Detection Of Toxoplasma Gondii In Animals Clinically And Verification Of The Relationship Between MiR-206 And Its Target Gene In Host Infected With Suscrofa Strains

Toxoplasma gondii(T.gondii)is a zoonotic parasitic protozoan with a wide range of hosts.Feline animals are the only ultimate host and other warm blooded animals including humans being are the intermediate hosts.Animal toxoplasmosis is usually present latent infections,but it can cause acute disease and death in pigs,as well as reproductive disorders in pregnant sows;Chronic infection can form cysts in pig tissues,it’s a threat to pork safety and harm for human health.In recent years,T.gondii has gradually become an important pathogen that endangers the development of China’s pig industry.Currently,the prevention and control of swine toxoplasmosis still rely on chemical drugs,but used drugs such as sulfonamides or pyrimidines only have therapeutic effect on acute infection in pigs commonly,but useless for toxoplasmosis cysts in tissues.With the promotion of China’s non antibiotic breeding policy,the prevention and control of this disease will face greater difficulties.Therefore,it is necessary for finding new drug targets to develop new prevention and control technologies for pig toxoplasmosis.T.gondii strain of Chinese Ⅰ genotype is a prevalent dominant genotype in humans and animals in China,but it’s pathogenesis not fully understood.Our previous research found that T.gondii strain of Chinese Ⅰ genotype from pigs not only caused acute disease in piglets,but also formed cysts in brain tissue.The induced immunopathology characteristics were closely related to changes in the miRNA expression profile of the host,suggesting that miRNA play a crucial role in the important biological process of the host during T.gondii infection.MiRNAs have been shown that have great potential as important molecular diagnostic biomarkers and therapeutic drug targets,and this field has become a research hotpot currently.Therefore,this study firstly conducted an epidemiological investigation of toxoplasmosis in diseased pigs,pet dogs,and cats in clinics who visited the Animal Hospital of Yangzhou University;And then functional cluster analysis of miRNAs in pig spleen tissue infected with T.gondii strain of Chinese Ⅰ genotype was carried out by K-means,SOM and hierarchical clustering methods;Subsequently,culture system for T.gondii strain of ChineseⅠ genotype in vitro was constructed to evaluate the regulatory roles of T.gondii in expressions of miR-206 and its potential target genes using qPCR technology;Finally,the targeting relationship between miR-206 and ARAF gene was verified through the dual Luciferase reporter gene system.The research results will help identify and discover important targets for biological processes such as host cell apoptosis regulated by T.gondii,and lay a foundation to find crucial drug targets for the prevention and control of pig Toxoplasmosis.1.Investigation of animals infected T.gondii clinically in Jiangsu regionA total of 198 clinically diseased pigs and 819 tissue samples from the diseased pigs as well as 155 blood samples collected from sick pet dogs or cats were detected for T.gondii by PCR and ELISA method,and the risk factors of T.gondii infection in pigs were statistically analyzed.The results showed that 115 pigs were positive for T gondii detection,with a positive rate of 58.08%;367 tissue samples were positive for T.gondii infection,with a positive rate of 44.81%.Compared to liver,spleen and inguinal lymph nodes,the detection rate of T.gondii in heart and lung tissues was higher.The risk analysis showed that age was a risk factor for T.gondii infection in sick pigs.The infection rate of T.gondii was 10.20%(5/49)in pet cats and was 0(0/106)in pet dogs,respectively.The research results provide data support for the clinically diagnosis and control of animal toxoplasmosis in Jiangsu region.2.Functional clustering analysis of microRNAs in spleen tissue of piglets with acute and chronic infection of T.gondii isolated from suscrofaSmall RNA library and sequencing analysis of laboratory-preserved spleen tissues of T.gondii strain of Chinese Ⅰ genotype acute and chronic infection;based on the dynamic expression levels of miRNAs in the spleen tissue of piglets infected with T.gondii at different time points,K-means,SOM and hierarchical methods were used for the miRNAs clustering,and bioinformatics were used to carry out functional enrichment analysis of different miRNA groups.The sequencing analysis showed that 252 miRNAs were identified in pig spleen tissues,which were divided into 22(K1-K22),29(SOM1-SOM29)and 6(H1-H6)miRNA subclusters by K-means,SOM and hierarchical clustering methods,respectively;functional enrichment highlighted biological processes such as metabolic regulation,ion transport,biosynthesis,and cell apoptosis induced by T.gondii,and successfully revealed that different miRNA groups control a large number of immune regulatory signaling molecules to play key roles in immune regulatory mechanism against T.gondii infection in the host.Among them,miRNA members of subclusters K1 and K2 were found to regulate Notch signaling,thereby mediating IL-1 cell response and Th1/Th2 cell differentiation,the subcluster K15 involving to innate immune responses including neutrophil degranulation and TLR4 cascade signaling;the subclusters’ SOM 17,SOM1 and SOM25 participating in B cell activation,SOM9 relating to white cell migration and chemokine activity,H2 involving in regulating the interaction between cytokines and cytokine receptors,and miRNA members in subcluster H3-K17-SOM1 regulating interleukin production,chemotaxis of immune cells,chemokine and C-type lectin receptor signaling pathway.The above results reflected the regulatory characteristics of the different miRNA groups in spleen tissues,as well as important features of host immune regulation induced by acute and chronic infection of T.gondii.3.Research on the express correlation between miR-206 and the potential target genes in the host cells regulated by Chinese Ⅰ genotype T.gondii infectionPig kidney epithelial cells(PK-15)were used as the infection model to construct an culture system in vitro for T.gondii strain of Chinese Ⅰ genotype(YZ-1 strain)isolated from a diseased pig;potential target genes for miR-206 were screened through miRanda and RNAhybrid databases,and qPCR method was used to evaluate the regulatory role of T.gondii in expressions of miR-206 and its potential target genes in PK-15 cells;miR-206 mimics and inhibitors were designed and synthesized,then transfected cells and detected the expression correlation between miR-206 and its potential target genes.The results showed that the potential target genes(WDR48,LAMC2,CASP1 and ARAF)involved in regulating cell apoptosis signaling pathway were obtained through miRanda and RNAhybrid.qPCR detection results showed that T.gondii infection significantly decreased the expression levels of miR-206 in PK-15 cells at the different time points(P<0.01).Among the four potential target genes,the expression levels of ARAF and WDR48 genes were significantly downregulated at all time points(P<0.01),LAMC2(24 and 27 hours)and CASP1(6,12 and 18 hours)showed the significant upregulated expression(P<0.01).miR-206 mimics or inhibitor were transfected into PK-15 cells.The qPCR detection results showed a significant negative correlation in expression levels between miR-206 and the potential target genes(WDR48,LAMC2,CASP1 and ARAF),and the expression levels between miR-206 and ARAF had the most significant correlation.4.Research on the validation and application analysis of the targeting relationship between miR-206 and ARAF gene in PK-15 cellsBioinformatics method was applied to predict the binding sites between miR-206 and the 3’ UTR region of the ARAF gene sequence;the dual Luciferase reporter gene vectors of ARAF wild type(wt)and mutant type(mut)were constructed by point mutation technique based on PCR method;the constructed vector was co-transfected with miR-206 mimics into PK-15 cells,and the activities of Firefly Luciferase and Renilla Luciferase were detected by microplate reader to evaluate the target binding effect of miR-206 and ARAF gene.The results showed the potential target binding of 11 base sites between miR-206 and the 3’ end sequence region of ARAF gene by miRanda and RNAhybrid databases;based on the predicted binding sites,the recombinant double Luciferase reporter gene vectors psiCHECK2-ARAF wt and psiCHECK2-ARAF mut were constructed successfully;after miR-206 mimics transfected into PK-15 cells,psiCHECK2-ARAF wt group inhibited the expression of Luciferase,and led to a significant decrease in fluorescence intensity(P<0.05);however,NC and psiCHECK2-ARAF mut group had no effect on the expression of Luciferase(P>0.05),indicating that miR-206 targeted binding with the 3’ end of ARAF gene.In conclusion,this study indicated the infection situation of toxoplasmosis in animals in Jiangsu Province.Through cluster analysis of miRNAs in spleen tissues,the important features of host immune regulation induced by acute and chronic infection of T.gondii were revealed.By functional screening,expression correlation and target binding of miR-206 and ARAF gene,the potential roles of T.gondii in regulating the expression of apoptosis-related genes through miR-206 and thereby affecting the apoptosis process of host cells was preliminarily identified.The results not only provide data support for the clinical diagnosis and control of animal toxoplasmosis in this region,but also lay a theoretical foundation for the search for key drug targets for the prevention and control of porcine toxoplasmosis,and then develop new prevention and control technologies for this disease.