Isolation, Identification And Preliminary Application Of E.coli Bacteriophage

The chickling colibacillosis which prevails in the poultry at large is one of main bacterial diseases which affect the development of poultry product in the world. Bacteriophages were isolated using 35 strains of virulent E.coli as host cells and their biological properties were determined. The therapy for chickling colibacillosis with the phage isolate was also conducted.Antibiotic resistance of 4 E.coli strains was determined by disk diffusion susceptibility test. The pathogenicity and LD50 of E.coli strain was determined by animal test. The results showed that E. coli O₁₅ and O₂₄ had the strongest resistance to antibiotics. Their resistance rate was 70.6%. The E.coli strains were all very sensitive to Cefazolin and resistant to ampicillin, amoxicillin, kanamycin, tetracycline, nalidixic-acid, enrofloxacin, ciprofloxacin and neomycin. The E.coli O₂₄ had the strongest virulent and its LD₅₀ was 6.6×10⁷ cfu for 7 days chicken.Phages were isolated, identified and purified by double-layer agar plate method with 35 strain of E. coli as host cells. Bacteriophage enumeration was conducted by soft agar overlay procedures. The phage lysis scope was determined. The results showed that 5 phage strains were isolated from the dung of poultry. They were named Bp2 Bp3 Bp4 Bp5 and Bp6 respectively. The concentration of the phages were all over 10⁸pfu/mL. Bp4 had the broadest lysis scope and its lysis rate was about 34.3%. The lysis rate of Bp5 or Bp6 was about 8.6% and that of Bp2 or Bp3 was about 3.8%.Phage Bp6 was examined by electron microscope after the particles were amplified and purified. Phage thermostability, pH stability, optimal multiplicity of infection (MOD and one-step growth curve of phage were determined. The results indicated that BP6 belonged to Myoviridea family with an isometric head about 48nm in diameter and a tail about 100nm in length. Bp6 still remained high lisis activities after being incubated at 60℃ for 60min or in pH 4-9 for 2h. The MOI of Bp6 was 0.001. The latent period and rise period were 25min²7min and 15min, respectively. The average burst size was about 83.8.Toxicity experiment was conducted to examine the security of Bp6. Bp6 was used in the therapy experiment for the chickling colibacillosis. It showe that phage Bp6 solution was safe for chickens. Chickens were inoculated with a dose of 6×10⁸ pfu phages by thoracic air sacinoculation at 3 hours before infection and the death rate decreased from 90% to 20%, whilethe death rate of chicken decreased from 90% to 70% when they were inoculated with thesame dose by subcutaneous infection at 3 hours before infection. The results indicated thatthoracic air sac inoculation was the most perfect infection method to prevent chicklings fromcolibacillosis.