Isolation,Identification,Biological Characteristics And Preliminary Application Of Bacteriophages Bp16 From Chicken Escherichia Coli

Avian colibacillosis is an infectious disease caused by pathogenic Escherichia coli,which is one of the important causes of morbidity and mortality of poultry,causing huge economic losses to poultry industry.At present,it has become an inevitable trend to limit and prohibit the use of antibiotics in aquaculture.Phage therapy is expected to become a new method to replace antibiotics.In this study,several E.coli phages were isolated and purified from the sewage of a chicken farm in Chengdu,Sichuan Province,and one E.coli phage with wide cleavage spectrum was screened.The biological characteristics,whole genome bioinformatics analysis and preliminary control test of the phage were carried out,which provided the basis for the clinical application of E.coli phage.1.Phage isolation and identification,biological characteristics and genome-wide bioinformatics analysisIn this study,55 strains of E.coli from poultry were used as host bacteria,and a wide cleavage spectrum E.coli phage named Bp16 was isolated and purified from chicken farm wastewater by double agar plate method.The phage could form round,transparent and neat edge plaque on double agar plate.After negative staining with2% phosphotungstic acid,its morphology was observed by transmission electron microscope The results of transmission electron microscope showed that the head of phage Bp16 was icosahedral structure,with tail and tail filament.The length of the phage was about 220 nm.According to the morphological structure,the phage Bp16 was a member of the order urophagoidea and myococcidae.The lysis efficiency of Bp16 was 12.73% by drop method,and the titer of phage lysate was 6.7×10¹⁰ PFU /m L by double agar plate method and plate counting method.The biological characteristics of Bp16,such as stability to temperature and p H,optimal complex number of infection(MOI),and one-step growth curve,were determined.The results showed that the phage could react at 37?⁶0? for 30 min or 1 h The results showed that the titer of phage remained unchanged,and the titer could still reach above 1×10⁴ PFU/m L at 80? for 1 h.The titer remained unchanged between p H 5and 10.The optimal MOI was 1.According to the one-step growth curve,the incubation period was 20 min,the outbreak period was 50 min,and the cleavage amount was 73% The results of bioinformatics analysis showed that the nucleic acid type of the phage was double stranded DNA,and the total genome length was 170004 There were two kinds of t RNA,which transported arginine(ARG)and methionine(MET)respectively.140 ORFs were predicted.The results of genetic evolution analysis showed that the phage belonged to the order of tailphages,myococcidae,tevenvirinae subfamily and mosigvirus genus.The above results showed that Bp16 had the characteristics of thermal stability,acid and alkali resistance and strong cleavage ability,which laid the foundation for subsequent phage therapy experiments.2.Evaluation of therapeutic effect of E.coli bacteriophageIn this study,1.5×10⁸ PFU of phage suspension was injected intraperitoneally 6h,3 h before infection,at the same time,3 h and 6 h after infection to verify the prevention and treatment effect of the phage on E.coli disease,and to study the best dosage of therapeutic agent.The results showed that the median lethal dose of the strain was 1.5 × 10⁸ CFU;in the treatment test,the protection rate of chickens inoculated with bacteriophage suspension 3 hours and 6 hours before E.coli infection reached 100%,while the protection rates of chickens inoculated with bacteriophage suspension at the same time,3 hours and 6 hours after E.coli infection were 80%,80% and 70% respectively,and the protection rate of antibiotic treatment group was70%,indicating that the bacteriophage has a certain preventive and therapeutic effect on E.coli infection The best therapeutic dose was 1.5×10⁹ PFU.The above results showed that phage Bp16 could prevent and cure E.coli,it provides a good experimental material for the treatment of the disease.