Study On Gene Expression Difference Of Bovine Kidney Cells Infected By Toxoplasma Gondii And Neospora Caninum

Neospora caninum and Toxoplasma gondii are two kinds of protozoal parasites which are very similar in morphology,biological characteristics and life history.N.caninum is one of the main causes of bovine abortion,which brings serious economic losses to the cattle industry.T.gondii can cause abortion in pregnant woman,pig,sheep and other livestock,but cattle are obviously not susceptible to it.There are no reports of clinical toxoplasmosis in adult cattle.If we can identify the key factors that cattle are susceptible to N.caninum not to T.gondii,it can provide important data for further revealing the pathogenic mechanism of N.caninum and T.gondii.The purpose of this project is to detect and analyze the differences of Madin darby bovine kidney cells(MDBK)infected by N.caninum and T.gondii and screen significant differential genes.MDBK cells were cultured and infected with a 1:3 MOI of N.caninum and T.gondii respectively.The growth difference of MDBK cells were observed.The results showed that most N.caninum tachyzoites had invaded the host cells at 12 hours after infection,and half amount of T.gondii tachyzoites were still outside of cells.However,some T.gondii vacuoles were broken at 56 hours after infection,and a large number of tachyzoites were released,and the cells were damaged and shedded,however,the number of ruptured N.caninum vacuoles was very small,and the degree of cell shedding was relatively mild.Anyway,there was some difference in the growth of N.caninum and T.gondii in MDBK cells,but the difference is not very significant.MDBK cells were infected with a 1:1 MOI of N.caninum and T.gondii,respectively.at 20 hours after infection,MDBK cells(control group)and MDBK cells infected by N.caninum and T.gondii(Nc-group and Tg-group)were sequenced by Illumina Novaseq 6000 sequencing platform.Transcriptome differences among the three ones were analyzed,and the key differential genes were screened.The results showed that compared with the control group,176 genes of MDBK cells in Nc-group were significantly up-regulated and 80 genes were significantly down-regulated.Among them,131 genes were unique to Nc-group,which were mainly related to intercellular space,serine,chemokine,tumor necrosis factor and other functions,and played a role through chemokine signal pathway and TNF signal pathway.6 upregulated genes and 2 down-regulated genes were obtained by further screening,among which the up-regulated genes were mainly related to hemoglobin,myometrial contraction,vascular development and immune response,while the down-regulated genes were mainly related to macrophages and neural structure.Compared with the control group,1071 genes in Tg-group were significantly upregulated and 1154 genes were significantly down-regulated.Among them,2100 DEGs were unique in g-group,which were mainly related to plasma membrane composition,cell surface receptors,T cell differentiation and other functions,and played a role through p53 signal pathway,NF-κ B signal pathway,MAPK signal pathway and so on.In further screening,8 DEGs were obtained from the up-regulated genes and down-regulated genes,among which the up-regulated genes were mainly related to cytokines,immune response,surface antigen,immune regulation and stress resistance,while the down-regulated genes were mainly related to neutrophils,epithelial cell structure,erythrocyte structure,actin,cell membrane and so on.Compared with Nc-group,Tg-group 1252 genes were significantly up-regulated and 986 genes were significantly down-regulated.A total of 65 DEGs were related to the infection process of N.caninum and T.gondii,which were mainly related to ribonucleotide transport,hormone regulation,cell proliferation and other functions,and played a role through the signal pathways of many molecules such as NF-κ B and the interaction between cytokines and their receptors.7 up-regulated genes and 9down-regulated genes were obtained by further screening,among which the upregulated genes were related to transmembrane transport,ectoderm development,cell membrane,innate immune system,liver and intestinal diseases,and down-regulated genes were related to heart,embryonic development,cell proliferation and so on.The results laid a foundation for further study of the mechanism of difference in susceptibility of cattle to Neosporidium and Toxoplasma gondii,and had very important guiding significance.