| Yellow scours and white scours are the common infectious diseases of newborn piglets produced by enterotoxigenic E. coli that result in severe diarrhea, dehydration and a massive death, and seriously affect the survival rate of piglet. At present, Escherichia coli multivalent vaccine and antibiotics were used to prevent and treat the diseases, but the expected effects were not be observed. 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 new piglet diarrhea diseases.At present, the natural fimbirae as mostly immunogen was used to produce the egg yolk anatibodies against to yellow sours and white sours, however, it was difficult to extract and purify the fimbirae, so that the yield were not enough to meet the needs of mass production. The objective of this study was to construct the recombinant expressing vector to efficiently express the recombinant protein of fimbirae in E. coli by genetic engineering technology, and to explore a new method to produce a larger number of immunogen.The F4 and F5 natural fimbria were extracted from the enterotoxigenic E. coli, and the recombinant fimbriae protein of F4 and F5 were expressed in E. coli Rosetta, respectively. Then the natural fimbriae and recombinant protein of F4 and F5 were used as immunogen to immunize the 9 months old Highland laying hens.Chickens were divided into 4 groups of 9 cages,3-4 chickens in each cage, named as group A (F4 fimbriae), group B (F4 protein), group C (F5 fimbriae) and group D (F5 protein). Group A was divided into 3 subgroups of 3 cages, named as A1, A2 and A3. The every chicken in A1, A2 and A3 were injected with 0.25mg,0.5mg, or 0.75mg of F4 fimbriae via pectoralis, respectively. Two weeks after the first immunization, the second immunity was performed. The chickens in the 3 cages (A1-1, A1-2, and A1-3) of subgroup A1 were injected with 0.25mg,0.5mg, or 0.75mg of F4 fimbriae, respectively. The chickens in the subgroup A2 and A3 were immunized with the same dose and method as subgroup A1. Two weeks after the second immunization, the eggs were collected and the yolk antibodies were extracted, then the titers of the yolk antibodies were detected by tube agglutination test and ELISA. The chickens in the cage which had the highest titer of the yolk antibody were selected, then the third immunization to be performed with dose of 1.00mg per chicken. Grouping and immunizing of group B, group C and group D were the same as group A. According to the titers of yolk antibodies to determine the time of egg collection.The extract method of mass production of yolk antibody against to F4 and F5 fimbrial was determined by comparison among some extract methods, just like chloroform method, dilution with water and hydrochloric acid, bitter-ammonium sulfate law and polyethylene glycol precipitation. Some related detections were done according to "Chinese Veterinary Pharmacopoeia". In vitro adhesion assay and adhesion inhibition assay to the small intestine epithelial cells isolated from 3-day old piglet were performed using the extracted yolk antibodies.6 nests of newborn piglets were fed with anti-K88ab fimbriae, anti-K88ab expressed protein, anti-K99 fimbriae, anti-K99 expressed protein, anti-K88ab fimbriae+ anti-K99 fimbriae or anti-K88ab expressed protein+ anti-K99 expressed protein yolk antibodies, respectively. Every nest were divided into two groups, and half were fed with 1:9 dilution of egg yolk and saline solution, another half were fed with extracted yolk antibodies at the first 3 days of every week in the first 3 weeks from born, the dose was 5ml per piglet per day.The results of ELISA and tube agglutination test showed that the natural fimbrial and the expressed protein used as immunogen could stimulate the laying hens to produce high titer egg yolk antibodies. The highest titer of yolk antibodies were detected at the third week after the third immunization, the titer of ELISA was 1:10240, and that of tube agglutination test was 1:5120, the high level of titers can be maintained for 5 weeks. The results of safety experiment showed that egg yolk antibodies reached the standard of biological product from Chinese Veterinary Pharmacopoeia.The results of specific experiments showed that egg yolk antibodies agglutinated with relevant E.coli,but the egg yolk antibodies do not agglutinated with other E.coli.In vitro tests showed that E. coli (K88+ or K99+) adhesion to small intestine epithelial cells was inhibited with appropriate anti-fimbrial antibody preparations (anti-K88 or anti-K99) by preincubating the mixture of 1ml of bacteria and 1 ml of antibody solution for 30 min, adding 1ml of epithelial cells to the mixture, and then incubating at 37℃for 30 min, however, E. coli without pretreatment with antibody adhered to the epithelia cells surfaces. Clinical trials showed that the piglets appeared in good spirits after feeding the yolk antibody. Only a few piglets who were fed 1:9 dilution of anti-K99 expressed protein yolk antibodies and water emerged minor diarrhea, the diarrhea weekly rate was 2.86%,11.4%,0%, respectively, in the 1st-3rd week from born, and that of control group (without feeding yolk antibody) was 3.57%,10.71% and 3.57%. The remaining experiment groups had no diarrhea at all. There was significant deviation on average weight gain between group of feeding anti-K99 fimbriae of 1:9 diluted egg yolk and control group, so did the group of feeding extracted yolk antibodies against K99 fimbriae. There were no significant deviation on average weight gain between other experimental groups and control group.Results of this study showed that expressed protein as an immunogen can produce high titer yolk antibodies. In this study, the preparations of egg yolk antibodies showed good effects in prevention and treatment against yellow and white scours, and it can be applied in clinical setting. |
