| Pullorum disease caused by Salmonella enterica serovar pullorum(S.Pullorum)is vertical transmission disease and results in a seriously losses to poultry industry.Eradication of Pullorum disease in breeding chickens has been raised in the mid-long term national control plans of animal diseases(2012-2020).Antimicrobials have been taken to control Pullorum disease in China and other developing countries for a long time,but it causes the problem of multiple drug resistance of bacteria and drug residues.Vaccination is one of the effective measures to prevent and control Pullorum disease,a live attenuated vaccine is commonly used vaccine.S.Pullorum infection is usually quarantined by using the whole blood plate agglutination test to eliminate positive chickens in practice.Therefore,development of a vaccine strain with the characteristics of DIVA(Differentiation of Infected and Vaccinated Animals,DIVA)has a positive effect on clean-up of S.Pullorum.S.pullorum mutations S6702?rfaL and S6702?rfaG strains were constructed in our laboratory with characteristics of low pathogenicity,high immunogenicity,and DIVA.Biofilm is a structural bacterial community formed by S.pullorum to resist adverse environments,which can lead to persistent infection.In order to further reduce the pathogenicity and enhance the safety of Salmonella vaccine candidate.In this study,we constructed the deletion mutants of biofilm systhesis associated gene on the basis of mutant strains of LPS by X-Red homologous DNA technology,and the biological characteristics of those attenuated S.pullorum strains were determined,which provide a basis for the feasibility of the attenuated Salmonella vaccine in practice.1.The construction of the deletion mutantsThe mutant strains including S6702?rfaL?csgA,S6702?rfaG?csgA,S6702?rfaL?csgAAompR and S6702?rfaG?csgA?ompR were constructed on the basis of S6702?rfaL and S6702?rfaG strains by ?-Red homologous DNA technology,and confirmed by PCR identification and sequencing.The biological characteristics of the mutant strains were identified.Growth curve of these strains revealed that the growth rates of single gene and double genes mutant strains were similar to S6702,and the growth rate of the three genes mutant strains was slightly slower than other strains.The biofilm-forming ability test showed that S6702ArfaL AcsgA?S6702?rfaG?csgA?S6702?rfaL?csgA?ompR and S6702?rfaG?csgA?ompR mutant strains had no biofilm-forming ability.The antimicrobial susceptibility test showed that all strains were sensitive to 7 drugs,and the sensitivity of wild-type strain S6702 was similar with that of mutant strains,but different with another S.pullorum S004 and S.enteritidis vaccine Sm24.The genetic stability test revealed that the mutant strains were stable,and the mutant strains could not obtain the deleted genes by natural recombination when co-cultured with S004.These date indicate that the mutant strains constructed in this study have stable biological characteristics and high safety.2.Pathogenicity and Immune efficacy of S.Pullorum mutantsThe pathogenicity of mutant strains was evaluated by the median lethal dose(LD 50)in 2-day-old SPF chickens.The LD50 of S6702 was 5.5×108CFU,the LD50 of S6702?rfaL and S6702?rfaG increased at least 16-folds compared with S6702,and the LD50 of double genes mutant strains and the three genes mutant strains were increased further.In immune protection test,2-day-old SPF chicks were orally immunized with 1×108 CFU mutant strains and Sm24.There was were no significantly difference in chickens' body weights among immune groups and control group.The results of the plate agglutination test showed that the immune serum of the mutant strains had no agglutination reaction with the laboratory-made staining antigen or staining antigen from Beijing Zhonghai at 14 days post immunication(dpi).Humoral immune responses were evaluated through determination of IgG levels by indirect ELISA,the IgG titers of all groups were low at 14 dpi.Chickens were challenged intraperitoneally with S.Pullorum S004 at 14 dpi.The protective efficacy showed that the single gene mutant strains provided 66.7%protection,double genes mutant strains and three genes mutant strains provided 66.7%protection,Sm24 strain provided 50%protection against S004 challenge.The antibody level and body weight of chickens at 7 dpc and 14 dpc were analyzed,the average antibody level and body weight of S6702?rfaG,S6702?rfaG?csgA and S6702?rfaG?acsgA?ompR groups were heavier than S6702?rfaL,S6702?rfaL?csgA and S6702?rfaG?csgA?ompR groups.To further verify the immune efficacy of S6702?rfaG?csgA and S6702?rfaG?csgA?ompR,chickens were challenged intraperitoneally or orally with S.Pullorum S004 at 14 dpi.The results of the intraperitoneal challenge showed that the protection rates of S6702?rfaG,S6702?rfaG?csgA and S6702?rfaG?csgA?ompR groups were all 66.7%,and the protection rate of Sm24 was 50%.The results of the oral challenge showed that from 3 dpc to 5 dpc,the isolation rate of the challenge control group is about 100%,the isolation rates of the immunized groups were significantly decreased,and the isolation rates was only about 30%at the 5 dpc.In summary,these data indicate that the attenuated strains S6702?rfaG?csgA and S6702?rfaG?csgA?ompR have the advantages of low virulence,high immunity and DIVA,it will helpful to develop the attenuated Salmonella vaccine. |
PDF Full Text Request