Immunogenicity And Protective Effects Of An Oral Vaccine Microparticle Against Aeromonas Punctata In Grass Carp (Ctenopharyngodon Idella)

The paper included three experiments which studied isolation, identification , determination of LC₅₀ about Aeromonas punctata,then preparing the microparticles containing the killed whole cells (WC) of Aeromonas punctata and evaluated their immunoprotective effects on grass carp. First, determination of LC₅₀ about Aeromonas punctata, to evaluate their immunoprotective effects on grass carp. The vaccine microparticles were prepared by the solvent evaporation method., To making sure the suit microparticles by adopting different condition. The sticking pont about the paper was to make sure the microparticles can or not be used as effective fish vaccines against Aeromonas punctata infection, which taking two method to determine it. First, In the process of inmunition, Collection blood serum of grass carp, Serum antibody titers determined after oral immunization by ELISA. Second, Protective effects of oral vaccine microparticles on grass carp against Aeromonas punctata.1. Isolation, identification, dfetermination of LC₅₀ about Aeromonas punctataBacteria was isolated from dying grass carp, then checking with lab techniques by identification tube to make sure its the case of Aeromonas punctata. And we also found that its LC₅₀ against grass carp was 10^6.2CFU/ml by injecting different bacteria concentration in healthy grass carp.2. Preparing the microparticles for vaccine sustained releaseMicroparticles were prepared by a modified w/o/w double emusion-solvent diffusion method with Polyacrylic resin HI as carrier materials. Scaning microscopy was used for the observation of microspheres. Size distribution was determined also by ruler of microscopy. The loading capacity and efficiency of vaccine in the microparticles were determined by Coomassie brilliant blue G-250, Study how the concentration of PVA, the concentration of Polyacrylic resin III, volue ratio of internal and outer phase, the power of sonication ,the homogenization speed to affect the particle sizes, entrapment efficiency and bacterial entrapment of microparticles. Results found that the real experimentation condition as follows: the concentration of PVA is 6 %, the concentration of Polyacrylic resin III is 60mg/ml, volue ratio of internal and outer phase is 1:6, the power of sonication is 400 W, the homogenization speed is 9500 r/min. which almost smooth microparticles 10um in diameter containing relatively high loading capacity(6.32%) and encapsulation efficiency of vaccine(62.35%). Inconclusion the vaccine microparticles were found to own a good loading capacity and efficiency, were markablly sustained release of vaccine.3. Immunogenicity and protective effects of the microparticle against Aeromonas hydrophilaAeromonas punctata from grass carp was isolated to prepare oral vaccine microparticle, and heat-killed whole cells, and the adjuvant vaccine of Freund's Adjuvant Incomplete,The immunoprotective effects of these vaccine candidates were investigated in grass carp by oral administration and the othe two by intraperitoneal injection according to three times and interval fourteen days. Antibodies in serum were detected by ELISA method every fourteen days from the first immunization. The results showed that the three vaccine candidates could elicit serum antibody response, but differently. The serum antibody peak was observed in the response triggered by the dissolved protein first (P <0.05), and then heat-killed whole cells, last was the oral vaccine microparticles. But the degressive rate of serum antibody was heat-killed whole cells > the adjuvant vaccine > oral vaccine microparticle. Fish vaccinated with each of the vaccines exhibited significant protective immunity when challenged with the pathogenic strain and the relative protection values ranged from 37.5%, 55% to 70%, which indicated that these vaccine candidates had protective effects on grass carp. They were all higher than that of comparison group (P<0.05).