The Role And Mechanism Of Gut Microbiota During Leptopsira Infection

Leptospirosis is an acute,natural,zoonotic infectious disease caused by pathogenic Leptospira interrogans.More than 1 million people worldwide are infected with Leptospira,resulting in about 60,000 deaths every year.The infection rate of leptospirosis in livestock(pigs,horses,cattle,sheep,etc.)was also above 10%.Leptospirosis not only greatly harms the development of animal husbandry,but also threatens the global public health security.At present,the main prevention and control measures of leptospirosis rely on vaccines and antibiotics.However,due to the numerous serotypes of Leptospira,more than 300 serotypes have been discovered,and the operating system of leptospiral virulence is relatively difficult,and the research progress is slow,leading to the shortcomings of leptospiral vaccine,such as poor crossprotection effect and short protection period.With the promulgation of the “Antibiotic prohibition order”,the use of antibiotics in the breeding industry will be further restricted,leading to the prevention and control of bacterial infectious diseases form more pressure.Therefore,it is urgent to develop new and effective alternative prevention and control products to replace antibiotics that cure the disease.Gut microbiota plays an important role in maintaining body health and determining the occurrence,development and outcome of diseases.However,the role of microbiota in leptospirosis remains unclear.Therefore,from the perspective of microbiota,this thesis explore the role and mechanism of microbiota in leptospirosis based on the sensitive model(hamster)and resistant model(mouse),and then explore the function of Lactobacillus animalis and its metabolite D-lactate as alternative product in resisting leptospirosis.The results showed that different ways of infection did not affect the proliferation of Leptospira in the gut,and the time of death caused by intraperitoneal injection was shorter.Therefore,intraperitoneal injection was used in subsequent experiments in the hamster model of leptospirosis.Fecal 16 S r RNA sequencing results showed that Leptospira infection could change the composition of microbiota of hamsters,and the proportion of Lactobacillus in the dying group was significantly reduced.Leptospira infection lead to colonic bleeding,extensive inflammatory cell infiltration,and could lead to significantly increased intestinal permeability and decreased expression of related tight junction genes.In addition,Leptospira infection resulted in a large number of bacterial translocation,but no bacteria were isolated by blood smear.In order to further clarify the role of microbiota during infection,microbiota was depleted by antibiotic cocktail for infection experiment.The results showed that the depletion of microbiota(the cocktail of ampicillin,metronidazole,neomycin and vancomycin)did not affect the proliferation of Leptospira in organs at early stage after infection,but significantly prolonged the survival time.Detection of LPS in blood showed that the LPS level in blood of microbiota-depleted hamsters was significantly lower than that of microbiota-untreated hamsters at the late stage of infection,suggesting that disturbed microbiota may aggravate the process of leptospirosis by producing excessive LPS.Leptospira proliferated briefly in the gut and were cleared at the late stage of infection without increasing intestinal permeability in the mouse model of leptospirosis.However,Leptospira infection could also change the composition of microbiota in mice,leading to a significant increase in the proportion of Lactobacillus in the late stage of infection.The microbiota of mice was helpful to resist Leptospira infection by microbiota depletion and fecal transplantation experiments.In order to further explore the protective mechanism of microbiota in leptospirosis,bone marrow derived macrophages and peritoneal resident macrophages were isolated for infection experiment.These results suggested that microbiota contributed to the phagocytosis and inflammatory response of bone marrow derived macrophages,but had no effect on the phagocytosis and inflammatory response of peritoneal resident macrophages during infection.Then,in vivo experiments were performed to detect the changes of microenvironment in the abdominal cavity during infection.Results showed that the microbiota depletion had no impact on the inflammatory environment,but the proportion and numbers of free macrophages were increased(macrophage disappearance reaction),further using inhibitors experiments showed that the microbiota helped maintain the macrophage disappearance reaction during infection,promoting the clearence of Leptospira.Finally,in vivo macrophage clearance and replenishment experiments showed that the effect of macrophages against Leptospira infection depended on the homeostasis of microbiota.Finally,L.animalis was identified as the differential strain by species level analysis of microbiota in the process of infection in mice.The results showed that supplementing with L.animalis could help mice resist Leptospira infection.In order to further explore the potential mechanism of the effect of L.animalis,it was found that the intestinal D-lactate content was significantly increased after the end of the gavage cycle.The results showed that the intestinal physiological concentration of D-lactate could effectively inhibit the growth of Leptospira,and the pathogenicity of Leptospira was significantly reduced with the increased concentration of D-lactate,but the morphology of Leptospira was not affected.RNA-seq results showed that D-lactate treatment could enhance the anti-infection ability of macrophages.D-lactate treatment significantly enhanced the phagocytosis and intracellular bactericidal ability of macrophages,but did not affect the inflammatory response after infection with Leptospira.Finally,animal experiments showed that supplementation with D-lactate could significantly reduce the leptospiral load in mice after infection.In summary,this study indicated that microbiota played an important role in the leptospirosis of hamster and mouse.In hamsters,Leptospira infection resulted in microbiota dysbiosis,accompanying with excessive LPS being absorbed into the blood and exacerbating disease progression.In mice,microbiota plays an anti-leptosipral role by regulating macrophage function and reducing leptospiral colonization in organs.Supplementation with L.animalis and D-lactate could significantly improve the antileptospirosis ability of mice,suggesting that supplementation with probiotics or prebiotics could reduce the occurrence of leptospirosis in clinical practice.