【作者】 王永生；

【导师】 张乃生；

【作者基本信息】 吉林大学 ， 兽医， 2011， 博士

【摘要】 奶牛子宫内膜炎是危害畜牧业生产发展的主要产科疾病,每年在世界范围内造成巨大的直接损失和间接损失。迄今为止,对其研究已经有近百年的历史,但由于其病原不明确、病理学机制尚未阐述清楚等多方面的原因,导致此疾病的防控及治疗仍然没有有效的方法。因此,寻找快速有效的病原检查方法,探索相应的治疗措施,选择有针对性的治疗药物等措施,是对本病研究的关键。本研究通过对奶牛子宫内膜炎真菌病原的巢式PCR-DGGE检测方法的建立和临床诊断效果的研究,既为奶牛子宫内膜炎病原学的临床检查建立快速、准确、有效的技术手段,也为以后有针对性的药物研究提供了指导和理论帮助,为奶牛子宫内膜炎有效的防控及治疗提供科学依据。本论文分四个阶段完成,首先利用传统的方法对子宫内膜炎患牛子宫内的真菌进行分离鉴定,并以此菌建立检测方法；其次在实验室条件下,建立奶牛子宫内膜炎真菌病原的巢式PCR-DGGE检测方法,并对其检测限、重复性等指标进行检测；然后结合临床实际应用,进一步完善该检测方法,并与传统方法进行比较；最后有针对性地给药,观察治疗效果。主要试验和结果如下：1.以传统方法分离到的病原性真菌作为参考菌种,在实验室条件下建立方法。利用真菌固有的18S rDNA保守区域设计通用引物,确立靶基因进行分析。结果表明：巢式PCR-DGGE方法检测灵敏度高、特异性强,且具有良好的可重复性。与传统方法比较,快速准确。临床应用该方法检测真菌病原,比传统方法更为优越,分离鉴定到的致病性真菌更为全面,适合应用于奶牛子宫内膜炎真菌病原的检测。2.利用药物对此方法的诊断效果进行检验,结果显示：根据巢式PCR-DGGE分析方法给以药物治疗,针对性强。对于真菌性子宫内膜炎奶牛的治疗具有确实的效果,实验动物病情的转归十分明确,缩短了治疗时间,降低滥用抗生素的风险。3.研究中应用巢式PCR-DGGE分析方法对吉林市某奶牛场奶牛子宫内的主要致病性真菌进行了流行病学调查,结果发现：念珠球菌属的白色念珠球菌、热带念珠菌、土曲霉为主要致病菌。一年四季子宫内膜炎均有发生,在秋冬季节真菌性子宫内膜炎的发生比例最高。更多还原

【Abstract】 Cow endometritis is a major obstetric disease which is harmful to the development of livestock production. It results huge direct and indirect losses in the worldwide each year. So far it has been studied for 100 years, but because its variable pathogens and pathological mechanisms were not clear and so many other causes, there still have no effective methods to the prevention of this disease. Therefore, the main points of this research are to find fast and effective methods of pathogen diagnosis, to explore the relative treating measures, and to give drugs more targeted. Through establishing the nest PCR-DGGE diagnosis method to cow endometritis fungal pathogen and studying the effect of clinical diagnosis, i.e., to establish accurate, rapid and effective techniques and tools for the clinical diagnosis of the etiology of cow endometritis. This research gives guidance and theory assistance for the targeted drug research and affords helps for the effective prevention and treatment of cow endometritis.This paper is divided into four parts, which the first part is that used a traditional method to isolate and to indentify the fungi in the cow’s womb which the cow has suffered from endometritis, regarded this as a comparison, and provided bacteria for the establishment of method. The second part is that established a nest PCR-DGGE diagnosis method to cow endometritis fungal pathogen under the present laboratory conditions, and tested its detection limit, repeatability, and so on. The third part is that combined with the actual clinical condition to apply this method and to improve it, and compared with the traditional method and made it more suitable for practical applications. The last part is that tested the effect of diagnosis by giving drugs purposely, observed the treating condition after giving medicine, and combined with the clinical symptoms to assure the disease prognosis. The main test and result are as follow:1、Regarded pathogenic fungi which was isolated by traditional method as reference strain, and established method under the present laboratory condition. Designed universal primers by the public 18S rDNA conserved regions of the fungi, and established target genes for analysis. The result shows that nest PCR-DGGE method was with highly sensitive detection, strong specificity and good reproducibility. Compared with the traditional method, it is more quickly and more accurate. It is superior to the traditional method on clinical application, and the isolation and identification of pathogenic fungi were more comprehensive. This method is adapted to the diagnosis of cow endometritis fungi pathogen.2、Tested the diagnosis effect of drug by using this method, the result shows that it had a stronger direction to give treating drug according to nest PCR-DGGE analysis method. This method had exact effect on the treatment of fungal endometritis cows, the disease prognosis of experimental animals were very clear, it greatly shorten the treating time, and it could reduce the risk of the abuse of antibiotics.3、The researchers applied nest PCR-DGGE analysis method to make epidemiological survey on the main pathogenic fungi in cow’s womb from Jilin dairy cow uterus. From the survey we found that the main pathogenic fungi were white candidiasis,candida tropicalis, which were belong to candidiasis, and aspergillus terreus. Endometritis have occurred throughout the year. Autumn and winter were the higher occurrence times of fungal endometritis.更多还原