| Pseudomonas aeruginosa has been a difficult problem in wild animal breeding, which is one of the relative difficult to control and prevention of the bacterial diseases. This bacterium has strong drug resistance and can produce a variety of virulence factors which has strong pathogenicity when infect the host, which results in the treatment effect is not ideal by single using of antibiotics and limits the extensive use of antibiotics. Therefore, reasonable vaccination plays an important role in the prevention of the disease. Currently, there is no vaccine of Pseudomonas aeruginosa which has got approval in our country. According to the distribution of pseudomonas aeruginosa in the farms of Moschus berezovskii, a strongest virulence strain was successfully screened as test strain by animal challenge study. After the extraction and purification of the outer membrane protein, the immunization was conducted and the immunogenicity was evaluated from the cellular immunity, humoral immunity and challenged protection, aiming to provide a reference basis for the control and prevention of Pseudomonas aeruginosa disease. The studies and results in this article are as follows:1. Screening of the test strainsSeven Pseudomonas aeruginosa strains isolated from Moschus berezovskii in distinct farms were selected in this experiment. For animal challenged study and comparing their toxicity using the same bacterium concentration to screen the strongest virulence strain.Then, a strain was got, named sp-19. At last, the LD50 was detected against the mice about this strain, indicated that LD50 is 6.7114×106 CFU2. Purification and identification of outer membrane protein of Pseudomonas aeruginosaThe outer membrane proteins were extracted and purified by outer membrane protein extraction kit. By SDS-PAGE, we could know there were 8 protein bands with approximate molecular weight 104 KD,71 KD,52 KD,43 KD,36 KD,22 KD,18 KD, and 9 KD, respectively. The result of western blotting showed that these proteins all could react with mice anti-sp-19 serum, which indicated that they had strong reactionogenicity.3. Immune effect evaluation of outer membrane protein of Pseudomonas aeruginosaThe immunizations were conducted by sp-19 inactivation whole-cell and the sp-19 outer membrane proteins which purified by outer membrane protein extraction kit and ultrasonication, respectively. After forth immunization, the antibody levels were detected by indirect ELISA. The results showed that the antibody levels along with immune process continue to rise in each vaccine groups. The antibody titers of the groups of kit extraction, inactivation whole-cell, and ultrasonication decreased in turn with 1:12500,1:2500, and 1:500, respectively. The lymphocyte proliferation test performed that the outer membrane proteins could promote lymphocyte proliferation in kit extraction, inactivation whole-cell, and ultrasonication groups with stimulus index 3.05,2.51, and 3.05, respectively. The levels of cytokines were detected by cytokines detection kit against IL-4 and IFN-y at day of 0,15,29, 43, and 50. The highest contents of IL-4 and IFN-y in kit extraction, inactivation whole-cell, and ultrasonication groups were 104 pg/mL and 134 pg/mL,87 pg/mL and 131 pg/mL,66 pg/mL and 97 pg/mL.4. Study on immune protection of outer membrane protein of Pseudomonas aeruginosaThe immunizations were conducted by sp-19 inactivation whole-cell and the sp-19 outer membrane proteins which purified by outer membrane protein extraction kit and ultrasonication, respectively. After second immunization, the mice were challenged with sp-19 activation whole-cell (5×LD50) by intraperitoneal injection. The results showed that the protection rates of kit extraction, inactivation whole-cell, ultrasonication, and control groups were 87.5%,62.5%,12.5%, and 0, respectively. Then, the passive immunity was conducted in this study. Briefy, the mice in each group were immunized with the hyperimmune serum of each group after 56 days by intraperitoneal injection. After 2 h, the mice were challenged with sp-19 activation whole-cell (2xLD50). The protection rates of kit extraction, inactivation whole-cell, ultrasonication, and groups showed 100%,87.5%, and 25%, respectively. The control group was 0. |
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