Development And Preliminary Application Of Assays For Diagonosis Of Pullorum Disease And Fowl Typhoid

Pullorum Disease?PD?,caused by Salmonella enterica subsp.enterica serovar Gallinarum biovar Pullorum?S.Pullorum?,is an acute systemic disease more commonly found in young birds,and adult birds can also be infected.Fowl Typhoid?FT?,caused by Salmonella enterica subsp.enterica serovar Gallinarum biovar Gallinarum?S.Gallinarum?,is an acute or chronic septicaemic disease that usually affects adult birds,although birds of all ages may be susceptive.PD and FT have seriously affected the development of poultry breeding in China,and were included in the national regulations of bacterial diseases of "National Medium and Long Term Animal Epidemic Prevention and Control Plan" in 2012.Both PD and FT can be detected serologically by use of a macroscopic tube agglutination test,which is the most commonly used and most convenient assay to identify whether chickens have been infected or not.However,the results of agglutination test often cross-reacted with other Salmonella in the D group,such as Salmonella Enteritidis.Therefore,the purpose of this study is:?1?to establish new PCR assays,respectively based on SPUL 2693 and SPUL₂694 for detecting the serovar Gallinarum.?2?based on the characteristics of the serovar Gallinarum lack of flagella,to prepare the monoclonal antibodies against S.Enteritidis flagellin,then establish a preliminary competitive ELISA method for the identification of S.Enteritidis,and combine the 09 competitive ELISA method for detecting birds infected with the serovar Gallinarum.It provides important biological materials and techniquess for detecting the bird infected with the serovar Gallinarum in the near future.1.Development and preliminary application of PCR assays for the detection of the serovar GallinarumIn this study,one step PCR methods for rapid detection of the serovar Gallinarum were established by using the specific gene SPUL₂693 and SPUL₂694 as the target gene respectively.Specificity of the PCR assays were evaluated by testing 27 serotypes of Salmonella and 6 non-Salmonella strains.The results demonstrated that only the serovar Gallinarum showed 2160 bp band in the SPUL₂693-PCR assay,2619 bp in the SPUL₂694-PCR assay,while other bacteria had no specific bands,which indicated that both two PCR methods had a good specificity.Sensitivity of the two PCR assays were evaluated by S.Pullorum RKS5078,and the results showed that the detection limit of the SPUL₂693-PCR was 2.143 pg/?L or 6 CFU,SPUL₂694-PCR was 4.03 pg/?L or 24 CFU.Furthermore,clinical samples were detected by the two PCR assays and traditional Salmonella serotype identification method simultaneously,and the results of PCR showed that 5 strains of S.Pullorum were detected,which were consistent with the results of traditional Salmonella serotype identification method.In summary,SPUL₂693-PCR,SPUL₂694-PCR established in this study for detection of the serovar Gallinarum showed a good specificity and high sensitivity,and provided new assays for detecting the serovar Gallinarum.2.Development and preliminary application of mAbs specific for flagellin antigen H:g of SalmonellaThe flagellin was extracted from the Salmonella Enteritidis C50041 in M-broth by acid hydrolysis method,the results of SDS-PAGE showed that the target band was about 57 kDa,which was consistent with the expected size.Western Blot revealed that the flagellin had a good immunoreactivity.The six weeks old female BALB/c mice were immunized with the purified flagellin,and after three times of immunizations,the cell fusion was conducted by using the lymphocyte hybridoma technology.After four times of subcloning,seven hybridoma cells were obtained by using indirect ELISA method,which was developed with the purified flagellin as detecting antigen.Seven mAbs were named as 9E3,12F5,12G3,12E3,7E9,7D7 and 7E2,respectively.Their subtypes were IgGl by using "Mouse Monclonal Antibody Isotyping Reagents".The results of Western Blot showed that seven mAbs could react with the flagellin of S.Enteritidis specifically.The results of indirect ELISA showed that the supernatant titers of seven mAbs were higher than 1.0×104,even up to higher than 2×105,which also showed that the ascites titers of seven mAbs were higher than 4×106,and the ascites titers of 12E3 and 7E2 were higher than 1×107.Specificity results showed that seven mAbs could react specifically with Salmonella serotypes with H:g,such as S.Enteritidis?H:g,m?,S.Dublin?H:g,p?,S.Agona?H:f,g,s?,S.Montevideo?H:g,m,[p],s?,S.Rissen?H:f,g?,S.Derby?H:f,g?,while showed no reaction with other Salmonella serotypes tested and non-Salmonella bacteria.The inhibition ELISA results further confirmed that seven mAbs could react with natural flagellin of S.Enteritidis.First,09 competitive ELISA method established in the laboratory was used to detect the positive serum of S.Pullorum,S.Enteritidis and SPF chicken,and then the competitive ELISA method initially established by 12E3 monoclonal antibody was used to identify them.The results combined with two competitive ELISA method could identify the positive serum of the serovar Gallinarum?09 inhibition rate:88%-95%;H:g inhibition rate:8%-23%?and the positive serum of S.Enteritidis?09 inhibition rate:72%-92%;H:g inhibition rate:65%-73%?.In conclusion,these results suggested that seven mAbs have a good application potential and provide important biomaterials for the diagnosis and control of pullorum disease and fowl typhoid.