Sea Lice Vaccines for Salmonid Aquaculture

Microtek Research and Development Inc. (Pfizer Animal Health)

Table of contents

• Executive Summary
• Introduction
• Methods
• Development and husbandry of a lice culture system
• The development of a sea lice infection model
• Validate proof of concept.
• Results
• Development and husbandry of a lice culture system
• The development of a sea lice infection model
• Validate proof of concept
• Conclusion
• References

Executive Summary:

Sea lice infestations present a significant challenge to aquaculturists, veterinary clinicians and vaccine developers. Sea lice tend to feed on blood, skin and mucus resulting in severe damage to the skin and even death [1-3]. These parasites have been estimated to cost the salmon aquaculture industry as much as US$480 million world-wide based on the FAO Fisheries and Aquaculture Information and Statistics Service (2008) [4]. It is believed that this estimate represents 6% to 10% of the value of production, and the problem has considerably worsened since for the countries affected. Currently chemical treatments are primarily used to control infestations, however this also presents several disadvantages including toxicities, drug tolerance, environmental impacts, negative consumer perception, availability, and not the least of which is cost. Vaccination presents an alternative to chemical use that addresses many of these disadvantages [5].

Recent R&D at Microtek International Inc. (now Pfizer Animal Health, Pfizer Canada Inc.) has identified a number of candidate vaccine antigens. The goal of this project was to complete the remaining development, pre-commercial manufacturing, and regulatory work required to license a vaccine for the control of sea lice infestations of farmed salmon in Canada. The initial trials were to be completed be completed at Microtek’s (now Pfizer Animal Health, Pfizer Canada Inc.) laboratories located at the University of Victoria and field trials at West Coast aquafarm(s).

After a significant effort we were unable to fully develop an in-house challenge model to complete the remaining studies. A number of obstacles such as episodic infestations, limited sea lice harvest sites, technical issues such as hatching systems, water turbulence, and oxygen levels resulted in a delay to the establishment of a cost-effective challenge model. However, we were able to determine that it is not feasible to establish an in house model at our facility. This has resulted in the successful development of alternative strategies: Contract Research Organizations (CRO), and the recent identification of an alternative Canadian facility. Consequently we have overcome initial difficulties and are proceeding with infestation trials of candidate vaccines.

Introduction:

The success of aquaculture is largely dependent on the control of fish pathogens, which includes a diverse spectrum of viruses, bacteria, fungi and parasites. Successful management strategies generally require a variety of approaches for disease control one of the most important being the deployment of efficacious and cost-effective vaccines. Vaccines range in complexity from inactivated cells to highly complex biotechnology-derived products. The most difficult pathogens such as parasites require complex vaccine designs for which few vaccine models have been developed. Currently there are commercially available fin-fish vaccines for some bacterial and viral diseases but none for parasites. Historically Microtek (now Pfizer Animal Health, Pfizer Canada Inc.) has excelled at the development of cost-effective subunit vaccines for aquaculture.

Microtek R&D (now Pfizer Animal Health, Pfizer Canada Inc.) has identified and tested several targets  that we strongly believe would form the basis of a novel vaccine against the most prevalent salmon louse, L. salmonis. This basic research work has taken place over the course of several years working collaboratively with our research partners and with considerable financial investment. The logical next steps in the development of a commercial vaccine would be to systematize the proof of concept, manufacture commercial scale pre-license serials and field test a commercial product.

Methods:

1) Development and husbandry of a lice culture system.

Sea lice egg strings were collected from local waters and transported to our laboratory. Egg strings were placed in specially designed tanks, containing ocean water  at the optimal salinity and temperature, and aerated until the eggs hatched and developed though the nauplius stage I and II to the infectious copepodid stage. Once sea lice reach the copepodid stage, they were harvested and added to tanks containing Atlantic salmon. The initial breeding studies were to involve the use of small tanks to ensure the capability of breeding sea lice on a smaller scale. Based on the breeding success of the initial studies, we could then scale up the production of sea lice to larger tanks.

2) The development of a sea lice infection model.

Based on the current literature on sea lice infection (reviewed in [6]) an average of roughly 100 sea lice per fish were to be added to the tanks. After a period of 24 hours, a number of Atlantic salmon were to be removed, anaesthetised, and the attached sea lice counted. After the counts have been completed the fish are returned to their holding tanks. To institute an infection model, we anticipated several trials would have to be completed in order to establish a consistent model that represents natural infections found in the field. This would involve using different amounts of sea lice per fish as well as testing Atlantic salmon of varying weights.

3) Validate proof of concept.

Confirmation trials of preliminary results following vaccination with the candidate target antigens. For the vaccination experiments, Atlantic salmon were to be anaesthetised and injected with the vaccine or with placebo. Sea lice challenge was to be carried out after proper immunity development to the vaccine.  After infection, a number of Atlantic salmon were to be removed from the tanks, anaesthetised, and the number of sea lice attached determined. In such a system we believe that we could easily and inexpensively test a number of vaccines and various combinations and doses. To further test our optimal dose in a larger setting, we proposed to use the facilities at DFO PBS in Nanaimo through our collaboration with Dr. Stewart Johnson.

Results:

1) Development and husbandry of a lice culture system.

After consultation with a number of experts in the field, it is believed that in order to develop a self-sustaining laboratory lice culture, a large number of resources must be directed toward this project alone. It is understood that several large tanks (significantly bigger than any available at our facility) needed to be specifically dedicated to breeding sea lice, as well as full time personnel with expertise must be put in place. Another large obstacle will be the ability to breed and raise enough copepodids at anyone time, to be capable of infecting Atlantic salmon in our in-house model. Therefore, after careful consideration and input from several sources we believe this objective cannot be achievable with the limited resources available for our sea lice project.

2 The development of a sea lice infection model.

The original AIMAP contribution application, submitted by Microtek International Inc. in June 2009, was completed prior to its acquisition by Pfizer, Inc. Over the last year, Microtek International Inc has been going through the integration process with Pfizer Canada Inc. This lengthy and involved integration process lead to a number of project delays as the company adapted and become familiarized with Pfizer policies.

The first year of the AIMAP-2010-P034 contribution was spent on developing and standardizing an in-house sea lice model. A number of obstacles were encountered throughout this period. The first and most important was the availability of sea lice and the establishment of contacts which to regularly use as a source of sea lice. Since the AIMAP grant only received approval by the end of summer, limited sampling due to low numbers was our first major obstacle. However, during this time several trips were taken and numerous egg strings were harvested and brought back to our facilities.

The next obstacle was the proper hatching of sea lice egg strings followed by their upkeep to ensure the healthy upbringing through the nauplius stage I and II to the infectious stage (copepodid). One of largest problems with developing a sea lice model is how variable the literature is when is comes to hatching and infecting protocols. A number of obstacles such as proper hatching systems, the depth at which our sea water was collected, water temperature, water turbulence,  oxygen levels, sea water purification and filtration methods, were encountered.

Although we were not capable of developing the sea lice challenge model, each collection and hatching event moved us further ahead in its development. Each obstacle taught us different aspects which are critical and must be properly maintained for a successful model.

3) Validate proof of concept.

At this time we were not to validate our proof of concept since we requires an in-house working model to complete this objective.

Conclusion:

After a significant effort we have determined that it will not be feasible to establish an in-house model at our facility. However, we have approached our original objective by two other strategies. The first will be to contract this work out to a Contract Research Organization (CRO) with a fully established sea lice breeding program and challenge model. We have recently established a full relationship with a CRO which specializes in Lepeophtheirus salmonis (northern species) and a CRO specializing in Caligus rogercresseyi (Southern species).

As our second strategy, we have identified and have begun developing relations with an alternative Canadian facility with a fully established Lepeophtheirus salmonis model. After preliminary discussions it appears that our previously proposed objectives will be achievable in the upcoming year.

References:

1. Pike, A.W. and S.L. Wadsworth, Sealice on salmonids: their biology and control. Adv Parasitol, 1999. 44: p. 233-337.
2. Dawson, L.H., et al., Effects of salmon lice Lepeophtheirus salmonis on sea trout Salmo trutta at different times after seawater transfer. Dis Aquat Organ, 1998. 33(3): p. 179-86.
3. Krkosek, M., et al., Sea lice and salmon population dynamics: effects of exposure time for migratory fish. Proc Biol Sci, 2009. 276(1668): p. 2819-28.
4. Costello, M.J., The global economic cost of sea lice to the salmonid farming industry. J Fish Dis, 2009. 32(1): p. 115-8.
5. Raynard, R.S., et al., Development of vaccines against sea lice. Pest Manag Sci, 2002. 58(6): p. 569-75.
6. Wagner, G.N., M.D. Fast, and S.C. Johnson, Physiology and immunology of Lepeophtheirus salmonis infections of salmonids. Trends Parasitol, 2008. 24(4): p. 176-83.