| Toxoplasma gondii(T.gondii)is a protozoan parasite that lives in nucleated cells.Toxoplasmosis is an important zoonotic parasitic protozoan disease that infects all warm-blooded animals and humans and can have a global epidemic distribution.In recent years,it has been reported that in some mountainous areas where the economic development is backward and the cultural development is low,there is still a bad habit of hunting and eating wild animals,and because of the diversity of food sources of wild animals,the risk of infection with viruses,bacteria,and parasites is higher compared with that of captive animals,and once humans accidentally eat positive tissues infected with T.gondii,it can cause human toxoplasmosis.Currently,China has a mature treatment system and prevention and control measures for the treatment and control of toxoplasmosis in captive poultry and livestock,but the research on the genes that differentially change in the host after infection with T.gondii in wild species and the genes that can be used as targets for the diagnosis of T.gondii infected is very unclear,which seriously limits the development and application of molecular diagnostic techniques for T.gondii infected in wild species.In this paper,we propose to replace wild species with domesticated civets,and investigate brain tissue from civets chronically infected with T.gondii for transcriptome sequencing and bioinformatics analysis to obtain differentially expressed genes in civets chronically infected with T.gondii and to screen and identify genes related to immune regulation.(1)A total of 2808 differential genes were analyzed,among which 860 differential genes were up-regulated and 1948 differential genes were down-regulated compared with the control group,and GO analysis showed that cellular processes and catalytic activity were the most abundant biological processes.KEGG analysis screened the enriched pathways related to immune regulation with higher expression of related genes.(2)Deep mining of the measured differential genes combined with bioinformatics was performed to analyze and screen the significantly expressed differential genes,and 10 differential genes with high expression in the relevant immunomodulatory metabolic pathways were screened as CCL5,CCL8,CCL23,CASP8,TLR1,TLR4,STAT1,ROS1,NOS1,and NOS2.(3)The collected differential genes presenting Toxoplasma-positive wild animals were collec ted and genotyped using PCR-RFLP technique,and the results were bat(Toxo DB#19,Toxo DB#66,Toxo DB #1),bamboo rat(Toxo DB#1,Toxo DB#111),pheasant(Toxo DB#2),hedgehog(Toxo DB#111,Toxo DB#1,Toxo DB#52),and brown house mouse(Toxo DB#10).(4)Brain tissues of T.gondii infected wild animals were taken separately,and q RT-PCR was performed to verify the expression trends using the primer sequences of the corresponding differe ntial genes of each species as templates.The results showed that CCL5 and TLR1 genes were up-r egulated in all five Toxoplasma-positive wild animals,the CCL8 gene was up-regulated in Toxopl asma-infected bats,hedgehogs,and brown house mice,NOS2 gene was up-regulated in bats,The NOS2 gene was down-regulated in bats,hedgehogs,and pheasants,and ROS1 was down-regulate d in hedgehogs,brown house,mice and pheasants.The experimental results indicated that the five differentially expressed genes,CCL5,TLR1,CCL8,NOS2 and ROS1,could be used as targets for genetic testing after T.gondii infection in wild animals. |
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