Mechanism Of Pasteurella Multocida Infection Promoted By Serum

Pasteurella multocida(Pm)is a kind of Gram-ball or short rod-shaped negative bacteria that can cause many diseases in mammals,poultry,and humans.According to the specificity of their capsular antigens,they are divided into five types of capsular serotypes:A,B,D,E and F.According to lipopolysaccharide antigens,there are 16serotypes from 1 to 16.In recent years,with the rapid development of the cattle industry in my country,the transportation stress caused by frequent transportation has caused the incidence of cattle pasteurises to increase continuously,which has brought huge economic losses to the breeding industry.Serum is one of the important raw materials in biomedical technology products.Adding a proper amount of serum to cell culture can provide growth hormone and adhesion factors for cell growth,shorten the cell growth cycle,speed up the vaccine production process during the vaccine production process,and increasing the yield of vaccines.At the same time,some pathogenic bacteria also need to add serum to grow well during the cultivation process.Our previous research found that the injection of Fetal Bovine Serum(FBS)while using attenuated P.multocida for mouse infection can promote the infection of attenuated P.multocida.In order to explore its internal mechanism,this article investigates and analyzes the effects of different serums on P.multocida infection,analyzes the effect of FBS on host immune function with the help of transcriptome sequencing,explores the target cells that serum may affect through in vitro cell experiments.and studies the active ingredients in the serum,to provide theoretical guidance for clinical application of serum.The main research methods and results are as follows:1.Effects of different serum on P.multocida infectionP.multocida Pm CQ6 is a low-virulence pathogen with LD50=1.14×10~8 CFU.This study found that animal serums such as fetal bovine serum,newborn bovine serum,goat serum,stressed mouse serum,and stressed bovine serum can promote P.multocida Pm CQ6(the infectious dose is 106 CFU,which is less than the minimum lethal dose,the injection method was intramuscular injection),which eventually led to the death of mice.The number of bacteria in the lung,liver,spleen,and peripheral blood showed a significant upward trend over time,and the inflammatory pathological changes of lung tissue were significant.2.Transcriptome sequencing analysis of mouse spleen tissueThis part mainly uses FBS as the research object.According to the number of bacteria in the lungs,liver and spleen,we select the FBS group(only injection of FBS),Na Cl group(only injection of Na Cl),and FBS-Pm CQ6 group(simultaneous injection of FBS and Pm CQ6),Na Cl-Pm CQ6 group(simultaneous injection ofNa Cl and Pm CQ6)transcriptome sequencing of the spleens of four groups of mice was performed to analyze the effect of FBS on host immune function.According to GO enrichment and KEGG enrichment analysis,the differential genes(DEGs)in the Na Cl vs FBS group and the Na Cl-Pm CQ6 vs FBS-Pm CQ6 group are mainly enriched in immune-related pathways,suggesting that FBS and FBS-Pm CQ6 mainly affect the host’s immunity.2.2 Analysis of FBS in immune responseTo explore the influence of FBS on host immunity,this study first screened the immune-related DEGs in the Na Cl vs FBS group based on GO enrichment,followed by cluster analysis and cell enrichment analysis on these DEGs,and finally based on KEGG enrichment analysis to determine the target cells of FBS.The results showed that a total of 119 immune-related DEGs were screened from the Na Cl vs FBS group,of which 51 DEGs were enriched in the innate immune response,24 DEGs were enriched in the adaptive immune response,and these DEGs were significantly down-regulated.Among the 119 DEGs,116 DEGs were significantly down-regulated,and cell enrichment analysis of 116 down-regulated DEGs showed that T cells were mainly enriched.KEGG enrichment analysis of 116 down-regulated DEGs found that they were significantly enriched in the pathways of T cell receptor signaling pathway,Th1and Th2 cell differentiation,and Th17 cell differentiation.In addition,24 DEGs of 119DEGs were enriched in T cell activation,and 21 DEGs were enriched in T cell proliferation,and these genes were significantly down-regulated.In addition,CD3,CD4,PCKθ,IL-12,STAT4,IL-6R,IL-2Ra,IL12Rb,RORγt,etc.related to the differentiation of CD4~+T into Th1,Th2,and Th17 were significantly down-regulated,suggesting that FBS may inhibit auxiliary CD4~+T The cells differentiate into Th1 cells,Th2 cells,Treg cells and Th17 cells,the body’s immune function is reduced,and the host’s ability to eliminate pathogens is reduced.2.3 The effect of simultaneous injection of FBS and Pm CQ6 in immune responseTo study the mechanism of mice death caused by Pm CQ6 infection and FBS injection,this study first used GO enrichment to analyze the DEGs of Na Cl-Pm CQ6 vs FBS-Pm CQ6 group and FBS vs FBS-Pm CQ6 group,and then enriched by KEGG.Analysis to determine the main pathways activated by the body after the mice were injected with FBS and Pm CQ6 at the same time.The results showed that there were 75immune-related up-regulated DEGs in the Na Cl-Pm CQ6 vs FBS-Pm CQ6 group,and380 immune-related up-regulated DEGs in the FBS vs FBS-Pm CQ6 group.Cell enrichment analysis of these DEGs found that they were all significant.Enriched on macrophages.Compared with other groups,the TNF/TLR/NLR/IL-17/NF-κB signaling pathway in the Na Cl-Pm CQ6 vs FBS-Pm CQ6 group was extremely significantly activated,suggesting that FBS and Pm CQ6 acted on mice at the same time and may cause macrophages to be activated,and through the TNF/TLR/NLR/IL-17/NF-κB signaling pathway to cause the production of a large number of pro-inflammatory cytokines such as IL-1β,TNF-α,IL-6,IL-12,IFN-γ,leading to an increased inflammatory response,aggravated tissue damage in mice,and ultimately led to death of mice.3.Effect of serum on mouse peritoneal macrophagesAccording to the analysis of transcriptome sequencing results,mouse macrophages activated under the combined action of FBS and Pm CQ6,indicating that the functional status of macrophages plays a more important role in Pm CQ6 infection.The neutral red method was used to detect FBS in vivo and in vitro effects on the phagocytic function of macrophages.The results showed that the phagocytosis of mouse peritoneal macrophages after 24 h of FBS was added to mice was inhibited,while the addition of FBS to macrophages cultured in vitro had no effect on their phagocytic function,indicating that FBS has no effect on macrophages.The regulation of phagocytic function is carried out by other cells,rather than directly acting;after adding FBS to the mouse body for 24 h,the peritoneal macrophages were taken,and 4 h after infection with Pm CQ6,the peritoneal macrophages were detected by q RT-PCR The expression of cytokines in the cells showed that the expressions of cytokines TNF-α,IL-1β,IFN-γ,CCL2,CCL3,CCL4 and CXCL9 were significantly up-regulated.The above results suggest that the inhibition of macrophage function will aggravate the inflammatory response of Pm CQ6 infection,which is consistent with the results of animal experiments.In addition,goat serum can also inhibit the phagocytic function of macrophages in vivo.After 4 hours of infection of Pm CQ6,the expression of cytokines TNF-α,IL-1βand IFN-γof macrophages whose phagocytic function is inhibited by goat serum is significantly increased.4.Reach components of SerumTo explore whether the hormones in FBS have immunosuppressive effects,stress hormones(epinephrine,isoproterenol,and norepinephrine)were used to act on mice simultaneously with Pm CQ6,and none of them could lead to death of the mice;using carbon adsorption FBS It acts on mice at the same time as Pm CQ6,and the mice die;the results of measuring the concentration of glucocorticoid show that the concentration of glucocorticoid in different animal sera,different brands of FBS and 10 kinds of stressed bovine sera is low,and there is no significant difference between the sera The difference indicates that the immunosuppressive component in FBS is not a hormone.To explore whether the protein components in FBS have immunosuppressive effects,the products obtained by treating the serum at different temperatures and Pm CQ6 can cause the mice to die at the same time;using proteinase K to dissolve the protein in the serum at the same time as Pm CQ6 after acting on mice,the mice will die;products greater than 30 k D,50 k D and 100 k D and Pasteurella multocida Pm CQ6 at the same time will also cause the mice to die.The above suggests that the immunosuppressive component in FBS is a heat-resistant macromolecular substance that is not dissolved by proteinase K,but which substance needs to be further explored.