doi:10.1111/jfd.12284
Journal of Fish Diseases 2014
Host tropism of infectious salmon anaemia virus in marine and freshwater fish species M Aamelfot1, O B Dale1, A McBeath2 and K Falk1 1 Norwegian Veterinary Institute, Oslo, Norway 2 Marine Scotland Science, Aberdeen, UK
Abstract
The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net-pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod-like and perch-like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists. Keywords: 4-O-Acetylated sialic acid, Atlantic salmon, virus histochemistry, virus reservior, orthomyxovirus. Introduction
Infectious salmon anaemia (ISA) is a serious, OIE-listed disease of farmed Atlantic salmon, Correspondence K Falk, Norwegian Veterinary Institute, lsveien 68, P.O. Box 750 Sentrum, 0106 Oslo, Norway Ulleva (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Salmo salar L., caused by an enveloped aquatic orthomyxovirus, ISAV. ISA is characterized by anaemia and circulatory disturbances such as oedemas, ascites and bleedings (Aamelfot, Dale & Falk 2014a). Since the major ISA epidemic in the 1990s, Norway has experienced 2–20 annual disease outbreaks. These have a tendency to cluster in time and space (Jarp & Karlsen 1997; Scheel et al. 2007). Phylogenetic studies confirmed that virus isolates from these clusters are related (Lyngstad et al. 2008), suggesting horizontal transmission. However, in many outbreaks, no obvious sources of infection have been identified. The questions remain on how ISAV is maintained in the ocean and where it came from originally, and whether a wild host exists, as has been hypothesized (Nylund et al. 2003). Atlantic salmon are farmed at sea in open netpens, with no barriers to pathogen exchange with the environment. Thus, wild fish could maintain ISAV in the environment and act as a reservoir of the virus. Open net-pen production systems are known to attract a high diversity of wild species due to the free availability of feed (Dempster et al. 2009). These may move between farms acting as carriers, thus contributing to the spread of pathogens. Saithe, Pollachius virens L., the most abundant fish species associated with Norwegian salmon farms (Dempster et al. 2009), has been suggested to act as a natural reservoir of salmonid alpha virus (SAV) (Graham et al. 2006) and is a carrier of infectious pancreatic necrosis virus (IPNV) (Wallace et al. 2008). However, it appears resistant to ISAV and clears the virus rapidly after infection (Snow et al. 2002; McClure et al. 2004). Finally, sea lice (Lepeophtheirus salmonis and Caligus sp.) may act as mechanical vectors, penetrating the skin
M Aamelfot et al. Host tropism of ISAV
Journal of Fish Diseases 2014
and could deposit virus into the blood (Nylund et al. 1994). Experimental trials have detected ISAV replication, without disease, in many fish species including brown trout (Nylund, Alexandersen & Rolland 1995; Nylund & Jakobsen 1995); rainbow trout (Nylund et al. 1997; MacWilliams et al. 2007); arctic char, Salvelinus alpinus L. (Snow, Raynard & Bruno 2001); herring, Clupea harengus L. (Nylund et al. 2002); and Atlantic cod, Gadus morhua L. (Grove et al. 2007). Thus, many species may harbour and spread ISAV, and a latent carrier status was suggested for salmonids (Nylund & Jakobsen 1995). ISAV has been detected, in the absence of disease, in wild salmonids, including brown trout, Salmo trutta L. and rainbow trout, Oncorhynchus mykiis (Walbaum), in Norway (Plarre et al. 2005) and Scotland (Raynard, Murray & Gregory 2001). Mortality and ISA-like signs were reported in one rainbow trout study (Biacchesi et al. 2007), suggesting ISAV may represent a threat to rainbow trout. Experimentally infected Pacific salmon showed no signs of disease (Rolland & Winton 2003). In Chile, however, ISAV was detected in jaundiced Pacific coho salmon, Oncorhynchus kisutch (Walbaum) (Kibenge et al. 2001). In addition, mortality was reported in Amago trout, Oncorhynchus masou (G€ unther) (Ito, Oseko & Ototake 2014); however, no virus transmission to cohabiting Atlantic salmon was detected. Thus, while several fish species are susceptible to ISAV, and subclinical infections could be common, the major disease problems appear restricted to farmed Atlantic salmon. Knowledge on ISAV susceptible species is important to understand ISAV epidemiology. However, investigation of susceptibility via screening for natural infection, or by experimental challenges, is difficult and costly. Thus, methods to select potential carrier species are needed. Host susceptibility and the ability to cause disease are determined by complex interactions between the virus virulence, the host and the environment. Every step in the infection cycle relies on the host and virus being compatible. Viral entry, for example, is dependent on the appropriate cell receptor (Helenius 2007). ISAV uses 4-O-acetylated sialic acids as its cellular receptor (Hellebø et al. 2004). Recently, we demonstrated the cell tropism of ISAV in Atlantic salmon by detecting the virus receptor in situ (Aamelfot et al. 2012). The receptor was found on endothelial cells, red blood cells (RBCs) and some epithelial cells. Ó 2014 John Wiley & Sons Ltd
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Here, we extended the investigations from Atlantic salmon to investigate various cold-water fish species that naturally encounter farmed Atlantic salmon for the ISAV receptor. In addition, we tested binding of RBCs from a small number of fish species to cultured cells expressing the haemagglutinin-esterase (HE) virus protein on the cell surface. Although the host tropism is dependent on multiple virus and host factors in addition to receptor specificity, screening fish for the receptor could narrow down the number of potential hosts that may have a role in the epidemiology of ISA.
Materials and methods
Tissue sampling Heart samples were collected from 53 cold-water fish species (Table 1). Tissues from heart, gill, liver, spleen, kidney, gut, skin and muscle were collected from 15 fish species (Table 2). Lepeophtheirus salmonis (sea lice) were collected and sectioned to ensure the presence of haemolymph and gut. The samples were collected from various sources. The fish species were obtained from Norwegian lakes, rivers and marine coastline (North Sea), Canadian lakes and the Pacific Ocean off the coast of Canada and Chile. The fish represented species from fresh water, saltwater and anadromous species. The samples were generous gifts from colleagues at the Norwegian Veterinary Institute, Norwegian University of Life Sciences, University of Tromsø, Institute of Marine Research, Norway, and Fisheries and Oceans, Canada. Detection of the ISAV receptor with ISAV histochemistry Virus histochemical detection of the ISAV receptor employed ISAV antigen on formalin-fixed paraffinembedded organ sections and was performed as previously described (Aamelfot et al. 2012). Briefly, labelling was performed with ISAV Glesvær/2/90 ISAV HE antigen preparations (100 HAU mL 1), MAb to ISAV HE (Falk, Namork & Dannevig 1998) and a HRP-conjugated anti-mouse Ig amplified detection system (EnVision) with DAB substrate. To test for binding specificity, sections were treated with 0.1 M NaOH for 30 min for saponification and with sialidase from Vibrio cholera (Sigma) for 8, 12 or 24 h at 37 °C (Schauer 1982; Reuter & Schauer 1994; Klausegger et al. 1999).
M Aamelfot et al. Host tropism of ISAV
Journal of Fish Diseases 2014
Table 1 Distribution of the ISAV receptor in heart tissue from fish species representing the three fish vertebrate classes Class
Order
Family
Species
Habitat
Agnatha Chondrichthyes
Myxiniformes Rajiformes
Myxinidae Rajidae
Hagfish, Myxine glutinosa L. Thorny scate, Amblyraja radiata (Donovan) Longnosed skate, Dipturus oxyrinchus L. Thornback ray, Raja clavata L. Spiny dogfish, Squalus acanthias L. Silver Herring, Clupea harengus harengus (L.) Herring, Clupea harengus L. Common carp, Cyprinus carpio L. Common Roach, Rutilus rutilus L. Pike, Esox lucius L. Silvery pout, Gadiculus argenteus L. Atlantic cod, Gadus morhua L. Haddock, Melanogrammus aeglefinus L. Whiting, Merlangius merlangus L. Blue whiting, Micromesistius poutassou L. Atlantic pollack, Pollachius pollachius L. Saithe, Pollachius virens L. Norway pout, Trisopterus esmarkii L. Poor cod, Trisopterus minutus L. Tusk, Brosme brosme L. Blue ling, Molva dypterygia L. Common ling, Molva molva L. Roundnose grenadier, Coryphaenoides rupestris (Gunnerus) Monkfish, Lophius piscatorius L. €ller) Capelin, Mallotus villosus (Mu Greater argentine, Argentina silus (Ascanius) Small sandeel, Ammodytes tobianus L. Atlantic wolf fish, Anarhichas lupus L. Largemouth bass, Micropterus salmoides L. Ballan wrasse, Labrus bergylta L. Goldsinny wrasse, Ctenolabrus rupestris L. Corkwing wrasse, Symphodus melops (L.) European perch, Perca fluviatilis L. (Atlantic ocean) Perch, Perca fluviatilis L. (Pacific Ocean) Witch flounder, Glyptocephalus cynoglossus L. American plaice, Hippoglossoides platessoides L. European hake, Merluccius merluccius L. Lemon sole, Microstomus kitt L. Plaice, Pleuronectes platessa L. Megrim, Lepidorhombus whiffiagonis L. Chinook salmon, Oncorhynchus tshawytscha (Walbaum) Coho salmon, Oncorhynchus kisutch (Walbaum) Rainbow trout, Oncorhynchus mykiss (Walbaum) Sockeye salmon, Oncorhynchus nerka (Walbaum) Atlantic salmon, Salmo salar L. Brown trout/sea trout, Salmo trutta L. Arctic char, Salvelinus alpinus L. Lake Trout, Salvelinus namaycush L. Grayling, Thymallus thymallus L. Lumpfish, Cyclopterus lumpus L. Norway Redfish, Sebastes viviparus L. Redfish, Sebastes mentella L. Grey Gurnard, Eutrigla gurnardus (L.)
S S S S S S S F F F S S S S S S S S S S S S S
Osteichthyes
Squaliformes Clupeiformes
Squalidae Clupeidae
Cypriniformes
Cyprinidae
Esociformes Gadiformes
Esocidae Gadidae
Lotidae
Macrouridae Lophiiformes Osmeriformes Perciformes
Lophiidae Osmeridae Argentinidae Ammodytidae Anarhichadidae Centrarchidae Labridae
Percidae
Pleuronectiformes
Pleuronectidae
Salmoniformes
Scophthalmidae Salmonidae
Scorpaeniformes
Cyclopteridae Sebastidae Triglidae
EC, endothelial cells; RBCs, red blood cells; +: positive; ECs positive, not all.
A plasmid containing the segment 6 coding region (HE) from the Nevis 390/98 (GenBank acc. no. 3
F S S S S S S A A A A A A A F F S S S S
RBCs
+ + + +
+ + +
+
+
+ + + +W
+ + + +W +W + + + + + + +W
+
+ + +W +¤
+ +
+ + + +
+ + + +
+
+
+
+
+ + + + + +# + +
+ + + + + + + +
+ +W +
+ +W +
: negative; S, salt water; F, fresh water; A, anadromous; W, weak; ¤: endocard only; #: some
RBC binding to transfected cells expressing HE
Ó 2014 John Wiley & Sons Ltd
S S S S S F S S S F
EC
AJ276859) isolate was constructed, propagated and transfected into Atlantic salmon kidney (ASK) cells as described previously (McBeath et al. 2011). A solution of 0.05% RBCs was prepared from whole
M Aamelfot et al. Host tropism of ISAV
Journal of Fish Diseases 2014
Table 2 Distribution of the ISAV receptor in heart, liver, spleen, kidney, gills, gut, skin and muscle in selected fish species Fish Agnatha Chondrichthyes Osteichthyes
EC Hagfish, Myxine glutinosa L. Thorny scate, Amblyraja radiata (Donovan) Arctic char, Salvelinus alpinus L. Atlantic cod, Gadus morhua L. Atlantic salmon, Salmo salar L. Ballan wrasse, Labrus bergylta L. Chinook salmon, Oncorhynchus tshawytscha (Walbaum) Coho salmon, Oncorhynchus kisutch (Walbaum) Common Roach, Rutilus rutilus L. Corkwing wrasse, Symphodus melops (L.) Goldsinny wrasse, Ctenolabrus rupestris L. Lumpfish, Cyclopterus lumpus L. European perch, Perca fluviatilis L. (Atlantic ocean) Rainbow trout, Oncorhynchus mykiss (Walbaum) Sockeye salmon, Oncorhynchus nerka (Walbaum)
+ + + + + + + +
+ +
RBC
+ + + + + +
+ +
Other labelling 1 – – – 2, 4 2, 2, – – 2, – – 2, 2,
3, 4 3, 4 3, 4
5, 6
3, 4 3, 4
EC, endothelial cells; RBC, red blood cell; +: positive labelling; : negative labelling; 1: labelling of leucocytes; 2: labelling of lamellar epithelial cells (EP) in gills; 3: basal keratinocytes in skin; 4: labelling of EP in gut; 5: labelling of cells in the gastrointestinal tract in association with the epithelium and of mucus cells; 6: labelling of EP in kidney tubuli.
blood of Atlantic salmon; rainbow trout; Atlantic cod; haddock, Melanogrammus aeglefinus L.; whiting, Merlangius merlangus L.; and saithe. Haemadsorption was performed as previously described (Smail et al. 2000). Briefly, transfected cells were washed once in PBS and incubated in 0.5 mL blood solution at room temperature. After 45 min, cells were washed twice with L15 medium and once with PBS to remove all unbound RBCs. Cells were checked visually using an inverted fluorescent microscope (Nikon) for the presence/absence of RBC binding. Haemoglobin content was measured approximately 10 min after blood removal. RBCs were permeabilized by adding 150 lL 0.05 M ammonium chloride (NH4Cl) (Wang & Iorio 1999) and incubated for up to 45 min at room temperature with gentle agitation until all RBCs had been destroyed. Haemoglobin-containing supernatant was transferred to a 96-well plate for absorbance reading at 540 nm on a Powerwave X plate reader (Bio-Tek Instruments). All plates contained a set of background controls consisting of cells transfected with plasmid with no additional insert. Results are presented as a ratio increase (%) over the controls. All reactions were performed in at least duplicate.
Ó 2014 John Wiley & Sons Ltd
Results
In some species, the receptors were also detected on epithelial cells in the gill, luminal epithelial cells in the hindgut and basal keratinocytes in the epidermis. The results are listed in Tables 1 and 2. Positive labelling was found on endothelial cells in 62% of the fish (33 of 53) examined. The most primitive fish, the jawless hagfish (class Agnatha), was negative on endothelial cells and RBCs, but positive on leucocytes. All the cartilaginous fish (class Chondrichthyes) were positive, mainly on endothelial cells. Of the bony fish (class Osteichthyes), 60% (29 of 48) tested positive. Using available information (www.fishbase.org), no association was found between the presence of the receptor and the different habitats, natural diet or living conditions. However, a taxonomic pattern was revealed. All salmonids (order Salmoniformes), thus all anadromous fish species tested, were positive. The salmonids were also positive on RBCs and some epithelial cells in the gills, hindgut and skin. All the flatfish (order Pleuronectiformes) were negative. Both perch-like fish (order Perciformes) and cod-like fish (order Gadiformes) had a mix of positive and negative species. Interestingly, haddock and saithe were negative on endothelial cells in general, however positive on the heart valves, indicating why saithe clears the ISAV infection rapidly. L. salmonis was negative.
In situ distribution of the ISAV receptor demonstrated by ISAV histochemistry
Haemadsorption of RBCs
The ISAV receptor was mainly found on endothelial cells and on RBCs in the positive species.
Blood from several fish species was also tested to provide further evidence of receptor specificity
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Journal of Fish Diseases 2014
(Fig. 1). Red blood cells from Atlantic salmon, rainbow trout and saithe were readily haemadsorbed by transfected cells expressing HE protein. Absorbance measurements indicated a greater than 25% increase over background controls. Atlantic cod and whiting RBCs haemadsorbed to a slightly reduced degree (>20% and >10% over background, respectively). There was little evidence of haddock RBCs binding to HE-expressing cells. Discussion
ISA is a significant disease in farmed Atlantic salmon, but is largely under control. Nevertheless, isolated outbreaks, for example along the Norwegian coastline, indicate a virus reservoir in the marine environment. Recently, we presented the in situ distribution of the ISAV receptor in Atlantic salmon and its association with ISA pathogenesis (Aamelfot et al. 2012). Here, we documented the distribution of the ISAV receptor in selected cold-water fish species. These receptor-positive species may potentially act as reservoirs or carriers of ISAV to the farmed Atlantic salmon. About two-thirds of the fish species expressed the ISAV receptor. The typical distribution in positive species was confined to endothelial cells and RBCs. Hagfish (class Agnatha) (evolutionary the most primitive fish examined) was negative on endothelial cells, but positive on leucocytes. A high prevalence of species positive on endothelial cells and RBCs was, however, found in other fish groups considered ‘evolutionarily primitive’ like
Figure 1 Absorbance ratio increase (%) over background controls demonstrating haemoglobin quantities obtained from bound red blood cells (RBCs) of several fish species to transfected ASK cells expressing ISAV HE protein 10 min after blood removal. n = 4 SD, except for saithe, whiting and haddock where n = 2. Ó 2014 John Wiley & Sons Ltd
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M Aamelfot et al. Host tropism of ISAV
the cartilaginous fish (class Chondrichthyes) (80%) and the salmonids (100%) (bony fish, class Osteichthyes). Apart from this taxonomic relationship, we have so far not been able to identify any common denominator characterizing the ISAV receptor-positive fish species, and other groups were more variable. As the receptor has also been detected in some mammals and birds (Aamelfot et al. 2014b), this was not surprising. Interestingly, the pathogenic Norwegian ISAV Glesvær/2/90 isolate used to detect the receptor haemagglutinate RBCs from various species including Atlantic salmon, rainbow trout and Atlantic cod to varying degrees but not those of brown trout (Falk et al. 1998). This was also demonstrated with the Nevis 390/98 HE protein in the transfection system described here, along with the positive haemadsorption of blood from saithe and whiting. This indicates that the two viruses have the same receptor affinity. In addition, an ISAV HPR0-type HE used in the same transfection/haemadsorption system showed no difference in binding capacity to the Nevis HPR7 HE (data not shown). The little evidence of haddock RBCs binding to the HE-expressing cells in the haemadsorption assay could be explained by the weak positive RBC labelling in situ in this species. Receptor binding preference is thought to be an important species barrier for different strains of influenza virus (Schneider-Schaulies 2000). Thus, differences in receptor distribution and receptor specificity among fish species could account for variations in susceptibility to ISAV infection. However, on its own, the presence of the appropriate virus receptor is not evidence of host potential. Further unknown restrictive mechanisms might be important. For example, here, saithe were positive for the virus receptor in RBCs which was confirmed by the positive haemadsorption; however, experimental studies have indicated this species is not susceptible to ISAV (Snow et al. 2002). Interestingly, there was good correlation between receptor-positive species and species where ISAV detection has been reported, including coho salmon, brown trout/sea trout, rainbow trout, arctic char and Atlantic cod (Nylund & Jakobsen 1995; Nylund et al. 1995, 1997; Snow et al. 2001; Smith et al. 2006; Biacchesi et al. 2007; Grove et al. 2007; MacWilliams et al. 2007). The low level of ISAV disease in these species could be related various factors including virus uptake. In fact, a limited
Journal of Fish Diseases 2014
ability to pass the external barriers of rainbow trout has been shown for strains of VHSV (Brudeseth, Skall & Evensen 2008). The wide distribution of the ISAV receptor across multiple fish species suggests it plays an important role at the cellular level. However, it is also vulnerable to pathogen exploitation as a receptor. Here, we show that the ISAV receptor, 4-O-acetylated sialic acids, are conserved in many including evolutionary primitive fish species, thus must have been successful evolutionarily for a long period of time. Being so conserved, the cost is probably huge for the fish to remove or change it, making receptor-positive species vulnerable to pathogens that can use the structure as a receptor. In fish farms, the density of susceptible hosts is unnaturally high. Once a virus is introduced to a farm, the chance of an infected host encountering a susceptible host increases compared with in the wild (Krkosek 2010). The risk increases further because of limited genetic diversity of the fish (Ebert 1998), which reduces the chance of a host having an advantageous genetic composition and therefore could survive the infection. In addition, the fish may be immune compromised due to stressful environmental conditions (Sniezko 1974) and with no natural predators to remove weak or sick fish. This provides the virus with excellent conditions to spread and evolve within the farm and may trigger the emergence of infectious diseases by increasing virulence through adaptation. Even though direct evidence for ISAV spread through wild fish in Norway is currently lacking, the aggregations of wild fish around and movement between fish farms indicate that this is as a potential risk. Interestingly, several viruses have transmitted from wild to farmed fish, including infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). Increased virulence of VHSV (Einer-Jensen et al. 2004) and ISAV (Nylund et al. 2003) has been observed in farmed fish compared with wild. However, whether these transmissions were due to pathogen evolution or the farming conditions is unclear. VHSV has its origin in the marine environment, and studies indicate it was present in marine species before fish farming was established (Raynard et al. 2007). It has been isolated from many different freshwater and marine species (Skall, Olesen & Mellergaard 2005). Marine strains of VHSV are non-virulent to rainbow trout; however, in 2007, VHS was detected in a Ó 2014 John Wiley & Sons Ltd
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M Aamelfot et al. Host tropism of ISAV
rainbow trout farm in Storfjord in mid-Norway (Dale et al. 2009). The adaptation was associated with breaches in biosecurity, including continuous production without fallowing, frequent movement of fish and a co-localization of rainbow trout with marine species. IHNV also has a broad host range and herring is thought to be the cause of virus transmission to farmed Atlantic salmon in Canada (Saksida 2006). The disease is seen primarily in salmonids and is therefore considered a major threat to the Atlantic salmon farming industry in Norway, Scotland, and other Atlantic salmon producing countries. Previously, we investigated fish species using an Atlantic salmon endothelial and RBC antibody marker (10E4) (Aamelfot et al. 2013), detecting a structure associated with the ISAV receptor. Surprisingly, only two species, in addition to Atlantic salmon, were positive, that is, grayling, Thymallus thymallus L., and spiny dogfish, Squalus acanthias L. Thus, while ISAV histochemistry detects all the receptor-positive species, 10E4 is only applicable on three species. The expression of the specific MAb epitope could explain species that are susceptible to ISA disease better than the ISAV histochemistry, and it would be interesting to know whether grayling or spiny dogfish could get ISA. Variation in receptor distribution and specificity among fish species could explain differences in susceptibility to ISAV infection. Here, we demonstrated the presence of the ISAV receptor in several common fish species in Norwegian coastal waters and in fresh water lakes, narrowing down the number of potential hosts that could be important in spread and maintenance of ISAV in the environment. This approach could be used in a more complete survey of fish species and form the basis of further investigations to determine whether wild hosts of ISAV exist. Acknowledgements The authors thank T. Poppe at the Norwegian Veterinary Institute (NVI), A.K. Jøranlid presently at the Norwegian Environment Agency, A. Frøyse and M. Bakke at the Norwegian University of Life Sciences, A. Lynghammar at the University of Tromsø and W.R. Bennett and K. Garver at the Fisheries and Oceans Canada for providing samples from various species. The authors also thank B.S. Nordvik at NVI and M. Fourrier at Marine Scotland Science for technical assistance. The work was
Journal of Fish Diseases 2014
funded by the Atlantic Innovation Fund, Canada Inc. and Novartis Animal Health. Conflict of interest
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Nylund A., Kvenseth A.M., Krossøy B. & Hodneland K. (1997) Replication of the infectious salmon anaemia virus (ISAV) in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 20, 275–279. Nylund A., Devold M., Mullins J. & Plarre H. (2002) Herring (Clupea harengus): a host for infectious salmon anemia virus (ISAV). Bulletin of the European Association of Fish Pathologists 22, 311–318. Nylund A., Devold M., Plarre H., Isdal E. & Aarseth M. (2003) Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis. Diseases of Aquatic Organisms 56, 11–24. Plarre H., Devold M., Snow M. & Nylund A. (2005) Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway. Diseases of Aquatic Organisms 66, 71–79. Raynard R.S., Murray A.G. & Gregory A. (2001) Infectious salmon anaemia virus in wild fish from Scotland. Diseases of Aquatic Organisms 46, 93–100. Raynard R., Wahli T., Vatsos I. & Mortensen S. (2007) Review of disease interactions and pathogen exchange between farmed and wild finfish and shellfish in Europe. DIPNET report, http://www.revistaaquatic.com/dipnet/docs/ doc.asp?id=48 p. 459, Aberdeen. Reuter G. & Schauer R. (1994) Determination of sialic acids. Methods in Enzymology 230, 168–199. Rolland J.B. & Winton J.R. (2003) Relative resistance of Pacific salmon to infectious salmon anaemia virus. Journal of Fish Diseases 26, 511–520. Saksida S.M. (2006) Infectious haematopoietic necrosis epidemic (2001 to 2003) in farmed Atlantic salmon Salmo salar in British Columbia. Diseases of Aquatic Organisms 72, 213–223. Schauer R. (1982) Chemistry, metabolism, and biological functions of sialic acids. In: Advances in Carbohydrate Chemistry Biochemistry (ed. by R.S. Tipson & D. Horton), pp. 131–234. Academic Press, Inc., New York. Scheel I., Aldrin M., Frigessi A. & Jansen P.A. (2007) A stochastic model for infectious salmon anemia (ISA) in Atlantic salmon farming. Journal of the Royal Society Interface 4, 699–706.
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Smail D.A., Grant R., Ross K., Bricknell I.R. & Hastings T.S. (2000) The use of haemadsorption for the isolation of Infectious Salmon Anaemia virus on SHK-1 cells from Atlantic salmon (Salmo salar L.) in Scotland. Bulletin of the European Association of Fish Pathologists 20, 212–214. Smith P.A., Larenas J., Contreras J., Cassigoli J., Venegas C., Rojas M.E., Guajardo A., Perez S. & Diaz S. (2006) Infectious haemolytic anaemia causes jaundice outbreaks in seawater-cultured coho salmon, Oncorhynchus kisutch (Walbaum), in Chile. Journal of Fish Diseases 29, 709–715. Sniezko S. (1974) The effect of environmental stress on outbreaks of infectious diseases of fishes. Journal of Fish Biology 6, 197–208. Snow M., Raynard R.S. & Bruno D.W. (2001) Comparative susceptibility of Arctic char (Salvelinus alpinus), rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) to the Scottish isolate of infectious salmon anaemia virus. Aquaculture 196, 47–54. Snow M., Raynard R.S., Bruno D.W., Van Nieuwstadt A.P., Olesen N.J., Løvold T. & Wallace C. (2002) Investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission. Diseases of Aquatic Organisms 50, 13–18. Wallace I.S., Gregory A., Murray A.G., Munro E.S. & Raynard R.S. (2008) Distribution of infectious pancreatic necrosis virus (IPNV) in wild marine fish from Scottish waters with respect to clinically infected aquaculture sites producing Atlantic salmon, Salmo salar L. Journal of Fish Diseases 31, 177–186. Wang Z. & Iorio R.M. (1999) Amino acid substitutions in a conserved region in the stalk of the Newcastle disease virus HN glycoprotein spike impair its neuraminidase activity in the globular domain. Journal of General Virology 80, 749–753. Received: 16 May 2014 Accepted: 6 May 2014
Journal of Fish Diseases 2014
Host tropism of infectious salmon anaemia virus in marine and freshwater fish species M Aamelfot1, O B Dale1, A McBeath2 and K Falk1 1 Norwegian Veterinary Institute, Oslo, Norway 2 Marine Scotland Science, Aberdeen, UK
Abstract
The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net-pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod-like and perch-like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists. Keywords: 4-O-Acetylated sialic acid, Atlantic salmon, virus histochemistry, virus reservior, orthomyxovirus. Introduction
Infectious salmon anaemia (ISA) is a serious, OIE-listed disease of farmed Atlantic salmon, Correspondence K Falk, Norwegian Veterinary Institute, lsveien 68, P.O. Box 750 Sentrum, 0106 Oslo, Norway Ulleva (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Salmo salar L., caused by an enveloped aquatic orthomyxovirus, ISAV. ISA is characterized by anaemia and circulatory disturbances such as oedemas, ascites and bleedings (Aamelfot, Dale & Falk 2014a). Since the major ISA epidemic in the 1990s, Norway has experienced 2–20 annual disease outbreaks. These have a tendency to cluster in time and space (Jarp & Karlsen 1997; Scheel et al. 2007). Phylogenetic studies confirmed that virus isolates from these clusters are related (Lyngstad et al. 2008), suggesting horizontal transmission. However, in many outbreaks, no obvious sources of infection have been identified. The questions remain on how ISAV is maintained in the ocean and where it came from originally, and whether a wild host exists, as has been hypothesized (Nylund et al. 2003). Atlantic salmon are farmed at sea in open netpens, with no barriers to pathogen exchange with the environment. Thus, wild fish could maintain ISAV in the environment and act as a reservoir of the virus. Open net-pen production systems are known to attract a high diversity of wild species due to the free availability of feed (Dempster et al. 2009). These may move between farms acting as carriers, thus contributing to the spread of pathogens. Saithe, Pollachius virens L., the most abundant fish species associated with Norwegian salmon farms (Dempster et al. 2009), has been suggested to act as a natural reservoir of salmonid alpha virus (SAV) (Graham et al. 2006) and is a carrier of infectious pancreatic necrosis virus (IPNV) (Wallace et al. 2008). However, it appears resistant to ISAV and clears the virus rapidly after infection (Snow et al. 2002; McClure et al. 2004). Finally, sea lice (Lepeophtheirus salmonis and Caligus sp.) may act as mechanical vectors, penetrating the skin
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and could deposit virus into the blood (Nylund et al. 1994). Experimental trials have detected ISAV replication, without disease, in many fish species including brown trout (Nylund, Alexandersen & Rolland 1995; Nylund & Jakobsen 1995); rainbow trout (Nylund et al. 1997; MacWilliams et al. 2007); arctic char, Salvelinus alpinus L. (Snow, Raynard & Bruno 2001); herring, Clupea harengus L. (Nylund et al. 2002); and Atlantic cod, Gadus morhua L. (Grove et al. 2007). Thus, many species may harbour and spread ISAV, and a latent carrier status was suggested for salmonids (Nylund & Jakobsen 1995). ISAV has been detected, in the absence of disease, in wild salmonids, including brown trout, Salmo trutta L. and rainbow trout, Oncorhynchus mykiis (Walbaum), in Norway (Plarre et al. 2005) and Scotland (Raynard, Murray & Gregory 2001). Mortality and ISA-like signs were reported in one rainbow trout study (Biacchesi et al. 2007), suggesting ISAV may represent a threat to rainbow trout. Experimentally infected Pacific salmon showed no signs of disease (Rolland & Winton 2003). In Chile, however, ISAV was detected in jaundiced Pacific coho salmon, Oncorhynchus kisutch (Walbaum) (Kibenge et al. 2001). In addition, mortality was reported in Amago trout, Oncorhynchus masou (G€ unther) (Ito, Oseko & Ototake 2014); however, no virus transmission to cohabiting Atlantic salmon was detected. Thus, while several fish species are susceptible to ISAV, and subclinical infections could be common, the major disease problems appear restricted to farmed Atlantic salmon. Knowledge on ISAV susceptible species is important to understand ISAV epidemiology. However, investigation of susceptibility via screening for natural infection, or by experimental challenges, is difficult and costly. Thus, methods to select potential carrier species are needed. Host susceptibility and the ability to cause disease are determined by complex interactions between the virus virulence, the host and the environment. Every step in the infection cycle relies on the host and virus being compatible. Viral entry, for example, is dependent on the appropriate cell receptor (Helenius 2007). ISAV uses 4-O-acetylated sialic acids as its cellular receptor (Hellebø et al. 2004). Recently, we demonstrated the cell tropism of ISAV in Atlantic salmon by detecting the virus receptor in situ (Aamelfot et al. 2012). The receptor was found on endothelial cells, red blood cells (RBCs) and some epithelial cells. Ó 2014 John Wiley & Sons Ltd
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Here, we extended the investigations from Atlantic salmon to investigate various cold-water fish species that naturally encounter farmed Atlantic salmon for the ISAV receptor. In addition, we tested binding of RBCs from a small number of fish species to cultured cells expressing the haemagglutinin-esterase (HE) virus protein on the cell surface. Although the host tropism is dependent on multiple virus and host factors in addition to receptor specificity, screening fish for the receptor could narrow down the number of potential hosts that may have a role in the epidemiology of ISA.
Materials and methods
Tissue sampling Heart samples were collected from 53 cold-water fish species (Table 1). Tissues from heart, gill, liver, spleen, kidney, gut, skin and muscle were collected from 15 fish species (Table 2). Lepeophtheirus salmonis (sea lice) were collected and sectioned to ensure the presence of haemolymph and gut. The samples were collected from various sources. The fish species were obtained from Norwegian lakes, rivers and marine coastline (North Sea), Canadian lakes and the Pacific Ocean off the coast of Canada and Chile. The fish represented species from fresh water, saltwater and anadromous species. The samples were generous gifts from colleagues at the Norwegian Veterinary Institute, Norwegian University of Life Sciences, University of Tromsø, Institute of Marine Research, Norway, and Fisheries and Oceans, Canada. Detection of the ISAV receptor with ISAV histochemistry Virus histochemical detection of the ISAV receptor employed ISAV antigen on formalin-fixed paraffinembedded organ sections and was performed as previously described (Aamelfot et al. 2012). Briefly, labelling was performed with ISAV Glesvær/2/90 ISAV HE antigen preparations (100 HAU mL 1), MAb to ISAV HE (Falk, Namork & Dannevig 1998) and a HRP-conjugated anti-mouse Ig amplified detection system (EnVision) with DAB substrate. To test for binding specificity, sections were treated with 0.1 M NaOH for 30 min for saponification and with sialidase from Vibrio cholera (Sigma) for 8, 12 or 24 h at 37 °C (Schauer 1982; Reuter & Schauer 1994; Klausegger et al. 1999).
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Journal of Fish Diseases 2014
Table 1 Distribution of the ISAV receptor in heart tissue from fish species representing the three fish vertebrate classes Class
Order
Family
Species
Habitat
Agnatha Chondrichthyes
Myxiniformes Rajiformes
Myxinidae Rajidae
Hagfish, Myxine glutinosa L. Thorny scate, Amblyraja radiata (Donovan) Longnosed skate, Dipturus oxyrinchus L. Thornback ray, Raja clavata L. Spiny dogfish, Squalus acanthias L. Silver Herring, Clupea harengus harengus (L.) Herring, Clupea harengus L. Common carp, Cyprinus carpio L. Common Roach, Rutilus rutilus L. Pike, Esox lucius L. Silvery pout, Gadiculus argenteus L. Atlantic cod, Gadus morhua L. Haddock, Melanogrammus aeglefinus L. Whiting, Merlangius merlangus L. Blue whiting, Micromesistius poutassou L. Atlantic pollack, Pollachius pollachius L. Saithe, Pollachius virens L. Norway pout, Trisopterus esmarkii L. Poor cod, Trisopterus minutus L. Tusk, Brosme brosme L. Blue ling, Molva dypterygia L. Common ling, Molva molva L. Roundnose grenadier, Coryphaenoides rupestris (Gunnerus) Monkfish, Lophius piscatorius L. €ller) Capelin, Mallotus villosus (Mu Greater argentine, Argentina silus (Ascanius) Small sandeel, Ammodytes tobianus L. Atlantic wolf fish, Anarhichas lupus L. Largemouth bass, Micropterus salmoides L. Ballan wrasse, Labrus bergylta L. Goldsinny wrasse, Ctenolabrus rupestris L. Corkwing wrasse, Symphodus melops (L.) European perch, Perca fluviatilis L. (Atlantic ocean) Perch, Perca fluviatilis L. (Pacific Ocean) Witch flounder, Glyptocephalus cynoglossus L. American plaice, Hippoglossoides platessoides L. European hake, Merluccius merluccius L. Lemon sole, Microstomus kitt L. Plaice, Pleuronectes platessa L. Megrim, Lepidorhombus whiffiagonis L. Chinook salmon, Oncorhynchus tshawytscha (Walbaum) Coho salmon, Oncorhynchus kisutch (Walbaum) Rainbow trout, Oncorhynchus mykiss (Walbaum) Sockeye salmon, Oncorhynchus nerka (Walbaum) Atlantic salmon, Salmo salar L. Brown trout/sea trout, Salmo trutta L. Arctic char, Salvelinus alpinus L. Lake Trout, Salvelinus namaycush L. Grayling, Thymallus thymallus L. Lumpfish, Cyclopterus lumpus L. Norway Redfish, Sebastes viviparus L. Redfish, Sebastes mentella L. Grey Gurnard, Eutrigla gurnardus (L.)
S S S S S S S F F F S S S S S S S S S S S S S
Osteichthyes
Squaliformes Clupeiformes
Squalidae Clupeidae
Cypriniformes
Cyprinidae
Esociformes Gadiformes
Esocidae Gadidae
Lotidae
Macrouridae Lophiiformes Osmeriformes Perciformes
Lophiidae Osmeridae Argentinidae Ammodytidae Anarhichadidae Centrarchidae Labridae
Percidae
Pleuronectiformes
Pleuronectidae
Salmoniformes
Scophthalmidae Salmonidae
Scorpaeniformes
Cyclopteridae Sebastidae Triglidae
EC, endothelial cells; RBCs, red blood cells; +: positive; ECs positive, not all.
A plasmid containing the segment 6 coding region (HE) from the Nevis 390/98 (GenBank acc. no. 3
F S S S S S S A A A A A A A F F S S S S
RBCs
+ + + +
+ + +
+
+
+ + + +W
+ + + +W +W + + + + + + +W
+
+ + +W +¤
+ +
+ + + +
+ + + +
+
+
+
+
+ + + + + +# + +
+ + + + + + + +
+ +W +
+ +W +
: negative; S, salt water; F, fresh water; A, anadromous; W, weak; ¤: endocard only; #: some
RBC binding to transfected cells expressing HE
Ó 2014 John Wiley & Sons Ltd
S S S S S F S S S F
EC
AJ276859) isolate was constructed, propagated and transfected into Atlantic salmon kidney (ASK) cells as described previously (McBeath et al. 2011). A solution of 0.05% RBCs was prepared from whole
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Journal of Fish Diseases 2014
Table 2 Distribution of the ISAV receptor in heart, liver, spleen, kidney, gills, gut, skin and muscle in selected fish species Fish Agnatha Chondrichthyes Osteichthyes
EC Hagfish, Myxine glutinosa L. Thorny scate, Amblyraja radiata (Donovan) Arctic char, Salvelinus alpinus L. Atlantic cod, Gadus morhua L. Atlantic salmon, Salmo salar L. Ballan wrasse, Labrus bergylta L. Chinook salmon, Oncorhynchus tshawytscha (Walbaum) Coho salmon, Oncorhynchus kisutch (Walbaum) Common Roach, Rutilus rutilus L. Corkwing wrasse, Symphodus melops (L.) Goldsinny wrasse, Ctenolabrus rupestris L. Lumpfish, Cyclopterus lumpus L. European perch, Perca fluviatilis L. (Atlantic ocean) Rainbow trout, Oncorhynchus mykiss (Walbaum) Sockeye salmon, Oncorhynchus nerka (Walbaum)
+ + + + + + + +
+ +
RBC
+ + + + + +
+ +
Other labelling 1 – – – 2, 4 2, 2, – – 2, – – 2, 2,
3, 4 3, 4 3, 4
5, 6
3, 4 3, 4
EC, endothelial cells; RBC, red blood cell; +: positive labelling; : negative labelling; 1: labelling of leucocytes; 2: labelling of lamellar epithelial cells (EP) in gills; 3: basal keratinocytes in skin; 4: labelling of EP in gut; 5: labelling of cells in the gastrointestinal tract in association with the epithelium and of mucus cells; 6: labelling of EP in kidney tubuli.
blood of Atlantic salmon; rainbow trout; Atlantic cod; haddock, Melanogrammus aeglefinus L.; whiting, Merlangius merlangus L.; and saithe. Haemadsorption was performed as previously described (Smail et al. 2000). Briefly, transfected cells were washed once in PBS and incubated in 0.5 mL blood solution at room temperature. After 45 min, cells were washed twice with L15 medium and once with PBS to remove all unbound RBCs. Cells were checked visually using an inverted fluorescent microscope (Nikon) for the presence/absence of RBC binding. Haemoglobin content was measured approximately 10 min after blood removal. RBCs were permeabilized by adding 150 lL 0.05 M ammonium chloride (NH4Cl) (Wang & Iorio 1999) and incubated for up to 45 min at room temperature with gentle agitation until all RBCs had been destroyed. Haemoglobin-containing supernatant was transferred to a 96-well plate for absorbance reading at 540 nm on a Powerwave X plate reader (Bio-Tek Instruments). All plates contained a set of background controls consisting of cells transfected with plasmid with no additional insert. Results are presented as a ratio increase (%) over the controls. All reactions were performed in at least duplicate.
Ó 2014 John Wiley & Sons Ltd
Results
In some species, the receptors were also detected on epithelial cells in the gill, luminal epithelial cells in the hindgut and basal keratinocytes in the epidermis. The results are listed in Tables 1 and 2. Positive labelling was found on endothelial cells in 62% of the fish (33 of 53) examined. The most primitive fish, the jawless hagfish (class Agnatha), was negative on endothelial cells and RBCs, but positive on leucocytes. All the cartilaginous fish (class Chondrichthyes) were positive, mainly on endothelial cells. Of the bony fish (class Osteichthyes), 60% (29 of 48) tested positive. Using available information (www.fishbase.org), no association was found between the presence of the receptor and the different habitats, natural diet or living conditions. However, a taxonomic pattern was revealed. All salmonids (order Salmoniformes), thus all anadromous fish species tested, were positive. The salmonids were also positive on RBCs and some epithelial cells in the gills, hindgut and skin. All the flatfish (order Pleuronectiformes) were negative. Both perch-like fish (order Perciformes) and cod-like fish (order Gadiformes) had a mix of positive and negative species. Interestingly, haddock and saithe were negative on endothelial cells in general, however positive on the heart valves, indicating why saithe clears the ISAV infection rapidly. L. salmonis was negative.
In situ distribution of the ISAV receptor demonstrated by ISAV histochemistry
Haemadsorption of RBCs
The ISAV receptor was mainly found on endothelial cells and on RBCs in the positive species.
Blood from several fish species was also tested to provide further evidence of receptor specificity
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Journal of Fish Diseases 2014
(Fig. 1). Red blood cells from Atlantic salmon, rainbow trout and saithe were readily haemadsorbed by transfected cells expressing HE protein. Absorbance measurements indicated a greater than 25% increase over background controls. Atlantic cod and whiting RBCs haemadsorbed to a slightly reduced degree (>20% and >10% over background, respectively). There was little evidence of haddock RBCs binding to HE-expressing cells. Discussion
ISA is a significant disease in farmed Atlantic salmon, but is largely under control. Nevertheless, isolated outbreaks, for example along the Norwegian coastline, indicate a virus reservoir in the marine environment. Recently, we presented the in situ distribution of the ISAV receptor in Atlantic salmon and its association with ISA pathogenesis (Aamelfot et al. 2012). Here, we documented the distribution of the ISAV receptor in selected cold-water fish species. These receptor-positive species may potentially act as reservoirs or carriers of ISAV to the farmed Atlantic salmon. About two-thirds of the fish species expressed the ISAV receptor. The typical distribution in positive species was confined to endothelial cells and RBCs. Hagfish (class Agnatha) (evolutionary the most primitive fish examined) was negative on endothelial cells, but positive on leucocytes. A high prevalence of species positive on endothelial cells and RBCs was, however, found in other fish groups considered ‘evolutionarily primitive’ like
Figure 1 Absorbance ratio increase (%) over background controls demonstrating haemoglobin quantities obtained from bound red blood cells (RBCs) of several fish species to transfected ASK cells expressing ISAV HE protein 10 min after blood removal. n = 4 SD, except for saithe, whiting and haddock where n = 2. Ó 2014 John Wiley & Sons Ltd
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M Aamelfot et al. Host tropism of ISAV
the cartilaginous fish (class Chondrichthyes) (80%) and the salmonids (100%) (bony fish, class Osteichthyes). Apart from this taxonomic relationship, we have so far not been able to identify any common denominator characterizing the ISAV receptor-positive fish species, and other groups were more variable. As the receptor has also been detected in some mammals and birds (Aamelfot et al. 2014b), this was not surprising. Interestingly, the pathogenic Norwegian ISAV Glesvær/2/90 isolate used to detect the receptor haemagglutinate RBCs from various species including Atlantic salmon, rainbow trout and Atlantic cod to varying degrees but not those of brown trout (Falk et al. 1998). This was also demonstrated with the Nevis 390/98 HE protein in the transfection system described here, along with the positive haemadsorption of blood from saithe and whiting. This indicates that the two viruses have the same receptor affinity. In addition, an ISAV HPR0-type HE used in the same transfection/haemadsorption system showed no difference in binding capacity to the Nevis HPR7 HE (data not shown). The little evidence of haddock RBCs binding to the HE-expressing cells in the haemadsorption assay could be explained by the weak positive RBC labelling in situ in this species. Receptor binding preference is thought to be an important species barrier for different strains of influenza virus (Schneider-Schaulies 2000). Thus, differences in receptor distribution and receptor specificity among fish species could account for variations in susceptibility to ISAV infection. However, on its own, the presence of the appropriate virus receptor is not evidence of host potential. Further unknown restrictive mechanisms might be important. For example, here, saithe were positive for the virus receptor in RBCs which was confirmed by the positive haemadsorption; however, experimental studies have indicated this species is not susceptible to ISAV (Snow et al. 2002). Interestingly, there was good correlation between receptor-positive species and species where ISAV detection has been reported, including coho salmon, brown trout/sea trout, rainbow trout, arctic char and Atlantic cod (Nylund & Jakobsen 1995; Nylund et al. 1995, 1997; Snow et al. 2001; Smith et al. 2006; Biacchesi et al. 2007; Grove et al. 2007; MacWilliams et al. 2007). The low level of ISAV disease in these species could be related various factors including virus uptake. In fact, a limited
Journal of Fish Diseases 2014
ability to pass the external barriers of rainbow trout has been shown for strains of VHSV (Brudeseth, Skall & Evensen 2008). The wide distribution of the ISAV receptor across multiple fish species suggests it plays an important role at the cellular level. However, it is also vulnerable to pathogen exploitation as a receptor. Here, we show that the ISAV receptor, 4-O-acetylated sialic acids, are conserved in many including evolutionary primitive fish species, thus must have been successful evolutionarily for a long period of time. Being so conserved, the cost is probably huge for the fish to remove or change it, making receptor-positive species vulnerable to pathogens that can use the structure as a receptor. In fish farms, the density of susceptible hosts is unnaturally high. Once a virus is introduced to a farm, the chance of an infected host encountering a susceptible host increases compared with in the wild (Krkosek 2010). The risk increases further because of limited genetic diversity of the fish (Ebert 1998), which reduces the chance of a host having an advantageous genetic composition and therefore could survive the infection. In addition, the fish may be immune compromised due to stressful environmental conditions (Sniezko 1974) and with no natural predators to remove weak or sick fish. This provides the virus with excellent conditions to spread and evolve within the farm and may trigger the emergence of infectious diseases by increasing virulence through adaptation. Even though direct evidence for ISAV spread through wild fish in Norway is currently lacking, the aggregations of wild fish around and movement between fish farms indicate that this is as a potential risk. Interestingly, several viruses have transmitted from wild to farmed fish, including infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). Increased virulence of VHSV (Einer-Jensen et al. 2004) and ISAV (Nylund et al. 2003) has been observed in farmed fish compared with wild. However, whether these transmissions were due to pathogen evolution or the farming conditions is unclear. VHSV has its origin in the marine environment, and studies indicate it was present in marine species before fish farming was established (Raynard et al. 2007). It has been isolated from many different freshwater and marine species (Skall, Olesen & Mellergaard 2005). Marine strains of VHSV are non-virulent to rainbow trout; however, in 2007, VHS was detected in a Ó 2014 John Wiley & Sons Ltd
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M Aamelfot et al. Host tropism of ISAV
rainbow trout farm in Storfjord in mid-Norway (Dale et al. 2009). The adaptation was associated with breaches in biosecurity, including continuous production without fallowing, frequent movement of fish and a co-localization of rainbow trout with marine species. IHNV also has a broad host range and herring is thought to be the cause of virus transmission to farmed Atlantic salmon in Canada (Saksida 2006). The disease is seen primarily in salmonids and is therefore considered a major threat to the Atlantic salmon farming industry in Norway, Scotland, and other Atlantic salmon producing countries. Previously, we investigated fish species using an Atlantic salmon endothelial and RBC antibody marker (10E4) (Aamelfot et al. 2013), detecting a structure associated with the ISAV receptor. Surprisingly, only two species, in addition to Atlantic salmon, were positive, that is, grayling, Thymallus thymallus L., and spiny dogfish, Squalus acanthias L. Thus, while ISAV histochemistry detects all the receptor-positive species, 10E4 is only applicable on three species. The expression of the specific MAb epitope could explain species that are susceptible to ISA disease better than the ISAV histochemistry, and it would be interesting to know whether grayling or spiny dogfish could get ISA. Variation in receptor distribution and specificity among fish species could explain differences in susceptibility to ISAV infection. Here, we demonstrated the presence of the ISAV receptor in several common fish species in Norwegian coastal waters and in fresh water lakes, narrowing down the number of potential hosts that could be important in spread and maintenance of ISAV in the environment. This approach could be used in a more complete survey of fish species and form the basis of further investigations to determine whether wild hosts of ISAV exist. Acknowledgements The authors thank T. Poppe at the Norwegian Veterinary Institute (NVI), A.K. Jøranlid presently at the Norwegian Environment Agency, A. Frøyse and M. Bakke at the Norwegian University of Life Sciences, A. Lynghammar at the University of Tromsø and W.R. Bennett and K. Garver at the Fisheries and Oceans Canada for providing samples from various species. The authors also thank B.S. Nordvik at NVI and M. Fourrier at Marine Scotland Science for technical assistance. The work was
Journal of Fish Diseases 2014
funded by the Atlantic Innovation Fund, Canada Inc. and Novartis Animal Health. Conflict of interest
No conflict of interest declared. References Aamelfot M., Dale O.B., Weli S.C., Koppang E.O. & Falk K. (2012) Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus. Journal of Virology 86, 10571–10578. Aamelfot M., Weli S.C., Dale O.B., Koppang E.O. & Falk K. (2013) Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor. Journal of Anatomy 222, 547–557. Aamelfot M., Dale O.B. & Falk K. (2014a) Infectious salmon anaemia – pathogenesis and tropism. Journal of Fish Diseases 37, 291–307.
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