Development of new vaccines to combat aquaculture pathogens in South East Asian countries

Aquaculture is a rapidly growing sector, with large economic value and potential. However, aquaculture is severely hampered by disease outbreaks, which cause large losses in production. Antibiotics are often used in South East Asian countries to control disease outbreaks, due to their low cost and ease of use, nevertheless they are ineffective against viral infections and give rise to antibiotic resistant strains. This research sought to develop more convenient and low-cost alternatives, using bacteria and microalgae to express proteins and double stranded RNA (dsRNA) which could then be administered orally. Soluble and insoluble expression of Glyceraldehyde-3phosphate dehydrogenase (GapA) from Streptococcus agalactiae, a bacterial pathogen which causes high mortality in fish, was achieved in Escherichia coli in shake flask and fed-batch fermentation. Purified soluble and insoluble protein were administered orally to red hybrid tilapia fish and challenged with S. agalactiae resulting in a 51.1% and 75.6% survival respectively, compared to 13.3% in the control group. GapA was expressed in the chloroplast of the microalgae Chlamydomonas reinhardtii and later optimised with yields of up to 18 mg/L being achieved. Freeze dried transgenic C. reinhardtii showed that protein could be detected up to 54 days when stored at room temperature, showing large potential for microalgae for the production and oral application of vaccines. In addition, two dsRNA expressing C. reinhardtii strains were generated targeting key viral genes of the white spot syndrome virus, which causes large losses in shrimp aquaculture. Part of the VP9, thought to be involved in viral replication, and ORF366, part of the nucleocapsid, genes were expressed in the chloroplast as dsRNA reaching yields of 3.86±1.07 µg/L (VP9) and 11.63±5.84 µg/L (ORF366). However, challenge trial data appeared to show no real benefit to survival rates to shrimp over the control (dsRFP expressing) strain. Finally, an alternative strategy to help identify biomarkers for disease resistance was also developed using an antibody generated to target Alpha2-macroglobulin (A2M) in Brown-marbled grouper. Blood serum samples from fish more resistant to infections of Vibrio parahaemolyticus showed higher levels of A2M, indicating A2M as a potential disease resistance biomarker.