Developing the First Live Modified Vaccine against Columnaris Disease for Aquaculture in Asia

Description

Flavobacterium covae (previously known as F. columnare genomovar II) is the causative agent of columnare disease that affects many freshwater fish species worldwide. This bacterium attaches to the fish, leads to lesions and ulcers, and causes a mortality rate of 90% in fish fry and up to 60% in freshwater fishes. Despite its significant impact on freshwater aquaculture worldwide, there is no effective vaccine to treat columnaris disease in Asia yet. One of the major challenges in the F. covae live-modified vaccine study is the lack of a suitable strain as the model for targeted genetic manipulation and virulence evaluation. In our previous study, we generated a mutant using the antibiotic random mutagenesis method (i.e., rifampin-resistance selection). The mutant has completely lost virulence while maintained high immunogenic properties. Furthermore, we identified 14 point mutations between the wild and the mutant strains, which were located in regions involved in 8 protein-coding genes, and 5 intergenic regions. However, the specific critical genes or regulatory elements responsible for the virulent loss have not been determined yet. In this project, our first objective is to construct the mutants containing each point mutation using site-directed mutagenesis and assess how each point mutation affects the bacterial phenotypic characteristics. Our second objective is a safety test by evaluating the mutant virulence by artificial infection and identifying the critical mutations responsible for virulence attenuation. Furthermore, when a bacterial mutant contains multiple mutations in addition to the virulence-determining ones, the immunogenicity is likely compromised because of the altered protein structure of the wild-type epitopes. We hypothesize that the relative percentage survival (RPS) of new vaccine candidates will be further increased by generating targeted virulence-relevant mutations (while avoiding any non-virulence-relevant changes). Thus, our third objective is to evaluate the immune protection rate of the newly constructed mutants and determine if the avirulent mutant demonstrated improved immune protection as a superior live vaccine candidate. This study will help us understand the pathogenesis of F. covae, and our work will lay the foundation for developing the first live-modified vaccines against columnaris disease caused by F. covae in aquaculture.