Coordination and development of three priority innovation projects for salmon farming in BC

Final Activity Report to AIMAP

Submitted by: The British Columbia Centre for Aquatic Health Sciences Society

Activity Period: September 15 2008 to September 15 2009

Table of contents

• Proposal Objective
• Component 1: Pilot trial of ATPase testing of smolt readiness
• Component 2: Pilot trial of rapid detection of IHNv technique
• Component 3: Selective Breeding Program
• Overall Outcome

Proposal Objective:

The overall objective of this initiative was to implement the scientific infrastructure necessary to facilitate multi-disciplinary and multi-stakeholder aquaculture innovation projects for industry, including three specific projects with direct economic relevance to industry. These projects were based on industry priorities and demonstrated potential for specific economic returns and support for ongoing commercial aquaculture activities (as defined under Expected Outcomes, pg. 13 of 31 of AIMAP contribution agreement dated September 15 2008).

Component 1: Pilot trial of ATPase testing of smolt readiness

For biological organisms, internal salt balance is a lifelong challenge. This challenge becomes especially dramatic in the life of a salmon during the time they migrate between freshwater and saltwater environments. The salt content of the ocean at 50°N latitude is 30-35% (Thomson, 1981) while that of rivers and streams is considerably less. The internal salinity of the salmon must all the while remain constant. How does the salmon achieve this? Through osmoregulation. The kidneys and gills are the two main osmoregulatory organs. Gill physiology, in particular, changes to maintain internal salt balance.

At the beginning of its life in freshwater, the salmon has a high salt content relative to the surrounding stream environment. Thus, water diffuses in along the osmotic gradient. During migration to seawater, the salt content of the salmon is low relative to the surrounding environment; therefore, a concentration gradient is established whereby the direction of flow is in favour of bringing salt into the salmon. So the salmon must constantly pump out salts against the concentration gradient via active transport. Active transport requires energy. This energy is in the form of ATP and is performed by Na+/K+ pumps that line the secondary lamellae of the gills.

ATPases are enzymes that catalyze the reaction of ADP à ATP and so will be found where active transport is occurring. During smolting (the physiological changes which occur in salmonids during migration between freshwater and saltwater environments), there is increased active transport occurring across the gill surface and therefore more ATPase enzymes present.

With this knowledge of the salmon natural life cycle, we are able to farm salmon by growing them in freshwater hatchery facilities then timing their entry into seawater net pens. To reduce stress and mortality, this timing must be precise. Historically, behavioural cues and visual appearance have been used to time saltwater entry. Visually, salmon change colour from greenish-brown, which helps them stay camouflaged in the stream, to a shiny silver which helps them remain unseen by predators in the ocean. More recently, the saltwater challenge has been used to gauge seawater readiness. This method uses measurements of plasma ion levels or osmolality as an indicator of smolt readiness after the fish are immersed in saltwater for a 24- or 48-hour period. This technique has welfare concerns as it requires fish to be exposed to high saline environments (>30ppt) when they may not be ready which would result in significant distress. Killing of the fish is required for this technique.    

The sodium-potassium ATPase assay (adapted from McCormick, 1993) has been developed in an attempt to time more precisely the window of saltwater entry in a humane manner. However the current procedure still requires fish to be euthanized to collect samples.   

The BC CAHS introduced the assay as developed by Europharma AS (formally MariCal) to the salmon farming industry of British Columbia. The assay involved collecting gill tissue from a sub-sample of a population (e.g. 5-20 fish) into an assay solution and transporting the samples on dry ice to the laboratory. Once at the laboratory samples are stored at-80C to the point of time that the analysis can be run. All samples collected from a population are tested at the same time to ensure consistency of the results. For quality control, both positive and negative controls are run during each evaluation. During the period of test validation, duplicate samples were collected and the assay was conducted at both BC CAHS and the MariCal Maine USA facility. This inter-lab validation continues to occur intermittently. 

The assay has been tested on Atlantic Salmon, Pacific Salmon, cultured and wild. Interpretation of the results appear to be most consistent in cultured Atlantic salmon. In general what we have found that this test requires that a population is monitored over a period of weeks to be able to detect change in ATPase levels. Weekly or fortnight testing provides the most reliable results with a recommended minimum of 3 discrete sampling periods. Furthermore there appears to be some problems with the test if the fish are treated for fungus just prior to collection of gill samples. 

There has been some preliminary testing at BC CAHS to decrease processing time of the tissue in the laboratory. This involves the use of sonication (ultrasound agitation) in place of manual crushing of the gill tissue, this testing is ongoing. At BC CAHS we have also conducted preliminary test to determine whether there is any significant difference in results based on killing methods - percussive stunning (standard method) which may be difficult on small fish compared to use of an overdose of anesthestic (TMS). Initial data appears that there may be very little difference in the assay results.

It can be concluded that ATPase testing can provide the producer with invaluable information as to the saltwater competency of their populations and when used in conjunction with observed behavioural and physical changes provides the most accurate measure of smoltification to date. As a consequence this service has become a staple fish performance screening parameter being conducted prior to juvenile fish being delivered to marine grow-out facilities. Between September 2008 and 2009 BC CAHS analyzed 4135 samples for ATPase activity and has established ongoing contracts with industry to continue to provide this service to them. Indications from fish health personal, particularly Marine Harvest the main user of this new technology, is that there is an improved health in the populations entered into the sea as a consequence of use of this assay (see attached letter of support).

References
McCormick, S.D. 1993. Methods for nonlethal gill biopsy and measurement of Na+, K+-ATPase activity. Can J. Fish. Aquat. Sci. 50:656-8

Thomson, R.E. 1981. Oceanography of the British ColumbiaCoast. Ottawa: DFO, pp. 20-23.

Component 2: Pilot trial of rapid detection of IHNv technique

In British Columbia, infectious hematopoietic necrosis virus (IHNv) is the most economically important viral pathogen of salmonids. 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 (IHN) in farmed Atlantic salmon: 1992-1996 and 2001-2003 (Armstrong et al. 1993; Traxler et al., 1993; St-Hilaire, 2000; Saksida, 2006). During the latest epizootic, mortalities were greater than 70% in fish less than 1 kg and averaged 40-50% when fish were larger than 1 kg. Thirty-six farm sites were diagnosed with IHNV during this epizootic (Saksida, 2006) and the estimated economic loss resulting from both epizootics was $40 million in inventory representing $200 million in lost sales. 

The source of the IHNV introduction to farmed salmon is unknown, but epidemiological investigations have identified sockeye salmon and herring as likely sources (St. Hilaire et al., 2002; Saksida, 2006). Due to the potential devastating effect of IHNV on the economic sustainability of the BC salmon aquaculture industry, companies have developed biosecurity action plans for viral containment in the event of another outbreak. However, effectiveness of any containment plan depends on rapid diagnosis of the index case. Therefore, rapid and accurate diagnosis of IHNV is essential. The traditional method of diagnosing IHNV was through recognizing necrosis of cells grown in tissue culture – a technique requiring between 5 and 21 days for confirmation of virus (Thorburn, 1996). 

Quantitative PCR (QPCR) is rapidly replacing more traditional methodologies as a diagnostic test. QPCR offers many advantages over other diagnostic techniques including a fast turn-around time as well as reduced frequency of false positives, increased sensitivity, low requirements for tissue and high sample through-put (Bilodeau et al., 2003; Cavender et al., 2004; Corbeil et al., 2003; del Cerro et al., 2002; Funk et al. in press; Kelley et al., 2004). This technology can also be employed in the detection of IHNV but must include an additional step owing to the fact that the genome of IHNV is composed of single-stranded, negative-sense RNA. Therefore, a reverse transcription step is required to convert genomic and mRNA to cDNA. Quantitative reverse transcription PCR assays (qRT-PCR) have been developed for viral pathogens of aquatic organisms including gill-associated virus of the black tiger prawn Penaeus monodon (De la Vega et al., 2004) and IHNV (Overturf et al., 2001; Purcell et al., 2006). While this work validates the use of this technology for estimating viral loads within infected tissues additional work is required for the reliable detection of IHNV.

This proposal seeked to develop a qRT-PCR assay for detection of IHNv. The primers and probe were designed using conserved regions of the N-gene to allow detection of as many strains of IHNV as possible so that the test can be employed over a broad geographical range. 

The study produced a rapid diagnostic test diagnostic capable of providing rapid IHNv diagnosis. Trials using experimentally infected animals as well as naturally infected suggest that this test is highly sensitive and specific to IHNv. It is estimated that turnaround time for this test is 48 hours this is compared to cell culture (5-21days); 50 samples can be processed in 72 hours. Inter-lab validation was conducted to ensure that the technique results are similar between BC CAHS and other diagnostic laboratories (e.g. Animal Health Laboratory -Abbotsford BC).      

There is considerable advantages in having this new technology available at BC CAHS including

• Faster turnaround time as a result of the new technology
• Location in Campbell River provides the salmon farms easy access to the BC CAHS facility and a faster turn over time
• Improved biosecurity of samples as a result retained chain of custody  (less need for couriers)

As a consequence of the assay - the aquaculture veterinarians have asked the BC CAHS to be part of the IHNv contingency plans as a approved diagnostic laboratory for samples to be submitted in the event of an IHNv outbreak in the future (see attached letters of support). 

References
Armstrong, R., Robinson, J., Rymes, C., Needham, T. (1993) Infectious hematopoietic necrosis
in Atlantic salmon in British Columbia. Canadian Veterinary Journal 34:312-313.

Bilodeau, A.L., Waldbieser, G.C., Terhune, J.S., Wise, D.J., Wolters, W.R. (2003) A real-time polymerase chain reaction assay of the bacterium Edwardsiella ictaluri in channel catfish. J. Aquat. Anim. Health 15:80-86.

Cavender, W.P., Wood, J.S., Powell, M.S., Overturf, K., Cain, K.D. (2004) Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolous cerebralis in rainbow trout Oncorhynchus mykiss. Dis. Aquat. Org. 60:205-213.

Corbeil, S., McColl, K.A., Crane, M.St.J. (2003) Development of a TaqMan quantitative PCR assay for the identification of Piscirickettsia salmonis. Bull. Eur. Ass. Fish Pathol. 23:95-101.

De la Vega, E., Degnam, B.M. Hall, MR. Cowley, J.A., Wilson, K.J. (2004) Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon. Dis. Aquat. Organ. 59: 195-203.

del Cerro, A., Mendoza, M.C., Guijarro, J.A. (2002) Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum. J. Appl. Microbiol. 93:149-156.

Funk, V.A., Raap, M., Sojonky, K., Jones, S., Robinson, J., Falkenberg C. and Miller, K.M. (2007) Development and validation of an RNA- and DNA-based quantitative PCR assay for determination of Kudoa thyrsites (Gilchrist) infection levels in Atlantic salmon (Salmo salar L.). Dis. Aquat. Org. accepted.

Kelley, G.O., Zagmutt-Vergara, F.J., Leutenegger, C.M., Myklebust, K.A., Adkison, M.A., McDowell, T.S., Marty, G.D., Kahler, A.L., Bush, A.I., Gardner, I.A., Hedrick, R.P. (2004)

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis. J. Vet. Diagn. Invest. 16:202-211.

Overturf, K., LaPatra, S., Powell, M. (2001). Real-time PCR for the detection and quantitative analysis of IHNV in salmonids. J. Fish Dis. 24: 325-333.

Purcell, M.K., Hart, S.A., Kurath, G., Winton, J.R. (2006) Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus. J. Virol. Meth. 132: 18-24.

Saksida, S.M. (2006) Infectious haematopoietic necrosis epidemic (2001 to 2003) in farmed Atlantic salmon Salmo salar in British Columbia. Dis. Aquat. Org. 72: 213-223.

St-Hilaire, S. (2000). Epidemiology of infectious hematopoietic necrosis disease in net-pen reared Atlantic salmon in British Columbia, Canada.  PhD thesis, University of Guelph, Guelph, Ontario.

St-Hilaire, S., C. S. Ribble, C. Stephen, E. Anderson, G Kurath, M.L. Kent. (2002). Epidemiological investigation of infectious hematopoietic necrosis virus in salt water net-pen reared Atlantic salmon in British Columbia, Canada. Aquaculture 212: 49-67.

Traxler, G.S., Roome, J.R. and Kent, M.L. (1993) Transmission of infectious hematopoietic necrosis virus in sea water. Dis. Aquat. Org. 16:111-114.

Thorburn, M.A. (1996). Apparent prevalence of fish pathogens in asymptomatic salmonid populations and its effect on misclassifying populations infectious status. J. Aquat. Anim. Health 8: 271-277.

Component 3: Selective Breeding Program

The BC CAHS had submitted a proposal to Genome BC that would support the development of a selective breeding program for the BC salmon farming industry. Administration of the Selective Breeding Program was included in this original AIMAP proposal however the Genome BC proposal did not proceed and an amendment was submitted to AIMAP April 8 2009 reflecting this. The largest component of this project comprised of liaising with industry, transferring knowledge and identifying key areas for innovation and funding sources, these same activities were applied to developing new innovation projects in addition to facilitating other aspects of establishing a selective breeding program in BC. As reported in Annex D (June 30th 2009) the administrative portion of the selective breeding component was also allocated towards developing alternative initiatives for improving the performance of the aquaculture industry.

The funding application to Genome BC was lead by a former researcher at BC CAHS, Dr. Kevin Butterworth. After his departure to pursue other research activities and the passing of the Lab Manager and Research Scientist Dr. Val Funk, administration of the Selective Breeding component was taken over by then CAHS CEO Ms. Linda Sams followed by the Business Manager Tracy Burgess. To improve selective breeding program in BC’s aquaculture industry Ms. Sams facilitated ongoing discussions with industry and genome BC to implement the project, and after considerable work to bring industry together with the purpose of establishing a selective breeding program the development of a genetic broodstock management tool has yet to be developed. Although there have been a number of discussions about the use of genetic markers and the value of using this technology in a selective breeding program. This approach is novel and until genotypic characteristics are correlated with desired heritable phenotypic traits – more work still needs to be done. However, the groundwork for developing such a program has been established for providing the industry tools and expertise in the eventuality that an industry-wide selective breeding program is established.

Alternate technologies for facilitating a selective breeding program have also been explored including, technology transfer initiatives to improve the cryopreservation success of BC aquaculture technology as a reliable technical service will be essential for a genetic-based selective breeding program to be viable. To facilitate this, two hatchery technicians form New Zealand King Salmon came to Canada and toured BC salmon hatcheries to demonstrate a cryopreservation technology with 70% viability rate compared to that of the BC industry at less than 25% viability. There was no immediate direct improvement to BC hatchery milt viablility after this technology transfer and the establishment of a laboratory-based cryogenetics faclility at BC CAHS was explored. More recently, consultation between BC CAHS and Cryogenetics AS in Norway have begun to explore a potential partnership agreement for transferring Cryogeneics proprietary cryopreservation technology to BC as Cryogenetics AS has 90-95% viability for Atlantic salmon species and is conducting ongoing research and development to optimize the same technology for Pacific Salmon species (along with Tri-gen technologies and Sea Spring Hatchery).

Additional alternative initiatives for improving the performance of the aquaculture industry that were developed included developing new AIMAP and ACRDP proposals for local industry stakeholders as follows: Soft-flesh suppression technology with Marine Harvest Canada, U.V. Treatment of processing plant effluent with Walcan Seafoods Ltd., and development of the Aquamax net manger system with Deanne Larson. ACRDP components were developed for two of the AIMAP projects in which BC CAHS would be directly participating including the soft-flesh suppression and UV treatment initiatives. Also conducted was a pilot evaluation of the effects of stress during fish-harvest on the manifestation of a parasite that causes soft-flesh in farmed Atlantic salmon (see also annex D report to AIMAP June 30th 2009) This is an urgent problem for Canada’s aquaculture industry as the potential effects of this parasite on product quality have created a negative stigma towards the quality of farmed salmon in national and international markets and because of this innovation opportunity BC CAHS was able to help industry address it in a timely manner.

Collectively these components delivered the following activities (as per AIMAP contribution agreement page 14 of 31):

• Provided a scientific support and advisory role to industry on topics of production efficiencies e.g. reduced costs and increased value of aquaculture products and improving environmental performance.

• Supported the development of new innovation initiatives in BC’s aquaculture industry through meeting attendance, information collection and dissemination on topics of interest for industry and government. The BC CAHS collected information from government, industry, academic, and community sources to support the design of projects that promote innovation and implementation of solutions.

• Coordinated researchers and industry end-users for innovation project partnerships.

• Provided linkages and assistance upon request as industry queries arose by facilitating access to stakeholders, networks and resources, in particular for knowledge transfer and evaluations of issues relating to sea lice and aquaculture (please see annex D for details).

• BC CAHS provided a framework for scientific exchange; nationally and internationally such as forming communication networks among members of the scientific community on fish parasite challenges affecting the aquaculture industry.

• BC CAHS has maintained and optimized use of local, national and international networks.

• The development of new innovation initiatives, global innovation practices were reviewed in order to introduce them to the aquaculture industry, e.g. the development of a high hydrostatic pressure processing methodology for soft-flesh suppression in farmed salmon.

• BC CAHS catalyzed the move of industry innovation priorities —such as a rapid screening method for viral outbreaks and suppressing the effects of a soft-flesh inducing parasite in farmed salmon — to concrete projects ready for implementation.

• Projects such as the ATPase testing of smolt readiness were moved from the pilot research stage to the commercialization stage.

Overall Outcome:

This project provided the scientific and network support needed for the development of major innovation initiatives of economic importance to the salmon aquaculture industry in three main areas including: 1) the implementation of a commercial-scale enzyme activity assay as an industry standard for minimizing fish losses at seawater entry; 2) testing and refining a rapid disease detection method for diagnosing IHNv to improve management of disease outbreaks and reduce economic losses to industry; and 3) progress towards developing a selective breeding program for BC’s aquaculture industry and the development and implementation of three new innovation projects for aquaculture industries and entrepreneurs.