| Aeromonas hydrophila widely distributed in warm water environment is a critical pathogen that can cause fatal disease of fish and amphibians, which shall give rise to serious economic losses in aquaculture. In addition, the contaminated aquatic animals and aquatic products may threaten human health. Therefore, the control operation against A.hydrophila takes a significant position in freshwater aquaculture.Usually oral vaccine is made in feed with less stress action, easier mass immunization for animals. Compared with the traditional immune routes, the oral vaccine has other potential advantages such as stability and safety, as well as long-term storage. Therefore, it shows a good prospect.The recombinant Escherichia coli with outer membrane protein of A. hydrophila in our lab had been taken to get the optimal conditions. They were cultured in LB medium at 37℃to OD 0.8, and then induced by 1.0mmol/L of isopropy-β-D-thiogalactoside (IPTG) for 4.5-5h at 28℃.We prepared mass of the protein under the optimal conditions. Microsphere oral vaccine consists of two parts. Core material is the prepared protein, and the outer wall material is sodium alginate which appeared good biocompatibility, coated with chitosan. Continuous improvement had been done with the method of microsphere preparation, including spray forming, divalent solution collected (liquid - gel conversion), high-speed homogenizer parcels, and preserving method such as freeze-drying agent. The orthogonal analysis of four factors in three levels had been taken. The results showed that the optimal conditions in microsphere preparation were:0.5% CaCl2,1200rpm of stirring speed,15mL/min of feeding rate and 2.0% sodium alginate (SA).83.07% of encapsulation efficiency was got, and the expansion rates of microsphere were 605.89%,652.01% and 559.36% in the 8.5% NaCl, distilled water and pH7.4 PBS, respectively. The concentration of total protein was 0.5786mg/10mg in the microspheres. The microspheres had good release ability under the acid-base conditions, good feeding security and good storage stability.To investigate the immune response after the oral vaccination with outer membrane protein of Aeromonas hydrophila microsphere (OMP-MS), mice were divided into groups with the different oral doses (GroupⅡ,Ⅲ,Ⅳ,Ⅴ). Injected group (GroupⅠ) and control group (GroupⅥ,Ⅶ) were set as positive control and negative control. RT-PCR, ELISA, paraffin section and other methods were taken to analyze the changes in some idexes, such as serum antibodies, mucosal antibodies, peyer’s patches and other factors. These immunizations were finally evaluated with challenge test. The results showed that IgA in serum and sIgA in intestine with oral immunization were significantly higher than those in injected group (P<0.05), which suggested that oral immunization induces local and systemic specific immunity. But for IgG level in serum, the result by oral route was significantly lower than that by injection. RT-PCR analysis showed mRNA expression in IFN-y and IL-4 had risen during the immune phase in both oral immunization and injected immunization. In all the oral immune groups, the number and the measurement of peyer’s patches in ileum increased. It suggested that the OMP-MS vaccine increased the immune response by oral route; Challenge test showed that the oral administrations of inactivated bacteria microspheres and three OMP-MS groups received 50.00%(3/6),33.33%(3/9), 75.00%(6/8) and 33.33%(3/9) of the relative of protective rates, respectively.Parabramis pekinensis cultured in ponds under the conditions of domestication were vaccinated by injection and oral routes, respectively. Serum and hindgut were collected at each week after immunization for the detection of IgM level in serum/mucus and lysozyme (LSZ) & SOD activities in serum. Phagocytic activity of lymphocytes in head kidney was detected. Then we carried out a challenge test in the 45th day. The results showed:after the immunization, IgM antibody levels in serum/intestinal mucus in oral immunization groups (Ⅲ,Ⅳ) were significantly higher than those in the injection groups (Ⅰ,Ⅱ), and there was no difference between the bacterial microsphere group (Ⅲ) and OMP-MS group (Ⅳ); at the 43rd day, the detection rates of IgM antibody from mucous in oral immunization groups were higher than those in the injected groups. The activity of lysozyme (LSZ) & SOD in the serum of immuned groups increased during the immunization period. The challenge test showed that the relative protection rates were 50.00%(16/30),55.67%(17/30),25.00%(9/30) and 21.43%(8/30), respectively in the different immuned groups (Ⅰ,Ⅱ,Ⅲ,Ⅳ). It suggested that immunization efficiency of OMP-MS was weaker, but it still had protective immune effect.In summary, we prepared A. hydrophila OMP-MS oral vaccine at the optimal conditions, then we immuned mice and Parabramis pekinensis. This vaccine could induce the immune respones in experimental animals, and protected them from infection to some degree. This study provided the basis for the application of genetic engineering subunit oral vaccine in aquaculture. |
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