Development And Application Of A SpiC Protein Based Indirect ELISA For The Detection Of Salmonella Antibody In Chicken

Salmonella is an important zoonotic pathogen.Which can infect not only livestock but also humans.Certain Salmonella serotypes,such as S.Pullorum,S.Enteritidis and S.Typhimurium can infect chicken and cause Salmonellosis as the latent infection even causing death of the infected.Salmonellosis in chicken causes great economic losses in poultry industry.At the same time,Salmonella is also one important food borne pathogen,the livestock and poultry products infected or contaminated by Salmonella may cause Salmonellosis in human,which threatens public health seriously.Therefore,effective methods for Salmonella detection must be established to control the spread of Salmonella in chicken.Detection of antibody in chicken serum is one of the most important methods to determine Salmonella infection.For a long time,the plate agglutination test(PAT)is often used to detect Salmonella antibody in chicken serum,but this method has a serious drawback of low sensitivity,and thus may fail to detect the Salmonella antibody.The PAT cannot satisfy the effective and comprehensive detection of the Salmonella antibody in chicken.Therefore,it is necessary to develop a new method for detection of Salmonella antibody in chickens.SpiC protein is a conservative protein in Salmonella which is widely found and has potential as a target antigen for detection of Salmonella infection.In this study,a SpiC protein based indirect ELISA for the detection of Salmonella antibody was developed and applied preliminarily.IPTG was used to induce His-SpiC expression,and then the His-SpiC was purified by affinity chromatography.SDS-PAGE identification demonstrated that there was an obvious target band at 20 kDa,indicating that the His-SpiC protein was correctly expressed with high purity.The SpiC protein were identified in all the mice infected with different serotypes of Salmonella,indicating that SpiC protein can be used as antigen to detect Salmonella antibodies in serum widely.Then,an indirect ELISA method was developed by using SpiC protein as an antigen to detect Salmonella antibody in chicken,and the parameters,including the concentration of SpiC protein,coat buffer,coat time,block buffer,blocking time,serum dilution and reaction time,concentration of HRP-labeled rabbit anti-chicken IgG and reaction time were optimized.The resultus demonstrated that the experiment with the following pipeline showing best results.The parameters in the pipeline were as follows:the concentration of His-SpiC protein was 2 ?g/ml,using CBS as coating buffer,then incubated at 37? for 2 h;using 5%skim milk as the blocking solution,blocked at 37? for 2 h;then the serum was diluted with 1:640 by using 2%BSA and incubated at 37? for 2 h;the rabbit anti-chicken IgG which labeled with HRP was diluted in 1:30000,incubated 45 min at 37?;in the last step TMB was added and incubated for 5 min,and the reaction was read at OD450 after stop solution was added in each well.Forty-eight SPF chicken serum were tested using the above experimental procedure.Through calculation,we got serum cut-off value was 0.26,which means that OD450? 0.26 were judged to be a Salmonella antibody positive serum.Salmonella antibody negative chicken serum(Newcastle disease,infection bursal disease,avian influenza,avian leukosis,proteus,Colibacillosis)and SPF chicken serum by the Salmonella Group B and D Antibody Test Kit also showed negative results by the SpiC indirect ELISA,indicating that the indirect ELISA method developed in this study possessed high specificity and had no cross reaction with other avian disease antibodies.The repeatability of this indirect ELISA was evaluated by intra and inter test.The results showed that the coefficient of variation(CV)in intra and inter test were all below 15%,indicating that the indirect ELISA detection method for chicken Salmonella antibody was reproducible.We used the indirect ELISA and PAT to detect serum from chicken which were infected by S.Pullorum via oral administration.The results showed that the antibody in chicken serum could be detected after 9 days of Salmonella infection by the indirect ELISA detection method,while the PAT could not detect Salmonella antibody until 15 days after Salmonella infection,which suggests that the ELISA detection method has higher sensitivity.The results further demonstrated that the antibodies of SpiC protein produced after Salmonella infection could persist in serum for at least 3 months.Then the sensitivity and specificity of the indirect ELISA detection method developed in this study and the commercial Salmonella B Group and D Group Antibody Test Kit were evaluated simultaneously.The results showed that the positive rate of these two methods was 100%and the negative rate was 94.3%.It indicated that the indirect ELISA detection method in this study could more effectively detect Salmonella antibody in chicken serum,and the sensitivity of this indirect ELISA method was higher than that of commercial test kit.We used the indirect ELISAmethod developed in this study to detect the chicken serum which were immunized by S06004?spiC and S06004?spiC?rfbL.The results showed that all the serum was negative,which mean that the indirect ELISA has the potential to distinguish between the chickens immunized by the spiC gene deletion vaccine and infected by wild Salmonella.The indirect ELISA method developed in this study was used to detect Salmonella antibody in chicken serum samples collected from three different chicken farms from Yangzhou,Heibei and Nantong.The results showed that the positive rate of the chicken farm in Nantong was the highest,followed by the chicken farm in Hebei and the lowest positive of sera was the chicken farm in Yangzhou.The results indicated that the method can be applied to detected clinical serum samples and provide an effective,reliable and wide detection method for detecting Salmonella antibodies in chicken serum.