| Salmonella enterica serovar pullorum(S.Pullorum)is one of the important pathogens that currently endanger the poultry industry in China.It has a wide epidemic range,and the morbidity and mortality are high,which has caused severe economic losses to the poultry industry.The National medium-and-long term animal disease control plan(2012-2020)clearly pointed out the eradication of Salmonella Pullorum in breeding poultry farms.Vaccination is an effective strategy to reduce infection of Salmonella Pullorum in chickens.Compared with other vaccines,live attenuated vaccines not only have the characteristics to produce persistent immune response,but also can obtain DIVA characteristics by gene knockout strategy for distinguishing natural infections from vaccine-immunized animals,Which is of great significance for the eradication of Salmonella Pullorum in poultry.Firstly,this study carried out an epidemiological investigation of Salmonella isolates from poultry clinical samples in East China during 2019-2020 and evaluated the drug resistance of the isolates.Then,the immune efficacy of the DIVA attenuated Salmonella Pullorum candidate strain S004ΔaroAΔsipBΔrfaG was evaluated,which will lay a foundation for the development of safe and effective live attenuated Salmonella Pullorum vaccine.1.Isolation,identification and drug resistance analysis of Salmonella from Poultry in East China during 2019-2020 and the pathogenicity investigation of quail Salmonella Pullorum isolate.During May 2019 and May 2020,clinical samples from poultry in East China was collected,isolated,and identified.Then serotypes were determined by multiplex PCR and glass plate agglutination assay,and the drug resistance of all the isolates against 21 common antibiotics was determined by Kirby-Bauer method.Meanwhile,one strain of Salmonella Pullorum isolated from quail was selected to study its pathogenicity in quails.76 strains of Salmonella were successfully isolated,including 3 5 strains of Salmonella Pullorum(46.05%),40 strains of Salmonella Typhimurium(52.63%),and 1 strain of Salmonella Potsdam(1.32%).Furthermore,36 strains of Salmonella were from chickens(4 7.37%),and therein Salmonella Pullorum was the main,accounting for 86.11%(31/36).22 strains of Salmonella from geese(28.95%),11 strains of Salmonella from ducks(14.47%)and 2 strains of Salmonella from pigeons(2.63%),which were mainly infected with Salmonella Typhimurium,accounting for 95.5%(21/22),90%(9/10),and 100%(2/2),respectively.There were 5 isolates(6.58%)from quail,and there in 60%(3/5)of Salmonella Pullorum and 40%(2/5)of Salmonella Typhimurium.The results of drug susceptibility assay showed that the antibiotic resistance rates of the isolates against penicillin,oxacillin,erythromycin,and rifampin were more than 80%,especially the resistance rates against oxacillin were 100%,and the multiple drug resistance rates were 77.63%,the multiple drug resistance rates of Salmonella Typhimurium and Salmonella Pullorum were 87.5%and 68.57%,respectively.The pathogenicity of Salmonella Pullorum from quail showed that the infected quails had enlarged and khaki-colored liver and bleeding lungs.The median lethal dose(LD50)of quail via airbag inoculation was 9.5×10~5CFU.2.Establishment of a dual fluorescent probe quantitative PCR method for detection of aroA and invA genes in SalmonellaIn this study,a nucleic acid detection method was established to distinguish wild-type strain from Salmonella Pullorum vaccine strain with aroA gene deletion.Specific fluorescent quantitative PCR primers and probes targeting invA gene and aroA gene were designed.Furthermore,the standard plasmids of invA gene and aroA gene were successfully constructed and the standard curves were drawn.The specificity results showed that the method can simultaneously amplify aroA and invA genes of the wild-type strain S004,while only amplify invA gene for Salmonella Pullorum vaccine candidate strain S004ΔaroAΔsipB.The sensitivity results showed that the sensitivity of both aroA gene and invA gene reached 10~1 CFU/mL.The colonization levels of S004ΔaroAΔsipB,Sm24,and S004 strains in duodenal mucosa,jejunal mucosa and liver were evaluated and the results suggested that the established dual fluorescence quantitative PCR probe method was consistent with the bacterial plate count method.Meanwhile,the probe method can distinguish between aroA gene deletion strains and wild strains.These results indicated that the established dual fluorescent probe quantitative PCR method based on Salmonella aroA and invA genes was specific and sensitive,and had characteristics for distinguishing between aroA gene deletion strains and wild strains.3.Evaluation of the immune efficacy of an attenuated Salmonella Pullorum candidate strain S004ΔaroAΔsipBΔrfaG.Two-day-old SPF chickens were orally immunized with attenuated vaccine strain S004ΔaroAΔsipBΔrfaG,wild strain S004,commercial vaccines Sm24,and PBS.Weight monitoring showed that the body weight of S004ΔaroAΔsipBΔrfaG immunization group was equivalent to that of PBS healthy control group and sm24 vaccine control group during the 21d immunization period(P>0.05).The established double fluorescent probe quantitative PCR method was used to detect the distribution of bacteria in vivo.On day 14 after immunization,invA gene was not detected in liver and duodenal mucosa of S004ΔaroAΔsipBΔrfaG and Sm24 immunized groups,while the invA gene can still be detected in S004 on day 21 after immunization,indicating that the attenuated vaccine strain with high safety can be eliminated in vivo at an early stage.Meanwhile,aroA gene and invA gene can be detected in both S004 and Sm24 groups,while aroA gene can be only detected in S004ΔaroAΔsipBΔrfaG group,indicating a success of DIVA strategy.Furthermore,the serum IgG level of S004ΔaroAΔsipBΔrfaG immunized group was equivalent to that of Sm24 immunized group at the third week(P>0.05),and significantly higher than that of PBS control group(P<0.05).The serum glass plate agglutination assay showed that the serum of S004ΔaroAΔsipBΔrfaG immunized group had no agglutination reaction at 7,14 and 21 days after immunization,which was in line with its DIVA characteristics.On day 14 after immunization,S004 strain was used for oral challenge.The results showed that the body weight of S004ΔaroAΔsipBΔrfaG group was equivalent to that of PBS healthy control group(P>0.05),but the body weight of Sm24 group and PBS challenge control group was significantly lower than that of PBS healthy control group on day 5 after challenge(P<0.05).On day 3 and 5 after challenge,the invA gene copy number of S004ΔaroAΔsipBΔrfaG immunized group was equivalent to the aroA gene copy number(P>0.05),indicating that there was only wild strain S004 and no S004ΔaroAΔsipBΔrfaG vaccine strain in duodenum and liver.Moreover,the invA gene and aroA gene copy number of S004ΔaroAΔsipBΔrfaG immunized group were significantly lower than those of challenge control group on day 5 after challenge(P<0.05),indicating that the vaccine strain provided a protection to inhibit the colonization of wild strain S004 in duodenum and liver.In conclusion,S004ΔaroAΔsipBΔrfaG strain had DIVA characteristics,good safety,and immune efficacy,which can be used as a candidate strain for developing an attenuated vaccine against Salmonella Pullorum. |
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