奶牛乳房炎病例中金黄色葡萄球菌RAPD-PCR基因分型的研究

用随机扩增多态性DNA 技术（RAPD）对来自2个集约化奶牛场不同奶牛乳房炎病例分出的16株金黄色葡萄球菌进行了基因分型。分别用3个寡核苷酸引物扩增，仅OLP11引物可扩增出质量良好、具有RAPD特征的条带图谱。对反应体系的退火温度、模板浓度进行了筛选，模板原倍浓度、33℃的退火温度能使所有检测菌株产生清晰、可分辨、具有较好重复性的带谱。分型结果表明，16株菌可分为2个类群，其中14株位于同一类群，大多数菌株的相关系数差距仅为0.04，这一结果提示这些菌株可能具有相同的来源和相似的遗传背景。     

4种不同来源溶菌酶对引起奶牛乳房炎葡萄球菌的抑制作用

本试验分别采用微球菌比浊法、PAGE电泳法、考马斯亮蓝法、紫外分光光度法与MIC法测定了4种不同来源的溶菌酶样品(SK、GK、ZD、LY)的酶活力、酶含量、样品中是否含有抗生素及溶菌酶对奶牛乳房炎葡萄球菌的抑制作用.结果发现,4种溶菌酶样品中都不合有抗生素,其中细菌性溶菌酶SK的酶活力最高,对奶牛乳房炎葡萄球菌的抑菌效果最好.可作为奶牛乳房炎无抗治疗药物开发的重点.     

奶牛乳房炎源链球菌裂解噬菌体的分离鉴定

奶牛乳房炎对奶牛产业的影响严重,传统抗菌疗法面临细菌耐药和药物残留的问题,因此急需研发一种能解决现有抗菌药物弊端与危害的新型疗法.论文以奶牛乳房炎源链球菌为宿主菌,分离获得裂解性噬菌体,对其进行生物学特性分析,为奶牛乳房炎无抗防治提供生物学材料.试验采用常规方法分离、纯化噬菌体,通过双层琼脂平板法观察噬菌斑的形态并测定...     

噬菌体内溶素对奶牛乳房炎致病菌的溶解作用

奶牛乳房炎一直是困扰养牛业的顽疾。诱发奶牛乳房炎的致病菌通常包括金黄色葡萄球菌、无乳链球菌等致病菌,停乳链球菌、乳房链球菌、大肠埃希菌等环境致病菌以及表皮葡萄球菌、腐生葡萄球菌、模仿葡萄球菌等机会性致病菌。噬菌体是一类细菌依赖性的病毒,可通过内溶素抑制肽聚糖的合成,或利用穿孔素-内溶素系统水解肽聚糖。在体外试验中,噬菌体及其内溶素可溶解、抑制细菌生物膜,裂解乳房炎致病菌;在体内试验中,可清除感染动物体内的乳房炎致病菌,对奶牛乳房炎具有治疗效果。因此,噬菌体及其内溶素在奶牛乳房炎治疗方面具有良好的应用前景。     

甘肃地区牛源金黄色葡萄球菌分子鉴定及RAPD分型

本研究目的是分离鉴定引起甘肃地区奶牛乳房炎的金黄色葡萄球菌,掌握其基因型情况。利用16S、23SrRNA保守序列PCR扩增对乳房炎奶样中的金黄色葡萄球菌进行鉴定,并进行RAPD基因分型。结果表明,310份奶样中共分离出金黄色葡萄球菌100株,RAPD结果显示这100株金黄色葡萄球菌均可得到清晰的RAPD指纹图谱,扩增产物在2～7条带之间,具有多种带型组成。通过聚类分析100株菌产生11个基因型,其中Ⅰ型4株,Ⅱ型4株,Ⅲ型10株,Ⅳ型13株,Ⅴ型7株,Ⅵ型24株,Ⅶ型16株,Ⅷ型6株,Ⅸ型4株,Ⅹ型10株,Ⅺ型2株。Ⅵ型为该地区的流行优势菌群,不同牛场各基因型菌株分布有明显差异。本研究说明牛场的环境与养殖条件对病原菌流行传播有明显的影响,这一结果对地区性奶牛乳房炎的防治提供了可靠的理论依据。     

利福昔明对奶牛乳房炎常见病原菌的体外抗菌活性试验

旨在测定利福昔明对奶牛乳房炎常见病原菌的体外抗菌活性。采用琼脂二倍稀释法测定利福昔明对奶牛乳房炎常见病原菌大肠杆菌、葡萄球菌和链球菌的最小抑菌浓度（MIC）。结果显示,利福昔明对19株大肠杆菌的MIC值为8~16μg/m L,对25株葡萄球菌的MIC值为0.002~0.06μg/m L,对18株链球菌的MIC值为0.06~2μg/m L。大部分临床分离菌的MIC值与标准菌的相近,表明分离菌株对利福昔明较为敏感,提示利福昔明对奶牛乳房炎常见病原菌具有较强的体外抗菌活性。     

Investigation of Biofilm Formation,Virulence Genes and agr Typing of Staphylococcus aureus from Bovine Mastitis Cases

The objective of the present study was to investigate the biofilm formation, antimicrobial resistance, virulence genes, and agr genotypes of Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases, and to analyze the correlations between agr genotypes and virulence genes. Biofilm formation, antimicrobial resistance, and virulence genes for 336 strains of S. aureus were detected by microtiter plate method, disk diffusion method, and PCR respectively, and the agr typing of tested strains was determined by multiplex PCR. The results showed that all 336 strains of S. aureus from bovine mastitis were biofilm producers, among which 52.1% and 47.9% of isolates tested were moderate (++) and strong (+++) biofilm producers, respectively. Antimicrobial susceptibility testing demonstrated that S. aureus strains were highly resistant to penicillin, with a resistance rate of 91.7%, followed by erythromycin (89.6%), kanamycin (72.9%), clindamycin (66.7%) and gentamicin (60.4%). However, all isolates were sensitive to nitrofurantoin and linezolid. PCR results showed that the prevalence of fnbA gene was the highest (99.7%), followed by icaD (98.2%), icaA (89.6%), clfA (86.0%), cna (56.0%), and bap (14.6%) genes. Moreover, the sea, seb, sec and tst genes were found in 26.5%, 8.3%, 6.8% and 8.3% of the isolates, respectively. The agr typing results showed that S. aureus strains belonging to agr Ⅰ was predominant in our study, accounting for 77.1% of the isolates, and the frequencies of agr Ⅱ, agr Ⅲ and agr Ⅳ genotypes were 14.0%, 4.8% and 2.1%, respectively. Statistical analysis indicated that the strains of S. aureus belonging to agr Ⅰ genotype have the potential to carry more virulence genes, while no toxin genes could be found in any of the strains belonging to agr Ⅳ. The results revealed high antimicrobial resistance to common antimicrobial agents in S. aureus isolated from bovine mastitis milk samples. Moreover, agr Ⅰ was the predominant genotype with diverse toxin genes in S. aureus from bovine mastitis, and the potential hazard should be of concern.     

牛源金黄色葡萄球菌生物被膜与毒素基因检测及agr分型

旨在对牛源金黄色葡萄球菌（Staphylococcus aureus,S.aureus）的生物被膜、耐药性、毒素基因和agr基因型进行研究,并分析agr基因型与毒素基因之间的相关性。分别用微量滴定板法、药敏纸片法和PCR对S.aureus的生物被膜、耐药性和毒素基因进行检测,用多重PCR对S.aureus进行agr分型。结果表明,336株牛源S.aureus均能形成生物被膜,其中,形成中等（++）和强（+++）生物被膜的S.aureus分别占52.1%和47.9%。药敏试验结果显示,S.aureus对青霉素耐药最为严重,耐药率达91.7%,其次是红霉素、卡那霉素、克林霉素和庆大霉素,耐药率分别为89.6%、72.9%、66.7%和60.4%,而所有S.aureus对呋喃妥因和利奈唑胺均表现为敏感。PCR检测结果显示,黏附素基因fnbA检出率最高,达99.7%,其次是icaD、icaA、clfA和cna,检出率分别为98.2%、89.6%、86.0%和56.0%,bap基因检出率最低,为14.6%。肠毒素基因sea的检出率为26.5%,其次是seb（8.3%）和sec（6.8%）,毒素基因tst的检出率占8.3%。分型结果显示,agr Ⅰ型S.aureus是主要的流行菌株,占77.1%,agr Ⅱ、agr Ⅲ和agr Ⅳ型S.aureus流行率分别为14.0%、4.8%和2.1%。统计分析结果表明,agr Ⅰ型S.aureus更具有携带多种毒素基因的潜力,而agr Ⅳ型S.aureus无毒素基因携带潜力。综上表明,牛乳腺炎性S.aureus对常见的抗菌药物耐药严重,毒素基因分布多样,agr Ⅰ型是奶牛乳腺炎性S.aureus主要的基因型,且具有携带多种毒素基因的能力,其潜在威胁应引起重视。     

Clinical Bovine Mycoplasmal Mastitis: An Epidemiologic Study of Factors Associated with Problem Herds

The California Dairy Herd Improvement Association records of 29 California dairies which experienced clinical mycoplasmal mastitis between January 1975 and December 1977 were examined and compared to the records of selected control herds. A 15-fold greater risk of clinical mycoplasmal mastitis was found among large herds as compared to small herds. On average, herds with clinical mycoplasmal mastitis culled 5 % more cows than did control herds (33 % vs 28 %). No difference was found in average milk production. These findings compare closely with the findings of a previous report where infected herds were identified by the presence of pathogenic mycoplasma in bulk tank milk. The similarity of results support the use of frequent bacteriologic culture of bulk tank milk as a routine surveillance strategy for mycoplasmal mastitis in endemic areas. The similarity of results also supports the use of routine clinical diagnostic data in the study of the epidemiology of diseases of veterinary importance.