Isolation And Identification Of Avian Salmonella In Parts Of The Yangtze River Delta Areas During 2021-2022 And Establishment Of A Competitive ELISA Based On Adhe And Bfr MAb

Salmonella is an important zoonotic Gram-negative pathogen that causes diarrhoea and food poisoning in humans,as well as septicaemia and abortion in animals.Infection of chickens can lead to diarrhoea,increased mortality and reduced egg production,resulting in serious economic losses.Due to the large number of Salmonella serotypes and increasing drug resistance,continuous monitoring of the epidemic distribution and drug resistance of Salmonella is of great significance for the prevention and control of the disease.Salmonella Pullorum can be transmitted both horizontally and vertically,it is necessary to establish a specific and sensitive antibody detection method for the clean up of S.Pullorum in breeding farms.In this study,the specific proteins Adhe and Bfr were screened by western blotting and indirect enzyme linked immunosorbent assay(ELISA),and the monoclonal antibody was used to establish a competitive inhibition ELISA.This provides a diagnostic and detection tool for the S.pullorum clean up in chicken breeding farms.1 Isolation and identification of avian-origin Salmonellain parts of the Yangtze River Delta areas during 2021-2022During July 2021 to July 2022,Salmonella was isolated and identified from clinical samples in parts of Jiangsu and Anhui areas,YangZhou University Animal Hospital and wild bird samples in parts of the Yangtze River Delta areas.Salmonella was cultured and purified by Mac conkey medium,Salmonella chromogenic medium and SS medium.The serotypes were determined by multiplex PCR(mPCR)and glass plate agglutination assay.Resistance of identified Salmonella from poultry to 21 antibiotics was detected by means of drug-sensitive paper.A total of 650 poultry-orgin clinical samples were tested and 69 strains of Salmonella(10.62%)were finally identified,S.Pullorum accounted for 52.17%(36/69),2 S.Typhimurium accounted for 34.78%(24/69)and S.Enteritis accounted for 13.04%(9/69).The results of the drug sensitivity test showed that 69 isolates were sensitive to aminoglycosides,sulfonamides and acylamides,and resistant to β-lactams and macrolides.51 strains of Salmonella were resistant to penicillin,with a resistance rate of 73.91%(51/69),and 66 strains of Salmonella were resistant to erythromycin,with a resistance rate of 95.65%(66/69).Notably,all isolates showed 100%resistance to benzocillin and rifampicin(69/69).Among the 69 Salmonella isolates,57 strains were multi-drug resistant strains,with a multi-drug resistance rate of 82.61%.A total of 1472 wildbird-origin samples were detected and 14 strains of Salmonella(0.95%)were finally identified,including S.Pullorum accounted for 50%(7/14),S.Typhimurium accounted for 28.57%(4/14),S.Enteritis accounted for 14.28%(2/14)and S.Aguna accounted for 7.14%(1/14).The results of the drug sensitivity test showed that wildbird-origin isolates were 92.86%(13/14)resistance to benzocillin and rifampicinsensitive,but sensitive to ciprofloxacin and amikacin,the resistance rate was less than 20%,and the multiple resistance rate was 14.28%(2/14).The above findings suggest that the dominant serotype of Salmonella in parts of the Yangtze River Delta areas in 2021-2022 were S.Pullorum.Most strains of poultry-origin are resistant to three or more antibiotics.Therefore,the prevalence and drug resistance of Salmonella should be monitored continuously.2 Expression and screening of specific proteins of Salmonella PullorumThe anti-inflammatory effector protein Ipaj,bacterial ferritin Bfr,heat shock protein 60 Groel and ethanol dehydrogenase Adhe are screened as the candidate antigenic of S.Pullorum with reference to the articles.Bfr,Groel,Adhe and Ipaj genes were cloned into the prokaryotic expression vectors pET28 or pET32,and the proteins were collected and purified after induced expression.Infectious sera against Salmonella spp.such as S.Pullorum,S.Typhimurium and S.Enteritis and from Escherichia coli,Pseudomonas aeruginosa and Klebsiella pneumoniae were used as primary antibodies,and screening for chicken S.Pullorum specific proteins using western blotting and indirect ELISA.The results showed that Adhe and Bfr proteins were specific proteins for S.Pullorum and have strong and good specific responses to positive serum of S.Pullorum,which laid a foundation for the subsequent preparation of monoclonal antibodies to establish a monoclonal antibody competitive inhibition ELISA.3 Establishment of a competitive ELISA based on monoclonal antibodies against Adhe and Bfr of Salmonella PullorumAdhe and Bfr,specific proteins of S.Pullorum,were purified and then immunized in 6～8 weeks BALB/c mice.Positive hybridoma cell lines stably secreting proteins specifically against Adhe and Bfr,named as 6G11 and 5B8,were successfully obtained using monoclonal antibody technique.The isotype identification results showed that both monoclonal antibodies were of the IgG 2a isotypes.The western blotting results showed that both 6G11 and 5B8 could specifically recognize S.Pullorum.Purified GST-Adhe and GST-Bfr proteins were used as coated antigens,respectively,and the optimal concentrations of the coated antigens were found to be 0.9375 μg/mL and 0.3125 μg/mL,respectively,using a square matrix experiment.The dilution of ascites was 1:160 and 1:140,respectively.Serum dilution was 1:2.The protein coating condition was 4℃ for 12 h.The blocking condition was 37℃ for 1.5 h,respectively.The action condition of primary antibody was 37℃ for 1.5 h.The action condition of enzyme-labeled secondary antibody was 37℃ for 1 h.The color development time of TMB was 10 min.For monoclonal antibodies against Adhe protein,the cut off value of positive serum was set up as 29.34%and that of negative serum was 22.6%,and for monoclonal antibodies against Bfr protein,the cut off value of positive serum was set up as 20.99%and that of negative serum was 16.55%,anything in between needed to be redetected.The specificity results show that the established method has good specificity.The sensitivity results showed that the established methods were more sensitive than the plate agglutination test,while the sensitivity of the competitive ELISA against Adhe monoclonal antibody was higher than that of Bfr.The deteceted results of positive serum samples showed that all 31 sera tested positive by plate agglutination test were detected by the competitive ELISA test against Adhe and Bfr mAb.The deteceted results of the clinical serum samples showed the positive rates of Adhe and Bfr competitive ELISA assay and glass plate agglutination test were 72%,60%and 84%,respectively.The coincidence rates of Adhe and Bfr competitive ELISA assay and glass plate agglutination test were 85.71%and 71.42%,respectively.Therefore,the competitive ELISA against Adhe monoclonal antibody is more suitable for clinical serum sample detection,with higher coincidence rate,providing diagnostic and detection tools for the clean up of chicken S.Pullorum,which has good application value.