Influence Of Intestinal Microorganisms On Mice Infected With Bovine Pasteurella Multocida Type A

Pasteurella multocida(Pm)is a gram-negative opportunistic pathogen that can be divided into A,B,D,E and F serotypes.Serotype A P.multocida(Pm A)mainly causes bovine pneumonia in cattle,and can cause severe respiratory syndrome when co-infected with other pathogens,which brings huge economic losses to the cattle industry.At present,the pathogenic mechanism of bovine-derived P.multocida(bovine Pm A)is unclear.In recent years,many studies have shown that gut microbes have a very important influence on the occurrence and development of diseases.The previous research of our laboratory found that within a certain dose range,after artificial mice infected with bovine Pm A,some mice died of severe illness,and some mice showed mild illness and survived(other pathogens also have similar phenomenon),but its specific mechanism is unknown.This study focused on the influence of intestinal microbes on the infection of P.multocida in mice.The specific experimental results are as follows:1.Establishment of a model for challenge of Pm CQ6Ten SPF female Kunming mice were randomly selected and injected with2.65×10⁷CFU of Pm CQ6 intraperitoneally to obtain a relatively stable survival rate of about 50%.At the same time,observe the clinical manifestations,bacterial colonization of lungs and pathological damage of lungs in mice,the results were found.After infection,the weight of mice generally decreased in the first 2-4 days,and then the weight of mice with severe disease continued to drop until death(severe death group,DG),while the weight of mice with mild disease recovered after 4-5 days,gradually approaching that of the control group,and finally returned to a healthy state(mild disease survival group,SG).Pm CQ6 colonization in the lungs of different mice also showed significant differences.DG mice had an average lung colonization of7.4×10⁷CFU/g,while SG mice had an average lung colonization of 6.5×10⁶CFU/g.The results of HE staining of mouse lung tissues showed that Pm CQ6 infection caused tissue structural lesions and inflammatory lesions on mouse lung tissues.DG mice had obvious epithelial cell shedding,tissue congestion and inflammatory exudation;Furthermore,ELISA was used to detect the expression of inflammatory factors in the lung tissue of Pm CQ6 infected mice.It was concluded that the expression of some inflammatory factors in the lung tissue of Pm CQ6 infected mice,such as IL-6,IL-12,IL-17,IL-1?,IFN-?,TNF-?increased,and the expression in DG group was extremely significantly higher than that in SG mice.The above results indicate that the experimental modeling is successful.2.Effect of intestinal microorganisms on mice infected with Pm CQ62.1 Intestinal flora clearance testIn order to understand whether intestinal microbes play a role in Pm CQ6 infected mice,SPF mice(n=6)were randomly selected,and broad-spectrum antibiotics(ampicillin,neomycin,metronidazole,and vancomycin)were added to drinking water for treatment 7 day,followed by intraperitoneal injection of Pm CQ6 two days later to observe the death of the mice.The results showed that 7 days after adding antibiotics to drinking water,the number of fecal microorganisms in mice dropped sharply(from an average of 10¹⁰ CFU/g to an average of less than 10³CFU/g),indicating that the intestinal microbes were successfully depleted.The mortality of mice infected with Pm CQ6 increased significantly from less than 20%to 60%.2.2 Fecal transplantation testThrough fecal transplantation tests,the role of intestinal microorganisms in mice infected with bovine Pm A was further determined.Thirty SPF mice were selected and randomly divided into experimental group(n=20)and control group(n=10).The mice were infected with 2.65×10⁷CFU Pm CQ6,and the feces of mice(DG group and SG group)were collected 32 hours after infection,while collecting the feces of mice(non-infected)in the control group(NC group).The feces were filtered with a 0.1um cell strainer to collect bacteria,and 100?L of normal saline suspension containing 1×10⁸CFU of bacteria(DG group,SG group and NC group)and 100?L of normal saline were added to the antibiotic pretreated mice by gavage(10 mice per group).One day later,these groups of mice were infected with Pm CQ6(2.65×10⁷CFU)comparing the mortality.The results showed that all mice with DG feces died within 4 days,the mice with normal saline survived 20%.The death time and number of mice with NC feces were consistent with the model,and they died after 30h,with a survival rate of 50%.Mice with SG feces were infected with Pm CQ6 and the death time was delayed and the survival rate reached 80%.The above results indicate that the intestinal microorganisms play an important role in the disease process of mice infected with bovine Pm A.3.Analysis of sequencing results of mouse gut microbiome and the effect ofLactobacillus on host infection with Pm CQ6After 32 hours of infection with 2.65×10⁷CFU of Pm CQ6,the intestinal contents of the infected mice(n=28)and injected with sterile normal saline mice(n=10)were collected for Microbiome and Metabolome sequencing and analysis.At the same time,the observation group(n=30)was designed to undergo the same challenge treatment,and the mouse mortality was calculated for 12 days of continuous observation.The results showed that the survival rate of mice was 60%,which was consistent with the previous modeling results,indicating the stability of the challenge model and the credibility of subsequent experiments.3.1 Analysis of Microbiology Sequencing ResultsThe quality assessment of the microbiome sequencing results shows that the CG content is between 51.22%-54.92%and the Q30 score(the percentage of bases with a quality value greater than or equal to 30 to the total number of bases)is above 95%.Therefore the sequencing results are stable and reliable.Statistical analysis of the original sequencing data found that after the mice were infected with Pm CQ6,at the phylum level,the control group was mainly composed of Firmicutes and Bacteroides,which accounted for more than 90%of the total,of which Firmicutes accounted for about 70%.Actinomycota and Proteobacteria account for less than 10%of the total.The experimental group accounted for more than 50%of Firmicutes,more than 30%of Bacteroides,less than 15%of Proteobacteria,and Actinomycota accounted for less than 0.1%.At the species level,the control group accounted for approximately 70%of lactobacillus,bifidobacterium and desulfovibrio each accounted for approximately 5%.Compared with the control group,lactobacillus in the experimental group accounted for less than 30%,bifidobacterium and desulfovibrio were significantly down-regulated,while Bacteroides,Lachnospiraceae,and Escherichia were significantly up-regulated.3.2 Target microorganism screeningAccording to the microbial genus horizontal species clustering heat map,combined with a series of pathological conditions such as the death of infected mice,the intestinal bacteria related to the host infection with Pasteurella were further screened.The samples of infected mice that were closer to the control group samples were grouped into one group(mild disease group,SG),and the infected mice samples that were farther away from the control group were grouped into another group(severe disease group,DG).Comparing the intestinal flora of SG,DG and control mice,it is found that the SG intestinal flora is similar to the control group,and the structure of the DG flora is complex,which is quite different from the SG and control group.Compared with SG,DG was significantly down-regulated by Lactobacillus and Bifidobacterium,and bacteria such as Prevotella,Bacteroides,Desulfovibrio,and Blautia were up-regulated.In this regard,we speculate that these differences in intestinal flora may be the reason that affects the host's development of severe or mild disease after Pm CQ6infection.3.3 Metabolome sequencing analysis resultsThe metabolome sequencing results were analyzed by orthogonal partial least squares discriminant analysis(OPLS-DA),which showed that the sequencing results were stable and reliable.Compared with the control group,galactol was significantly down-regulated in the experimental group,while short-chain fatty acids(3-hydroxypropionic acid,3-methylphenylacetic acid)were significantly up-regulated in the experimental group.KEGG analysis showed that there were significant differences between the experimental group and the control group in the galactose metabolic pathway.4.Function verification of LactobacillusBased on the screening results of target microbiota,5 strains of Lactobacillus were isolated from the intestinal contents of healthy mice.The isolated Lactobacillus were identified by 16Sr DNA sequencing,and they were mainly classified as Lactobacillus johnsonii(L.johnsonii)and one strain of Lactobacillus reuteri(L.reuteri).SPF mice were selected and randomly divided into a test group and a control group(10 in each group),and successively gavage L.reuteri and two strains of L.johnsonii(L.johnsonii MH33 and L.johnsonii LB-19).In the control group,after intragastric administration of MRS liquid medium for 1 week.Pm CQ6 was injected intraperitoneally to compare the clinical manifestations and immune response of several groups of mice.The results showed that the survival rate of Pm CQ6 infection of mice in the experimental group after continuous intragastric administration of L.johnsonii MH33 was 70%significantly higher than that of the control group(the control group survival rate was 20%),while the remaining Lactobacillus strains had no obvious effect.The colonization test results showed that Pm CQ6 colonized the lung tissue of L.johnsonii MH33 gavaged mice at 2.0×10⁷CFU/g after 32 hours of infection,while the average bacterial content in the lung tissue of the control group reached3.2×10⁸CFU/g.The ELISA test also found that after Pm CQ6 infection,the expression of IL-6,IL-12,IL-17,IL-1?,IFN-?,and other inflammatory factors was down-regulated in intragastrically administered L.johnsonii MH33 mice compared to the control group.The above results show that the addition of L.johnsonii MH33 can protect the mice by reducing the inflammatory response caused by Pm CQ6 infection.