| Objectives:Porcine pleuropeumonia has caused very serious loss and damage in the modern high-stocking density large-scale breeding farms. The etiology, epidemic characteristics, test methods and other research of porcine pleuropneumonia has been a focus of attention of the various countries’researchers and scholars; while the systematic studies of tissue damage caused by bacteria infection and immune-related indexes on the body of pig are rare. Therefore, this study aimed at:(1) Establishing the pleuropneumoniae pathological model of artificial infection;(2) Observating and studying the swine symptoms, blood indicators and biochemical parameters, antioxidant indicators, immune parameters (red blood cell immunity, T cell-mediated immunity, humoral immunity and cytokine levels) and pathological changes post-infection with APP;(3) Using the Agilent swine genome-wide microarray containing43,603probes to detect the changes in gene expression of infected pigs’lungs and hilar nodes, to explore the molecular mechanisms of injury infection.Results:1. The porcine pleuropneumonia disease model were builded succesfully by nasal spraying0.25ml diluent contained3.5-4×107CFU/ml APP every kilogram weight.2. The number of leukocytes, and the numbers and the percentages of granulocyte and monocytes were all increased significantly (P<0.01) while the number and the percentage of lymphocytes were decreased significantly (P<0.01). The content of GLB in infected-APP pigs serum was increased significantly (0.01<P<0.05), and ALB, A/G were decreased significantly (0.01<P<0.05); the activity of AST were increased (P<0.01) while LT/ST was decreased (0.01<P<0.05); the contents of TBil, IBil and IB/DB were increased extremely significantly (P<0.01); the contents of CRE and BUN were both increased slightly; the concentration of GLU, the activity of AKP, the content of Ca and Ca/P were decreased significantly (P<0.01), the content of P was increased significantly (0.01P<0.05)3. The ratios of RBC-C3b rosette were increased significantly (P<0.01) and the ratios of IC rosette were decreased (P<0.01); the percentage of CD3+T cells in the peripheral blood and CD3+CD4+T cells were decreased extremely significantly (P<0.01) while the percentage of CD3+CD8+T cells and CD4+/CD8+were decreased significantly (0.01<P<0.05); the SI was reduced evidently(0.01<P<0.05); the contents of IgA and IgE were increased while the count of IgG was decreased. The counts of serum SAA and c-reactive protein were increased slightly; The count of TGF-β was decreased significantly (0.01<P<0.05), and IL-1, IL-2and IL-6were decreased; The counts of PDGF, IFN-y and TNF-a were increased.4. In the lung tissues of infected-APP pig, SOD and GSH-PX activities were decreased (P<0.01), and CAT activities were increased (0.01<P<0.05); MDA and-OH contents were decreased (0.01<P<0.05or P<0.01); T-AOC were decreased (P<0.01). In the HLN of infected-APP pig, CAT and GSH-PX activities were were increased (P<0.01);-OH count and T-AOC contents were decreased (P<0.01).5. The main results of pig genome-wide expression analysis of the chip are as follows:(1)Pig-chip scan results were showed that the sampling point was uniform and inerratic, the signal intensity was high, the signal to noise ratio was high, the specificity of probe was strong, detection rate of chip was high, and it concentrated in the range53%～74%.(2) T-tests were used to detect the differentially expression with a pig’s lung and HLN in infected and non-infected:having got the DE genes and these intersection in the infected and non-infected lung and HLN.(3) The SOM of DE genes revealed that the genes were classified into9kinds, included the processes of immune enhancement, Toll-like receptor signaling pathway, phosphatidylinositol signaling pathway, and adhesion connecting pathway. The HCL of DE genes revealed that lung samples and HLN samples each clustered together; infected and non-infected samples of the same tissue each clustered together.(4) PCA revealed that the main expression pattern in the same kind of tissue was that DE genes in disease animals were higher than those in healthy ones; while in the same individual, DE genes in lymphoid tissue were higher than those in lung tissue.(5) GCT based on GO analysis revealed that89GO terms that the degree of genetic variation reached significant and highly significant (PErmineJ<0.05and0.01) were found in lung, including27GO terms related with immune.272GO terms that the degree of genetic variation reached significant and highly significant (PErmineJ<0.05and0.01) were found in HLN, including50GO terms related with immune.(6) GSEA revealed that genes were enriched significantly in a number of infection-related pathway including Fc epsilon RI (one of IgE receptors) signaling transduction pathway, NOD-like receptor signaling pathway, acute myeloid leukemia, cancer, progesterone-mediated oocyte maturation, spliceosome, Ⅱ diabetes, steroid hormones biosynthesis, and have got the direction (upward or downward) of these pathway that plays a regulatory role in each group. By Leading edge analysis found several potential genes appeared in a different pathway in the high-frequency and may had an important role in the regulation of body resistance to infection and the repair of damaged tissue by Leading edge analysis found.(7) The reliability evaluation results showed that the largest relative Standard Deviation (RSD) was5.50%and the smallest RSD was only2.57%in the gene chip. QRT-PCR verification results showed that there were a positive correlation between two detection results from different experimental methods in lung and lymphoid tissue and correlation coefficient averaged at0.861±0.127.Conclusion and innovation1. Initially reveal the molecular basis of the APP infection damage to the lung and HLN by using the Agilent pigs genome-wide microarray expression profiling.31genes were selected which might have a significant impact and important role for the pigs’ immune, anti-infection and anti-tissue damage, as well as19pathways of greater research value.2. The cellular immune and humoral immune function of the infected-APP pigs decreased, while the erythrocyte immune function increased. The T-AOC of infected-APP pigs were decreased. |
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