【作者】 张园；

【导师】 剡根强；

【作者基本信息】 石河子大学 ， 预防兽医学， 2008， 硕士

【摘要】 奶牛乳房炎是引起奶牛业最严重的疾病之一,引起奶牛乳房炎的主要病原体是细菌已被人们所认识,但目前很多国家和地区发现了真菌引起的奶牛乳房炎逐渐增多,给本病的防治带来难度。本试验对新疆北疆部分地区临床型乳房炎奶样进行了真菌病原分离鉴定,调查了真菌在我国新疆北疆部分地区奶牛乳房炎的感染情况,并对分离株进行了形态学、生化特性及分子生物学鉴定,药物敏感性和动物致病性试验,调查结果表明:1、从121团、142团、150团、126团、128团、129团、130团、西部牧业牛场、伊犁地区及克拉玛依地区10个规模化奶牛场临床型和隐性型乳房炎采集的923份奶样中,检出250份真菌,真菌的检出率为27.09%,各个牛场酵母样真菌的检出率为100%,各个牛场检出率为42.41%-7.0%,以白色念珠菌为主,部分牛场同时检出丝状真菌,检出率为17.40%-7.41%,说明各个地区规模化养殖场奶牛均有不同程度的真菌性乳房炎存在。2、对检出比例最高的酵母样真菌,进一步做芽管试验、微培养法观察出芽和厚壁孢子,并结合发酵和同化试验对分离株进行鉴别,结果均为念珠菌属及念珠菌属中的白色念珠菌。3、根据GenBank中白色念珠菌18SrRNA序列(AB013586),采用Oligo6.0设计了一对白色念珠菌的特异性引物。提取29株分离株、标准白色念珠菌的染色体DNA,扩增出310bp的特异性条带,阴性对照大肠杆菌、金黄色葡萄球菌均无扩增带,表明扩增的特异性。最小可以检测到4.1pg的DNA,敏感性较高,可用于临床乳房炎奶样中白色念珠菌的快速检测。4、对各地区主要分离酵母样真菌进行药物敏感性和动物致病性试验,药敏试验结果表明:64株酵母样真菌对制霉菌素表现高敏,两性霉素B表现敏感,对伊曲康唑、氟康唑、酮康唑分别有4、22、10株表现敏感,分别有7、10、27株菌株表现高敏,分别有7、25、21株菌株表现敏感,对头孢芬噻肟、青霉素钾、庆大霉素、硫酸链霉素、5%盐酸土霉素、恩诺沙星、乙酰螺旋霉素、四环素、氟哌酸、氨苄青霉素、磺胺等抗生素均不敏感;用60只小鼠接种酵母样真菌培养物,结果表明:临床乳房炎分离到的真菌60株有56株无致病性,有致病性的4株分离株对氟康唑、酮康唑耐受,这就给临床乳房炎的治疗造成一定的困难。更多还原

【Abstract】 Cow mastitis can cause dairy industry one of the most serious diseases ,causing cow mastitis is the major bacterial pathogens have been recognized, but many countries and regions found that the cow mastitis caused by fungus gradually increased, it bring difficulty of disease Control.The test to parts of the North of Xinjiang clinical mastitis milk samples be Isolated and identified of the fungal pathogen, survey epiphyte infection in our in North Xinjiang parts of the cow mastitis, and it be the morphologic、biochemical properties and molecular biology Identification, drug sensitivity and animal pathogenic test, the survey showed that:1、from 121 Farm, 142 Farm, 150 Farm, 126 Farm, 128 Farm, 129 Farm, 130 Farm, the west Animal Husbandry cattle ,YiLi area and KeLaMaYi area 10 large-scale dairy farm clinical and recessive mastitis Acquisition 923 of the milk samples , fungi were founded of 250, the detection rate of fungi is 27.09 percent, all cattle yeast-like fungi detection rate is 100%, the detection rate is 42.41% -7.0%, the main fungi is Candida albicans;parts of cattle filamentous fungi were detected at the same time, the detection rate is 17.40% -7.41%, it show that fungal mastitis has different levels of exist in all parts of the cow farms.2、to the highest proportion the detection of of yeast-like fungi, do further germ tube experiments, observe the budding and thick-walled spores, conjunction with fermentation and assimilation test to identified, the results are the Candida and Candida albicans of Candida.3、according to the GenBank sequence of Candida albicans 18 SrRNA (AB013586), using Oligo6.0 to design Candida albicans specific primers. Extraction of 29 isolates, standards Candida albicans chromosome DNA, 310 bp of the specificity strip was extended , the negative comparison Escherichia coli、Staphylococcus have no specificity strip.Minimum 4.1 pg DNA can be detected, it has high sensitivity and can be used for clinical mastitis in dairy rapid detection of Candida albicans.4、to do the separation of the different regions of main yeast-like fungi drug sensitivity and animal pathogens testing, drug sensitivity test results showed that: to nystatin 64 yeast-like fungi are high-sensitivity, amphotericin B are sensitive, for Itraconazole, fluconazole, and ketoconazole it respectively have4、22、10strains are sensitive, 7、10、27 strains are high-sensitivity, and 7、25、21strains are sensitive, to cefotaxime Sodium、benzylpenicillin potassium、Gentamicin、streptomycin sulfate、oxytetracycline hydrochloride、enrofloxacin、Acetylspiramycin、Tetracycline、norfloxacin、ampicillin、sulfanilamide are not sensitive;60 mice inoculated with yeast-like fungi culture, the results showed that: the 56 strains isolated from clinical mastitis have no pathogenic, the 4 strains with pathogenic are tolerance to fluconazole ketoconazole, which gives clinical mastitis causing certain difficulties of treatment.更多还原