| Salmonella is widespread in the world,with more than 3,000 serotypes.It is one of the most important foodborne pathogens.It is a zoonotic disease that can contaminate all parts of the food supply chain and seriously threaten livestock and poultry farming and public health.Antibiotics have been the most effective weapon against human pathogens since they were invented.They have been widely used or even abused in the past few decades.The bacterial resistance caused by them has become increasingly serious.The emergence of multi-resistant strains and even"super bacteria"has become a major threat worldwide.In order to curb the emergence and development of drug resistance,people have been constantly looking for green and effective alternatives to antibiotics.Among them,probiotics such as Clostridium butyricum are prepared with wide sources,good effects,low production costs and high safety.By the attention.Existing research has proved that Clostridium butyricum can improve the performance of livestock and poultry and improve its physiological indexes,but the research on the role of Clostridium butyricum in antagonizing Salmonella enteritidis in broiler models is not comprehensive enough,and it is immune to Clostridium butyricum.Research on the mechanism of effects is not enough.Therefore,this study investigated the protective effects of Clostridium butyricum on Salmonella enteritidis-infected broilers and their immune effects.The main contents are as follows:1.Study on the effects of Clostridium butyricum against Salmonella enteritidisA 1-day-old broiler chicken(AA)was selected as the research object to explore the protective effect of Clostridium butyricum on Salmonella enteritidis-infected broiler chickens.AA broiler chickens totaled 240,and were randomly divided into 4 groups at the age of 1 day,with 60 in each group,including 3 replicates.During the entire breeding cycle,the blank control group continued to be fed the basic diet without any other treatment;the positive control group was continuously fed the basic diet,and Salmonella enteritidis was administered at 9 days of age to construct a pathological model(5×10~4 CFU/0.2 mL);Clostridium butyricum control group added Clostridium butyric acid(3×10~8 CFU/kg)to the basic diet and continued feeding;Clostridium butyricum experimental group added Clostridium butyric acid to the basic diet(3×10~8 CFU/kg),and Salmonella enteritidis(5×10~4 CFU/0.2mL)was administered at 9 days of age.The experimental results show that the weight ratios of the control group and the Clostridium butyricum control group are 1.60 and 1.46,the Salmonella group is 1.90,and the Clostridium butyricum experimental group is 1.73 at the age of 1 to 30 days.Compared with the control group,the weight-to-weight ratio of the Clostridium butyricum control group showed a downward trend,and the average daily gain(ADG)showed an upward trend.There were differences between the groups but not statistically significant(P>0.05);The weight ratio of Salmonella group increased,while the data of the weight ratio of the Clostridium butyricum treatment group was between that of the Salmonella group and the control group.Impact.From the results of tissue sections,both the negative control group and the Clostridium butyricum control group showed complete intestinal villous structure,and no obvious intestinal villous structure damage occurred;the Salmonella group had obvious intestinal villous rupture,and Ding The intestinal epithelium structure of the experimental group of Clostridium acid is relatively complete,indicating that Clostridium butyricum can protect the integrity of intestinal tissues.Compared with the control group,broiler chickens in the experimental group of Clostridium butyricum had increased HI antibody titers to Newcastle disease(ND)vaccine(P>0.05).The antibody titer level decreased,and the antibody titer of the C.butyricum treatment group was higher than that of the Salmonella group.This shows that Clostridium butyricum can promote the immune response after the body is immunized with Newcastle disease vaccine,thereby increasing the levels of ND and AI-H9 antibodies and their protective efficacy.There was no significant difference in serum IgA content between the treatment groups of 30-day-old broiler chickens,and IL-1βshowed a downward trend compared with the control group.-8 showed a downward trend,suggesting that Clostridium butyricum could reduce IL-8 in broiler serum.The IFN-γin Clostridium butyricum control group was lower than that in the blank control group,and the Clostridium butyricum experimental group was lower than the Salmonella group,indicating that Clostridium butyricum can reduce the serum IFN-γeffect in broilers.On the 30th day of the test,the intestinal floraα-diversity Shannon index of the dietary treatment groups was not significantly different(P>0.05),and the Simpson index of the Clostridium butyricum group was significantly lower than the control group(P<0.05),indicating that Clostridium acid can regulate the structure of intestinal flora and establish a stable and healthy intestinal flora.There was no significant difference in ACE index and Chao1 index between the bacterial flora richness index and dietary treatment group(P>0.05).The results of this study indicate that Clostridium butyricum can protect broiler chickens infected with Salmonella enteritidis,and has the potential to be one of the alternatives to antibiotics for antagonizing Salmonella enteritidis infection in livestock and poultry farming.2.Immune effect mechanism of Clostridium butyricumA 3-week-old C57 strain of mice was used as a research object to investigate the immune effects of Clostridium butyricum on healthy organisms.During the test period,36 healthy 3-week-old C57 mice were selected,and the mice were randomly divided into four groups of 9mice each in 3 cages.The grouping is as follows:each mouse in the blank control group was fed a normal diet and ordinary drinking water;each mouse in the sodium butyrate control group was fed a normal diet,and 0.1 g/mL sodium butyrate was added to the drinking water;In the Clostridium acid group,each mouse was fed with a common diet,and 1×10~7 CFU of Clostridium butyricum was added to each ml of drinking water;in the high-concentration Clostridium butyricum group,each mouse was fed with a common diet,in each ml 1×10~9CFU of Clostridium butyricum was added to the drinking water.After 3 weeks of breeding,the serum and cecum contents were collected to detect the contents of short-chain fatty acids and the diversity of intestinal microorganisms.Spleen and subcutaneous adipose tissue were collected to prepare single-cell suspensions and tested by flow cytometry.The results showed that the concentrations of acetic acid(P<0.05),propionic acid,butyric acid,and valeric acid(P<0.05)in the sodium butyrate group all had an upward trend,but there were large differences among individuals;The concentration was increased,and the trend of the low-concentration Clostridium butyricum group was more obvious.Flow cytometry results showed that the sodium butyrate in spleen lymphocytes and the ratio of CD4+/CD8+in the Clostridium butyricum treated group showed a downward trend(P>0.05),while the ratio of CD4+/CD8+in subcutaneous adipose tissue cells was in sodium butyrate It was up-regulated in the high-concentration Clostridium butyricum group,and there was no significant change in the low-concentration Clostridium butyricum group.The number of Tregs in the subcutaneous adipose tissue cells of the Clostridium butyricum experimental group increased,indicating that the feeding of Clostridium butyricum can promote Tregs accumulate in subcutaneous adipose tissue,and then regulate various physiological indicators of the body,which has implications for the regulation of body obesity and fatty inflammation by Clostridium butyricum.In this study,16S rRNA high-throughput sequencing technology was used to analyze the characteristics of microbial flora in the intestinal contents of mice.At the level of Class classification,feeding both Clostridium butyricum and sodium butyrate reduced the abundance of Bacteroides and increased the abundance of Microbacterium verrucosa.Clostridium butyricum also increased the abundance of Campylobacter and Desulfovibrio degree.At the Family level,both C.butyricum and sodium butyrate treatment reduced the abundance of S24-7,and C.butyricum increased the abundance of Helicobacter.Combined with Metastat analysis,it was found that in the relative abundance of certain specific bacteria,the sodium butyrate group and the low-concentration Clostridium butyricum group had basically the same trend as the blank control group.For example:The relative abundance of Bacteroides,Anaerotruncus,Prevotella,and Lactobacillus Lactobacillus was higher than that of the control group,and the relative abundance of Desulfovibrio was reduced.The above results indicate that after ingestion of sodium butyrate and Clostridium butyricum in drinking water,the intestinal flora composition of mice changed similarly,suggesting that butyric acid may be one of the effector molecules of Clostridium butyricum to exert immune effects.After annotating the KEGG function,observing the relative abundance of the KEGG second-class functional groups with differences between the groups,it was found that the C.butyricum group was more consistent with the blank control group,while the sodium butyrate group showed a different Abundance:The abundance of functional groups related to nucleic acid metabolism and carbohydrate metabolism decreases,and the abundance of functional groups related to metabolism of heteromorphic biomass and terpenes and polyketide metabolism increases.Combining the structure of the flora and the structure of functional annotations,it is speculated that Clostridium butyricum,as a living bacteria that can be colonized in the intestine,may interact with more bacteria and play a probiotic role through more pathways and pathways.In summary,Clostridium butyricum can protect broilers infected with Salmonella enteritidis,can promote the immune function of healthy mice,exert immune effects,and provide Clostridium butyricum to antagonize pathogenic bacteria and reduce the use of antibiotics in breeding.Important theoretical basis. |
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