【作者】 杨洪森；

【导师】 刘长松； 曹随忠；

【作者基本信息】 四川农业大学 ， 临床兽医学产科， 2011， 硕士

【摘要】 乳房炎是奶牛业生产中危害最大的疾病之一。目前,应用动物模型进行研究是解决细菌性奶牛乳房炎的发病机理和临床症状研究的重要手段。通过动物模型进行乳房炎的研究,不仅可以为疾病的发生、发展以及由此病引发的动物机体组织的改变提供重要线索,而且为新型抗菌药物的开发提供有效信息。为了寻找更为典型廉价、易于建立的乳房炎动物模型,同时也为乳房炎的发病机理研究、诊断、药物治疗效果评价及预后提供科学依据,进行了以下研究：1.家兔乳头管灌注金黄色葡萄球菌和大肠杆菌建立乳房炎动物模型研究48只分娩后7d的健康新西兰泌乳家兔,随机分成空白对照组(T1组)、金黄色葡萄球菌组(T2组)和大肠杆菌组(T3组)。分别按每个乳腺1.5×105CFU/0.5mL量,向家兔乳头管内灌注无菌肉汤、金黄色葡萄球菌菌液和大肠杆菌菌液,于灌注无菌肉汤和细菌前2h,灌注后.24h、48h、72h、5d和7d,测定家兔体温,观察活体家兔乳房临床质地变化,并采集家兔耳缘静脉血和乳腺组织,测定全血中LYM和GRA总数,以及血清中CRP和TNF-α含量与乳腺组织中TNF-α含量,制作乳腺组织病理切片并观察乳腺组织的动态病理变化。结果显示：(1)家兔乳头管灌注两种细菌后体温均出现了升高趋势,血液中WBC、LYM和GRA总数也出现了相应的变化,表明机体出现炎症反应。(2)T2组和T3组血清TNF-α含量在灌注细菌后均呈下降趋势,T2组组织中TNF-α含量在灌注细菌后6h时显著下降(P<0.05),48h时显著升高(P<0.05),T3组组织中TNF-α含量在灌注细菌24h时显著升高(P<0.05)；机体炎症出现时,血清中CRP含量相应程度的升高。(3)在家兔乳头管灌注细菌12h后均出现了乳房红肿、质地变硬等临床症状,乳腺组织12h时开始出现大量乳腺上皮脱落和炎性细胞增多等病理变化。上述三点表明成功建立了家兔乳房炎模型。2.家兔乳头管灌注金黄色葡萄球菌和大肠杆菌对血清中SOD、GSH-PX和NOS活性及NO含量的影响在研究1的基础上,测定血清中SOD、GSH-PX和NOS活性及NO含量,分别分析不同细菌感染的乳房炎指标之间的差异性。结果显示：(1)灌注细菌后,T2组血清SOD和GSH-PX活性显著升高(P<0.05),T3组血清SOD活性显著下降(P<0.05),而GSH-PX活性显著升高(P<0.05),T1组血清中SOD活性变化差异不显著(P>0.05)。这表明,家兔感染金黄色葡萄球菌和大肠杆菌性乳房炎时,机体内SOD和GSH-PX活性不同。(2)灌注细菌后,T2组血清NO含量和iNOS活性显著下降(P<0.05), TNOS活性显著升高(P<0.05)；T3组血清中NO在72h时显著下降(P<0.05), TNOS活性稍有升高趋势,iNOS活性12h时显著下降(P<0.05)；T1组血清中NO含量和TNOS活性显著下降(P<0.05),iNOS活性在48h时显著升高(P<0.05)。这表明,在金黄色葡萄球菌和大肠杆菌所引起的乳房炎中,NOS活性不同,对机体的NO含量影响不同。3.家兔乳头管灌注金黄色葡萄球菌和大肠杆菌对血清和组织中HP、SAA及S100A12含量的影响在研究1的基础上,测定血清和组织中SAA、HP和S100A12含量,分别分析金黄色葡萄球菌和大肠细菌感染的乳房炎指标之间的差异性。结果显示：(1)T2组血清和组织中SAA含量在接种细菌后6h时显著升高(P<0.05)；T3组血清中SAA含量接种细菌后6h时显著升高(P<0.05),组织中SAA含量接种细菌后6h时显著下降(P<0.05),7d时显著升高(P<0.05)；T1组血清中SAA含量在接种无菌肉汤后稍微升高,组织中SAA含量变化不明显。表明SAA对炎症反应敏感。(2)接种细菌后,T2组血清HP含量组内差异不显著(P>0.05),组织中HP含量升高；T3组血清中HP含量再接种细菌后6h时显著下降(P<0.05),7d时显著升高(P<0.05),组织中先升高,之后缓慢下降；T1组变化不明显。表明机体损伤时组织和血清中HP含量出现相应变化。(3)T2组在接种细菌48h后血清中S100A12含量显著升高(P<0.05),组织中S100A12含量在12h时显著升高(P<0.05)；T3组在接种细菌后血清中S100A12含量变化不明显,组织中S100A12在48h时显著升高(P<0.05),T2组和T3组差异显著；T1组血清中S100A12显著升高(P<0.05),组织中S100A12变化不明显。表明在家兔金黄色葡萄球菌和大肠杆菌引起的乳房炎中,组织和血清中的S100A12含量均出现一定变化,金黄色葡萄球菌组和大肠杆菌组差异显著(P<0.05)。更多还原

【Abstract】 Bovine mastitis is one of the most harmful diseases in dairy productions. Recently, It detected that when researching the pathologic mechanism and clinical symptoms of bovine bacterial mastitis. The research by applying animal models is an important trick to work on mastitis. The methods not only offer important clews for the nosogenesis and development of mastitis, then in result of fomenting the alteration of animal tissues, but also offer effective information for the exploition of neotype antibacterials through trial and error with animal. With the purpose of searching for the more classic, low-cost, and easily established animal mastitis model, and simultaneously in order to supply scientific accordance of studying the pathologic mechanism, diagnose, pharmacotherapy effectives and prognosis of mastitis, the following sequentiase researches were carried out.Research 1. Establishment of experimental mastitis model by infusing Staphylococcus Aureus and Escherichia coli via teat duct in rabbit.48 healthy rabbits of parturition 7d were divided into control group (T1), Staphylococcus Aureus group (T2) and Escherichia coli group (T3) randomly,16 rabbits per group. Staphylococcus Aureus and Escherichia coli,1.5×105 CFU, were infused into glands of rabbit. The ear venous blood and breast tissue were collected at 2h before, and 6h,12h,24h,48h,72h,5d,7d after infusion with asepsis broth and bacterial respectively. Breast textures of rabbit were observed at different time point. Body temperature and WBC, LYM, GRA, contents of CRP, TNF-αof serum and TNF-αof breast tissue, and the variations of dynamic pathology of gland tissue through making pathological section were observed. The result showed that (1) Body temperature of rabbit showed up step-up tendency, and WBC, LYM, GRA also present variety correspondingly, this make clear that engine block had determinate inflammatory reaction. (2) The concentration of TNF-αin serum submit downturn in T2 and T3, in tissue, TNF-αincreased significantly(P<0.05), the concentration of CRP in serum increased correspondingly. (3) Rabbits appeared with clinical symptoms of acute mastitis after infusing bacterial via teat candal. Breast tissue also showed up athological change correspondingly. So it was indicated that experimental mastitis model had been successfully established.Research 2. Effects of infusing Staphylococcus Aureus and Escherichia coli via teat duct in rabbit on activities of SOD, GSH-PX, NOS, contents of NO in serum.The method of dividing groups of rabbits, infusing bacterial via teat and collecting blood were same as research 1. The activities of SOD, GSH-PX and NOS, content of NO in serum were determined at different time point. Then analyzed the variability within index from different bacterial. The results showed that (1) The activities of SOD and GSH-PX in serum significantly increased in T2 (P<0.05), SOD descent and GSH-PX increased significantly in T3(P<0.05). The change of group T1 was not significant(P>0.05). It makes clear that inflammation is different when rabbits infected mastitis caused by Staphylococcus Aureus and Escherichia coli because of the different contents of ROS. (2) The concentration of NO and activities of iNOS decreased significantly(P<0.05), activities of TNOS increased significantly(P<0.05) in T2. In T3, the concentration of No decreased significantly at 72h(P< 0.05). The activities of iNOS decreased significantly at 12h(P<0.05) and TNOS increased slightly. In T1, the concentration of NO and activities of TNOS increased significantly(P<0.05), iNOS increased significantly at 48h(P<0.05). It makes clear that engine block catalysis NO synthesis of NOS is different. The damage to engine block is different when rabbits infected mastitis caused by Staphylococcus Aureus and Escherichia coli.Research 3. Effects of infusing Staphylococcus Aureus and Escherichia coli via teat duct in rabbit on contents of SAA, HP, S100A12 in serum and tissue.The method of dividing groups of rabbits, infusing bacterial via teat and collecting blood were same as research 1. The contents of SAA, HP, S100A12 in serum and tissue were determined at different time point, then analyzed the variability within index from different bacterial. The results showed that (1) The concentration of SAA increased significantly in serum and tissue in T2(P<0.05). In T3, SAA increased significantly in serum and descent significantly in tissue(P< 0.05). In T1 SAA increased slightly in serum, the change was not significant(P>0.05). This make clear that SAA is sensitive fairly in inflammation. As long as inflammation occurrence, the concentration of SAA increase. (2)The concentration of HP in serum was not significant and increased in tissue in T2. HP decreased significantly in serum in T3(P< 0.05), increased significantly at 7d(P<0.05), the change was not significant in tissue. T1 was not obvious. This make clear that organism released multiplicity of Hb, Hb bind with HP result that the content of HP descent. (3)After 48h of infusion bacterial the content of S100A12 increased significantly in serum in T2(P<0.05), increased significantly at 12h in tissue(P<0.05). In T3, the change high or low of S100A12 showed alternately in serum, increased significantly in tissue at 48h(P<0.05).T1 increased significantly in serum (P<0.05)and not obviously in tissue. This make clear that the change showed up in serum and tissue when inflammation. T2 and T3 was different significantly(P<0.05). Account that, mastitis infect from Staphylococcus Aureus and Escherichia coli are different.更多还原