The Mechanism Study Of Intestinal Dysbiosis Induces The Inflammation In The Respiratory And Intestine Tracts Of Broiler Under Ammonia Exposure

Ammonia(NH₃)affects the respiratory health,however,the role and way of intestinal microbiota in respiratory tract and intestinal health caused by ammonia exposure are unclear.In order to prove the role of intestinal microbiota dysbiosis in the respiratory tract and intestinal inflammatory injury under inhaled ammonia exposure,this study is divided into the following three parts:Experiment 1:Effects of different concentrations of ammonia exposure on respiratory and intestinal inflammatory cytokines and microbiota of broilers288 male AA broilers were randomly divided into 4 groups with 6 replicates in each group.12broilers in each replicate were exposed to 0,15,25 and 35 ppm ammonia respectively for 21 days.The results showed that compared with the control group,the increase of ammonia concentration significantly increased cytokines IL-1?,IL-6 and IL-10(P<0.05).The above-mentioned cytokines were positively correlated in trachea and ileum(P<0.05).With the increase of ammonia concentration,the number of harmful bacteria in respiratory tract and intestine were increased significantly,and the number of beneficial bacteria were reduced.The change trend of some key bacteria in respiratory tract and intestine was similar with the increase of ammonia concentration.The results showed that the inflammation and microbiota of respiratory tract and intestine were crosstalk under different concentrations of ammonia.Experiment 2:Study on the relationship between intestinal microbiota and inflammatory injury under ammonia exposureOne hundred and forty-four male broilers were randomly divided into two groups with 6 replicates in each group and 12 broilers in each replicate.They were exposed to 35 ppm ammonia in control group(0ppm)and treatment group respectively for 21 days.The study found that,ammonia exposure caused intestinal injury,inflammatory cell infiltration and so on.Then,we also observed the decrease of muc2,claudin-1,IL-6 and IL-10,accompanied by the significant increase of TLR4,My D88,NF-?B,TNF-?,IL-1?and caspase 3.Ammonia exposure increased the diversity of intestinal microbiota,changed the structure of intestinal microbiota,and changed the abundance of intestinal microbiota,including Firmicutes,Proteobacteria and Bacteroides at the phylum level,Streptococcus,Escherichia Shigella,Faecalibacterium,[Ruminococcus]?torques?group,Ruminococcaceae?UCG-014,unclassified?f?Lachnospiraceae,Rothia,unclassified?f?Ruminococcaceae at the genus level.Correlation analysis showed that there was a significant positive correlation between the altered microorganism and TLR4 and TNF-?.It was also found that ammonia exposure destroyed the respiratory barrier,decreased the expression of claudin,and increased the expression of MUC5AC,TLR4,My D88,NF-?B,TNF-?,IL-1?,IL-6,IL-10,caspase 3.Correlation analysis showed that the intestinal microbiota was positively correlated with TLR4,My D88,TNF-?and IL-1?in the trachea.The results indicate that TLR4/TNF-?signaling pathway maybe an important regulatory mechanism of intestinal microbiota induced the inflammation in the respiratory tract and intestinal tract.Experiment 3:Intestinal microbiota induced inflammatory injury of the intestine and respiratory tract through TLR4/TNF-? signaling pathway under ammonia exposureIn order to determine whether the intestinal microbiota dysbiosis is involved in the intestinal injury caused by ammonia,we conducted two experiments:antibiotic elimination of intestinal microbiota and bacterial transplantation.1)From 3 weeks before ammonia exposure,broilers were fed with antibiotic mixture(100 mg/g streptomycin,100 mg/g vancomycin,100 mg/g metronidazole,100 mg/g amoxicillin)until sacrifice.2)The ileal contents of broilers exposed to ammonia were suspended in 15 ml PBS,centrifuged,and the supernatant was obtained,which was immediately given to healthy broilers(1 ml for each broiler).It was found that transferring intestinal microbiota of ammonia exposed broilers into healthy broilers could cause intestinal injury and increase TLR4 and TNF-?concentrations of recipient broilers.In addition,the elimination of intestinal microbiota by antibiotics attenuated the intestinal injury induced by ammonia and reduced the production of TLR4 and TNF-?.Moreover,through the test of significant difference of the respiratory tract microbiota of the control group and the ammonia exposure group,the microbiota transplantation group and PBS group,the microbiota transplantation group and the ammonia exposure group,PBS and control group,the ammonia exposure group and the antibiotic treatment group,[Ruminococcus]?torques?group is an important microbiota bridge between intestinal microbiota dysbiosis and respiratory tract injury caused by ammonia exposure.In addition,the concentration of TLR4 and TNF-?in the intestine of broiler chickens in the microbiota transplantation were increased.In addition,the depletion of intestinal microbiota by antibiotics attenuated the respiratory tract injury caused by ammonia and reduced the production of TLR4 and TNF-?.In conclusion,TLR4/TNF-?signaling pathway is an important regulatory mechanism of inflammation injury in the respiratory and intestine tract mediated by intestinal microbiota dysbiosis under ammonia exposure.[Ruminococcus]?torques?group is an important bacterial communication pathway between intestinal tract and respiratory tract under ammonia exposure.