Transcript Profiling Of The Infection Interactions Between Mus Musculus And A. Pleuropneumoniae Serotype 7

Actinobacillus pleuropneumoniae can cause the highly contagious respiratory disease,with the characteristics of acute hemorrhage and chronic fibrinous pleuropneumonia.This disease is a worldwide epidemic with high morbidity and mortality,bring serious impact to the porcine industry.The evolution of the drug resistance mechanism and antigenicity led to the poor efficacy of antibiotics and low vaccine immune protection.Scientific prevention and control measures should be based on the pathogenic mechanism of pathogens,so that the effective ways to cut off the transmission and control infection can be taken.With the development of high-throughput sequencing technology,gene research has entered into the era of huge data and the post genome era,which accelerates the process of system mechanism research.In this study,RNA-seq technique was taken to study the transcriptomes of App serotype 7 and the infected mice lung tissue,which was helpful to reveal the immunological interactions of the pathogen and host.1.Establishment of a mouse model infected with App serotype 7SPF Kunming mouse named Mus musculus was selected to successfully conduct the intraperitoneal injection infection model and intranasal infection model.And a real-time quantitative PCR method based on 16SrDNA was established to detect the content of App in different organs of two infection models at different infection periods,and the histopathology changes were analyzed by HE staining.The results showed that death of mouse and the content of App had the charicteristics of dose dependent and sequential changes in the two model,but there was a big difference between the different groups of intranasal infection model.All organs studied were detected as positive,indicating that App achieved the proliferation and spread to other tissues in the model,and it was infectious to mouse.Among them,the App content of spleen and liver in intraperitoneal injection infection model were higher than lung tissue,and the heart was the lowest.In the intranasal infection model,the App content of lung tissues were the highest,and had the most serious pathological changes.The intranasal infection model was optimized,and results showed that when the bacterial dose was lO×LD50(4.26×109cfu)the lungs had high content of App and typical pathological changes in the model,which provided a foundation for the further RNA-seq on infected lungs.2.Transcript Profiling of the Immunological Interactions between Mus musculus and A.pleuropneumoniae Serotype 7Five RNA samples in 8-hour group with high content of App were selected and balanced mixed for RNA-seq,which was named Illumina Hiseq 2500 RNA-seq.And the App that cultured in vitro and healthy lungs were selected as control groups.Four cDNA libraries were constructed,including the cDNA library of App in infected lungs(T01),the cDNA library of App cultured in vitro(T02),the cDNA library of infected lungs(T03)and the cDNA library of healthy lungs(T04),and the mApped rates to reference genome were 15.68%,97.99%,67.68%and 85.01%respectively.A total of 333 differentially expressed genes of App were detected in the infected lungs compared with in vitro conditions,comprising 113 that were up-regulated and 220 that were down-regulated.And a total of 2428 differentially expressed genes were found in infected lung tissue compared with healthy tissue,of which 1484 genes were up-regulated and 944 were down-regulated.In addition,8 genes identified in RNA-seq with high differences in expression were chosen from the eukaryotic and prokaryotic groups,respectively,to validate the gene expression findings by qPCR to prove the reliability of the data.The results showed that there was a strong correlation(R2=0.989)in gene expression between RNA-seq and qRT-PCR.Analysis on the gene expression and functional annotation of the App differentially expression genes were taken.The results showed that large number of metabolism genes had high expression,and majority of them were related to anaerobic metabolism,such as adhE,sdaC,dmsA,B,C,D,torY,Z,and encoded oxidoreductase and hydrogenase,participating in the anaerobic respiration in bacteria.On the other hand,the main virulence genes were down-regulated or had no significant differences in expression.Among them,apxIA,D,ompA,tbpA,B,metQ1,2 etc.were down-regulated.In addition,some virulence genes were up-regulated.Genes like murC,G,lrgB,glmS and ALR,were involved in the formation of bacterial cell wall and membrane,and genes UreB,C encoded urease took part in the immune evasion of App.Analysis on the enrichment of mouse differentially expression genes on signaling pathways were taken.The results showed that large number of immune signaling pathways were activated,such as NLR signaling pathway,TLR signaling pathway,RLR signaling pathway,cytosolic DNA-sensing pathway,TNF signaling pathway,BCR signaling pathway,TCR signaling pathway,Jak-STAT signaling pathway,MAPK signaling pathway etc.and caused the release of cytokines in multi species and large-scale,such as Csf2,Cxc12,Cxc110,Cxc13,IL-6,IL-1?,IL-23? and IL-1?,IL-10,IL-36?,IL-17F,IL-17A,Cc14,Cc13,Cc120,Cc12,Cc17,TNF and IFNy etc.Some cytokines had positive feedback to the signaling pathways and further promoted the expression of cytokine,inducing the cascade of cytokines.Most of these cytokines participated in the regulation of IL-17/IL-23 net,which suggests that it played an important role in the anti-infection of App.