Evaluation Of Safety And Protective Efficacy Of The Attenuated Salmonella Gallinarum SG02

Salmonella Gallinarum,a kind of pathogenic bacterium which only infects fowls,can be transmitted horizontally through the alimentary tract and red mites in the poultry farms,and can also be transmitted vertically through developing eggs.Chicken infections are characterized by depression,diarrhea,decrease in the egg production,reduced hatchability and so on.Although the disease has been basically eradicated or markedly controlled in most developed countries,the disease incidence rate in some developing countries is still high resulting in the severe morbidity and mortality.At present,a large number of antimicrobial resistance(AMR)of pathogenic bacteria have been detected in Salmonella Gallinarum isolated from poultry farms,and the drug-resistant genes were found to be transmitted to other enterobacteriaceae bacterial strains,which induce direct impacts on the human and animal health.Therefore,it is of great economic significance to find effective methods to prevent and control fowl typhoid,in which vaccination is one of the effective methods to prevent Salmonella Gallinarum infection.In this study,Identifications and culture technology explorations were performed using a healthy chicken-derived Salmonella Gallinarum isolate,SG02.Then,the safety of the SG02 strain and the protective efficacy against Salmonella Gallinarum wild strain were evaluated in order to provide reference for the development of Salmonella Gallinarum vaccine candidate in the future.1.Preliminary study on culture conditions of the SG02 strainThis study preliminarily explored the culture conditions of SG02 strain through the screening of culture medium,sterilization conditions and pH value.The results showed that SOC medium was more suitable for the growth of SG02 strain,and the optimal sterilization conditions of SOC medium were 121? for 20 min,and the optimal pH value was 7.5.2.Safety evaluation for the SG02 strainIn order to evaluate the safety of SG02 strain in chickens,we established the chicken immune model.The chickens were orally inoculated with 5×109 CFU at the age of 5 days.Then,liver and spleen samples of chickens were collected on 2,3,4,5,6,8,10 and 12 days post immunization to detect the dynamic distribution of bacteria in vivo.The results showed the bacterial load of livers underwent an upward trend followed by a downward trend and reached the peak on the 5 days post immunization.And the bacterial load of spleens showed two peaks on the 5 and 10 days post immunization.According to the above results,the ability of virulence reversion for the SG02 strain was assessed through the bacterial passage in vivo.The first generation chickens were orally inoculated with 5 ×109 CFU at the age of 5 days.The second and third generation chickens were orally inoculated with the mixture of liver and spleen of the first and second generation chickens respectively The results showed that no clinical symptoms including depression and diarrhea and no gross pathological changes involved in the heart,liver and spleen were observed in each chicken inoculated with the SG02 strain of different generations.At the same time,SG02 strain was isolated only in the chickens inoculated with the first generation of SG02 strain,indicating that SG02 strain did not show the pathogenicity and the virulence reversion ability within passages vivo.In addition,in order to explore the safety of overdose immunization of the SG02 strain in chickens,42 fiveday-old chickens were randomly divided into 3 groups(n=14,respectively),including the immune group 1,the immune group 2 and the control group.Two immunized groups were orally immunized SG02 strain with 1 ×1010 CFU and 5 × 1010 CFU,respectively,while the control group orally inoculated with sterile PBS.All chickens were observed for 21 days after immunization.Then we evaluated the safety of SG02 strain according to the weight gains,clinical symptoms and pathological changes.The results showed that SG02 strain did not induce significant effects on the daily weight gains of chickens and did not cause clinical symptoms including depression and diarrhea.Autopsy showed that the overdose immunization only caused slight damage to the heart and liver in chickens.3.Evaluation of protective efficacy of SG02 strainIn order to explore the protective efficacy of the SG02 strain against Salmonella Gallinarum U20,30 five-day-old chickens were randomly divided into three groups:immune group,challenge group and control group.The chickens in the immune group were orally inoculated with 2.5×109 CFU at the age of 5 days,chickens in the challenge group and the control group were orally inoculated with sterile PBS.On the 21 days after immunization,all chickens from the immune group and challenge group were challenged with 1×1010 CFU of the U20 strain by the intraperitoneal injection route.The protective efficacy of SG02 strain against U20 strain was evaluated by monitoring the daily weight gains,clinical symptoms and pathological changes of chickens.The results showed that there was no significant difference in the daily weight gains and clinical symptom scores between the immune group and the control group(p>0.05).The mean pathological change scores of heart,liver and spleen were lower than that of the challenge group after the autopsy,and the mean pathological change scores of liver of chickens(p<0.05)from the immune group were significantly lower than that of the challenge group after the autopsy.The morbidities of the immune group and the challenge group were 40%,100%,respectively.These results suggested that a single oral immunization of the SG02 strain with the dose of 2.5 × 109 CFU could prevent and control the infection of fowl typhoid in chickens to a certain extent.