| Pasteurella multocida(Pm)is a spherical or short rod gram negative bacterium.According to the capsule antigen,it can be divided into five capsule serotypes,A,B,D,E and F.It is a conditional pathogen of respiratory tract in animals.When the body is stimulated or injured by various stress factors,it can cause severe respiratory infection.With the continuous development of beef cattle breeding in China,Beef cattle suffer from various stress factors such as transport stress and cold stress caused by weather changes,which cause the occurrence of bovine Pasteurella multocida,which has brought great harm to the development of meat industry.When the animal body is subjected to cold stimulation,it will provide favorable conditions for the invasion of pathogenic bacteria such as bacteria and viruses and induce a variety of respiratory diseases.At present,under cold stress conditions,the cause and mechanism of pulmonary infection caused by Pasteurella multocida are not clear enough.The aim of this study was to investigate the effects of Nos.infection of bovine A type Pasteurella multocida on lung and peripheral blood Th17/Treg cell immune balance and related cytokines expression in cold stressed mice.1.By using different concentrations(5?10~4 CFU/mL?5?10~3 CFU/m L?5?10~2CFU/m L)of bovine Pasteurella multocida capsular serotype A nasal drops to infect mice,and carrying out cold stress treatment(temperature:2-5?,humidity:60%,wind speed:4-5,10 hours a day,for 7 days),to explore the construction conditions of the model of Pm drops infection in cold stressed mice.The results showed that the mortality of 5?10~4 CFU/m L Pm was 100%,that of 5?10~3 CFU/mL Pm was 90%,and that of 5?10~2 CFU/mL Pm was 60%.Therefore,the conditions of the model were as follows:5?10~2 CFU/m L ml,2-5?,60%humidity,4-5wind speed,10 hours a day for 7days.2.To study the effect of different treatment conditions on the mortality and pathological changes of lung tissue of mice by cold stress and intranasal infection with Pm.The results showed that there was no death in the control group and cold stress group,20%in Pm group and 60%in cold+Pm group.At the same time,the results of gross anatomy and pathological section of lung tissue of mice showed that:In the control group,the lung tissue and pathological sections were normal;in the cold-stress group,the appearance of lung tissue appeared bleeding spots,a small amount of bleeding in the alveoli cavity;in the Pm group,there were small areas of necrosis on the lung tissue surface,obvious bleeding in the alveoli cavity,fusion of the alveoli wall,and a small amount of inflammatory cell infiltration;In the cold+Pm group,the results showed that there were large areas of necrotic foci and bleeding spots in the lung tissue of mice,the walls of alveoli were thickened,and inflammatory cells and lymphocytes were infiltrated.3.The ratio of Th17 and Treg cells in peripheral blood of mice was detected by flow cytometry after cold stress treatment,Pasteurella multocida intranasal infection or both treatment respectively.The results showed that the Th17 cells in peripheral blood of mice induced by cold stress were significantly higher than those in the control group(P<0.05),but significantly lower than those under cold stress of Pasteurella multocida intranasal infection,there was no significant change in the number of Treg cells(P<0.05);Nasal infection with Pasteurella multocida can induce Th17 cells in the peripheral blood of mice to be significantly higher than that in the control group(P<0.05),and there was no significant change in the number of Treg cells;the number of peripheral blood Th17 cells induced by cold stress+Pm nasal drip infection was significantly higher than that of the control group(P<0.05);The number of cells decreased significantly(P<0.05),resulting in the imbalance of Th17/Treg cells in the peripheral blood of mice.Therefore,in the cold stress state,intranasal infection with Pasteurella multocida of bovine origin can cause severe imbalance of Th17/Treg cells in the peripheral blood of mice.Therefore,under cold stress,intranasal infection with Bovine Pasteurella multocida can cause severe imbalance of Th17/Treg cell balance in the peripheral blood of mice.4.After cold stress treatment,Pasteurella multocida intranasal infection or combined treatment,the changes of cytokines(IL-17A,IL-6,IL-10,TGF-?)in peripheral blood of mice in each group were detected by ELISA.The results showed that IL-6 in cold stress group was significantly higher than those in the control group(P<0.05),but significantly lower than those in the combined effect group(P<0.05),IL-10 and IL-17 were significantly lower than those in the control group(P<0.05).The content of TGF-?was not significantly different from that of the control group;IL-6 in the Pasteurella multocida infection group was significantly higher than that in the control group and the Cold group(P<0.05),but significantly lower than that in the combined effect group(P<0.05),IL-10 was significantly lower than that in the control group(P<0.05),IL-17A and TGF-?had no significant difference with the control group,IL-17A and IL-6 in cold+Pm group were significantly higher than those in the control group(P<0.05),IL-10 and TGF-?in serum in cold+Pm group were significantly lower than those in the control group(P<0.05).It shows that cold stress can induce Pasteurella multocida nasal infection in mice to cause the expression of Th17 cell-related factors and the decrease of Treg cell-related cytokine expression,resulting in an inflammatory reaction.5.The m RNA expression of Th17/Treg cell-related cytokines(IL-17A,IL-6,IL-10,TGF-?,ROR?t,Foxp3)in the lung tissue of mice was detected by f real-time RT-PCR after cold stress treatment,Pasteurella multocida intranasal infection or both treatments.The results showed that the expression of IL-17A and ROR?t mRNA in lung tissue of mice in cold stress group was significantly higher than that in control group(P<0.05),TGF-??IL-10 and Foxp3 were significantly lower than that in control group(P<0.05),and the expression of IL-6 was not significantly different from that in control group(P>0.05);the expression of IL-17A and ROR?t in lung tissue of mice in Pasteurella multocida infection group was significantly higher than that in control group(P>0.05)The expression of IL-6 mRNA was significantly higher than that of the control group(P>0.05),but the expression of TGF-?,IL-10 and Foxp3 mRNA was significantly lower than that of the control group(P>0.05);The expression of IL-17A,IL-6 mRNA and ROR?t mRNA in cold stress+Pm nasal drops infection group was significantly higher than that in the control group(P>0.05);and the anti-inflammatory factors IL-10 and TGF-?mRNA and Treg cells were related The expression level of Foxp3 mRNA of the transcription factor decreased significantly(P>0.05).Therefore,cold stress,Pasteurella multocida nasal drops infection and cold stress+Pasteurella multocida infection can cause changes in the expression of Th17/Treg cell-related cytokines in mouse lungs.Pasteurella spp.Infections have the most severe effects and can cause inflammatory reactions in the lung tissue of mice.In summary,mice were inoculated with 5?10~2 CFU/m L bovine Pasteurella multocida capsular serotype A in 2-5?,60%humidity,4-5 wind speed,and fed with cold stress for 7 days continuously.The mice model of bovine Pasteurella multocida in cold stress was successfully constructed.At the same time,it was clear that under this condition,Induces imbalance of Th17 cell/Treg cell immune balance in peripheral blood and lungs of mice,the proportion of Th17 cells increased,the expression of Th17 related cytokines(IL-17A?IL-6?ROR?t)was up-regulated,the proportion of Treg cells was down regulated,the expression of related cytokines(IL-10?TGF-??Foxp3)was down regulated,and induce systemic and pulmonary inflammatory responses in mice. |
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