Isolation, Identification And Preliminary Application Of Chicken Coli Phage

Avian colibacillosis is several quite different disorders,which is caused by pathogenic E.coli, and mainly including sepsis, airbag inflammation, granuloma, enteritis, peritonitis, salpingitis, synovitis, panophthalmitis, omphalitis diseases, etc. This disease can occur throughout the year, but winter and spring multiple, which caused large economic loses for the livestock farming. Antibiotics are used to prevent and control the disease in clinic, but the problems of antibiotic resistance, resistant strains increasing are becoming more serious with useing of antibiotics.Phage are the natural enemies of bacteria, which can crack the host bacteria with strong specificity, but not infect body or produce resistance. It's a new idea to treat avian colibacillosis with phage and it's also a direction of the livestock and poultry disease research.This study collected stool samples from a large chicken farm, and several strains of E. coli were isolated and identified from feces. Then continue to isolated phage from feces hosted with the E. coli, and measured the biological characteristics for one of the phage. The study built the model of avian colibacillosis with the E. coli, and carried out the phage therapy experiment.The isolation and identification of E.coli. The study put the samples in macconkey and EMB in turn using plate streaking method, and purified the single colony with EMB. After that, gram's method and biochemical reaction were used to identify the E.coli which were purified. The results are that 20 strains of E.coli were separated from feces, all strains were red or pink on macconkey and dark purple with green metallic luster on EMB, all strains were red and short by the gram stain, all strains were E.coli by biochemical reaction.The isolation and identification of E.coliphage. The study separated E.coliphage from feces by double agar plate with host 20 strains of the E.coli, purified these E.coliphage by liquid enrichment. Phage host cross infection experiment was carried out by the method of topical application. Then the morphology of the the phage was observed by transmission electron microscope after 2% phosphotungstic acid dye solution for negative dye. The results are that 6 strains of phage were separated, which are Bp1, Bp2, Bp3, Bp4, Bp5, Bp6, and their corresponding host are E1, E2, E3, E4, E5, E6, all phage can form a unified plaque on double agar plate, Bp1 and Bp3 can infect each other host bacteria, Bp5 and Bp6 have no apparent tail, their head show irregular polyhedron shape and 50 nm in diameter.The biological characteristic research of coliphage. The phage titer of Bp1, Bp2, Bp3, Bp4, Bp5 and Bp6 were measured with double agar plate method and the plate count method. At the same time, the temperature stability, p H stability,MOI and one-step growth curve of Bp5 were measured. The results are that phage titer of Bp1, Bp2, Bp3, Bp4, Bp5 and Bp6 were 2×10¹⁴, 1.65×10¹⁴, 1.9×10¹⁰, 4.5×10¹⁴, 2.4×10¹⁰ and 1.35×1011. Bp5 keep a high titer between 40 and 60 degree centigrade, while between 5 and 10 in PH, the best MOI was 1,the step growth curve showed that the incubation period was 20 min, cracking period was 50 min, burst size was 61.6.The construction of chicken colibaciuosis model. The study attacked chicken with E1, E2, E3, E4, E5 and E6 using oral, neck subcutaneous injections and intraperitoneal injection, to find the best strains for modeling and best way for attack test. The results are that the experimental chickens sickened with the symptoms of pericarditis,perihepatitis and air sacculitis which used E5 by intraperitoneal injection.The treatment trials of phage for chicken colibaciuosis. The therapy was given ahead of 6h and 3h, the same time with infection, delayed for 3h and 6h by oral and intraperitoneal injection Bp5. The results are that percentage of illness of ahead of 3h and 6h were 12.5% and 25% in oral phage therapy experiment, which had certain preventive and therapeutic effect compared with the 62.5% of the control group. All the percentage of illness of ahead of 6h, 3h and the same time with infection were 0 in intraperitoneal injection phage therapy experiment, which showed that all the phage had a very good prevention effect. Besides, the percentage of illness of delayed for 6h was 37.5%, which also had a certain therapeutic effect.