Differential Expression Of TLRs And Its Signaling Pathway Factors Induced By Mo Infection In Different Breeds Of Sheep

Mycoplasma pneumoniae(Mo)is the main pathogen of Mycoplasma pneumonia of sheep and goats(MPSG)and 3 months old goats and sheep are the susceptible animals.MPSG is characterized by fibrinous exudation inflammation in the lungs.The susceptibility to mycoplasma pneumoniae was different in different breeds of sheep,which was correlated with the immune system response of different breeds of sheep.Toll-like receptors(TLRs)are the body's immune related important molecules,mainly distributed in epithelial cells,immune cells,etc.By identifying pathogen associated molecular patterns,the signaling pathways factors transfer signal along the MyD88 pathways or TRIF ways to regulate and control NF-?B,AP-1,etc.Finally the immune cell proliferation and inflammatory cytokines help the body to resist and eliminate pathogen infection.However,uncontrolled inflammation can cause tissue damage.Based on the analysis of different breeding sheep toll-like receptor gene polymorphism and expression,the samples of lung and blood of different breeds of goat and sheep(black goat,white goat,Ma sheep,Boer goat and Hu sheep)artificially infected by Mo were collected.TLR1-10 DNA content,TLR1-10 gene and MyD88 or TRIF signaling pathway factors transcription levels were detected by TaqMan real time PCR to explore the Mo susceptibility difference of different breeds of sheep.The results of detection and analysis lays a foundation for the study of the relationship between Toll-like receptors and susceptibility to mycoplasma pneumonia.1.Study on the difference of TLRs gene level in different breeds sheep infected with MoThe lung and blood samples of black goats,white goats,Ma goats,Boer goats and Hu sheep artificially infected with Mo were collected.The TLR1-10 gene content of the samples were detected by TaqMan real-time PCR and the difference was analyzed.The results showed,there were some sheep dead in the infected group of black goat,white goat,Ma goat,boer goat at 96 h-144 h and no death in the infection process in Hu sheep infected group and control groups of each breeds sheep.The typical pneumonia symptoms were seen in all infected groups.The quantitative analysis of TLRs gene in lung samples of sheep showed that the content of TLR2/4/6/8/9 gene in lung tissue of the same breed sheep infected group were significantly higher than that of the control group(P<0.05),and there was no significant difference in TLR3/7/10 between the infected group and the control group(P>0.05).The content of TLR1/5 had difference between breeds in infected group and control group.The results of intraspecific comparison suggested that TLRs were involved in the process of Mo infection,and there were differences among breeds.The difference analysis between breeds of TLR1-10 gene content in infected group lung tissue showed that the content of TLR2 gene in white goat was significantly higher than that in other breeds(P<0.01).The content of TLR10 in black goat were significantly higher than those in other breeds(P<0.01).The content of TLR6/8 in black and white goat were significantly higher than those in Ma goat,Boer goat and Hu sheep(P<0.01).The content of TLR4/5 in Boer and Ma goat and TLR5 in Hu sheep were significantly higher than those in white goat and black goat(P<0.01).The results of inter-specific comparison suggest that TLR2/4/6/8 were involved in the rapid development of inflammation and death in the early stage of infection in black goat,white goat and Ma goat.The quantitative analysis of TLRs gene in blood samples of the same breeds sheep showed that the content of TLR1/2/4/6/10 in infected group were higher than those of the control group at different time and 48 h,96 h samples were higher than 0h.There was no significant difference in TLR7 of black goat and TLR5 of Hu sheep between infected group and control group.Intraspecific comparison suggested that TLRs could be involved in the occurrence and development of inflammation and there were some differences in TLR5/7/8 among different breeds of sheep.The quantitative analysis of TLRs gene in blood samples of different breeds sheep showed that there were no significant difference in TLR1/2/4/5/6 between the five breeds 0 h samples in infected group(P>0.05).The content of TLR1 gene in 48 h and 96 h blood samples of Boer goat and Hu sheep were significantly higher than those of black goat and white goat(P<0.01).TLR2/4 in 48 h and 96 h blood samples of black goat and white goat were significantly higher than those of Ma goat,Boer goat and Hu goat(P<0.01).TLR6 gene in 96 h blood samples of white goat and Boer goat were significantly higher than those of black goat,Ma goat and Hu sheep(P<0.05).The inter-species comparison suggested that TLR1/2/4/6 were involved in the process of systemic inflammation in all breeds sheep.The level of TLR1 might be negatively correlated with the occurrence of lung inflammation in the experimental sheep,while the TLR2/4/6 positively correlated with the development of inflammation.2.Study on the difference of TLRs transcription induced by Mo infection in different breeds sheepThe transcription levels of TLR1-10 in 25 lung samples and 126 blood samples from the five breeds sheep were detected and analyzed by TaqMan real-time PCR.The analysis in lung samples of experimental sheep showed that the transcription level of TLRs gene in lung tissue of sheep infected group were significantly higher than those of control group(P<0.05).The transcription level of TLR1/2/4/5/6/8/9gene in lung tissue of black goat and white goat were higher than that of TLR3/7/10.The transcription level of TLR4/5/7/8 gene in lung tissue of Ma goat,Hu sheep and Boer goat were higher than that of TLR1/2/3/6/9/10.The results of intra-specific comparison suggested that the transcription levels of TLRs gene were similar to that content and there were correlation between TLRs gene level and inflammatory process.Transcriptional levels of TLRs gene in lung samples of different breeds of sheep showed that the TLR1/2/9 of black goat and white goat were significantly higher than that of Ma goat,Boer goat and Hu sheep(P<0.05).The transcriptionallevels of TLR6 and TLR4 of white goat were the highest(P<0.01).The transcriptional level of TLR5/7/10 was the highest in the lung tissue of Boer goat(P<0.01).The results of interspecific comparison suggested that there were interspecific differences in TLRs transcription levels involved in the regulation of mycoplasma inflammatory process in sheep.The analysis of TLR1-10 transcription level in blood samples of sheep showed that the TLR1/2/4/6/7/8 gene transcription level of 48 h and 96 h samples of the same breeds sheep infected with Mo were higher than that of 0 h and control group.There was no significant difference in TLR5/9 between infected group and the control group at each time point for every breeds.The transcription level of TLR3 gene of Ma goat,Boer goat and Hu sheep was higher than that of the control group and TLR10 of Ma goat infected group was higher than that of the control group.The results of intra-specific comparison suggested that TLR1/2/4/6/7/8 was generally involved in systemic inflammation.There were inter-specific differences in TLR3/5/9/10.There was no significant interspecific difference in the transcription level of TLR1 gene at 0h and 48 h in infected group(P>0.05).The transcription level in 96 h blood sample of Hu sheep was significantly higher than that of other breeds(P<0.05).The TLR4 transcription level of black goat,white goat,Boer goat and Hu sheep was significantly higher than that of Ma goat at 96 h(P>0.01).The levels of TLR6 transcription in 48 h and 96 h blood samples of white goats were significantly higher than those of the other 4 sheep(P<0.01).The transcription level of TLR7 gene in blood samples of white goats and Boer goats was significantly higher than that of black goats,Ma goat and Hu sheep at different time points.The transcription level of TLR8 gene at 48 h and 96 h blood samples of white and black goats was significantly higher than that of Ma goat,Boer goat and Hu sheep.The results of interspecific comparison suggest that TLR1 negatively regulated the inflammatory process at the transcriptional level and TLR2/4/6/7/8 transcription were involved in the regulation of inflammatory process.3.Study on the difference of transcription level of TLRs MyD88 pathway linker induced by Mo infection in different breeds of sheepTLRs MyD88 pathway factors transcription level were detected and analyzed in25 lung samples and 126 blood samples of 5 breeds of sheep by TaqMan real-time quantitative PCR method.The results showed that the transcription levels of MyD88,TRAF6,NF-?B,FOS and JUN genes in the lung tissues of the 5 breeds of sheep infection groups were significantly higher than those in the control group(P<0.01).The interspecific difference analysis showed that the NF-?B gene transcription level in the lung tissue of white goat was significantly higher than that of the other 4 sheep(P<0.01).FOS gene transcription level of black and white goats was significantly higher than that of Ma goat,Boer goat and Hu sheep(P<0.01).There was no significant difference in JUN gene transcription between species(P>0.05).Differential analysis of transcription level of MyD88 pathway factor in lung tissue of experimental sheep suggested that Mo infection could activate the TLRs MyD88 pathway to promote inflammatory response,among which white and black goats had a strong response.Analysis of the transcription levels of MyD88,TRAF6,NF-?B,FOS and JUNgenes in the blood samples showed that the 5 sheep infection groups were significantly higher than the control group(P<0.01)and the transcription level of 48 h and 96 h samples of infected group was higher than that of 0 h.The transcription levels of MyD88,TRAF6 and FOS in black and white goats were significantly higher than those in Ma goat,Boer goat and Hu sheep(P<0.01).The NF-?B gene transcription level in the 5 breeds sheep infection groups increased continuously from0 h to 96 h and had no difference.There was no significant difference in JUN gene among the 5 sheep infection groups(P<0.01).The differential analysis of the transcription levels of the MyD88 pathway factors in the blood samples suggested that the activation of the TLRs MyD88 pathway by Mo infection promoted the inflammatory response,and that the TLRs/MyD88/ NF-?B pathway was different in the 5 sheep species,and the black and white goats presented stronger responses.4.Study on the difference of transcription level of TLRs TRIF Pathway linker induced by Mo infection in different breeds of SheepTaqMan real-time PCR detection method was used to detect the transcription level of TLRs TRIF pathway factors in 25 lung samples and 126 blood samples of 5breeds of sheep.The results showed that TRIF,TRAF3,IRF3,IRF7 gene transcription in lung of the infected group were significantly higher than that in the control group(P<0.01).Interspecific difference analysis showed that TRIF and TRAF3 genes were significantly different from each other(P<0.05),and IRF3 gene transcription level was the highest in the lung tissue of Ma goat(P<0.01).The transcriptional level of IRF7 in lung tissues of black and white goats was significantly higher than that of Ma goat,Boer goat and Hu sheep(P<0.01).Differential analysis of transcriptional levels of TRIF pathway factors suggested that Mo infection could initiate TLRs TRIF signaling pathway in sheep,especially in black and white goats.The transcriptional levels of TRIF,IRF3 and IRF7 genes in the blood samples of the 5 breeds of sheep infection groups were significantly higher than those in the control group(P<0.01).The interspecific difference analysis showed that the transcriptional level of TRIF gene in black goat and white goat infection group was significantly higher than that in Ma goat,Boer goat and Hu sheep at 96 h(P< 0.01).At 48 h and 96 h,the transcription level of IRF3 gene in the Ma goat infection group was significantly higher than that in the black goat,white goat,Boer goat and Hu sheep(P <0.01).At 48 h and 96 h,the transcription level of IRF7 in black goat and white goat infection group 48 h and 96 h samples was significantly higher than that in Ma goat,Boer goat and Hu sheep.Differential analysis of transcriptional levels of TRIF pathway factors in blood samples suggested that Mo infection activated the TLRs TRIF pathway,and black and white goats showed strong expression.