| Salmonella enterica serovar pullorum (S. Pullorum) causes an acute or chronic infectious disease for chickens. The disease can spread widely by horizontal and vertical transmission, and cause a huge threat to the development of the poultry industry. Eradication of S. Pullorum in breeding chickens was raised in the mid-long term national control plans of animal diseases. The decrease of the bacterial carrying rate is an important issue, and the elimination of positive chickens is the first key step. Therefore, the development of gene-deleted vaccine to differentiate natural infections and vaccination is an important strategy to be considered. Lipopolysaccharide has been considered as an essential component for outer membrane biogenesis and important factor in virulence of Gram-negative organisms such as Salmonella. With the continuous deepening in research on pathogenesis, virulence gene, lipopolysaccharide, and bacterial vaccine, the application of Salmonella vaccine by genetic engineering method achieved a lot of progress, and showed a good prospect in practice.In this study, we constructed the deletion mutants of LPS by λ Red homologous DNA technology, and their biological characteristics and immune efficacy were detected, providing a basis for the feasibility of the attenuated Salmonella vaccine in practice.1. The construction of the deletion mutantsThe rfbA, rfaL, rfaJ, rfaI, rfaG, and rfaF gene of S. pullorum S6702 were amplified according to the sequence deposited in GenBank. The mutant strains including S6702△rfbA, S6702△rfaL, S6702△rfaJ, S6702△rfaI, S6702△rfaG, and S6702△rfaF were constructed by λ-Red homologous DNA technology, and further confirmed by PCR identification and sequencing. Growth curve of these strains revealed that the growth rates of mutant strains were similar to that of wild-type strain S6702. A quantitative microtitre assay with crystal violet staining in 96-well plate showed that S6702△rfaJ and S6702△rfaF had similar bio film-forming ability with wild-type strain S6702. The bio film-forming ability of S6702△rfbA and S6702△rfal were significantly enhanced, while the bio film-forming ability of S6702△rfaL and S6702△rafG were decreased compared with S6702. LPS map by SDS-PAGE showed that the LPS of 6 mutant strains showed a decrease in molecular weight, which suggested that the synthesis of glycan structure was blocked. Therefore, these data further confirmed that mutant strains were constructed successfully.2. Pathogenicity and Immunogenicity of LPS mutants in SPF chickensTo determine the 50% lethal dose LD50 of mutants,2-day-old SPF chickens were inoculated intramuscularly with the mutants and wild type strain. The LD50s of S6702, S6702△rfbA, S6702△rfaL, S6702△rfaJ, S6702△rfaI, S6702△rfaG, and S6702△rfaF were as follows: 3.2×10O8,>1.5×109,>1.5×109,1.4×109,>2.2×109,>1.0×109, and>2.5×109, suggesting that the pathogenicity of mutant strains were significantly declined compared with S6702. Chickens’ body weights were no significantly different among the groups of S6702△rfbA, S6702△rfaL, and S6702△rfaG. However, there were significantly decreases of chickens’ body weights of groups S6702△rfaJ, S6702△rfaI, and S6702△rfaF compared with the control group. Humoral immune responses were evaluated through determination of specific IgG levels by indirect ELISA, using whole S. Pullorum bacteria as coating antigen. The level of IgG was low on day 10. Chickens were immunized at 2 day-old and then challenged with 5. Pullorum S004 at 10 days post inoculation. The protective efficacy assay showed that the mutant strains provided 100% protection against S004 challenge. The positive rate of S6702△rfaL was higher and more stabilization than other mutants in 1 month-old chickens. Slide agglutination test demonstrated that serum antibodies induced by the mutants did not react with antigen of wild-type strain S6702, indicating that the mutants S6702ArfbA, S6702△rfaL, and S6702△rfaG were immuno genic.3. Immune efficacy of LPS mutants in SPF chickensAccording to the protective efficacy assay, S6702△rfbA, S6702ArfaL, and S6702△rfaG were selected for further assession of immune efficacy. On day 7,14,21 after immunization, the blood samples were collected and detected by indirect ELISA. The level of IgG could be detected at 1 week and rose to the peak at 3 week. The proportion of CD4+and CD8+T cells from the group of mutant S6702△rfaL was increased compare with control group, indicating a strong cellular immunity. Liver and spleen samples of chickens from each group were aseptically collected on day 7,14,21 for bacterial recovery. On day 21, the mutant strains have been eliminated from liver and spleen. Challenge study showed that the protection percentages of S6702△rfbA,S6702△rfaL,and S6702△rafG were 80%、80% and 70%,respectively.In summary, these data indicated that S6702△rfbA, S6702 ArfaL, and S6702△rfaG had better immune efficacy to significantly induce specific humoral and cellular immune responses without agglutination antibody, it will helpful to develop the attenuated Salmonella vaccine in practice. |
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