羊支原体性肺炎两标准株对抗菌药物敏感性的研究

本实验测定了环丙沙星、氧氟沙星、单诺沙星、红霉素、罗红霉素、泰勒菌素、泰妙菌素、四环素等8种药物对羊肺炎支原体两个标准株Y98和Y-goat的体外抑菌浓度以及红霉素与氧氟沙星、泰勒菌素对Y-goat和四环素与氧氟沙星、泰勒菌素对Y98的联合药敏作用。对羊肺炎支原体两个标准株对抗菌药物的敏感性进行了分析。     

山羊传染性胸膜肺炎支原体和绵羊肺炎支原体对抗菌药物敏感性的研究

测定了环丙沙星、氧氟沙星、单诺沙星、红霉素、罗红霉素、泰乐菌素、泰妙菌素、四环素等8种药物对羊肺炎支原体两个标准株Y-98和Y-goat的体外抑菌浓度以及红霉素与氧氟沙星、泰乐菌素对Y-goat和四环素与氧氟沙星、泰乐菌素对Y-98的联合药敏作用.结果表明,这8种抗菌药物对Y-goat和Y-98的MIC(μg/mL)分别为:环丙沙星0.223、0.002 23,氧氟沙星0.281、0.014 0,单诺沙星0.136、0.014 0,红霉素0.021 8、无效,罗红霉素0.032 7、无效,泰乐菌素0.042 2、0.039 0,泰妙菌素0.021 7、0.052 0,四环素0.195、0.052 0.红霉素与氧氟沙星的联合药敏指数为1,是相加作用;红霉素与泰乐菌素对Y-goat的联合药敏指数为1.5,是无关作用;四环素与氧氟沙星、泰乐菌素对Y-98的联合药敏试验指数均为0.375,是协同作用.     

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

AIM: To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae (MP)-infected rat lung tissues and to explore its mechanism. METHODS: SD rats (n=40) were randomly divided into 4 groups:blank control group, model group, Jiedu-Qingfei group and positive control group, with 10 rats in each group. The rats in experimental groups were slowly dripped with 1×10⁹ CFU/L MP solution into their nostrils for 4 d. One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation, and the other rats were given gavage therapy. The rats in blank control group and model group were intragastrically given the same volume of normal saline, the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks, and the rats in psoitive control group were given dexmethasone sodium phosphate (0.5 mg·kg^-1·d^-1). After the experiment, the rats were killed. The serum and bronchoalveolar lavage fluid (BALF) were collected for detecting the levels of interleukin-12 (IL-12), IL-13 and TNF-α by ELISA. The right lung tissues were used for pathological observation and HE staining, while the left lung tissues were used to detect the expression of NF-κB p50, I-κBα and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels. RESULTS: The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive, indicating that the rat model of MP infection was successfully established. On the 1st day of the treatment, the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group (P<0.05). After treatment, the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group. The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection (P<0.05), while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group (P<0.05). The levels of IL-13 and TNF-α in the serum and BALF of MP-infected rats were increased significantly, while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group (P<0.05). The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly (P<0.01). After treatment, the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group (P<0.01). The mRNA expression level of I-κBα in model group was significantly lower than that in control group. After treatment, the mRNA expression of I-κBα in Jiedu-Qingfei group was significantly higher than that in model group (P<0.05). The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group. After treatment, the protein expression of NF-κB p50 and p38 MAPK was decreased significantly. The protein level of I-κBα in model group was significantly lower than that in blank control group, and after treatment with Jiedu-Qingfei mixture, the protein expression level of I-κBα was increased significantly (P<0.05). CONCLUSION: Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.     

Mycoplasma pneumoniae induces IL-1β production through activating NLRP3 inflammasome by ROS in RAW264.7 cells

AIM: To investigate whether Mycoplasma pneumoniae (Mp)-induced interleukin-1β (IL-1β) production in RAW264.7 cells is through the activation of NLRP3 inflammasome via reactive oxygen species (ROS). ME-THODS: RAW264.7 cells were randomly divided into 3 groups. In normal group, RAW264.7 cells were treated without Mp. In model group, RAW264.7 cells were treated with 1∶ 10 multiplicity of infection (MOI) of Mp. In NAC group, RAW264.7 cells were pretreated with N- acetylcysteine (NAC) at a concentration of 5 mmol/L for 30 min before infection with Mp. The RAW264.7cells were infected with Mp (1∶ 10 MOI) for 4, 8, 16 and 24 h in model group and NAC group, respectively. The intracellular ROS level was analyzed by flow cytometry. The mRNA expressions of NLRP3, ASC and caspase-1 were detected by real-time PCR. The protein levels of NLRP3, ASC and caspase-1 p20 were determined by Western blot. The levels of pro-inflammatory cytokine IL-1β in the supernatant were measured by ELISA. RESULTS: Compared with normal group, the production of ROS were significantly increased at 4, 8, 16 and 24 h after infection, the mRNA expression of NLRP3, ASC and caspase-1 were increased at 8, 16 and 24 h after infection, the protein levels of NLRP3, ASC and caspase-1 p20 were increased at 16 and 24 h after infection, and the releases of IL-1β were increased at 24 h after infection in model group (P<0.01). Compared with the model group, the level of ROS in NAC group decreased, so as the expression of NLRP3, ASC and caspase-1 at mRNA and protein levels and the releases of IL-1β in the supernatant at the corresponding time points. CONCLUSION: Mp may stimulate the ROS production to activate NLRP3 inflammasome in RAW264.7 cells.     

Immunogenic properties of Landrace pigs selected for resistance to mycoplasma pneumonia of swine

Mycoplasma pneumonia of swine (MPS) lung lesions and immunogenic properties were compared between a Landrace line that was genetically selected for reduced incidence of pulmonary MPS lesions, and a non‐selected Landrace line. The MPS‐selected Landrace line showed significantly lower degrees of pulmonary MPS lesions compared with the non‐selected Landrace line. When changes in immunity before and after vaccination were compared, the percentage of B cells in the peripheral blood of the MPS‐selected Landrace line was significantly lower than that of the non‐selected line. Furthermore, the concentration of growth hormone and the mitogen activity of peripheral blood mononuclear cells in the MPS‐selected Landrace line showed significantly (P < 0.05) lower increases after vaccination than the non‐selected line. Conversely, the concentration of peripheral blood interferon (IFN)‐γ and salivary immunoglobulin A (IgA) after Mycoplasma hyopneumoniae vaccination was significantly higher in the MPS‐selected Landrace line than in the non‐selected line. Gene expression of toll‐like receptor (TLR)2 and TLR4 was significantly higher in the MPS‐selected Landrace line in immune tissues, with the exception of the hilar lymph nodes. The present results suggest that peripheral blood IFN‐γ, salivary IgA TLR2, and TLR4 are important immunological factors influencing the development of MPS lesions.     

Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics of pig lines generated by crossing an MPS pulmonary lesion selected Landrace line and a highly immune capacity selected Large White line

To understand the influence of crossbreeding on Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics, two crossbred lines were characterized. One crossbred line, LaWa, was generated by crossing the MPS pulmonary lesion selected Landrace line (La) and the highly immune‐selected Large White line (Wa). The second crossbred line, LaWb, was generated by crossing the La line and the nonselected Large White line (Wb). The crossbred LbWb line (nonselected Landrace line × nonselected Large White line) and the La line were used as controls. The LaWa and LaWb lines had an intermediate level of MPS lung lesions between La and LbWb lines, although the difference was not statistically significant. After stimulation with sheep red blood cells (SRBCs), the LaWb and LaWa lines showed immune characteristics similar to that of the La line; the number of monocytes in peripheral blood increased, while B cells, T cells, secretion of SRBC‐specific immunoglobulin G, and interleukin (IL)‐13 decreased. Additionally, the number of natural killer (NK) cells and the expression of IL‐4 and IL‐17 were significantly higher in the LaWb and LaWa lines, respectively. These data suggested that crossbreeding of La and Wa lines resulted in the inheritance of some of the selected immune responses.     

猪源肺炎克雷伯菌的分离与鉴定

从9只病死猪肺、肝脏中分离到4株革兰氏阴性杆菌,并对分离菌株进行形态学、培养特性、生化特性和分子生物学的鉴定,确定分离的菌株为肺炎克雷伯菌肺炎亚种。根据GeneBank上肺炎克雷伯菌16S rRNA可变区序列设计1对引物,4株细菌基因组DNA均可扩增到1段大小为127 bp特异性片段,经测序与已知肺炎克雷伯菌相应序列的同源性为100%,鉴定4株细菌均为猪源肺炎克雷伯菌。动物毒性试验证明其对小鼠有较强的致病性。     

石河子地区某规模化奶牛场肺炎克雷伯菌的分离鉴定及耐药性分析

为了阐明引起石河子地区某规模化奶牛场犊牛呼吸道症状的主要细菌性病原体及其生物学特性，本研究采集2~6月龄犊牛鼻拭子与肛拭子各39份，通过细菌分离培养、形态学观察、生化分析、PCR扩增16S rRNA基因和溶血酵素（khe）基因、药敏试验和小鼠致病性试验等方法对分离菌株的生物学特性进行分析。结果显示，分离菌株中有3株在MIAC平板上形成紫红色带有沉淀环的菌落（鼻拭子1株，肛拭子2株），且镜检为革兰氏阴性短杆菌，疑为肺炎克雷伯菌。全自动微生物分析系统显示，分离株与肺炎克雷伯菌相似性均为96%；PCR扩增16S rRNA基因测序结果显示，分离株与GenBank数据库中肺炎克雷伯菌核苷酸相似性在96.5%~99.8%之间，肺炎克雷伯菌特异性基因khe阳性且相似性达99%以上；药敏结果显示，分离菌株对青霉素类、头孢菌素类、一代和二代氨基糖苷类、四环素类、一代大环内酯类、磺胺类、多烯类、林可酰胺类抗菌药呈现出不同程度的耐药；对三代氨基糖苷类、二代大环内酯类、氯霉素类、多肽类、喹诺酮类抗菌药敏感，且呈现不同程度的多重耐药性；致病性试验结果表明，分离菌株均可不同程度导致小鼠死亡且以鼻拭子分离株致病性较强。本研究成功分离鉴定石河子地区规模化奶牛场引起犊牛呼吸道症状的肺炎克雷伯菌3株，并阐明分离菌的部分生物学特性，为新疆地区牛源肺炎克雷伯菌病的检测、诊断及临床治疗提供技术支撑。     

白虎汤对鸡毒支原体体外抑制作用及其超微结构的影响

目的:观察鸡毒支原体(MGPG31)形态和超微结构变化,探讨白虎汤体外对MGPG31抑制作用的机理。方法:(1)用二倍稀释法测定白虎汤对MGPG31的MIC;(2)在MIC(0.094g/ml)和亚MIC(0.0235g/ml)的条件下取白虎汤与鸡毒支原体培养48h后的样品,通过透射电镜观察支原体的形态和超微结构变化。结果:白虎汤对鸡毒支原体的MIC1和MIC2分别为0.047g/ml和0.094g/ml。白虎汤主要作用于繁殖期支原体,影响细胞膜通透性和形态而起到抑制作用。