Study On The Interaction And Intervention Mechanism Between Mycoplasma Gallisepticum Infection And Mucosal Immunity Of Respiratory Tract Microbiota

Mycoplasma gallisepticum(MG)is a wall-less prokaryotic microorganism.Chronic respiratory disease caused by MG infection is one of the most common diseases in the poultry industry.MG infection can lead to the growth retardation of chicks,the reduction of egg production of laying hens and the weight loss of broilers,causing major economic losses to the poultry industry.The main strategy for the prevention and treatment of MG infection is antibacterial drugs.However,the continued use of antibacterial drugs has caused serious problems such as the growing levels bacterial resistance and drug residues.Therefore,it is the significant research direction of veterinary clinical research to seek an ideal antibacterial drug to replace the MG prevention strategy.Respiratory microbiota could enhance host respiratory immunity,and many studies have shown that the disturbance of respiratory microbiota is closely related to respiratory diseases.The mucosal immune system of the respiratory tract plays a crucial role in the recognition an d elimination of pathogens.The mechanism of Th1/Th2 immune balance is an important immunological basis for the body in the pathogenesis of disease.In this study,respiratory microbiota-related microbial regulation or immunomodulatory therapy was introduc ed into the prevention and treatment of chronic respiratory disease caused by MG infection.To explore the correlation between MG infection and respiratory microbiota disturbance and the mechanism of Lactobacillus salivarius remodeling the structure of respiratory microbiota to alleviate tracheal inflammation injury caused by MG infection.The aim of this study is to provide a basis for the prevention and treatment of MG infection and new microbial treatment options.(1)Correlation and mechanism between MG infection and respiratory microbiotaFirstly,the MG infection models with different infection duration were established.The results showed that MG infection could cause tracheal inflammation injury,lead to Th1/Th2immune imbalance,the reduction of tracheal antioxidant enzyme activity and CD4~+T/CD8~+T lymphocyte ratio in peripheral blood(p<0.01)and the disturbance of respiratory microbiota.In addition,MG infection caused pathological damage and oxidative stress in the lung,and lead to Th1/Th2 immune imbalance.MG infection also caused abnormal ultrastructural pathologies in HD11 cells.MG infection activated the JAK/STAT signaling pathway to induce the pro-inflammatory response in HD11 cells at 6 h post-infection and inhibited the activation of the JAK/STAT signaling pathway at 10 h post-infection.Disturbance of respiratory microbiota was involved in MG infection-induced tracheal inflammation injury.Compared with Con-T1d group,the obvious pathological changes and oxidative stress in the trachea,th e expression levels of Th1cytokines were significantly decreased,and the expression levels of SIg A and Th2 cytokines were obviously increased,and the JAK/STAT signaling pathway was inhibited in MG-T1d group.The expression levels of Th1 cytokines were significantly increased,the expression levels of SIg A and Th2 cytokines were obviously reduced,and the JAK/STAT signaling pathway was activated in MG-T7d group.These results showed that the disturbance of respiratory microbiota caused by MG infection induced Th1/Th2 immune imbalance,host immunosuppression,and destruction of the respiratory mucosal barrier.The above results indicated that the role of respiratory microbiota in MG infection-induced tracheal inflammation injury,which broadens new ideas fo r the prevention and treatment of MG infection.(2)The effect and mechanism of respiratory microbiota disturbance on MG infectionChickens were intranasally treated with antibiotics cocktail including ampicillin(1 g/L),vancomycin(0.5 g/L),metronidazole(1 g/L),and neomycin(1 g/L)twice a day to selectively deplete respiratory bacteria.It has been found that depletion of respiratory bacteria promoted MG infection,as reflected in the form of the increase of MG colonization,destruction of the respiratory mucosal barrier integrity,and severe tracheal damage compared to the control group.These results suggested that the intact respiratory tract microbiota contributed to the resistance of MG infection.In addition,on the basis of respiratory microbiota disturbance,chickens were intranasally treated with chicken-derived IL-4 and IL-12 recombinant proteins confirmed that respiratory microbiota disturbance promoted the MG colonization in the trachea by inducing Th1/Th2 immune imbalance in respiratory mucosa.However,the r IL-4 restored the anti-MG ability of the respiratory mucosa,suggesting that the respiratory microbiota may play an anti-MG role by regulating the Th1/Th2immune balance.MG infection induced the significant decrease in the abundance of Parabacteroides distasonis.In this experiment,it was found that Parabacteroides distasonis significantly reduced MG colonization,and improved the integrity of the respiratory mucosal barrier by increasing the expression of tight junction protein.The above results suggested that the disturbance of respiratory microbiota increased MG colonization in the trachea,and promoted tracheal inflammation injury by destroying the integrity of the respiratory mucosal barrier.(3)The study on the effect of Lactobacillus salivarius remodeling the structure of respiratory microbiota to alleviate MG-induced inflammatory injury in the tracheaThe previous study confirmed that intestinal-derived Lactobacillus salivarius as a feed additive which could significantly reduce inflammato ry injury in the lung caused by MG infection.However,the key mechanism of Lactobacillus salivarius on MG infection is unclear.The results showed that Lactobacillus salivarius significantly reduced MG colonization,relieved tracheal pathological damage and improved immunosuppression by MG infection.Lactobacillus salivarius played an anti-MG infection role by regulating the Th1/Th2 immune balance of respiratory mucosa,inhibiting the activation of JAK/STAT signaling pathway,and enhancing mucosal barrier function.In addition,Lactobacillus salivarius significantly improved the disturbance of respiratory microbiota caused by MG infection,increased the content of L-Iditol,L-Isoleucine and Dimethyl sulfone,and decreased the content of Pyridoxine,Indole a nd Pantothenol.The above results indicated the role of respiratory microbiota and its related metabolites in MG infection,and confirmed that Lactobacillus salivarius effectively inhibited MG-induced tracheal inflammation injury by remodeling the homeostasis of respiratory microbiota and its related metabolites.(4)Methylsulfonylmethane alleviated MG-induced inflammatory injury and its anti-inflammatory mechanismThe previous results of this experiment have confirmed that the content of methylsulfonylmethane(MSM)was significantly reduced by MG infection,and Lactobacillus salivarius restored the abnormal changes of methylsulfonylmethane content.However,the effect of MSM on MG infection is unclear.The results showed that MSM significantly alleviated MG-induced pathological damage in the trachea,reduced oxidative stress and inhibited the production of inflammatory cytokines by MG infectio n.MSM obviously improved the abnormal ultrastructural pathologies in HD11 cells,inhibited cell de ath and alleviated the inflammatory injury under MG infection.The specific mechanism was that MSM inhibited the activation of NF-κB/MAPK signaling pathway by downregulating the expression level of NF-κB signaling pathway-related proteins(TLR4,p-IκBα/IκBα,p-p65/p65,IL-1β,TNF-α)and MAPK signaling pathway-related proteins(p-JNK/JNK,p-ERK/ERK).The above results revealed the important role of MSM in defense against MG-induced inflammatory injury,and verified that it has an excellent anti-inflammatory effect.In conclusion,the inflammatory injury of respiratory tract and HD11 cells and the Th1/Th2immune imbalance caused by MG infection were closely related to the respiratory microbiota.Lactobacillus salivarius and methylsulfonylmethane could play an anti-MG infection role by regulating the Th1/Th2 immune balance of respiratory mucosa,inhibiting the activation of JAK/STAT and NF-κB/MAPK signaling pathways,and enhancing the mucosal barrier function,which provide a new strategy for the prevention and treatment of MG infection.