| Toll like receptor(TLRs) is a kind of pattern recognition receptors which is composed of 14 members of the TLR combination, plays an important role in innate and adaptive immunity. At present, we known TLR2, TLR3, TLR4 and TLR7, TLR8,TLR9 can trigger antiviral defense mechanism. OPA(ovine pulmonary adenomatosis,)is a chronic contagious disease, lung cancer, and caused by JSRV(Jaagsiekte sheep retrovirus, JSRV), which is seriously affecting the world sheep industry development.At present, the polymorphism of Toll like receptors and other immune response genes have been discovered that is closely related with the disease resistance and susceptibility. This study used the PCR-SSCP technology analyze the polymorphism of Xinjiang Kazak sheep TLR8 gene, chose 3 mutational sheep and 3 non mutational sheep to make the infection experiments for JSRV, and detected levels of inflammatory cells and cytokines on the early infection. After 5 months, we progressed necropsy observations and tissue observation to further explore the relation of susceptibility between Kazak sheep TLR8 gene mutation and JSRV, and provide theoretical support for the sheep breeding of disease resistance.1. Analysis of Xinjiang Kazak sheep TLR8 gene polymorphismWe used PCR-SSCP method detecte the mutations, found three pairs of primers’ amplified fragments exist polymorphism, and detected four mutation sites, which is respectively G170 A, G1307 A, A1628 G and T1604 C. G170 A and G1307 A is synonymous mutations, A1628 G T1604C is missense mutations, and the change of amino acid is Leu496 Pro and Gln504 Arg. The genotype frequency and allele frequencies were calculated, the results for hardy Weinberg equilibrium test is non-equilibrium state.2. Kazakh sheep TLR8 gene SNP mutation effect on cytokine after infection of JSRVUsing sheep lung tissues to prepare JSRV suspension to make the infection experiments, then detected the changes of the blood count, and used the ELISA method to detect the inflammatory factor levels on early infection. The results showed that the diameter of virus is about 80-100 nm. after inoculation JSRV, the determination of respiration, pulse and other physiological indexes were not abnormal every 3 hours. Blood count results showed that the white blood cell, lymphocytes and other inflammatory cells are increased for mutation group and the control group of sheep, but the difference between groups was not significant. ELISA results showed that the content of gamma-IFN, IL1-beta and TNF- alpha beta in serum for the mutation group is lower than control group, the difference between groups was significant. And the content of TLR8 and My D88 for mutation was also lower than the control group, but no significant difference between the groups. This experiment selected mutant sheep. This study chose mutational sheep to make the infection experiments for JSRV, and detected levels of inflammatory cells and cytokines on the early infection, that found the level of inflammatory cytokines for sheep which is TLR8 gene mutation was lower than non mutation group, that is presumably due to TLR8 gene SNP mutation prevents the immune response and that in virus infection and immunity was blocked, leading to immunoregulation abated after virus infection.3.Pathological observation of artificial infection for JRSVAfter artificial infection of JRSV for 5 months, we dissected the sheep of mutation group and control group to observe the pathological changes of lung,selected the lung tissue of pathological lesions and make sections to observe in optical microscope. Contract the pathological damage of mutation group and control group comparison after infection. The results found that the lungs of mutation group sheep were soild in texture change, there were many gray nodules,that is tumor shape, which grew hyperplasia lumps. While the lungs of control group sheep only appeared in a few of suspected tumor nodules, the injury level of lung was less than the mutation group sheep. Pathological observation found that the lung tissue of mutation group sheep had a large number of irregular adenoma lesions, while the control group only appeared a small amount of bronchial adenoma lesions, most alveolus pulmonis still maintain the original structure. |
