| In recent years, with the improvement of pig farming industry, the long distance transport of pigs becomes more and more general. But subsequently, the transportation, as a kind of stress, brings more and more negative effects on the swine, such as the sudden death during the transportation. Many studies had showed that the pig deaths in transport process were directly associated with transport stress on the suppression of the immune function. Stress can affect the function of the immune system of animals by neuron-endocrine-immunity network, this research will explorate about transport stress on the immune function and its control mechanism in pigs from the perspective of cytokines.19 pigs castrated F2 offspring of Pietrain and Erhualian parents and kept at the same environment (weight,50±2 kg, kept in Jiangsu Academy of Agricultural Sciences) were randomly divided into 4 groups (A, B, C, D). on the day of the transportation trial, control group pigs (group A, n=4) were kept under normal housing conditions, the other group pigs (n=5, respectively) were sub-packed in two separate wooden boxes (2 and 3 pigs per box) and used for animal transport vehicles forl h (group B),2 h (group C) and 4 h (group D) transport stress, Transport stress was performed under conditions identical to those found in China(outdoor temperature was 18-25℃) about 30-40 km.h-1. There was no drinking water and feeding during the transport stress. At the completion of specific transport stress, the pigs were slaughtered to take blood, serum used for lymphocyte proliferation index and the quantitative detection of dynamic change on cytokines, mesenteric lymph nodes, spleen and thymus were quickly removed and fixed in 4% paraformaldehyde for NF-κB localization by confocal laser scanning microscope, the other two same tissue samples snap frozen in liquid nitrogen, then frozen at-80℃for the quantitative detection of cytokine mRNAs and P38MAPK. To investigate the correlation of peripheral blood lymphocyte proliferation index, serum cytokine levels and mRNA expression of cytokines in the immune organs in the transport-stressed pigs, we hope to find the key cytokines which can affect the immune function during the process of transport stress, and preliminary understanding of its regulatory mechanism.In order to evaluate the influence of transport stress on humoral immunity and cellular immunity totally, the peripheral blood lymphocytes (PBLs) proliferation of normal pigs were stimulated by the serum of stressed pigs alone in different dilution times or with plant haemagglutinin A (PHA) and Lipopolysaccharides (LPS) separately. Lymphocyte proliferation index was determined by MTT colorimetric assay. Results show that,1:16, 1:32 and 1:64 serum dilution, which are able to increase the normal rate of pigs in peripheral blood lymphocyte proliferation, and reached its peak, but there was no significant difference compared with the control group (P>0.05), then began to decline in the capacity of lymphocyte transformation to minimize in 4 h, and which was significantly decreased compared to 1 h (P＜0.05). For the same dilution of serum, add after PHA and LPS can significantly enhance the normal pigs peripheral blood T, B lymphocyte proliferation activity (P<0.01), compared with the control group,1:32 diluted serum together with PHA when in the transport 1h significantly enhanced in peripheral blood T lymphocyte proliferation activity (P<0.05). The rest of the transport of different time periods of different dilution of the serum and the control group showed no significant difference. For different transport time, the proliferation activity of T lymphocytes reach to maximum in 1 h, then decreased rapidly, when the value dropped in 4 h and below the control group.1 h group and 4 h group showed significant difference (P<0.05) in 1:16 and 1:32 diluted serum. Compared with the control group,1:32 diluted serum and the combined effect of LPS in the transport 1h can significantly enhance the peripheral blood B lymphocyte proliferation activity (P<0.05). There was no significant difference in 1:16 and 1:64 dilution of serum during the process of transport stress.In order to explore the transport stress on the mechanism of serum T, B lymphocyte proliferation effect in pigs, the concentration of IL-1β, IL-2, IL-4, IL-6, IL-10 and TNF-αin sera was measured with homologous specific Enzyme-Linked Immunosorbent Assay. The results showed that the levels of IL-2, IL-6 and IL-10 in sera were increased with transportation stress and reached to the peak after 1 h of transportation, then slowly decreased and reached the lowest level at 4 h after transportation. The levels of IL-6 and IL-10 in the sera at 4 h were significantly decreased (P<0.01) compared with control. The level of IL-2 in sera at 4 h was significantly decreased (P<0.05) compared with 1 h of transportation stress, but there were no significant changes with respect to the IL-1β, IL-4 and TNF-a. Humoral and Cellular immunity have been changed during transportation stress in pigs, that is, Thl and Th2 type cytokines are obvious response, the dynamic balance of Thl/Th2 responses is the guarantee of a normal immune status during transportation stress in pigs.In order to understand the mechanism of the dynamic changes of three cytokines (IL-2, IL-6 and IL-10) significantly changed in serum of transport-stressed pigs, using real-time fluorescence quantitative RT-PCR technology, the IL-2, IL-6 and IL-10 and its corresponding receptor mRNAs transcription levels were determined in the mesenteric lymph node, spleen and thymus of transport-stressed pigs. The results showed that after 1 h, 2 h and 4 h transport stress, the expression of cytokines in the mesenteric lymph node have occurred significant changes compared with control group and during transportation stress, and got to maximum at 4 h during transportation stress, and significantly higher than control,1 h and 2 h groups (P＜0.01), receptors mRNAs have also changed dramatically, but the trend vary to the expression of cytokines, the expression levels of IL-6, IL-10, IL-6R and IL-10R mRNAs in spleen were significantly changed compared with control group and during transportation stress. In the thymus, in addition to IL-6RmRNA, the three cytokines IL-2RmRNA and IL-10RmRNA have dramatically changed compared with control group and during transportation stress. Therefore, the expression levels of cytokines and its receptors in various immune organs are different in transportation-stressed pigs, the change of serum cytokine levels is the result of co-regulation of these three immune organs.In order to find the main mediator reflection change of immune function during transportation stress in pigs, using the one-tailed analysis in Spass to analyze the Pearson correlation coefficient of lymphocytes proliferation effect, cytokine expression levels in serum and cytokines and receptors mRNAs levels in mesenteric lymph node, spleen, thymus. Results showed that the correlation coefficients between IL-6 in serum and T,B lymphocyte transformation rate in serum diluted 1:32 were 0.823 and 0.764 in pigs respectively, The correlation coefficient between IL-10 in serum and T lymphocyte transformation rate in serum diluted 1:32 was 0.789 in pigs, and showed statistically significant differences (P<0.01). In the mesenteric lymph nodes, IL-6mRNA levels and IL-10 levels in serum were negatively correlated (r=-0.719, P=0.004), IL-2mRNA expression levels with IL-10 in serum was significantly negatively correlated (r=-0.720, P=0.004). In the spleen, IL-6mRNA levels and IL-6 in serum was obviously a positive correlation (r=0.669, P=0.009), there is an obvious positive correlation between IL-6mRNA expression and IL-10 in serum (r=0.663, P=0.009).In the thymus, IL-10 concentration in serum were positively correlated with IL-10mRNA(r=0.712, P=0.000) and IL-lORmRNA (r=0.698, P=0.002), IL-6mRNA level was positively correlated with IL-2mRNA (r=0.768, P=0.000). Statistical analysis showed that IL-6 can be used as the change of immune function marker during transport stress in pigs, and in three immune organs, thymus and spleen play a major role in the secretion of IL-10 and IL-6 respectively, and mesenteric lymph node play the role of negative regulatory factors to IL-10.To explore the regulation of various immune organs on cytokine expression levels during transportation stress in pigs, using ELISA method for P38MAPK quantitative detection in mesenteric lymph nodes, spleen and thymus cells in pigs, using laser scanning confocal microscopy for NF-κB cell positioning, the initial number of positive cells and penetration nuclear rate statistics of three immune organs. The results showed that the P38MAPK expression in 2 h was significantly higher than 1 h,4 h and the control group (P<0.05) in transported-stressed pigs, there was no significant change of P38MAPK in mesenteric lymph nodes and spleen before and after transport stress. The distribution of NF-κB in three immune organs both in the cytoplasm and nuclei during the transportation stress, according to random statistics, the number of positive cells gradually increased with the transport time and peak at 4 h, penetration nuclear rate was also the same trend. The results showed that P38MAPK-NF-κB is an important signaling system on the regulation of cytokine expression in immune organs during the transportation stress.The above test results show that the effect of transport stress on the immune function has time mechanism, which is not necessarily immunosuppressive,1 h and 2 h transport stress can lead compensatory enhancement on immune function, transport stress caused the immune suppression at 4 h. Statistical analysis showed that IL-6 can be used as the change of immune function marker during transport stress in pigs, and in three immune organs, thymus and spleen play a major role in the secretion of IL-10 and IL-6 respectively, and mesenteric lymph node play the role of negative regulatory factors to the upper two cytokines. P38MAPK-NF-κB is an important signaling system on regulation of cytokine expression in immune organs during the process of transport stress in pigs. |
PDF Full Text Request