| Mycoplasma pneumonia of swine (MPS), is caused by Mycoplasma hyopneumoniae (Mhp) in pigs with chronic respiratory disease. It exists widely in the world and China also for a long time and can cause serious harm to the pig industry. There are many papers about MPS which has gradually been paid great attention by scholars at home and abroad.These reports are mostly focused on vaccine development, etiology, medicine, and diagnosis.The typical pathological changes of MPS include the peripheral inflammation, interstitial pneumonia, and the change of the lung and the like.Typical clinical symptoms include breathing, wasting and cachexia. The typical pathological changes of MPS include the peribronchitis, lymphocyte infiltration and proliferation, pulmonary carnification and interstitial pneumonia; the clinical symptoms of MPS are gasping, emaciation and cachexia. It was little to be observed relevant reported that MPS typical pathological changes, clinical symptoms and the mechanism of signal transduction and the role of cytokines in the pathogenesis of the disease. This experiment will be carried out from the following two aspects.1. Pathogenic epidemiological investigation of MPS in Changsha city and its surrounding areas.1260 pig samples, including 45 nasal swabs,81 tracheal swabs,469 bronchoalveolar lavage fluid (BALF),625 lungs and 40 blood samples, which were collected from Changsha City and its surrounding areas, were detected for Mycoplasma hyopneumoniae (Mhp) and Mycoplasma hyorhinis (Mhr) by PCR and sequence analysis. It were carried out to Separate and to preliminary identificate of Mhr. It shows that the best samples of detecting Mhr and Mhp in pigs in Changsha City and the surrounding areas are: BALF (Mhp) and nasal swab (Mhr) collected from pigs at slaughter in winter. The results of this study provide a new epidemiological basis for the prevention and control of MPS in Changsha and its surrounding areas and the detection of Mhp and Mhr.2. Differential genes expression profile Study of the porcine alveolar macrophages (PAM) induced by Mhp. Mhp strain 232 was incubated with PAM cultured in vitro in RPMI-1640 medium, while PAM which incubated in the mudium alone was used as the control. PAM was harvested after incubated for 12 hours and 24 hours and then total RNA were extracted and cDNA library was constructed. Digital-gene-expression profiling technology was used to detect the PAM transcriptome, data statistics and the results were analyzed by programes of GO, KEGG, protein interaction network and IPA. By DGE it were found that 86 genes of PAM transcription group were significantly differentially expressed at 12 hours after PAM induced by Mhp (hapm), which 49 upregulated and 37 downregulated.; 889 genes of PAM transcription group were significantly differentially expressed at 24 hapm, which 517 upregulated and 312 downregulated.GO analysis showed that the majority of differentially expressed genes (DEG) were involved in 54 GO enrichment items at 12 hours, include:1. Biological process(46items):its function was mainly described as nInflammatory response, Immune response, Regulation of signal transduction.2. Molecular function (11 items):its function was mainly described as Chemokine activity, Cytokine receptor binding and Tumor necrosis factor receptor binding. 3. Cellular component (3 items):its function was mainly described as Extracellular region, Extracellular space and Cell surface. GO analysis showed that the majority of DEGs were involved in 128 items at 24 hours, include:1. Biological process (106 items):its function was mainly described as Inflammatory response, Immune response and Leukocyte activation. 2. Molecular function (11 items):its function was mainly described as Chemokine activity, Chemokine receptor binding and Protein binding.3. Cellular component (11 items):its function was mainly described as Cytoplasm, Cell surface and Extracellular region.The analysis found that Mhp induced PAM after 24 hours (compared with 12 hours of induction), the number of enrichmen items increased, genetic differences between the same GO enrichment items increase in different degrees. After induced for 12 hours by Mhp, the extracellular space of PAM can may only be effect. Mhp use the cells and cell receptor interactions for the initial signal transmission and immune response. After induced by Mhp for 24 hours, Mhp may be in the PAM cells at the same time, through the signal transduction and immune response mediated by the body of the corresponding pathological symptoms.KEGG analysis showed that after PAM was induced by Mhp for 12 hours, these differentially expressed genes were mainly enriched in the 101 KEGG enrichment items. It suggested that these DEGs closely related with MPS mainly focused on the following 4 KEGG enrichment items:Toll-like receptor signaling pathway, Cytokine- cytokine receptor interaction pathway, T cell receptor signaling pathway and antigen presentation pathway. After PAM induced by Mhp for 24 hours, these DEGs were mainly enriched in the 250 KEGG enrichment items. It suggested that these DEGs closely related with MPS mainly focused on the following 7 KEGG enrichment items:NF-KB signaling pathway, endocytosis pathway, chemokine signaling pathway, Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction pathway, T cell receptor signaling pathway and antigen presentation pathway. After PAM induced by Mhp for 24 hours (compared with for 12 hours after induction), the number of of KEGG enrichment item increased, and the numbers of DEG of the same KEGG enrichment item increased in varying degrees. After induced by Mhp for 12 hours, PAM may be the first to start the Toll receptor signaling pathway, to transmit signals to the PAM, and to stimulate the immune response. After induced by Mhp for 24 hours, Mhp mediated phagocytosis and chemokine activation through the signal transduction to be anti regulation, the formation of the state of the interaction between the two.IPA analysis revealed that induced by Mhp for 24 hours, the DEGs were connected to each other to form 25 internal interacting sub networks (sub network 1-25). It reported by the documents, there were 6 and 8 genes respectively in the network 7 and in the network 15 to relate closely with Mhp. In the sub network 1-25, DGEs closely related to clinical symptoms and pathological changes of MPS mainly are:TNF, IL8, CCL3, CCL5, CCL8 and TGF61, etc.Protein interaction network analysis revealed 131 proteins encoded by DEGs involved in network interaction. There are 13 proteins encoded by DEGs to interact to form CCL sub network. There were 8 of 13 peoteins encoded CCL sub network to be involved in the gene interaction network.According to the results detected by DGE technology and the result of GO analysis, KEGG analysis, IPA analysisand STRING analysis, it was analysed that the typical pathological changes and clinical symptoms of MPS are closely related to PAM regulation related gene differential expression in this experiment. Mhp infected pigs to form Bronchial inflammation, and then PAM gathered to the site of inflammation and be activated. PAM can secrete IL1 (3.64 times,12h; 8.37times,24h), IL8 (2.91 times,24h) and other inflammatory cytokines, causing the body to appear inflammatory reaction. Inflammatory cells (Lymphocytes and Neutrophils) seep out of the blood vessels, under the action of CCL3 (24h expression increased 5.76 times) and CCL5 (24h expression increased 3.72 times) and CCL8 (24h expression increased 23.45 times), Lymphocyte enriched and infiltrated to the site of the inflammatory lesion (the bronchial and the fine bronchus), further leads to the formation of lymphoid follicles. Mhp infected pigs to form bronchial inflammation, Activated PAM gather to the site of inflammation and then secreted and released TGFB1 (24 hours 2.91 times), prompting pulmonary fibrosis, and then leading to the emergence of interstitial pneumonia. After infected pigs, Mhp induced a large number of PAM secreting IL1 (12h expression increased 3.64 times,24 h) to furtherly secret TNF (24 hours expression increased 3.68 times) and IL8 (24h expression increased 2.91 times). Increased expression of IL8 and TNF can promote the animal’s body appeare cachexia, fatigue, appetite loss, and to gain weight loss.This study laid the foundation for the pathogenesis of MPS, and it helpful to improve the molecular mechanism of Mhp effect on PAM function, which provides a new clue and basis for the further elucidation of Mhp promoting the formation of MPS through PAM. |
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