Development Of An Indirect ELISA Method For The Detection Of Chicken Salmonellosis Antibodies

Chicken Salmonellosis which seriously do harm to poultry industry is still widespread in our country. Poultry infected with Salmonella were apparently healthy but bacteria which contaminated eggs and chicken products were easily discharged from sicken poultry. Contaminated products caused food poisoning threatened food safety and public health.Salmonella carrier state could be life-long, and vertical transmission is a very important route for Salmonellae transmission. So identification and elimination of infected breeding hens are particularly important. Plate agglutination test has been used to detect the specific antibody caused by Salmonella enterica serovar Gallinarum/pullorum for many years with a low sensitivity and accuracy. Intermittent or no emissions of bacteria of infected chicken induced the traditional isolated bacteria methods not to be sensitive enough. Because numerous serotype of infectious Salmonella, the traditional ELISA experiment can’t meet the actual demand.Salmonella surface presentation of antigens protein InvJ is widespread and conserved in Salmonella. The protein can be used to detect Salmonella antibodies and effectively screen out from infected chicken. Therefore, the molecular cloning methods were used to get InvJ protein, the protein was used as antigen in this indirect ELISA method. The results showed below.1. The amplification and fusion expression of Salmonella InvJThe Salmonella protein InvJ have a low homology with other bacterial, and it is a conserved protein in Salmonella. Primers were designed according to the sequences of Salmonella enterica subsp. enterica serovar Gallinarum/pullorum published in Genbank (CP003047) and successfully amplified from Salmonella enterica subsp. enterica serovar Enteritidis strain CVCC3375. Then insert InvJ gene into prokaryotic expression pET-28a(+) vector, structure the fusion plasmid pET-in4. The plasmid was transformed into E. coli BL21(DE3) and fusion protein was successfully expressed after induction. Western-blot results showed that the protein had good immunogenicity.2. Establishment of indirect ELISA assay method for the detection of InvJ antibodiesThe purified protein was used as coating antigen to detect specific antibodies in indirect ELISA. Optimum conditions was determined below:Concentration of coating antigen was0.17μg/mL; Phosphate(20mmol/L, pH7.0) buffer was used as coating buffer; coating temperature and time was4℃and12hours;5%skim milk was selected as blocking buffer, and was incubated for90minutes. The best serum dilution buffer was PBST and the dilution ratio of serum sample was1:800. Incubation time of serum sample and HRP-labeled goat-anti-chicken IgY antibodies was45min, and coloration time was15min. Cut-off value for the indirect ELISA was0.162.The repeatability test showed good reproducibility of the method. A total of655sera from nine farms were tested by this method, the lowest positive rate is0, and the highest rate is100%.This method provides a convenient and reliable tool for Salmonellosis diagnosis in chicken farm.