Canadian Aquaculture R&D Review 2013

Fish Health

Continued market access for Alberta commercial Aquaculture producers through the development of fish health management plans

Alberta commercial fish culturists held discussions with their Provincial government regulators in late 2010, on the topic of compulsory requirements for Fish Health Management Plans (FHM Plan).

The goal of this project has been to cooperatively assist commercial fish culturists in Alberta to complete an accredited Fish Health Management Plan, following the developed template, as well as a document of “Required Elements” (to help explain each section of the FHM Plan) and a background document on related aspects of certification and marketing.

The objectives of the project were to:

1) Set up a new Alberta Fish Health Management Plan by modifying the existing Province of British Columbia’s Fish Health Management Plan template.

2) Coordinate the development of Alberta’s Fish Health Management Plan with Alberta Aquaculture Association (AAA), Alberta Agriculture and Rural Development, (AARD), Alberta Sustainable Resource Development (ASRD), and Canada Food Inspection Agency (CFIA), maintaining close liaison throughout the process.

3) Assist each fish culturist with fine-tuning of their individual plan.

Alberta’s commercial fish culturists now have a completed Fish Health Management Plan for each of its members, and a Template available for new entrants. The updated template will be accessible to aquaculture producers across Canada, in particular, the Prairie Provinces, who may wish to use it for their facility operations.

MAY 2011 – MAR. 2012

Funded by: DFO – Aquaculture Innovation and Market Access Program (AIMAP) co-funded by: Alberta Agriculture and Rural Development

project lead: Mark McNaughton (Alberta Aquaculture Association)

Project team: Mark McNaughton (Alberta Aquaculture Association); Eric Hutchings (Lethbridge Consulting Company)

Contact: msmcnaughton@lincsat.com

www.affa.ab.ca

Identification and treatment of gyrodactylid infections in cultured Wolf Eels

Wolf Eels (Anarrhichthys ocellatus) are considered an appropriate new species for development in the Canadian aquaculture industry. Recent research has looked at the potential to move this culture species from experimental to commercial production. During their studies, researchers identified the Gyrodactylus spp. as a commonly occurring parasite that is responsible for recurring outbreaks in captive reared Wolf Eels and which impedes production. Wolf Eels are cultured in high density to prevent innate aggressive behaviour. They also have a high reproduction rate. These two factors are thought to contribute to the fast spread of the parasite amongst cultured fish. The objective of this project is to investigate Gyrodactylus outbreaks occurring in captive reared Wolf Eels, identify the species responsible and develop an efficacious treatment protocol.

The first phase of research will involve the identification of the species of Gyrodactylus responsible for the disease outbreaks. This will include the collection of information on parasite reproduction and life cycle which will be used to develop treatment protocols. The second phase will be to conduct controlled studies to investigate the effectiveness of different treatments (e.g., freshwater, hydrogen peroxide, formalin) and treatment protocols (dose, duration, frequency) in reducing and eliminating infections. Additional information will also be collected on various aspects of Gyrodactylus infections in Wolf Eels including the behavioural, physiological, and immunological responses of the Wolf Eels to infections and parasite site preferences (i.e.,
gill vs skin).

This research is needed to assess risk, develop treatment protocols and to provide new information that will be essential for a successful Wolf Eel aquaculture industry.

OCT. 2012 – MAR. 2013

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP); Kyuquot SEAfoods Ltd.

project lead: Simon Jones (DFO)

Project team: Shannon Balfry, Denis Thoney (Vancouver Aquarium)

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

Identification and quantification of Kudoa thyrsites-specific DNA in seawater

The parasite Kudoa thyrsites can cause soft-flesh syndrome that affects the flesh quality in post-harvest Atlantic Salmon farmed in British Columbia. Although fish infected with the parasite show no clinical signs of disease, muscle in the processed fillet rapidly deteriorates, resulting in economic losses to the industry. Knowledge is limited on the life cycle of K. thyrsites and there are no vaccines or approved treatments for the parasite; however, researchers are exploring candidate treatments. Most species of Kudoa have only been identified from within the specific cells or tissues of their fish hosts. All other aspects of their life cycle, including how the parasite enters and migrates within the fish and the alternate host species are unknown; however, it is hypothesized that the infective stages occur in seawater

The proposed research will develop a test for the detection of the waterborne fish-infective stage of K. thyrsites. The method will be validated and used to screen water samples from commercial production sites historically known to be at risk for K. thyrsites infection. These samples will be taken at various depths and times. This will help assess the seasons and water depths when changes occur in the abundance of the infective stage, in the marine environment.

OCT. 2012 – FEB. 2013

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP); Marine Harvest Canada; BC Centre for Aquatic Health Sciences (CAHS)

Project lead: Simon Jones (DFO)

Project team: Wyth Marshall, Ahmed Siah (BC CAHS); Diane Morrison (Marine Harvest Canada)

Contact: simon.jones@dfo-mpo.gc.ca

www.affa.ab.ca

Plasmodia of Kudoa thrysites within the muscle

Low pathogenic Infectious Salmon Anemia virus (ISAv) in vivo: a comparative genomic study

Since the initial identification of 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 Infectious Salmon Anaemia virus (ISAV) portrait: 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.

MAR. 2011 – MAR. 2015

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) co-funded by: Kelly Cove Salmon Ltd.

project lead: Nellie Gagné (DFO)

Project team: Mark Laflamme, Francis Leblanc, Brian Glebe, Mélanie Roy, Steve Leadbeater (DFO); Keng Pee Ang (Kelly Cove Salmon Ltd.)

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

Drawing blood for ISAv analysis

Implementation of real time PCR for fish pathogen screening at the BC Centre for Aquatic Health Sciences

This project aims at implementing a qPCR screening test for fish health diagnostic at the BC Centre for Aquatic Health Sciences (BC CAHS). Although cell culture remains the “Gold Standard” screening technique for fish health, real-time qPCR allows a high-throughput, sensitive and rapid turn-around time screening. 

Due to its close location to the fish farms, strong collaboration with fish farm industry and its involvement in research project on wild fish surveillance programs, BC CAHS in collaboration with Mainstream Canada has implemented a qPCR screening assay for fish health surveillance. BC CAHS is currently performing a qPCR screening for IHNv, VHSv, ISAv and Renibacterium salmoninarum. In addition, experiments have been performed to evaluate the Elongation Factor alpha 1 mRNA as a suitable indicator of Atlantic Salmon tissue quality control.

Further assays are currently under development in order to screen for Aeromonas salmonicida, Yersinia ruckeri. Furthermore, BC CAHS is starting the process of ISO 17025 accreditation for fish health diagnostic.

sept. 2011 – sept. 2013

Funded by: Mainstream Canada; Western Diversification (ISO 17025 Accreditation)

project lead: Ahmed Siah (BC CAHS)

Project team: Henrik Duesund, Kathleen Frisch, Peter McKenzie (Mainstream Canada); Kathryn Temple, Sonja Saksida (BC CAHS)

Contact: Ahmed.siah@cahs-bc.ca

Juvenile Sockeye Salmon

Health assessment of juvenile Fraser River Sockeye Salmon in the Strait of Georgia and adjacent waters

It is generally accepted that pathogen transfers occur between wild and farmed salmonids, and that these transfers occur in both directions. What is not understood, however, is the magnitude of these transfers and the risk that they pose to both wild and farmed salmonids. For the last 3 years (2010 – 2012) we have monitored the health of juvenile Sockeye Salmon during their migration down the Fraser River and through the Strait of Georgia and Johnstone Strait. We have used traditional diagnostic methods and validated molecular diagnostic techniques to screen sockeye for a variety of pests, including sea lice (Lepeophtheirus salmonis and Caligus clemensi), and viruses and have used histology to look for signs of disease. Sea lice are also being counted on other species of juvenile salmon and non-salmonids. In addition to improving our understanding of pathogens and diseases of Sockeye Salmon, our surveys are providing important information on the distribution, routes of migration and stock makeup of juvenile salmon in the Strait of Georgia and Johnstone Strait. Taken together all of these data along with data from the salmon farming industry will help in the assessment of risks associated with the transfer of pathogens between wild and farmed salmon.

may 2010 – july 2012

Funded by: DFO – Program for Aquaculture Regulatory Research (PARR) co-funded by: BC Pacific Salmon Forum; Pacific Salmon Commission Southern Fund (Apr. – July 2012)

project lead: Stewart Johnson (DFO)

Project team: Kyle Garver, Simon Jones, Chrys Neville, Marc Trudel (DFO)

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

Refinement of an Infectious Hematopoietic Necrosis virus dispersion model for the Discovery Islands area and an extension to west coast of Vancouver Island

Since the introduction of Atlantic Salmon to the BC coast in the mid 1980’s there have been two serious outbreaks of Infectious Hematopoietic Necrosis virus (IHNV) in farmed Atlantic Salmon: 1992 – 1996 and 2001 – 2003. In the latter outbreak, thirty-six farm sites representing both east and west coast regions of Vancouver Island were diagnosed with IHNV. The estimated economic loss resulting from both epizootics was $40 million in inventory representing $200 million in lost sales. A central question regarding outbreaks in farmed Atlantic Salmon is the role of natural waterborne transmission in the spread of virus between farms. Studies investigating spatial and temporal patterns of the IHNV outbreaks suggest that farming practices themselves contributed significantly to the spread of disease both within and between areas; however, the extent to which waterborne transmission contributes to virus dispersal during an outbreak is unclear. As research conducted under ACRDP # P-09-03-006 provided a baseline for the establishment of the first viral dispersion model, this study seeks to further these studies as described herein to refine the biological measurements of the established viral dispersion model.

june 2011 – mar. 2013

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) co-funded by: Grieg Seafoods BC Ltd., Mainstream Canada, Marine Harvest Canada Inc.

project lead: Kyle Garver (DFO)

Project team: Mike Foreman, Dario Stucchi, Ming Guo, Darren Tuele, Peter Chandler (DFO)

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

Weather monitoring station
Map of ocean currents around Discovery Islands, BC

Genomic characterization of jaundice-associated mortality events in cultured Chinook Salmon

This project was undertaken to determine whether a jaundice syndrome associated with low-level mortality in Chinook Salmon farmed in Tofino Inlet was more likely caused by a viral infection or an environmental toxin. Our project combined genomics, histopathology, epidemiology, and standard veterinary diagnostic techniques to determine which of these etiologies was more likely involved. Prevalence of jaundice syndrome was consistently greater at farm A than B over multiple years. The most significant lesions included tissue necrosis and fibrin deposition, primarily in kidney and liver. Genomic signatures comprised of thousands of differentially regulated genes occurred in both kidney and liver, with strong effects on immune response, proteolysis, metabolism, and cell cycle. The types of immune processes elicited were highly consistent with a viral etiology (response to virus, response to exogenous dsRNA, Stat signaling, type-I interferon response, viral replication); conversely, there was no signal that could be construed as toxicant-response. Based on a PCR survey of infectious agents, fish with jaundice syndrome commonly had greater loads of piscine reovirus than did healthy fish. This virus is purported to cause heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon in Europe, but the lesions associated with HSMI are very different from lesions in Chinook Salmon with jaundice syndrome. Tissue tropism is not uncommon with reovirus infections, so it is possible that this virus could affect different tissues in different species. As a whole, this research supports a viral etiology, however, more research will be required to determine if the piscine reovirus is causative of, associated with, or merely a bystander to the jaundice syndrome.

apr. 2011 – apr. 2012

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) co-funded by: Creative Salmon

project lead: Kristi Miller (DFO)

Project team: Karia Kaukinen, Brad Davis (DFO); Sonja Saksida (CAHS)

collaborators: Gary Marty (BC Ministry of Agriculture)

Contact: Kristi.Miller@dfo-mpo.gc.ca

Validation of dietary medication and sterilised seawater 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, 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. An earlier ACRDP project demonstrated the efficacy of dietary Nicarbazin, a compound used to prevent coccidiosis in poultry, against K. thyrsites in Atlantic Salmon. The present study will use laboratory-reared Atlantic Salmon to compare the efficacy of Nicarbazin and ultraviolet irradiation of seawater against K. thyrsites, and to obtain data relating to the longevity of Nicarbazin in Atlantic Salmon tissues following cessation of treatment.

apr. 2011 – mar. 2012

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) co-funded by: Marine Harvest Canada; Mainstream Canada Ltd.; Grieg Seafood BC Ltd.

project lead: Simon Jones (DFO)

collaborators: Sharon DeDominicis (Marine Harvest Canada); Barry Milligan (Mainstream Canada Ltd.); Peter McKenzie (Grieg Seafood BC Ltd.)

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

Viral hemorrhagic septicemia virus (VHSV) and Great Lakes fish

The purpose of the study was to provide data to predict the effect of Viral Hemorrhagic Septicemia Virus (VHSV) genotype IVb on Walleye and other fish in the Great Lakes. The first objective was to predict morbidity/mortality in naive Walleye using a biologically relevant waterborne infection and re-infection model at temperatures permissible for VHSV replication (~10-12°C). The reference strain of Walleye was not very susceptible to experimental infection via waterborne exposure (1×107 virions; <5% mortality) and was only moderately impacted by a large dose of virus given intraperitoneally (i.p.) (1×107 virions; 30-40% mortality). Walleye were more resistant than Fathead Minnows but more susceptible than Rainbow Trout using similar experimental conditions. In addition, Walleye previously waterborne-exposed to VHSV were almost completely resistant to re-infection via i.p.-injection five months later. The second objective was to determine the relative susceptibility of four genetically distinct Great Lakes Walleye strains, of which one was found to be significantly less susceptible to experimental i.p.-infection. To date, therefore, it appears likely that there is a low impact of infection with VHSV on stocked Walleye, and that strain selection would further reduce any impact. A third objective was to create new tools for the culture of VHSV and numerous Walleye cell lines have been created. In addition, the behaviour of VHSV IVa and IVb has been compared in Rainbow Trout gill epithelial and splenic macrophage cell lines. Research in Rainbow Trout and in wild fish species including Freshwater Drum and Fathead Minnow demonstrated that the virus (viral antigen and RNA) was present in eggs and sperm. Ongoing research is examining innate immune mechanisms in gill epithelium and the role of virus neutralizing antibody in protection of Walleye.

oct. 2008 – ongoing

Funded by: Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA); NSERC Strategic; NSERC Discovery; Great Lakes Fisheries Commission; FRST New Zealand Postdoctoral Fellowship; Ontario Veterinary College Fellowship

Project lead: John S. Lumsden (U. Guelph)

Project team: Lowia Al Hussinee, Alex Reid, Jessica Grice, Lincoln Tubbs, Paul Huber, (U. Guelph); Brian Dixon, John Pham, Nguyen Vo, Niels Bols (U. Waterloo)

Contact: jsl@uoguelph.ca

VHSV in trout

Development of novel RNA-based treatments against ISAV

The purpose of the project is to develop a novel RNA interference-based vaccine against ISAV. RNA interference (RNAi) has been successfully used to combat viral infections in many vertebrate and invertebrate species, and offers the distinct advantage of being used both as a prophylactic vaccine, and as a treatment to combat the virus at the first signs of infection. We have identified genetic sequences that are common to all strains of ISAV, and we have designed, cloned and evaluated the efficacy of a number of RNAi-based gene inhibitors using model cell lines. Our results are encouraging and suggest that RNAi-based therapies could be developed against ISAV. We have noticed, however, that fish cells kept in culture over long periods of time become non-permissive to ISAV; this has caused problems in our analyses. As such, further testing will need to be done to best determine how RNAi therapies could be applied to live fish. If successful, this would represent the first and only treatment against ISAV infection.

nov. 2009 – mar. 2013

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) co-funded by: Kelly Cove Salmon Ltd.

project lead: Mark Laflamme (DFO)

Project team: Nellie Gagné (DFO)

collaborators: Keng Pee Ang (Kelly Cove Salmon Ltd.); Gilles Robichaud (U. Moncton)

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

Deoxynivalenol and disease susceptibility to cold water disease and immunity in Rainbow Trout

The increasing demand for fish protein has led to aquaculture being the fastest growing food producing sector in the world. This evolution has led to numerous modifications in production to sustain the demand. The replacement of fishmeal with more economical plant-based by-products in aqua feeds has led to an increased risk of exposure to mycotoxins in aquatic species. Deoxynivalenol (DON), a Fusarium mycotoxin, is of concern as it is the most commonly occurring mycotoxin worldwide. However, the mechanisms of action of DON and its potential role in fish health are largely unknown. Therefore, the objective of the current research was to determine if DON had any impact on the susceptibility of Rainbow Trout to Bacterial Cold Water Disease and immunity, including immunoglobulin production and macrophage function. Rainbow Trout were fed a nutritionally complete diet naturally contaminated with two concentrations of DON (4 or 6 ppm). After four weeks of feedborne exposure to DON, Rainbow Trout were experimentally infected via intraperitoneal injection with a virulent isolate of Flavobacterium psychrophilum. A significant reduction (p<0.05) in mortalities in DON-fed groups was observed in comparison to control and pair-fed groups at 21 days post infection. These findings warrant further investigation of the effects of DON on the bacterium and on Rainbow Trout immunity.

MAy 2010 – ongoing

Funded by: NSERC Discovery Grant

Project team: Ian Ryerse, Jamie Hooft, Dominic Bureau, Maureen Jarau, John S. Lumsden (U. Guelph)

Contact: jsl@uoguelph.ca

Deoxynivalenol (DON) molecular structure

Evaluation of membrane filtration and UV disinfection for the control of Flavobacterium psychrophilum in recirculation aquaculture systems

Recirculation aquaculture systems (RAS) utilize disinfection technologies to remove pathogens from wastewater and improve fish health. Ultraviolet (UV) irradiation is a disinfection treatment commonly used in RAS. However, Flavobacterium psychrophilum, the causative agent of Bacterial Cold Water Disease (BCWD), is tolerant of recommended UV doses used in aquaculture systems. Membrane filtration (MF) has been used as a disinfection technology in many industries, but has not been thoroughly tested as a disinfection treatment in RAS. The objective of this study was to evaluate MF as a disinfection treatment in RAS and its ability to remove F. psychrophilum. Total bacterial removal efficiencies were assessed between MF and UV treatments over 30 days in a RAS that reared Nile Tilapia (Oreochromis niloticus). MF and UV had equivalent bacterial removal efficiencies and achieved 98.5% and 99.6% removal, respectively. MF also exhibited an additional benefit of removing 95.4% of total suspended solids and experienced low rates of membrane fouling. Under laboratory conditions, we challenged MF with concentrated doses of F. psychrophilum and achieved 5.7-log reductions of the pathogen. Therefore, MF represents an effective alternative to UV disinfection and can be used to remove F. psychrophilum from RAS, potentially reducing outbreaks of BCWD.

sept. 2010 – dec. 2012

Funded by: Environment Canada; Ontario Ministry of Food, Agriculture and Rural Affairs

project lead: David C. Huyben (U. Guelph)

Project team: Richard D. Moccia (U. Guelph)

Contact: dhuyben@uoguelph.ca

www.aps.uoguelph.ca/aquacentre/

Membrane filter
Recirculation system at the Alma Aquaculture Research Station
Checking water flow rates

Innate immunity of teleosts; pattern-recognition receptors and acute phase response

Two plasma lectins, intelectin and ladderlectin, were isolated by their ability to bind to a wide range of infectious agents (PRRs) that cause disease in Rainbow Trout. Ladderlectin was found to have two isoforms and the genomic sequence encompassed six exons and five introns, with exon 2 encoding 14 amino acids that were exclusive to one isoform. Two-dimensional PAGE and western blots demonstrated multiple electrophoretic forms of both lectins. Enzyme immunoassays showed that there was significant group and individual variation in plasma lectin concentrations. Neither lectin was an acute phase reactant nor were the concentrations substantially altered during bacterial infection, however, both lectins were localized by immunohistochemistry in intimate association with microbes in vivo. Both lectins were widely distributed on mucosal surfaces and both were also identified on the cell surface of leukocytes and on trout cell lines. Ladderlectin, but not intelectin, also bound to VHSV, the first demonstration to our knowledge of a virus-binding PRR in fish.

Acute phase proteins were also examined in Rainbow Trout and Walleye using 1 or 2D-PAGE and mass spectrometry. Partial amino acid sequences were identified and 3' RACE was used to isolate cDNA sequences specific to these unknown proteins. A 9.5 kD Rainbow Trout acute phase protein, that underwent a 75-fold upregulation, was found to contain a conserved apolipoprotein A-I domain. A Walleye plasma protein that bound Flavobacterium columnare had 66% similarity to apolipoprotein A-I from Striped Bass (Morone saxatilis). There is therefore evidence indicating that proteins in the apolipoprotein family are involved in the acute phase response and or are PRRs of both fish. These proteins are known to contain lipid-binding domains and may act as PRRs to bind lipid components of fish pathogens during the innate immune response.

MAR. 2004 – ongoing

Funded by: NSERC Discovery and Strategic grants; Chemaphore Inc.; NSERC Fellowship; Ontario Veterinary College Fellowship

Project Lead: John S. Lumsden (U. Guelph)

Project team: Spencer Russell, Karrie Young, Andrew Peterson, Alex Reid, Lowia Al-Hussinee, Adrian Di Natale (U. Guelph); John Pham, Niels Bols, Brian Dixon (U. Waterloo)

Contact: jsl@uoguelph.ca

Epitheliocystis in salmonids

Intracellular gram-negative bacteria associated with epitheliocystis, including several Chlamydia-like organisms, have been found in many fish species. In Ontario, this condition affects Arctic Charr and Lake Trout, and it has also emerged in farmed Rainbow Trout in the last two years. Diagnosis is a challenge as the organisms cannot be cultured and bacterial inclusions are present before clinical signs appear but largely disappear by the time fish are submitted for examination by light microscopy. The histological lesions are fairly consistent, however, and include prominent single-cell necrosis of leukocytes and epithelial cells, in addition to thickening and blunting of lamellae. Identification of the agent from fresh and formalin-fixed gill tissue of affected salmonid species is a priority of this project. Primers described for detection of the 16S rRNA gene of Chlamydiales and 16S rRNA universal bacterial primers have generated a 300 bp and 1500 bp products, respectively. Two sequences have been consistently identified; one with 88% similarity to an uncultured Neochlamydia sp. isolated from a cat with ocular disease and another with 90% similarity to Candidatus brachiomona cysticola, which was also recently identified from Atlantic Salmon with epitheliocystis in Norway. Localization of organisms in affected gills using in situ hybridization and laser microdissection are in progress. Transmission trials and attempts to culture the organism using a Rainbow Trout gill epithelial cell line are also underway.

MAY 2009 – PRESENT

Funded by: Ontario Ministry of Natural Resources (OMNR); Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA - AHSI); Chilean Government Graduate Scholarship

Project team: Elena Contrador, Salvatore Frasca, Brandon Lillie, John S. Lumsden (University of Guelph); Paul Methner, Elizabeth Wright (OMNR)

Contact: John S. Lumsden (jsl@uoguelph.ca)

Epitheliocystis in gills of seawater farmed Atlantic Salmon

Detection and pathogenesis of Spring Viremia of Carp Virus (SVCV) in Ontario baitfish

Spring viremia of carp is a World Organization of Animal Health (OIE) reportable viral disease. SVCV has been recently isolated from carp in Hamilton Harbour, Ontario, Canada. The first aim of the study is to assess the ability of SVCV to infect Ontario baitfish, including Emerald Shiner, Fathead Minnow, and White Sucker. These fish are commonly moved between watersheds and may spread SVCV to new locations. Experimental infection trials have been performed using the Canadian isolate of SVCV (HHOcarp06) using intraperitoneal injection of fish of interest. SVCV caused morbidity and mortality in Flathead Minnow, Emerald Shiner, Koi, and White Sucker, but not in Rainbow Trout. The pathogenicity of disease is being assessed using histopathology and immunohistochemistry. The second aim is to discover if SVCV is more widespread in Ontario than presently realized. A retrospective study is underway using RT-qPCR to detect viral RNA from numerous fish species collected by the Ontario Ministry of Natural Resources (OMNR) from 2008 to 2012. The sensitivity of the RT-qPCR was assessed in tissues spiked with virus dilutions. In conclusion, at least three Ontario baitfish species are at some risk from SVCV, however, further analysis of tissues and results is required.

MAy 2010 – ongoing

Funded by: OMNR; Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA); Egyptian Government Graduate Scholarship; FRST New Zealand Postdoctoral Fellowship; NSERC USRA

Project lead: John S. Lumsden (U. Guelph)

Project team: Ehab Misk, Eva Nagy, Lincoln Tubbs, Adrian Di Natale, Shelby Isaac (U. Guelph); Elizabeth Wright, Kevin Loftus (OMNR); Kyle Garver (DFO)

Contact: jsl@uoguelph.ca

Improved management strategies for bacterial cold water diseases

Bacterial Cold Water Disease (BCWD) caused by Flavobacterium psychrophilum is the most important infectious disease for Ontario Rainbow Trout aquaculture. Several aspects of BCWD have been studied in this research but most are focused on improved management options for farmers. Isolate phenotypic and genotypic variability and antimicrobial susceptibility profiles were compared between Ontario and BC isolates and Ontario isolates were more diverse, however, an association was not found to correlate a bacterial characteristic to clinical presentation. The effect of erythromycin, available only as an emergency drug, was compared with that of florfenicol in a farm trial and both appeared to be equally effective. Autogenous vaccines were trialed on-farm and in the laboratory but they have not been effective. Cold-induced proteins and genes were identified that may be virulence factors and several of the gene products have been cloned and expressed as recombinant proteins for further research. A range of Ontario isolates was used to demonstrate a wide variability in virulence in experimental trials. Morbidity and mortality are being correlated with splenic bacterial colony forming units and quantitative PCR to provide an endpoint before mortality and shorten the length of trials. The breeding program at Lyndon Hatcheries is presently supplying family fish that are being tested for susceptibility to experimental infection with a virulent Ontario isolate. These families are also being examined for MH and immune genes.

MAy 2007 – ongoing

Funded by: Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA); NSERC Strategic and CRD (with the Freshwater Fisheries Society of British Columbia and Microtek Inc.); DFO – Aquaculture Collaborative Research and Devlopment Program (ACRDP) (with Lyndon Hatcheries Ltd.); Ontario Veterinary College Fellowship

Project team: Shohreh Hesami, Arman Yazdanpanah, Maureen Jarau, Lowia Al-Hussinee, Paul Huber, Glen Soltes, Jan MacInnes, John S. Lumsden (U. Guelph); Samantha Hodgins, Calvin Kellendonk, Brian Dixon (U. Waterloo); Sean Pressey, Clarke Rieck (Lyndon Hatcheries)

Contact: jsl@uoguelph.ca

Antigen presentation in teleost fish

Antigen processing is the pathway in which proteins from pathogens are targeted by the host immune response. Despite a fairly detailed knowledge of the genes and proteins that are involved in this process in mammals, the proteins involved in this pathway and their specific interactions are unknown. This project has cloned the genes for those genes involved in this pathway which have not been isolated to date, characterized them and has produced antisera for them. Immunohistochemistry and co-immunoprecipitation are employed to investigate the protein interactions within this pathway.

To date in this project calreticulin has been cloned and characterized. It is located in the endoplasmic reticulum (ER) and responds to immune stimulation, but not to ER stress. ERP57 has also been cloned and characterized. This gene is duplicated in salmonids, with one version having what may be a functional ER retention signal, while the other has a nuclear localization signal. This suggests a division of the roles undertaken by mammalian ERP57. Antibodies have also been generated to MHC class I, beta-2 microglobulin, TAP, and Calnexin. The regulation of these proteins during immunostimulation is being investigated, but initial co-immunoprecipitation studies have shown that tapasin and MHC class I interact within the ER of the RTS11 Rainbow Trout macrophage cell line.

Understanding the process by which pathogenic proteins are identified by fish immune systems will assist in designing vaccines that can elicit strong, effective immune responses in fish. This will hopefully prevent losses to disease, enhancing profitability of aquaculture operations.

MAR. 2008 – ongoing

Funded by: NSERC Discovery

PROJECT LEAD: Brian Dixon (U. Waterloo)

Project team: Lital Sever, Niels Bols, Nguyen Vo, Brian Dixon (U. Waterloo)

Contact: bdixon@uwaterloo.ca

The localization of Rainbow Trout ERp57

Early detection of “Soft flesh” in Atlantic Salmon

Kudoa thyrsites is a marine myxosporean parasite, endemic to the West Coast of Canada, which infects fish and causes post mortem myoliquefaction. Atlantic Salmon are susceptible to infections and this has resulted in significant economic losses to the aquaculture industry. There are several diagnostic tests that can be used to detect infections in fish including molecular tests; however, most of these tests require lethal sampling and results are not available for several days. We proposed to evaluate the sensitivity and specificity of ultrasound for detecting fish infected with K. thyrsites. This technology has been used to detect changes in muscle structure in other animals and provides a rapid assessment of soft tissues. We hypothesize that using computer image analysis of ultrasound images we will be able to identify fish that will develop post mortem myoliquefaction before they develop the condition. If successful this technology has the potential to be used on a large scale at processing plants to identify fish that are not well suited for the “fresh” market.

sept. 2012 – sept. 2013

Funded by: UPEI CERC grant

PROJECT LEAD: Sophie St-Hilaire (UPEI)

Project team: LeeAnn Pack, Tim Burnley, Sohrab Ameli (UPEI); Sonja Saksida (CAHS)

coLlaborators: Centre for Aquatic Health Sciences (CAHS); Marine Harvest

Contact: ssthilaire@upei.ca

Kudoa thrysites infection in salmon

DNA vaccine models against ISA

The goal of this project is to explore the feasibility of new DNA model vaccines against ISAV. It is a novel approach using plasmid constructs expressing ISAV protein subunits combined to an HSP binding sequence, with a suitable linker and signal sequence. Several antigenic ISA proteins and peptides will be selected, from full length protein to single epitopes. The various plasmid constructs will be tested in vivo, and RPS values (relative protection survival) will be determined by comparison to vector alone. To achieve this goal we will: 1) produce recombinant vectors for the expression of specific ISAV genes; 2) prepare sufficient quantities of recombinant vectors for a vaccine trial; 3) do a vaccine trial using different vaccine formulation; and 4) detect DNA vaccine, in situ transcription and measure genomic markers of immune response after vaccination.

july 2009 – dec. 2012

Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP) co-funded by: Atlantic Canada Fish Farmers Association (ACFFA)

project lead: Nellie Gagné (DFO)

Project team: Mark Laflamme, Nathalie Simard (DFO); Lisa Phillips, Kira Salonius (Novartis Animal Health Canada Inc.)

collaborators: Novartis Animal Health Canada Inc.

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