| Chicken colibacillosis is one of the most common bacterial diseases in the process of breeding and production,especially enteritis chicken colibacillosis,which damages the intestinal mucosa and causes diarrhea in diseased chickens.With the emergence of drug-resistant bacteria,drug residues,public health safety and other problems,bacteriophages with strong specificity and not easy to produce resistance have been paid more and more attention.In this paper,Escherichia coli bacteriophage was isolated and identified from sewage by drop method and double plate method,and its biological characteristics,bacteriostatic effect in milk and chicken were analyzed,and finally its effect in the treatment of chicken colibacillosis was explored.The results were as follows:Isolation and identification of E.coli bacteriophage: Using 25 strains of Escherichia coli as host bacteria,5 strains of Escherichia coli bacteriophages were isolated from sewage.Host spectrum detection showed that HC-39 could lyse 9strains of Escherichia coli,HC-33 and HC-34 could lyse 7 strains of Escherichia coli,HC-26 could lyse 6 strains of Escherichia coli,HC-25 could only lyse its host bacteria,and the plaque produced was bright and large in diameter,and the titer was 5×1010PFU/m L.HC-25 was selected for the experiment.HC-25 is a tailed phage order and short-tailed phage family,and the best infection complex is 0.01.the latent period is30 min,the lytic phase is 60 min,and the cleavage amount is 105 PFU/cell.The activity is stable in the range of p H 5.0~11.0,and the activity is high at 45℃.The nucleic acid type of HC-25 is DNA,the genomic length 70 042 bp,the GC content is42.14%,encoding 110 ORFs is perforin and ORF17 is lysozyme,which is the main protein of lytic bacteria.GO functional annotation analysis of HC-25 showed that 44 genes were annotated to 1 cell component,12 molecular functions and 8 biological processes respectively.The results of genome alignment showed that HC-25 had the highest homology with Escherichia coli phage IME267,and the genome sequence had been submitted to Gen Bank with accession number OQ555404.Bacteriostatic effect of Escherichia coli bacteriophage in milk and chicken:Milk at 4℃,18℃ and 37℃ was treated with Escherichia coli and bacteriophage for 0,1,3,5,8 h.In milk at 4℃ and 18℃,the number of Escherichia coli decreased with the extension of bacteriophage action time,and the bacteriostatic effect depended on the concentration of bacteriophage,while in milk at 37℃ for 1 and 3 h,the number of bacteria decreased significantly,and the number of bacteria increased at 3 h,5 h and 8h,but it was significantly lower than that of the control group.At the same time,chicken at 4℃ and 18℃ were treated with 0,1,2,4,8,16,24 h.It was found that the number of Escherichia coli decreased significantly after phage treatment at 4℃ and18℃ for 1,2 and 4 h.The number of Escherichia coli began to increase at 8,16 and24 h,and the increase at 4℃ was slightly lower than that at 18℃,but significantly lower than that of the control group.Efficacy of Escherichia coli phage in the treatment of colibacillosis in chickens: After intraperitoneal injection of Escherichia coli into 7-day-old chicks,it was found that there were obvious clinical symptoms of colibacillosis,including bowed head and eyes,anal paste and poor diet.After autopsy,it was found that the intestinal tract of diseased chickens had obvious bleeding and swelling;histopathological examination showed that the villus height of cecum and duodenum decreased significantly,the muscle layer thickened significantly,and the levels of IL-1β and IL-6 in blood increased significantly.Next,bacteriophages were given for prevention and treatment before and after E.coli injection.The results showed that compared with the control group,after administration of phage and florfenicol,the mental state and appetite of diseased chickens were recovered,the intestinal bleeding and swelling of diseased chickens were not obvious,the villus height of cecum and duodenum was significantly increased,the muscle layer was significantly thinner,and the levels of IL-1β and IL-6 in blood decreased significantly.The therapeutic effect of bacteriophage 3 hours after Escherichia coli injection was significantly better than that of florfenicol group.In summary,5 bacteriophages were isolated from 25 host bacteria.The host spectrum of bacteriophage HC-25 is narrow,and the plaque produced is bright and large,which is tailed phage order,short-tailed bacteriophage family,alkali-resistant;the main protein of lytic bacteria encoded by phage HC-25 genome is perforin and lysozyme;it has obvious bacteriostatic effect in milk and chicken,and has a good effect in the treatment of chicken colibacillosis. |
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