Characteristics Of Distribution And Adhesion Of Porcine Intestinal Coliphages And Protective Effect Of Mucosal Adhering Coliphages On Gut

Bacteria and their bacteriophages(phages)are the main members of the gut microbe.As the natural enemies of bacteria,the species and quantity of phages affect the gut microflora and animal physiology.Studies have shown that phages can adhere to epithelial cells and protect cells against bacteria.As the first line of immune defense in the body,the mucosal barrier plays an important role in maintaining the health of the animal.Therefore,the adhesion characteristics of intestinal phages to the intestinal epithelium and its biological barrier effect on intestinal mucosa remain to be explored.This study aims at addressing the serious problem of diarrhea caused by pathogenic Escherichia coli(E.coli)in piglet production by studing coliphages,which have a "predator-prey" relationship with intestinal E.coli.In this study,the distribution of porcine intestinal coliphages was explored,and their biological characteristics,adhesion characteristics to intestinal epithelium,and prevention effect of E.coli infection were studied.This study can increase our understanding of the ecological effects of intestinal coliphages and provide a theoretical basis for the application of phages in the prevention and treatment of E.coli infection.1.Distribution,isolation,identification and biological characteristics of porcine intestinal coliphagesIn this study,90 strains of E.coli were used to explore the phage distribution in the intestinal mucosa and digesta of healthy pigs from three farms.Subsequently,coliphages were isolated from the samples,and the biological characteristics including cleavage spectrum determination,electron microscope observation,genome sequencing,and bacteriostatic efficacy were studied.The results showed that the coliphage diversity of intestinal digesta was higher than that of the intestinal mucosa,and there were 'core phages'with high detection rate and specific phages only in digesta or mucosa in each farm.A total of 75 coliphages were isolated,among which 7 phages could lyse Salmonella or Citrobacter while infecting E.coli and 8 phages could infect pathogenic E.coli.Of those 12 phages capable of lysing enterotoxigenic E.coli(ETEC)but not producing progeny by infecting ETEC,7 phages could significantly inhibit the growth of ETEC at high multiplicity of infection.The genome of phage S164 isolated from farm ? shared quite high homology with phage L42 isolated from farm ?(99.98%),however,the efficiency of plating of these two phages were different.The phage S192 isolated from farm ? and phage L122 isolated from farm ? were absolutely identical.This study investigated the distribution of coliphages of pig gut and confirmed the presence of bacteriophages that can lyse pathogenic bacteria in the gut,which provided a new perspective for preventive measures for piglet diarrhea.2.Characteristics of phage adhesion to the intestinal epitheliumTo understand the characteristics of coliphages adhesion to the intestinal epithelium,studies on IPEC,Caco-2,and MDCK cell lines were carried on in vitro with several phages isolated from different sources,and phage S143₂ and phage W143 were selected to explore the adhesion characteristics in mice.The results in vitro showed that the number of phages adhering to mucus-producing cells was significantly higher than that of non-mucus-producing cells.The adhesion abilities of phages were different,but they did not correlate with the source of phage isolation.Phage adhesion to intestinal epithelium was dose-dependent and loosely connected to cells.The results in vivo showed that the concentrations of S143₂ and W143 in the mucosa were higher than those of digesta in mice,and S143₂ has better adhesion ability than W143.3.Protective effect of phage adhesion to the intestinal epithelium in advance against infection of EPEC143 on gutThe purpose of this study was to investigate the protective effect of phage adhesion to intestinal epithelium on intestinal mucosa against pathogenic E.coli.First,phages S143₂,S144 2 and W143 infecting EPEC143 were selected to conduct in vitro studies on the IPEC cell line.The results in vitro showed that all phages adhering to IPEC in advance could significantly reduce the number of bacteria in cell culture medium and on the cells.Interestingly,the bacteriostatic effects of phages on cells were weaker than those in the LB medium.Therefore,we further compared the bacteriostatic efficacy of phages under different culture conditions,and the results showed that 5%CO2 did not affect the bacteriostatic efficacy of phages in LB medium,whereas the bacteriostatic efficacies of phages were significantly reduced in DMEM/F12 medium under 5%CO2.Subsequently,S143₂ and W143 were selected for the mice experiment.To evaluate the effect of phages on the prevention of E.coli infection in mice,the body weight of the mice was determined and EPEC143 in fecal sample was quantified.The results showed that the administration of phage S143₂ and W143 could alleviate EPEC143-induced weight loss and reduce the amount of fecal EPEC143 in mice,and S143₂ showed the batter protective effect than W143.