| Enterotoxigenic Escherichia coli(ETEC) is a kind of pathogenic E. coli, as a result of contaminated food and water,it always infects humans and animals, especially young animals. It has high morbidity and mortality, affecting the stable development of animal husbandry and aquaculture, as well as a threat to human health. Study found that the main pathogenic factor of ETEC is adhesin and enterotoxin. Etp A is a kind of adhesins,and it is a high molecular weight glycoprotein adhesins. Etp A is not only play a role in conjunction with the conserved regions of bacterial flagellin molecules, also improtant in binding to specific receptors on the intestinal epithelial cells. It mediates adhesion between bacteria and host intestinal like an adhesion molecules bridge. In different serotypes of ETEC, Etp A has a high conservativeness and high homology. Study confirmed, Etp A has a good immunogenicity, can effectively reduce the adhesion of ETEC on the intestinal epithelial cell. The finding provides new ideas for to prevent diarrhea caused by ETEC.In order to reduce the economic losses and health damage of animals caused by the ETEC, enhancing immune function in animals is also improtant while looking for effective prevention measures. Animals’ intestines has a huge area, which gathered a large number of microbial flora called intestinal flora. Intestinal mucosa is also rich in immune cells and immune molecules which involved constitute bodys’ intestinal mucosal immune system that against the incursion of pathogenic microorganisms. Increase the beneficial intestinal flora can regulate the stability and diversity of the intestinal microflora. It can promote the growth of the intestinal mucosa, stimulate the secretion of intestinal mucosal immune molecules and improve intestinal mucosal immune function while can helps the body to resist the invasion of pathogenic microorganisms.Saccharomyces cerevisiae is a member of Saccharomycodes that belongs to Saccharomycetaceae, it is also a kind of probiotic. In the study of its prebiotic effect found that it is rich in nutrients and unknown growth factor, they can improve animal performance, and promote the development of the intestinal mucosa, then increase the number of beneficial bacteria, and enhance the immune function of intestinal mucosal. Our group successfully constructed the Enterotoxigenic Escherichia coli adhesion Etp A-expressing engineered Saccharomyces cerevisiae strain and made it had a high expression in Saccharomyces cerevisiae by technologies of gene cloning and homologous recombination. And in cellular assay we found that the engineered strain can effectively prevent adhesion of ETEC on intestinal epithelial cells, and it have a role in protecting epithelial cells.In view of above-mentioned, the following experiment in this study were carried out:1. This study selected 120 healthy SD rats, they were 21 d of weaning age, and half of them were male and another half were female. They were randomly divided into three groups: control group, Saccharomyces cerevisiae group, engineered strain group(hereinafter referred to as Etp A group), each group had 40 r ats and half of them were male and another half were female. During the test, gavaged normal saline to control group then given gavaged107 CFU/m L S. cerevisiae broth to S. cerevisiae group and gavaged 107 CFU/m L engineered strain to Etp A group. Once a day and each 2m L lasted 28 days, then gavaged 108 CFU / m L of E. coli(H10407) broth once a day,eac htime gavaged 5 m L for each, lasted 3days. After gavaging for 7, 14, 21, 28 days and after gavaged ETEC for 3 days, we got feces from each group. We studied the effects of the engineered strain on OTU and p H of intestinal microflora by T-RFLP and the test of p H. Also studied the number of Bacteriodes, Clostridium, Enterococcus, Bifidobacterium and Lactobacillaceae by Quantitative Real-time-PCR.2. At the same time, we collect intestinal tissues of each group. Then we observed the effects of the engineered strain on villus length, width, crypt depth and villus length/crypt depth(V/C) of duodenum, jejunum,and ileum. Studied the number of SIg A, IL-2, IL-4 and IFN-γ by ELISA and Quantitative Real-time PCR.From the above tests these conclusions were obtained:1.Effects of Enterotoxigenic Escherichia coli adhesion Etp A engineering –Saccharomyces cerevisiae strain on intestinal microflora:(1) The effect on diversity of intestinal microflora: At 7th, 14 th, 21 st, 28 th days the numbers of S. cerevisiae group and Etp A group were more than control group, and reached max at 14 th day, they were reached a very significant level with control group(P<0.01). But the difference between S. cerevisiae group and Etp A group were not distinct(P>0.05), After gavaged ETEC EtpA group had no distinct difference with before(P>0.05), it was more than S. cerevisiae group in a significant level(P<0.05) and in a very signi ficant level with control group(P<0.01).(2) The effect on some improtant flora of intestinal microflora: From the 7th day to 28 th day the intestinal microflora of S. cerevisiae group and Etp A group were more than control group.(1)At the 21 st day and 28 th day, the number of Bacteriodes from S. cerevisiae group and Etp A group were more than control group in a in a significant level(P<0.05), the difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC Etp A group had no distinct difference with before(P>0.05),but it was more than S. cerevisiae group and control group in a very significant level(P<0.01).(2)At the 21 st day, the number of Clostridium from S. cerevisiae group and Etp A group reached max, and at the 14 th day and the 28 t h day they were more than control group in a very significant level with(P<0.01). The difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC Etp A group and S. cerevisiae group had no distinct difference with before(P>0.05), only control group had a very significant level with before(P<0.01).(3)At the 14 th day, the number of Enterococcus from S. cerevisiae group and Etp A group had significant growth, at the 14 th day they were more than control group in a very significant level with(P<0.01). The difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC Etp A group had no distinct difference with before(P>0.05), control group and S. cerevisiae group had distinct difference with before(P<0.05),but the difference among each group were not distinct(P>0.05).(4)From the 14 th day, the number of Bifidobacterium from S. cerevisiae group and Etp A group had significant growth, at the 14 th day they were more than control group in a very significant level with(P<0.01). The difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC Etp A group had no distinct difference with before(P>0.05), it had distinct difference with S. cerevisiae group(P<0.05)and had very significant difference with control group(P<0.01).(5)At the 14 th day and the 21 st day, the number of Lactobacillaceae from S. cerevisiae group and Etp A group had a very signi ficant level with control group(P<0.01), The difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC each group had a decrease, Etp A group was significant distinct with another two groups(P<0.01).(3) The effect on p H of intestinal microflora: During the study, from the 7th to 28 th day, the p H of S. cerevisiae group and Etp A group had a downtrend, at the 21 st day they two had a very significant level with control group(P<0.01), The difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC control group had a significant decrease(P<0.01),and had a very significant level with S. cerevisiae group and Etp A group(P<0.01), The difference between S. cerevisiae group and Etp A group had a significant distinct(P<0.05).2. Effects of Enterotoxigenic Escherichia coli adhesion Etp A engineering –Saccharomyces cerevisiae strain on mucosal immunity:(1) The effect on structure of intestinal mucosa:(1)From the 7th to the 28 th day, the villus length of S. cerevisiae group and Etp A group had a growth trend and reached to the max at the 28 th day, the villus length of jejunums had a very significant level with control group(P<0.01), the difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC the villus length of S. cerevisiae group and control group decreased significantly(P<0.01) and had a very significant level with Etp A group(P<0.01).(2)During the test the villus width of intestine in S. cerevisiae group and Etp A group had no distinct(P>0.05).(3)At the 28 th day, the crypt depth of jejunums in S. cerevisiae group and Etp A group had a significant level with control group(P<0.05), the difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC the crypt depth of each group increased significantly(P<0.01), Etp A group had a very significant level with control group and S. cerevisiae group(P<0.01).(4)At the 28 th day, the V/C of jejunums and ileums in S. cerevisiae group and Etp A group reached the max and they had a very significantly growth with control group(P<0.01), the difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC, the V/C of jejunums in control group increased significantly(P<0.01) and had a very significantly growth with S. cerevisiae group and Etp A group(P<0.01), the V/C of ileums in S. cerevisiae group had a significantly decrease with before and Etp A group(P<0.05).(2) The effect on SIg A in intestinal mucosa: From the 7th to the 28 t h day, the amount of SIg A in S. cerevisiae group and Etp A group had a growth trend and reached to the max at the 21 st day, they had a very significantly increase with control group(P<0.01), the difference between S. cerevisiae group and Etp A group were not distinct(P>0.05). After gavaged ETEC, each group increased significantly(P<0.01), Etp A group had a very significant level with control group and S. cerevisiae group(P<0.01).(3) The effect on significant cytokines in intestinal mucosal immunity: From the 7th to the 28 th day, the amount of IL-2, IL-4 and IFN-γ in S. cerevisiae group and Etp A group had a growth trend. Then the 14 th day, the amount of IL-2 of they two increased significantly and had a very significantly increase with control group(P<0.01). From the 7th day the amount of IL-4 and IFN-γ increased significantly and at the 21 st day and the 28 t h day, they had a very significantly increase with control group(P<0.01). After gavaged ETEC the amount of IL-2, IL-4 and IFN-γ in control group decreased significantly and all had a very significantly difference with Etp A group(P<0.01).Based on the results got these conclusions:1. Enterotoxigenic Escherichia coli adhesion Etp A-expressing engineered Saccharomyces cerevisiae strain had beneficial effects like Saccharomyces cerevisiae :it could promote the growth of the intestinal mucosa, enhance the absorption function and rich flora diversity, then increase the amount of significant flora while decrease the p H, and also could increase the amount of SIg A and significant CKs then enhance the intestinal mucosal immune function.2. From the 7th day, with the increase of the number of days the villus length and V/C gradually increase, at the 14 th day probiotics and important cytokines increased significantly then increased slowly, at the 21 st day the SIg A increased significantly then at the 28 th day reached max.3. Enterotoxigenic Escherichia coli adhesion Etp A-expressing engineered Saccharomyces cerevisiae strain had the immunogenic from Etp A: while the invasion of ETEC came to host, it could prevent the adhesion from ETEC, protect the integrity of the intestinal mucosa structure and maintain the stability of the intestinal microflora also improve intestinal mucosal immune function. |
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