Isolation And Identification Of Salmonella From Poultry In Parts Of Jiangsu And Anhui And Establishment Of A Competitive ELISA Method Based On Monoclonal Antibodies Against O₉ And O₁₂ Antigens

Salmonella is an important zoonotic pathogen and one of the major pathogens of foodborne infectious diseases all over the world.Salmonella infection can not only cause the death of chicks,but also cause a decline in the egg-laying capacity of adult chickens,resulting in serious economic losses in the poultry industry.Therefore,it is of great significance to understand the distribution and prevalence of Salmonella for the control and prevention of Salmonellosis.Meanwhile,it is necessary to develop a sensitive,rapid,and accurate method to detect Salmonella antibody for achieving the eradication of Salmonella Pullorum infection in chicken farms.Enzyme-Linked immunosorbent assay（ELISA）is considered as one of the most common laboratory antibody detection methods.However,there are few specific and effective ELISA kits for detection of Salmonella Pullorum at present.This study established a competitive ELISA method to detect Salmonella Pullorum antibody based on monoclonal antibody against O9 and O12 of Salmonella,which will provide an effective tool for the eradication of Salmonella Pullorum in China.1.Isolation,identification,and drug resistance analysis of Salmonella from poultry in parts of Jiangsu and Anhui during 2020-2021During July 2020 to June 2021,clinical samples from poultry in parts of Jiangsu and Anhui,such as liver,gallbladder,and cecum,was collected,and then Salmonella were isolated and identified.The serotypes were determined by multiplex PCR（mPCR）and glass plate agglutination assay,and the drug resistance of all the isolates against 21 common antibiotics was determined by Kirby-Bauer method.A total of 620 samples were collected and 76 strains of Salmonella were identified.The total separation rate was 12.26%,Salmonella included 43 strains of Salmonella Typhimurium（56.58%）,26 strains of Salmonella Pullorum（34.21%）,5 strains of Salmonella Enteritis（6.58%）,1 strain of Salmonella Kentucky（1.32%）,and 1 strain of Salmonella Thompson（1.32%）.Animal origins showed that 32 Salmonella strains（42.11%）were isolated from chickens,and Salmonella Pullorum was the main isolates,accounting for 71.88%（23/32）.3 strains of Salmonella from duck（3.95%）,33 strains of Salmonella from goose（43.42%）,7 strains of Salmonella from pigeon（9.21%）and 1 strain from quail（1.32%）were mainly infected with Salmonella Typhimurium,accounting for 66.67%（2/3）,96.97%（32/33）,85.71%（6/7）,and 100%（1/1）,respectively.The results of drug sensitivity test showed that the isolates were generally resistant to β-lactam（penicillin and benzoxacillin）and macrolide（rifampicin and erythromycin）,especially to benzoxacillin and rifampicin,the drug resistance rate were 100%and 98.68%,respectively,while sensitive to quinolones,aminoglycoside,sulfonamides,and aminoalcohols,all the drug resistance rate was below 20%.The multiple drug resistance rate of 76 strains was 78.95%（60/76）.The multiple drug resistance rate of Salmonella Typhimurium and Salmonella Pullorum were 76.74%（33/43）and 76.92%（20/26）in particular,respectively.These results indicated that Salmonella Typhimurium and Salmonella Pullorum were the main Salmonella isolates from poultry in parts of Jiangsu and Anhui during 2020 to 2021,and multiple drug resistance existed widely,which should be continuously monitored.2.Establishment of a competitive ELISA method based on monoclonal antibodies against O9 and O12 antigensIn this study,the monoclonal antibody was prepared,purified,and labeled from previously hybrioma cells,named O9-2C8 and O12-1C5.The checkerboard method was used to explore reaction conditions of the competitive ELISA and the coating conditions of LPS from Salmonella Pullorum S6702.Firstly,the coating concentration of LPS based on O9 and O12 competitive ELISA were determined as 1.25 μg/mL and 1.875 μg/mL,respectively.Secondly,the working concentration of peroxidase-labeled monoclonal antibody O9 and O12 were determined as 11.3 μg/mL（1:100）and 2.988 μg/mL（1:80）,respectively.Thirdly,the dilutions of serum were determined as 1:4 and 1:2,respectively.The competitive ELISA conditions were further optimized,antigen coating conditions were determined at 4℃ for 12 h,the work conditions of peroxidase-labeled monoclonal antibody O9 and O12 were setted at 37℃ for 2 h,and the work conditions of substrate were setted at 37℃ for 5 min.Finally,two competitive ELISA methods were established.In O9 competitive inhibition ELISA,the critical value of positive serum was confirmed as 35.51%and that of negative serum was 24.70%,and the values between 35.51%and 24.70%,were suspicious.In O12 competitive inhibition ELISA,the critical value of positive serum was confirmed as 32.27%and that of negative serum was 21.71%,and the values between 32.27%and 21.71%were suspicious.Meanwhile,the inhibition rates of serum of Escherichia coli,Pasteurella,Pseudomonas aeruginosa,Klebsiella pneumoniae,and Proverendella were all less than 20%by both O9 and O12 competitive ELISA methods,were negative reactions,suggesting the specificity of these two methods is well.3.Assembly and verification of a competitive ELISA antibody detection kit based on monoclonal antibodies against O9 and O12 antigensThe weekly antibody levels were detected by O9 and O12 competitive ELISA methods under the simulated natural infection of Salmonella Pullorum.We compared the number of positive samples in Salmonella Pullorum S6702 group with different methods at the early stage of infection（14 d and 21 d）,and the results showed glass plate agglutination>O9 competitive ELISA>O12 competitive ELISA.Another Salmonella Pullorum 0322J1 group was as follows:glass plate agglutination>O9 competitive ELISA=O12 competitive ELISA.We also compared the number of positive samples in Salmonella Pullorum S6702 group with different methods at a later stage（>28 d）,and the result showed glass plate agglutination=O9 competitive ELISA>O12 competitive ELISA,and another Salmonella Pullorum 0322J1 group was as follows:glass plate agglutination=O9 competitive ELISA>O12 competitive ELISA.In a word,O9 competitive ELISA was more effective than O12 competitive ELISA in both the group of S6702 and clinical strain 0322J1.Therefore,we focused on the assembly of O9 competitive ELISA kit.The ELISA kit can be stored at 4℃ for at least 3 months by using Proclin300 and HRP protector.The variation coefficient of intra-batch and inter-batch repeats were less than 10%,indicating that the competitive ELISA kits had good stability.Compared with the commercial kit（Biocheck）in a third-party laboratory,the detection results of 36 serum samples with known backgrounds,including 34 Salmonella Pullorum positive serum samples and 2 negative serum samples,showed that the antibody positive rate of our kit was 44.44%,which was 50.00%coincidence rate with the results of glass plate agglutination test.The antibody positive rate of Biocheck kit was 77.78%,and the coincidence rate with the results of glass plate agglutination test was 83.33%.The coincidence rate between the competitive ELISA kit and the Biocheck kit was 44.44%.Compared with the Biocheck kit,the number of positive samples measured by our kit was less,but there were 4 samples with positive glass plate agglutination results,which can be detected by our kits but not by the Biocheck kit.Therefore,our competitive ELISA kit has potential application prospects after systematic optimization.