奶牛乳房炎金黄色葡萄球菌基因多态性分型试验

利用随机引物AP-7,建立引物随机多态性扩增(RAPD)体系对71株引起内蒙古和贵州地区奶牛乳房炎的金黄色葡萄球菌分离株进行基因分型研究。结果表明,71株金黄色葡萄球菌均得到清晰的RAPD指纹图谱,扩增产物为2～9条带,产物大小为240～4 500bp。菌株共分为6个基因型,其中Ⅰ型17株(占23.9%)、Ⅱ型3株(占4.2%)、Ⅲ型33株(占46.5%)、Ⅳ型15株(占21.1%)、Ⅴ型2株(占2.8%)、Ⅵ型2株(占2.8%)。Ⅰ型为内蒙古地区的流行优势菌群,Ⅲ型为贵州地区的流行优势菌群。两地区各基因型菌株比例有明显差异,这可能与奶牛养殖业水平和环境差异有关。     

牛源金黄色葡萄球菌16SrRNA分子鉴定与随机多态性扩增分型

利用16S rRNA保守序列对引起内蒙古地区奶牛乳房炎的金黄色葡萄球菌进行分离鉴定,并且建立引物随机多态性扩增(RAPD)体系对金黄色葡萄球菌分离株进行基因分型研究.结果表明,共分离出金黄色葡萄球菌35株,RAPD结果显示这35株菌均可得到清晰的RAPD指纹图谱,扩增产物在1～8条带之间,产物大小在350～4 500bp之间.菌株分为6个基因型,Ⅳ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,这与牛场的环境和病原菌在牛场间流行传播情况有关.研究结果为地区性奶牛乳房炎的防治及新疫苗的研制提供了可靠的理论依据.     

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。     

乳源金黄色葡萄球菌耐药性分析与分型

为研究内蒙古及上海部分地区金黄色葡萄球菌流行病学特征,对两个地区的分离株进行生化鉴定、16S r RNA鉴定,并对鉴定为金黄色葡萄球菌等分离株进行药敏试验及RAPD随机扩增多态性分型。结果表明:内蒙古及上海部分地区的分离菌中有91株被鉴定为金黄色葡萄球菌;药敏试验结果显示两个地区分离的金黄色葡萄球菌均有较严重的多重耐药现象,且上海地区分离菌的耐药情况比内蒙古地区严重,但两个地区的分离菌均对喹诺酮类抗生素高度敏感;RAPD结果显示菌株共分为12个基因型,其中1型5株(5.5%),2型6株(6.6%),3型15株(16.5%),4型10株(11.0%),5型13株(14.3%),6型、9型和12型各2株(2.2%),7型8株(8.8%),8型24株(26.4%),10型3株(3.3%),11型仅1株(1.1%),8型菌株占总菌株数的26.4%,为主要流行的优势菌群。地域环境可能是影响病原菌耐药及基因型分布差异的重要原因。     

我国部分地区牛源金黄色葡萄球菌基因多态性分型研究

为了解我国牛源金黄色葡萄球菌(S.aureus)的基因多态性,本研究利用随机引物多态性扩增(RAPD)体系对174株分离自贵州、内蒙古、四川、上海、甘肃等地奶牛乳房炎的S.aureus及一株标准菌CVCC2246进行基因分型。结果表明,175株S.aureus均得到清晰的RAPD指纹图谱,扩增产物为1~9个片段,产物大小为240 bp~4 500 bp。所有菌株共分为8个基因型,其中1型16株;2型和3型各37株;4型15株;5型18株;6型14株;7型13株;8型8株。2型和3型菌株占总菌株42%以上,在贵州、内蒙古、四川、上海、甘肃分布广泛,为流行优势基因型;但5型是内蒙古地区的流行优势基因型。各地区菌株基因型比例有明显差异,可能与奶牛饲养水平和环境差异有关。     

牛源金黄色葡萄球菌凝固酶基因的分型

为了了解我国不同地区奶牛场金黄色葡萄球菌基因型的分布情况,采用凝固酶基因（Coa）扩增的分型方法,对在奶牛乳房炎奶样中所分离到的26株金黄色葡萄球菌进行了分型。结果显示,26株金黄色葡萄球菌共分为5个基因型,黑龙江省分离株以PCR 3型为主（10/16）。发现在同一牛群中的分离株可以存在相同的基因型,也可以存在不同的基因型;而不同牛群中的分离株也可属于同一基因型。这种结果提示,在某个或某些牛群中存在某一金黄色葡萄球菌的流行型,但也可能存在多个金黄色葡萄球菌的克隆。     

奶牛乳房炎克雷伯氏菌的基因分型

采用随机扩增多态性DNA（RAPD）技术对内蒙地区37株奶牛乳房炎克雷伯氏菌进行了基因分型。试验结果表明：利用引物（P1）将37株菌分7个基因型，37株的56．8％（21／37）是同一个基因型。本结果对克雷伯氏菌性乳房炎的防治研究有重要的参考意义。     

奶牛乳房炎样品金黄色葡萄球菌分离株的基因分型

为了掌握上海地区引起奶牛乳房炎的金黄色葡萄球菌基因型,本文采用ERIC-PCR分型方法,对从患有乳腺炎的奶牛乳汁中分离所得的74株金黄色葡萄球菌进行了基因分型。结果显示,74株金黄色葡萄球菌可扩增出4~9条带,共分为4个聚类群,亲缘关系相同或相近的菌株占97.7%。发现在同一牛群中的分离株可以是相同的基因型,也可以存在不同的基因型,而不同牛群中的分离株也可属于同一基因型。但总的来说,上海地区引起奶牛乳房炎的金黄色葡萄球菌多为同一基因型,在某个或某些牛群中存在金黄色葡萄球菌的优势基因型,而不同地区、不同环境和条件下同一种病原菌的基因型也存在一定差异,可能存在多个金黄色葡萄球菌基因型。     

奶牛乳房炎金黄色葡萄球菌脉冲场凝胶电泳分型研究

为探讨新疆北疆奶牛乳房炎致病菌的流行规律,本研究采用Sma Ⅰ酶切,脉冲场凝胶电泳(PFGE)分型的方法对43株分离自新疆6个规模奶牛场隐性乳房炎奶样的金黄色葡萄球菌进行了分子分型比较研究.结果表明,所有菌株都能被PFGE法分型,43株金黄色葡萄球菌可分为A、B、C、D和E 5个基因型.A型株(22株,51.2%)有13个亚型,相似度在81.8%～96.8%之间,在5个奶牛场均分离到,是主要的流行株;B型(25.6%)、C型(14.0%)、D型(7.0%)各型别内菌株间的相似度为100%,E型仅有1株.不同地区主要流行株有差别:乌鲁木齐主要流行A型菌株,昌吉以A型和B型菌株为主,而奎屯主要流行C型和B型.有2个奶牛场流行株只有1个基因型,也有2种基因型(3个牛场)或3种基因型(1个牛场)同时存在,但以1种为优势株.这些结果提示,不同地区主要流行株有差别,多数奶牛场以1种流行株感染为主,不同牛场可能流行相同的菌株,在较大地域范围内某些流行株具有侵染优势.     

奶牛乳房炎金黄色葡萄球菌的分子流行病学调查

实验对从内蒙古自治区分离到的63株引起奶牛乳房炎的金黄色葡萄球菌进行了随机扩增多态性分析（RAPD），可扩增出2—13条条带，主要分为12个基因型，亲缘关系相同或相近的菌株占73％。     

Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds

To determine the distribution of genes that encode enterotoxins A, B and C, 36 strains of Staphylococcus aureus isolated from goat mastitis and 64 isolated from bovine mastitis were analyzed by Multiplex PCR. Of the total strains studied, 37 (37%) were detected to have some of the SEs genes. From the bovine mastitis strains, 4 (6.3%) co-amplified the sea and seb genes and 2 (3.1%) were positive for the sec gene. From the goat mastitis strains, 31 (86%) tested positive to the Multiplex, and the sec gene was detected in all of them. The production of SE was detected in all strains harboring the corresponding gene. The results demonstrated that S. aureus isolated from goat mastitis had a higher enterotoxigenic potential than those isolated from bovine mastitis. Additionally, the presence of the sec gene in the majority of goat mastitis strains suggests a possible involvement of SEC in goat mastitis pathogenesis.     

多重PCR快速检测奶牛乳房炎3种主要病原体

奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。     

ARDRA and RAPD analyses of human and animal isolates of Streptococcus gallolyticus

A total of 23 Streptococcus gallolyticus strains, consisting of 12 strains from feces of healthy animals and 11 from clinical cases of human or cow mastitis milk, were examined genealogically. Four strains of S. bovis "biotype II/1" and 3 strains of S. equinus, the closely related organisms to S. gallolyticus, were also analyzed for outgroup comparison. Neither the amplified ribosomal DNA restriction analysis (ARDRA) nor the randomly amplified polymorphic DNA (RAPD) analysis that had been designed to recognize S. gallolyticus strains virulent in pigeons could differentiate clinical strains from the others of S. gallolyticus. No correspondence between the DNA profile in either analysis and the host animal species was detected.     

Identification of Escherichia coli recovered from milk of sows with coliform mastitis by random amplified polymorphic DNA (RAPD) using standardized reagents

A standardized-reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis was used for typing 58 Escherichia coli strains that were recovered from the milk of sows, having coliform mastitis, within a single swineherd in Sweden. Previously, the 58 E. coli strains were characterized serologically and profiled biochemically. They were also evaluated for their serum resistance and their ability to adhere to fibronectin and bovine fetal fibroblasts. The RAPD analysis was fast, easily performed, and required only a nanogram of DNA. The indistinguishable banding patterns obtained with repeated analyses of 2 isolates from each strain demonstrated that RAPD analysis using standardized beads is a technique that provides reproducible results for typing E. coli strains that cause mastitis in sows. The results of the RAPD analyses demonstrated that E. coli sow mastitis strains are highly variable in serotype, biochemical profiles, virulence factors, and RAPD type, and that all 58 strains can be differentiated by means of the RAPD technique. The strains grouped into 24 RAPD types by combining the results of 2 primers, and into 38 groups by combining the results of serotype and RAPD type. No relationship between serotypes, virulence factors and RAPD types was found.     

金黄色葡萄球菌α-溶血素基因的克隆与序列分析

参考Gary S Gray和Michael Kehce在《传染与免疫》杂志上发表的关于金黄色葡萄球菌α溶血素蛋白的基因序列,设计合成1对引物,从分离自牛体的野生株金黄色葡萄球菌中提取细菌总DNA,对α溶血素蛋白基因进行PCR扩增,产物经琼脂糖凝胶电泳分析,呈现1条约960 bp的条带,回收纯化后,将其克隆至pMD18 T质粒载体中,进行核苷酸序列分析,然后与报道的α溶血素蛋白基因进行比较。结果表明:所鉴定的野生株金黄色葡萄球菌的α溶血素蛋白基因序列与报道的核苷酸的同源性高达99.99%以上,证实为金黄色葡萄球菌Wood 46株α溶血素蛋白基因。     

Characterization of 26 isolates of Staphylococcus aureus, predominantly from dairy sheep, using four different techniques of molecular epidemiology.

Little information is available regarding the molecular epidemiology of Staphylococcus aureus-induced mastitis in dairy sheep. In this study, 4 different typing techniques were compared in typing 26 S. aureus isolates, predominantly from cases of subclinical mastitis in dairy ewes. The 4 techniques were pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) on 2 genes (coagulase and clumping factor B), randomly amplified polymorphic DNA-polymerase chain reaction (PCR) (RAPD-PCR), and multilocus sequence typing (MLST). On the basis of discriminatory power as the key parameter of typing systems, MLST and PFGE were found to be the most powerful techniques. The MLST and PFGE could contribute to epidemiological surveillance and evaluation of mastitis control programs, by documenting prevalence and dissemination of endemic clones in infected populations. The results of this study show that a single clone of S. aureus is widely distributed in infected ewe mammary glands.     

Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis.

This study was conducted to investigate polymorphism of the coagulase gene of Staphylococcus aureus causing bovine mastitis. One hundred eighty-seven strains of S. aureus were isolated from bovine mastitic milk samples obtained from 187 different Danish dairy farms. The isolates were characterised for restriction fragment length polymorphism (RFLP) of the coagulase gene. A variable region of the coagulase gene was amplified using the polymerase chain reaction (PCR) followed by AluI restriction enzyme digestion. A total of 15 different RFLP patterns were observed. The predominant pattern was found in 35% of the isolates. The ease of analysing coagulase gene polymorphisms among a large number of strains, and the multiple distinct polymorphic patterns generated, supports the use of this technique in epidemiological investigations of bovine mastitis. The predominating variants may have predelection for causing intramammary infections.