Establishment And Application Of Indirect Elisa For Detection Of Salmonella D Antibody Based On LPS

Salmonella is an important zoonotic pathogen widely existing in nature.which can cause food-borne poisoning and cause serious harm to human beings.There are many kinds of Salmonella,among which Salmonella Pullorum,Salmonella Enteritidis and Salmonella Typhimurium are the most harmful to poultry.Among them,S.Pullorum is an avian pathogenic bacterium,which is harmful to poultry breeding industries in China seriously.S.Pullorum can be transmitted vertically and horizontally,and it is difficult to be eradicated.The eradication of pullorum disease in breeding chicken farms is still the main work at present.Regarding the purification of salmonella,the National Medium-and Long-Term Animal Disease Prevention and Control Plan(2012-2020)requires that all breeding chicken farms in China should meet the purification standards by 2020.The key work for eradication of pullorum disease is to eliminate the infected chickens.However,the traditional bacterial isolation and identification technology is complicated and takes a long time,so it is not suitable for clinical detection.Most detection methods based on molecular biology need expensive instruments and professional operators,and are not suitable for clinical popularization.Immunology-based detection technology is the most widely used detection technology at present,and the immunological method is simple and convenient.In the clinical detection of pullorum disease,the plate agglutination test of whole blood or serum is the most widely used method.However,the strains used for preparation of agglutination antigen are unstable,and the antigen is not extracted and purified,there may be a deficiency in accuracy,which lead to false positives.In addition,the sensitivity of plate agglutination is poor,so there may be missed detection in practical applications.Therefore,a rapid detection method with strong specificity and high sensitivity is needed.O antigen,the main component of lipopolysaccharide(LPS),is the determinant of antigen,which is the basis of serological classification of Salmonella.Based on the specificity of O antigen,OIE thinks that ELISA with LPS as coating antigen is the most specific method to determine pullorum and typhoid.Therefore,this study aims to extract LPS from S.Pullorum,and establish a method for detecting antibodies against group D Salmonella infections,including pullorum disease.In this study,a S.Pullorum strain(TC 3 strain)with good immunogenicity was selected as the preparation strain,and LPS from S.Pullorum was extracted and purified by hot phenol-water method,with a yield of 1.463%.The protein concentration was 0.0995mg/m L after digestion with protease.The polysaccharide content was 18.80% after purification.The gel electrophoresis and silver stain identification of the extracted LPS were carried out after dissolution,and the results showed that LPS had a stepped structure,which proved that the LPS structure was complete.Using the extracted LPS as the coated antigen,a series of reaction conditions were determined by the square matrix titration method.The antigen coating concentration was determined to be 2.233 mg/L.The optimal dilution of the serum sample was 1:200,and the action time was 60 min.The most suitable blocking condition was to block using 5% skimmed milk for 60 min and 37 °C.The optimum working mass concentration of HRPlabeled rabbit anti-chicken Ig G antibody was 1:8 000,and acted for 60 min at room temperature.The chromogenic condition is to react in dark for 15 min.The positive determination standard was S/P ≥ 0.5.The positive serum of Escherichia coli and multiple serum groups of Salmonella,such as S.typhimurium,were tested.There was no obvious cross reaction,which proved that the specificity of this detection method was good.The detection sensitivity was tested using series diluted positive serum,and the result showed that the sensitivity of our method was 400 times higher than agglutination plate.The variation coefficients of intra-batch and intra-batch repetition are all less than 10%,which showed good repeatability.Finally,283 clinical serums were tested,the positive compliance rate of our method was 94.67% by comparing with Biochek’s Group D antibody detection kit,the negative compliance rate was 98.11%,and the total compliance rate was 98.59%.The established indirect ELISA method and plate agglutination were used to detect1185 clinical serum samples from five breeding chicken farms of different parents in Hubei province.The results showed that the individual positive rate of S.Pullorum antibody detected by indirect ELISA ranged from 4.35% to 21.88%,and the overall individual positive rate was 17.03%.The individual positive rate of S.Pullorum antibody detected by plate agglutination was 0% ～ 4.35%,and the overall individual positive rate was 7.43%.The positive rate of indirect ELISA was significantly higher than that of plate agglutination.In summary,an LPS-based Salmonella Group D antibody ELISA detection method was established in this study.This method has strong specificity and good sensitivity,and it can be used for clinical antibody detection of S.Pullorum and other Group D Salmonella infections.This study will provide effective techniques and tools for the detection and eradication of pullorum disease.