THE TRANSMISSION AND PREVENTION OF AQUATIC DISEASES IN MAINE

Project Methods
Goal 1Goals Investigate the factors that govern the transmission of infectious disease between wild and farmed aquatic organismsThis component of the project will be predominately field based. However, there is a significant component of this research that will be developmental to develop a proof of concept of the principles of continuous monitoring of eDNA. Working with the colleagues the principals of the npvrl eDNA collector need to be fully developed and tested and because this has commercial potential the Intellectual Property needs to be fully protected before details can be published. The collection efficiently also needs to be determined for the concentrate. Effectively the sensitivity and specificity of the new sampling method need to be evaluated and compare to the traditional method of extracting the DNA out of a field sample onto a nitrocellulose membrane in the field in the laboratory before the eDNA analysis can begin. Once this proof of concept has been established. It will be extended into the field in Cobscook Bay, Maine, to collect the samples that will subsequently be analysed for the present, relative abundance and species of sea lice present using the probes developed by McBeath et al 2006Goal 2The overwintering strategy of sea lice The overwintering strategy of sea lice will be assessed using these three approaches: 1. Stable isotopes Analysis (SIA): The use of stable isotopes is a well-established tool to identify the trophic levels of an organism within a food web. A less well-known application of stable isotopes is their use to determine the temperatures in which an organism is living. The technique was established for the determination of temperature profiles in paleoenvironments in regards to the tests of carbonaceous (sub) fossils such as Foraminiferans using the ratio of oxygen16 to oxygen18. Recently, this technique has been applied to marine plankton to help understand the impact of climate change on planktonic communities.2 Distribution within the water column: Sea lice were assumed to be limited to the top 4m of the ocean for many years 40. However, a recent paper by Nelson et al (2017)41 demonstrated that the infectious nauplii and copepodids can be found at depth in the Bay of Fundy (BoF 41). Currently, it is unknown if this is due to a unique set of oceanographic environments or a previously unknown strategy where the larvae migrate to warmer waters at depth41 to avoid the external cold surface water of the BoF and the GoM during winter months. Indeed, the thermal expansion and compressibility of wax esters and lipids in thier tissues may allow the lice larval stages to be neutrally buoyant in cold waters, and they can thus avoid spending energy to remain at depth 41.3 Physiology of development: In conjunction with objective 2, it is proposed to look at the effect temperature has on the physiology of sea lice eggs and larvae. Johnston and Albright 33,34 and Boxaspen 42 wrote seminar papers that showed the speed of development to the infectious stage and sexual maturity are temperature dependent. However, the actual physiological impact temperature has on respiration and the larvae's lipid reserves is unknown. L. salmonis larvae are entirely lecithotrophic, relying for energy upon maternally derived lipid reserves stored in the cells of the developing gut. This will allow for us to assess the total lipid volume using a variation of the method of Cook et al (2012)43, by measuring the lipid droplets seen in the eggs histologically. A pilot study has already established the validity of the histological method and the lipid droplets in the eggs of can be seen in Figure 4.Goal 3Develop new technology that allows the vaccination and the activation of the mucosal immune system of fish, in particular farmed Atlantic salmon, to protect them from ectoparasite infections and bacterial and viral infections that are taken up by the host via the mucosa.One limitation of aquaculture vaccines is they are often formulated with powerful oil-based adjuvants that cause serious granulomas to form (see figure 1, proposal, p.3). Adjuvants that trigger mucosal responses are far less harsh and often produce a transient inflammation that rapidly disappears. It is proposed that both epidermal presentation of vaccine to fish and new adjuvant formulations are developed, not least those based on Crystalline Nannocellulose (CNC), a product that has many biomedical applications (and where Maine is a leader in its application) and immunogenic copolymers e.g.poly (lactic-co-glycolic acid) (PLGA). At the very least, CNC and PLGA will act as a depot for the vaccine in the epidermis increasing the time the vaccine is presented to the mucosal immune system. Rather than being transported to the pronephros as happens with intraperitoneal (IP) injected vaccines. To assess the potential of both the epidermal vaccine delivery system and the mucosal immune response, the zebrafish tool box will be very useful in assessing these new methods. The Caspar line of zebra fish is the perfect model animal as many of its immune cells can be tagged with RFP or GFP to observe the cellular processes directly and combined with assays looking at key epithelial immune genes and in combination with immune assays such as IgZ/IgM ELISA's against the model vaccine. This would be a key breakthrough in being able to use the zebrafish model to evaluate commercial fish vaccines. Successful completion of this project component would provide the groundwork for a new class of adjuvants for use in aquaculture that specifically target the skin. It is anticipated that epithelial adjuvants will provide the two fundamental roles of an adjuvant; that of a depot for a vaccine allowing presentation to the immune system over a long period of time, and as a platform for immunomodulation, leading to upregulation of Th1 and/or Th2 responses in the host. It is also anticipated that due to the documented safety profile of the adjuvant that will be investigated such a CNC and PLGA, there will be minor to no adverse effects of the adjuvant leading to a reduction in adhesions and injection site granulomas which cause welfare and production issues.Cellulose nanocrystals and PLGA are sophisticated, biological derived, nanoparticles that are made from readily renewable plant materials such as those derived from forestry, or straw after grain harvest. CNCs were first produced in the 1980s and there has been considerable work with them in food technology, pharmacology and tissue engineering fields. They are being considered for generally recognized as safe (GRAS) status by the American Food and Drug Administration, which if successful, will exempt CNC from routine testing under the Federal Food, Drug, and Cosmetic Act.Although CNC and PLGA have a role in pharmacology, including drug depot applications, e.g. antimicrobial surfaces and wound dressings 44, there has been no investigation into their role as an adjuvant or antigen depots for immunotherapeutics.Goal 4Host parasite interactionsThe host parasite interface remains crucial to the control of parasitic infections. It is well known that sea lice secrete immunomodulators to overcome the hosts' defenses and to establish a successful infection. Indeed, my previous HATCH study produced a patent for a sea lice vaccine based on some of these products. The issue is that all current fish vaccines are delivered by injection into the peritoneal cavity (IP). This is an excellent mechanism for inducing a systemic antibody and cell mediated immune response, but does very little to protect the mucus membranes and skin where sea lice and many other pathogens and parasites target for infection (e.g. F. columnare). Building on the findings of the previous HATCH project it is proposed to look at delivering vaccines to the mucosal immune system.