Sample collection
Between August 2016 and November 2019, 425 sampling events were conducted opportunistically (based on availability and accessibility) at 64 Atlantic salmon net-pen productions sites in British Columbia, Canada. We grouped the sites into 9 geographically distinct regions to encompass groups of farm sites that were within 10 Nautical miles (NM) of one other, while regional boundaries have at least 15 NM (often larger) between the closest sites in adjacent regions (Fig. 1; Additional file 1).
Fish tissues were collected as part of 302 of the 425 total sampling events from all three commercial Atlantic salmon producers operating in the study region. Typically, 3–20 fish were collected per sampling event and tissues from each fish were screened for the presence of PRV-1 RNA. Seawater was collected as part of 151 sampling events (typically 1–2 samples per event) and benthic sediment was collected as part of 3 sampling events (3–4 samples collected per event) which were also screened for PRV-1 RNA. All seawater and benthic samples were collected from sites operated by a single producer (designated Producer A in this study). Organs were sampled for histopathology as part of 169 sampling events; these included sites operated by all three producers and was used to identify heart inflammation in populations with known or deducible PRV-1 positive or negative status (i.e., individuals from populations testing or previously testing positive for PRV-1 were presumed positive, and individuals from populations at or prior to testing negative were presumed negative). A detailed inventory of all sampling events is provided in Additional file 1. All sampling was conducted under the supervision of an industry fish health manager and/or veterinarian consistent with animal welfare practices of Canadian salmon aquaculture.
In addition to commercial site visits, samples were also collected as part of an experimental ocean net-pen study conducted between June, 2018 and February, 2019 at the Fisheries and Ocean Canada (DFO) Pacific Biological Station (49.2079° N, 123.9599° W) as previously described [15]. During this experimental trial, blood was collected from cohorts of Atlantic salmon held in either duplicate 4,300 L tanks of UV-sterilized seawater, duplicate 4,300 L tanks of raw seawater sourced from Departure Bay, or in a 100 m3 experimental net-pen site approximately 300 m offshore in Departure Bay [15]. The experimental net-pen site had been devoid of cultured Atlantic salmon for more than 10 years and is geographically separated from the nearest commercial Atlantic salmon net-pen by more than 37 nautical miles (> 70 km). This provided an opportunity to identify putative non-aquaculture related PRV-1 transmission within a 9-month time frame (Fig. 1). Blood samples were collected at 28-day intervals (n = 20 per population per time point) for a period of 252 days and screened for the presence of PRV-1. Animal care and sample collection in the DFO experimental systems was performed in strict accordance with the recommendations set out by the Canadian Council on Animal Care (CCAC) guide to the care and use of experimental animals and all live animal protocols were approved by the Pacific region animal care committee (animal use protocol number: 18–010).
Blood and tissue collection
All specimens of Atlantic salmon were collected as either recently deceased commercial net-pen mortalities or as live fish dip-netted directly from the commercial or experimental populations. Netted fish were euthanized by a percussive blow to the head and fresh mortalities (denoted by the industry as ‘silvers’) were selected based on gill pallor indicative of death having occurred less than 12 h prior to collection. In all instances, either blood, heart or a combination of spleen, heart and head kidney were aseptically removed for the purpose of PRV-1 molecular screening. Sample type was variable between sites and years; however, in all instances, samples were collected and processed similarly. Blood (100–1000 µL) was obtained from a caudal puncture using a 1 mL syringe and 22-gauge needle and transferred to a 1.5 mL microtube on ice and frozen at -80 °C within 4–6 h. Internal organ samples (50–100 mg) were aseptically excised and preserved in either 1 mL of 70% ethanol or 1 mL of RNAlater™ storage solution (Sigma Aldrich) and frozen at -80 °C within 4–6 h. For histopathology, samples of heart and skin/skeletal muscle (including the lateral line) were preserved directly in 10% Neutral Buffered Formalin and stored at room temperature.
Seawater and benthic sediment collection
Seawater sampled from within Atlantic salmon net-pen farm sites were collected over the top 10 m using a Watermark® 2.2 L vertical polycarbonate water sampler. Immediately upon collection, the seawater was transferred into a sterile 1 L Nalgene bottle. Either a 250 uL subsample was transferred to a 2 mL sterile screwcap tube or the entire 1 L Nalgene was placed on ice depending on transportation and logistic limitations, and taken to the laboratory where it was frozen at -80 °C until further processing. Benthic sediment samples were acquired from underneath the net-pens using a grab sampler. Upon retrieval, sediment samples were transferred to sterile 50 mL tubes and kept on ice until being placed at -80 °C upon receipt at the laboratory.
PRV-1 RNA detection
Blood and tissue screening
PRV-1 L1 RNA was detected in blood, heart or pooled spleen, heart and head kidney samples by real-time quantitative PCR (qPCR) at one of four diagnostic laboratories. The Fisheries and Ocean Canada Pacific Biological Station Aquatic Animal Health Research Laboratory (DFO-AAH) in Nanaimo British Columbia screened all samples collected from Producer A. The Washington State University Washington Animal Disease Diagnostic Laboratory (WADDL) in Pulman, Washington, USA screened all samples collected from Producer B. Samples collected from Producer C were screened at either the British Columbia Animal Health Centre (BC-AHC) in Abbottsford, British Columbia, Canada or the British Columbia Center for Aquatic Health Sciences (BC-CAHS) in Campbell River, British Columbia, Canada.
In all instances, laboratory screening was conducted based on previously published methods [7, 16, 17]. Specific to screening conducted at DFO-AAH, total RNA was extracted from 100 μL blood or ~ 50 mg tissues in TRIzol Reagent (Life Technologies) as per manufacturer’s instructions that implemented a 5 mm steel bead and TissueLyser II (Qiagen) operating for 2 min at 25 Hz. A portion of eluted RNA (1.0 μg) was denatured for 5 min at 95 °C, immediately cooled to 4 °C, and reverse-transcribed using a High-Capacity cDNA Reverse Transcription kit (Life Technologies) following the manufacturer’s instructions. Resulting cDNA was used directly as template for qPCR analysis in a StepOne-Plus real-time detection system (Applied Biosystems) using previously described primers and TaqMan probe [16]. Each reaction contained 400 nM primers and 300 nM TaqMan probe, 1X TaqMan Universal Master Mix and 1 μL cDNA template within each 15 μL reaction. Cycling conditions included an initial incubation of 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Samples were assayed in duplicate and were considered positive if both technical replicates reported a Ct value < 40 cycles, inconclusive if only one technical replicate reported a Ct value < 40 cycles, or negative if both technical replicates failed to fluoresce beyond the preset threshold (∆ Rn 0.01) in 40 cycles. PRV RNA quantification was determined in each positive instance by serial dilution of a 482 bp double-stranded DNA gBLOCK fragment (Integrated DNA Technologies) consisting of sequence targeted by the qPCR primer and probe [2]. A seven-step tenfold dilution series of the gBLOCK fragment spanning a dynamic range of 10–107 target copies per reaction was incorporated in duplicate into each run.
Molecular detection of PRV-1 RNA conducted at WADDL, BC-AHC and BC-CAHS was obtained as a fee-for-service. Reports were provided to DFO scientists distinguishing samples as either positive or negative for PRV-1 RNA as determined by qPCR. Relative target quantities (e.g., Ct or estimated target copy number) were not consistently provided by these laboratories and therefore not considered in this study.
Seawater screening
All seawater samples were collected at Producer A sites and screened for the presence of PRV-1 L1 RNA at the DFO-AAH by one of two methods. (1) Frozen water samples were thawed at 4 °C and 0.25 mL was transferred to a 1.5 mL tube containing 0.75 mL of TRIzol LS (Life Technologies) with RNA extracted following the manufacturers protocol and PRV testing completed as described above. To increase sensitivity, a second (2) method was used on some samples which utilized thawing the samples at 4 °C and combining 10 mL of sample with 30 mL of ammonium sulphate preservation solution (4 M ammonium sulphate, 25 mM sodium citrate, 10 mM EDTA; pH 7.5). Samples were then inverted 30-40X to mix and centrifuged at 12,000 × g for 10 min at 4 °C. Supernatant was discarded and the pellet containing viral particles was suspended in 1 mL of TRIzol from which RNA was purified, reverse transcribed, and assessed by qPCR as described above. The efficiency of PRV-1 RNA recovery was verified for both methods via spike-in additions of cesium chloride purified PRV-1 in a four-step tenfold dilution spanning a dynamic range of 105–102 spike-in copies per triplicate seawater sample.
Benthic sediment screening
All sediment samples were screened for the presence of PRV L1 RNA at the DFO-AAH. PRV-1 RNA was extracted from approximately 2–4 g of sediment using a RNeasy PowerSoil Total RNA Kit (QIAGEN) following the manufactures instructions and eluted in 50 µL of Solution SR7. Approximately 1 µg of RNA was reverse transcribed and subjected to qPCR analysis as described above. The efficiency of PRV-1 RNA recovery was verified by spike-in additions of small volumes of highly infected Atlantic salmon blood into raw presumptively PRV-free sediment collected from the Nanaimo River estuary in a four-step tenfold dilution series spanning a dynamic range of 105–102 estimated spike-in copies per sample.
Histopathology
Organs preserved in 10% NBF were routinely embedded into paraffin, sectioned at 3 µm, and stained with H&E for light microscopy [4]. Organs from most fish included heart, liver, head kidney, trunk kidney, spleen, intestinal ceca, mesenteries, brain, gill, and skin/skeletal muscle. Samples were submitted by salmon farm fish health staff for routine histopathology as part of the normal diagnostic caseload of the BC-AHC. During the 3 years that project data were collected, five different pathologists provided diagnostic results; none of the pathologists were told that their diagnoses were part of a research project until > 2 years after the last report was competed. Of the 779 hearts subjected to histopathology, 126 (16%) included PRV PCR results as part of the case; pathologists examined the other 653 hearts while blinded to information about the PRV-1 infection status. For heart histopathology, thirteen indices were scored as either being none/not present (0), mild (1), moderate (2) or severe (3). This included the two inflammatory indices of lymphohistiocytic epicarditis (EPL) and endocarditis (ENL) that are most commonly associated with a diagnosis of HSMI; we combined these to give a cumulative heart inflammatory score (0 = none; 1–2 = mild, 3–4 = moderate, 5–6 = severe). HSMI-like heart inflammation has previously been defined as having cumulative heart scores of 4–6 by this method [7, 14]. Micrographs of heart and skeletal muscle inflammation at each score defined in this study are demonstrated in Zhang et al. [12] and Polinski et al. [7]. Also, for each fish, the lead pathologist had listed the lesions or infectious agents that fit in the summary category “Cause/marker of significant morbidity/death.” For this study, we identified all cases in which the pathologists included inflammatory heart lesions in this summary category.
Statistical analysis
A Pearson correlation coefficient (r) and associated p-value was calculated for time at sea relative to PRV-1 prevalence for the 302 sampling events that sampled fish tissues. PRV-1 relative blood loads (Ct) were compared between normal, moribund, and mortality Producer A specimens by one-way analysis of variance. Relative PRV-1 loads were compared between heart and blood samples in fish which had both samples taken by a paired t-test. Relative PRV-1 blood loads were compared in individuals with known heart inflammation scores (none, mild, moderate, or severe) by one-way analysis of variance. For individuals with matched heart histopathology and PRV-1 RNA blood load results, heart inflammation severity scores were compared between PRV positive and PRV negative populations by Mann–Whitney U and Kolmogorov Smirnov nonparametric tests to assess relative ranks and cumulative distributions, respectively. All statistical analyses were conducted using Graphpad Prism 9.