Protective immunity against Vibrio harveyi in Grouper induced by nano-bacterin plus recombinant protein GAPDH

• Main Authors: Yun-Pei Yang, 楊昀珮
• Other Authors: Chung-Da Yang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/18866251736262581841

碩士 === 國立屏東科技大學 === 動物疫苗科技研究所 === 101 === Orange-spotted grouper (Epinephelus coioides) is an economically important farmed fish in southeast Asia, including China and Taiwan. Infecting with diseases is causing huge loss in aquaculture industry. In Taiwan, the disease rate of farming fish has increased tremendously due to the way of high-density farming fishery, which resulted in high death rate of fishes and shells. The economic consequence has been devastating. Vibriosis is one of the most serious diseases, especially Vibrio harveyi, a major pathogen of marine fish in this region, research related to vibrosis of farming fish has been intensively carried out worldwide. Clinical signs induced by V. harveyi included gastroenteritis and eye lesions. The infection causes high mortality in fish. The vaccination against Vibrio species is a viable strategy for controlling vibriosis. However, no commercial vaccine is currently available. Current studies have shown that the outer membrane proteins (OMPs) of pathogenic Vibrio bacteria are protective antigens to induce effective protection against Vibrio infection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one OMP of Vibrio species and its gene was cloned into pET24a plasmid to express recombinant GAPDH (rGAPDH) in BL21 (DE3) Escherichia coli. After immunoblotting assay, we found that sera from grouper infected with V. harveyi obviously recognized the purified rGAPDH. Six groups of 25 grouper each were peritoneally vaccinated twice at a 21-day interval with PBS, rGAPDH (50 μg/fish), microfluidizer nano-bacterin (MF) (50 μg/fish) only, MF (50 μg/fish) plus rGAPDH (50 μg/fish), MF (150 μg/fish) plus rGAPDH (50 μg/fish), formalin-killed V. harveyi. Fish sera were collected on days 14 and 35 after the first vaccination. ELISA was used to determine serum responses against V. harveyi lysate. Results showed that specific antibody titers were significant increased in fish vaccinated with rGAPDH (50 μg/fish), microfluidizer nano-bacterin (MF) (50 μg/fish) only, MF (50 μg/fish) plus rGAPDH (50 μg/fish), MF (150 μg/fish) plus rGAPDH (50 μg/fish), formalin-killed V. harveyi. Three weeks after booster, fish were challenged with 4.45× 106 CFU V. harveyi (AOD99094-1) and then monitored for mortalities for 21 days. The relative percent of survival (RPS) for each group was calculated. Fish vaccinated with rGAPDH (50 μg/fish) showed 9.1％ of RPS. Fish vaccinated with MF (50 μg/fish) showed 93.75％ of RPS. Fish vaccinated with MF (50 μg/fish) plus rGAPDH showed 93.75％ of RPS. Fish vaccinated with MF(150 μg/fish) plus rGAPDH (50 μg/fish) showed 87.51％ of RPS. Fish vaccinated with formalin-killed V. harveyi showed 45.5％ of RPS. These results demonstrated that vaccination with MF or MF mixed with rGAPDH could provide a potent protection against V. harveyi infection in grouper. In conclusion, we successfully applied microfluidizer nano-bacterin on the Vibrio vaccine development in grouper.