doi:10.1111/jfd.12310
Journal of Fish Diseases 2015, 38, 271–281
An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater I Cano, C Joiner, A Bayley, G Rimmer, K Bateman, S W Feist, D Stone and R Paley Aquatic Animal Disease, Centre for Environment, Fisheries and Aquaculture Science, The Nothe Weymouth, Dorset, UK
Abstract
A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 9 105 TCID50 mL 1 for SAV1 and 3 9 104 for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 102 to 107 TCID50 mL 1. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 102 TCID50 mL 1. Clearly, salmon fry are susceptible to SAV infection and pancreas disease. Keywords: bath infection, histology, in situ hybridisation, RT-PCR, salmon pancreas disease, salmonid alphavirus. Correspondence I Cano, Aquatic Animal Disease, Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe Weymouth, Dorset DT4 8UB, UK (e-mail: [email protected]) Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
This article is published with the permission of the Controller of HMSO and the Queen’s Printer for Scotland.
271
Introduction
Salmon pancreas disease (PD) is a significant concern to the Atlantic salmon Salmo salar L. farming industry in Europe (Kristoffersen et al. 2009). Initially identified in European fish farms as long ago as the 1980s, recently the cumulative loss to Norwegian fish farms from 2004 to 2009 was estimated at €554.8 million (Jensen 2010). The cause of the disease is infection with the alphavirus, salmon pancreas disease virus (SPDV) or salmonid alphavirus (SAV) (Nelson et al. 1995). SAV belongs to the genus Alphavirus (family Togaviridae) and has a single-strand, positive sense RNA genome of 12 kilobases (Hodneland et al. 2005). Based on partial sequences from nsP3 and E2 genes, Fringuelli et al. (2008) classified SAV into six subtypes which were geographically and genotypically different. SAV subtype 1 has been identified from Ireland and Scotland; SAV3 has found exclusively from Norway; SAV4 both in Scotland and Ireland; SAV5 found in Scotland; and SAV 6 from Ireland (Graham et al. 2012). SAV2, named sleeping disease virus (SDV) (Castric et al. 1997) typically affects freshwater cultured rainbow trout Oncorhynchus mykiss (Walbaum) causing sleeping disease (SD). It has been identified in France, Spain, Italy, UK, Germany and Croatia (Graham et al. 2003, 2007a; Bergman et al. 2008; Vardic Smrzlic et al. 2013). In 2008, isolates grouping with SAV2 were also reported from PD in farmed marine salmon in Scotland (Graham et al. 2011) and more recently numerous outbreaks of PD in Norway have been attributed to infection with isolates grouping with subtype 2 SAV (Hjortaas et al. 2013). Clinical signs associated with PD include inappetence, lethargy and an increased number of
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faecal casts in cages. In natural outbreaks of PD in the marine phase of production mortalities varied from 10% to 30% (Graham et al. 2010). Pancreas disease is characterised by initial viraemia that normally lasts for 2 weeks (Houghton 1995), followed by pancreatic acinar cell loss, cadiomyocytic degeneration and subsequent skeletal muscle degeneration and fibrosis (McVicar 1987; McLoughlin & Graham 2007). In response to infection, the host produces neutralising antibodies and displays long-lasting acquired immunity (Houghton & Ellis 1996; McLoughlin, Rowley & Doherty 1998). Pancreas disease experimental infection trials have been traditionally carried out using two different methods of virus transmission: by intraperitoneal (i.p.) injection and cohabitation. Salmon have been challenged at different life stages including by i.p. injection challenge in parr (Raynard & Houghton 1993; Boucher et al. 1995; McLoughlin et al. 1995; Desvignes et al. 2002) and presmolts in freshwater (Christie et al. 2007) and post-smolts in sea water (Raynard & Houghton 1993; McLoughlin et al. 1996). Cohabitation challenge has been developed for parr (Graham et al. 2011) and post-smolts (Raynard & Houghton 1993; McLoughlin et al. 1996). Mortalities were not recorded in any of these challenges, although reisolation of the virus in cell culture was achieved from 7 to 21 days post-inoculation (dpi) in i.p. injected presmolts (Christie et al. 2007) and smolts (McLoughlin et al. 1996) and in cohabitant smolts from 14 to 21 dpi (McLoughlin et al. 1996). Typical PD histopathological lesions in sampled fish were described in all of the studies. The aim of this study was to develop a novel PD bath challenge for salmon fry in fresh water. Two different subtypes of SAV: SAV1 (an isolate from Ireland) and SAV5 (an isolate from Scotland) were used. The pathogenesis of the disease was studied by recording mortalities, virus isolation, RT-PCR, histology and in situ hybridisation (ISH).
Materials and methods
Salmonid alphavirus cell culture
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Virus isolates were obtained from sera or tissues of salmon from PD outbreaks in Ireland (isolate G28) and Scotland (isolate V4638). Sequencing and phylogenetic analysis, based on the sequence 272
of a 493 bp fragment of the nsP3 gene (Fringuelli et al. 2008), confirmed that the Irish isolate belongs to the SAV 1 subtype and the Scottish isolate to SAV5 (data not shown). Chinook salmon embryo (CHSE-214) cells were used to propagate and titrate the SAV isolates. Cells were inoculated by adsorption of virus for 3 h, followed by addition of maintenance media [L-15 supplemented with 1 mM L-glutamine, 10% foetal bovine serum, 100 U mL 1 penicillin and 100 lg mL 1 streptomycin (Gibco, Life Technologies)] and incubation at 15 °C. Development of cytopathic effects (CPE) was observed at soon as 6 dpi, and the virus suspension was harvested after 12 dpi, clarified by centrifugation at 1000 g and titrated in CHSE-214 cells in 96-well cell culture plates and the TCID50 calculated by the method of K€arber (1931). Bath challenge Triplicate tanks of 450 Atlantic salmon fry, of average weight 0.9 g, were bath challenged by immersion in one of two different SAV subtypes (Irish isolate SAV1 or Scottish isolate SAV5). A negative control tank of 450 fish was sham challenged with culture medium only. For each isolate, triplicate static immersion vessels were prepared by adding 100 mL of virus stock (at maximum titre, harvested from cell culture) to 6 L of tank water. The titres of the virus in the immersion bath were 3 9 105 TCID50 mL 1 for SAV1; and 3 9 104 TCID50 mL 1 for SAV5 and the passage number in CHSE cells of the challenge inoculums was P2 and P8, respectively. Fish were held in the immersion vessel for a static challenge for 4 h, then returned to holding tanks and maintained at a flow rate of 0.2–0.5 L min 1 per tank. The water temperature was maintained at 10 1 °C throughout. Prior to the challenge, stock salmon fry were randomly sampled and tested for the presence of viral and/or bacterial pathogens. None were detected. Sampling regime Fry were held and observed for 8 weeks post-challenge (wpc), with daily observation for abnormal behaviour, systemic reactions or mortality. Fry challenged with each viral isolate were sampled at three different points: 3, 5 and 7.5 wpc. At 3 wpc, only one tank of each replicate was
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sampled, 30 fish were sampled for virus re-isolation and 10 fish for histology. At 5 wpc, 75 fry per isolate (25 fry per replicate tank) were processed for virus re-isolation and viral RNA detection by RT-PCR, and 15 fish per isolate from only one replicate tank were processed for histology and ISH studies. At the final sampling (7.5 wpc), 15 fish per replicate tank were processed for virus recovery. Ten fish from the negative control were processed to use as negative reference in the virology and histology analysis at the same sampling points. At the end of the challenge, 8 wpc, fry from one tank SAV1 subtype challenged and from the negative control tank were individually weighed. For the rest of the experimental groups, mean fry weight was determined from pooled group weights. For virological analysis, sampled whole fry were homogenised 1:10 in maintenance media supplemented with 1% antibiotic–antimycotic solution, 5% glutamax, 0.16% Trizma base solution (Sigma) and 0.48% sodium bicarbonate. Homogenates were clarified by centrifugation for 10 min at 2500 g, and the supernatants were titred in CHSE-214 cells. RNA extraction and RT-PCR
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Viral RNA was extracted from 100 lL of homogenised tissue using an EZ1 Virus mini Kit v2.0 and EZ1 extraction robot (Qiagen), eluting the RNA in 60 lL of elution buffer. 3.5 lL of RNA was reverse transcribed in a 20 lL reaction containing 1 mM dNTP, 500 ng of random primers and 200 units M-MLV reverse transcriptase (Promega) at 37 °C for 1 h. SAV nsP4 RNA was detected by nested PCR using the following primers: Alphavirus generic For: 5-GAYATGA AACGCGAYGTNAAR-3 and Alphavirus generic Rev: 5-GTCCTGGCTCTTRTCGAANG-3) for first round followed by Alphavirus generic For int: AGTGACGCCRGGCACNAAR and Alphavirus generic Rev int: GAGGAGATGTCCGTYTCNA G for second round, giving fragments of 276 and 236 bp, respectively. These primer sets have been shown to detect at least three of the six genotypes of SPDV: SAV1, SAV3 and SAV5; and the SDV genotype SAV2 (data not shown). PCR reactions were performed in a 50 lL reaction volume consisting of 19 GoTaq flexi buffer (Promega), 2.5 mM MgCl2, 1 mM dNTP mix, 50 pmol of the forward and reverse primers, 1.25 273
I Cano et al. PD fry bath challenge
units of GoTaqâ DNA Polymerase (Promega) and 2.5 lL of the purified DNA. The reaction mix was overlaid with mineral oil and after an initial denaturing step (5 min at 95 °C) was subjected to 35 temperature cycles (1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C) in a PTC225 Peltier thermal cycler (MJ Research) followed by a final extension step of 10 min at 72 °C. PCR products were visualised on 2% agarose gels stained with ethidium bromide. Histopathology and in situ hybridisation Sampled salmon fry were fixed in 10% neutralbuffered formalin for 24 h. Animals were embedded in paraffin wax using a vacuum infiltration processor using standard protocols. Embedded blocks were sectioned at 3 4 lm thickness using a rotary microtome, and sections were stained with haematoxylin and eosin (H&E). Sections were examined using a Nikon E800 light microscope with images captured using LuciaTM software. The tissues sections were examined as a blind study and the presence or absence of lesions recorded in the following target organs: pancreas tissue, heart and skeletal muscle. A scoring system was used to semi-quantify the distribution and severity of the tissue lesions, similar to that used in previous PD studies (McLoughlin et al. 2006; Christie et al. 2007; Graham et al. 2011). Sequential sections, mounted in silane-treated slides (Sigma-Aldrich), were used for ISH of viral genome. ISH assays were carried out as previously described (Cano et al. 2009). Briefly, deparaffinised and rehydrated sections were permeabilised with Proteinase K (100 lg mL 1 in DEPC H2O; Promega) for 30 min at 37 °C. Hybridisation was performed using a 276 bp SAV-specific digoxigenin (DIG)-labelled probe obtained by PCR (Roche) using the Alphavirus generic For/Rev primers described above. The DIG-labelled probe was denatured at 95 °C for 5 min prior to hybridisation overnight at 42 °C. Based on previous optimisation, two post-hybridisation washes were performed with 19 saline sodium citrate buffer (SSC) with 6 M urea and 0.2% (w v 1) bovine serum albumin (BSA) for 15 min at 42 °C for each one. Tissue sections were blocked with 6% (w v 1) skimmed milk (Sigma) in TB buffer and incubated with an anti-DIG monoclonal antibody conjugated to
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alkaline phosphatase (Roche) for 1 h. The endogenous phosphatase activity was removed by a treatment with 1 mM levamisole (Sigma), and the hybridisation signal was detected using NBT/ BCIP (Roche). Tissue sections were dehydrated and mounted with cover slip and mounting medium (Entellan; Merck). Electron microscopy For examination by transmission electron microscopy (TEM), heart from moribund salmon fry were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature and rinsed in 0.1 M sodium cacodylate buffer (pH 7.4). As positive control, CHSE-214 cells were inoculated with SAV1 isolate. After 6 dpi cells were harvested, rinsed in 0.1 M sodium cacodylate buffer (pH 7.4) before being fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 15 min. Cells were then scraped from the culture flask using a cell scraper and centrifuged to form a pellet, pellets were then processed in the same manner as for heart. Tissues were post-fixed for 1 h in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Samples were washed in three changes of 0.1 M sodium cacodylate buffer before dehydration through a graded acetone series. Samples were embedded in Agar 100 epoxy (Agar Scientific; Agar 100 premix kit medium) and polymerised overnight at 60 °C in an oven. Semi-thin (1 2 lm) sections were stained with toluidine blue for viewing with a light microscope to identify suitable target areas. Ultrathin sections (70 90 nm) of these areas were mounted on uncoated copper grids and stained with 2% aqueous uranyl acetate and Reynolds’ lead citrate (Reynolds 1963). Grids were examined using a JEOL JEM 1400 transmission electron microscope and digital images captured using an AMT XR80 camera and AMTv602 software.
Results
Morbidity-mortality and weight records
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
Cumulative mortality in salmon fry challenged with either alphavirus subtype SAV1 or SAV5 was 1.2% or less (compared with 0% in the control group). Moribund fry swam poorly and stayed at 274
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the bottom of the tank before dying within approximately 2 h of the first signs being observed. There were no obvious external or internal clinical signs in the mortalities examined, other than severe exophthalmia. The first mortality was recorded in a tank challenged with SAV1 as early as 11 days post-challenge (dpc), and the final mortalities were at 50, 55 and 57 dpc. Virus was recovered from the majority of mortalities (24 of 30). Titres of virus recovered from the mortalities ranged from 102 to 107 TCID50 mL 1. Histopathological changes consistent with PD pathology were observed in some of the moribund fish which were killed and examined. TEM studies of the hearts of moribund fish showing cell necrosis revealed the presence of icosahedral virus-like particles in the cytoplasm of epicardium cells. The average size of these coincides with the size of viral particles observed in infected CHSE-214 cells, with a diameter ranged between 62 to 75 nm and an electro-dense core of approximately 37.5 nm (Fig. 1). Virus-like particles were also associated to vesicular structures in the cytoplasm. A general loss of appetite was observed from the third week after challenge. Weights were determined at the end of the challenge both individually (for control and one of the SAV1 groups) or en masse (all other groups). Although there was a notable degree of intratank variation in final weights of fry, including within the control tank, salmon fry that had been challenged with the SAV1 subtype weighed significantly less than those in the control tank (Student’s t-test, P < 0.001), 1.63 g vs. 2.19 g. SPD infection in salmon fry No external signs or gross pathology were observed in sampled fry at 3, 5 or 7.5 wpc. However, at 3 wpc, infectious SAV1 was recovered from 80% of the samples analysed. CHSE-214 cells inoculated with fry homogenate developed characteristic CPE, consisting of small groups of pyknotic and rounded cells. Viral titres ranged from 102 to 106 TCID50 mL 1. At the same time point, SAV5 was recovered from 43% of the samples, with viral titres ranging from 102 to 103 TCID50 mL 1. For both viral subtypes, early histopathological lesions at the 3 wpc sample point were observed in the pancreatic tissue around the pyloric caeca, mainly consisting of multifocal atrophy and
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Journal of Fish Diseases 2015, 38, 271–281
(a)
(b)
Figure 1 Transmission electron micrographs of (a) CHSE-214 cells inoculated with SAV1 at 6 days post-inoculation (scale bar 100 nm) and (b) heart of moribund salmon fry showing histopathology consistent with PD (scale bar 500 nm). Virus-like particles can be observed both in the cytoplasm of infected cells and associated with vesicular structures. Nu: nucleus.
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
necrosis of exocrine pancreatic cells in 30% of fry sampled for SAV1 and 20% for SAV5. At this time, red muscle lesions were observed in 30% of SAV1-infected fry, and a single fish challenged with the Scottish isolate SAV5 showed cardiac lesions consisting of focal myocardial degeneration and proliferation of endothelial cells. In the 5 wpc sample, infectious virus was recovered from 60% of the samples analysed for SAV 1; and 24% of those sampled for SAV5, with titres for both isolates ranging from 102 to 103 TCID50 mL 1. Viral RNA, however, was detected by specific nested RT-PCR in a higher number of samples: 74.6% and 26.6% for SAV1 and SAV5, respectively. Also at the 5 wpc sample point, pancreas lesions were more generalised, showing in some cases total absence of pancreatic acinar cells (Fig. 2b), affecting up to 80% of salmon fry challenged with SAV1, and 33% for fry challenged with the SAV5 isolate. More of the salmon fry sampled showed heart lesions in both ventricle and atrium than at 3 weeks (Fig. 2d), with 50% of positive samples for SAV1 and 20% for SAV5 showing diffuse myocardial degeneration, necrosis and inflammatory cell infiltration. At this time, severe skeletal muscle degeneration and inflammation, including white and red muscle, were observed in fry challenged with SAV1 isolate (Fig. 2f,h) affecting 66% of fry sampled. Myocytic degeneration in red muscle was observed in 20% of fry infected with SAV5 (Fig. 2g), however, degeneration of white skeletal muscle was 275
not observed in fry infected with SAV5 at this sampling point. Finally at 7.5 wpc, it was still possible to detect infectious virus in 13% of the samples for both SAV isolates by culture, with titres of 102 TCID50 mL 1. A summary of the findings is provided in Tables 1 and 2. In situ hybridisation In situ hybridisation studies were performed on sections of whole fry sampled at 5 wpc. No labelling was observed in either of the negative controls used: tissues from non-infected fish and infected fish without specific probe. The labelling in the pancreas tissue was observed in exocrine pancreas cells (Fig. 3b), however, the samples analysed showed an advanced state of degeneration of the pancreas tissue. In situ hybridisation positive hearts showed patches of labelled endocardial cells in the stratum spongiosum of the ventricle. Multifocal areas of labelling in the skeletal muscle were observed at 5 weeks in SAV1-infected fish. Cytoplasm of infected myocytes showed presence of numerous dark blue precipitate suspected to be areas of viral replication (Fig. 3d,f). Discussion
To our knowledge, this is the first published study describing a bath challenge model of SPDV in
I Cano et al. PD fry bath challenge
Journal of Fish Diseases 2015, 38, 271–281
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
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Journal of Fish Diseases 2015, 38, 271–281
Figure 2 Histopathology of Atlantic salmon fry infected by SAV. (a, b) H&E stain of pancreas section of healthy (a) and bath challenged (b) sampled fish showing dramatic loss of exocrine cells. (c, d) H&E stain of atrium section from heart of healthy (c) and infected (d) fish showing proliferation of endothelial cells in the myocardium. (e, f) Cross-section of negative control (e) and SAV1 infected (f) fry showing region from the spine to the lateral line. (g) Red skeletal muscle lesion in SAV5-infected fry. Insert: low magnification of transverse section of fry through the dorsal fin. (h) Detail of white skeletal muscle lesion in SAV1-infected fry. D: dorsal fin; asterisk: myocytic degeneration. Bar 50 lm.
Table 1 Detail of histophatological damage caused by SAV in target tissues: pancreas, heart and skeletal muscle, of bath challenged salmon fry sampled at 3 and 5 weeks Week sampling
Viral isolate
Pancreas
Heart
Skeletal muscle
3
SAV SAV SAV SAV
2 2 2 2
0 2 1 1
1 0 2 3
5
1 5 1 5
(2/10); 3 (1/10) (2/10) (6/15); 3 (6/15) (4/15); 3 (1/15)
(6/6) (1/6) (3/10); 2 (2/10) (2/10)
(1/10); 2 (2/10) (10/10) (6/15); 3 (4/15) (3/15)
The number of fish with lesions in each organ and severity of the lesions was expressed as follow: lesion score following of parenthesis expressing number fish showing lesion of this scoring/number of total fish examined. Score system: pancreatic lesions: 0: normal appearance; 1: focal pancreatic acinar cell necrosis; 2: multifocal necrosis-atrophy of pancreatic acinar tissue; 3: total absence of pancreatic acinar cells. Heart lesions: 0: normal appearance; 1: focal myocardial degeneration inflammation (100 fibres affected). Skeletal muscle lesions: 0: normal appearance; 1: focal myocytic degeneration inflammation; 2: multifical myocytic degeneration inflammation; 3: severe myocitic degeneration inflammation.
Table 2 Summary of sampling data. Results of virus re-isolation in cell culture, viral RNA detection by specific nested PCR, and pancreas lesions consistent with PD at 3, 5 and 7.5 weeks sampling of salmon fry infected with SAV 1 and SAV 5 at 3 9 105 and 3 9 104 TCID50 mL 1, respectively Wpc 3 5 7.5
Isolate
Virus isolation
SAV SAV SAV SAV SAV SAV
24/30 (80%) titre 10 –10 13/30 (43%) titre 102–103 44/75 (60%) titre 102–103 18/75 (24%) titre 102–103 2/15 (13.3%) titre 102 2/15 (13.3%) titre 102
1 5 1 5 1 5
2
6
Viral RNA detection
Pancreas lesions
nd nd 56/75 (74%) 20/75 (26%) nd nd
3/10 2/10 12/15 5/15 nd nd
(30%) (20%) (80%) (33%)
x/y means number of positives/total fish sampled; in parenthesis is given the percentage of positive fish. Viral titre expressed in TCID50 mL Wpc, Week post-challenge.
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
Atlantic salmon at the fry stage in freshwater. Pathogenicity of two SPDV subtypes was investigated; an Irish isolate of SAV subtype 1 and a Scottish isolate of SAV subtype 5. The development of an SAV transmission model by bath exposure provides a good surrogate for natural transmission of PD (Stene et al. 2013). The present study shows that salmon at the fry stage are susceptible to infection with SPDV and develop disease consistent with PD following bath challenge. Mortality was not observed in any of the previous experimental infections carried out by i.p. injection and cohabitation for parr, smolts and post-smolts (Raynard & Houghton 1993; Boucher et al. 1995; McLoughlin et al. 1995, 1996; Desvignes et al. 277
1
2002; Christie et al. 2007; Graham et al. 2011; Grove et al. 2013), although all these authors reported typical PD histopathological lesions in the heart and pancreas, demonstrating infection. Natural subclinical SAV infection in marine-reared Atlantic salmon has also been reported in Scotland (Graham et al. 2007b), suggesting that environmental factors such as water temperature may influence the severity of a PD outbreak (McLoughlin & Graham 2007). Mortality rates in natural PD outbreaks in Ireland have ranged from 1% to 48% in Atlantic salmon smolts during their first year at sea (McLoughlin & Graham 2007). Currently, there are no records of natural PD outbreaks caused by SAV subtypes in salmon in the freshwater phase. Although, the SAV2 subtype has been
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(a)
(b)
(c)
(d)
(e)
(f)
Figure 3 In situ hybridisation (ISH) of SAV genome in challenged salmon fry. The labelling is observed microscopically as dark blue staining. (a, b) Section of pancreas tissue of non-infected salmon fry (a) and SAV 1-infected fry at 5 weeks post-challenge (b). Necrotic acinar cell shows staining (asterisk). (c, d) Skeletal muscle section of negative fish (c) and SAV 1-infected fish (d) showing extensive areas of myocitic cells labelled at 5 weeks. (e, f) Detail of muscle. (f) Dark blue precipitates locate in the cytoplasm of positive myocytic cell (asterisk). Bar 50 lm.
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demonstrated to cause sleeping disease in rainbow trout in freshwater and pancreas disease in salmon in sea water (Hjortaas et al. 2013). Re-isolation of virus from sera is possible up to 14 dpi (Christie et al. 2007). In the present study, the re-isolation of the virus was achieved from 278
whole fry homogenate, so it is not known if those positive fry were also viraemic. The time-scale of the formation of lesions reported in this study correlates with previous experimental infections and natural PD outbreaks (McLoughlin et al. 2002), with the observation of
Journal of Fish Diseases 2015, 38, 271–281
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
multifocal lesions in pancreatic tissue at 3 wpc. Heart lesions became more generalised at 5 wpc compared to 3 wpc, confirming previous reports that acute heart lesions occur concurrently or slightly later than acute pancreatic acinar necrosis (McLoughlin & Graham 2007). The percentage of infected fish at 3 wpc was higher with Irish SAV1 isolate than the Scottish SAV5. Moreover, the severity of the lesions and number of fish affected observed at 5 wpc was also higher in those salmon fry infected with the SAV1 isolate, with a dramatic difference in the severity of lesion observed in the white skeletal muscle between both isolates. Graham et al. (2011) suggested differences in the dynamics of infection with SAV1 causing more extensive histopathological damage than SAV5 and our results appear to support that finding. However, The 1 log of difference in the viral titre of the bath challenge (105 TCID50 mL 1 for SAV1 vs. 104 for SAV5) may also explain the difference in the observed pathogenicity of the isolates. Salmonid alphavirus particles were observed in the epicardium of moribund fry by TEM studies. Virions were associated with vesicular structures or cytopathic vacuoles identified as alphavirus RNA replication complexes (Herath et al. 2012). The preliminary ISH study has revealed that the technique is able to detect the SAV genome in infected tissues and can be used in future pathology studies. Moreover, the technique could support differential diagnosis of archive histology samples of SAV diseases and of other pathologies that cause similar lesions such as infectious pancreatic necrosis (IPN), cardiomyopathy syndrome (CMS), heart and skeletal muscle inflammation (HSMI) and nutritional myopathies (McLoughlin & Graham 2007). Surviving fry challenged with SAV1 showed significantly lower weight than control fish (up to 28% less in two of the three triplicates compared to control fish at 8 wpc). Christie et al. (2007) also reported that i.p. challenged pre smolt weight was significantly lower than control fish. Recent studies showed that loss of pancreatic tissue may result in poor nutritional status that may explain loss of weight in surviving fish that have suffered an asymptomatic infection (Larsson et al. 2012). The number of remaining infected salmon fry at 7.5 weeks was similar for both isolates, 13.3% of sampled fish, with viral titre recovered from salmon fry homogenate of 102 TCID50 mL 1. 279
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These results show that clearance of the virus did not occur in all challenged fish, suggesting that carrier state at salmon fry stage is possible. PD is endemic in most salmon marine sites in Ireland and tends to recur in each successive generation of fish introduced onto the site, suggesting a substantial reservoir of infection in the marine environment, or a possible freshwater carrier state (McLoughlin & Graham 2007). Some alphaviruses infecting mammals can persist after appearance of an immune response, clearance of virus from the circulation and apparent clearance of infectious virus from tissue (Levine, Hardwick & Griffin 1994). Those alphavirus can be transmitted transplacentally (Shinefield & Townsend 1953). However, vertical transmission for SAV has not been demonstrated (Kongtorp et al. 2010). Furthermore, whilst Bratland & Nylund (2009) detected SAV by PCR in ova and fry sampled from commercial farms, in a study of 46 Norwegian freshwater production sites Jansen et al. (2010) failed to detect SAV in salmon in the freshwater phase. The presence of SAV in salmon in freshwater is probably not common but may occur. Differential susceptibility to SPDV infection in different strain of Atlantic salmon has been reported (Crockford et al. 1999; McLoughlin et al. 2006). The production of PD resistant salmon will reduce the impact of PD (Norris 2008), in the same way that breeding resistance to IPN is expected to reduce losses in salmon (Moen et al. 2009; Houston et al. 2010). The present challenge model in fry will benefit from further refinement which could include challenging smaller fry, infection with different and potentially more virulent isolates or genogroups of SAV and increased thermal stress on the fry, to increase severity of the challenge and the number of fish presenting severe lesions. Nevertheless, the promising results in transmitting PD to fry by bath challenge allow for the development of cost-effective selective breeding programs for PD resistance. Acknowledgements The authors want to thank Dr Eann Munro, Marine Scotland, for kindly providing the Scottish isolate SAV5 (V4638); and Dr Neil Ruane, Marine Institute, for kindly providing the Irish isolate SAV1 (G28) and especially to Andy Payne for his comments and review of the manuscript. This
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study was supported by Cefas DP325 Seedcorn project and Defra contract FB002. Publication History Received: 2 May 2014 Revision received: 5 August 2014 Accepted: 6 August 2014
This paper was edited and accepted under the Editorship of Professor Ron Roberts. References Bergman S.M., Fichtner D., Riebe R. & Castric J. (2008) First isolation of sleeping disease virus (SDV) in Germany. Bulletin of the European Association of Fish Pathologists 28, 148–155. Boucher P., Raynard R.S., Houghton G. & Laurencin F.B. (1995) Comparative experimental transmission of pancreas disease in Atlantic salmon, rainbow trout and brown trout. Diseases of Aquatic Organisms 22, 19–24. Bratland A. & Nylund A. (2009) Studies on the possibility of vertical transmission of Norwegian salmonid alphavirus in production of Atlantic salmon in Norway. Journal of Aquatic Animal Health 21, 173–178. Cano I., Ferro P., Alonso M.C., Sarasquete C., Garcia-Rosado E., Borrego J.J. & Castro D. (2009) Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L. Journal of Fish Diseases 32, 143– 150. Castric J., Baudin Laurencin F., Bremont M., Jeffroy J., Le Ven A. & Bearzotti M. (1997) Isolation of the virus responsible for sleeping disease in experimentally infected rainbow trout (Oncorhynchus mykiss). Bulleting of the European Association of Fish Pathologists 17, 27–30. Christie K.E., Graham D.A., McLoughlin M.F., Villoing S., Tood D. & Knappskog D. (2007) Experimental infection of Atlantic salmon Salmo salar pre-smolts by i.p. injection with new Irish and Norwegian salmonid alphavirus (SAV) isolates: a comparative study. Diseases of Aquatic Organisms 75, 13–22.
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Houghton G. & Ellis A.E. (1996) Pancreas disease in Atlantic salmon: serum neutralisation and passive immunisation. Fish & Shellfish Immunology 6, 465–472. Houston R.D., Haley C.S., Hamilton A., Guy D.R., MotaVelasco J.C., Gheyas A.A., Tinch A.E., Taggart J.B., Bron J.E., Starkey W.G., McAndrew B.J., Verner-Jeffreys D.W., Paley R.K., Rimmer G.S., Tew I.J. & Bishop S.C. (2010) The susceptibility of Atlantic salmon fry to freshwater infectious pancreatic necrosis is largely explained by a major QTL. Heredity 105, 318–327. Jansen M.D., Wasmuth M.A., Olsen A.B., Gjerset B., Modahl I., Breck O., Haldorsen R.N., Hjelmeland R. & Taksdal T. (2010) Pancreas disease (PD) in sea-reared Atlantic salmon, Salmo salar L., in Norway; a prospective, longitudinal study of disease development and agreement between diagnostic test results. Journal of Fish Diseases 33, 723–736. Jensen B.A. (2010) PD Costs Norway €550 million. Intrafish, article no. 270569 www.intrafish.com (accessed 07/06/2010). K€arber G. (1931) Beitrag zur kollektiven Behandlung pharmakologischer. Naunyn-Schmiedebergs Archiv f€ ur experimentelle Pathologie und Pharmakologie 162, 480–483. Kongtorp R.T., Stene A., Andreassen P.A., Aspehaug V., Graham D.A., Lyngstad T.M., Olsen A.B., Olsen R.S., Sandberg M., Santi N., Wallace C. & Breck O. (2010) Lack of evidence for vertical transmission of SAV 3 using gametes of Atlantic salmon, Salmo salar L., exposed by natural and experimental routes. Journal of Fish Diseases 33, 879–888. Kristoffersen A.B., Viljugrein H., Kongtorp R.T., Brun E. & Jansen P.A. (2009) Risk factors for pancreas disease (PD) outbreaks in farmed Atlantic salmon and rainbow trout in Norway during 2003-2007. Preventive Veterinary Medicine 90, 127–136. Larsson T., Krasnov A., Lerfall J., Taksdal T., Pedersen M. & Mørkøre T. (2012) Fillet quality and gene transcriptome profiling of heart tissue of Atlantic salmon with pancreas disease (PD). Aquaculture 330–333, 82–91. Levine B., Hardwick J.M. & Griffin D.E. (1994) Persistence of alphaviruses in vertebrate hosts. Trends in Microbiology 2, 25– 28. McLoughlin M.F. & Graham D.A. (2007) Alphavirus infections in salmonids – a review. Journal of Fish Diseases 30, 511–531. McLoughlin M.F., Nelson R.T., McCormick J.I. & Rowley H. (1995) Pathology of experimental pancreas disease in freshwater Atlantic Salmon parr. Journal of Aquatic Animal Health 7, 104–110. McLoughlin M.F., Nelson R.T., Rowley H.M., Cox D.I. & Grant A.N. (1996) Experimental pancreas disease in Atlantic salmon Salmo salar post-smolts induced by salmon pancreas
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disease virus (SPDV). Diseases of Aquatic Organisms 26, 117–124. McLoughlin M.F., Rowley H.M. & Doherty C.E. (1998) A serological survey of salmon pancreas disease virus (SPDV) antibodies in farmed Atlantic salmon, Salmo salar L. Journal of Fish Diseases 21, 305–307. McLoughlin M.F., Nelson R.N., McCormick J.I., Rowley H.M. & Bryson D.B. (2002) Clinical and histopathological features of naturally occurring pancreas disease in farmed Atlantic salmon, Salmo salar L. Journal of Fish Diseases 25, 33–43. McLoughlin M.F., Graham D.A., Norris A., Matthews D., Foyle L., Rowley H.M., Jewhurst H., MacPhee J. & Todd D. (2006) Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus. Diseases of Aquatic Organisms 72, 125–133. McVicar A.H. (1987) Pancreas disease of farmed Atlantic salmon, Salmo salar, in Scotland: epidemiology and early pathology. Aquaculture 67, 71–78. Moen T., Baranski M., Sonesson A.K. & Kjoglum S. (2009) Confirmation and fine-mapping of a major QTL for resistance to infectious pancreatic necrosis in Atlantic salmon (Salmo salar): population-level associations between markers and trait. BMC Genomics 10, 368. Nelson R.T., McLoughlin M.F., Rowley H.M., Platten M.A. & McCormick J.I. (1995) Isolation of a Toga-like virus from farmed Atlantic salmon Salmo salar with pancreas disease. Diseases of Aquatic Organisms 22, 25–32. Norris A. (2008) Heritability of mortality in response to a natural pancreas disease challenge in Atlantic salmon postsmolts on a West of Ireland sea site. Aquaculture 31, 913–920. Raynard R.S. & Houghton G. (1993) Development towards an experimental protocol for the transmission of pancreas disease of Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 15, 123–128. Reynolds E.S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208–212. Shinefield M.R. & Townsend T.E. (1953) Transplacental transmission of western equine encephalitis. Journal of Pediatrics 43, 21–25. Stene A., Viljugrein H., Yndestad H., Tavornpanich S. & Skjerve E. (2014) Transmission dynamics of pancreas disease (PD) in a Norwegian fjord: aspects of water transport, contact networks and infection pressure among salmon farms. Journal of Fish Diseases 37, 123–34. Vardic Smrzlic I., Kapetanovic D., Valic D., Teskeredzic E., McLoughlin M.F. & Fringuelli E. (2013) First laboratory confirmation of sleeping disease virus (SDV) in Croatia. Bulletin of the European Association of Fish Pathologists 33, 78–83.
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Journal of Fish Diseases 2015, 38, 271–281
An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater I Cano, C Joiner, A Bayley, G Rimmer, K Bateman, S W Feist, D Stone and R Paley Aquatic Animal Disease, Centre for Environment, Fisheries and Aquaculture Science, The Nothe Weymouth, Dorset, UK
Abstract
A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 9 105 TCID50 mL 1 for SAV1 and 3 9 104 for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 102 to 107 TCID50 mL 1. Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 102 TCID50 mL 1. Clearly, salmon fry are susceptible to SAV infection and pancreas disease. Keywords: bath infection, histology, in situ hybridisation, RT-PCR, salmon pancreas disease, salmonid alphavirus. Correspondence I Cano, Aquatic Animal Disease, Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe Weymouth, Dorset DT4 8UB, UK (e-mail: [email protected]) Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
This article is published with the permission of the Controller of HMSO and the Queen’s Printer for Scotland.
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Introduction
Salmon pancreas disease (PD) is a significant concern to the Atlantic salmon Salmo salar L. farming industry in Europe (Kristoffersen et al. 2009). Initially identified in European fish farms as long ago as the 1980s, recently the cumulative loss to Norwegian fish farms from 2004 to 2009 was estimated at €554.8 million (Jensen 2010). The cause of the disease is infection with the alphavirus, salmon pancreas disease virus (SPDV) or salmonid alphavirus (SAV) (Nelson et al. 1995). SAV belongs to the genus Alphavirus (family Togaviridae) and has a single-strand, positive sense RNA genome of 12 kilobases (Hodneland et al. 2005). Based on partial sequences from nsP3 and E2 genes, Fringuelli et al. (2008) classified SAV into six subtypes which were geographically and genotypically different. SAV subtype 1 has been identified from Ireland and Scotland; SAV3 has found exclusively from Norway; SAV4 both in Scotland and Ireland; SAV5 found in Scotland; and SAV 6 from Ireland (Graham et al. 2012). SAV2, named sleeping disease virus (SDV) (Castric et al. 1997) typically affects freshwater cultured rainbow trout Oncorhynchus mykiss (Walbaum) causing sleeping disease (SD). It has been identified in France, Spain, Italy, UK, Germany and Croatia (Graham et al. 2003, 2007a; Bergman et al. 2008; Vardic Smrzlic et al. 2013). In 2008, isolates grouping with SAV2 were also reported from PD in farmed marine salmon in Scotland (Graham et al. 2011) and more recently numerous outbreaks of PD in Norway have been attributed to infection with isolates grouping with subtype 2 SAV (Hjortaas et al. 2013). Clinical signs associated with PD include inappetence, lethargy and an increased number of
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faecal casts in cages. In natural outbreaks of PD in the marine phase of production mortalities varied from 10% to 30% (Graham et al. 2010). Pancreas disease is characterised by initial viraemia that normally lasts for 2 weeks (Houghton 1995), followed by pancreatic acinar cell loss, cadiomyocytic degeneration and subsequent skeletal muscle degeneration and fibrosis (McVicar 1987; McLoughlin & Graham 2007). In response to infection, the host produces neutralising antibodies and displays long-lasting acquired immunity (Houghton & Ellis 1996; McLoughlin, Rowley & Doherty 1998). Pancreas disease experimental infection trials have been traditionally carried out using two different methods of virus transmission: by intraperitoneal (i.p.) injection and cohabitation. Salmon have been challenged at different life stages including by i.p. injection challenge in parr (Raynard & Houghton 1993; Boucher et al. 1995; McLoughlin et al. 1995; Desvignes et al. 2002) and presmolts in freshwater (Christie et al. 2007) and post-smolts in sea water (Raynard & Houghton 1993; McLoughlin et al. 1996). Cohabitation challenge has been developed for parr (Graham et al. 2011) and post-smolts (Raynard & Houghton 1993; McLoughlin et al. 1996). Mortalities were not recorded in any of these challenges, although reisolation of the virus in cell culture was achieved from 7 to 21 days post-inoculation (dpi) in i.p. injected presmolts (Christie et al. 2007) and smolts (McLoughlin et al. 1996) and in cohabitant smolts from 14 to 21 dpi (McLoughlin et al. 1996). Typical PD histopathological lesions in sampled fish were described in all of the studies. The aim of this study was to develop a novel PD bath challenge for salmon fry in fresh water. Two different subtypes of SAV: SAV1 (an isolate from Ireland) and SAV5 (an isolate from Scotland) were used. The pathogenesis of the disease was studied by recording mortalities, virus isolation, RT-PCR, histology and in situ hybridisation (ISH).
Materials and methods
Salmonid alphavirus cell culture
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Virus isolates were obtained from sera or tissues of salmon from PD outbreaks in Ireland (isolate G28) and Scotland (isolate V4638). Sequencing and phylogenetic analysis, based on the sequence 272
of a 493 bp fragment of the nsP3 gene (Fringuelli et al. 2008), confirmed that the Irish isolate belongs to the SAV 1 subtype and the Scottish isolate to SAV5 (data not shown). Chinook salmon embryo (CHSE-214) cells were used to propagate and titrate the SAV isolates. Cells were inoculated by adsorption of virus for 3 h, followed by addition of maintenance media [L-15 supplemented with 1 mM L-glutamine, 10% foetal bovine serum, 100 U mL 1 penicillin and 100 lg mL 1 streptomycin (Gibco, Life Technologies)] and incubation at 15 °C. Development of cytopathic effects (CPE) was observed at soon as 6 dpi, and the virus suspension was harvested after 12 dpi, clarified by centrifugation at 1000 g and titrated in CHSE-214 cells in 96-well cell culture plates and the TCID50 calculated by the method of K€arber (1931). Bath challenge Triplicate tanks of 450 Atlantic salmon fry, of average weight 0.9 g, were bath challenged by immersion in one of two different SAV subtypes (Irish isolate SAV1 or Scottish isolate SAV5). A negative control tank of 450 fish was sham challenged with culture medium only. For each isolate, triplicate static immersion vessels were prepared by adding 100 mL of virus stock (at maximum titre, harvested from cell culture) to 6 L of tank water. The titres of the virus in the immersion bath were 3 9 105 TCID50 mL 1 for SAV1; and 3 9 104 TCID50 mL 1 for SAV5 and the passage number in CHSE cells of the challenge inoculums was P2 and P8, respectively. Fish were held in the immersion vessel for a static challenge for 4 h, then returned to holding tanks and maintained at a flow rate of 0.2–0.5 L min 1 per tank. The water temperature was maintained at 10 1 °C throughout. Prior to the challenge, stock salmon fry were randomly sampled and tested for the presence of viral and/or bacterial pathogens. None were detected. Sampling regime Fry were held and observed for 8 weeks post-challenge (wpc), with daily observation for abnormal behaviour, systemic reactions or mortality. Fry challenged with each viral isolate were sampled at three different points: 3, 5 and 7.5 wpc. At 3 wpc, only one tank of each replicate was
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sampled, 30 fish were sampled for virus re-isolation and 10 fish for histology. At 5 wpc, 75 fry per isolate (25 fry per replicate tank) were processed for virus re-isolation and viral RNA detection by RT-PCR, and 15 fish per isolate from only one replicate tank were processed for histology and ISH studies. At the final sampling (7.5 wpc), 15 fish per replicate tank were processed for virus recovery. Ten fish from the negative control were processed to use as negative reference in the virology and histology analysis at the same sampling points. At the end of the challenge, 8 wpc, fry from one tank SAV1 subtype challenged and from the negative control tank were individually weighed. For the rest of the experimental groups, mean fry weight was determined from pooled group weights. For virological analysis, sampled whole fry were homogenised 1:10 in maintenance media supplemented with 1% antibiotic–antimycotic solution, 5% glutamax, 0.16% Trizma base solution (Sigma) and 0.48% sodium bicarbonate. Homogenates were clarified by centrifugation for 10 min at 2500 g, and the supernatants were titred in CHSE-214 cells. RNA extraction and RT-PCR
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Viral RNA was extracted from 100 lL of homogenised tissue using an EZ1 Virus mini Kit v2.0 and EZ1 extraction robot (Qiagen), eluting the RNA in 60 lL of elution buffer. 3.5 lL of RNA was reverse transcribed in a 20 lL reaction containing 1 mM dNTP, 500 ng of random primers and 200 units M-MLV reverse transcriptase (Promega) at 37 °C for 1 h. SAV nsP4 RNA was detected by nested PCR using the following primers: Alphavirus generic For: 5-GAYATGA AACGCGAYGTNAAR-3 and Alphavirus generic Rev: 5-GTCCTGGCTCTTRTCGAANG-3) for first round followed by Alphavirus generic For int: AGTGACGCCRGGCACNAAR and Alphavirus generic Rev int: GAGGAGATGTCCGTYTCNA G for second round, giving fragments of 276 and 236 bp, respectively. These primer sets have been shown to detect at least three of the six genotypes of SPDV: SAV1, SAV3 and SAV5; and the SDV genotype SAV2 (data not shown). PCR reactions were performed in a 50 lL reaction volume consisting of 19 GoTaq flexi buffer (Promega), 2.5 mM MgCl2, 1 mM dNTP mix, 50 pmol of the forward and reverse primers, 1.25 273
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units of GoTaqâ DNA Polymerase (Promega) and 2.5 lL of the purified DNA. The reaction mix was overlaid with mineral oil and after an initial denaturing step (5 min at 95 °C) was subjected to 35 temperature cycles (1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C) in a PTC225 Peltier thermal cycler (MJ Research) followed by a final extension step of 10 min at 72 °C. PCR products were visualised on 2% agarose gels stained with ethidium bromide. Histopathology and in situ hybridisation Sampled salmon fry were fixed in 10% neutralbuffered formalin for 24 h. Animals were embedded in paraffin wax using a vacuum infiltration processor using standard protocols. Embedded blocks were sectioned at 3 4 lm thickness using a rotary microtome, and sections were stained with haematoxylin and eosin (H&E). Sections were examined using a Nikon E800 light microscope with images captured using LuciaTM software. The tissues sections were examined as a blind study and the presence or absence of lesions recorded in the following target organs: pancreas tissue, heart and skeletal muscle. A scoring system was used to semi-quantify the distribution and severity of the tissue lesions, similar to that used in previous PD studies (McLoughlin et al. 2006; Christie et al. 2007; Graham et al. 2011). Sequential sections, mounted in silane-treated slides (Sigma-Aldrich), were used for ISH of viral genome. ISH assays were carried out as previously described (Cano et al. 2009). Briefly, deparaffinised and rehydrated sections were permeabilised with Proteinase K (100 lg mL 1 in DEPC H2O; Promega) for 30 min at 37 °C. Hybridisation was performed using a 276 bp SAV-specific digoxigenin (DIG)-labelled probe obtained by PCR (Roche) using the Alphavirus generic For/Rev primers described above. The DIG-labelled probe was denatured at 95 °C for 5 min prior to hybridisation overnight at 42 °C. Based on previous optimisation, two post-hybridisation washes were performed with 19 saline sodium citrate buffer (SSC) with 6 M urea and 0.2% (w v 1) bovine serum albumin (BSA) for 15 min at 42 °C for each one. Tissue sections were blocked with 6% (w v 1) skimmed milk (Sigma) in TB buffer and incubated with an anti-DIG monoclonal antibody conjugated to
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alkaline phosphatase (Roche) for 1 h. The endogenous phosphatase activity was removed by a treatment with 1 mM levamisole (Sigma), and the hybridisation signal was detected using NBT/ BCIP (Roche). Tissue sections were dehydrated and mounted with cover slip and mounting medium (Entellan; Merck). Electron microscopy For examination by transmission electron microscopy (TEM), heart from moribund salmon fry were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature and rinsed in 0.1 M sodium cacodylate buffer (pH 7.4). As positive control, CHSE-214 cells were inoculated with SAV1 isolate. After 6 dpi cells were harvested, rinsed in 0.1 M sodium cacodylate buffer (pH 7.4) before being fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 15 min. Cells were then scraped from the culture flask using a cell scraper and centrifuged to form a pellet, pellets were then processed in the same manner as for heart. Tissues were post-fixed for 1 h in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Samples were washed in three changes of 0.1 M sodium cacodylate buffer before dehydration through a graded acetone series. Samples were embedded in Agar 100 epoxy (Agar Scientific; Agar 100 premix kit medium) and polymerised overnight at 60 °C in an oven. Semi-thin (1 2 lm) sections were stained with toluidine blue for viewing with a light microscope to identify suitable target areas. Ultrathin sections (70 90 nm) of these areas were mounted on uncoated copper grids and stained with 2% aqueous uranyl acetate and Reynolds’ lead citrate (Reynolds 1963). Grids were examined using a JEOL JEM 1400 transmission electron microscope and digital images captured using an AMT XR80 camera and AMTv602 software.
Results
Morbidity-mortality and weight records
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Cumulative mortality in salmon fry challenged with either alphavirus subtype SAV1 or SAV5 was 1.2% or less (compared with 0% in the control group). Moribund fry swam poorly and stayed at 274
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the bottom of the tank before dying within approximately 2 h of the first signs being observed. There were no obvious external or internal clinical signs in the mortalities examined, other than severe exophthalmia. The first mortality was recorded in a tank challenged with SAV1 as early as 11 days post-challenge (dpc), and the final mortalities were at 50, 55 and 57 dpc. Virus was recovered from the majority of mortalities (24 of 30). Titres of virus recovered from the mortalities ranged from 102 to 107 TCID50 mL 1. Histopathological changes consistent with PD pathology were observed in some of the moribund fish which were killed and examined. TEM studies of the hearts of moribund fish showing cell necrosis revealed the presence of icosahedral virus-like particles in the cytoplasm of epicardium cells. The average size of these coincides with the size of viral particles observed in infected CHSE-214 cells, with a diameter ranged between 62 to 75 nm and an electro-dense core of approximately 37.5 nm (Fig. 1). Virus-like particles were also associated to vesicular structures in the cytoplasm. A general loss of appetite was observed from the third week after challenge. Weights were determined at the end of the challenge both individually (for control and one of the SAV1 groups) or en masse (all other groups). Although there was a notable degree of intratank variation in final weights of fry, including within the control tank, salmon fry that had been challenged with the SAV1 subtype weighed significantly less than those in the control tank (Student’s t-test, P < 0.001), 1.63 g vs. 2.19 g. SPD infection in salmon fry No external signs or gross pathology were observed in sampled fry at 3, 5 or 7.5 wpc. However, at 3 wpc, infectious SAV1 was recovered from 80% of the samples analysed. CHSE-214 cells inoculated with fry homogenate developed characteristic CPE, consisting of small groups of pyknotic and rounded cells. Viral titres ranged from 102 to 106 TCID50 mL 1. At the same time point, SAV5 was recovered from 43% of the samples, with viral titres ranging from 102 to 103 TCID50 mL 1. For both viral subtypes, early histopathological lesions at the 3 wpc sample point were observed in the pancreatic tissue around the pyloric caeca, mainly consisting of multifocal atrophy and
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(a)
(b)
Figure 1 Transmission electron micrographs of (a) CHSE-214 cells inoculated with SAV1 at 6 days post-inoculation (scale bar 100 nm) and (b) heart of moribund salmon fry showing histopathology consistent with PD (scale bar 500 nm). Virus-like particles can be observed both in the cytoplasm of infected cells and associated with vesicular structures. Nu: nucleus.
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
necrosis of exocrine pancreatic cells in 30% of fry sampled for SAV1 and 20% for SAV5. At this time, red muscle lesions were observed in 30% of SAV1-infected fry, and a single fish challenged with the Scottish isolate SAV5 showed cardiac lesions consisting of focal myocardial degeneration and proliferation of endothelial cells. In the 5 wpc sample, infectious virus was recovered from 60% of the samples analysed for SAV 1; and 24% of those sampled for SAV5, with titres for both isolates ranging from 102 to 103 TCID50 mL 1. Viral RNA, however, was detected by specific nested RT-PCR in a higher number of samples: 74.6% and 26.6% for SAV1 and SAV5, respectively. Also at the 5 wpc sample point, pancreas lesions were more generalised, showing in some cases total absence of pancreatic acinar cells (Fig. 2b), affecting up to 80% of salmon fry challenged with SAV1, and 33% for fry challenged with the SAV5 isolate. More of the salmon fry sampled showed heart lesions in both ventricle and atrium than at 3 weeks (Fig. 2d), with 50% of positive samples for SAV1 and 20% for SAV5 showing diffuse myocardial degeneration, necrosis and inflammatory cell infiltration. At this time, severe skeletal muscle degeneration and inflammation, including white and red muscle, were observed in fry challenged with SAV1 isolate (Fig. 2f,h) affecting 66% of fry sampled. Myocytic degeneration in red muscle was observed in 20% of fry infected with SAV5 (Fig. 2g), however, degeneration of white skeletal muscle was 275
not observed in fry infected with SAV5 at this sampling point. Finally at 7.5 wpc, it was still possible to detect infectious virus in 13% of the samples for both SAV isolates by culture, with titres of 102 TCID50 mL 1. A summary of the findings is provided in Tables 1 and 2. In situ hybridisation In situ hybridisation studies were performed on sections of whole fry sampled at 5 wpc. No labelling was observed in either of the negative controls used: tissues from non-infected fish and infected fish without specific probe. The labelling in the pancreas tissue was observed in exocrine pancreas cells (Fig. 3b), however, the samples analysed showed an advanced state of degeneration of the pancreas tissue. In situ hybridisation positive hearts showed patches of labelled endocardial cells in the stratum spongiosum of the ventricle. Multifocal areas of labelling in the skeletal muscle were observed at 5 weeks in SAV1-infected fish. Cytoplasm of infected myocytes showed presence of numerous dark blue precipitate suspected to be areas of viral replication (Fig. 3d,f). Discussion
To our knowledge, this is the first published study describing a bath challenge model of SPDV in
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Journal of Fish Diseases 2015, 38, 271–281
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
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Figure 2 Histopathology of Atlantic salmon fry infected by SAV. (a, b) H&E stain of pancreas section of healthy (a) and bath challenged (b) sampled fish showing dramatic loss of exocrine cells. (c, d) H&E stain of atrium section from heart of healthy (c) and infected (d) fish showing proliferation of endothelial cells in the myocardium. (e, f) Cross-section of negative control (e) and SAV1 infected (f) fry showing region from the spine to the lateral line. (g) Red skeletal muscle lesion in SAV5-infected fry. Insert: low magnification of transverse section of fry through the dorsal fin. (h) Detail of white skeletal muscle lesion in SAV1-infected fry. D: dorsal fin; asterisk: myocytic degeneration. Bar 50 lm.
Table 1 Detail of histophatological damage caused by SAV in target tissues: pancreas, heart and skeletal muscle, of bath challenged salmon fry sampled at 3 and 5 weeks Week sampling
Viral isolate
Pancreas
Heart
Skeletal muscle
3
SAV SAV SAV SAV
2 2 2 2
0 2 1 1
1 0 2 3
5
1 5 1 5
(2/10); 3 (1/10) (2/10) (6/15); 3 (6/15) (4/15); 3 (1/15)
(6/6) (1/6) (3/10); 2 (2/10) (2/10)
(1/10); 2 (2/10) (10/10) (6/15); 3 (4/15) (3/15)
The number of fish with lesions in each organ and severity of the lesions was expressed as follow: lesion score following of parenthesis expressing number fish showing lesion of this scoring/number of total fish examined. Score system: pancreatic lesions: 0: normal appearance; 1: focal pancreatic acinar cell necrosis; 2: multifocal necrosis-atrophy of pancreatic acinar tissue; 3: total absence of pancreatic acinar cells. Heart lesions: 0: normal appearance; 1: focal myocardial degeneration inflammation (100 fibres affected). Skeletal muscle lesions: 0: normal appearance; 1: focal myocytic degeneration inflammation; 2: multifical myocytic degeneration inflammation; 3: severe myocitic degeneration inflammation.
Table 2 Summary of sampling data. Results of virus re-isolation in cell culture, viral RNA detection by specific nested PCR, and pancreas lesions consistent with PD at 3, 5 and 7.5 weeks sampling of salmon fry infected with SAV 1 and SAV 5 at 3 9 105 and 3 9 104 TCID50 mL 1, respectively Wpc 3 5 7.5
Isolate
Virus isolation
SAV SAV SAV SAV SAV SAV
24/30 (80%) titre 10 –10 13/30 (43%) titre 102–103 44/75 (60%) titre 102–103 18/75 (24%) titre 102–103 2/15 (13.3%) titre 102 2/15 (13.3%) titre 102
1 5 1 5 1 5
2
6
Viral RNA detection
Pancreas lesions
nd nd 56/75 (74%) 20/75 (26%) nd nd
3/10 2/10 12/15 5/15 nd nd
(30%) (20%) (80%) (33%)
x/y means number of positives/total fish sampled; in parenthesis is given the percentage of positive fish. Viral titre expressed in TCID50 mL Wpc, Week post-challenge.
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
Atlantic salmon at the fry stage in freshwater. Pathogenicity of two SPDV subtypes was investigated; an Irish isolate of SAV subtype 1 and a Scottish isolate of SAV subtype 5. The development of an SAV transmission model by bath exposure provides a good surrogate for natural transmission of PD (Stene et al. 2013). The present study shows that salmon at the fry stage are susceptible to infection with SPDV and develop disease consistent with PD following bath challenge. Mortality was not observed in any of the previous experimental infections carried out by i.p. injection and cohabitation for parr, smolts and post-smolts (Raynard & Houghton 1993; Boucher et al. 1995; McLoughlin et al. 1995, 1996; Desvignes et al. 277
1
2002; Christie et al. 2007; Graham et al. 2011; Grove et al. 2013), although all these authors reported typical PD histopathological lesions in the heart and pancreas, demonstrating infection. Natural subclinical SAV infection in marine-reared Atlantic salmon has also been reported in Scotland (Graham et al. 2007b), suggesting that environmental factors such as water temperature may influence the severity of a PD outbreak (McLoughlin & Graham 2007). Mortality rates in natural PD outbreaks in Ireland have ranged from 1% to 48% in Atlantic salmon smolts during their first year at sea (McLoughlin & Graham 2007). Currently, there are no records of natural PD outbreaks caused by SAV subtypes in salmon in the freshwater phase. Although, the SAV2 subtype has been
I Cano et al. PD fry bath challenge
Journal of Fish Diseases 2015, 38, 271–281
(a)
(b)
(c)
(d)
(e)
(f)
Figure 3 In situ hybridisation (ISH) of SAV genome in challenged salmon fry. The labelling is observed microscopically as dark blue staining. (a, b) Section of pancreas tissue of non-infected salmon fry (a) and SAV 1-infected fry at 5 weeks post-challenge (b). Necrotic acinar cell shows staining (asterisk). (c, d) Skeletal muscle section of negative fish (c) and SAV 1-infected fish (d) showing extensive areas of myocitic cells labelled at 5 weeks. (e, f) Detail of muscle. (f) Dark blue precipitates locate in the cytoplasm of positive myocytic cell (asterisk). Bar 50 lm.
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
demonstrated to cause sleeping disease in rainbow trout in freshwater and pancreas disease in salmon in sea water (Hjortaas et al. 2013). Re-isolation of virus from sera is possible up to 14 dpi (Christie et al. 2007). In the present study, the re-isolation of the virus was achieved from 278
whole fry homogenate, so it is not known if those positive fry were also viraemic. The time-scale of the formation of lesions reported in this study correlates with previous experimental infections and natural PD outbreaks (McLoughlin et al. 2002), with the observation of
Journal of Fish Diseases 2015, 38, 271–281
Ó 2014 Crown Copyright. Journal of Fish Diseases Ó 2014 John Wiley & Sons Ltd
multifocal lesions in pancreatic tissue at 3 wpc. Heart lesions became more generalised at 5 wpc compared to 3 wpc, confirming previous reports that acute heart lesions occur concurrently or slightly later than acute pancreatic acinar necrosis (McLoughlin & Graham 2007). The percentage of infected fish at 3 wpc was higher with Irish SAV1 isolate than the Scottish SAV5. Moreover, the severity of the lesions and number of fish affected observed at 5 wpc was also higher in those salmon fry infected with the SAV1 isolate, with a dramatic difference in the severity of lesion observed in the white skeletal muscle between both isolates. Graham et al. (2011) suggested differences in the dynamics of infection with SAV1 causing more extensive histopathological damage than SAV5 and our results appear to support that finding. However, The 1 log of difference in the viral titre of the bath challenge (105 TCID50 mL 1 for SAV1 vs. 104 for SAV5) may also explain the difference in the observed pathogenicity of the isolates. Salmonid alphavirus particles were observed in the epicardium of moribund fry by TEM studies. Virions were associated with vesicular structures or cytopathic vacuoles identified as alphavirus RNA replication complexes (Herath et al. 2012). The preliminary ISH study has revealed that the technique is able to detect the SAV genome in infected tissues and can be used in future pathology studies. Moreover, the technique could support differential diagnosis of archive histology samples of SAV diseases and of other pathologies that cause similar lesions such as infectious pancreatic necrosis (IPN), cardiomyopathy syndrome (CMS), heart and skeletal muscle inflammation (HSMI) and nutritional myopathies (McLoughlin & Graham 2007). Surviving fry challenged with SAV1 showed significantly lower weight than control fish (up to 28% less in two of the three triplicates compared to control fish at 8 wpc). Christie et al. (2007) also reported that i.p. challenged pre smolt weight was significantly lower than control fish. Recent studies showed that loss of pancreatic tissue may result in poor nutritional status that may explain loss of weight in surviving fish that have suffered an asymptomatic infection (Larsson et al. 2012). The number of remaining infected salmon fry at 7.5 weeks was similar for both isolates, 13.3% of sampled fish, with viral titre recovered from salmon fry homogenate of 102 TCID50 mL 1. 279
I Cano et al. PD fry bath challenge
These results show that clearance of the virus did not occur in all challenged fish, suggesting that carrier state at salmon fry stage is possible. PD is endemic in most salmon marine sites in Ireland and tends to recur in each successive generation of fish introduced onto the site, suggesting a substantial reservoir of infection in the marine environment, or a possible freshwater carrier state (McLoughlin & Graham 2007). Some alphaviruses infecting mammals can persist after appearance of an immune response, clearance of virus from the circulation and apparent clearance of infectious virus from tissue (Levine, Hardwick & Griffin 1994). Those alphavirus can be transmitted transplacentally (Shinefield & Townsend 1953). However, vertical transmission for SAV has not been demonstrated (Kongtorp et al. 2010). Furthermore, whilst Bratland & Nylund (2009) detected SAV by PCR in ova and fry sampled from commercial farms, in a study of 46 Norwegian freshwater production sites Jansen et al. (2010) failed to detect SAV in salmon in the freshwater phase. The presence of SAV in salmon in freshwater is probably not common but may occur. Differential susceptibility to SPDV infection in different strain of Atlantic salmon has been reported (Crockford et al. 1999; McLoughlin et al. 2006). The production of PD resistant salmon will reduce the impact of PD (Norris 2008), in the same way that breeding resistance to IPN is expected to reduce losses in salmon (Moen et al. 2009; Houston et al. 2010). The present challenge model in fry will benefit from further refinement which could include challenging smaller fry, infection with different and potentially more virulent isolates or genogroups of SAV and increased thermal stress on the fry, to increase severity of the challenge and the number of fish presenting severe lesions. Nevertheless, the promising results in transmitting PD to fry by bath challenge allow for the development of cost-effective selective breeding programs for PD resistance. Acknowledgements The authors want to thank Dr Eann Munro, Marine Scotland, for kindly providing the Scottish isolate SAV5 (V4638); and Dr Neil Ruane, Marine Institute, for kindly providing the Irish isolate SAV1 (G28) and especially to Andy Payne for his comments and review of the manuscript. This
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study was supported by Cefas DP325 Seedcorn project and Defra contract FB002. Publication History Received: 2 May 2014 Revision received: 5 August 2014 Accepted: 6 August 2014
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