Study On The Some Biological Properties And The Immunization Efficiency Of SLT-â…¡e Gene Mutant Strain Of Escherichia Coli Causing Edema Disease Of Swine

Edema disease of swine (ED) is mainly caused by Hemolytic Escherichia coli which lead to edema of the head and the 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. Edema disease is transmitted via the alimentary infection. According to the feature of mucosa infection, effective oral vaccine which can induce local and systematic immunity is of great significance for the prevention and control.In this study, some biological properties of SLT-â…¡e(Shiga-like toxinâ…¡variant), gene mutant strain of Escherichia coli O₁₃₉, and its immune efficiency as oral vaccine were investigated.The results of the growth curves of the mutant strain and the E.coli O₁₃₉ showed that the mutant strain grew well and had no significant difference with the E.coli O₁₃₉. The median lethal dose of the toxin from the mutant strain and the E.coli O₁₃₉, to Kunming mice and vero cell were determined, the LD50 of the toxin from E.coli O₁₃₉ to Kunming mice is 287.17μg/ml, the CD50 of the same one to vero cell is 0.547μg/ml, but the mutant strain toxin had no pathogenicity to Kumming mice and vero cell, this result showed that the toxicity of the mutant strain was lower than that of the E.coli O₁₃₉ obviously.The mutant strain was serial passage for 50th generative in LB broth without Nalidixic acid, and the SLT-â…¡e* gene of the 50th generative mutant strain were identified by PCR and restriction digestion, the gene sequencing results showed that, it was 100% homologous with the SLT-â…¡e* gene of the first generative mutant strain. The mice was live and had no untoward reaction after drenched 2×10⁹CFU mutant strain, it indicated that the strain was safe for mice. The detection result of the time that the mutant strain colonized in the mice's intestinal tract showed that the mutant strain can live at least 3 days in the mice's intestinal.Balb/c mice were immunized via oral with the mutant strain. The weight of mice, the IgG level in the serum, the sIgA level in fecal material and intestine mucus of the immunized mice were determined and detected by ELISA. The results showed that the oral immunization didn't effect the weight gain of mice. On the days 7, 21, 28, 35 and 42 after the first immunization, anti-O₁₃₉ and anti-SLT-â…¡e B antibody IgG were detected in the serum of immunized mice. On the days 7, 14, 21, 28, 35 and 42 after the first immunization, anti-F18ab antibody IgG were detected in the serum of immunized mice. On the days 35 after the first immunization, anti-O₁₃₉ antibody sIgA were detected in fecal material of immunized mice, anti-SLT-â…¡e B and anti-F18ab antibody sIgA were close to positive value. On the 14th day after the third immunization, anti-O₁₃₉, anti-SLT-â…¡e B and anti-F18ab antibody sIgA were detected in intestine mucus of immunized mice. The results showed that the gene mutant strain had a good immunogenicity and induced the mice to produce IgG and sIgA.The T lymphocyte proliferative respongse of immunized mice was also detected, and the immune protection rate of the immunized Kunming mice were examined. Useing protein SLT-â…¡e B and ConA as antigen stimulant, the stimulation index of splenic lymphocyte were detected, the result showed that the difference between oral immunization group and control group was significant (P<0.05), it indicated that the mutant strain induced proliferative respongse of the T lymphocyte. The results of immune protection experiment showed that the gene mutant strain can provide 75% (15/20) protection to the mice after challenged intraperitoneally with SLT-â…¡e.The all results indicated that the gene mutant strain can stimulate humoral immunity, mucosal immunity and cellular immunity response in mice, and it had a good protect effection to the mice, the mutant strain can be used as a candidate of oral virulence gene deletion vaccine to against the edema disease of swine.