| Salmonella can adhere to intestinal epithelium,invade the mucosal immune system and release endotoxin,resulting in diarrhea in piglets and increasing the mortality rate of piglets which cause huge losses to the pig industry,and the negative effects of antibiotic treatment are getting worse.As virus that infects bacteria,phage is one of the most promising green substitutes of antibiotics.Studies have shown that phages can adhere to epithelial cells and protect cells against bacteria,however the protective effect of gut phages on intestinal mucosa is still unclear.Therefore,in order to addresses the problem of piglet diarrhea caused by Salmonella,we isolated and identified Salmonella phages from pig intestines,and studied the characteristics of phage adhesion to intestinal epithelium and the protective effect of phages adhesion on the intestinal epithelium.This study provides new insight for the development of preventive measures for piglet diarrhea and provides a research basis for phage to become a new antibiotic alternative.This paper mainly carried out the following work:1.Isolation and Characterization of Salmonella choleraesuls phagesIn this study,using S.choleraesuls 13122 as indicator bacteria,three phages were isolated from 40 pig intestinal chyme and mucosa samples from two farms,and were named Salmonella phage L6jm,S19cd and Lv5cm.Morphology of phages were observed and genome were sequenced,and phages' nd biological characteristics such as host range,efficiency of plating(EOP),pH and thermal stability,optimal multiplicity of infection(MOI),one-step growth curve and bacteriostatic efficacy were determined.The results showed that Salmonella phage L6jm could infect E.coli 244 and lyse 9 E.coli strains including one enterotoxigenic E.coli(ETEC)strain.S19cd could infect E.coli 44 and E.coli 172,and lyse 7 E.coli strains in addition.Lv5cm could lyse 2 E.coli strains but could not infect them.The optimal MOI of three phages against S.choleraesuls 13122 were 10-2,10-6 and 10-2 respectively.All the three phages had strong tolerance to the environment with maintaining activity under pH 3-11 and temperature lower than 50?.All the three phages have significant(P<0.05)bacteriostatic effects on S.choleraesuls 13122 in liquid LB medium,and L6jm could inhibit the growth of ETEC 104 at MOI of 100.2.The characteristics of phage adhesion to intestinal epithelium and its bacteriostasis in vitroTo explore the adhesion characteristics and bacteriostatic effect of phage on intestinal epithelium,IPEC-1,Caco-2 and MDCK cell lines were selected for experiments in vitro.Compared with MDCK cells that did not produce mucus,all three phages were more inclined to adhere to mucous intestinal epithelial(IPEC-1 and Caco-2)cells.Adhesion ability were different among the three phages,while the adhesion numbers of L6jm and S19cd on the three cell lines were significantly(P<0.05)higher than Lv5cm.Phages adhering to intestinal epithelial cells were in a loosely connected way and dose-dependent.The protective effects of phages pre-adhered to IPEC-1 cells against the S.choleraesuls 13312 were further determined.The results showed that the pre-adhesion of phage S19cd and Lv5cm to cells can significantly(P<0.05)reduce the number of bacteria in the cell culture medium and those adhered on the cells,and the bacteriostatic effect of S19cd was better than Lv5cm,while L6jm had no bacteriostatic effect.In order to investigate whether nutritional conditions affect the bacteriostatic effect of phage L6jm,the bacteriostatic effect of phages in the presence or absence of CO2 and LB or DMEM/F12 medium were determined.It was found that these different nutritional conditions had little effect on the bacteriostatic effect of S19cd and Lv5cm,but DMEM/F12 medium made L6jm lose its bacteriostatic ability,suggesting that nutritional conditions may affect the interaction between phages and bacteria.Meanwhile,during the process of phage bacteriostasis,the 5 mutant bacteria resistant to phage were isolated and identified,one of them named ST 12 had significantly(P<0.05)lower growth capacity than S.choleraesuis 13312.3.The characteristics of phage adhesion to intestinal epithelium and its protective effect in vivoTo further explore the characteristics of phage adhesion to intestinal epithelium and its bacteriostatic protective effect in vivo,6-week-old Balb/c mice were chosen to take experimentation.The results showed that both Salmonella phages S19cd and Lv5cm were excreted in large amounts through feces after 8 hours of intragastric administration,but could still be detected in small intestine mucosa and chyme samples,and the quantities of phage on the mucosa were significantly(P<0.05)higher than those in chyme,suggesting that phages could adhere to intestinal epithelium.Compared the adhesion ability of two phages by "mucosa phage count/chyme phage count",there was no significantly(P>0.05)difference between the two phages.To evaluate the protection of administration of phage on mice against S.choleraesuls 13312,the body weight of the mice was determined and the quantities of S.choleraesuls 13312 in fecal samples were quantified.The results showed that administration of S19cd in advance could significantly(P<0.05)alleviate the weight loss caused by S.choleraesuls 13312. |
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