| Salmonella enterica serovar Gallinarum biovar Pullorum(Salmonella Pullorum)is one of major pathogens that brings up serious damage to poultry industry in China,it causes young chickens high morbidity and high mortality,and adult chicken latent infection and being transmitted vertically to eggs or offspring.Although flock clearance and antibiotics are often used to control infection of Salmonella Pullorum,these measures are not well suitable to the need for prevention and control of Salmonellosis in developing countries including China,because of their high cost,drug residues and Salmonella antimicrobial resistances.Vaccination is considered to be one of effective measures to control and reduce Salmonella Pullorum infection.Previous studies in our laboratory showed that Salmonella Pullorum S06004?spiC match the"Safety Assessment of Genetically Modified Organisms(GMO)”in China.And so,in this study Salmonella Pullorum vaccine seed bank was constructed to avoid the decrease in safety and efficacy because of genetic variation,and further vaccine was prepared using the working seed bank and its safety and immunoprotection efficacy were evaluated.1.Construction and genetic stability of seed bank for Salmonella Pullorum vaccine strain S06004?spiCAs a biological product,the vaccine strain seed must be stored in a form of "seed bank" to prevent phenotypic or genotypic changes when it was sub-cultured.According to the "Chinese Veterinary Pharmacopoeia",three-stage seed banks(including primary seed bank,master seed bank and working seed bank)were constructed,and the features of strains from these seed banks were comprehensively analyzed.The strains in three-stage seed banks are pure and free of other bacteria.The genetic stability of the strains in the seed bank was determined including the characteristics of growth,serology,morphology,biochemistry,target genes,immunogenicity and level of LD50.The results showed that the genetic stability of the strains in these seed banks are consistent,the sequence of target gene is same without basepair mutant,and the virulence is unchanged.The 25th generation passage strains in vivo and in vitro are still consistent with the original vaccine strain,which reveal they have good genetic stability.2.Evaluation of the safety and immunoprotection efficacy of Salmonella Pullorum vaccine strain S06004?stpiCIn this study,vaccine was prepared using the working seed bank and further evaluated for safety and immunoprotection efficacy.After the vaccine S06004?spiC was inoculated into the 3-day chickens,the body weights of chickens in the vaccination group were not significantly different compared to those in the blank control group,but significantly higher than those in the infection group with wild-type strain S06004(P<0.05).No significant lesions were found in the liver,spleen and cecum by histopathological sections in the vaccination group,compare to those in the infection group.The loads of vaccine strains in the liver,spleen and feces in the vaccination group were significantly lower than those in the infection group at the same timepoint(P<0.05),vaccine strain could not be isolated in liver at 21 days,in spleen at 28 days and in feces at 35 days after inoculation.The results of non-target animal experiments showed that even the high-dose vaccine strain did not cause any clinical symptoms and pathological changes in murine model,and so the vaccine strain does not infect non-target animals and is not a hazard for public safety.The vaccine could provide at least 90%immunoprotective efficacy against the different pathogenic clinical isolates of Salmonella Pullorum,and could effectively reduce the colonization of the challenge strains(P<0.05).Its cross-protection effectiveness against Salmonella Gallinarum and Salmonella Enteritidis was over 60%.The levels of serum antibodies IgG and mucosal antibodies IgA increased continuously after inoculation of vaccine strain,which were significantly higher than those in the control group(P<0.01),indicating that the vaccine strain could induce both humoral and mucosal immune responses in chickens.Cytokine mRNA in spleen was detected by qPCR,the results showed that the mRNA levels of IFN-y and IL-2 were significantly higher than those in the infection group(P<0.01).The mRNA level of IL-1? increased in the early stage and then decreased significantly,and the mRNA levels of IL-4,IL-10 were significantly increased at the late stage(P<0.05).It turned out that the vaccine strain could significantly stimulate cellular immune response.All results indicated that the vaccine has good safety and immunoprotection efficacy. |
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