| Enterotoxigenic Escherichia coli (ETEC) is the major pathogenic E coli caused diarrhea diseases in neonatal and post-weaning piglets. Five fimbrial antigens including K88, K99, 987P, F41and F18 have been recognized to be associated with E coli diarrhea in pigs. A polymerse chain reaction (PCR) technology was developed to detect different fimbrial gene of ETEC. The prevalence of these five fimbrial antigens and their variants in some pig farms in Hubei province were investigated by PCR, and K88 and F18 was confirmed to be the predominant variants associated with post-weaning diarrhea. Recombinant vectors were constructed by inserting the faeG gene and faeG-linker-fedF fusion gene into prokaryotic expression vector pET-28a (+). The FaeG protein and FaeG-FedF fusion protein were efficiently expressed in E. coil Immunity and ability of induced egg yolk antibody of these recombinant proteins were also analyzed. The active efficiency of egg yolk antibodies against ETEC infection was evaluated by in vivo and in vitro experiments. The results were as follows:1. PCR technology was developed to detect fimbrial antigens, detection of reference stains by PCR with special primers showed target bands. To determine the most common fimbrial antigens of ETEC in piglets with diarrhea, two investigations were carried out on intensive pig farms in Hubei province, central China. In 2002-2003, 227 fecal samples from neonatal and post-weaning piglets with diarrhea were tested for the presence of the fimbrial antigen K88 and K99 of ETEC by PCR. Twenty-three (10.1%) of 227 fecal samples were found to contain fimbrial antigen K88, which was identified as K88ac variant; and 13 (5.7%) samples containing K99. In 2004, another 179 fecal samples from diarrheic piglets, 1 day to 6 weeks of age, were tested for prevalence of fimbrial antigen K88, K99, 987P, F41 and F18. Sixty (33.52%) of the 179 samples carried at least one of the ETEC fimbrial antigens. K88 antigen was detected in 60% of sixty positive samples. In the 36 samples known to carry fimbrial antigen K88, 32 (88.90%) contained K88ad; and 4 (11.10%) contained K88ac; none of them carried K88ab. Fimbrial antigen F18 was found in 16(26.67%) samples, 5 (8.33%) of which was F18ab, the other 11(18.34%) was F18ac. In addition, the detection ratio of fimbrial antigens K99, 987P and F41 were 3.33%, 18.33% and 5.00%, respectively. These results indicate that PCR is a quick, sensitive and specific methodology for the detection and epidemiological investigation of ETEC; K88 and F18 are the most common fimbrial antigen of ETEC associated with diarrhea in post-weaning piglets in Hubei province.2. Fusion gene encoding faeG and fedF of ETEC fimbrial antigens was linked with the (Gly4Ser)3 linker by PCR amplification, faeG gene and the fusion gene faeG-linker-fedF were cloned into pET-28a(+) , and the constructed recombinant plasmid was then introduced into E coli DH5 a and BL21(DE3). After induction by IPTG they were well expressed, and the percentage of recombinant protein in complete bacterial protein was about 30%. SDS-PAGE of the bacteria lysed by ultrasonic showed that both recombinant FaeG and fusion protein FaeG-FedF were insoluble.3. Response surface analysis (RSA) was applied to optimize the main parameters on expression of recombinant K88 subunit protein FaeG in Escherichia coli. The results showed that these three factors and their interactions all had significant effects on expression of recombinant (P＜0.01). Further simulated optimization revealed that under the conditions of induction starting time 2.5h after culture, IPTG concentration 0.8mmol/L and inducing time 5h, the percentage of recombinant FaeG protein in complete bacterial protein were 35.4%, improved by 28.7% compared with the production(27.5%) obtained under the conditions optimized by one-variable-at-a-time. It is concluded that RSA is an effective method to optimize the parameters such as induction starting time, IPTG concentration and inducing time in improving the expression of recombinant protein in Escherichia coli.4. By use of immunoblotting test with corresponding monoclonal and polyclonal antibodies, the immunological specificity and reactivity of rFaeG and FaeG-FedF have been verified. The results showed that rFaeG could be recognized by rabbit anti-K88ac serum antibody and FaeG-FedF could be recognized by both rabbit anti-K88ac serum antibody and anti- F18 "a" antigen factor serum antibody. It suggested that recombinant protein rFaeG and confusion protein FaeG-FedF have homology antigen site with natural fimbrial protein. Immunizations of rFaeG and FaeG-FedF can excite immune response in hens, and induce production of egg yolk antibodies. Titer of egg-yolk antibodies rose quickly in a week after the second immunization, average titer can be above 1:210, and maintained for long time. Recombinant protein rFaeG and confusion protein FaeG-FedF can be as antigens used in production of egg yolk antibodies.5. Eggs obtained from chickens immunized with K88ac purified fimbrial antigen, rFaeG and FaeG-FedF, were spray dried to prepare Anti-K88ac Spray dried egg powder(SDEP), Anti-rFaeG SDEP and Anti-FaeG-FedF SDEP, respectively. Adhesion assay in vitro was developed to determine availability of spray dried hyperimmun egg powder inhibiting homologous or heterologous bacteria adhesion to piglet small intestine epithelial cells. The results showed that the average numbers of adherent bacterial cells per enteroepithelial cell for control without antibodies was 7.6, Anti-K88ac SDEP, Anti-rFaeG SDEP and Anti-FaeG-FedF SDEP could significantly inhibite the adhesion of ETEC K88ac to piglet epithelial cell(p＜0.01); there were no significant effect on the adhesion of ETEC K88ac between three hyperimmue SDEP. Anti-FaeG-FedF SDEP also could significantly inhibit the adhesion of F18ab (P＜0.01) and F18ac (P＜0.01). Hyperimmun SDEP induced by fusion protein FaeG-FedF with two different fimbrial antigens, has been shown to be across-protective against heterologous bacteria.6. The protective effects of egg yolk antibodies obtained from hens immunized with recombinant protein were evaluated in three field trials, and effects on growth performance of post-weaning pigs fed diet with hyperimmun SDEP were also studied. One hundred and forty pigs weaned at 21±2d of age were assigned to five dietary treatments with ten pigs per pen; each group has three pens except control group with two pens. The protective effect of egg yolk antibodies obtained from hens immunized with rFaeG recombinant protein were evaluated in experimentâ… . The data showed that during 0-28d postweaning, piglet fed diet with Anti-K88ac SDEP and Anti-rFaeG SDEP, had significantly lower morbility and minimal diarrhea (P＜0.05), whereas there were no significant differences between protective effect of Anti-K88ac SDEP and Anti-rFaeG SDEP. Anti-rFaeG inclusion body SDEP has not shown distinct protective effect against diarrhea. There was no significant difference in average daily gain (ADG), average daily feed intake (ADFI) and feed: gain ratio (F/G) among all the groups in this experiment. Anti-K88ac SDEP and Anti-rFaeG SDEP had similar effect on controlling piglet diarrhea; it indicated that renatured rFaeG could be used as antigen in production of specific egg yolk antibodies.One hundred and twenty pigs weaned at 21±3d of age were randomly assigned to three dietary treatments, each treatment group has eight pens with five pigs per pen in experimentâ…¡. Diet supplemented with 3% spray dried plasma protein (SDPP) was positive control diet. Effects on protecting piglets from diarrhea and growth performance were evaluated, as Anti-rFaeG SDEP displacing partial SDPP in diet. Optimal level of Anti-rFaeG SDEP in diet was also be determined in experimentâ…¡. Diarrheic rate in treatment with Anti-rFaeG SDEP was significantly lower than SDPP (P＜0.05). Diarrheic index of Anti-rFaeG SDEP treatment was also significantly lower than SDPP (P＜0.01). There was no significant difference in ADG between Anti-rFaeG SDEP group and SDPP group in this experiment, piglets fed diet with Anti-rFaeG SDEP had a high growth velocity as well as SDPP, and average body weight was above 15kg at 7-week-old. Furthermore, pigs fed 1% or 1.5% Anti-rFaeG SDEP diet had no significant difference in diarrhea rate, ADG, ADFI and F/G. The results suggested that Anti-rFaeG SDEP displacing partial SDPP in diet, could more effectively decreased diarrhea of piglets, and could result in similar growth performance, and 1% was therefore considered to be the economically optimal level.Three hundred and sixty pigs weaned 21±3d of age were randomly assigned to five dietary treatments, each treatment group has six pens with twelve pigs per pen in experimentâ…¢. Effect of amino acids balance diet with supplement of hyperimmun SDEP on incidence of diarrhea and performance of post-weaning piglets were evaluated in this experiment. Diet with 3% SDPP was positive control, while diet with 1.5% SDEP was negative control. 1-2 weeks post weaning, incidence of piglet diarrhea in group fed Anti-K88ac SDEP, Anti-rFaeG SDEP and Anti-FaeG-FedF SDEP significantly lower than group fed SDEP without any specific antibodies(P＜0.05), it was consistent with SDPP. There was significant differences among all the treatments (P＜0.05). Both SDPP and hyperimmune SDEP could relive diarrhea disease. There was no significant difference in ADG between Anti-rFaeG SDEP group and SDPP group in this experiment. Piglets fed diet with Anti-rFaeG SDEP had a high growth velocity as well as SDPP, and average body weight was above 15kg at 7-week-old. Feed: gain ratio in SDPP, Anti-K88 SDEP and Anti-FaeG-FedF SDEP were significantly lower than SDEP group(P＜0.05). No significant difference in ADFI was observed among all the groups (P＞0.05). Supplement of Anti-K88 SDEP and Anti-FaeG-FedF SDEP in amino acids balance diet can improve growth performance and increase gain: feed ratio of post-weaning piglets.In conclusion, recombinant ETEC fimbrial protein could be used as antigen in production of specific egg yolk antibodies, and the early-weaned piglets that fed diets supplemented with these specific egg yolk antibodies could be protected against ETEC infection. |
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