Fish and rearing conditions
The fish used in the present study were non-vaccinated Atlantic salmon post-smolts from the Aquagen strain, which had been produced as under-yearling smolts [25, 26] at the Institute of Marine Research (IMR) in Matre, Western Norway. The fish population was screened and confirmed to be negative for SAV3 and piscine reovirus (PRV). Two separate batches of fish from the same production cycle were transported to the rearing and disease challenge facility at IMR in Bergen. The first batch of freshwater smolts (average weight of 40.9 ± 6.5 g) was transferred into seawater upon arrival in Bergen (in early September 2013) and acclimatized in seawater for two weeks before infection experiments commenced. Thus, this first batch of experimental fish was challenged with SAV3 in the initial period after seawater transfer, and is hereafter referred to as Phase-A fish. The second batch of post-smolts (average weight of 88.8 ± 14.0 g) was transferred to Bergen (in October 2013) after being reared in seawater at Matre for seven weeks. Hence, this second batch of post-smolts was in a later period after seawater transfer and is hereafter referred to as Phase-B fish.
At IMR in Bergen, the fish from each batch were randomly distributed into 12 identical 250 L seawater tanks (12 °C, 34.5‰). Each tank contained 65 fish. The tanks were supplied with aeration (oxygen saturation of >85 %) and the water flow was maintained at 400 litres per hour (Lh−1). The fish were acclimatized in the experimental tanks for one week before SAV3 infection of shedders and for two weeks before SAV3 experimental challenges. The fish were fed twice daily to satiation with a commercial salmon feed (Spirit Supreme, Skretting, Norway). The fish were starved for 24 h prior to each sampling. The fish were bath anaesthetized with a mixture of metomidate (10 mg L−1) and benzocaine (60 mg L−1) before handling and injection, and euthanized with a mixture of metomidate (10 mg L−1) and benzocaine (160 mg L−1) before tissue sampling.
Salmonid alphavirus (SAV)
Chum salmon heart-1 (CHH-1) cells were cultivated in 75 cm2 plastic cell culture flasks containing Leibovitz's L-15 medium (L-15) (Life Technologies, UK) supplemented with 10 % (v/v) foetal bovine serum (FBS) (PAA, France) at 20 °C. A SAV3 isolate from Atlantic salmon heart [27] was cultivated using CHH-1 cells in L-15 supplemented with 2 % FBS at 15 °C. The virus was harvested at 7 days post-infection (dpi) when the cytopathic effect (CPE) was observed. Quantification of virus stock was performed using the end-point dilution assay, and 50 % tissue culture infectious dose (TCID50) was calculated [28]. After this experiment was completed we discovered that the SAV3 isolate used was contaminated with low levels of infectious pancreatic necrosis virus (IPNV), as the isolate had earlier been passed through one round of infection in fish. However, IPNV contamination levels were relatively low (6 Ct values higher than SAV3). Anterior kidneys of IM Phase-A and BI Phase-A fish at 7, 14, 21 and 28 dpi were, therefore, analyzed for the presence of IPNV [29]. Twenty-five percent of the tested IM Phase-A fish were positive for relatively low levels of IPNV (average Ct value of 36), but all of the tested BI Phase-A fish were negative. Hence, co-infection with IPNV is not likely to have a major effect on the interpretation of the results of this study.
Experimental design
Both Phase-A and Phase-B experiments had a similar design, which is outlined in Fig. 1. One and eight weeks after seawater transfer of Phase-A and Phase-B fish, respectively, all the fish in 3 of the 12 tanks from each experiment were i.m. injected in the muscle in front of the dorsal fin and above the lateral line with 2 x 50 μl of SAV3. The titer of SAV3 in Phase-A and Phase-B was similar in the range of 2.0-3.7 x104 TCID50 per fish. These fish served as shedder fish, whose purpose was to shed SAV3 into the tank-water. The water in these tanks (referred to as shedder tanks) was monitored for the presence of SAV3 by RT-qPCR. On the day of challenge, 7 days (in Phase-A) and 8 days (in Phase-B) after injection of shedder fish, three treatment groups in triplicate tanks were created: (i) fish i.m. injected with cell culture medium from uninfected CHH-1 cells, serving as non-infected control (referred to as ‘CT group’); (ii) fish i.m. injected with SAV3 (referred to as the ‘IM group’); (iii) fish infected by bath immersion in seawater (as described below) containing SAV3 (referred to as the ‘BI group’). The IM groups in Phase-A and Phase-B were given relatively similar titer of SAV3 (2.6–3.7x104 TCID50 per fish). The experiments were carried out in strict accordance with guidelines and approved by Norwegian Animal Research Authority (NARA).
Bath immersion (BI) challenge model
The BI groups were bathed in seawater from the shedder tanks as follows. The water flow in the 3 shedder tanks was stopped for one hour, and the tanks were supplied with extra aeration. After this 1 h period, the shedder fish were removed from the tanks and replaced by new fish. These BI fish were kept in the aerated shedder tanks with no water flow for six hours. The oxygen level in all tanks was closely monitored when the water flow was stopped. After 6 h of bath immersion, the water flow was resumed with normal aeration, which diluted out and removed the remaining virus in the tanks within a short time.
Water sampling
Seawater sampling from the tanks, and up-concentration of SAV3 from the seawater samples, was performed [18] with some modifications. This water filtration technique is based on the VIRADEL (virus-adsorption-elution) method [30, 31] carried out by using electropositive, charge-modified, glass and cellulose (1MDS) filters [32, 33]. Briefly, one litre of seawater was collected from the tanks using sterile autoclaved screw-cap 1 L glass bottles. The water samples were vacuum filtered at a flow of 70–100 ml min−1 using a glass filtration system (MilliPore, USA.) through electropositive Zeta Plus™ 1MDS filters (Cuno Inc, USA.). After filtration, the filters were placed upside down in 55 mm petri-dishes containing 1 ml of lysis buffer (iPrep™ PureLink® Total RNA Kit, Invitrogen, USA.), and agitated at 500 rpm for 15 min on an orbital shaker. Four hundred microliters of the eluant was then transferred into a 1.5 ml tube and stored at -80 °C for RT-qPCR.
In the Phase-A experiment, water was collected from tanks containing IM fish at 2, 4, 6, 8, 10, 14, 18, 23 and 28 dpi, while water was collected from tanks containing the CT fish at 2, 4, 7, 10, 18 and 28 dpi. Water sampling in Phase-B was optimised based on the viral shedding profile seen in IM and CT groups in Phase-A. In the Phase-B experiment water was collected from tanks containing IM fish at 4, 8, 15 and 20 dpi, while water from tanks containing BI fish was collected at 1, 3, 5, 7, 10, 14 and 21dpi, and from tanks containing CT fish at 1, 8 and 15 dpi.
Tissue sampling
Eight fish per tank per sampling point were euthanized as previously described, and length and weight were recorded before blood and tissue sampling. Blood was collected at 1, 3, 7, 10, 14, 21, and 28 dpi in Phase-A with heparinized syringes (for plasma) and Phase-B without anticoagulant (for serum) from the caudal vein. Plasma and sera were collected by centrifugation (9500 g, 10 min) within 3 h of sampling or after storage at 4 °C overnight, respectively. Gills from Phase-A fish were sampled and analyzed for Na+, K+-ATPase (NKA) activity at 1, 3, 7, 10, 14, 21 and 28 dpi by collecting gill filaments in 100 μl of ice-cold SEI buffer (150 mM sucrose, 10 mM EDTA, 50 mM imidazole, pH 7.3) and snap freezing in liquid nitrogen. Since Phase-B fish were expected to be stabilized and little influence of infection was expected, gills from these fish were not analysed for NKA activity. Hearts were collected at 3, 7, 14, 21, and 28 dpi for RT-qPCR analysis. Hearts from day 7, 14, 21 and 28 were cut in two longitudinally and the half that included the ventricle with bulbus arteriosus was snap frozen in liquid nitrogen while the other half including the atrium from four fish per tank was fixed in 10 % neutral buffered formalin. Plasma, sera, gill filaments and hearts were stored at -80 °C until further analyses. In addition, pancreatic tissue associated with pyloric caeca was sampled from four fish per tank at 7, 14, 21, and 28 dpi and fixed in 10 % neutral buffered formalin.
RNA extraction and RT-qPCR
Total RNA was extracted from a homogenate of approx. 50 mg of heart tissue using an iPrep™ PureLink® Total RNA Kit (Invitrogen, USA.) with TRIzol® reagent (Ambion). The RNA isolation from filtered water eluants, plasma and serum samples was performed using the same kit with lysis buffer, according to the manufacturer’s instructions. The isolated RNA from plasma samples was lithium chloride precipitated to remove heparin which interfered with RT-qPCR. The AgPath-ID One-Step RT-qPCR Kit from Ambion, Life Technologies and a RT-qPCR assay targeting the SAV nsP1 gene were used for detection of SAV3 [34] with the following modification of the probe sequence FAM-5'-TCGAAGTGGTGGCCAG-MGB. The assay was performed with 200 ng of RNA (heart) or 2 μl of isolated RNA (plasma, sera and water), 400 nM of forward primer, 600 nM of reverse primer and 160 nM of probe in a total volume of 7 μl on a 384 well-plate. Amplification and fluorescence detection were measured by a 7900HT Fast Real-Time PCR system (Applied Biosystems) as recommended by the manufacturer. The threshold value for all samples was set to 0.1 and the amplification was run for 40 cycles. The elongation factor 1A (EFIAA) [35] was checked from a random selection of 25 % of heart samples from infected treatment groups representing all time-points of sampling to validate the quality of the RNA samples and all the samples showed stability and satisfactory levels of EFIAA (Ct value of 21–22).
Analysis of Na+,K + -ATPase (NKA) enzyme activity
In the Phase-A experiment, we analysed the NKA activity of gill filament samples from 12 fish per treatment at each time-point (1, 3, 7, 10, 14, 21, and 28 dpi) as representatives of each tank as described previously [15]. The NKA activity is expressed as μmol ADP mg protein−1 h−1.
Analysis of plasma cortisol
On each sampling day, plasma samples from 12 individual fish in each treatment group from both Phase-A and Phase-B experiments were analyzed for cortisol concentration by ELISA according to the manufacturer’s recommendations (IBL International, Hamburg, Germany).
Histology and immunohistochemistry (IHC)
The pancreas and heart tissues from all groups were fixed in 10 % neutral buffered formalin for 2 days before being processed and embedded in paraffin wax. Samples selected from fish with SAV3-positive hearts (identified using RT-qPCR) were sectioned at 3 μm using a Leica RM 2255 microtome (Leica Microsystems, Germany) and placed on SuperFrost® Plus slides (Menzel-Gläser, Germany). Selected histological sections (n = 3-9 from each group per sampling point of Phase-A and n = 2–4 from each group per sampling point of Phase-B) were stained with Haematoxylin-Erythrosin-Saffron (HES). The selected tissue sections were stained with an anti-E2 antibody to confirm the presence of SAV3. The paraffin sections were incubated at 60 °C for 30 min followed by de-waxing and dehydration with alcohol series and water. Antigen retrieval was carried out in 10 mM citrate buffer (pH 6.0) for 20 min followed by transferring to Tris-buffered saline (TBS, pH 7.4) using a 2100 Retriever model pressure cooker (Prestige Medical, England) according to the manufacturer’s instructions to unmask antigens. Blocking of non-specific antibody binding to sections was performed by using 5 % bovine serum albumin (BSA) in TBS for 20 min. All wash steps were carried out with TBS for 5 min until counterstaining. The primary antibody, a rabbit polyclonal anti-E2, was diluted 1: 200 in TBS containing 2.5 % BSA, and applied to the sections and incubated at 37 °C for 1 h, followed by biotinylated anti-mouse/rabbit immunoglobulin G, secondary antibody, and avidin-biotin alkaline phosphatase according to manufacturer’s recommendation (Vectastain universal ABC-AP kit; Vector Laboratories, California, USA). Sections were stained with the fuchsin substrate and chromogen system (Dako North America Inc., USA) and counterstained with Shandon’s haematoxylin. Positive immunostaining was visualised microscopically as a red/magenta colouration.
Data and statistical analysis
Fulton’s condition factor was calculated by 100 × weight (g)/ [fork length (cm)]3. The Ct values of heart samples, gill NKA activities and plasma cortisol levels were imported into Statistica 12 (StatSoft, Inc., OK, USA). Differences in SAV3 RNA levels in the hearts of IM Phase-A and Phase-B fish were tested with a Mann-Whitney U non-parametric test, whereas One-way ANOVA was applied to NKA activity and plasma cortisol data.