Development of mucoadhesive nanovaccine against columnaris disease in red tilapia (Oreochromis sp.)

Abstract

Tilapia (Oreochromis sp.) is very important and high production of freshwater fish in Thailand. Columnaris disease has been now recognized as one of the most serious infectious diseases in farmed tilapia. The disease is caused by Flavobacterium columnare. Among the prevention and control strategies, vaccination is one of the most effective approach. According to the pathogenesis of this bacteria, the characteristic lesion is almost at mucosal area of skin and gill. Therefore, hypothesized that the mucosal nanovaccine with mucoadhesive characteristic could be suitable vaccination method to control columnaris disease. In this study, we determined vaccine strain candidate by clinical field isolation, morphology and molecular characterization and virulent ability test. We prepared chitosan-complexed nanovaccines (CS-NE) through emulsification and homogenization techniques followed by coating with mucoadhesive polymer chitosan. The physiochemical properties of CS-NE were analyzed. Their mucoadhesive characteristics, vaccine efficacy and immune responses were also evaluated. The analysis of hydrodynamic diameter and zeta-potential also indicated the successful modification of CS-NE that were positively charged, nano-sized and spherical. In vivo mucoadhesive study demonstrated the excellent affinity of the CS-NE toward fish gills as confirmed by TEM, bioluminescence imaging, fluorescent microscopy, and spectrophotometric quantitative measurement. Following vaccination with the prepared nanovaccines by immersion 30 mins, the challenge test was then carried out 30-60-90- and 120-days post-vaccination and resulted in 89,91,71 and 61 % mortalities, respectively in the control fish. The RPS of CS-NE vaccinated fish was calculated at 78,61,50 and 36, respectively. As a result, the formulated biomimetic nanovaccine mocking the mucoadhesive characteristic of live F. columnare can help achieve better adsorption on mucosal surfaces and more efficient vaccine efficacy that revealed in MALT histology. We evaluated immune response of CS-NE fish vaccinated include serum bactericidal activity, ELISA-IgM specific F. columnare, MALT histology and relative gene expression. Significantly higher serum bacterial activity and ELISA-specific IgM antibodies in CS-NE was also seen. The MALT histology revealed a significant higher leucocyte cell accumulation and antigen uptake, in accordance with our result of up-regulation of IgT, IgM, TNF α, IL1β and MHC-1 genes in gill, kidney and spleen. Our study demonstrated the feasibility of mucoadhesive nanovaccine-immersion vaccination as an effective delivery method for prevention and control columnaris disease in tilapia.