Epidemiological Investigation Of Avian Salmonella In Eastern China During 2018 To 2019 And Preparation Of Lipopolysaccharide Monoclonal Antibody

Salmonella is not only an important zoonotic pathogen,but also is one of the main pathogens of foodborne infectious diseases in the world.According to statistics,more than 2,600 serotypes of Salmonella are globally distributed,and at least 292 serotypes of Salmonella have been reported in China.Salmonellosis with a characteristic of vertical and horizontal transmission seriously harms human and animal health.Salmonella is widespread in poultry farms,some can cause clinical disease,and some can lead to latent infections.Salmonella antibody testing can be used to determine wheather the animal is infected with Salmonella.The most common method for antibody detection is the plate agglutination test,the Enzyme-linked immunosorbent assay is considered as a sensitive and specific method.Lipopolysaccharide is an important part of the cell wall of Salmonella.O antigen is one of its components,most of the currently established ELISA antibody detection methods are directed against LPS antigen.Therefore,persist surveillance of the distribution and prevalence salmonella and development of a detection method basing on LPS monoclonal antibodies are of great significance for prevention and control of salmonellosis.In this study,an epidemiological survey of Salmonella and drug resistance analysis in East China from 2018 to 2019 were investigated,and monoclonal antibodies were prepared by using the immunization of inactivated bacteria combined with LPS,which will provide the foundation for the prevention and control of Salmonellosis.1.Isolation,identification,and resistance analysis of Salmonella from poultry in East China during 2018 to 2019To investigate the prevalence and antimicrobial resistance of Salmonella from poultry in East China,suspected samples were collected during 2018 to 2019,Salmonella were isolated and identified by mPCR and serum agglutination assay.The drug sensitivity test was used to analyze the bacterial resistance to 21 antibiotics by Kirby-Bauer method.The results showed that a total of 84 isolates was identified as Salmonella,including 36 strains of S.typhimurium,30 strains of S.pullorum,5 strains of S.enteritidis,5 strains of S.derby,1 strain of S.newport,2 strains of S.potsdam,2 strains of S.dublin,1 strain of S.Stanley,and 2 strains of S.choleraesuis biotype Kunzendorf.The results of the drug sensitivity test showed that the isolates were generally resistant to penicillin,oxacillin,rifampicin,and tetracycline,especially the resistance rate to oxacillin and rifampicin reached 100%.The sensitivity rates of Salmonella isolates to ciprofloxacin,gentamicin,and compound neonomine were higher,all of which were above 85%,and the multi-drug resistance rate was 90.48%.76 isolates with multi-drug resistance were tested for class ? integron,and the target bands of variable region could be amplified in 2 Salmonella isolates,which carried aadA22 gene and the aadA16-afdA27 combination gene cassette respectively,with resistantace to aminoglycosides and sulfonamides.The above results suggested that the isolates of poultry-derived Salmonella in East China were mainly S.typhimurium and S.pullorum,and the multi-drug resistance was serious,it need to be continuously monitored.2.Development of monoclonal antibodies against lipopolysaccharide from S.pullorum,S.typhimurium,and S.enteritidisThe lipopolysaccharide of S.pullorum(S44),S.enteritidis(C50041),and S.typhimurium(S016)extracted by the thermal phenol method,and bacteria inactivated by boiling were used as immunogens.Seven monoclonal antibodies were successfully prepered by hybridoma technique.Four monoclonal antibodies were obtained from the group of C50041,named as 1C2,1C5,2C7,and 2C8,respectively.Two monoclonal antibodies were obtained from the group of S016,named as 7F6 and 3H6,respectively.One monoclonal antibody were obtained from the group of S44,named as 1E4.The heavy chain subclasses of 1C2 and 7F6 were classified to IgG1,the heavy chain subclasses of 1C5,2C7,and 2C8 were belonged to IgG3,3H6 and the heavy chain subclass of 1E4 was belonged to IgM.The result of plate agglutination test showed the monoclonal antibodies could bind to antigens 05,09,and 012.The results of plate agglutination test,indirect ELISA test and Western-blot test showed that 1C5 specifically reacted with Salmonella that contained O12 antigen(S.pullorum,S.enteritidis,and S.typhimurium,etc.).This data will lay the foundation for establishing a high-sensitivity,high-specificity,and high-stability method to detect Salmonella with O12 antigen.