An investigation into the susceptibility of Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) to infectious salmon anaemia virus (ISAV)

The susceptibility of Atlantic cod (Gadus morhua) and Atlantic Halibut (Hippoglossus hippoglossus) to an Atlantic salmon (Salmo salar) pathogenic isolate of infectious salmon anaemia was investigated. No mortality or clinical signs of infectious salmon anaemia disease were recorded in either species following experimental challenge via intra-peritoneal (i.p.) injection or cohabitation with i.p. infected fish of the same species. Furthermore, the presence of virus in internal organs (kidney and spleen) could not be demonstrated in fish surviving at the end of the 42day challenge period. The implication of these findings with respect to the future commercial production of Atlantic cod and halibut is discussed. *Corresponding author’s email: [email protected] Introduction Infectious salmon anaemia (ISA) is an orthomyxoviral disease of cultured Atlantic salmon (Salmo salar) which has caused epizootics and extensive economic loss in Norway, Canada, Scotland, the USA, the Faroe Islands and Chile (reviewed by Kibenge et al., 2004). The serious impact of ISA is reflected by its status as the only List I disease within the European Union (EU Directive 93/53/ EEC). This legislation implements measures aimed at preventing the introduction and subsequent transmission of what is considered to be a disease, which is exotic to the EU. Eradication required by this legislation was implemented following the appearance of ISA disease in Scotland, resulting in the successful elimination of the disease in cultured Atlantic salmon (Stagg, 2003). Increasing evidence exists, however, to suggest the presence of a naturally occurring reservoir of infectious salmon anaemia virus (ISAV) in wild fish (Raynard et al., 2001a). Furthermore, molecular and epidemiological evidence suggests that ISA associated with aquaculture has repeatedly and independently emerged from such a reservoir. Indeed, based on alignments of a polymorphic region of the haemagglutinin-esterase gene, Mjaaland et al. (2002) suggested that all aquaculture-associated virus subtypes appeared to have derived via differential deletion events from a putative longer ancestral sequence. The subsequent detection of a virus with such an ancestral sequence from asymptomatic wild fish in Scotland lent further support to such an evolutionary model (Cunningham et al., 2002). This evidence, combined with the apparent independent Bull. Eur. Ass. Fish Pathol., 25(5) 2005, 190 emergence of ISAV in Europe and North America (Blake et al., 1999) and the fact that outbreaks have occurred in Norway with no apparent link to other infected sites, further supports the notion that ISA disease originated in wild fish. In addition to risks presented by a reservoir of ISAV in wild fish from which pathogenic ISAV variants appear to have emerged, virus can be also be directly transmitted from Atlantic salmon cages by both horizontal transfer via seawater and anthropogenic activity associated with aquaculture activity (Jarp & Karlsen, 1997). This was most clearly demonstrated during the emergence of ISA in Scotland (Murray et al., 2003). Such factors thus present a potential risk to other fish species. Previous studies aimed at identifying risks of transmission of ISAV to other host species have largely focussed on the role of wild fish species and their potential relevance in spreading disease within Atlantic salmon aquaculture. This has resulted in identification of a number of salmonid species which have been shown to be potential carriers of the virus (reviewed by Kibenge et al., 2004). Such studies have also included non-salmonids such as saithe (Pollachius virens) (Snow et al., 2002) and Atlantic herring (Clupea harengus) (Nylund et al., 2002), both of which are known to be associated with aquaculture. While no evidence was obtained to implicate saithe in the epidemiology of ISAV (Snow et al. 2002), replication of virus was demonstrated in herring which were shown to represent an asymptomatic carrier species (Nylund et al., 2002). The aim of the current study was to investigate the potential impact of ISAV on the commercial production of alternative species such as Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus). Increasing diversification within the mariculture industry has placed a considerable emphasis on such species in recent years. The potential for such species to be reared in an environment where ISAV may be present, including in proximity to Atlantic salmon culture operations, renders the susceptibility of these species to ISAV an important issue to address. The current study was thus instigated to determine the susceptibility of both cod and halibut to an isolate of ISAV, which originated in association with Scottish aquaculture and is pathogenic to Atlantic salmon. Materials and methods Cell culture and virus propagation The salmon head kidney (SHK-1) cell line was used for the propagation and isolation of virus in this study, according to previously described methods (Dannevig et al., 1995). Isolate 390/98, which was recovered from a clinical outbreak of ISA in West Scotland was used in all infection experiments. Virus was amplified by passage on SHK-1 cells, harvested and stored at –80°C. An aliquot of virus was titrated on SHK-1 cells using the tissue culture infectious dose method (TCID50) (Reed & Muench, 1938; Burleson et al., 1992) following a single freeze-thaw cycle. Both positive and negative cell cultures were tested for the presence of ISAV using haemadsorption with Atlantic salmon erythrocytes as previously described (Smail et al., 2000). Bull. Eur. Ass. Fish Pathol., 25(5) 2005, 191 RT-PCR Tissues (kidney and spleen) were removed from fish surviving at the end of the experiment and stored at -80°C until processing. Tissues were then thawed and disrupted in Trizol® (Life Technologies) (1ml per 100mg) using disposable pestles. Total RNA was extracted according to the protocol supplied by the manufacturer (Life Technologies) prior to resuspension in DEPCtreated water. One μg RNA was used as template for reverse transcription (RT) and PCR, which was conducted according to the method, previously reported by Mjaaland et al (1997). Fish Cod (mean weight +/SE, 45.7+/-3.8g) were obtained from the Seafish Marine Research Unit, Ardtoe, Argyll. Halibut (mean weight +/SE, 2.6+/0.2g) were obtained from Mannin Seafarms, Isle of Man. Atlantic salmon (mean weight +/SE, 56.0+/2.5g) were reared at the FRS Marine Research Unit, Aultbea, Rossshire, Scotland. Prior to infection experiments, fish were screened for the presence of infectious salmon anaemia virus, infectious pancreatic necrosis virus, infectious haematopoietic necrosis virus and viral haemorrhagic septicaemia virus as described previously (Dannevig et al., 1995; Snow & Smail, 1999). Fish were acclimated for at least 14 days after arrival to the laboratory to sea water conditions (sand filtered from Nigg Bay, Aberdeen) with aeration and flow rates of 40 l min-1 at a temperature of 10 ± 1°C. Fish were maintained on commercial pellet diets, but were starved for 24h before infection. Experimental infection of Atlantic cod with ISAV Atlantic cod were stocked into each of 6 x 30 l aquaria at a density of 20 fish per tank. Ten fish from each of tanks 1-3 were removed, anaesthetised by immersion in tricaine methane sulphonate (3-aminobenzoic acid ethyl ester of MS-222; Sigma) at a concentration of 100mg l-1, marked by clipping of the dorsal fin, and inoculated intraperitoneally with 2.5x107 TCID50 of ISAV in a volume of 50μl. Ten fish from tanks 4-6 were similarly anaesthetised, marked and mock injected with an equivalent volume of L-15 media containing no virus. Experimental infection of Atlantic halibut with ISAV Atlantic halibut were stocked into each of a further 4 x 30 l tanks (tanks 7-10) with similar conditions at a density of 40 fish per tank. Twenty individuals were removed, anaesthetised, marked and injected intraperitoneally with a dose of 1.25 x107 TCID50 of ISAV in a volume of 50 μl and returned to tanks 7 and 8. Twenty fish from each of tanks 9 and 10 were similarly marked and injected with L-15 media containing no virus. Experimental infection of Atlantic salmon with ISAV Atlantic salmon were stocked into each of a further 4 x 30 l tanks aquaria at a density of 10 fish per tank (tanks 11-16). All ten fish from each of tanks 11 and 12 were removed, anaesthetised, marked by clipping of the dorsal fin, and inoculated intra-peritoneally with 2.5x107 TCID50 of ISAV in a volume of 50μl. Ten fish from tanks 13 and 14 were similarly anaesthetised, marked and mock Bull. Eur. Ass. Fish Pathol., 25(5) 2005, 192 injected with an equivalent volume of L-15 media containing no virus. Fish in all aquaria were monitored daily for the duration of the experimental infection period (42d) and all dead fish removed and examined. Examination of fish surviving experimental challenge Tissues (kidney and spleen) were removed from both cod and halibut surviving at the end of the experimental infection period and examined for the presence of ISAV using tissue culture and RT-PCR using the methods described above. Material from groups of 5 control i.p. infected cod from each of tanks 4-6 was pooled for analyses by both tissue culture and RT-PCR. Five each of ISAV infected fish and cohabiting fish were sampled from each of tanks 1-3 and processed individually by tissue culture and RT-PCR (30 samples). Material from groups of 5 halibut injected with ISAV or cohabiting with these fish (tanks 7 and 8) was pooled for analysis in each case. Material from 10 control i.p or cohabiting halibut (tanks 9 and 10) was pooled for analysis. Atlantic salmon were not sampled for the presence of ISAV since they had been injected with virus and it is known that virus is detectable for at least 40d following administration via this method (Rimstad et al., 1999), but any gross pathological signs indicative of ISA disease were recorded. Results Pathogen testing of fish Cod, halibut and Atlantic salmon were confirmed by virological examination to be free from detectable ISAV, VHSV, IHNV and IPNV. Experimental infection of Atlantic cod and Atlantic halibut with ISAV No mortality or clinical signs normally associated with ISA disease were recorded in either i.p infected or fish cohabiting with these i.p infected fish of the same species for the duration of the experimental infection periods. The presence of ISAV was not identified using either RT-PCR or virological examination in any of the i.p infected or cohabiting fish surviving at the end of the challenge period. Experimental infection of Atlantic salmon Mortality in i.p infected salmon reached 20 and 40% in tanks 11 and 12 respectively. All dead fish exhibited clinical signs consistent with ISA disease. All 8 surviving fish in tank 11 displayed signs of severe exopthalmia and external haemorrhaging which is consistent with establishment of a chronic infection. Similarly, 4 survivors from tank 12 also displayed signs associated with ISA, while 2 appeared normal. Positive controls were not tested for the presence of ISAV due to the appearance of specific clinical signs consistent with ISA disease. Since ISAV may be detected for at least 40d in salmon following administration via intra-peritoneal injection (Rimstad et al., 1999), the detection of ISAV was considered highly likely regardless of clinical disease status. Discussion Mortality in positive control Atlantic salmon was lower than expected based on previous infection models established in our laboratory Bull. Eur. Ass. Fish Pathol., 25(5) 2005, 193 (Raynard et al., 2001b), despite administration of a high dose of the same isolate of ISAV. This experiment was conducted in late October, around a time where other workers have experienced difficulty in infecting and killing salmon with ISAV (Nylund et al, 2002). Such a phenomenon was not related to the viral stock used in this study since the same stock was used in an immersion challenge conducted in December of the same year which resulted in mortality of 100% in duplicate tanks (n= 10) with zero mortality recorded in respective controls (data not shown). Thus Atlantic salmon appear to show seasonal differences in their susceptibility to ISAV infection. The underlying mechanisms for this are unclear, but may be related to seasonal differences in host innate immunity against viral infection. Based on this work, Atlantic cod and halibut did not appear to be susceptible to ISA disease. The results of any experimental trial should be interpreted with caution, however. Indeed, the nature of such experiments permits the examination of a single life stage of a single genetic stock of fish, challenged with a single isolate of a pathogen. The apparent seasonal differences observed in challenging Atlantic salmon, a known ISAV susceptible species, serve to emphasise this fact. Furthermore, examples of multiple apparent independent emergence of ISAV in Atlantic salmon in Canada and Europe coupled to the diversity in subtypes of viruses identified in association with aquaculture suggest that this virus is highly capable of adapting to new environments and host species. This experiment was not designed to study the potential development of a carrier status in the species studied. Results from other workers have, however, suggested that Atlantic cod may harbour ISAV. Indeed, analysis of 24 pools of tissue (5 fish per pool) originating from Atlantic cod taken from a well boat holding salmon from a cage with clinically diseased fish in Cobscook Bay, Maine USA, identified one sample from which ISAV was isolated (MacLean et al., 2003). The authors were, however, unable to conclude whether this virus originated from exogenous particle adhering to the gill lamellae of sampled fish or if indeed the cod were truly infected with ISAV. In summary, based on this study Atlantic cod and Atlantic halibut do not appear to be susceptible to ISA disease. Furthermore, even fish injected with a high dose of the pathogen did not show any evidence of residual virus after approximately 7 weeks. The risk of development of clinical ISA disease in these species thus appears low. However, there have been other documented examples, where the increased exposure of pathogens to new host species, has lead to the apparent emergence of pathogenic variants of RNA viruses associated with aquaculture. Indeed, the exposure of rainbow trout to a naturally occurring marine source of viral haemorrhagic septicaemia virus (VHSV) is believed to have lead to the original evolution of a highly pathogenic variant, which subsequently spread throughout the rainbow trout aquaculture industry in Continental Europe (Einer-Jensen et al., 2004; Snow et al., 2004). Furthermore, VHSV associated with aquaculture has been shown to evolve at a Bull. Eur. Ass. Fish Pathol., 25(5) 2005, 194 higher rate than that naturally found in the marine environment (Einer-Jensen et al., 2004). Increasing evidence highlights the existence of an environmental reservoir, which may contain diverse and highly adaptable subtypes of ISAV. 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