| Salmonella infection is one of the most important public health problems worldwide.According to the US Centers for Disease Control and Prevention,more than one million cases of Salmonella infections occur annually in the United States alone.Salmonella Enteritidis(S.Enteritidis)is an important zoonosis that can use its virulence factors to destroy the intestinal barrier and cause severe inflammation in the gut,leading to enteritis in animals and humans,as well as systemic infections.As the use of antibiotics makes the prevention and control of S.Enteritidis more difficult.Probiotics,as a kind of antibiotic substitute,have been widely concerned for their effects of inhibiting intestinal inflammatory reaction,enhancing immunity and promoting intestinal flora homeostasis,common probiotics including Clostridium butyricum(C.butyricum),Lactobacillus,Bacillus subtilis and so on.Our previous studies showed that C.butyricum can inhibit the colonization of S.Enteritidis in chickens and increase the diversity of intestinal flora.However,the mechanism of C.butyricum inhibiting the colonization of S.Enteritidis in chickens remains unclear.Therefore,it is significant to explore the mechanism of C.butyricum inhibiting the colonization of S.Enteritidis in chickens.In this study,we first investigated the direct effect of C.butyricum on S.Enteritidis.In this study,different CFU C.butyricum and 10~6CFU/m L S.Enteritidis were co-cultured 1:1 to draw the growth curve of S.Enteritidis,the results showed that the inhibitory effect of C.butyricum on S.Enteritidis gradually increased with the increase of the concentration of C.butyricum.C.butyricum and S.Enteritidis with the same CFU were co-cultured 1:1 to draw the growth curve of S.Enteritidis.In order to further explore the mechanism of C.butyricum inhibiting S.Enteritidis,the sterile culture supernatant of C.butyricum was prepared to investigate the effect of different concentrations of the sterile culture supernatant on the proliferation of S.Enteritidis.The results showed that the number of S.Enteritidis decreased with the increase of the concentration of C.butyricum culture supernatant.The culture supernatant of C.butyricum(30%of the total system)was co-cultured with S.Enteritidis(10~6 CFU/m L)and the growth curve of S.Enteritidis was drawn,compared with the control group,the supernatant of C.butyricum aseptic culture inhibited the logarithmic growth of S.enteritidis,and the inhibitory effect was significant.Furthermore,C.butyricum sterile culture supernatants at a volume ratio of 30%inhibited S.enteritidis biofilm formation compared with controls,with an anti-biofilm formation effect.The results showed that the p H value of the culture supernatant was between4.7 and 4.9,suggesting that the p H value might be one of the factors of inhibiting the proliferation of S.Enteritidis.After adjusting the p H value of the sterile culture supernatant of C.butyricum to the p H value of the RCM liquid medium,the supernatant was co-cultured with S.Enteritidis(10~6 CFU/m L),while the control group was S.Enteritidis(10~6 CFU/m L)without any treatment,the growth curve of S.Enteritidis was drawn,and the inhibitory effect of culture supernatant of C.butyricum(p H=RCM)on the growth of S.Enteritidis and the effect of anti-biofilm disappeared compared with the control group.Therefore,C.butyricum can inhibit the proliferation of S.Enteritidis in vitro by regulating p H.The p H of cecal contents in chicks was measured to be between 7.3 and 7.5,so C.butyricum may inhibit the colonization of S.Enteritidis in chicks through other mechanisms.To further explore the reasons of C butyricum inhibiting the colonization of salmonella enteritidis in chickens through animal experiments,32 1-day-old SPF chickens were selected as study objects,after three-day-old,they were randomly divided into 4 groups,8 chicks in each group,and were divided into control group(CON).After the beginning of the experiment,they were given 0.5 m L normal saline once a day for three consecutive days.The C.butyricum group(CB)was given 0.5 m L of C.butyricum spore solution(4×10~8 CFU/m L)once a day for three days,while the C.butyricum group(CBS)was given 0.5 m L of C.butyricum spore solution(4×10~8 CFU/m L)once a day for three days,for three consecutive days,10~9 CFU/m L of S.Enteritidis per mouse was administered on the 4th day of the trial,and 0.5 m L of normal saline was given to the S.Enteritidis group(S)once a day for three consecutive days after the start of the trial,on the 4th day of the experiment,S.Enteritidis was challenged with 10~9 CFU/m L.The results showed that there was no significant difference in the average body weight of each group on the 6th day after S.Enteritidis infection.Compared with the control group,the liver index of the challenge group increased,but the difference was not significant(P>0.05)compared with the control group,the spleen index of the challenge group increased,but the difference was not significant(P>0.05).On the 3rd and 6th day after challenge,the S.Enteritidis in the liver and spleen were detected,in C.butyricum treated group,the number of S.Enteritidis in liver and spleen decreased significantly(P<0.05),but there was no significant difference in the number of S.Enteritidis on the 6th day after infection(P>0.05).The results of pathological section showed that the liver cells in the control group and the C.butyricum group were closely arranged,the white pulp and red pulp were mixed in the spleen tissue,and the structure of caecum villi was intact.At Day 3 and Day 6 of S.Enteritidis infection in the liver,there was a small amount of lymphocyte infiltration in the C.butyricum treated group and lymphocyte infiltration and neutrophil infiltration in the S.Enteritidis challenged group.On the3rd and 6th days after splenic infection,the red pulp of the C.butyricum treated group was slightly hyperemia with a small amount of eosinophil infiltration,while the red pulp of the virus challenged group was slightly hyperemia with a small amount of eosinophil infiltration.On the3rd and 6th days after cecal infection,goblet cells increased and secreted abundantly in the C.butyricum treated group,while the lamina propria in the challenged group was hyperemia,hemorrhage and infiltration of lymphocyte cells.The results of detection of intestinal oxygen content by immunohistochemistry on the 6th day after challenge showed that the control group showed anoxic status,but the anoxic status was eliminated after challenge with S.Enteritidis,treatment with C.butyricum restores intestinal anoxia.Analysis of intestinal microflora revealed that the relative abundance of obligate anaerobe in the gut at Day 6 after challenge was significantly increased in the C.butyricum treatment group compared with the S.Enteritidis challenge group.According to the relative abundances of the genera,Faecalis and Lactobacillus were the two most abundant bacteria on the third day after infection.The relative abundances of Ruminococcus increased significantly on the sixth day after infection in the challenge group.On the 6th day after challenge,the distribution of microbialβdiversity in the cecal contents of the four groups was not uniform on the PCOA map,the use of C.butyricum and S.Enteritidis had a greater effect on the cecum microbes of chicks than that of the control group.In terms of inflammatory factors,the relative expression level of IL-6 in the challenge group was significantly higher than that in the control group on the 6th day after infection(P<0.01).The relative expression level of PPAR-γin the C.butyricum group was significantly higher than that in the C.butyricum group(P<0.05)compared with C.butyricum treated group.The relative expression level of NF-κB in C.butyricum group was increased,but the difference was not significant(P>0.05).There was no difference in the relative expression of TNF-αamong the groups.The results indicated that C.butyricum could inhibit the proliferation of S.Enteritidis in vitro by regulating p H.C.butyricum can alleviate the pathological damage caused by S.Enteritidis,restore the reduction of anaerobic bacteria in the intestinal tract caused by S.Enteritidis infection,ensure the anaerobic environment in the intestinal tract,and inhibit the colonization of S.Enteritidis in chickens. |
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