【作者】 代敏；

【导师】 彭成；

【作者基本信息】 成都中医药大学 ， 中药学， 2010， 博士

【摘要】 目的通过研究TCMAI对奶牛乳腺炎耐药菌的体内外抗菌活性、临床疗效及对奶牛肝肾功能的影响及其肌肉刺激性和急性毒性,开展其在防治奶牛乳腺炎方面的有效性和安全性评价。分析TCMAI对大肠埃希菌和金黄色葡萄球菌生长及其超微结构的影响,探讨TCMAI的抗菌作用机制。方法全自动微生物分析系统和16S rDNA序列分析技术鉴定分离于四川成都、绵阳和眉山的奶牛乳腺炎病原菌,纸片扩散法分析其对8种抗菌药物的敏感性；琼脂平板二倍稀释法测定包括清热类、解表类、化湿类、收涩类等78味单味中药粗提物、4味中药抗菌活性部位和创新中兽药TCMAI对乳腺炎病原菌的体外抑菌活性；耐药金黄色葡萄球菌和大肠埃希菌感染小鼠保护试验和奶牛乳腺炎临床试验研究TCMAI防治奶牛乳腺炎的有效性；肌肉刺激性试验、小鼠急性毒性试验及奶牛肝肾功能等血生化指标分析研究TCMAI的安全性；生长曲线法和电子显微镜技术探讨TCMAI的抗菌作用机制。结果分离鉴定奶牛乳腺炎病原菌120株,其中15株(12.5%)对青霉素、链霉素、红霉素、四环素、庆大霉素、环丙沙星、头孢噻吩和头孢噻肟敏感,14株(11.67%)中介,91株(75.83%)耐药,以对青霉素(67.21%)和链霉素(64.81%)的耐药率最高,且多数耐药菌株呈多重耐药。清热解毒药拳参、清热燥湿药黄连、解表药香薷、化湿药厚朴、收涩药诃子和其它类药物地榆对奶牛乳腺炎耐药菌具有较强的体外抗菌活性。创新中兽药TCMAI除对受试的3株单胞菌无抑菌活性外,对其余15个种属的117株耐药菌和敏感菌均有较强的体外抑菌活性,MIC为0.26～8.17mg/ml; 0.49g/kg TCMAI肌肉注射对金黄色葡萄球菌耐药菌和大肠埃希菌耐药菌感染小鼠有100%的保护力,并呈量效关系；4.08mg/kg TCMAI乳池灌注和肌肉注射对19头奶牛26个乳区临床型乳腺炎奶牛的头治愈率和乳区治愈率分别为89.47%和88.46%,总有效率为100%；乳区肿胀平均消除时间为2.1d。TCMAI对4只兔肌肉刺激性试验的总反应分值为8分,肌肉注射和腹腔注射小鼠的LD5o分别为1.873g/kg和0.442g/kg。亚抑菌浓度TCMAI能明显延长金黄色葡萄球菌的迟缓期,延缓其生长速度,繁殖方式异常,出现三分裂、四分裂等多种分裂方式,胞壁和胞膜损伤,胞浆内容物溶解,细胞空化死亡；亦能明显延长大肠埃希菌的迟缓期,显著减少其生长量,损伤胞膜,胞浆内容物渗漏,细胞空化死亡结论TCMAI具有广谱抗菌活性,安全性高、毒副作小,对奶牛乳腺炎耐药菌具有极强的体内、外抗菌活性,对临床型乳腺炎的治愈率高,能用于治疗各种耐药菌和敏感菌引起的临床型乳奶牛乳腺炎。更多还原

【Abstract】 Objective:The objectives of the study were to evaluate the efficacy and safety of TCMAI which discovered from traditional Chinese medicine. Based on the antibacterial effect, the antibacterial mechanism of TCMAI was studied by electron microscope technique.Methods:Pathogenic bacteria for dairy cow mastitis from chengdu, mianyang and meishan were identified by methods of automatic microbe analytic system and sequence analysis of 16S rDNA, and susceptibility to 8 antimicrobial agents was determined by K-B method.In vitro agar plate diffusions was used in testing susceptibility to crude extract of 78 chinese medicines, antibacterial action of 4 Chinese medicines and TCMAI discovered. However, in vivo antibacterial activity of TCMAI was detected by mice infected by resistant staphylococcus aureus and escherichia coli, and clinical trial of dairy cow mastitis. The antimicrobial mechanism was processed by electron microscope technique and growth curves of S.aureus and E.coli. The availability of TCMAI was evaluated through analyzing antibacteria effect and clinical effect. The safety of TCMAI was evaluated through analyzing rabbit muscle stimulation test, acute toxicity test in mice, as well as the change of liver and renal function in cows with mastits.Results:120 strains pathogenic bacteria for dairy cow mastitis were obtained, and 87.5% isolates showed different degrees of resistance to 8 antibiotics. Among these 8 antibiotics tested, most bacteria showed higher resistance to penicillin(67.21%) and streptomycin(64.81%). Among these 78 Chinese medicines tested, Bistort rhizome, coptis chinensis, elsholtzia, magnolia officinalis, garden burnet and fructus chebulae showed higher antibacterial effect to pathogenic resistant bacteria from dairy cow with mastitis. Except for 3 strains maltophilia, TCMAI showed extremely strong antibacterial activity for 117 strains tested bacteria, and MIC was 0.26～8.17mg/ml. Moreover, for the mice infected by resistant S. aureus or E.coli, TCMAI had high protective capacity in dose-dependent manner if mice was given prevention medcine by muscular injection. On the basis of breast swelling and clinical mastitis history, and so on,26 quarters in 19 cows with clinical mastitis were treated with TCMAI by breast perfusion and muscular injection, and 13 quarters in 10 cows were treated with antibiotics. TCMAI therapy demonstrated cure rates at animal level and at quarter level was,89.47% and 88.46%, respectively. However, cure rates of antibiotic controls at animal level and at quarter level was 80.00% and 847.62%, respectively. In addition TCMAI could rapidly eliminate the breast swell caused by bacteria. LD50 of TCMAI was respectively 1.873g/kg and 0.442g/kg, as it were given into the mice by muscular injection and intraperitoneal injection. Low muscle stimulation showed that TCMAI could be used in injection. The results of study on antibacteiral mechanism suggested that TCMAI significantly prolonged retardation stage and decreased growth reproductive speed of S. aureus and E.coli. Cell content was gradually dissolved because of cell structure damaged, and lastly bacteria died of empty cell. In addition, reproductive mode of S. aureus was changed, It is that most binary fission was substituted by multiple division.Conclusions:For bovine mastitis caused by resistant and sensitive bacteria, TCMAI is extremely attractive candidates as therapeutic agents due to their wide spectrum of antimicrobial activity and mechanism of action. Further more, it has lower toxicity and higher safety.更多还原