Date of Award

Spring 5-9-2025

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

First Committee Advisor

Deborah Bouchard

Second Committee Member

Heather Hamlin

Third Committee Member

Dana Hill

Abstract

New and emerging diseases in aquaculture result in forty percent production loss annually (FAO 2024), presenting numerous challenges for the industry in terms of aquatic animal health, profitability, and sustainability. To improve the health of finfish aquaculture, advancements in novel diagnostic tools and efficacious prevention measures must be applied. For instance, new cell culture techniques and cell lines need to be developed to allow for the research and culture of currently unculturable viruses in aquaculture, such as the non-delete variant of infectious salmon anemia virus-hemagglutinin-esterase protein region 0 (ISAV-HPR0) and piscine reovirus (PRV) in salmonids. In addition, advancing the commercial availability of new prevention tools, such as vaccines and vaccine adjuvants, is critical for the prevention of disease outbreaks and optimizing aquatic animal health to sustain a food source for a growing population. Considering this, this research aimed to 1) optimize a primary Atlantic salmon gill cell isolation and culture method with the potential to allow further research into pathogens showing gill tropism and 2) test the efficacy of mannosylated chitosan as a potential novel vaccine adjuvant for advancing aquaculture immersion vaccine formulations. Cell culture is considered a gold standard tool for diagnostics and is necessary for the research and cultivation of various viruses of aquacultured finfish. As such, developing new methods for culturing tissue and cell lines is critical to improving the understanding of host-pathogen relationships at a cellular level. Chapter 2 outlines the results from a series of trials to develop a reproducible method for isolating and culturing Atlantic salmon (Salmo salar L.) primary gill cells from Atlantic salmon gill tissue. The following parameters were evaluated towards optimizing the isolation and culture procedure: 1) disinfection solutions for contamination prevention, 2) trypsin-EDTA concentrations for optimal tissue digestion and cell dissociation, 3) wet tissue weight, and 4) culture media additives for optimal cell growth and replication. These parameters yielded varying results and led to the foundation of an optimized method for gill cell isolation and culture with the potential to be applied to research into key salmonid aquaculture pathogens with gill tropism. In Chapter 3, an in vivo cohabitational challenge was performed to test the efficacy of a novel immersion vaccine adjuvant, mannosylated chitosan adjuvant (MCA) formulated with a bacterin for a mucosal vaccine against Aeromonas salmonicida subsp. salmonicida in Atlantic salmon parr. The in vivo vaccine efficacy study comprised of nine vaccine treatments consisting of a DPBS immersion negative control, a bacterin only immersion control, varying MCA concentrations with and without bacterin exposed via immersion vaccination, and a nasal vaccination of MCA + bacterin at a 1:10 dilution, in a cohabitation model. Six-hundred-degree days post-vaccination, salmon were challenged by cohabitation with A. salmonicida subsp. salmonicida. No significant differences in survival postchallenge were observed between any of the novel treatment groups and the salmon that received a negative DPBS sham or bacterin-only immersion vaccination. Due to the known correlation of specific antibody responses found in post-A. salmonicida i.p. vaccinated salmon and vaccine efficacy against furunculosis, the specific IgM response in serum and mucus, and specific IgT response in mucus following vaccination and challenge were tested for using pull-down assays with western blots. These results concluded that the bacterin-only and bacterin-MCA combination immersion vaccine formulations did not elicit an efficacious specific immune response. Furthermore, the lack of protection afforded by the bacterin-only immersion vaccine control made it difficult to conclude whether MCA can act as a mucosal adjuvant to enhance the efficacy of a bacterin immersion vaccine for finfish species. This necessitates additional investigations into the interactions of MCA with fish mucosal surfaces and bacterin are needed. Overall, the results of this research aimed to improve 1) diagnostic tools for pathogen research and 2) novel vaccine adjuvant formulations for immersion delivery in Atlantic salmon. Although this research laid the foundation for primary gill cell isolation and culture, further investigations into gill cell viability and optimal growth conditions are required to improve reproducibility for future viral research. Additionally, interactions between MCA and fish mucosal surfaces must be evaluated to improve pathogen prevention in aquaculture. Research investigating into the interactions of MCA with various antigen types (i.e., whole-killed bacterin, DNA/RNA, or inactivated virus and Virus-Like Particle (VLP) formulations) are needed to understand how effective MCA is for immersion vaccinations to advance knowledge of aquaculture vaccine technologies.

Download

Files over 10MB may be slow to open. For best results, right-click and select "save as..."

Share