Establishment And Application Of Rapid Detection Method Of Salmonella Pullorum Mediated By Saf Pili Monoclonal Antibody

Pullorum disease is an infectious disease caused by Salmonella pullorum.The mortality rate of chickens under 20 days old is as high as 80%-100%.Adult chickens can be recessively infected and vertically transmitted to chicks,resulting in a large-scale epidemic of the disease in chicken flocks,causing huge economic losses to the breeding industries in China.Therefore,the establishment of a rapid,sensitive and specific detection method is of great significance for the clinical detection,quarantine and purification of Salmonella pullorum.Through previous research,our research group found that Saf（sp）pili can mediate the adhesion between bacteria and host,and is specifically expressed in Salmonella pullorum.Based on this,this study took the Saf pili of Salmonella pullorum as the target.Firstly,the monoclonal antibody against Saf pili of Salmonella pullorum was successfully prepared by using the high-efficiency monoclonal antibody preparation platform developed in our laboratory.On this basis,using the anti-Saf pili antibody,a glass plate agglutination method for detecting Salmonella pullorum was established,and the simulated contaminated samples and clinical samples were detected,in order to provide a sensitive,specific,rapid and simple technical means for clinical detection,monitoring,quarantine and purification of Salmonella pullorum.The details are as follows:1 Preparation of monoclonal antibody against Salmonella pullorum Saf fimbriaeIn order to prepare monoclonal antibodies against Saf pili of Salmonella pullorum,the recombinant strain S9HH-Saf（sp）（S9HH is a generic inert carrier strain）carrying Saf（sp）target antigen independently constructed by our laboratory was used as the immunogen.Without any adjuvant,BALB/c mice aged 6 weeks were immunized with 5×108 CFU/mL.After four times of immunization,the spleen lymphocytes were collected and fused with myeloma cell SP2/0.The positive clones were screened by glass plate agglutination method（S9HH-Saf（sp）as positive control and S9HH as negative control）.Finally,three monoclonal antibodies that can stably secrete anti-Saf（sp）pili were obtained,named 4EB5,4FA11 and 22CF3 respectively.The results of subtype identification showed that 4EB5,4FA11 and 22CF3 were IgG1 subtypes.Then the ascites of mice was prepared and purified.The ascites titers of monoclonal antibodies were identified by glass plate agglutination test with positive standard strain S9HH-Saf（sp）with bacterial solution concentration of 5×109 CFU/mL.the agglutination titers of monoclonal antibodies 4EB5,4FA11 and 22CF3 were 1:128,1:128 and 1:256 respectively.After the three monoclonal antibodies were mixed in equal proportion,the titer was 1:256,and the reaction was stronger and faster.Then,the specificity of the McAbs was identified by glass plate agglutination test and Western blot:（1）glass plate agglutination took the target antigen recombinant strains constructed and verified in the early stage of our laboratory as the detection antigen.The results showed that the three McAbs only had positive agglutination reaction with strain S9HH-Saf（sp）expressing Saf（sp）pili,and had no cross reaction with seven strains such as S9HH-F18ab,S9HH-F18ac,S9HH-Peg,S9HH-K88ac,S9HH-K99,S9HH-987P and S9HH.（2）Western blot showed that the McAbs only reacted with S9HH-Saf（sp）strain and produced a specific band with a size of 16.4 KD,which was consistent with the expectation;It did not react with the negative control（S9HH）.The results showed that the three monoclonal antibodies had strong specificity and high sensitivity.In this study,monoclonal antibodies with agglutinating activity against the Saf pili of Salmonella pullorum were successfully prepared,which laid a foundation for the further basic research of the Saf pili of Salmonella pullorum and the establishment of the detection method of the strain.2 Establishment and preliminary application of glass plate agglutination method for Salmonella pullorumIn order to establish a method for rapid detection of Salmonella pullorum,the antiSalmonella pullorum Saf wool monoclonal antibody prepared in the above chapter was used as the detection antibody.By optimizing the conditions,a glass plate agglutination detection method of Salmonella pullorum was established.Then,the specificity and sensitivity of the method were tested:（1）specificity test.Through glass plate agglutination test,15 strains of Salmonella expressing saf gene（including Salmonella pullorum CVCC523,CVCC526,CVCC535,CVCC540,S07,S78,1-1,747-1,827-Y,727-5,829,829-I,755-L,AHC428 and T89）were used,Three strains of Salmonella that do not express saf gene（Salmonella typhimurium DT104,W32,Salmonella enteritis 50336）and three strains of non Salmonella（including Escherichia coli ETEC C83901,ETEC C83902,ETEC C83903）were tested for specificity.The results showed that only Salmonella pullorum had specific agglutination reaction,and other strains were negative,which proved that the glass plate agglutination method established in this study had good specificity.（2）Sensitivity test.The standard strain of Salmonella pullorum NCTC5776 was tested after dilution of the culture medium at OD600nm=1.The results showed that this method could detect at least 1×104 CFU/mL pure culture of Salmonella pullorum standard strain showed that the method was sensitive.In addition,this study uses the established glass plate agglutination detection method for preliminary application detection:（1）This method is used to detect artificially contaminated sterile chicken feed samples.Different concentrations of Salmonella pullorum NCTC5776 were added to the sterile feed samples and tested after pre enrichment.The results showed that when the initial concentration of pollution was 102 CFU/mL,Salmonella pullorum could be detected after pre enrichment for 10 hours.（2）The established glass plate agglutination method and national standard method were used to detect 10 clinical materials（liver）.The samples were prepared by dissecting and grinding the liver in a sterile environment.After pre enrichment,Salmonella pullorum was detected by the established glass plate agglutination method.After retesting by the national standard method（including bacterial culture,biochemical characteristic detection and serological detection）,the coincidence rate of the two results was 100%.It is worth mentioning that,compared with the cumbersome traditional national standard identification method,this method can be used for detection only by pre enrichment of the sample,and the glass plate agglutination reaction can get the results in only 2 minutes,which is fast and convenient.In conclusion,the glass plate agglutination detection method of Salmonella pullorum established in this study has high sensitivity and specificity,and can quickly and accurately detect Salmonlla pullorum in contaminated samples and sick chicken liver samples,which provides a fast and effective method for the detection and purification of Salmonella pullorum.