Research On Mucosal Immunity And Micro Ecological Function Of Attentuated Edema Disease-causing Escherichia Coli In Piglets

Edema disease of swine caused by Shiga toxin-producing Escherichia coli (STEC) is a common acute fatal enterotoxemia. The F18ab fimbriae and Shiga-like toxin type II responsible (Stx2e, also called Vero cytotoxin) for edema disease of swine is an important virulence factor. Disease incidence rate of Piglet Edema is about10%-35%, mortality rate as high as80%～100%. in recent years it is the leading cause of death in early weaned piglets, resulting in significant economic losses to the pig industry. In order to prevent edema disease of piglet, the text on the basis of has been successfully constructed2toxin gene induced edema disease of piglet Escherichia coli strain S451521knockout strains F1, F2(O139:F18), has carried on the following research. and reproductive ability.The general biological of the F2attentuated variants were further detected in this reseach:Recovery of F2strains, in TSB liquid medium at37℃, static culture of48h. Detection of F18ab pili gene:Using dual PCR amplification to the specific fragment of two size is about170bp and520bp, and the specific fragment size expected. Detection of F18ab pili protein: Using the above cultures do glass plate agglutination test and electron microscopy for F18ab pili protein, The glass plate agglutination test can see the agglutination blocks appear; Electron microscopy results display that a large number of fimbriae are visible around cells expressed.Those indicating that the bacterial pili protein was successfully. The30-day-old ICR mice were separated into three experimental groups and one control group. The animals of the experimental groups A, B and C were intragastric inoculated with E. coli S451521, F1and F2respectively, and the animals of the control group4were treated with normal saline (NS) in the same way. The data showed that the virulence of the attentuated variants, F1and F2, were all lower the wild type strain S451521, thereinto, F1strain was reduced60.73%while F2strain was reduced84.38%.Research in reproductive ability of the F2attentuated variants in piglet intestine:Fourteen piglets,24-day-old and non-immune, were separated into two groups, including experimental group Ⅰ were intragastric inoculated with attentuated strain F2while group Ⅱ were treated with NS as control. Following the fecal samples were collected from all the animals, bacteria were counted and identified. The result showed that the number of E. coli strain F2defecated from the tested piglets was increased with time, and can achieve70000CFU per gram of feces at28d after inoculation. The data indicate that the attentuated E. coli strain F2could colonizated and grow abundantly in the piglet’s intestine.The ability of stimulate piglet intestinal mucosal immunity and humoral immunity were further detected in this reseach: Thirty piglets,24-day-old and non-immune, were separated into three groups, including experimental group A were intragastric inoculated with attentuated STEC strain F2, group B were intragastric inoculated with attentuated STEC strain F2and attentuated heat-labile toxin protein, while group C were treated with NS as control; Following the intestinal mucus and serum samples were collected from the animals. The titer of the antibody specific to F18fimbriae were detected by Indirect ELISA. The result showed that the attenuated STEC strain F2could stimulate mucosal antibody of F18fimbriae in small intestine of piglets, furthermore also could stimulate the production of serum antibody. The attenuated LT could enhance the production of secretory and serum IgA, but the effect was not significant, while no effect on IgG. The titer of the antibody specific to edema disease-causing Escherichia coli were detected by micro agglutination. The result showed that the attenuated STEC strain F2could stimulate mucosal antibody of edema disease-causing Escherichia coli in small intestine of piglets, furthermore also could stimulate the production of serum antibody. The data suggested that the attenuated STEC strain F2could be used as oarl vaccine of edema disease.The adhesion of the wild strain S451521inhibited by its attentuated variant were further detected in this reseach: The distal duodenum sample was aseptic cellected from the piglet after a week of weaning, and separated into several intestinal segments (each2cm). Following each segment was adhered by attenuated STEC strain F2and/or wild type strain S451521, the same size samples were cut out from each segment and put into LB broth with same volume. The intestinal mucosa were fully scraped into LB broth, and daubed on the LB plates with serial dilutions. Following120colonies were picked out from each sample and submitted to PCR examination and kanamycin-resistant identification, the adhensive inhibition ability of the attenuated STEC to the wild type was determinated. The result showed that the attenuated STEC strain F2could obviously inhibit the wild-type bacteria adhesion to epithelial cells of piglets, and the inhibition rate were84.62%at least. The data suggested that the attenuated STEC strain F2could be used as oarl probiotics strain to prevent the edema disease.