Study On The Effect Of Phage Composition Against Intestinal Pathogen Infection In Rabbits

Enteropathogenic Escherichia coli（EPEC）is one of the main pathogens causing diarrhea in rabbits.Traditional antibiotic treatment could lead to many problems,such as drug resistance and chemical drug residues,etc.Searching for new antimicrobial agents is in urgent need.Bacteriophage is a kind of virus that can parasitize bacteria,using its own specificity to infect bacteria,causing the infected bacteria lyse to death.Bacteriophage therapy,which only acts against specific bacteria,and has no side effect on normal flora and cells,has the potential application prospects as an alternative to antibiotics.The purpose of this study was to isolate and identify rabbit E.coli phage.Its biological characteristics and genomic characteristics were studied.Besides,the prevention and control effect of rabbit E.coli were also evaluated,so as to lay a foundation for the prevention and control of rabbit E.coli using phages.In this study,one strain of E.coli was isolated and identified from a rabbit farm in Zhejiang Province,which was named as ZJE1.ZJE1 was sensitive to cephalosporins and tetracyclines.Five bacteriophages,named as ZRP1,ZRP2,ZRP3,ZRP4 and ZRP5,were later isolated from the host bacteria and their biological characteristics were studied.Their morphological characteristics were observed by transmission electron microscope.The bacteriophages were composed of hexagonal head,contractible tail sheath and tail tube.All the five strains belonged to Myococcyphage family.The host spectrum,optimal infection complex number,p H stability,thermal stability and one-step growth curve of phage were determined by double-layer plate method.The results showed that ZRP1,ZRP2,ZRP3,ZRP4 and ZRP5 all showed good activity in p H5 to p H7 under 55°C with high host specificity.The incubation period was 11-19 minutes.The in vitro anti-E.coli activity of the five strains was measured by lytic method.The results showed that among the five,four strains showed good lytic activity.ZRP5 was the best with a titer of 3.68×10¹¹PFU/m L.The whole genomes of 5 phages were sequenced by Illumina Hi Seq technology.The results showed all the five were virulent phages with double stranded DNA,belonging to Caulyphage order,Myocaulyphage family.ZRP1 belonged to Wifcevirus order,with a 6820 bp genome length and 46.16%GC content.ZRP2 and ZRP3 were Vequintavirus with genome length of 137511 bp and 136351 bp,respectively,and GC content of 43.61%and 43.70%,respectively.Phage ZRP4 and ZRP5 belonged to Tevenvirinae subfamily and Krischvirus genus,with genome length of 167091 bp and166580 bp,respectively,and GC content of 40.40%and 40.25%,respectively.Further comparative study was conducted on two T4-like phages,ZRP4 and ZRP5.Both the two were composed of head structural proteins,DNA packaging,caudal tube and caudal sheath structural proteins,basal plate structural proteins,long tail fibrous structural proteins and regulatory enzyme protein modules.The phylogenetic tree was constructed by using the conserved capsid protein gp23 gene of phage.The results showed that ZRP4 and KFS-EC belonged to the same clade,and ZRP5 and E26belonged to the same clade.When the phylogenetic tree of T4 phage was constructed using the distal tail filar protein gp37,the results showed that ZRP4 and RB49belonged to the same clade,and ZRP5 and E26 belonged to the same clade.There were 249 genes shared by ZRP4 and ZRP5 strains,with 23 genes specific to ZRP4and 18 genes specific to ZRP5.In vitro bacteriostatic experiments were conducted to select the combination ratio of ZRP1,ZRP2,ZRP3,ZRP4 and ZRP5,which was named as ZRP1-5.To compare the anti-ZJE1 effects of ZRP1-5,35-day-old New Zealand rabbits were divided into 4groups,namely group A（blank control group）,B（challenge group）,C（oral administration of ZRP5）and D（oral administration of ZRP1-5）.Rabbits in group B,C and D were orally challenged with 50 m L of E.coli ZJE1（4×10⁸ CFU/m L）and in the meantime administrated of ZRP5 or ZRP1-5.The results showed that the number of white blood cells,neutrophils and serum endotoxins in rabbits infected with E.coli ZJE1 showed an upward trend.The weight gain of rabbits in group C and D was significantly higher than that in group B（P<0.05）.Meanwhile,the number of white blood cells and neutrophils as well as the level of endotoxin in serum of group C and D were significantly lower than those of group B（P<0.05）.The results of the ileal section showed that ZJE1 infection caused intestinal epithelial villi damage in group B,while it remained intact in group C and D.In summary,ZRP5 and ZRP1-5 isolated in this study can resist E.coli both in vivo and in vitro,and the antibacterial effect of ZRP1-5 is better than that of ZRP5.In conclusion,this study confirmed the biological characteristics and genetic background of the phages,which provided a research basis data support for the treatment of rabbit enteropathogenic E.coli with bacteriophage.It also provided a potential application prospects in the treatment of rabbit colibacillosis.