A molecular assessment of infectious agents carried by Atlantic salmon at sea and in three eastern Canadian rivers, including aquaculture escapees and North American and European origin wild stocks
Abstract
Infectious agents are key components of animal ecology and drivers of host population dynamics. Knowledge of their diversity and transmission in the wild is necessary for the management and conservation of host species like Atlantic salmon (Salmo salar). Although pathogen exchange can occur throughout the salmon life cycle, evidence is lacking to support transmission during population mixing at sea or between farmed and wild salmon due to aquaculture exposure. We tested these hypotheses using a molecular approach that identified infectious agents and transmission potential among sub-adult Atlantic salmon at marine feeding areas and adults in three eastern Canadian rivers with varying aquaculture influence. We used high-throughput qPCR to quantify infection profiles and next generation sequencing to measure genomic variation among viral isolates. We identified 14 agents, including five not yet described as occurring in Eastern Canada. Phylogenetic analysis of piscine orthoreovirus showed homology between isolates from European and North American origin fish at sea, supporting the hypothesis of intercontinental transmission. We found no evidence to support aquaculture influence on wild adult infections, which varied relative to environmental conditions, life stage, and host origin. Our findings identify research opportunities regarding pathogen transmission and biological significance for wild Atlantic salmon populations.
Introduction
Infectious agents such as viruses, bacteria, and other microparasites are ubiquitous in aquatic and marine environments (Marcogliese 2008; Lafferty 2017), yet their diversity among wild fish hosts remains largely undescribed. Logistical constraints of studying pathogens in the wild have limited our understanding of naturally occurring infections in wild fishes (Miller et al. 2014). This knowledge is necessary to anticipate how changing environmental conditions may affect the virulence of endemic agents, the introduction of exotic agents, and associated disease development (Burge et al. 2014), which have the potential to drive host population dynamics and the economics of fisheries (Selakovic et al. 2014; Johnson et al. 2015; Lafferty et al. 2015). Most information on fish disease has been derived from aquaculture settings where fish are more easily observed in later stages of disease (Bakke and Harris 1998). However, extrapolation of this information to wild fishes may be misleading as conditions experienced by cultured fish differ greatly from wild fish. A prime example is the Atlantic salmon (Salmo salar), which is a key cultured species with well-established relationships between infection dynamics and survival in captivity (Bakke and Harris 1998). While farmed salmon are fed, handled, and held at high densities, wild salmon must hunt for prey, avoid predators, migrate across dynamic environments, and are generally found at relatively lower densities throughout most of their life history. These differences likely contribute to disparate disease outcomes between wild and cultured salmon, even for the same pathogen. To inform future research on how pathogens may influence host population dynamics, evaluations are needed that characterize infectious agents carried by wild and cultured Atlantic salmon.
Since the late 1980s, Atlantic salmon populations have experienced large declines in abundance over much of their range (Parrish et al. 1998; Klemetsen et al. 2003). Wild Atlantic salmon begin their lives in fresh water, rear in natal rivers for one to seven years, and then migrate to marine feeding areas in the North Atlantic Ocean; at maturity, adults return to natal rivers to spawn and then can migrate back out to sea (Jonsson and Jonsson 2011). Survival throughout marine migrations has been shown to be a key factor in observed population declines, yet these life stages remain relatively understudied and the causes of mortality unknown (Hansen and Quinn 1998; Sheehan et al. 2012). Pathogen transmission dynamics in marine habitats and how infections contribute to wild salmon mortality at sea is even less well known (Hansen and Quinn 1998). Substantial mixing of stocks occurs in the Labrador Sea near Greenland (Chaput et al. 2018), including both North American and European origin fish (Reddin and Friedland 1999; Sheehan et al. 2012). This mixing not only provides an opportunity for infectious agents to spread among individuals, but also geographical areas since salmon will eventually migrate back to natal freshwater systems to spawn (Madhun et al. 2018; Vendramin et al. 2019). Introduction of emerging pathogens or pathogen strains through intercontinental exchange may pose new risks to otherwise threatened populations in a way that is not easily controlled by anthropogenic activities. Although the transmission of agents may or may not result in disease or reduced host survival, understanding if and where pathogen exchange occurs is a crucial first step toward characterizing impacts on wild Atlantic salmon across their range, including the influence of human activities like aquaculture.
Many wild Atlantic salmon stocks come into contact with aquaculture facilities during seaward or spawning migrations, which may result in infectious agent exchange between farmed and wild fish (Heggberget et al. 1993; Garseth et al. 2018). There is also a persistent issue of escapees from salmon farms mixing with wild stocks, posing further opportunities for pathogen transmission in addition to potential interbreeding and fitness consequences for wild salmon (Castellani et al. 2018). Modeling studies of wild Pacific salmon (Oncorhynchus spp.) on the west coast of Canada have demonstrated decreased wild salmon productivity in association with exposure to Atlantic salmon farms with high macroparasite (sea lice) densities (Krkošek et al. 2011; Peacock et al. 2013). Microparasites such as viruses, bacteria, and various metazoan species certainly have the potential to impact wild salmon productivity, especially under poor environmental conditions (Burge et al. 2014).
Despite recent advances in our understanding of the diversity of infectious agents hosted by salmon on Canada’s west coast (Bass et al. 2017; Di Cicco et al. 2017, 2018; Nekouei et al. 2018; Tucker et al. 2018), our knowledge of infectious agents affecting wild Atlantic salmon on the east coast remains scant. Infectious agent surveys can be used to direct future research toward quantifying specific host interactions; for example, similarities in the composition of agent species and genotypes hosted by wild and cultured Atlantic salmon may indicate farm–wild pathogen exchange (Olivier 2002; Johansen et al. 2011). Importantly, coinfection (multiple agent species in one host) and superinfection (multiple agent genotypes in one host) are common in wild animals and the dynamics of these communities are linked to host fitness outcomes (Martin et al. 2012; Alizon et al. 2013; Sofonea et al. 2015). Host health and performance, especially in the wild, can be impacted by shifts in coinfection or superinfection prior to the occurrence of detectable or typical tissue changes (pathology) associated with disease (Brassard et al. 1982; Wiik-Nielsen et al. 2016; Downes et al. 2018). This complexity warrants an approach beyond traditional diagnostics to characterize transmission events that can influence disease processes in wild fish.
Molecular tools are rapidly increasing our ability to describe the pathogen dynamics of wild animal populations and can be cost-effectively applied to quantify an array of infectious agents (e.g., Miller et al. 2016). The minimal tissue requirements of molecular approaches allow for nonlethal tissue biopsy, which is especially useful for studying populations of conservation concern (Archie et al. 2009). Studies of wild Pacific salmon in British Columbia, Canada, have successfully applied high-throughput polymerase chain reaction (HT-qPCR) for infectious agent screening and host response characterization (Jeffries et al. 2014; Miller et al. 2014; Bass et al. 2017). As this HT-qPCR tool was developed to include assays to pathogens impacting salmon worldwide, the same approach is amenable to application in Atlantic salmon on the east coast of Canada. Additional molecular techniques, such as next generation sequencing (NGS), can provide further insight into how and where pathogens are exchanged in the wild; for example, hosts that carry phylogenetically similar viral strains likely share a transmission source and (or) location (Stimson et al. 2019). RNA viruses are especially useful for characterizing pathogen transmission dynamics due to their relatively high strain variability across spatial and temporal gradients (Stimson et al. 2019). By combining qPCR and sequencing approaches, we can not only quantify similarity in coinfection profiles based on location (marine sub-adults and freshwater adults) and source (wild and cultured escapees), but also conduct phylogenetic analysis of viruses to identify evidence of transmission at sea or between farmed and wild fish.
We tested two hypotheses using a molecular approach: (H1) the Labrador Sea will comprise a melting pot of European and North American origin salmon as a potential area where pathogen exchange between fish of different continents occurs, and (H2) adult salmon exchange infectious agents with cultured salmon during spawning migration, resulting in similarity in infection profiles based on the proximity of their natal rivers to aquaculture. Atlantic salmon were collected from marine feeding grounds near Greenland (sub-adults) and from three eastern Canadian rivers (mature adults) with variable aquaculture influences: distant from aquaculture (Restigouche River, nonthreatened wild population), proximal to aquaculture (St. John River, threatened wild population), and aquaculture escapees (Magaguadavic River, where resident wild population is extirpated). Tissue samples were evaluated for the presence and loads of 44 viruses, bacteria, and other microparasites known or expected to cause disease in salmon worldwide using a high-throughput Fluidigm BioMark platform (Fluidigm Corporation, San Francisco, CA, USA) and assay panel (Miller et al. 2014, 2016). To identify potential natural (marine stock mixing) and anthropogenic (aquaculture-wild) transmission routes, we compared viral isolates from this study with published sequences obtained from Atlantic salmon in previous studies worldwide. Our objective was to provide baseline data to inform future studies of the transmission and disease dynamics of wild Atlantic salmon.
Methods
Sample collection, preservation, and transfer
Biological sampling at all fish collection sites included a fork-length measurement, external morphology assessment, scale and tissue sampling, and external observation for macroparasites. Tissues were sampled from fish (∼0.5 mg) using sterile tools and fixed in RNAlater (Ambion, Austin, Texas, USA; 1.5 mL). Gill, heart, and kidney biopsy samples (multi-tissue) were taken from fish in Greenland and at the Magaguadavic and Restigouche rivers in 2017, whereas only kidney samples were taken from Greenland-sampled fish in 2016 and only nonlethal gill biopsies from fish of the threatened St. John River population in 2017. Gill biopsies have been shown to comprise most infectious agents detected in multi-tissue HT-qPCR analyses (Teffer and Miller, 2019).
To obtain samples from offshore marine waters where fish from North American and European stocks mix, wild salmon were collected over two years by commercial fishers using gill nets in the Labrador Sea along the coast of Greenland (Fig. 1). Direct acquisition of freshly landed fish and tissue sampling took place at local markets in Paamiut and Maniitsoq, Greenland, in September of 2016 (N = 43; kidney only, Paamiut) and 2017 (N = 30; multi-tissue, Maniitsoq), respectively. Tissues collected in Greenland in 2016 were stored at 4 °C for 30 d and then −20 °C for 13 months. Tissues collected in Greenland in 2017 were stored at 4 °C for 24 h and then −20 °C for 2 months. Greenland-collected samples were transported from Greenland to Quebec, Canada, on ice (stored at −20 °C at night during 3 d transport), and then shipped on dry ice to the Atlantic Salmon Federation (ASF) headquarters in Chamcook, New Brunswick, Canada, where they were stored at −80 °C until analysis.
Fig. 1.
Salmon bearing rivers were sampled in 2017, including the Restigouche (low aquaculture influence; N = 30), St. John (high aquaculture influence; N = 30), and Magaguadavic rivers (aquaculture escapees; N = 17; Table 1, Fig. 1). The Restigouche River sampling site is isolated from aquaculture influence and located near the head of tide. Restigouche fish were lethally sampled between 12 June and 1 July 2017 with the collaboration of Listuguj Mi’gmaq fishers following chain of custody procedures for sample preservation (see below). Restigouche tissue samples were stored at 4 °C for 24 h and then at −18 °C for ≤18 d, then transported on ice to the ASF headquarters in Chamcook, New Brunswick, (≤24 h transport) and stored at −80 °C.
Table 1.
| Source location | Continent of origin | Year | Tissue type(s) | N | Fork length, mm (mean ± SD) |
|---|---|---|---|---|---|
| Labrador Sea near Greenland | North America | 2016 | Kidney | 10 | 654 ± 24 |
| 2017 | Multi-tissuea | 26 | 672 ± 55 | ||
| European | 2016 | Kidney | 33 | 618 ± 25 | |
| 2017 | Multi-tissuea | 4 | 668 ± 20 | ||
| Restigouche River | North America | 2017 | Multi-tissuea | 30 | 836 ± 67 |
| St. John River | North America | 2017 | Gill | 30 | 572 ± 31 |
| Magaguadavic River | North America | 2017 | Multi-tissuea | 17 | 653 ± 47 |
a
Gill, heart, and kidney.
In the St. John River, returning adult wild and hatchery (released at the juvenile stage) Atlantic salmon were sampled nonlethally for gill tissue at the Department of Fisheries and Oceans (DFO) Biodiversity Facility near the base of Mactaquac Dam situated 3 km above head of tide; this facility is approximately 100 km upstream from the river mouth and is proximal to the commercial salmon aquaculture industry. The St. John tissue samples were transported to the ASF headquarters on ice (≤2 h transport), stored at 4 °C for 24 h, and then stored at −80 °C.
The Magaguadavic River collection site is proximal to commercial Atlantic salmon aquaculture operations in the Bay of Fundy, and escaped salmon are a regular occurrence in the river (Carr 1995; Morris et al. 2008). Escapees were collected from a trap in a head of tide fish ladder, identified as escapees using external morphology and scale characteristics (Carr 1995), euthanized, and then transported on ice to ASF headquarters (20 min) for tissue sampling. Tissue samples were stored at ASF headquarters at 4 °C for 24 h and then stored at −80 °C.
Tissue samples from all locations were stored at the ASF headquarters in a −80 °C freezer for 50–255 d. All samples were shipped on dry ice to the DFO Pacific Biological Station, Nanaimo, British Columbia, on 1 February 2018 (1 d transport) and stored at −80 °C until analysis.
Laboratory protocols
Greenland fish were genotyped using genome-wide single-nucleotide polymorphisms (Jeffery et al. 2018) at the DFO Salmonids Section Population Genomic Lab to assign North American or European origin (Table 1). Infection profiles were evaluated at the DFO Molecular Genetics Laboratory, Pacific Biological Station, using the Fluidigm BioMark HT-qPCR platform and assay panel to quantify the presence and relative loads of 44 infectious agents in RNA extracted from preserved tissues (Table 2). Most assays included in the panel for this study have been analytically validated for specificity, sensitivity, repeatability, and reproducibility between platforms (Miller et al. 2016), with the exception of Atlantic salmon calicivirus (ASCV) and salmon gill poxvirus (SGPV), which were added after the initial panel was developed and validated (only specificity and sensitivity validated). Tissue preparation, nucleic acid extraction and normalization, cDNA synthesis, specific target amplification, incorporation of artificial control standards and processing controls, and dynamic array preparations were completed according to protocols described by Miller et al. (2016). The primers and probes used in this screening are listed in Table 2. Artificial positive controls (Chinook embryo cell control nucleic acids, infectious agent artificial control standards) and negative controls were included in the protocol and a second fluorescent NED-labeled dye (Applied Biosystems, Foster City, CA, USA) was included in all reaction chambers to detect laboratory contamination by artificial control standards. All singleplex HT-qPCR assays were run in duplicate on dynamic arrays. Limits of detection (LOD) specific to each assay (Miller et al. 2016; Table 2) were applied to the data at 95% detection confidence, which provides a measure of analytical sensitivity corresponding to the amount of analyte in a sample that is expected to produce a positive result 95% of the time. To be incorporated into the analysis, infectious agents needed to be detected in both duplicates at a quantification cycle (Cq) within the 95% LOD. HT-qPCR results are reported as copy number calculated using sample Cq (average of duplicates) and standard curves for each assay. We characterized infections as emerging versus endemic from a review of peer-reviewed and publicly available grey literature (e.g., government and organization reports); we classified the designation “emerging” for agents not previously known to occur in eastern Canada, recognizing that some may be endemic but simply not previously assessed. Throughout, we were careful not to assume the detection of an infectious agent was equivalent to the detection of disease.
Table 2.
| Agent | Type | Abbreviation | Limits of detection | Primer and probe sequences | Accession no. | Assay reference |
|---|---|---|---|---|---|---|
Atlantic salmon calicivirusa | Virus | ascv | 27.14 | F: ACCGACTGCCCGGTTGT R: CTCCGATTGCCTGTGATAATACC P: CTTAGGGTTAAAGCAGTCG | — | Gideon Mordecai |
Infectious hematopoietic necrosis virus | Virus | ihnv | 27.64 | F: AGAGCCAAGGCACTGTGCG R: TTCTTTGCGGCTTGGTTGA P: TGAGACTGAGCGGGACA | NC_001652 | |
Infectious pancreatic necrosis virus | Virus | ipnv | 27.63 | F: GCAACTTACTTGAGATCCATTATGCT R: GAGACCTCTAAGTTGTATGACGAGGTCTCT P: CGAGAATGGGCCAGCAAGCA | — | |
Infectious salmon anemia virus | Virus | isav | 26.12 | F: TGGGCAATGGTGTATGGTATGA R: GAAGTCGATGAACTGCAGCGA P: CAGGATGCAGATGTATGC | EU118822 | |
Salmonid herpesvirus | Virus | omv | 26.59 | F: GCCTGGACCACAATCTCAATG R: CGAGACAGTGTGGCAAGACAAC P: CCAACAGGATGGTCATTA | — | |
Piscine myocarditis virus | Virus | pmcv | 26.29 | F: AGGGAACAGGAGGAAGCAGAA R: CGTAATCCGACATCATTTTGTGA P: TGGTGGAGCGTTCAA | HQ339954 | |
Piscine reovirus | Virus | prv | 26.11 | F: TGCTAACACTCCAGGAGTCATTG R: TGAATCCGCTGCAGATGAGTA P: CGCCGGTAGCTCT | — | |
Salmon alphavirus | Virus | sav | 26.28 | F: CCGGCCCTGAACCAGTT R: GTAGCCAAGTGGGAGAAAGCT P: TCGAAGTGGTGGCCAG | AY604235 | |
Salmonid gill poxvirus | Virus | sgpv | 25.15 | F: ATCCAAAATACGGAACATAAGCAAT R: CAACGACAAGGAGATCAACGC P: CTCAGAAACTTCAAAGGA | — | |
Putative Totiviridae | Virus | toti | 25.87 | F: TCTGCGCGCTGCACCTA R: ATGCGGAGGAACTCACACACT P: CAAGTGCTACACTGCG | — | Gideon Mordecai |
Viral erythrocytic necrosis virus | Virus | ven | 24.85 | F: CGTAGGGCCCCAATAGTTTCT R: GGAGGAAATGCAGACAAGATTTG P: TCTTGCCGTTATTTCCAGCACCCG | — | |
Viral encephalopathy and retinopathy virus | Virus | venv | 26.21 | F: TTCCAGCGATACGCTGTTGA R: CACCGCCCGTGTTTGC P: AAATTCAGCCAATGTGCCCC | AJ245641 | |
Viral hemorrhagic septicemia virus | Virus | vhsv | 26.86 | F: AAACTCGCAGGATGTGTGCGTCC R: TCTGCGATCTCAGTCAGGATGAA P: TAGAGGGCCTTGGTGATCTTCTG | Z93412 | |
Aeromonas hydrophila | Bacterium | ae_hy | 28.67 | F: ACCGCTGCTCATTACTCTGATG R: CCAACCCAGACGGGAAGAA P: TGATGGTGAGCTGGTTG | AY165026 | |
Aeromonas salmonicida | Bacterium | ae_sal | 25.61 | F: TAAAGCACTGTCTGTTACC R: GCTACTTCACCCTGATTGG P: ACATCAGCAGGCTTCAGAGTCACTG | M64655 | Keeling et al. 2013 (modified) |
Flavobacterium psychrophilum | Bacterium | fl_psy | 29.46 | F: GATCCTTATTCTCACAGTACCGTCAA R: TGTAAACTGCTTTTGCACAGGAA P: AAACACTCGGTCGTGACC | — | |
Ca. Piscichlamydia salmonisa | Bacterium | pch_sal | 30.72 | F: TCACCCCCAGGCTGCTT R: GAATTCCATTTCCCCCTCTTG P: CAAAACTGCTAGACTAGAGT | EU326495 | |
Piscirickettsia salmonis | Bacterium | pisck_sal | 23.32 | F: TCTGGGAAGTGTGGCGATAGA R: TCCCGACCTACTCTTGTTTCATC P: TGATAGCCCCGTACACGAAACGGCATA | U36943 | |
Renibacterium salmoninarum | Bacterium | re_sal | 25.91 | F: CAACAGGGTGGTTATTCTGCTTTC R: CTATAAGAGCCACCAGCTGCAA P: CTCCAGCGCCGCAGGAGGAC | AF123890 | |
Rickettsia-like organism | Bacterium | rlo | 25.23 | F: GGCTCAACCCAAGAACTGCTT R: GTGCAACAGCGTCAGTGACT P: CCCAGATAACCGCCTTCGCCTCCG | EU555284 | |
Ca. Syngnamydia salmonisa | Bacterium | sch | 27.9 | F: GGGTAGCCCGATATCTTCAAAGT R: CCCATGAGCCGCTCTCTCT P: TCCTTCGGGACCTTAC | FJ897519 | |
Tenacibaculum maritimum | Bacterium | te_mar | 26.71 | F: TGCCTTCTACAGAGGGATAGCC R: CTATCGTTGCCATGGTAAGCCG P: CACTTTGGAATGGCATCG | — | |
Vibrio anguillarum | Bacterium | vi_ang | 26.41 | F: CCGTCATGCTATCTAGAGATGTATTTGA R: CCATACGCAGCCAAAAATCA P: TCATTTCGACGAGCGTCTTGTTCAGC | L08012 | |
Aliivibrio salmonicida | Bacterium | vi_sal | 25.84 | F: GTGTGATGACCGTTCCATATTT R: GCTATTGTCATCACTCTGTTTCTT P: TCGCTTCATGTTGTGTAATTAGGAGCGA | AF452135 | |
Yersinia ruckeri | Bacterium | ye_ruc | 28.13 | F: TCCAGCACCAAATACGAAGG R: ACATGGCAGAACGCAGAT P: AAGGCGGTTACTTCCCGGTTCCC | — | |
Paramoeba perurans | Amoeba | ne_per | 25.39 | F: GTTCTTTCGGGAGCTGGGAG R: GAACTATCGCCGGCACAAAAG P: CAATGCCATTCTTTTCGGA | EF216905 | |
Ichthyophthirius multifiliis | Ciliate | ic_mul | 23.7 | F: AAATGGGCATACGTTTGCAAA R: AACCTGCCTGAAACACTCTAATTTTT P: ACTCGGCCTTCACTGGTTCGACTTGG | IMU17354 | |
Gyrodactylus salaris | Fluke | gy_sal | 26.42 | F: CGATCGTCACTCGGAATCG R: GGTGGCGCACCTATTCTACA P: TCTTATTAACCAGTTCTGC | — | |
Spironucleus salmonicida | Flagellate | sp_sal | 26.05 | F: GCAGCCGCGGTAATTCC R: CGAACTTTTTAACTGCAGCAACA P: ACACGGAGAGTATTCT | AY677182 | |
Nanophyetus salmincola | Fluke | na_sal | 24.3 | F: CGATCTGCATTTGGTTCTGTAACA R: CCAACGCCACAATGATAGCTATAC P: TGAGGCGTGTTTTATG | AY269674 | |
Sphaerothecum destruens | Mesomycetozoea | sp_des | 26.5 | F: GGGTATCCTTCCTCTCGAAATTG R: CCCAAACTCGACGCACACT P: CGTGTGCGCTTAAT | AY267346 | |
Facilispora margolisi | Microsporidian | fa_mar | 30.55 | F: AGGAAGGAGCACGCAAGAAC R: CGCGTGCAGCCCAGTAC P: TCAGTGATGCCCTCAGA | HM800849 | |
Loma salmonae | Microsporidian | lo_sal | 25.42 | F: GGAGTCGCAGCGAAGATAGC R: CTTTTCCTCCCTTTACTCATATGCTT P: TGCCTGAAATCACGAGAGTGAGACTACCC | HM626243 | |
Paranucleospora theridiona | Microsporidian | pa_ther | 28.16 | F: CGGACAGGGAGCATGGTATAG R: GGTCCAGGTTGGGTCTTGAG P: TTGGCGAAGAATGAAA | FJ59481 | |
Ceratonova shasta | Myxozoan | ce_sha | 28.5 | F: CCAGCTTGAGATTAGCTCGGTAA R: CCCCGGAACCCGAAAG P: CGAGCCAAGTTGGTCTCTCCGTGAAAAC | AF001579 | |
Myxobolus arcticus | Myxozoan | my_arc | 26.8 | F: TGGTAGATACTGAATATCCGGGTTT R: AACTGCGCGGTCAAAGTTG P: CGTTGATTGTGAGGTTGG | HQ113227 | |
Myxobolus insidiosus | Myxozoan | my_ins | 26.43 | F: CCAATTTGGGAGCGTCAAA R: CGATCGGCAAAGTTATCTAGATTCA P: CTCTCAAGGCATTTAT | EU346375 | |
Parvicapsula kabatai | Myxozoan | pa_kab | 25.58 | F: CGACCATCTGCACGGTACTG R: ACACCACAACTCTGCCTTCCA P: CTTCGGGTAGGTCCGG | DQ515821 | |
Parvicapsula minibicornis | Myxozoan | pa_min | 29.62 | F: AATAGTTGTTTGTCGTGCACTCTGT R: CCGATAGGCTATCCAGTACCTAGTAAG P: TGTCCACCTAGTAAGGC | AF201375 | |
Parvicapsula pseudobranchicolaa | Myxozoan | pa_pse | 25.16 | F: CAGCTCCAGTAGTGTATTTCA R: TTGAGCACTCTGCTTTATTCAA P: CGTATTGCTGTCTTTGACATGCAGT | AY308481 | |
Tetracapsuloides bryosalmonae | Myxozoan | te_bry | 24.98 | F: GCGAGATTTGTTGCATTTAAAAAG R: GCACATGCAGTGTCCAATCG P: CAAAATTGTGGAACCGTCCGACTACGA | AF190669 | |
Trypanoplasma salmositica | Protozoan | cr_sal | 24.34 | F: TCAGTGCCTTTCAGGACATC R: GAGGCATCCACTCCAATAGAC P: AGGAGGACATGGCAGCCTTTGTAT | — | |
Dermocystidium salmonis | Protozoan | de_sal | 25.49 | F: CAGCCAATCCTTTCGCTTCT R: GACGGACGCACACCACAGT P: AAGCGGCGTGTGCC | U21337 | |
Ichthyophonus hoferi | Protozoan | ic_hof | 24.17 | F: GTCTGTACTGGTACGGCAGTTTC R: TCCCGAACTCAGTAGACACTCAA P: TAAGAGCACCCACTGCCTTCGAGAAGA | AF467793 | |
Si:dkey-78d16.1 protein | Host reference | hkg | 45 | F: GTCAAGACTGGAGGCTCAGAG R: GATCAAGCCCCAGAAGTGTTTG P: AAGGTGATTCCCTCGCCGTCCGA | — |
a
Agents that have not been previously described as occurring in this region in peer-reviewed or publicly available grey literature.
Note: F, forward primer; R, reverse primer, P, probe
Sequencing
A subset of Atlantic salmon samples in which piscine orthoreovirus (PRV-1) or infectious salmon anemia virus (ISAV) were detected were selected for sequence analysis using NGS to validate HT-qPCR detections and conduct phylogenetic analyses. All PRV detections described in this study refer to the PRV-1 genotype. Two North American origin Atlantic salmon collected in offshore waters near Greenland in 2017 were positive for ISAV, but only one had sufficient sequencing coverage for analysis. Three Atlantic salmon samples in which PRV-1 was detected were sequenced: one North American origin, marine-collected fish sampled at the Maniitsoq market, Greenland, in 2017 (J3575_NAM, multi-tissue, PRV-1 Ct of 12.4); one European origin fish sampled at the Paamiut market, Greenland, in 2016 (J3611_EUR, kidney only; PRV-1 Ct of 13.3); and one aquaculture escapee collected in the Magaguadavic River (J3542_MAG, multi-tissue; PRV-1 Ct of 19.1). PRV-1 was chosen as an ideal candidate to evaluate transmission potential at sea and between wild and farmed Atlantic salmon as it has widespread prevalence across the range of Atlantic salmon (and beyond) and was detected in both European and North American origin fish at sea and in aquaculture escapees in this study. To our knowledge, this is the first publication of the full genome sequence for PRV obtained from hosts collected in eastern Canada.
All NGS samples were processed on the same v2 300 Illumina MiSeq sequencing run PRV-1. Samples (J3575_NAM, J3611_EUR, and J3542_MAG) generated ∼2.9, 3.0, and 2.4 mol/L post-trim reads, respectively, with average quality scores of 35.0 or greater. We applied target enrichment for all known viral genomes that infect salmon via the SureSelectXT RNA Direct NGS target workflow (Agilent, Santa Clara, California, USA). A custom set of RNA target enrichment probes (120 base pairs (bp) in length and staggered along the exome or viral RNA) were designed to the genomes of salmonid, relevant fish, and other emerging viruses that were included in our infectious agent HT-qPCR screening platform. These sequences (435.384 kbp) and subsequent bait oligonucleotides included the PRV-1 and ISAV genomes. In the case of ISAV, multiple sequences were included for some segments to represent the various genogroups, hyper polymorphic region (HPR0), and sequences that were <85% homologous. Baits that failed the SureSelect quality assurance or quality control parameters and (or) significantly matched salmonid genes via blast searches were removed, leaving the final set of enrichment probes at 15 609.
We prepared the RNAseq library with the SureSelect Strand-Specific RNA library Prep kit (Agilent, Santa Clara, California, USA) according to manufacturer’s instructions. The adaptor-ligated samples were purified with the Agencourt AMPure XP system (Beckman Coulter, Brea, California, USA). High sensitivity (HS) DNA chips were run on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California, USA) to determine the final library size and the Qubit dsDNA HS kit (Invitrogen, Carlsbad, California, USA) was used to determine the concentration. Hybridization of the adapted cDNA library with the viral SureSelect bait capture library (Agilent, Santa Clara, California, USA) was performed at 65°C for 24 h according to manufacturer’s instructions. The cDNA library or capture library hybrids were captured on streptavidin magnetic beads and purified with the Agencourt AMPure XP system (Beckman Coulter, Brea, California, USA). Index tags were added to the postcaptured libraries through 14 rounds of amplification and purified using the Agencourt AMPure XP system (Beckman Coulter, Brea, California, USA). HS DNA chips were run on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California, USA) to determine the final library size, and the concentration was determined using the Qubit dsDNA HS kit (Invitrogen, Carlsbad, California, USA). Sample libraries were normalized to 4 nmol/L and denatured and diluted to obtain a final library of 20 pmol/L. The Atlantic salmon enriched RNAseq libraries were processed on one paired end v2 300 bp kit on the Illumina MiSeq System (Illumina, San Diego, California, USA), which included a 10% PhiX Control v3 Library spike-in to improve overall run quality.
Sequence analysis was performed using the Partek Flow software (Partek Inc., St. Louis, Missouri, USA). Adaptors and bases with Phred quality scores <30 were trimmed from both ends and reads less than 25 bp were removed. The remaining reads were aligned to the PRV genome segments of the Norwegian isolate Salmo/GP-2010/NOR (Palacios et al. 2010) using the BWA-MEM (Burrows Wheeler Aligner) software and algorithm with default parameters (Li 2013). SAMtools variant caller was utilized to determine SNPs using the default settings (Li et al. 2009; Li 2011). The consensus sequences were compared against all available sequences in GenBank (Benson et al. 2003) using the BLAST program (blast.ncbi.nlm.nih.gov/Blast.cgi) via the National Center for Biotechnology Information (Altschul et al. 1990) to identify their closest matches across each segment.
ISAV sequences were de novo assembled to enable unbiased assembly of a deletion which is known to occur on segment 6 of the genome that has been associated with virulence (Gagné and LeBlanc 2018). Adapters were removed using Trimmomatic and host-associated reads were removed by alignment to the Atlantic salmon genome using the Burrows–Wheeler aligner (Davidson et al. 2010; Li and Durbin 2010; Bolger et al. 2014). Unmapped sequences were de novo assembled using SPAdes (Bankevich et al. 2012). The viral genomic sequences were aligned using MUSCLE (within Geneious) (Edgar 2004), and an approximately maximum-likelihood phylogenetic tree were constructed using FastTree (Price et al. 2010). The trees were displayed and annotated using Figtree (available at tree.bio.ed.ac.uk/software/figtree/) and ggtree (Yu et al. 2018). PRV and ISAV segment consensus sequences for all sequenced samples were deposited into GenBank under the accession number series MN106286 to MN106316.
Statistical methodology
To quantify and visualize differences in infectious agent communities based on host group membership (Restigouche, St. John, Magaguadavic, Greenland-collected North American origin, and Greenland-collected European origin), we used nonmetric multidimensional scaling (NMDS) analysis and permutational multivariate analysis of variance (PERMANOVA; Fig. 2). Infectious agent loads were normalized to the maximum copy number for each agent (i.e., the quotient of each load value and the maximum load value of that agent in the study) prior to NMDS and PERMANOVA analysis. Any agent detected in fewer than two individuals was removed from the analysis as well as any host with no agents detected to reduce statistical bias (N = 120 fish included in NMDS). Community composition was also visually represented by comparing how agent prevalence and community composition differed among groups; this was achieved by plotting the prevalence of each agent as a proportion of the cumulative prevalence (i.e., the sum of the proportional prevalence of all agents; Fig. 3).
Fig. 2.
Fig. 3.
As a cumulative infection metric, relative infection burden (RIB) was calculated for each fish as a composite score incorporating aspects of pathogen richness and loads:where for a given fish, the copy number of the ith infectious agent (Li) is divided by the maximum copy number within the population for the ith infectious agent (Lmaxi) and then summed across all agents (m) detected in the given fish (Bass et al. 2019). We used RIB as a community-level metric of cumulative infection burden, which comprises load, prevalence, and richness information when averaged across a host group to determine if fish sampled in the Labrador Sea in different years (2016 and 2017) could be pooled within continental stock assignment (North American or European origin). Linear regression was used to compare infection profiles between years within stock groups (i.e., identify any significant effect of sampling year within stock groups). Because of the right-skewed distribution of RIB, this variable was log-transformed to meet the assumptions of normality. Generalized linear models (GLM) were used to identify differences in infectious agent richness among groups (total unique agents per host). Where sample sizes allowed (≥10 detections in each group), analysis of variance was used to identify load differences between groups.
(1)
Results
Genotyping of marine-collected Atlantic salmon and annual infection differences
Among fish captured in the Labrador Sea near Greenland (marine-collected; Fig. 1), both European and North American stocks were represented in 2016 (kidney tissue only; European origin: N = 33, North American origin: N = 10) and 2017 (multi-tissue; European origin: N = 4, North American origin: N = 26). RIB did not differ significantly between years within continental stock groupings (p > 0.05 for both stock groups), so data from 2016 to 2017 were pooled within stock groups for subsequent analyses and reporting. Year-specific data for marine-collected fish can be found in Table S1.
Prevalence and load differences among collection locations and strains
Fourteen infectious agents were detected overall (both marine and freshwater samples), including four species of bacteria, five viruses, and five other microparasite species, commonly occurring as multiple infections within hosts (Table 3; Figs. 2, 3). PERMANOVA identified a significant effect of group (i.e., collection location and continental origin) on infection profiles (r2 = 0.35, p < 0.01). Three NMDS axes sufficiently comprised variation in infection community profiles (stress = 0.07), with the majority of group separation comprised by the first two axes (Fig. 2). River-collected wild fish (Restigouche, St. John) showed the highest degree of overlap in NMDS positioning and the largest 95% confidence interval areas, suggesting high individual variability in infection profiles of freshwater-collected wild adults relative to other groups. Greenland-collected North American and European origin fish had similar NMDS positioning, though European origin fish loaded higher on axis 1 (furthest from river-collected wild fish). The escapee group was isolated from other groups on the NMDS plot, largely due to strong viral agent influences on infection profiles.
Table 3.
| Marine: N. American origin (N = 36) | Marine: European origin (N = 37) | Magaguadavic River (N = 17) | Restigouche River (N = 30) | St. John River (N = 30) | Total | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Agent | N | Mean | SD | N | Mean | SD | N | Mean | SD | N | Mean | SD | N | Mean | SD | N | |
| Parasites | |||||||||||||||||
P. pseudobranchicola | 21 | 6463 | 15 914 | 4 | 538 | 384 | 7 | 1927 | 2716 | 14 | 1674 | 3924 | 3 | 1087 | 837 | 49 | |
| T. bryosalmonae | 12 | 31 547 | 71 331 | 18 | 351 773 | 656 993 | 5 | 71 395 | 99 215 | 0 | — | — | 0 | — | — | 35 | |
| P. theridion | 12 | 3395 | 5968 | 7 | 3697 | 5195 | 1 | 181 | — | 10 | 2807 | 6367 | 4 | 3527 | 6171 | 34 | |
| I. hoferi | 2 | 385 | 482 | 0 | — | — | 0 | — | — | 15 | 1 608 948 | 3 587 305 | 10 | 338 474 | 433 839 | 27 | |
| S. destruens | 1 | 1485 | — | 0 | — | — | 0 | — | — | 0 | — | — | 0 | — | — | 1 | |
| Bacteria | |||||||||||||||||
| Ca. P. salmonis | 3 | 83 350 | 130 482 | 2 | 248 725 | 194 166 | 9 | 148 466 | 255 231 | 11 | 1 989 479 | 4 397 990 | 9 | 109 302 | 221 325 | 34 | |
| F. psychrophilum | 0 | — | — | 0 | — | — | 0 | — | — | 11 | 3614 | 5175 | 19 | 2284 | 4426 | 30 | |
| A. salmonicida | 0 | — | — | 0 | — | — | 0 | — | — | 7 | 2 805 611 | 7 186 680 | 3 | 139 877 | 241 677 | 10 | |
| Ca. S. salmonis | 0 | — | — | 0 | — | — | 0 | — | — | 4 | 85 | 53 | 0 | — | — | 4 | |
| Viruses | |||||||||||||||||
| PRV | 2 | 5980 | 8363 | 1 | 18 799 | — | 13 | 24 | 33 | 0 | — | — | 0 | — | — | 16 | |
| ASCV | 0 | — | — | 0 | — | — | 10 | 188 248 | 383 841 | 1 | 10 | — | 0 | — | — | 11 | |
| ISAV | 2 | 218 | 264 | 0 | — | — | 0 | — | — | 0 | — | — | 0 | — | — | 2 | |
| SGPV | 0 | — | — | 0 | — | — | 2 | 227 | 81 | 0 | — | — | 0 | — | — | 2 | |
| VERV | 1 | 22 | — | 0 | — | — | 0 | — | — | 0 | — | — | 0 | — | — | 1 | |
| Richness | — | 1.6 | 1.1 | — | 0.9 | 0.9 | — | 2.8 | 1.3 | — | 2.4 | 1.3 | 1.6 | 1.3 | — | ||
| RIB | — | 0.23 | 0.5 | — | 0.14 | 0.3 | — | 0.21 | 0.34 | — | 0.36 | 0.4 | 0.12 | 0.23 | — | ||
Note: Total positive detections (N) and the mean and standard deviation (SD) of agent copy numbers, richness (total unique agents per host), and relative infection burden (RIB) are shown for each agent relative to the sampling location. RIB is a composite metric that incorporates richness and load information from all agents detected in each host; PRV, piscine orthoreovirus; ASCV, Atlantic salmon calicivirus; ISAV, infectious salmon anemia virus; SGPV, salmonid gill poxvirus; VERV, encephalopathy and retinopathy virus.
Among marine-collected adult Atlantic salmon, nine infectious agent species were detected, with greater richness among the North American origin group (nine agents; mean 1.6 agents per individual) than European origin fish (five agents; mean 0.9 agents per individual; GLM: p = 0.004; Fig. 3). Among marine-collected fish, all agents detected in the European origin group (Parvicapsula pseudobranchicola, Tetracapsuloides bryosalmonae, Paranucleospora theridion, Candidatus Piscichlamydia salmonis, and PRV-1) were also detected in the North American origin group; four additional agents were detected in the North American group (Ichthyophonus hoferi, Sphaerothecum destruens, ISAV, viral encephalopathy, and retinopathy virus (VERV)). Mean RIB was greater for North American origin marine fish (0.23) than European origin (0.14), but not significantly different (F = 0.81, p = 0.37). In the marine environment, prevalence among European origin fish was dominated by T. bryosalmonae, whereas P. pseudobranchicola was the most prevalent agent among North American fish (Fig. 3). Half of the European origin fish had positive detections of T. bryosalmonae (49%), whereas P. theridion (19%), P. pseudobranchicola (11%), Ca. P. salmonis (5%), and PRV-1 (3%) were detected at lower prevalence. North American origin fish also carried P. pseudobranchicola (58%), T. bryosalmonae (33%), and P. theridion (33%) at moderate prevalence, whereas Ca. P. salmonis (8%), I. hoferi (6%), PRV-1 (6%), ISAV (6%), VERV (3%), and S. destruens (3%) were detected at lower prevalence. Except for T. bryosalmonae (European origin loads were greater; F = 5.07, p = 0.032), agent loads were similar between continental origin groups at sea (nonsignificant at p > 0.05 or insufficient detections for comparison).
Adult Atlantic salmon were collected from freshwater and brackish sites in the Magaguadavic (N = 17), Restigouche (N = 30), and St. John (N = 30) rivers in eastern Canada (Fig. 1). Aquaculture escapees sampled in the Magaguadavic River (multi-tissue) were unique in their infection profiles, which included three viruses, one bacterial species, and three other microparasites (Fig. 3). The Magaguadavic infection profile more closely resembled that of marine-collected fish than the wild river-sampled groups. Among escapees, PRV-1 was the most prevalent agent (76%), followed by ASCV (59%), Ca. P. salmonis (53%), and P. pseudobranchicola (41%), T. bryosalmonae (29%), salmonid gill poxvirus (SGPV) (12%), and P. theridion (6%).
The infection profile of returning adults from the St. John population (gill tissue only, threatened population with high aquaculture influence) was similar to those from the Restigouche River population, but with slightly lower richness (six agents). Agents detected in St. John fish included Flavobacterium psychrophilum (63%), I. hoferi (33%), Ca. P. salmonis (30%), P. theridion (13%), P. pseudobranchicola (10%), and Aeromonas salmonicida (10%).
The Restigouche population (multi-tissue, low aquaculture influence) had the greatest infectious agent richness of freshwater-sampled groups (eight agents), which included all agents detected in the St. John population plus additional bacterial, viral, and myxozoan agents. Among Restigouche fish, I. hoferi (50%), and P. pseudobranchicola (47%) were detected in approximately half of the sampled population, whereas Ca. P. salmonis (37%), F. psychrophilum (37%), P. theridion (33%), and A. salmonicida (23%) occurred at moderate prevalence, and salmon Candidatus Syngnamydia salmonis (13%) and ASCV (3%) at low prevalence.
The primary characteristics that differentiated infection profiles among river-collected groups were the enhanced viral richness and T. bryosalmonae prevalence in the Magaguadavic River sampled escapees relative to wild populations and the lower infectious agent richness in the St. John population. Infectious agent richness of the St. John population (mean = 1.6) was significantly lower than that of the Restigouche population (mean = 2.5; GLM: p = 0.02), whereas the escapee richness (Magaguadavic: mean = 2.8) was most similar to the Restigouche group (p = 0.45), but with very different agent composition. No significant differences in individual agent loads were identified, either due to low sample sizes (low power due to few positive detections) or nonsignificant ANOVA results. RIB was lowest overall in the St. John population but did not significantly differ among river-sampled groups (ANOVA: p = 0.51; Table 3).
PRV and ISAV sequence analysis
Analysis of segment six of the ISAV genome revealed that the strain identity in two North American origin fish at sea belonged to the “European” genotype (Gagné and LeBlanc 2018). One isolate (J3577) included the full-length HPR0 (identified by the absence of a deletion on segment six of the genome); the other isolate (J3574) did not have HPR coverage to reveal type.
From the PRV NGS analysis, a reference-guided assembly of J3575_NAM generated 228 846 total alignments (7.8% of total reads) to the Norwegian PRV Salmo/GP-2010/NOR 10 segment reference genome (Palacios et al. 2010), which resulted in 100× coverage for >99% of the genome and an average depth of 2568 reads. J3611_EUR generated 635 925 total alignments (25.7% of total reads) to the reference genome, which resulted in 100× coverage for >99% of the genome with an average depth of 7009 reads. Finally, J3542_MAG generated 7762 total alignments (0.3% of total reads) to the reference genome, which resulted in 30× coverage for >79% of the genome with an average depth of 85 reads. Over all three samples, segments M2 (outer shell) and S1 (outer clamp/NS p13) displayed the greatest variation relative to the reference genome (97.2%/60–61 SNPs and 96.8%–97.2%/30–35 SNPs, respectively). For J3575_NAM and J3611_EUR, segments S3 (NS RNA) and L1 (core shell) displayed the least variation (99.5%/6 SNPs and 99.3%/28 SNPs, respectively), whereas for J3542_MAG, segments L3 (core RdRp) and L1 (core shell) displayed the least variation relative to the reference genome (99.6%/17 SNPs and 99.4%/23 SNPs, respectively).
BLAST searches and phylogenetic analysis of the S1 segment reveal that all three samples cluster with PRV-1a along with all other Canadian strains to date (Figs. 4, S1). The PRV genome sequences of J3575_NAM and J3611_EUR were virtually identical over all segments except for one SNP in both L3 (core RdRp) and S1 (outer clamp/NS p13; Fig. 4). These sequences were isolated from Atlantic salmon sampled at Maniitsoq market in September 2017 and Paamiut market in September 2016, respectively, after being caught in offshore marine waters near Greenland. The PRV genome sequence of J3542_MAG (aquaculture escapee caught in the Magaguadavic River in September 2017) was >99.0% homologous to the two Greenland-collected samples across all segments with the exception of segment M3 (NS factory), which was a 98.9% homologous. PRV isolates from the two marine-collected (Greenland) hosts clustered with wild and cultured Atlantic salmon from Norway (Garseth et al. 2013), wild fish from Denmark, and cultured fish in the Faroe Islands (Denmark). Alternatively, the escapee isolate J3542_MAG was most similar in the S1 segment (99.7%) to VT03022017-69 (accession No. MF946300) isolated from an Atlantic salmon recovered after escaping a farm near McNutts Island, Nova Scotia, Canada, in March 2017 (Kibenge et al. 2017; F. Kibenge, personal communication).
Fig. 4.
Discussion
We used HT-qPCR and viral sequencing to characterize variability in the infectious agent profiles and potential transmission dynamics of wild and cultured Atlantic salmon. We identified 14 agents in the tissues of salmon collected as sub-adults in the Labrador Sea near Greenland or as mature adults in three eastern Canadian rivers. Five of these agents, to our knowledge, have not been described as occurring in eastern Canada in peer-reviewed or publicly available literature, which included two bacteria (Ca. P. salmonis and Ca. S. salmonis), one virus (ASCV), one microsporidian (P. theridion), and one myxozoan parasite species (P. pseudobranchicola). SGPV and PRV-1 have only been reported to the International Council for the Exploration of the Sea (ICES) as occurring in this region (ICES 2018), and the S1 segment sequence of PRV-1 was isolated from a fish in Nova Scotia and is available in GenBank, but its geographic source is labeled only as “Canada” (Kibenge et al. 2017; F. Kibenge, personal communication 2019). To our knowledge, this is the first publication of the full genome sequence for PRV obtained from hosts collected in eastern Canada. Our results identify several opportunities for future research and a need to improve our knowledge of infectious agent transmission dynamics and disease potential among wild and cultured Atlantic salmon.
There were three key findings in this study. First, we identified both North American and European origin fish in marine waters off the coast of Greenland, providing further evidence to support this feeding area as a multi-continental melting pot (Hansen and Quinn 1998; Sheehan et al. 2012; Chaput et al. 2019). The similar infectious agent composition between North American and European origin hosts sampled in Greenland, and the finding of virtually identical PRV-1 genome sequences in fish of different continental origins supports the hypothesis of inter-continental transmission of pathogens in North Atlantic feeding areas where stocks mix (Madhun et al. 2018; Vendramin et al. 2019). Second, the high degree of similarity of the PRV-1 genome sequenced from two independent aquaculture escapees in eastern Canada suggests a common source or transmission of PRV-1 within aquaculture facilities that was distinct from the two wild fish sequenced in this study. Third, we found no significant effect of aquaculture proximity on infection profiles of wild returning adult salmon sampled in the St. John and Restigouche rivers of New Brunswick, Canada. Below we present an expanded discussion around these three findings.
Marine transmission potential between continental stocks
Phylogenetic analysis of the three PRV-1 detections sequenced in our study has uncovered potential transmission pathways for PRV-1 (and possibly other agents) between Europe and the Atlantic coast of North America. Nearly identical sequences of PRV-1 were isolated from European and North American origin fish sampled from marine feeding grounds near Greenland. This finding supports the hypothesis that ocean feeding grounds, where fish from different continents converge, provide a natural pathway of agent transmission between Europe and North America (Gagné and LeBlanc 2018; Madhun et al. 2018; Vendramin et al. 2019). We found high homology between sequences of PRV-1 isolated from two escaped farm salmon in eastern Canada in 2017, one collected from the Magaguadavic River in New Brunswick (this study) and the other recovered in Shelburne Harbour, Nova Scotia (Kibenge et al. 2017; F. Kibenge, personal communication). PRV-1 S1 sequences from both aquaculture escapees differed from those isolated from wild fish at sea (Greenland-collected), and PRV-1 was not detected in either wild river-sampled population in this study. It is worth noting that the PRV-1 variants observed both in Greenland and in eastern Canada all clustered with the Norwegian “wild-type” variant, classified in some studies as PRV-1a (Kibenge et al. 2019) that, based on the S1 segment, is divergent from PRV-1b, has been proposed to be of higher virulence (Dhamotharan et al. 2019) and has been shown to be the causative agent of heart and skeletal muscle inflammation (HSMI) (Wessel et al. 2017). However, HSMI has been diagnosed in farmed Atlantic salmon in western Canada in association with PRV-1a (Di Cicco et al. 2017). We propose that natural routes of transcontinental transmission favor movements of less virulent pathogen strains, allowing more time, for example, for European source hosts to migrate to sea and transmit the virus to North American hosts, and for infected North American hosts to then survive their migration back to natal rivers, thereby completing the cycle of intercontinental exchange.
We detected and confirmed with genome sequencing the avirulent HPR0 strain of ISAV in a North American origin fish collected at sea near Greenland. ISAV is in the family Orthomyxoviridae and the virulent form of the virus (HPRΔ) can be highly pathogenic in aquaculture settings (Lovely et al. 1999). ISAV was first detected in Atlantic Canada in 1996, with sequence analysis showing three separate emergences in North America, including avirulent (HPR0) and virulent (HPRΔ) forms (Gagné and LeBlanc 2018). The virus has been observed in wild and cultured salmon in eastern Canada and the USA (Bouchard et al. 1999 2001; Ritchie et al. 2001; Olivier 2002) and detected at low prevalence (<1%) in escaped aquaculture fish in Norway (Madhun et al. 2017). In wild fish, most if not all detections have shown no evidence of disease (including challenged hosts); therefore, it is assumed to be the avirulent HRP0 strain that is affecting asymptomatic wild hosts (Plarre et al. 2005; Gustafson et al. 2018). It is not known whether wild fish have been affected by virulent strains of ISAV, which can develop spontaneously from the avirulent strain through a deletion in segment 6 (Nylund et al. 2003; Godoy et al. 2013). The virulent strain causes acute disease and is therefore unlikely to be detected in sampling of wild fish. No detections of ISAV were found in European origin fish; therefore, we could not compare ISAV sequences between hosts of different continental origins to characterize the potential for its intercontinental exchange at marine feeding areas.
Another virus, VERV, was detected in one North American origin fish at sea. This piscine nodavirus has a wide geographic range, including coastal waters of New Brunswick, Canada, where it has been described at extremely low prevalence in wild winter flounder (Pleuronectes americanus) (Barker et al. 2002). Susceptibility of Atlantic salmon to VERV infection and disease has been demonstrated following intraperitoneal challenge (Korsnes et al. 2005) but not via cohabitation (Korsnes et al. 2012). The detection of VERV in an Atlantic salmon in eastern Canada and at low prevalence and variable loads in wild and farmed salmon in western Canada (Tucker et al. 2018; Laurin et al. 2019) warrants its continued monitoring in wild fish to confirm low susceptibility and virulence.
Among fish collected in Greenland, overall infection profiles were quite similar between continental stock origin groups, generally including the same agents but at a higher richness in North American origin fish. Key differences between European and North American stocks were associated with the prevalence of T. bryosalmonae and P. pseudobranchicola. Tetracapsuloides bryosalmonae is a prevalent parasite endemic in eastern Canada (Khan 2009) that can cause proliferative kidney disease, primarily at elevated water temperatures (Bettge et al. 2009). The absence of this agent in returning adult salmon in this study is interesting given its moderate prevalence in fish at sea. Parvicapsula pseudobranchicola is a prevalent myxosporean parasite originally characterized in Norway and newly detected in eastern Canada; it affects the pseudobranch of fishes as a generally low-virulence agent but can cause runting in cultured fish (Nylund et al. 2018). Both of these parasites cannot be horizontally transmitted at sea (Morris and Adams 2006; Nylund et al. 2018), so disparate relative prevalence of these two agents depending on continental origin (freshwater stage) is unsurprising.
Infection profiles of wild and escaped farm salmon in rivers
Among adult salmon in three New Brunswick rivers, the infection profile of a group of aquaculture escapees in the Magaguadavic River was unique relative to two wild salmon populations from the Restigouche and St. John rivers. Contrary to our expectation that proximity to aquaculture would enhance infection severity of wild populations through acquisition of agents that thrive in culture settings, infectious agent loads and richness were highest in the Restigouche population, which was furthest from aquaculture influence. St. John fish could only be nonlethally sampled for gill as opposed to gill, heart, and kidney from Restigouche and escapee fish. However, gill has been shown to have equal or greater infectious agent richness than multi-tissue pools (Teffer and Miller 2019). Only one virus, ASCV, was detected (in just one host) in the Restigouche River. The only other fish with ASCV detections in this study were aquaculture escapees. ASCV is common in Norwegian fish culture (Mikalsen et al. 2014) and is the most commonly detected virus in farmed salmon in western Canada (K. Miller, unpublished data). ASCV is often detected as a coinfection with other agents (e.g., with PRV), but studies to date have had variable and often inconclusive findings for its independent pathological effects (Mikalsen et al. 2014; Wiik-Nielsen et al. 2016). Interestingly, a related fish calcicivirus in baitfish was associated with clinical disease only when coinfected with a second virus (Mor et al. 2017). Given its widespread prevalence, future studies should evaluate sequence variation among ASCV isolates across geographic regions and examine the potential role ASCV plays in disease progression in coinfections.
Aquaculture escapees had the second highest overall infectious agent richness with few bacterial species and the highest prevalence of viruses of any group. Greater than half of escapees carried PRV-1 and ASCV, often as coinfections; for example, most fish positive for ASCV were also positive for PRV-1, and both SGPV-positive hosts were also positive for PRV-1 and ASCV. SGPV has previously been described in Norway and the Northeast Atlantic Ocean (Nylund et al. 2008; Garseth et al. 2018). In this study, we detected SGPV solely in escaped aquaculture fish, and its occurrence in eastern Canada has been reported to the ICES (ICES 2018). The composition of bacterial and microparasite species hosted by aquaculture escapees was more similar to marine-collected than river-sampled fish, despite hosts being collected in a freshwater environment (i.e., exposed to freshwater infectious agents). Closer alignment of infection profiles between escapees and marine-collected fish may be due to aquaculture practices that inhibit some infections (e.g., antibiotics for bacterial agents) as well as alternate dietary sources (e.g., fish feed versus wild, potentially infected prey) and extended coastal residence.
Microparasite species composition in wild Atlantic salmon in the Restigouche and St. John rivers was highly congruent with infection profiles described in adult Pacific salmon in western Canadian rivers (Bass et al. 2017; Teffer et al. 2017). Exceptions to this included T. bryosalmonae and S. destruens, which were absent in river-collected Atlantic salmon in this study. The consistent prevalence of P. theridion (aka Desmozoon lepeophtherii, a candidate causative agent of proliferative gill inflammation) across sampling locations in this study aligns with the widespread occurrence of its alternate sea louse host (Lepeophtheirus salmonis) in eastern Canada (Carr and Whoriskey 2004; Sveen et al. 2012). High prevalence among wild Atlantic salmon in this study suggests that P. theridion is not highly virulent in this system. Lower prevalence of this agent among marine-collected fish versus freshwater adults supports adult infections as enhanced in the nearshore environment (Hendricks 1972; Rand 1992). The relatively low prevalence (one fish) of P. theridion in aquaculture escapees is interesting and potentially due to farm practices that inhibit exposure or spore development (e.g., low-temperature environment) (Sanchez et al. 2000; Sveen et al. 2012). We were unable to find any peer-reviewed literature describing the presence of P. theridion in eastern Canada despite records of its occurrence and association with sea lice in Scotland, Norway, and the eastern Pacific (Freeman and Sommerville 2011; Nylund et al. 2011; Jones et al. 2012; Sveen et al. 2012; Miller et al. 2014). Ichthyophonus hoferi, a protistan parasite, was detected at moderate prevalence among wild salmon in both rivers and is endemic in the Northwest Atlantic Ocean and coastal waters of eastern Canada (Hendricks 1972; Rand and Cone 1990; Rand 1992). Ecological impacts of this agent should be explored as Ichthyophonus spp. infections can affect host swimming ability, especially under suboptimal environmental conditions (e.g., high temperature) (Tierney and Farrell 2004; Kocan et al. 2009).
The Restigouche and St. John populations hosted two Chlamydiae species (Ca. P. salmonis and Ca. S. salmonis) that have not yet been described in eastern Canada. Candidatus Syngnamydia salmonis is a newly described species potentially associated with gill disease in Norway (Nylund et al. 2015) and has been detected intermittently among wild and aquaculture salmon on the west coast of Canada (Miller et al. 2014; Thakur et al. 2018). Candidatus Piscichlamydia salmonis is known to affect Atlantic salmon in aquaculture (Norway, Ireland) and farmed Arctic char (Salvelinus alpinus) in the USA and Canada (Draghi et al. 2004, 2010). The loads of these bacteria described in this study are unlikely to have been pathogenic, as the density of epitheliocysts would need to be extremely high to affect cell function and host respiration (Pawlikowska-Warych and Deptuła 2016). Other bacterial species detected in this study included F. psychrophilum and A. salmonicida. Flavobacterium psychrophilum is a common bacterial agent with a global distribution across temperate zones; its virulent strains can be pathogenic at low temperatures (<16 °C) (Holt 1987; Nilsen et al. 2014). Aeromonas salmonicida is the causative agent of furunculosis, endemic in eastern Canada (Foda 1973) and can be highly virulent. Preventative vaccination for A. salmonicida is generally applied in aquaculture (Mitchell and Rodger 2011), which may explain the absence of these bacteria in escapees in this study. Both of these bacterial agents can contribute to secondary infections following dermal injury (Svendsen and Bøgwald 1997; Janda and Abbott 2010; Starliper 2011; Teffer et al. 2017).
Conclusions
We present for the first time a quantitative molecular screening of dozens of infectious agents in wild and escaped Atlantic salmon in offshore feeding areas of the Northwest Atlantic Ocean and three eastern Canadian rivers. Our results offer baseline coinfection and viral phylogenic data that provide insight into potential transmission dynamics among wild Atlantic salmon stocks at sea and evidence to support natural incursion of infectious agents from Europe to North America. Continued study of marine infection dynamics is warranted to confirm this natural transatlantic transmission route for infectious agents, which would introduce a managerial challenge for infectious disease control if pathogenic effects result from this exchange.
This study was undertaken to improve baseline knowledge of infectious agents carried by wild and escaped cultured Atlantic salmon and investigate transmission potential through the use of phylogenetic analysis of viral isolates. We cannot assign pathology to any infectious agents detected in our study as host health and performance were not evaluated. As with any study of wild animals, it is also possible that heavily infected fish or those carrying highly pathogenic agents died or were predated prior to sampling (Miller et al. 2014). We were limited in our comparative analysis because of disparity in tissue types sampled across sites but included these informative results as a starting point for future hypothesis testing on transmission dynamics in eastern Canadian waters. Our understanding of the mechanisms and frequency of infectious agent transmission among wild fishes is still in its infancy, especially for highly migratory and offshore marine hosts like Atlantic salmon and for pathogens that can cross continental borders via marine exchange. Molecular tools can be used to rapidly advance this knowledge and, combined with telemetry approaches and experimental studies, can improve our understanding of the disease ecology of Atlantic salmon throughout their range.
Acknowledgements
We would like to acknowledge and thank the fish market employees in Maniitsoq and Paamiut, Greenland, who provided us with a space to sample as well as their help and collaboration, and special thanks to the fishermen who participated in this study, including those in Paamiut (Jens Hanseeraq Kleist and Fritz Mikaelsen) and Maniitsoq (Efraim Heilmann, Frans Petersen, Daniel Heilmann, Hendrik Heilmann, and Qulutannguaq Moller). Restigouche River samples were provided by Listuguj fishers who collaborated closely with Dr. Gillis to meaningfully contribute to this study.
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Information & Authors
Information
Published In
FACETS
Volume 5 • Number 1 • January 2020
Pages: 234 - 263
Editor: Vance L. Trudeau
History
Received: 21 August 2019
Accepted: 28 January 2020
Version of record online: 23 April 2020
Copyright
© 2020 Teffer et al. This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Data Availability Statement
All relevant data are within the paper and Supplementary Material.
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Author Contributions
JC, EBB, and KMM conceived and designed the study.
JC, AT, AS, IB, DD, C-AG, and EBB performed the experiments/collected the data.
AKT, JC, AS, GM, and KMM analyzed and interpreted the data.
JC, IB, DD, C-AG, EBB, and KMM contributed resources.
AKT, JC, DD, C-AG, EBB, GM, and KMM drafted or revised the manuscript.
Competing Interests
The authors have declared that no competing interests exist.
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Amy K. Teffer, Jonathan Carr, Amy Tabata, Angela Schulze, Ian Bradbury, Denise Deschamps, Carole-Anne Gillis, Eric B. Brunsdon, Gideon Mordecai, and Kristina M. Miller. 2020. A molecular assessment of infectious agents carried by Atlantic salmon at sea and in three eastern Canadian rivers, including aquaculture escapees and North American and European origin wild stocks. FACETS.
5(1): 234-263. https://doi.org/10.1139/facets-2019-0048
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