Dynamic Changes In Lung Microbiota Of Broilers In Response To Aging And Ammonia Stress

The microbiome begins to colonize the lung at early animal development and it changes regularly with the development of animals,most of the existing studies focused on the changes in the mammal’s lung microbiota and link them to disease.In large-scale broiler breeding,most viruses invade broilers through their respiratory tract and some vaccines in broilers are administered through the nasal route,homeostasis of lung microbiota is particularly important for broiler health,however,there is a lack of research on the changes of broiler lung microbiome.Broiler breeding environments are complex and it subject to various external stimuli.Ammonia gas is one of external stimuli,and the existing international permissible level of NH3 in poultry houses is 25 ppm.However,few studies have reported on the changes of the lung microbiota and lung tissue when broilers exposure to the low concentrations of external ammonia.In this study,16S r RNA gene sequence,real-time PCR,HE tissues staining and other technologies were used to study the dynamic changes of white feather（Arbor Acres）broilers lung microbiome in different development stage,and by constructing the ammonia stimulation model,we further explored the broiler lung microbiota and lung tissue changes when exposure the ammonia with critical concentration.Trial 1 Age-dependent changes in the lung microbiota of broilers.In this experiment,100 1-day-old unvaccinated Arbor Acres broilers were randomly divided into 5 groups（3D,7D,14D,21D,and 35D）and all the birds were raised normally.Broilers were euthanized by cervical dislocation at 3 days（3D）,7 days（7D）,14 days（14D）,21days（21D）and 35 days（35D）,and lung lavage fluid was collected for 16S r RNA sequencing.The 16S r RNA sequencing analysis revealed that（1）Theαdiversity increased with age,and it tend to mature at 21 days.Moreover,the alpha diversity of 35-day-old was significantly higher than that of 3-day-old（P=0.0020）and 7-day-old（P=0.0040）;（2）Theβdiversity showed that there were some differences in the composition of broiler lung microbiome at different ages.The composition at 7-day-old and 14-day-old were similar,the 21-day-old and 35-day-old were relatively similar,and the bacterial community structure gradually stabilized after the 21-day-old.At the genus level,it was found that the relative abundance of Lactobacillus was lower at the age of 3 days,and it remained high in another four groups,while the relative abundance of Escherichia coli Shigella was high at the age of 3 days,and remained lower level at four age stage thereafter.（3）The microbiome phenotypes showed that the relative abundance of Gram-negative bacteria in broiler lung was higher at 3 days of age,lower at 7 and 14 days of age,and higher at 21 and 35 days of age.The trend of Gram-positive bacteria showed opposite trend.At the same time,it was also found that the relative abundance of aerobic bacteria gradually increased with age,while the anaerobic bacteria showed the opposite trend.Furthermore,the relative abundance of potentially pathogenic bacteria and stress-resistant bacteria were both higher at 3 days of age and remained low in the other four groups.（4）Correlation network diagrams showed the interaction status of the microbiota for each age group,and it was found that there were some differences in the main microbiome of the 5 groups.（5）Prediction of microbiota function showed changes in broiler lung microbiota function with age.Among them,at 21 days of age,pathways related to Transport and Catabolism,Cellular Processes and signaling,Glycan Biosynthesis and Metabolism were significantly enriched in broiler lung microbial functions,at 35 days of age,pathways related to Transport and Catabolism,Xenobiotics Biodegradation and Metabolism,Metabolism of Terpenoids and Polyketones,and lipid metabolism were significantly enriched.Trial 2 Stimulation of critical concentration of ammonia causes broiler lung damage but did not disrupt the lung microbiomeIn this experiment,40 1-day-old unvaccinated Arbor Acres broilers were selected and raised till 14-day-old,16 broilers with similar size and weight were selected and divided into two groups.All the two groups of birds were raised up to 21-day-old,and at the age of 22 days,the broilers in the NH3 group were stimulated with 25±2 ppm of ammonia.After 7 days of stimulation,lung tissue and lung lavage fluid were collected.By RT-q PCR,H&E staining and16S r RNA gene sequencing,it was found that（1）Compared with the CON group,there were inflammatory cell infiltration and local bleeding symptoms in the broiler lung tissue of NH₃group.（2）Compared with the CON group,the expression levels of inflammatory factors（IL-10 and IL-1β）were significantly increased.Short-term stimulation of critical concentration of ammonia leads to inflammation in the broiler lungs.（3）Theαdiversity andβdiversity of broiler lung microbiota was not changed by the critical concentration of ammonia.（4）At phylum and genus level,the composition of the broiler lung microbiome was not changed by the critical concentration of ammonia.These results strongly imply the short-term stimulation of critical concentration of ammonia is not enough to destroy pulmonary microbial homeostasis.In summary,this study analyzed the lung lavage fluid of 5 groups of broilers by 16S r RNA sequencing.The results showed that the stability and composition of broiler lung microbiome changed regularly with the development of broilers,and the structure of microbiota became stable gradually after 21 days of age.Using a critical concentration of ammonia to simulate a harsh environment and stimulate the broilers,it was found that the lungs of broilers were inflamed,but the stability of the flora was not destroyed.Conclusion:This study revealed the age-dependent changes and stress tolerance of broiler lung microbiota from the microbiota composition,microbiota classification and microbiota function.This study provides theoretical basis and practical guidance for the prevention and control of broiler diseases in large-scale breeding.