Canadian Aquaculture R&D Review 2015

Fish Health

PATHOGEN SUSCEPTIBILITY OF SOCKEYE SALMON – PHASE 1: INFECTIOUS SALMON ANEMIA (ISAV) AND ALPHAVIRUS (SPDV)

Sockeye Salmon (Oncorhynchus nerka) is the third most common Pacific salmon species, after Pink and Chum Salmon. The Fraser River salmon run has experienced declines in productivity since the 1990s. The reasons for this decline remain speculative. The Commission of Inquiry into the Decline of Sockeye Salmon in the Fraser River was launched as a consequence. One recommendation of the report is that “The DFO should undertake or commission research into the health of Fraser River Sockeye Salmon, including: the hypothesis that diseases are transmitted from farmed salmon to wild Sockeye”.

The CFIA-led, BC surveillance was launched in 2012. Thousands of samples were tested for ISAV and none was found. Although the testing meets the statistical requirements to eventually declare disease freedom, it does not look at the susceptibility of Pacific salmon species to the pathogens targeted. The risk of introduction is minimal but the consequences of an introduction or of an outbreak in a farm are not predictable without this data. We propose to examine disease resistance or susceptibility of Sockeye to various pathogens known to affect Atlantic Salmon, e.g., ISAV and SPDV (or Salmon Alphavirus). With the expertise in DFO, along with high level biocontainment facilities, this type of disease research can proceed and will generate essential knowledge for better management practices.

Apr. 2014–Mar. 2018

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP)

Project Lead: Nellie Gagné (DFO)

Project Team: Francis Leblanc, Phil Byrne (DFO); Diane Morrison (Marine Havest Canada)

Contact: Nellie.Gagne@dfo-mpo.gc.ca

Immunostained ISAV infected cells. Photo: Mélanie Roy

Bio-containment facility. Photo: Nellie Gagné

STUDY OF GENOMIC DIVERSITY IN AEROMONAS SALMONICIDA, THE ETIOLOGICAL AGENT THAT CAUSES FURUNCULOSIS, TO ESTABLISH ITS RESISTOME, EPIDEMIOLOGICAL MARKERS AND POTENTIAL TREATMENTS

Increased knowledge of the pathogen will aid Canadian aquaculture productivity while enabling growers to: (1) make appropriate use of antibiotics during treatment; (2) better track the pathogen’s location; and (3) suggest alternatives to antibiotics as treatment.

The A. salmonicida bacterium is the infectious agent that causes furunculosis in salmonids (salmon, trout, Arctic Charr, etc.). Controlling this disease, which is very harmful to the aquaculture industry, can prove to be quite demanding and fruitless, mainly because of the logistic constraints of the vaccination and the very frequent resistance of A. salmonicida to several antibiotics. We are therefore studying the genomic diversity of A. salmonicida to better understand its virulence and its antibiotic resistome. Through familiarization with this diversity, tools and alternative treatments for preventing or curing furunculosis can be created. In concrete terms, we are developing a kit for quickly diagnosing antibiotic resistance. We are also studying the action of mobile DNA elements in the evolution, host adaptation, and geographic distribution of A. salmonicida, and their potential activation by various treatments, including the effect of certain essential oils with a view to developing a treatment. It is also our intent to verify the potential of bacteriophages (viruses that infect bacteria) as a cure for furunculosis. Furunculosis is a recurrent disease that is hard to control and, as such, all of these approaches must be considered in order to control it.

Nov. 2012–Mar. 2019

Funded By: Natural Sciences and Engineering Research Council of Canada (NSERC) Co-Funded By: Ressources Aquatiques Québec (RAQ), MAPAQ (Innovamer program); Société de recherche et de développement en aquaculture continentale (SORDAC)

Project Lead: Steve Charette (U Laval)

Project Team: Antony Vincent, Valérie Paquet, Mélanie Trudel, Katherine Tanaka, Jean-Guillaume Edmond-Rheault, Sabrina Attéré (U Laval)

Collaborators: Nicolas Derome, Michel Frenette (U Laval); Andrée Lafaille (U Montréal)

Contact: Steve.Charette@bcm.ulaval.ca

www.amibe.org

ESTIMATING THE POTENTIAL FOR WATERBORNE TRANSMISSION OF INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) BETWEEN SALMON FARMS AND WILD SOCKEYE IN THE DISCOVERY ISLANDS, BRITISH COLUMBIA

Results from this study will provide further tools to manage infectious hematopoietic necrosis virus, and allow DFO to ensure the protection of farmed and wild fish health by determining optimal locations for aquaculture sites.

In British Columbia, infectious hematopoietic necrosis virus is responsible for major economic losses in Atlantic Salmon aquaculture operations. Due to the significance of this pathogen, it is important to understand the risk of, and factors affecting, viral dispersion from infected sites. To this end, this study is developing a viral dispersion model for the Discovery Islands, an area home to multiple net-pen salmon farms. Accurate geo-spatial predictions of risk for IHNV transmission are simulated through the coupling of IHNV transmission parameter estimates as reported (PLoS ONE 8(12):e82296) with a recently developed hydrodynamic ocean circulation model for the Discovery Islands (Atmosphere-Ocean 50(3):301-306). Additionally risk estimates are not only quantified for disease transmission potential between farms, but also between farmed Atlantic and wild Sockeye Salmon. Utilizing empirical data obtained through controlled laboratory exposure studies, the susceptibility of saltwater phase Sockeye Salmon to IHN disease is determined. Ultimately, temporally- and spatially-evolving viral concentration maps are combined with the lab-determined minimum infectious dosages to estimate the infection connectivity among farms and risk of virus spread to wild fish.

Jun. 2013–Apr. 2015

Funded By: DFO-Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Marine Harvest Canada; Grieg Seafood; Cermaq

Project Lead: Kyle Garver (DFO)

Project Team: Mike Foreman (DFO)

Collaborators: Barry Milligan, Diane Morrison, Peter McKenzie

Contact: Kyle.Garver@dfo-mpo.gc.ca

Discovery Islands aerial photo. Photo: Kyle Garver (DFO)

DETERMINATION OF CRYPTOSPORIDIUM SPP. OOCYST LEVELS IN THE HILLSBOROUGH RIVER, PRINCE EDWARD ISLAND

Contamination of oysters occurs predominantly in coastal or estuarine environments with wastewater sewage discharge and agricultural run-off from farms. Cryptosporidium spp. is a zoonotic protozoan parasite that has been detected in many shellfish species in both fecal-contaminated and clean oyster growing areas across the globe. Despite this fact, no data are available on oocyst concentrations in harvest-zone waters or in oysters harvested in Prince Edward Island. An assessment of the levels of this parasite in areas where the health authorities permit oyster harvesting is an important public health concern.

The primary objective of this project is to determine the concentrations of Cryptosporidium oocysts in water from three different shellfish harvest zones (prohibited, restricted, and approved) during the spring season after a rainfall event (which would indicate a higher risk of contamination). Some oyster sampling will be done concurrently to compare the concentrations of the parasite with findings from unpublished data by Willis et al. (2012) and to provide information about the accumulation of oocysts in oysters in the field.

We hypothesize that: (1) The number of oocysts will be greatest in the prohibited zones, followed by restricted zones, with zero prevalence in approved zones, and; (2) the level of oocysts in the water will be increased by rainfall events.

Our results will be linked to a risk assessment model for the probability of human illness due to consumption of contaminated oysters with Cryptosporidium spp. in PEI.

May 2014–Apr. 2015

Funded By: Canada Excellence Research Chair (CERC) – Aquatic Epidemiology, UPEI

Project Lead: Spencer Greenwood (UPEI)

Project Team: Javier Sanchez, Henrik Stryhn, T. McClure, Jeffrey Davidson, Jessica Willis (UPEI)

Collaborators: Juan Aguirre Garcia

Contact: sgreenwood@upei.ca

INFECTIOUS SALMON ANEMIA VIRUS SUSCEPTIBILITY AND HEALTH STATUS OF WILD VERSUS CULTURED ATLANTIC SALMON: A COMPARATIVE STUDY

There are serious concerns about the status of wild Atlantic Salmon in Atlantic Canada, and many populations are designated threatened or endangered by COSEWIC. The potential interactions between cultured and wild salmon in areas where they coexist are a primary concern. The health status and disease resistance of wild Atlantic Salmon (wAS) is unknown, whereas information for cultured Atlantic Salmon (cAS) is abundant. For example, while Infectious Salmon Anemia Virus (ISAV) remains a recurrent problem for the salmon aquaculture industry in Atlantic Canada, with outbreaks detected in Nova Scotia (NS) and Newfoundland (NL) since 2012, knowledge regarding the prevalence of this virus in wild populations as well as the potential transmission between wild and cultured stocks is lacking.

This project proposes to use in vivo disease challenges and next generation sequencing (NGS) technologies to compare the susceptibility of wild Atlantic Salmon stocks (Saint John River, Inner Bay of Fundy, Miramichi, and Margaree river stocks) and cultured stocks (Saint John River origin) to ISAV. The use of NGS (RNASeq) will enable us to look at genetic differences and measure immune responses and general health status, which could potentially explain differences in susceptibility (if observed). Additionally, we will examine the evolution rate of ISAV by looking at full ISAV sequences in tissues thoughout the course of the in vivo challenges.

Apr. 2014–Mar. 2017

Funded By: DFO – Program for Aquaculture Regulatory Research (DFO – PARR)

Project Lead: Nellie Gagné (DFO)

Project Team: Francis Leblanc, Gérald Chaput, Steven Leadbeater, Royce Steves (DFO)

Collaborators: John Whitelaw (DFO); Mark Hambrook (Miramichi Salmon Association); Darryl Murrant (NS Dept Fish & Aquaculture, Margaree fish hatchery)

Contact: Nellie.Gagne@dfo-mpo.gc.ca

Atlantic Salmon in quarantine (tanks). Photo: Nellie Gagné (DFO)

INVESTIGATING PROBIOTIC BACTERIA AND THEIR BACTERIOCINS AS PART OF A DISEASE MANAGEMENT STRATEGY IN SALMON AQUACULTURE

This research will offer the first comprehensive assessment of the antibiotic properties of known and unknown bacteriocins and, as such, constitutes an investigation into a novel category of drug treatment. The results of this research will help inform disease management strategies to minimize the impact of pathogens and ultimately improve fish health.

The susceptibility of farmed salmon to bacterial disease and sea lice (Lepeophtheirus salmonis and Caligus species) is a health management issue for the aquaculture industry. Currently, antibiotics and antiparasitics are used to treat bacteria and sea lice, respectively, however, there are concerns regarding the effectiveness and long term sustainability of these methods. This research project will evaluate the potential for probiotic bacteria (microorganisms associated with beneficial effects to humans and animals) and bacteriocins (antimicrobial, naturally occurring compounds produced by certain bacteria) to aid in the reduction of antibiotics currently used to treat bacterial diseases in salmon. Another point of focus will be on the potential of Bacillus and Paenibacillus bacterial species to aid in reducing the use of chemical therapeutants, such as emamectin benzoate (SLICE®), as a treatment of sea lice infections in salmon.

Apr. 2014–Mar. 2015

Funded By: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) Co-Funded By: Cermaq Canada; Marine Harvest Canada Inc.; National Strategic Research and Engineering Council (NSERC)

Project Lead: Simon Jones (DFO)

Project Team: Peter McKenzie (Cermaq Canada); Diane Morrison (Marine Harvest Canada Inc.); Marije Booman (DFO); John Vederas, Lynn McMullen (U Alberta)

Collaborators: Cermaq Canada; Marine Harvest Canada Inc.

Contact: Simon.Jones@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

Mature female sea louse (Lepeophtheirus salmonis) with egg strings. Photo: Emily Nelson (DFO)

DOES CHALLENGE WITH THE BRITISH COLUMBIA STRAIN OF PISCINE REOVIRUS AFFECT ATLANTIC OR SOCKEYE SALMON?

This study suggests PRV from the Pacific Coast of British Columbia is of low pathogenicity in Atlantic and Sockeye Salmon.

Piscine reovirus (PRV) was first discovered in Norwegian farmed Atlantic Salmon with the disease heart and skeletal muscle inflammation (HSMI). While fish with HSMI generally carry higher loads of PRV, clinically healthy fish in Norway also carry the virus. PRV is geographically widespread, occurring in the Atlantic, as well as in wild and farmed salmon in the Pacific Ocean where HSMI has not been reported. Consequently, the presence of PRV in asymptomatic fish raises questions concerning the exact relationship between PRV and HSMI. To evaluate the risk posed to native and cultured salmon residing in the Pacific, we utilized controlled laboratory exposures to determine virulence of PRV in Atlantic and Sockeye Salmon. Naïve Atlantic Salmon challenged by intraperitoneal injection of PRV positive tissue homogenate resulted in development of a sustained PRV infection; however, compared to control groups there were no significant differences in morbidity due to PRV exposure and no evidence of HSMI. We also demonstrated that PRV could spread to naïve Atlantic and Sockeye Salmon by waterborne transmission with nearly 100% of naive fish becoming positive after 4 weeks of exposure. Despite the development of persistent infections, no fish to date have showed any morbidity, gross signs of disease, or histological evidence of HSMI when compared to controls.

Apr. 2013–Mar. 2015

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Marine Harvest Canada

Project Lead: Stewart Johnson (DFO)

Project Team: Kyle Garver (DFO); Diane Morrison (Marine Harvest Canada)

Collaborators: Marine Harvest Canada

Contact: Stewart.Johnson@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/science/enviro/aquaculture/acrdp-pcrda/index-eng.htm

COMPARISON OF FIELD ISOLATES OF MORITELLA VISCOSA: CHARACTERIZATION AND IN VIVO CHALLENGE MODEL DEVELOPMENT TO ADDRESS WINTER ULCER MITIGATION IN CANADA

The knowledge obtained through this research will be useful in the development of future vaccines for Moritella viscosa, which would provide a non-antibiotic, proactive strategy to address winter ulcer disease. Access to a licensed vaccine would increase the sustainability of salmonid aquaculture through improved animal welfare and reduced economic losses due to mortality, antibiotic use, and the downgrading of the product due to ulcer damage.

The bacteria M. viscosa is considered to be the main cause of winter ulcer disease, which primarily affects marine farmed salmonid fish during cold periods. Winter ulcer disease is becoming a high priority problem for Canadian producers, and while mortalities vary according to site, a large number of the remaining fish are also affected by the disease. To date, there is no vaccine licensed for winter ulcer disease in Canada, and the demand for access to vaccines is growing. Current vaccines are based on European disease strains, and in order for the vaccine to be licensed for use in Canada, its efficacy against Canadian strains must be examined.

This project aims to establish a live challenge model (in which the animal is given the bacteria to assess their response) relevant to M. viscosa outbreaks in Canada which can be used for future vaccine development studies. This will involve: (1) a series of in vitro (cell culture) studies to better understand how the bacteria grow in the presence of salmon cells and determine immune response differences between Canadian strains; and (2) selecting two sources of bacteria to be used to develop a disease challenge model using live Atlantic Salmon. The challenge model will help to determine a good candidate strain that will produce testable predictable results, a challenge exposure method, and a culture exposure temperature.

Apr. 2014–Mar. 2015

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Cooke Aquaculture Inc., Novartis Animal Health Canada Inc.

Project Lead: Steven Leadbeater (DFO)

Project Team: Anthony Manning (RPC); Leighanne Hawkins (Cooke Aquaculture Inc.); Allison MacKinnon (Novartis Animal Health Canada Inc.)

Collaborators: Cooke Aquaculture Inc., Novartis Animal Health Canada Inc.

Contact: Steven.Leadbeater@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

LOW PATHOGENIC INFECTIOUS SALMON ANEMIA VIRUS (ISAV) IN VIVO: A COMPARATIVE GENOMIC STUDY

Since the initial identification of Infectious Salmon Anemia Virus (ISAV) in Norway in 1984, and in the Bay of Fundy in 1996, viral evolution and selective pressure, combined with improved detection have revealed an interesting and challenging ISAV portrait, that is, the presence of essentially avirulent strains such as the HPR0 variant, as well as highly virulent strains, such as HPR4 variants. Additionally there are many other strains identified which have varying degrees of virulence. The ISAV remains a looming threat to the salmon aquaculture industry, and ISAV continues to evolve. This study will contain an in-depth assessment of the etiology of ISAV to gain further understanding of the variable virulence and infection dynamics observed, in vivo, in salmon.

Apr. 2011–Mar. 2015

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Kelly Cove Salmon Ltd.

Project Lead: Nellie Gagné (DFO)

Project Team: Mark LaFlamme, Francis Leblanc, Mélanie Roy (DFO); Keng Pee Ang (Kelly Cove Salmon Ltd.)

Collaborators: Kelly Cove Salmon Ltd.

Contact: Nellie.Gagne@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

DESCRIPTION OF OCEANOGRAPHIC CONDITIONS WITHIN HERMITAGE BAY, NEWFOUNDLAND, AT SITES WITH AND WITHOUT THE OCCURRENCE OF ISA OUTBREAK

Should there be a correlation between environmental parameters and the occurrence of Infectious Salmon Anemia (ISA) outbreaks, the results of this project will help identify other areas of the region when and where there is a potential risk of outbreak. This would provide the aquaculture industry with an anticipatory tool on how to mitigate the risk and improve the sustainability of salmon farming.

The first reported case of ISA disease was in a salmon farm in Hermitage Bay, Newfoundland (NL), in the summer of 2012 and a few more cases of ISA outbreaks have been reported in the same region since that time. The optimum temperature survival conditions of the virus, 5 to 15°C in seawater, coincide with farmed rearing conditions in the regions but stressors to farmed species may lead to increases in the infection rates.

This project aims to understand the variability of the oceanographic conditions in Hermitage Bay, NL, and assess the potential link between those conditions (temperature, salinity, and dissolved oxygen) and the occurrence of ISA outbreaks. In that bay, some sites have reported ISA outbreaks, although at different periods of the year, while others did not and it is suggested that the possible differences in the physical parameters surrounding these sites might have had an effect on the overall health conditions of the fish (i.e., stress) and allowed for an outbreak of the virus. This research project will address the physical properties of the water and analyze the conditions before and during ISA outbreak, in order to identify the environmental conditions associated with the outbreak and assess the possible correlation between the conditions and the outbreak.

Apr. 2014–Mar. 2015

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Cold Ocean Salmon; Newfoundland Aquaculture Industry Alliance (NAIA)

Project Lead: Andry Ratsimandresy (DFO)

Project Team: Sebastien Donnet, Kevin Le Morzadec (DFO); Julia Bungay (Cold Ocean Salmon); Miranda Pryor (NAIA)

Collaborators: Cold Ocean Salmon, NAIA

Contact: Andry.Ratsimandresy@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

DEVELOPMENT OF A NOVEL RNA-BASED TREATMENT AGAINST THE INFECTIOUS SALMON ANEMIA VIRUS (ISAV)

This project has provided insight into the viral replication of Infectious Salmon Anemia Virus (ISAV) and provides support for the use of ribonucleic acid interference (RNAi) against marine viruses like ISAV. Further, this research has permitted the development of new tools that will greatly facilitate future work and help improve fish health management in the aquaculture industry.

ISAV, a significant viral pathogen, causes mass mortalities and represents a recurrent problem to the salmon aquaculture industry. Current vaccines for ISAV do not offer total protection for the entire lifespan of cultured salmon. Additionally, no post-vaccination treatments exist for infections during the later stages of salmon development. This study explored the development of a novel RNAi-based vaccine as a treatment against the ISA virus. It was found that viral replication may be 10–100 times lower within treated cells compared to untreated control cells. Unfortunately, it was also discovered that cultured cells grown in the laboratory over a long period of time develop a resistance to the virus, and this resistance makes it difficult to truly assess the efficacy of the treatment. Further studies will have to be done to address the age-related resistance in order to better assess the efficacy of RNAi-based vaccines.

Apr. 2009–Mar. 2013

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Kelly Cove Salmon Ltd.

Project Lead: Mark LaFlamme (DFO)

Project Team: Nellie Gagné (DFO); Gilles Robichaud (U Moncton); Keng Pee Ang (Kelly Cove Salmon Ltd.)

Collaborators: Kelly Cove Salmon Ltd.

Contact: Mark.LaFlamme@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

FIELD VALIDATION OF DIETARY MEDICATION TO REDUCE THE SEVERITY OF KUDOA THYRSITES IN FARMED ATLANTIC SALMON

Farmed Atlantic Salmon are at risk of infection with Kudoa thyrsites throughout British Columbia (BC), leading to an elevated risk of reduced fillet quality. The cost to the BC farmed Atlantic Salmon industry was over $15 million in 2010, adding to the difficulty for the BC industry to remain competitive in the global salmon market. Early screening of farmed stock is now often used for Kudoa detection. Neither vaccines nor medicines are currently available for the prevention or treatment of the infection. This project evaluated the efficacy of dietary nicarbazin against Kudoa in Atlantic Salmon held in a production environment.

The results of this study confirmed that under production conditions, treatment of seawater-reared Atlantic Salmon with a medicated diet containing nicarbazin results in elevated muscle residues of dinitrocarbanilide (DNC). The study also confirmed observations made in laboratory studies that DNC declines rapidly in muscle following cessation of medication. An alternative treatment regime will be required to ensure that DNC residues are sufficiently high over a longer duration during the grow-out phase of salmon production to maximize efficacy against Kudoa in harvest fish.

Apr. 2011–Oct. 2013

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Marine Harvest Canada

Project Lead: Simon Jones (DFO)

Collaborators: Marine Harvest Canada

Contact: Simon.Jones@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm/a>

IDENTIFICATION AND TREATMENT OF GYRODACTYLID INFECTIONS IN CULTURED WOLF-EELS (ANARRHICHTHYS OCELLATUS)

Wolf-eels (Anarrhichthys ocellatus) are considered an appropriate new species for development in the Canadian aquaculture industry and recent research has looked at the potential to move this culture species from experimental to commercial production. During their studies, researchers identified a commonly occurring parasite (Gyrodactylus spp.) that was responsible for recurring disease outbreaks in captive-reared wolf-eels which could ultimately impede production. The objective of this project was to investigate Gyrodactylus outbreaks occurring in captive reared wolf-eels, identify the species responsible, and develop an efficacious treatment protocol.

The parasite infecting wolf-eels at the CAER facility in West Vancouver was confirmed as Gyrodactylus corti, originally described from captive wolf-eels in California. This research suggested that G. corti is a common parasite of wolf-eels and that increased intensities of infection occur in captivity. Infections cause an acute reaction in the gills, which contributes to elevated mortality. Infection with G. corti caused mortality as high as 83% among captive wolf-eels.

This research demonstrated that the most effective treatment against G. corti was formalin baths and, to a lesser extent, freshwater baths. However, both treatments only provide temporary or partial benefits as infections have been observed to recur. Careful management of newly captured wolf-eels, including quarantine and treatment is recommended. This research was needed to assess risk, develop treatment protocols, and to provide new information that will be essential for a successful wolf-eel aquaculture industry. Further research will need to explore additional regimes of formalin, freshwater, or combined treatments, in order to optimize results.

Apr. 2012–Mar. 2013

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Kyuquot SEAfoods Ltd.; Vancouver Aquarium

Project Lead: Simon Jones (DFO)

Collaborators: Kyuquot SEAfoods Ltd.; Vancouver Aquarium

Contact: Simon.Jones@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

Micrographs of wolf-eel (Anarrhichthys ocellatus) histological sections. A, normal gill; B, gill infected with Gyrodactylus corti (arrows) showing thickened lamellae, indicative of epithelial hyperplasia. Photo: Simon Jones (DFO)

DOES INFECTION WITH PISCINE REOVIRUS (PRV) EFFECT HOW SALMON RESPOND TO CHALLENGE WITH AND VACCINATION AGAINST INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV)?

Although co-infection of fish with multiple pathogens has long been recognized, the consequences of such infections have received little attention. This project will examine the consequences of viral co-infections in salmon, in particular, the relationship between the Piscine Reovirus (PRV) and Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic and Sockeye Salmon. Specifically, the study will address how hosts, infected with viruses of no or low pathogenicity effect, will respond to vaccination against and to challenge with other viruses. Challenge trials will be used to examine IHN disease progression in naïve and IHNV vaccinated non-PRV (control) and PRV-infected Atlantic Salmon. An IHNV challenge trial will also be conducted with non-PRV (control) and PRV-infected Sockeye Salmon that are naïve with respect to IHNV. These challenge trials will be used to determine if there are differences between groups in morbidity associated with IHNV challenge, and to generate biological samples for gene and microRNA expression studies. Transcriptional responses will be quantified using a combination of RNA-seq, also called “Whole Transcriptome Shotgun Sequencing”, and Real Time Quantitative PCR. This research will help to determine what, if any, additional risk is posed to wild and/or farmed fish due to changes in their ability to respond to IHNV vaccination and/or challenge due to the presence of PRV.

Sep. 2014–Dec. 2016

Funded By: DFO – Program for Aquaculture Regulatory Research (DFO – PARR)

Project Lead: Stewart Johnson, Kyle Garver (DFO)

Project Team: Jon Richards, Julia Bradshaw (DFO)

Collaborators: Mathew Rise (MUN); Rune Adnreassen (Oslo and Akershus University College of Applied Sciences)

Contact: Stewart.Johnson@dfo-mpo.gc.ca, Kyle.Garver@dfo-mpo.gc.ca

THE EFFECTS OF PRIOR EXPOSURE AND BODY SIZE ON THE INTENSITY OF KUDOA THYRSITES INFECTIONS IN ATLANTIC SALMON

The knowledge gained through this research project will lead to a greater understanding of disease transmission and wild-farmed interactions. Additionally, this information will allow for better finfish cage siting and decision making and the development of tools to help further manage this disease.

The parasite, Kudoa thyrsites, is the cause of soft-flesh syndrome in post-harvest Atlantic Salmon farmed in British Columbia. Infected fish exhibit no clinical signs of disease, but the affected muscle rapidly deteriorates after processing. These infections can cause substantial economic hardship to the salmon aquaculture industry with some sites estimating a 10% loss of annual yield, resulting in losses of between $6–10 million. Currently, there are no vaccines or approved strategies for treatment (e.g., chemotherapeutic intervention) for K. thyrsites.

Earlier research revealed geographic variation in the prevalence and intensity of K. thyrsites in Atlantic Salmon reared at different seawater production sites. This research discovered that infections typically resolve between six and twelve months following laboratory exposure of fish to the parasite, and therefore, the occurrence of infections in harvested salmon after 18 months or more in seawater suggest multiple or ongoing exposures to the parasite during commercial net pen rearing.

This project will further investigate options for managing K. thyrsites infections. Firstly, it will test the efficacy of ultraviolet irradiation of seawater as a method to inactivate K. thrysites in the laboratory and further study the influence of prior exposure to K. thyrsites on parasite development during subsequent exposure. The project will also assess the influence of fish size on the prevalence and severity of the infection.

Apr. 2013–Mar. 2015

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Marine Harvest Canada; Mainstream Canada

Project Lead: Simon Jones (DFO)

Collaborators: Marine Harvest Canada; Mainstream Canada

Contact: Simon.Jones@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

FISH PEST AND PATHOGEN CULTURED TO WILD TRANSFER POTENTIAL: STOCKING DENSITY EFFECT

Salmon farms infected with Infectious Salmon Anemia (ISAV) from wild stocks, can become an amplified source of ISAV which can subsequently be shed back to wild fish. Given the waterborne transfer and dispersal of ISAV, the resulting plumes or zones of ISAV may contribute to the transfer of the pathogen between farms, and to migrating wild salmon that intersect these plumes. Although the risk of pathogen transfer is of general concern to the industry and the general public alike, the DFO Maritimes Regional Regulators are particularly interested in the role of stocking density in potentially increasing pest and pathogen transfer from farms to endangered wild salmon in the Region. This study sets out to generate new information on ISAV shedding and infection rates of captive Atlantic Salmon. Laboratory studies will be conducted to estimate the shedding rate of ISAV from infected Atlantic Salmon as a function of fish stocking/handling density, the longevity of the infective capacity of waterborne ISAV, and the exposure profile for naive Atlantic Salmon to become infected with ISAV. The resulting information will be used to develop geographically specific physical-biological models for predicting the potential of waterborne spread of ISAV from farmed to wild salmon in the Maritimes Region of Atlantic Canada.

Mar. 2014–Mar. 2017

Funded By: DFO – Program for Aquaculture Regulatory Research (DFO – PARR)

Project Lead: Fred Page, Nellie Gagné (DFO)

Project Team: Blythe Chang, Francis Leblanc, Steven Leadbeater, Kyle Garver (DFO)

Collaborators: Michael Beattie (NBAAF)

Contact: Fred.Page@dfo-mpo.gc.ca, Nellie.Gagne@dfo-mpo.gc.ca

IDENTIFICATION OF VECTORS OF MSX TO SUPPORT I&T DECISIONS RELATED TO INTER-PROVINCIAL MOVEMENTS OF MUSSELS: IS MUSSEL INTRA-VALVULAR LIQUID A VECTOR FOR MSX TRANSMISSION?

With the increased participation of new partners, the number of requests for transfers of mussel seed and market product, both intra- and inter-provincially, is growing. The transfers of live mussels are regulated under the Fisheries (General) Regulations, Section 56, and are reviewed on a case by case basis by the Introductions and Transfer Committees (ITC) of the receiving Province. Multinucleated sphere X (MSX) is an infectious disease that causes heavy mortality in shellfish although it does not affect human health. To date, DFO analysis of mussels collected from heavily infected MSX positive areas of the Bras d’Or Lakes, and from MSX positive areas outside of the Lakes, has not detected MSX in the soft tissues/intra-valvular liquid. Nevertheless, concern remains with the unidentified secondary host(s) that may be transferred with the epifauna on the mussel lines. Applications to transfer “raw or unprocessed lines” or “primary processed lines” into MSX negative areas are increasing. In the absence of information, epifauna is considered potentially positive for MSX and mitigation recommended by the ITCs. However, identifying the secondary host may allow targeted mitigation (cleaning/time of year) and an accurate risk assessment of these proposed activities.

Apr. 2013–Mar. 2015

Funded By: DFO – Program for Aquaculture Regulatory Research (DFO – PARR)

Project Lead: Mary Stephenson (DFO)

Contact: Mary.Stephenson@dfo-mpo.gc.ca

SALMON PANCREAS DISEASE (ALPHAVIRUS) CHALLENGE AND VALIDATION OF RT-QPCR AND VIROLOGY TESTING

Salmon pancreas disease virus (SPDV), the aetiological agent of pancreas disease (PD), is an alphavirus, belonging to the Togaviridae family. Diseased fish are often lethargic, with abnormal swimming behaviour and mortality can reach 50% in cages. Pancreas disease was first detected in farmed Atlantic Salmon, Salmo salar L., in Scotland in 1976. Since then it has also been described in Norway, France, and Spain.

Salmon aquaculture in Canada could be greatly affected by alphavirus if present. Diagnostic assays, including RT-qPCR, exist (Hodneland and Endresen, 2006) but have not been evaluated in full. Since fish origin greatly influences their susceptibility, an initial evaluation of the potential of PD to affect a Canadian strain of salmon (e.g., Saint John River) is warranted.

Project objectives completed in 2012 include: (1) the importation of live SPDV strains for the purpose of evaluating/developing a RT-qPCR assay; (2) initial growth in cell culture; (3) preparation of histological slides for training; and (4) determining the susceptibility of salmon to PD. A live challenge was performed at the The Gulf Biocontainment Unit – Aquatic Animal Health Laboratory (GBU-AAHL, a level three biocontainment facility). The project objectives for 2014/15 are: (1) to do individual viral culture assays for a subset of the tissues collected; (2) to compare viral culture and RT-qPCR, and obtain validation data (stage 1 and 2); and (3) to write a protocol and validation dossier.

The project provided relevant training for a virologist, histopathologist, and provides reference material for potential alphavirus cases and differential diagnostic. The information gathered during the live fish challenge, like tissue tropism and disease presentation, are essential for the eventual detection of SPDV in surveillance program.

Apr. 2012–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Nellie Gagné (DFO)

Project Team: Phil Byrne, Mark Laflamme, Melanie Roy (DFO)

Collaborators: Nils Stein, Ann Aas Eng (Pharmaq)

Contact: Nellie.Gagne@dfo-mpo.gc.ca

Alphavirus challenge at the Gulf Biocontainment Unit – Aquatic Animal Health Laboratory (GBU-AAHL).

VALIDATION OF A REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION (RT-QPCR) ASSAY TO DETECT INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV)

Infectious pancreatic necrosis (IPN) disease is a federally reportable aquatic animal disease in Canada. Although IPN virus (IPNV) is no longer an OIE listed pathogen, their recommended screening method for detection of the virus was virus isolation. Since the National Aquatic Animal Health Program (NAAHP) diagnostic labs use qPCR assays to screen samples, the need for a validated IPNV-specific qPCR assay was identified. The assays available in the published literature did not have the required performance characteristics so DFO pursued development of a new test method for detection of the virus.

DFO has developed and validated a new RT-qPCR test method for detection of IPNV. The assay is sensitive and is capable of recognizing IPNV isolates present in Canada as well as those that could be introduced into the country through international trade or domestic movement of aquatic animals. NAAHLS member labs are using the validated assay for IPNV testing of NAAHP samples.

DFO now has the capability to use RT-qPCR to detect the presence of IPNV in movements of fish within Canada and can respond to request from our trading partners for IPNV-free attestations.

Apr. 2012–Mar. 2014

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Sharon Clouthier (DFO)

Contact: Sharon.Clouthier@dfo-mpo.gc.ca

DESIGN OF PROTOCOLS FOR THE OZONE DISINFECTION OF FISH EGGS FOR ERADICATION OF VERTICALLY TRANSMITTED DISEASES

Pathogens can be introduced into a population by one of two methods, either through transmission from another infected individual (horizontal transmission) or by the passage of the pathogen from broodstock to the egg (vertical transmission). With vertical transmission, disinfection of eggs has become a critical step in aquatic husbandry, and is one of the most important methods of controlling transmissible diseases in an aquaculture facility. This project tested the effectiveness of ozonated water in preventing disease transmission while evaluating its effects on developing egg and larval performance.

Overall, salt-water ozone treatment showed no negative effects on cod eggs or larvae at levels tested (< 3.0 mg/L for 90 seconds) and has the potential to be a safe, effective disinfectant on Atlantic cod eggs. Similarly, ozone was also found to be an effective egg treatment against the fungus, Saprolegnia diclina for freshwater fish (Atlantic Salmon and Rainbow Trout), with negative growth of this fungus recorded in all treatments.

This project provided valuable insight into the use of ozone within hatcheries to directly disinfect fish eggs of three species of interest to aquaculture. This allows industry the opportunity to improve its overall environmental performance by limiting the traditional use of harsh chemicals to disinfect eggs. This investigation also provided an opportunity to scale up ozone trials from previous laboratory-scale models to a semi-commercial facility, thereby optimizing amounts of disinfectant to be used for larger quantity of eggs. While more research on the effectiveness of ozone on specific pathogens is necessary, the information gained through this project can help to greatly improve health management practices in controlling transmissible diseases within aquaculture facilities.

Apr. 2010–Mar. 2013

Funded By: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) Co-Funded By: Newfoundland Cod Broodstock Company

Project Lead: Dounia Hamoutene (DFO)

Project Team: Jessica Fry, Cyr Couturier (MUN); Juan Carlos Perez-Casanova, Lynn Lush (DFO); Andy Walsh (Sapphire Sea Farms)

Collaborators: Newfoundland Cod Broodstock Company

Contact: Dounia.Hamoutene@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/science/enviro/aquaculture/acrdp-pcrda/index-eng.htm

REFINEMENT OF AN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS DISPERSION MODEL FOR THE DISCOVERY ISLANDS AREA AND AN EXTENSION TO WEST COAST OF VANCOUVER ISLAND

In British Columbia, infectious hematopoietic necrosis virus (IHNV) is the most economically important viral pathogen of salmonids. To begin to assess the risk of viral dispersal through waterborne transmission from an infected site, it is necessary to understand, not only the biological parameters associated with virus dispersion, but also the physical components of the environment in which the virus is being dispersed. This project aimed to refine the physical and biological measurements of the established viral dispersion model. Specifically, the risk of virus transmission from APEX vaccinated Atlantic Salmon was determined, as well as investigated the susceptibility of Sockeye Salmon to IHNV.

The vaccinated Atlantic Salmon were highly protected against the development of the lethal IHN disease such that out of 100 APEX vaccinated Atlantic Salmon only two fish succumbed to IHN disease. This protection was afforded regardless of whether the vaccinated fish were exposed to IHNV via intraperitoneal injection or waterborne immersion. In preliminary trials on Sockeye susceptibility to IHNV, no mortality was observed in either the mock challenge group or the two lowest virus exposure levels. Mortality was only observed at the two highest IHN virus exposure levels tested with cumulative mortality ranging from 8% to 36%.

Quantification of the IHNV transmission parameters obtained through this project will enable accurate geospatial predictions of risk for IHNV transmission from marine salmon sites. Understanding how the virus is dispersed among salmon farms will ultimately increase the sustainability of the aquaculture industry through optimization of fish health.

Apr. 2011–Mar. 2013

Funded By: DFO – Aquaculture Collaborative Research and Development Program (DFO – ACRDP) Co-Funded By: Grieg Seafoods; Mainstream Canada; Marine Harvest

Project Lead: Kyle Garver (DFO)

Collaborators: Grieg Seafoods; Mainstream Canada; Marine Harvest

Contact: Kyle.Garver@dfo-mpo.gc.ca

www.dfo-mpo.gc.ca/aquaculture/acrdp-pcrda/index-eng.htm

Refuge Cove, Discovery Islands, BC. Photo: Darren Tuele (DFO)

RAPID DETECTION OF REPLICATING INFECTIOUS SALMON ANEMIA VIRUS – PHASE 2

Modern diagnostic laboratories call upon a limited number of assays for the detection of pathogens. These assays are most often validated, which means that their behaviour in most situations is well documented and understood. For example, validated assays have defined limits of specificity and sensitivity. The work involved in validating an assay is quite significant, and in some sense, is never ending. Indeed, an important part of the validation process is the re-assessment and, if possible, improvement of the assay.

The detection of the Infectious Salmon Anemia Virus (ISAV) in diagnostic labs is currently achieved by two main methods based on either RT-qPCR amplification or by the observation of cytopathic effect (CPE) in cell cultures. Each of these methods has both advantages and drawbacks.

This research combines the strongest aspects of both these techniques, and adds new tweaks to the detection process, in an effort to modernize and improve upon our validated assays. Four distinct methods will be evaluated to measure the ease and rapidity they offer for the detection of replicating virus: (1) secondary RT-qPCR; (2) Electric Cell-substrate Impedance Sensing (ECIS); (3) co-centrifugation of virus and cell lines; and (4) the use of chemical aids for viral entry, exit, and replication.

This represents an improvement of one of the most utilized assays within the National Aquatic Animal Health Program (NAAHP). Further, the automation of some processes via ECIS and the detection of replicating HPR0 (a strain of ISAV) represent significant assets to the NAAHP pathogen detection methodologies.

Apr. 2013–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Mark LaFlamme (DFO)

Project Team: Nellie Gagné, Jean-René Arseneau (DFO)

Contact: Mark.Laflamme@dfo-mpo.gc.ca

REFERENCE GENE MEASUREMENT AS A PROXY FOR VIRUS DEGRADATION

The National Aquatic Animal Health Program (NAAHP) recognized a need for a project that would provide a way to measure if a sample of viruses has decomposed too far for a reliable analysis.

Our approach was to get an index of viral decomposition by measuring the level of degradation in host species tissue. We evaluated the degradation of the host tissue and of the virus in the infected host tissue in parallel using RT-qPCR. Infected tissues were kept in various conditions. We were able to determine that the degradation of the RNAas indicated by the ref gene is also an indication of the degradation of ISAV in tissues. The choice of reference

gene assay is important to get a good indication of virus degradation.

We now have the critical information needed to allow sound decision making if samples have been stuck in transit or if samples from external laboratories require testing by the Canadian Food Inspection Agency (CFIA) and there is doubt as to their condition.

Apr. 2013–Mar. 2014

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Nellie Gagné (DFO)

Project Team: Mark Laflamme, Mélanie Robichaud-Haché (DFO)

Contact: Nellie.Gagne@dfo-mpo.gc.ca

SUB-CLINICAL EFFECTS AND METABOLISM OF THE FEED-BORNE FUSARIUM MYCOTOXIN DEOXYNIVALENOL (DON) IN RAINBOW TROUT

Elucidation of the histopathological effects and an improved understanding of detoxification processes will provide information necessary for the potential development of more effective strategies to minimize the negative impacts of deoxynivalenol (DON) on sensitive aquaculture species. Furthermore, these studies provide a robust framework for aquaculture feed manufacturers to establish regulatory guidelines.

Contamination of fish feeds with mycotoxins is an issue of concern due to the increased use of economical plant proteins. Highly repeatable findings from several growth trials have confirmed that Rainbow Trout are extremely sensitive to the ubiquitous Fusarium mycotoxin, deoxynivalenol. Accordingly, a series of studies were conducted to investigate the basis of this sensitivity and to determine the efficacy of potential mitigation strategies. No distinct histopathological lesions were associated with feeding Rainbow Trout practically-formulated diets containing purified or naturally occurring DON. Increases in the number of dead cells and decreases in the number of mitotic figures in the pyloric caeca were observed. Similarly, some changes in the number of mitotic figures in the liver and in the degree of hepatic vacuolation were noted. A commercial feed additive was not efficacious in preventing negative effects of DON on growth, carcass composition, and nutrient utilization of Rainbow Trout. Comparatively, Nile Tilapia were not adversely affected by low dietary levels of DON. Increased digestible starch content does not appear to be an effective nutritional stragtegy for reducing the effects of DON on Rainbow Trout. Furthermore, species-specific sensitivity does not appear to be related to differences in hepatic UDP-glucuronosyltransferase activity.

Sep. 2010–Nov. 2014

Funded By: Biomin (B.R.A.I.N. program)

Project Lead: Dominique Bureau (U Guelph)

Project Team: Jamie Hooft, Cristina Ferreira, John Lumsden, John Cant, James Squires, Yanping Lou (U Guelph)

Collaborators: Rudolf Krska, Michael Sulyok (U Natural Resources and Life Sciences, Vienna)

Contact: dbureau@uoguelph.ca

DETERMINING THE LONG TERM VIABILITY OF WHITE SPOT DISEASE IN LOBSTER EXPERIMENTALLY INFECTED THROUGH DIET

White Spot Disease (WSD) is a reportable disease of trade importance to the National Aquatic Animal Health Program (NAAHP) and the World Organisation for Animal Health (OIE). Knowledge of WSD’s oral infectivity in fed lobsters at set temperatures was required to support current trade negotiations and market access for Canada’s lobster industry. This project will determine if lobsters can contract White Spot Disease by eating infected shrimp tissue. An additional temperature profile was added at the request of the CFIA, as results are needed for trade negotiations.

The study found lobster tissue was negative for the presence of WSD genetic material. Shrimp bioassay samples were positive. The positive shrimp bioassay corroborates the fact that the PCR-positive shrimp material contained viable WSD virus (note that a PCR-positive test result does not provide information concerning the viability of the positive material). WSD cannot be cultured in the lab using cell culture techniques traditionally used for finfish virus isolation and propagation. Further analysis is pending.

The conclusion based on the molecular and bioassay results is that, at 10°C, adult market sized lobster fed WSD-infected shrimp material do not appear to be susceptible to developing disease associated with WSD infection. Long term carriage of the WSD virus in asymptomatic (i.e., ‘healthy’) lobster also does not occur at 10°C.

Apr. 2013–Mar. 2014

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Phil Byrne (DFO)

Project Team: Mark LaFlamme (DFO)

Contact: Philip.Byrne@dfo-mpo.gc.ca

CONFIRMATION OF THE VIABILITY AND INFECTIVITY OF WSSV-INFECTED SHRIMP USED IN A FEED CHALLENGE TRIAL INVOLVING AMERICAN LOBSTER

This project is a continuation of previous work that involved a WSSV (White Spot Symptom Virus) challenge of market-sized lobster using WSSV-infected shrimp (frozen) in which the shrimp were fed to lobster. The WSSV-fed lobsters were found to be negative. This is not unexpected, as lobsters are not the primary host species. However, in order to ensure adequate titre had been used, there was a need to confirm the viability and virulence of WSSV in the infected shrimp used as food. The objective of this work is to demonstrate the infected shrimp used as food in the lobster trial contained a sufficient viral load to cause infection when fed to other shrimp, the typical host species for this virus.

The susceptibility of a commercial species (Homarus americanus) to WSSV, a CFIA and OIE reportable disease, will directly inform risk assessment and trade negotiations.

May 2014–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Phil Byrne (DFO)

Project Team: Mark LaFlamme (DFO)

Contact: Phil.Byrne@dfo-mpo.gc.ca

Shrimp in rearing facilities grown for the study. Photo: DFO

(L-R) GBU – AAHL technician Phyllis Dixon (holding a lobster) and Danielle Gordon (Veterinary student) are examining a healthy American lobster (Homarus americanus) from a GBU aquatic animal holding unit. Live lobster are used to support an ongoing investigation that is evaluating the susceptibility and pathobiology of shrimp viral pathogens in lobster by using experimentally infected lobsters within the GBU high level biocontainment facility. Photo: DFO

THE USE OF RNALATER FOR THE INACTIVATION OF THE INFECTIOUS SALMON ANEMIA VIRUS

RNAlater (Ambion/Life Technologies) is perhaps the most well-known of the commercially available buffers for the protection of RNA, and has been found to completely stabilize the RNA in a variety of tissues and viruses. It is clear that RNAlater preserves nucleic acids in all tissue types. What is less clear is whether or not RNAlater provides protection to virus, or rather inactivates the virus while preserving its RNA. In 2005, the effects of RNAlater were tested on a variety of human viruses, and it was found that some viral preservation was possible. The research showed that following high level purification, the virus was still infectious.

A more important question is whether infected tissues remain infectious while in the presence of the RNAlater? To address this question, we are investigating the infectiousness of the Infectious Salmon Anemia Virus (ISAV) virus, which has been preserved in RNAlater. This project will determine if the product, RNAlater, kills the virus. This is important to support our laboratory biocontainment standards and quality management systems.

Apr. 2013–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Mark LaFlamme (DFO)

Project Team: Nellie Gagné, Jean-René Arseneau, Adrien Boudreau (DFO)

Contact: Mark.Laflamme@dfo-mpo.gc.ca

DEVELOPMENT AND DIAGNOSTIC VALIDATION OF A QPCR ASSAY TO DETECT MIKROCYTOS MACKINI

Under the Canadian Health of Animals Act (HAA), the occurrence of Microcytos mackini must be reported to the Canadian Food Inspection Agency (CFIA). The overarching goal of this multi-year project is the development and validation of a molecular diagnostic test for the listed pathogen of Pacific Oyster (Crassostrea gigas), M. mackini. The main aims of this study, which have been addressed sequentially since this project’s inception in 2007, are as follows: (1) to isolate a new, more quickly evolving genetic region to use for molecular diagnosis of M. mackini (completed); (2) to survey extant diversity of M. mackini throughout its range to ensure appropriate specificity of the new assay (completed); (3) to develop a sensitive and specific real-time PCR assay for this pathogen (completed); (4) to complete full diagnostic validation of this assay (in progress); and (5) to publish the work (not completed).

This phase of the work will lead to publication of the new qPCR assay to detect M. mackini. Its analytical and diagnostic characteristics will be fully assessed. Information on its performance is required to support its use for testing under National Aquatic Animal Health Program (NAAHP) within Canada, as well as its use internationally.

May 2007–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Cathryn Abbott (DFO)

Project Team: Gary Meyer, Geoff Lowe (DFO)

Collaborators: Charles Caraguel (U Adelaide); Nils Toft (Technical University of Denmark); Serge Corbeil, Axel Colling (CSIRO, Australia)

Contact: Cathryn.Abbott@dfo-mpo.gc.ca

MEGALOCYTIVIRUS/RED SEA BREAM IRIDOVIRUS QUANTITATIVE PCR ASSAY DEVELOPMENT: PHASE I – MEGALOCYTIVIRUS ISOLATE COLLECTION, VIRUS AMPLIFICATION, AND STAKEHOLDER CONSULTATION

The pathogen Red Sea Bream Iridovirus has been detected in over 30 species of wild and cultured marine and freshwater fish. International trade in live ornamental fish is considered a major route of entry for megalocytiviruses into new geographical areas. This pathogen has been identified by the Canadian Food Inspection Agency (CFIA) as a potential emerging disease that is now present in the United States.

The goal for the Megalocytivirus/Red Sea Bream iridovirus Phase I (i.e., 2014–15 of this project) is to collect and bioamplify isolates that will be used to establish the analytical specificity of the assay (e.g., isolates belonging to the Iridoviridae family or viruses co-localizing to the target tissue). In Phase II (i.e., 2015–16), a qPCR assay(s) will be developed for detection of megalocytiviruses and/or RSIV. The long-term goal of the project is validation of the molecular assay according to World Organization for Animal Health (OIE) standards and harmonization of this methodology for the detection of megalocytiviruses and/or RSIV in diagnostic labs across Canada and eventually in other countries as well.

Canada now has a collection of RSIV isolates that are required to support testing under the National Aquatic Animal Health Program (NAAHP) within Canada. They will also be made available for use internationally.

May 2013–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Sharon Clouthier (DFO)

Project Team: Tamara Schroeder, Adrian Zetner (DFO)

Contact: Sharon.Clouthier@dfo-mpo.gc.ca

ESTABLISH AN EXPERIMENTAL HOST-PATHOGEN MODEL INVOLVING RED SEA BREAM IRIDOVIRAL DISEASE AGENT (RSIV) IN FINFISH AND PRODUCE RSIV-INFECTED TISSUE

Red Sea Bream iridoviral disease (RSIV) is a significant cause of mortality of Red Sea Bream (Pagrus major) and dozens of other species. It is a reportable disease under the National Aquatic Animal Health Program (NAAHP), and the National Aquatic Animal Health Laboratory System (NAAHLS) is required to develop a validated assay for this pathogen. As RSIV propagation by cell culture is not always effective, alternative means of producing positive material for use in validation are required. The Gulf Biocontainment Unit – Aquatic Animal Health Laboratory (GBU-AAHL) will use injection trials to try and produce positive material for use in subsequent assay validation.

Virus derived from this type of initial animal trial can then be harvested and prepared for more elaborate animal-based trials as needed as well as properly characterized and titred using molecular techniques.

May 2013–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Phil Byrne (DFO)

Contact: Phil.Byrne@DFO-mpo.gc.ca

Schematic representation of critical elements of biocontainment at the Gulf Biocotnainment Unit (GBU) in Charlottetown. The diagram portrays selected physical barriers including the precise management of room air pressure that enables directional air flows, HEPA-filtration of all exhaust air, multi-room entry/exit corridors (between areas of no containment and highest containment) and waste water sterilization in the basement using large autoclaves. GBU is certified to the highest level of biocontainment standard in Canada for work with aquatic animal pathogens and live animals, and is designated ‘AQC3 in vivo’. This level of biocontainment is required for lab-based investigations where high risk imported pathogens are experimentally introduced into live animals and includes work with agents such as RSIV, White Spot Syndrome Virus (WSSV) and Salmon Alpha Virus (SAV). Photo: Phil Byrne (DFO)

REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION (RT-QPCR) DIAGNOSTIC ASSAY FOR DETECTION OF SPRING VIREMIA OF CARP VIRUS (SVCV) – DIAGNOSTIC VALIDATION PHASE IIA (SAMPLE GENERATION AND DISTRIBUTION)

Spring viremia of carp, also known as acute infectious dropsy, is a highly contagious disease found primarily in cultured and wild populations of Common Carp, Cyprinus carpio. The causative agent Spring viremia of carp virus, (SVCV) has been found in Canada (Lake Ontario).

In general, mortality is most likely to occur in Cyprinus carpio, although Asian Carps may also develop the disease. SVCV can be extremely virulent (70–100% mortality) to juvenile carp in their first year and die-offs are typically observed in the spring when the water temperature is between 15°C and 17°C.

Phase I (i.e., 2013–14) of the project was to develop a molecular assay capable of detecting spring viremia of carp virus during the acute phase of the disease. Phase II will be used to generate samples for use in validation of the molecular assay.

Information on the assay’s performance is required to support its use for testing under National Aquatic Animal Health Program (NAAHP) within Canada, as well as to make it available for use internationally.

May 2013–Apr. 2016

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Sharon Clouthier (DFO)

Project Team: Adrian Zetner, Tamara Schroeder (DFO)

Collaborators: Carol McClure (AVC)

Contact: Sharon.Clouthier@dfo-mpo.gc.ca

Melisa Lindsay (DFO) holding a Common Carp, Cyprinus carpio.

THE EPIDEMIOLOGY OF WINTER ULCER DISEASE IN FARMED ATLANTIC SALMON IN CANADA

The overall goal of this project is to better understand the epidemiology of Moritella viscosa on Atlantic Salmon fish farms on the East Coast of Canada.

Winter ulcer disease caused by M. viscosa is the primary reason for antibiotic usage in aquaculture on the east coast of Canada. Anecdotal reports suggest mortality from this disease is increasing and it is unknown whether this is due to treatment resistance, increased virulence, reduction in host immunity, increased exposure, or a combination of these factors.

Identification of risk factors for this disease and management strategies to control the bacterial pathogen are needed to improve the sustainability of the industry. To date, there is no published descriptive epidemiology for M. viscosa in Canada. This basic information is essential for developing hypotheses on control and prevention strategies.

Specific objectives are to: (1) describe disease outbreaks with respect to specific environmental and husbandry factors at pen and farm levels; (2) assess antibiotic treatment efficacy for this pathogen over the last five years; and (3) develop a proposal to address potential management strategies for vaccination and treatment applications, based on the epidemiology of the disease.

Sep. 2014–Jun. 2015

Funded By: Canada Excellence Research Chair (CERC) – Aquatic Epidemiology, UPEI

Project Lead: Sophie St. Hilaire (UPEI)

Project Team: Allison MacKinnon (Novartis)

Collaborators: Novartis

Contact: ssthilaire@upei.ca

RAPID DETECTION OF REPLICATING INFECTIOUS SALMON ANEMIA VIRUS

Much of the aquaculture industry depends on regular diagnostic testing to assure the health of its animals. In the finfish aquaculture industry, viral pathogens are of particular concern, as they can cause significant mortalities. As such, fish are tested frequently.

RT-qPCR is a modern molecular technique that is rapidly gaining favour in diagnostic labs of all types, and is often used for the detection of the infectious salmon anemia virus. Perhaps the only drawback of this technique is that while it detects the nucleic acids of the virus, it provides no information regarding the replicative ability of the virus. In other words, both replicating and non-replicating viruses are detected; this can bring into question the biological significance of RT-qPCR findings. Conversely, classical viral isolation techniques provide strong evidence of replicating virus, but are generally slower, more labour intensive and less sensitive than RT-qPCR.

The current project seeks to combine the strongest aspects of both these techniques, while adding tweaks to the detection process, in an effort to modernize and improve upon our validated assays. We believe that fusion of proven classical techniques with modern molecular techniques will result in increased capacity and quicker turnaround times for testing labs.

This project will provide a new more rapid testing option for use in certain conditions.

Apr. 2013–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Mark LaFlamme (DFO)

Project Team: Jean René Arseneau, Nellie Gagné, Mélanie Roy (DFO)

Contact: Mark.Laflamme@dfo-mpo.gc.ca

DFO biologist, Mélanie Roy, preparing cells for viral isolation assays. Photo: Mark LaFlamme (DFO)

DETERMINE IF THE PRESENCE OF HPR0 GENOMIC MATERIAL (DETECTED USING RT-QPCR IS ASSOCIATED WITH THE CONCURRENT PHYSICAL PRESENCE OF VIRAL MATERIAL (I.E., DISTINCT VIRIONS OR PARTIAL VIRUS ASSEMBLY, EVIDENCE OF VIRUS REPLICATION AND LOCALIZED TISSUES OR CELL-SPECIFIC PATHOLOGICAL CHANGES) THAT IS CONSISTENT WITH INFECTIOUS SALMON ANAEMIA VIRUS (ISAV)

The causative agent of infectious salmon anemia (ISA) is the ISA virus (ISAV). ISAV affects mainly Atlantic Salmon (Salmo salar). It is a Canadian Food Inspection Agency (CFIA) as well as World Organization for Animal Health (OIE) reportable disease. Both virulent and non-virulent forms of the virus exist, the latter often is identified as HPR0. To date HPR0 has only been detected successfully using molecular techniques. This limitation has proven problematic for regulatory bodies as molecular tests are not conclusive in determining if a virus is viable, thereby making risk assessments and regulatory decisions regarding HPR0 ambiguous at best. Adding to the complexity of the issue is the unknown link between HPR0 and virulent forms of the virus.

This project proposes to bring together several testing approaches in order to help to visualize HPR0 using more informative traditional methods such as transmission electron microscopy. In order to successfully apply traditional methods, a population with a high prevalence of HPR0 must be found. The objective for the first phase of the project is to identify a suitable population for study. If this research is successful, the definitive identification of ISAV HPR0 would inform the diagnostic approach used for HPR0 as well as facilitate risk assessments, not only for Canada’s NAAHP, but also for other countries.

May 2013–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Phil Byrne (DFO)

Project Team: Nellie Gagné (DFO)

Contact: Phil.Byrne@dfo-mpo.gc.ca

DEVELOPMENT OF ARTIFICIAL REFERENCE MATERIAL FOR ASSESSING IHNV AND VHSV RT-QPCR ASSAYS

The use of artificial positive control (APC) have an advantage over traditional controls because they are unrelated to the target pathogen and can be easily differentiated from each other thereby facilitate the identification of false positive results. RNA transcripts generated from APC are also ideal candidates for use in proficiency testing panels.

To this end, the aims of this project are threefold: (1) Establish procedures for generating large batches of IHNV and VHSV artificial reference material (ARM) for use as standards, controls, and proficiency testing; (2) identify optimal storage practices and shelf life of bulk reference materials; and (3) develop characterization procedures for IHNV and VHSV bulk reference materials.

Results from small scale pilot studies at the DFO Pacific Biological Station (PBS) Aquatic Animal Health Laboratory proved promising for proficiency testing of IHNV and VHSV RT-qPCR in that the artificial transcripts mimicked the biological target, could be accurately quantified, and present extremely low false positive. Due to the advantages of APC, a VHSV-APC has also been established. Other DFO National Aquatic Animal Health Laboratory System (NAAHLS) labs are beginning to implement the use of APC for other National Aquatic Animal Health Program (NAAHP) listed pathogens.

May 2014–Mar. 2015

Funded By: DFO – Centre for Aquatic Animal Health Research and Diagnostics (DFO – CAAHRD)

Project Lead: Kyle Garver (DFO)

Contact: Kyle.Garver@dfo-mpo.gc.ca

A quantitative polymerase chain reaction (PCR) machine, used to detect Viral Hemorrhagic Septicemia Virus (VHSV) genetic material. Photo: DFO