Isolation, Identification And Treatment Of The Lytic Bacteriophage Of Escherichia Coli

Calf bacterial diarrhea, which is mainly harm to the calf within ten days especially the one to three days old, is one of the foremost reasons of the death of calf. It brings a great loss to the cattle farming. Enterotoxigenic Escherichia coli is one of the main pathogenic bacterias that cause calf bacterial dirrhoea. However,since the invention of antibiotics, they were consider as the most effect weapon to control bacterial infection. Because of this, abuse of antibiotics has become increasingly popular, and it leads to increasing resistance of pathogens.About phage therapy as a new method of treatment, related researchers have been more and more concern. In order to discuss the feasibility of the usage of phage treatment to the infection of Enterotoxigenic Escherichia coli, this study used Enterotoxigenic Escherichia coli O101 as the host bacteria, and one strong lytic bacteriophage was isolated from sweage samples and was characterized. This study lays the groundwork for the bacteriophage treatment for Escherichia coli O101 infection. The results are as follows:Thirty bateriophages of Escherichia coli O101 were separated and purified from ten sewage samples using double-agar plate method. Three bacteriophages of strong lysis were selected from the thirty bacteriophages, and named respectively: P3, P7 and P23; Concentration of the bacteriophages was all over 109pfu/mL. P3's Plaque diameter is of the largest, up to 6mm. The plaques of the bacteriophage P3 is clear and with clear edge, its bacteriophage titer was 5.5×109 pfu/mL. It was a stronglysis bacteriophage as the focus of the study.The biological characteristics of bacteriophage P3 were determined, and the result showed that: the optimum temperature of the bacteriophage infecting to Enterotoxigenic Escherichia coli O101 is 37℃; the optimum pH of is pH 7.0; P3 still remained high lysis activities at 60℃for 60min or in pH 4-11 for 2h; The bacteriophage was resistent to aether and chloroform; The MOI of P3 is 1; The latent period and rise period are 20min and 80min respectively. The average burst size is about 55.Virion buoyant density in CsCl of P3 is 1.32g/mL.Electronic microscope showed that the bacteriophage had icosahedral heads of diameter about 20 nm, no tail and no envelope. SDS-PAGE profiles indicated P3 contained two structural proteins at least, and the molecular weights of the two capsid proteins estimated at 49KDa and 23KDa.The nucletic acid of the bacteriophage was prepared for agrose eletro phoresie. The result showed that the genome of the P3 was about 15kb, and the DNA of bacteriophage P3 could be degradation by DNaseⅠ, and not by RnaseA and Mung Bean Nuclease. It explained that the nucletic acid of the bacteriophage was linear duplex DNA. Base on the Eighth Bulletin of International classify commission of virus, the bacteriophage may belongs to Tectivirus.Toxcity experiment was conducted to examine the security of P3. It showed that the bacteriophage was safe for mice. P3 were used in the therapy experiment of the infection of Escherichia coli O101 through oral inoculation. The therapy was given ahead of 3h, the same time with infection and delayed for 3h.The oral dose was about 109pfu/mL. Feces were collected after 6h and 12h, and weight,then colony counting to calculate the number of the bacteria per gram of feces. The results showed that the three cases could be significantly reduced the number of host bacteria in the feces. However, the effect of oral phage therapy was better when the therapy was given at the same time with infection or three hours after inowlation. The experiment lays a foundation for the phage therapy of ETEC infections in calves.