with Feulgen stain for DNA. tion, therefore they remain unassigned. Transmission electron microscopy The stickleback Gasterosteus aculeatus is widely Blocks from samples detected positive by his- used as an experimental model, particularly for tological assessment were sent to the Centre carcinogenesis, endocrinology and chemical for Environment, Fisheries and Aquaculture testing studies (Katsiadaki et al., 2006). However, Science, Weymouth, UK, for TEM analysis. there is a paucity of information on viral infec- A small portion of the wax embedded mate- tions in this species (Mao et al., 1999). rial was removed from the block, de-waxed in xylene and rehydrated through a series of al- The present study reports a megalocytivirus cohols to water. The samples were then fixed in infection in stickleback where histology re- 1% osmium tetroxide in 0.1M phosphate buffer vealed numerous hypertrophic cells distrib- (pH 7.4) for 1 hour and rinsed in the same buffer uted throughout the body, predominantly in before dehydration through a graded acetone the subcutis, musculature and kidney. The af- series. They were then embedded in Agar 100 fected cells showed pronounced cytomegalia, epoxy resin (Agar Scientific, Agar 100 pre-mix a pathological change compatible with certain kit, medium) and polymerised overnight at viral agents such as megalocytiviruses. Material 60°C. Semi-thin sections (1-2 μm) were stained was analysed by transmission electron micro- with Toluidine Blue for viewing by light microscopy (TEM), which confirmed the suspected scopy and selected regions were identified for megalocytivirus infection. ultrastructural study. Ultrathin sections (70-90 nm) were obtained and stained with uranyl Materials and methods acetate and Reynold’s lead citrate. Grids were Sample collection and histology examined on a JEOL JEM 1210 transmission Wild caught sticklebacks, 68.7 mm and 3.19 electron microscope and digital images cap- g on average, were reared in an experimental tured using a Gatan Erlangshen ES500W camera facility in British Columbia, Canada, in ambient and Gatan Digital MicrographTM soVware. sea water conditions (8 to 10.5ºC). A batch of 180 sticklebacks were sampled for histologi- Results cal analysis as part of a sea lice experimental Histopathology challenge. Fish were fixed whole in 10% (w/v) Examination revealed numerous cytomegalic neutral buffered formalin, and a transversal cells in a single fish. These cells occupied the portion of the body at the dorsal fin level of each subcutis, especially around the lateral line, and individual was routinely processed into paraffin invaded the adjacent dermis and skeletal muscle wax blocks. Blocks were sent to Marine Scotland (Figure 1A). They were also observed through- Science, Marine Laboratory, Aberdeen, UK, out the main horizontal septum between the where 25 of the blocks were sectioned (3-5 μm), epaxial and hypaxial musculature, at the stained with haematoxylin and eosin (H&E) median septum surrounding the spinal cord, and analysed under light microscopy. Samples vertebral column and dorsal vessels (Figure 1B), 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 Affected cells showed hypertrophy of the cy- whole kidney parenchyma (Figure 1C) and the toplasm and contained large numbers of sub- gastrointestinal submucosa (Figure 1D), and in spherical and hexagonal viral particles distrib- the peritoneum surrounding the gastrointestinal uted throughout the cytoplasm (Figure 3A). tract. Other internal organs were not included Viral particles appeared unenveloped, were in the sample. The cytomegalic cells appeared between 120 and 145 nm in diameter, and com- enlarged, basophilic, with a pale granular ap- prised empty capsids and nucleocapsids (Figure pearance and frequently with an inconspicuous 3B). In a few cells, structured arrays of particles eccentric nucleus. The Feulgen stain revealed were present. In most cases, the host cell nucleus positive red-purple inclusions in the cytoplasm was not apparent, although accumulations of of the affected cells and in the nucleus of all coarse electron dense material, suggestive of cells (Figure 2). The cytoplasm of uninfected chromatin, were observed. cells stained green. 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. Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 231 Discussion with the viral infection in the affected organs Histology and TEM were the approaches used to (Rodger et al., 1997; Gibson-Kueh et al., 2003). characterise the pathology and identify the aeti- In this case, mild haemorrhage was observed ology of the megalocytivirus infection. Viruses in the kidney, but this is thought to be induced are classified according to their virion architec- by damage of the vascular endothelium due to ture, morphology, the nature and structure of pressure from the enlarged cells. No necrosis or their genetic material, and the type of pathology inflammation was noticed. In the current report, they induce. Iridoviruses are large, 120-300 nm although detected only in a single individual, in diameter, icosahedral, and double-stranded the pathological changes, the abundance of DNA viruses (Smail and Munro, 1989; Whit- virions in affected cells and their widespread tington et al., 2010) that assemble in the cy- distribution make this case significant at the toplasm of the host cells, where they appear individual level. The severity of the infection unenveloped. Unlike other virus families, the is likely to have compromised the host, as the 3 genera of Iridoviridae that affect fish can be infected cells would have been functionally distinguished by histology according to the impaired. However, in the original

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