Detection Fimbriae Gene Of Enterotoxigenic Escherichia Coli From Porcine By Multi-PCR And The Preparation Of Antibodies Against Enterotoxigenic Escherichia Coli Fimbriae

Enterotoxigenic Escherichia Coli (ETEC) is the main pathogen ofcolibacillosis of neonatal and post weaned piglets. The pathogenesis of ETEC is strongly related to adhesive fimbriae and enterotoxin. Mechanisms in the pathogenesis of piglets diarrhea caused by ETEC is that ETEC attach to the small intestine by means of the adhesive fimbriae and replicate and elaborate enterotoxin that interfere the biochemistry of small intestine cell. In this experiment, four kinds of Enterotoxigenic Escherichia coli fimbriae were extracted by thermoextract method. For K99,F41 and 987p fimbriae , vaccines were made from each fimbriae and Freund's adjuvant. For K88 fimbriae, vaccines were made by Freund's adjuvant, white oil adjuvant, A1(OH)3 adjuvant and propolis adjuvant respectively. And vaccine containing four fimbriae was made by Freund's adjuvant. Laying chickens were immunized with different vaccines. The result indicated that all four fimbriae could cause the immunity reaction. K88 fimbriae could induce the highest level egg yolk antibodies which could sustain a long time. And the Freund's adjuvant and the White oil adjuvant vaccine could induce the highest level egg yolk antibodies and could keep stable within a long time. Trial of adhesion in vitro indicated that the attachment of bacterial cells to intestinal epithelial cells was strongly inhibited when homologous anti-fimbrial antibody solutions were used. The anti-K88 antibody reduced considerably the severity of diarrhea in piglets challenged with K88+ETEC. All results indicate that the chicken egg yolk antibody against ETEC fimbriae was effective. For detection, adhesive fimbriae and enterotoxin are the markers of ETEC. To detect four kinds of adhesive fimbriae genes of ETEC from porcine, four pairs of primers are designed and synthesized according to the conserve sequence region of four kinds of fimbriae structural gene. Four pairs of primers could amplify 201bp,314bp,380bp and 459bp segment respectively. The length and the results of restriction enzyme digestion indicate that the amplified products are respected. A multiplex polymerase chain reaction(PCR) system for the rapid screening of K88,K99,F41 and 987p genes was developed. The specificity of thismulti-PCR system was confirmed by PCR of ETEC reference strains and negative control bacterial strains. Results also show that the multi-PCR was sensitive. It will be useful for identification of K88, K99JF41 and 987p genes and diagnosis of procine colibacillosis.