| Salmonellosis is a general term for various animal and human diseases caused by Salmonella,which not only brings great losses to livestock industry,but also has great public health significance for human being.Avian salmonellosis is caused by Salmonella Gallinarum(S.Gallinarum),Salmonella Pullorum(S.Pullorum)and avian paratyphoid.Paratyphoid infection in chickens rarely causes systemic diseases,but this kind of Salmonella often causes food borne diseases in humans.S.Pullorum and S.Gallinarum are important pathogenic bacteria that harm the development of poultry industry.These pathogenic bacteria can spread horizontally and vertically,mainly infecting chickens within 3 weeks old.They also have adverse effects on young and adult chickens,such as diarrhea symptoms and lower egg production rate.Although S,Gallinarum and S.Pullorum have been eradicated in poultry farm in some European and American countries,the incidence rate and mortality rate in chickens induced by S.Gallinarum and S.Pullorum in China,still cause serious economic losses.In the past,antibiotic treatments could lead to the production of multidrug-resistant bacteria.These multi drug resistant pathogens could be transmitted to humans through the food chain.Vaccination is one of the most effective ways to prevent Salmonella infection.A nature-derived attenuated S.Gallinarum strain(named SG01,patent number:ZL201811491200.6)was isolated and identified previously in the laboratory.Based on early studies on safey evaluation of SG01 strain,we speculate that the SG01 strain possess potential as the attenuated S.Gallinarum vaccine candidate.In this study,the cross protection efficacy of SG01 against S.Gallinarum and S.Pullorum and the immune duration of SG01 were evaluated in order to lay a foundation for the development of a nature-derived attenuated live vaccine against S.Gallinarum infection.1.Evaluation of cross protection efficacy of nature-derived attenuated vaccine candidate SG01A nature-derived attenuated strain of S.Gallinarum was isolated and identified previously in the laboratory.According to results of safety evaluation for theSG01 strain in early studies,we speculate that the SG01 strain possess potential as the attenuated S.Gallinarum vaccine candidate.For applications in the clinical practice,so it is necessary to select a suitable medium for SG01 strain to expend production.We found the growth speed of the SG01 vaccine candidate in SOC medium is much higher than that of ordinary LB and LB(containing 2.5%fetal new-born calf serum).To evaluate the cross-protection efficacy of SG01 strain as a nature-derived attenuated live vaccine candidate against S.Gallinarum and S.pullorum,which belongs to the same Salmonella serogroup D to S.Gallinarum,a total of 50 five-day-old chickens were selected and divided into 5 groups:immunization group 1,S.Gallinarum challenge group 1,immunization group 2,S.Pullorum challenge group 2 and control group(n=10,respectively).Chickens from both immunization group 1 and 2 were vaccinated with 5×109 CFU of SG01 vaccine candidate by oral route.After immunization,clinical symptoms were continuously observed for 14 days and all chickens were weighed on the 7th and 14th days after immunization.On the 15th day after immunization,chickens from the immunization group 1 and S.Gallinarum challenge group 1 were challenged with virulent S.Gallinarum U20 strain(1×1010 CFU)by intraperitoneal route;chickens from the immunization group 2 and S.Pullorum challenge group 2 were challenged with virulent S.Pullorum SP03 strain(1×1010 CFU)by intraperitoneal route.Clinical symptoms were continuously observed for 14 days after challenge.On the 15th day after challenge,all chickens were sacrificed humanely and autopsied for pathological changes.The scoring system based on clinical symptoms including depression and diarrhea symptoms and pathological changes involved in hearts,livers and spleens was used to determine the morbidity rate according to the established criteria for determination of morbidity.The results showed that the vaccination of SG01 candidate(5×109 CFU)by oral route had no significant effect on the daily weight gain of the chickens and did not induce clinical symptoms including depression and diarrhea on chickens.After challenge with S.Gallinarum or S.Pullorum for different groups.Mean scores of clinical symptoms including and pathological changes for the chickens from both two immunization groups were significantly lower than that of challenge groups.In addition,the morbidities of chickens from the immunization group 1 and S.Gallinarum challenge group were 30%(3/10)and 100%(10/10),while the morbidities of chickens from the immunization group 2 and S.Pullorum challenge group 2 were 50%(5/10)and 100%(10/10).The results of the cross-protection efficacy against S.Gallinarum and S.Pullorum showed that vaccination of SG01 strain orally could prevent the artificial high-dose infection with S.Gallinarum and S.pullorum to a certain extent.It is expected to be effective for cross-protection in poultry production.2.Immune duration efficacy and antibody detection of nature-derived attenuated S.Gallinarum vaccine candidate strain SG01In order to explore the duration of systemic antibodies after immunization and the relationship between antibodies induced by SG01 strain and its protective efficiency,the serum antibody levels after immunization with SG01 strain orally was monitored and the corresponding relationship between serum antibody levels and the protection efficacy was evaluated.A total of 160 five-day-old SPF chickens were divided into three parts:immunization group(n=60),challenge group(n=50)and control group(n=50).Chickens from immunization groups were divided into 6 small groups(n=10,respectively),and all chickens from immunization groups were immunized with 5×109 CFU of SG01 candidate orally on five-day-old.Chickens from the five immunization groups were challenged with 1×1010 CFU of virulent S.Gallinarum strain U20 intraperitoneally at 1,2,3,7 and 10 weeks after immunization.Clinical symptoms were continuously observed for 14 days after challenge and all chickens were sacrificed humanely and autopsied for pathological changes on the 15th day after challenge.Morbidity rates of different groups were determined using the scoring system based on clinical symptoms and pathological changes.Another group of the chickens immunized with SG01 strain was set up without challenge.Blood samples of this group of chickens were collected every week from 1 to 10 weeks after immunization to identify serum antibody agglutination titers by S9-peg agglutination antigens developed in the laboratory(patent number:ZL202010400163.4).The result of the protest tests showed that,the morbidities of chickens from the immunization group and challenge group were 60%(6/10)and 100%(10/10),respectively,for challenge on the first week after immunization;30%(3/10)and 100%(10/10),respectively,for challenge on the second week after immunization;10%(3/10)and 90%(9/10),respectively,for challenge on the third week after immunization;0%(0/10)and 80%(8/10),respectively,for challenge on the 7th week after immunization;0%(0/10)and 60%(6/10),respectively,for challenge on the 10th week after immunization.In addition,the detection for serum antibody agglutination titers of 10 chickens which were not challenged with U20 strain showed that the anti-Salmonella serogroup D agglutination antibody titers were not detected in chickens on the first and second weeks after immunization.Three chickens(3/10)were identified agglutination titers shown as 1:1 for each chicken on the third week after immunization,while all chickens(10/10)were identified 1:8 agglutination titers on the 4th week after immunization.Agglutination titers of chickens reached the peak on the 7th week after immunization shown as 1:16 for 8 chickens(8/10)and 1:8 for 2 chickens(2/10).Seven chickens(7/10)maintained the high level of agglutination titers shown as 1:8,while only one chicken(1/10)was not identified the agglutination titer on the 10th week after immunization.These results indicated that serum antibody titers could be detected with S9-peg agglutination antigens on the third week after immunization with 5×109 of SG01 strain by oral route.Together with the result that SG01 strain provided 80%protection efficacy for challenge with virulent S.Gallinarum strain on the third week after immunization,indicating that prevention against S.Gallinarum infection induced by SG01 candidate vaccination could emerge under the emergence of antibody agglutination titers for chickens.Moreover,vaccination of the SG01 strain orally could provide over 8 weeks of protection period for young chickens. |
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