| Diarrhea in piglets is one of the most challenging problems in swine farming worldwide, which causes huge economic loss annually. Enterotoxigenic Escherichia coli (ETEC) is one of the most prevalent pathogens responsible for piglet diarrhea. The pathogenicity of ETEC depends on the co-effect of fimbriae and enterotoxins. At present, vaccination is the most effective way for controlling ETEC diarrhea in piglets. However, conventional vaccines showed some limititions, especially safety issue, limited application area and limited efficacy.To improve the protective spectrum and efficacy of vaccine, in this study, the two major virulent factors of ETEC, fimbriae and enterotoxin, were targeted to block the two arms account for diarrhea. Genetic recombination technology was employed to fuse three enterotoxin genes to improve the immunogenicity and avoid the toxicity of these enterotoxins.At the very beginning, diarrhea condition in pig farms was surveyed in Desheng, Dalian. E.coli strains were isolated from diarrheal feces by MacConkey Agar. Through PCR, all candidate E.coli strains were identified with STb and F41. Most of these strains were LTB positive.Firstly, the protocol for isolation and purification of recombinant enterotoxins SLSwas optimized with a novel dilution-dialysis-ultrafiltration-centrifugation method, through which the purity of SLS was increased from11.2%to nearly100%. Meanwhile, the recovery of SLS was improved. Then, in an attempt to obtain high-expressing of fimbriae from ETEC strains, Minca broth was shown with more efficiency than TSB or LB. Finally, several novel vaccines were developed, including the vaccines composed of recombinant SLS and fimbriae, recombinant SLS alone, fimbriae alone, and inactivated whole cell of ETEC. Then after these vaccines were proved safe in mice, they were injected to sows to passively immunize the piglets in a field study. Piglets born and bred by sow immunized with multivalent vaccine showed a2.94%diarrhea rate versus29.4%in piglets of blank control group. Meanwhile, the score of synthetic diarrhea index of immunization group was far lower than the blank control’s(p<0.01). Indirect ELISA results of serum and breast milk from sowsshowed that the titre of specific antibody in serum continued to rise after the first and second immunization and the titres of anti-F4, anti-F5and anti-SLS IgG were1:10000,1:8000and1:16000, respectively, and the titre of anti-SLS IgG in breast milk was1:12000three days after delivery. Together, the results of this study sugguested vaccines developed in this study had great potential in the prevention of diarrhea in piglets. |
