| In recent years, the reports of aquatic animals infected by Aeromonas veronii were common, it caused a serious economic losses on aquaculture. At present, the treatment of the disease caused by the bacteria is mostly used by chemical drugs, however, It can easily lead to water pollution and drug residues. Bacterial inactivated vaccine is green, safe, making simple and low cost, compared with other vaccines for fish. Inactivated vaccine used with adjuvant, the body can continue to produce the specific antibodies, to improve the disease resistance of aquatic animals. Based on the research of bacteria inactivated vaccine is still shallow, this test used Aeromonas veronii to make the inactivated vaccine; and abstracted membrane protein extract made of different vaccines, immune grass carp with Aluminum hydroxide gel adjuvant, to explore the immune responses of several vaccines and the adjuvant immune enhancement.1 Isolation and identification of Aeromonas veronii and analysis characteristic.The pathogenic strain of this test was isolated from the onset Argus in some fish farms, separating and pure culture the main strains after regressing experiment, then named is as"WLJD-1". Identified it was Aeromonas veronii after physiological and biochemical identification. Obtain a fragment of 1483bp after the amplification of 16S rRNA. The phylogenetic tree shows this strain gets the closest relationship of Aeromonas veronii, the similarity of the nucleotide is over 99%. Final identified it as the Aeromonas veronii. Gram staining shows Gram-negative short bacillus. The lethal concentration of Grass Carp is 2.31×107 CFU/ml. The DST of 40 kinds of antibiotics shows the bacteria is sensitive to Ceftriaxone, Cephalothin, Gentamicin and other 17 kinds of drugs. It shows drug resistance of penicillin G, Ampicillin, Carbenicillin and other 10 kinds of drugs.2 Research on Aluminum adjuvant on the enhancement effect of Aeromonas veronii inactivated vaccine.Set different temperature and different final formalin concentration to inactivate test. Get the optimum temperature is 28 ℃, the final formalin concentration is 0.2% inactivation time is 24h. Add Aluminum hydroxide gel respectively 0%,25% and 50% by volume to the whole-cell inactivated vaccine to make three kinds of vaccine, the vaccine is divided into three different vaccine groups, negative group and the control group. Inject the grass carp after safety testing. Determine the Agglutination serum antibody titers and the other index atl,7,14,21,28,29,32,35,42d. Poison attack at the 28th day. The result shows:the vaccine serum antibody titers peak at 21d, the 50% addition group reached the highest, up to 1:640; the 50% addition group has the biggest RPS, reached 85.71%; the vaccine group was significantly greater than the negative group and the control group (P<0.01)on the number of white blood cells, the vaccine was significantly more than the whole cell group (P<0.05); On the on percentage of the lymphocytes, granulocytes, monocytes, the average of the 50% adjuvant group was maximum,25% adjuvant group ranks the second. And both the two goups were significantly higher than the whole-cell group, negative group and the control group. (P<0.05); The phagocytosis rate of each vaccine group was significantly higher than the negative group and the control group (P<0.01), both in the 14th day reaches the maximum value,50% adjuvant group is 47.4% which reached the largest; the adjuvant activity of CAT was significantly higher than the control (P<0.01), and significantly higher than the whole-cell group (P<0.05), the vaccine group AKP activity was significantly higher (P<0.05), the 50% adjuvant group’s activity of CAT, AKP were highest in average. In summary, the aluminum adjuvant has the better immunity enhancement funtion, the strength of the immune enhancement is: All bacteria without adjuvant group<25% adjuvant group <50% adjuvant group.3 Four kinds of Aeromonas veronii inactivated vaccines on the immunogenicity of Grass Carp.Extract the outer membrane protein of the Aeromonas veronii (OMP), SDS-PAGE electrophoresis Detect its bands and the nominal concentration. Use Aeromonas veronii inactivate whole-cell vaccine and OMP made adjuvanted and non-adjuvanted vaccine in 4 kinds, adjuvant group adds the al hydro gel asl:1 volume, it can be used after safety test. Immunized carp, and determine its immune effects, immune cells and enzymes indicators index at its 1,7,14,21,28,35,42,43,46,49,56d. Set the second immunization at the 14th day, poison attack at the 42th day. The result shows: The antibodies were significantly higher in the vaccine group after immunization, the average level of OMP adjuvant group is the highest of all, reached at 1:1280 in the 14th and 35th day, OMP adjuvant group was significantly higher than other vaccine group (P<0.05); The RPS of the OMP adjuvant is the highest, reaching 89.8%.In terms of immune cells indexes, the average number of four vaccine groups were significantly higher than the negative group and control group (P<0.05) on leukocytes, adjuvant groups was significantly higher than non-add adjuvant groups (P<0.05);The number of granulocytes, lymphocytes, monocytes in OMP adjuvant group were significantly higher than the other groups (P<0.05), the vaccine group were significantly higher than the negative group and the control group (P<0.05); On phagocytosis rate, the vaccine groups were significantly higher than the negative group and the control group (P<0.01), two adjuvant groups were significantly higher than non-adjuvanted groups (P<0.05) on average level, the OMP adjuvant group reached 46.09% which is the highest one; the first immunization vaccine group after 48h, the S phase of lymphocytes was significantly different in the negative group and the control group (P<0.05)in four vaccine group. the second immunization 48h later, the G0/G1 phase, S phase and G2/M phase of four vaccine groups were significant differences from negative group and the control group (P<0.05). After challenge, the lymphocyte of the OMP adjuvant group replicated the fastest. In terms of enzyme activitythe average activity of T-SOD, CAT, AKP in vaccine groups were significantly higher than the negative group and the control group (P<0.05), adjuvant groups were significantly higher than non-adjuvant group (P<0.05), the average levels of the OMP adjuvant were highest both in T-SOD and CAT activities, the whole cell adjuvant reached the highest on AKP vitality. In summary, the OMP vaccine adjuvant provoked the strongest non-specific immunity, immune best,the F-Av adjuvant followed. |
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