Isolation And Characterization Of Enteropathogenic Escherichia Coli From Piglets And Development Of An Indirect Elisa For Detecting Specific Antibody To Pilus Protein

Enterotoxigenic Escherichia coli,(ETEC) is the major pathogenic E.coli caused diarrhea diseases in neonatal piglets. E.coli with fimbrial genes were isolated from live diarrheic piglets of some industrial pig farms of Jiangsu and Jiangxi province in order to investigate the prevalence of F4、F5、F6 fimbrial genes.Fimbrial subunit genes faeG、fanC、 fas A were expressed in E.coli respectively by the genetic engineering technology and the immunogenicity of the fusion proteins was verified. An indirect enzyme-linked immunosorbent assay(ELISA) was developed based on a purified recombinant F4 pili protein of ETEC and applied to preliminary clinical detection.The detailed experimentations were described below:1 Isolation and characterization of enteropathogenic Escherichia coli from pigletsSince Oct 2012 to Sep 2013,146 fecal samples were obtained from live diarrheic piglets of 13 industrial pig farms of Jiangsu and Jiangxi province, a total of 116 Escherichia coli were isolated and identified. After designing three set of primers according to the sequences of the fimbrial genes that published in the GenBank,PCR was performed to detect the fimbrial genes of all the 116 isolates.The data showed that 20 isoloates(17%) were F4 positive,6 isolates(5.2%) were F5 positive,19 isolates(16%) were F6 positive and 5 isolates(4.3%) carried two fimbrial genes meanwhile.In addition, hemolytic assays showed that 13 isolates were β-hemolytic and 6 isolates were a-hemolytic.By analyzing the relevance between adhesion genes and hemolysis,it provided a certain basis for evaluation of pathogenicity of E. coli.2 Prokaryotic expression of subunits of F4、F5、F6 fimbrial proteins of enterotoxigenic Escherichia coliTo express the fimbrial subunits of enterotoxigenic Escherichia coli (ETEC) in vitro and laid a foundation of further preparation of ETEC multivalent vaccines and diagnostic kits.The faeG、fanC、fasA genes without signal peptide were cloned into the prokaryotic expression vector pET-28a respectively and the correct recombinant plasmids pET-28a-faeG、pET-28a-fanC、pET-28a-fasA were transformed into E.coli BL21.The fusion proteins F4、F5、F6 with a relative molecular mass of about 27kDa、20kDa and 22kDa mainly existed in a form of inclusion and showed nice antigenicity justified by Western-blot. The fusion proteins were purified by protein purification kit with Ni-Agarose His tag and refolded.3 Development of an indirect ELIS A for detecting specific antibody to F4 pilus protein of enterotoxigenic Escherichia coliAn indirect enzyme-linked immunosorbent assay(ELIS A) was developed based on a purified recombinant F4 pili protein of enterotoxigenic Escherichia coli and the serum from the immuned New Zealand long ears rabbit. The assay was established using 3.5 ng of F4 protein as coating antigen per well, with the optimal dilution of 1:3200 for testing serum and 1:5000 for HRP-labeled SPA.50 g/L skimmed milk powder blocked for 2 h,and the reaction time of serum and HRP-labeled SPA were both 1 h. It was proved that the method was rather specific and repeatable by cross experiment, blocking test and repeated experiment. The coincidence rate between the indirect ELISA and PCR was determined as 96.2%.The 312 swine serum samples without ETEC immune were detected by the established indirect ELISA, the positive rate was 9.9%.