| ObjectiveThe purpose of this study was to isolated a strong lytic and wide spectrum of E.coli phage from hens feces,to analyse its biological characteristics.Chicken colibacillosis model was established and antibacterial experiments was be carried out by this phage.The changes of intestinal flora in chicks of different groups were analyzed by PCR-DGGE.Laying experiment and theory foundation for cure colon bacillus diseases by bacteriophage in future.MethodsAccording to the E.coli germline and phylogenetic relationship,the different groups and the molecular polymorphism of 26 chicken-derived Escherichia coli were identified and representative multi-plant Escherichia coli were selected as the host for isolating phage.The phages were isolated from the feces of the chicken in different areas using the host bacteria and then purified them.Select a high titer,strong lytic and wide spectrum of phage.The stability of the thermostability,p H stability,stability of ether and chloroform,optimal infection number(MOI)and one-step growth curve were measured.The chicken colibacillosis model was established and colibacillosis model pathogenic dose(absolute lethal dose)was determined.The 16 S r DNA V6 regions of chicken fecal flora from different groups(control group,Escherichia coli infection group,phage group)were separated by PCR-DGGE.The antibacterial effect of phage on chicken colibacillosis was evaluated.ResultsAccording to RAPD,phylogenetic group and E.coli serotype,14 strains of chicken-derived E.coli were identified as host bacteria.Nine phages were isolated using host bacteria from the chicken named SP1~SP9.After purification the titer of phage SP2 was 3.6×1010pfu/m L and the plaques were more than 2mm with a clear edge.The cleavage rate of SP2 was 46.1% by cross cleavage test,and the complete cleavage rate was 33.3%.Compared with the other 8 strains,the cleavage rate was the highest and the cleavage spectrum was the most extensive,so the SP2 was used in follow-up study.The MOI of the phage was 0.1,the latency of the infected host was 20 min,the lytic cycle was 70 min,the burst size was 36 and the activity was retained after 60 min at 70?,the titer of phage SP2 was the highest at p H 7,acidic environment on its impact is relatively large,In addition,the effect of chloroform is quite small.Electron microscopy showed the head of phage was icosahedral with tail structure.E.coli chicken colibacillosis was established by chicken-derived Escherichia coli(O78).The absolute lethal dose was determined as 3×109CFU.DGGE showed that the diversity of intestinal flora increased from 1 to 10 days.The intestinal flora has the most abundant content at 10 days.The DGGE atlas was changed after chicken infected with E.coli.The flora was maladjusted.The phage group showed that the individual bands returned to normal.In addition,it is find that some bacteria are always exist in each group unaffected by E.coli.Clustering analysis showed that the intestinal flora diversity began to recover after two days of phage effect.ConclusionsA strong lytic and wide spectrum of E.coli phage is isolated from chicken manure using chicken-derived Escherichia coli named SP2.Thermal stability is good,suitable for neutral or slightly alkaline environment growth and development,with a relatively long cleavage period and a large amount of cracking.Phage SP2 has a good antibacterial effect on chicken colibacillosis. |
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