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.     

贵州地区牛源金黄色葡萄球菌分离株16S rRNA分子鉴定及RAPD分型研究

利用16S rRNA保守序列对引起贵州地区奶牛乳房炎的金黄色葡萄球菌进行分离鉴定,建立随机多态性扩增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,69份奶样中共分离出金黄色葡萄球菌37株,DNA随机多态性扩增（random amplified polymorphic DNA,RAPD）结果显示这37株金黄色葡萄球菌扩增产物在1～7条带之间,产物大小为400～4500 bp。菌株可分为7个基因型,其中Ⅰ型7株（18.9%）、Ⅱ型2株（5.4%）、Ⅲ型2株（5.4%）、Ⅳ型1株（2.7%）、Ⅴ型19株（51.4%）、Ⅵ型1株（2.7%）、Ⅶ型5株（13.5%）,Ⅴ型为该地区的流行优势菌群。地理和气候环境对病原菌流行传播的影响是病原菌基因型分布差异的重要原因。     

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。     

宁夏地区规模牧场金黄色葡萄球菌的分离鉴定与药敏试验

乳房炎是奶牛最常见的疾病之一,金黄色葡萄球菌是导致奶牛乳房炎的重要细菌之一.为了调查宁夏地区规模牧场金黄色葡萄球菌在奶牛中的流行情况和耐药特点.本研究通过流行病学调查、常规微生物学及PCR鉴定方法,对宁夏地区6个规模牧场中的856份奶样中的病原微生物进行分离鉴定和耐药性分析.结果表明,856份奶样中,共获得467株金黄...     

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。     

广东一奶牛场临床乳房炎致病菌分离及其药敏试验

为了解广东某牛场奶牛乳房炎的细菌感染情况及其对常用药物的敏感性,本试验对从8头临床乳房炎患牛采集的8份奶样中分离细菌,纯化、鉴定后再进行药敏试验。结果表明,8份奶样中分离出3株大肠杆菌、5株金黄色葡萄球菌和3株链球菌。其中分离得到的大肠杆菌和链球菌对硫酸头孢喹肟最敏感,金黄色葡萄球菌对复方氨苄西林和阿莫西林同样敏感。本试验可为临床乳房炎治疗用药提供参考。     

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.     

邯郸地区奶牛乳房炎病原菌的分离鉴定及耐药性分析

奶牛乳房炎是奶牛养殖中的常见病和多发病,严重危害我国奶牛养殖业的发展.为确定邯郸地区奶牛乳房炎主要致病菌,本试验采集邯郸地区临床型奶牛乳房炎奶样31份,进行了病原菌分离鉴定、分子生物学检测和耐药性分析.结果,分离出金黄色葡萄球菌9株、大肠杆菌9株、沙门氏菌8株、链球菌9株,分离率分别为29.03％、25.81％、29....     

北京地区奶牛隐性乳房炎金黄色葡萄球菌的分离鉴定及药敏试验

为了解北京地区奶牛隐性乳房炎中金黄色葡萄球菌的感染情况及对常用药物的敏感性,用科玛嘉显色培养基和16SrRNA PCR检测方法对5个奶牛场的100份隐性乳房炎奶样进行金黄色葡萄球菌的分离鉴定,采用K-B纸片扩增法进行药敏试验。结果表明,显色培养基分离到24株疑似金黄色葡萄球菌,经16SrRNA PCR鉴定,15株为金黄色葡萄球菌;药敏试验结果显示,15株金黄色葡萄球菌均对β-内酰胺类中的氨苄西林产生普遍耐药性,对克林霉素和环丙沙星的耐药率较高,分别为53.33%和40%;对氧氟沙星、庆大霉素、头孢噻肟和头孢曲松的耐药性较低,耐药率为6.67%。说明15株金黄色葡萄球菌分离株对7类9种抗菌药物都有不同程度的耐药性。     

新疆部分地区奶牛乳房炎金黄色葡萄球菌荚膜多糖分型的研究

为调查新疆奶牛乳房炎金黄色葡萄球菌主要流行血清型,本研究采用玻板凝集和双重PCR方法对不同地区奶牛乳房炎乳样中分离的117株金黄色葡萄球菌进行血清分型.结果表明荚膜多糖5型占10.26％(12/117),8型占13.68％(16/117),336型占64.96％(76/117),未分型菌株占11.11 ％(13/117).该研究为新疆奶牛乳房炎金黄色葡萄球菌疫苗菌株的筛选提供了依据.     

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。     

Induced staphylococcal infections in the bovine mammary gland

In a study to develop and define a practical model of bovine mastitis caused by Staphylococcus aureus, induced infections were attempted in 203 bovine mammary glands of 41 cows, using 12 strains of S aureus. Approximately 100 colony-forming units of S aureus in saline solution were injected after milking, and milk samples were collected daily from test glands for 14 days to monitor the progress of infections and inflammatory responses. Relationships were examined for cow-related factors and for various characteristics of the strains of S aureus used to the development of a persistent intramammary infection. A dairy cow that was useful in this model was defined as follows: (1) the 2nd to 7th month in the 1st to 5th lactation; (2) producing milk from all mammary glands that contained less than 6 x 10(5) somatic cells/ml; and (3) having mammary glands that were free of any primary mastitis pathogen, as well as micrococci and Corynebacterium bovis. From the present study, it was not possible to define clearly a strain of S aureus which would be useful in the model, but 5 strains of S aureus were identified as being capable of producing persistent subacute infections with a high degree of repeatability.     

乳房炎奶牛金黄色葡萄球菌毒素基因的检测及PFGE分型研究

作者针对临床及亚临床乳房炎奶牛乳汁中金黄色葡萄球菌分离株的毒素基因进行检测和脉冲场凝胶电泳(PFGE)基因分型,比较2种类型乳房炎金黄色葡萄球菌分离株的差异.无菌法采集奶样,采用国际标准方法从中分离金黄色葡萄球菌,用多重PCR方法扩增nuc基因和mecA基因以确证金黄色葡萄球菌(SA)和耐甲氧西林金黄色葡萄球菌(MRSA).进一步用PCR方法检测SA的各种毒素基因(SEs、ETs、TSST 1和PVL基因等).利用限制性内切酶Sma Ⅰ对SA基因组DNA进行酶切和PFGE分析,最后利用BioNumerics软件进行聚类分析.结果:19.3％(23/119)的临床乳房炎奶样和14.8％(26/176)的亚临床乳房炎奶样确定为金黄色葡萄球菌阳性样品,分别从中分离鉴定出43株和26株金黄色葡萄球菌,其中临床乳房炎分离株中有5株为mecA基因阳性.临床乳房炎奶牛奶样中检测到SA的SEA、SEB、SED、SEJ和PVL毒素基因,检出率分别为3.8％(1株)、11.5％(3株)、19.2％(5株)、7.7％(2株)和31.2％(10株);亚临床乳房炎奶牛乳样中仅检测到SA的SEA和PVL毒力基因,检出率分别为7.0％(3株)和84.1％(37株).表明临床与亚临床乳房炎奶牛乳汁中SA菌株携带的毒素基因不一样,SEs可能是临床乳房炎菌株的重要致病基因,PVL可能是亚临床乳房炎菌株的重要致病基因.69株SA使用Sma Ⅰ酶切分型后,可分为7个大簇、50个基因型,来源相同的SA分型后大部分位于同一簇内.临床乳房炎奶牛乳汁中检测到MRSA菌株,PVL基因在亚临床乳房炎中的检出率为临床乳房炎的2.7倍.PFGE方法能较好的区分临床乳房炎和亚临床乳房炎的SA分离菌株.     

Validation of DNAse reaction for identification of Staphylococcus aureus strains in routine mastitis diagnosis

In routine mastitis diagnostic S. aureus strains are often identified using DNAse reaction. However, this enzyme activity is not limited only to S. aureus strains. Furthermore, the strength of the DNAse reaction between different strains varies strongly. These factors lead to the fact that the results are often not comparable between laboratories. The aim of these investigations was to validate the DNAse reaction for routine identification of S. aureus in mastitis diagnostic and to set a critical limit for interpretation of DNAse reaction. The results of 189 strains isolated from bovine mastitis milk samples show that colonies with beta delta or delta-haemolysis and DNAse reactions of > or = 2 mm can reliably be identified (sensitivity 91.2%, specificity 96.9%) as S. aureus.     

Induction of anti-phagocytic surface properties of Staphylococcus aureus from bovine mastitis by growth in milk whey

The respiratory burst activity of bovine polymorphonuclear (PMN) cells in response to milk whey- and TSB-grown S. aureus strains isolated from bovine mastitis was studied in whole blood chemiluminescence (CL) and in a CL system with purified bovine neutrophils. In both cases milk whey-grown S. aureus strains elicited significantly less CL than homologous strains grown in TSB. Ingestion of milk whey-grown S. aureus strains by bovine neutrophils was also considerably lower than that of the corresponding homologous organisms grown in TSB. Binding of complement factor C3 to serum-opsonized milk whey-grown S. aureus strains was lower compared with TSB-grown homologous organisms. Moreover, 5 of 6 S. aureus strains grown in milk whey were significantly more resistant to in vivo clearance from the peritoneal cavity of mice compared with homologous bacteria grown in TSB. S. aureus strains grown in TSB exhibited hydrophobic surface properties, whereas homologous strains grown in milk whey were hydrophilic.     

Milk whey induction of agglutination in ovine and bovine mastitis Staphylococcus aureus

A total of 59 mastitis staphylococcic strains were tested for growth agglutination upon supplementation of growth media with ovine and bovine milk whey and mammary secretions from dry cows. Differences were observed when comparing bacterial species or origins (ovine vs. bovine) of bacteria and whey. All of the ovine and bovine S. aureus strains tested, but only 4 among 22 other ovine mastitis staphylococcic strains, showed growth agglutination in Todd Hewitt broth (THB) supplemented with greater than or equal to 30% (v/v) ovine milk whey. None of the strains agglutinated during growth in regular THB medium. Ovine whey had an agglutination induction capacity higher than bovine whey (P less than 0.005), concerning the number of responsive ovine and bovine S. aureus strains. There were no differences between whey samples from different ewes with regard to their capacity to induce agglutination. Ovine S. aureus strains were more responsive than bovine strains of this bacterial species, concerning the number of responsive strains (P less than 0.001) to bovine whey (greater than or equal to 30% in THB), the proportion of responsive strains at low (10%) ovine whey concentration (P less than 0.001), and the strength of reaction (precipitation timing and clump size). Secretions from dry cows systematically induced agglutination in all of the bovine and ovine S. aureus strains tested.     

多重PCR检测奶牛乳腺炎金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母菌方法的建立与应用

参照GenBank发表的序列，在金黄色葡萄球菌、无乳链球菌和停乳链球菌16SrRNA与23SrRNA之间的区域设计了3对引物，参照念珠菌和隐球菌的18SrRNA的序列设计1对引物，建立了检测金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母真菌4种乳腺炎主要致病菌的多重PCR方法。参照Skladny的方法制备模拟了细菌感染l临床标本。结果表明：本试验建立的多重PCR方法具有较好的特异性，多重PCR方法检测乳样中的金黄色葡萄球菌的细菌最小浓度为10^4CFU／mL，检测无乳链球菌、停乳链球菌和酵母真菌的细菌最小浓度分别为10^4CFU／mL、10^3CFU／mL和10^3CFU／mL。通过对采自临床型乳腺炎（46个）和隐性乳腺炎（167个）动物共计213个乳样分别用传统细菌学培养法和多重PCR方法进行检测，多重PCR对金黄色葡萄球菌和酵母真菌的检测具有更高的检出率（P〈0．01），但该方法对无乳链球菌和停乳链球菌的检出率与培养法差异不显著（P〉0．05）。     

Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences

Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.     

Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea

One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea.