| Edema disease of swine (ED) is mainly caused by Hemolytic Escherichia coli which lead to edema of head, stomach submucosa, ataxia, spasm and tetraplegia. ED is an acute disease. Because the onset of disease is often sudden and the course rapid, treatment is often ineffective, and mortality rate is very high. Following the researches of these years, people become grasp the pathopoiesis mechanism of edema disease. F18ab fimbriae and Stx2e toxin were two pathogenic factors of edema disease. At present, Escherichia coli multivalent vaccine and antibiotics were used to prevent and treat this disease, but the expected effects were not well. Egg yolk antibody as a kind of effective alternative to antibiotics in biological control showed the obvious effect and also opened a new way in the prevention against to the edema disease of swine.In this study, F18ab fimbriae and Stx2e toxin were extracted, and the recombinant protein FedF and Stx2eB, which had neutralizing epitope were obtained respectively by prokaryotic expression technology. Then the four different proteins were used as immunogen to immunize the six months old Highland laying hens for three times. Each time interval was two weeks. Chickens were divided into four groups. The chicken of Stx2e toxin group were divided into two groups and each group were immunized 0.20mg per chicken and 0.40mg per chicken respectively in the first immunization. Two weeks later, the second immunity was performed. Then the two groups of first immunization were divided into two subgroups respectively, the dose of subgroups were the same as the first immunization and twice as the first immunization respectively. In the third immunization, each chicken was immunized as much as the highest dose of the second immunization. The chicken of Stx2eB recombinant protein group were divided into two groups and each group were immunized 0.50mg per chicken and 1.00mg per chicken respectively in the first immunization. The second and the third immunization were immunized with the same method as Stx2e toxin group. The chicken of F18ab fimbriae protein group were divided into two groups and each group were immunized 0.38mg per chicken and 0.76mg per chicken respectively in the first immunization. The second and the third immunization were immunized with the same method as Stx2e toxin group. The dose of FedF recombinant protein group of three immunity times was 1.00mg per chicken, 2.00mg per chicken and 2.00mg per chicken respectively. One week after the second immunization, the eggs were collected and the yolk antibodies were extracted, then the titers of the yolk antibodies were detected by indirect ELISA. The highest titer of the Stx2e toxin group was 1:5120. The highest titer of the Stx2eB recombinant protein group was 1:5120. The highest titer of the F18ab fimbriae protein group was 1:20480. The highest titer of FedF recombinant protein group was 1:1280.And the high level of titers could maintain for six weeks, two weeks, four weeks and three weeks respectively. The results of safety experiment showed that egg yolk antibodies reached the standard of biological product from Chinese Veterinary Pharmacopoeia. In vitro tests showed that E. coli (F18ab+) adhesion to small intestine epithelial cells was inhibited with appropriate anti-F18ab fimbriae antibody or anti-FedF recombinant protein antibody preparations by preincubating the mixture of 1 ml of bacteria and 1 ml of antibody solution for 30 min, however, E. coli (F18ab+) without pretreatment with antibody adhered to the epithelia cells surfaces. The neutralization test in vitro showed that both anti-Stx2e toxin egg yolk antibody and anti-Stx2eB recombinant protein egg yolk antibody could inhibit the pathogenic affection to Vero cell. Comparing with control group the groups with appropriate anti-Stx2e toxin or anti-Stx2eB egg yolk antibody had little dead cell instead of most normal growth cell. The neutralization test in vivo showed that anti-Stx2e toxin egg yolk antibody stock solution, 1:2 dilution and 1:4 dilution had protection to Kunming mice and the rate of protection was 100%, 60% and 60% respectively. So did the anti-Stx2eB recombinant protein egg yolk antibody to Kunming mice and the rate of protection was 100%, 60% and 40% respectively. While the mouse which were injected physiological saline or egg yolk antibody only were normal growth and the mouse which were injected Stx2e toxin only were dead in 48h. The mortality was 80%.This study also established a loop-mediated isothermal amplification (LAMP) method for the detection of Shiga-like toxin II variant producing Escherichia coli. According to 12 Stx2e sequences and 9 Stx2 sequences available in GenBank, a set of four primers were designed based on the Stx2e conservative sequence. DNA was extracted from an overnight culture of the E. coli (Stx2e+) by boiling method to do LAMP assay. Product of positive reaction with LAMP became turbid and the negative control was no change. Product of positive reaction with LAMP showed ladder-like pattern on gel electrophoresis and the negative control had no ladder-like pattern. After addition of SYBR Green I, the products can be visualized directly by the naked eye or under UV light, which showed green while the negative control showed light orange. The optimal reaction temperature and time of the LAMP were investigated. LAMP assays, which were under isothermal condition of 63℃for 1h had the best results. The specificity test showed that ladder-like products were produced with those Stx2e positive samples by LAMP, while no product was generated with other controls(LT+,ST+,Stx2+). The sensitivity assay of LAMP had a detection limit equivalent to 38cfu/tube, was 100 times sensitive than PCR method (3.8×103cfu/tube). The agreement rate between LAMP and PCR was 100% in detecting simulation samples. Thus the LAMP assay was a rapid, specificity and sensitivity method.Results of this study indicated that the preparations of egg yolk antibodies could be produced aimed at Stx2e toxin and F18ab fimbriae. And these offered material basis for preventing edema and experiment basis for applying in clinical setting. The LAMP assay was rapid, sensitive, specific and judging easily so that it had the potential to be developed into a rapidly diagnosis tool for edema disease of swine and detection of Escherichia coli producing Stx2e. |
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