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1.
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.  相似文献   

2.
甘肃地区牛源金黄色葡萄球菌分子鉴定及RAPD分型   总被引：1，自引：0，他引：1  
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。  相似文献   

3.
奶牛乳房炎金黄色葡萄球菌脉冲场凝胶电泳分型研究   总被引：1，自引：0，他引：1  
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.  相似文献   

4.
安晶  王晓  王新  郭刚  王雅春  俞英 《中国畜牧兽医》2014,41(10):213-218
本研究旨在检测并对比分析中国北方地区3个荷斯坦牛场乳源金黄色葡萄球菌(Staphylococcus aureus,简称金葡菌)的流行情况及乳汁中总菌群的抗生素耐药性。试验采集北方地区3个荷斯坦牛场的生产群奶样、大罐奶样和临床型乳房炎牛奶样共181份,首先采用Baird-Parker选择性培养基对金葡菌进行分离纯化;再采用特异性PCR方法鉴定含金葡菌特异基因nuc的金葡菌菌株;最后采用琼脂稀释法检测奶样中总菌群及金葡菌对14种抗生素的耐药性。结果发现,181份奶样带菌率为100%;金黄色葡萄球菌检出率为16.03%,分离纯化了48株金葡菌。奶样总菌群耐药性检测结果表明,中国北方地区3个荷斯坦牛场对于β-内酰胺类抗生素(苯唑西林、氨苄西林、头孢西丁、头孢哌酮)耐药性均较高,达89%以上;对红霉素、利福平、四环素、万古霉素、甲氧苄啶/磺胺甲噁唑、阿莫西林/克拉维酸的耐药性达80%以上;对氯霉素的耐药率过半;仅对阿米卡星耐药率较低(18%~58%)。对金葡菌耐药性检测结果显示,3个牛场金葡菌对环丙沙星、头孢西丁和甲氧苄啶/磺胺甲噁唑有较高耐药性,但均对阿米卡星、氯霉素、加替沙星和万古霉素敏感。3个牛场绝大多数菌株都产生了多重耐药谱。研究结果为奶牛场乳源总菌群、金葡菌耐药性情况及科学用药提供试验数据。  相似文献   

5.
<正>奶牛乳房炎金黄色葡萄球菌的分型对预防菌株在牛体之间传播、了解菌株地域性分布特点及针对性疫苗的研制均有重要的意义。本研究旨在将多位点可变数目串联重复序列分析(MLVA)法应  相似文献   

6.
奶牛乳房炎,尤其是由金黄色葡萄球菌(金葡菌)感染引起的隐性乳房炎是最难以防控的奶牛常见病。近五年来,本课题组对中国北方六个省市九个荷斯坦牛场的生产群高体细胞数(SCC)和低SCC奶牛以及临床乳房炎奶牛进行了十一批次采样,共采集奶样1 112头份,分离出191株金葡菌,所检测牛的平均金葡菌感染率达11.7%,其中生产群奶牛金葡菌感染率为12.4%,临床乳房炎牛为10.8%。对这些金葡菌进行药敏试验以及毒素基因检测,发现氯霉素和阿米卡星对金葡菌有较强抑制性,毒素基因检出率最高者是杀白细胞素基因PVL(70.2%)。依据金葡菌PFGE分型结果绘制中国北部及华东地区奶牛乳源金葡菌的流行图谱。结合国内外相关研究结果,对比分析了国内外奶牛金葡菌乳房炎的感染情况以及金葡菌的耐药性、毒素基因和流行类型,并提出了降低奶牛感染金葡菌乳房炎的相关策略。  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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。  相似文献   

10.
为调查北京某牛场乳房炎金黄色葡萄球菌的耐药性,采集该牛场48头奶牛的乳头涂抹点、牛乳、粪便以及卧床土等200份样品进行金黄色葡萄球菌的分离鉴定及药敏试验,共检出可疑菌株142株,并通过耐热核酸酶试验、触酶试验、凝固酶试验等确定出致病性金黄色葡萄球菌47株。药敏试验结果表明,分离的47株菌株对多种药物具有不同程度的耐受性,对牛场常用药青霉素的耐药率高达83%,对氨苄西林、链霉素的耐药率分别为60%、53%,已具有较高的耐药性,建议不再经常使用;氧氟沙星、诺氟沙星、环丙沙星、庆大霉素、万古霉素、头孢哌酮、头孢唑啉、头孢噻吩、阿米卡星等具有较强的抗菌活性(S﹥80%),可用于奶牛乳房炎的临床用药。本研究结果为该牛场在对乳房炎致病性金黄色葡萄球菌的诊断及治疗药物的选择方面提供了技术服务。  相似文献   

11.
Staphylococcus aureus is a common cause of bovine mastitis. A simple and efficient typing method would be helpful in understanding S. aureus sources and spread. Ninety-six S. aureus strains, isolated between 1961 and 2003 from the milk of 90 dairy cows belonging to 75 French herds, were subjected to multiple-locus variable-number tandem repeats analysis (MLVA) by PCR. The conjunction of clfA, clfB, SAV1078 and fnb gene tandem repeats (TRs) enabled the definition of 61 types. When coa, spa, sdrC, sdrD and sspA TRs were used individually as additional markers, 63, 68, 67, 65 and 67 types were defined, respectively, versus 77 types when they were all included in the method. These additional TRs did not improve the differentiation of isolates collected in the same farm. The MLVA procedure using the tandem repeats embedded in clfA, clfB, SAV1078 and fnb loci as a basic combination at the herd level or associated with other TRs such as spa, sdrC, sdrD, sspA and coa can be a valuable tool for bovine S. aureus epidemiological studies.  相似文献   

12.
Clostridium perfringens is a well-characterized bacterial species which can be both commensal and pathogenic in humans and many animals. Genetic typing of the bacterium is often used for molecular epidemiological purposes, and can be useful for observing population structures as well. Analysis of the variable number of tandem repeats (VNTRs) within the genome, called multiple-locus VNTR analysis (MLVA) provides genetic information useful for molecular typing. A MLVA typing method has been developed recently by Sawires and Songer [Sawires, Y.S., Songer, J.G., 2005. Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe 11, 262-272] for C. perfringens. A novel MLVA protocol is described here, with the aim of investigating the discriminatory potential of the method, and to obtain preliminary data on the population structure of C. perfringens from a wide variety of C. perfringens sources. This protocol uses new loci in noncoding regions of the chromosome, and also makes use of capillary electrophoresis for more precise results and for high-throughput typing. DNA sequencing of amplicons was performed to ensure inclusion of conserved tandem repeats within each locus. Fifty-four epidemiologically unrelated isolates from a local collection obtained from 11 different animal species were typed at 6 loci. Thirty-five unique MLVA types were obtained, resulting in a Simpson's index of diversity of 0.975. Epidemiologically related isolates (n=27) previously typed by pulsed-field gel electrophoresis (PFGE) were also examined with MLVA and the congruency of the two methods was found to be very high. All 81 isolates were successfully typed with MLVA, and polymerase chain reactions (PCR) were automated using robotics and 96-well plates, with PCR product sizes determined using capillary electrophoresis. Reproducibility was also shown to be very high.  相似文献   

13.
The study concerns 130 Staphylococcus aureus strains isolated from different raw-milk dairy products (122 isolates) and human samples (eight isolates). Four different typing techniques were applied: biochemical profiles (Biolog GP), restriction fragment length polymorphism of coagulase gene (coaRFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA). Moreover multiplex-PCR was used to study the distribution of genes encoding staphylococcal enterotoxins. The results of this study reveal marked genomic and phenotypic variability among the tested S. aureus. The considered techniques were all found useful for strain typing, but, based on discriminatory power as the key parameter of the typing system, MLVA and Biolog GP were found to be the most powerful techniques. The methods showed little concordance in terms of discerning the clusters of related strains.  相似文献   

14.
During a previous longitudinal study, performed on four farrow-to-finish farms (A to D), samples were taken from twelve sows, their offspring, and the environment on various occasions over six months to study the MRSA presence. During the present study, a selection of the obtained MRSA isolates were typed by multiple-locus variable-number tandem-repeat analysis (MLVA), Pulsed Field Gel Electrophoresis (PFGE), spa typing, and SCCmec typing to study the genetic diversity of LA-MRSA isolates and to determine possible MRSA sources for pig(let)s. PFGE, spa typing, and SCCmec typing revealed the presence of one or few dominant genotype(s) per farm. In contrast, 212 MLVA types were detected on the four farms, forming one cluster on farm A, three on farm B, four on farm C and two on farm D. The genotype, found on farm A was unique for this farm. Farms B, C and D shared one cluster. In general, MLVA types from these clusters were isolated from piglets, sows, and the environment on various sampling events. Piglets carried MLVA types both related and unrelated to their mother sows’ MLVA types at farrowing and onwards. In conclusion, molecular typing revealed that within a farm one or a few dominant strain(s) are widespread. Potential MRSA sources for piglets were mother sows, the environment and other piglets.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0089-4) contains supplementary material, which is available to authorized users.  相似文献   

15.
The molecular diversity, antibiotic resistance patterns and presence of resistance genes were determined in Staphylococcus aureus isolates from cases of bovine mastitis in a dairy cattle herd in China. Multiple locus variable number tandem repeat analysis was used for molecular typing. Resistance was determined through minimum inhibitory concentrations and resistance genes were detected by PCR. There was low molecular diversity; one predominant strain (type I) accounted for the majority of cases of S. aureus mastitis in the herd and this strain had a high frequency of resistance to penicillin and tetracycline. The most prevalent resistance genes were blaZ, ermC and tetM.  相似文献   

16.
Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, a severe disease of poultry, responsible for heavy economic losses. Epidemiologic investigation of fowl typhoid significantly benefits from molecular typing tools, RAPD and PFGE have been proposed for this purpose. PFGE, a well established technique, is still the gold standard among typing methods for most bacteria, including salmonella. Nevertheless, it has some limitations regarding execution and reproducibility, in particular it is labour intensive and requires good technical expertise. Furthermore, it needs accurate standardization and results can be ambiguous to interpret. Such limitations can hamper reproducibility and transfer of results. As a possible alternative to PFGE, multilocus variable-number of tandem-repeats analysis (MLVA) has recently emerged as an effective genotyping method for many bacterial pathogens showing high discriminatory power associated to robustness. We developed a six-loci MLVA protocol for Salmonella Gallinarum and compared it to PFGE performed with SpeI, XbaI and NotI on fifty isolates. The proposed MLVA has a high discriminatory power, equivalent to that of the three-enzyme PFGE (Simpson's index 0.94 for MLVA, 0.93 for three-enzyme PFGE) but it is simpler to perform and straightforward in genotype identification, allowing unambiguous exchange of results. Stability of selected VNTR loci, assessed in vitro and in vivo, is good but not absolute, reflecting the sensitivity of MLVA to detect evolutionary changes of bacteria. Clustering of the isolates as determined by MLVA typing is substantially confirmed by PFGE.  相似文献   

17.
The phage typing and cultural characteristics of 574 strains of S. aureus of poultry origin in Australia were examined. With the avian phage set of Shimizu (1979) it was possible to type 74.2% of strains. A number of significant variations in the phage typing patterns of Australian strains compared to those reported from Japan and Europe were observed. A lower proportion of Australian strains were of avian phage group I and a higher proportion of group III. A high proportion of strains were of mixed lytic groups. No locally isolated phages were able to increase significantly the percentage of typeable strains, although four local phages appeared to be of greater value for phage typing poultry strains of S. aureus than some other phages of the avian phage set. The international (human) phage set was of limited value in typing Australian strains of poultry origin although four strains were identified which were indistinguishable from strains of human origin. Using cultural characteristics of the strains in conjunction with phage typing, the Australian strains of S. aureus were assigned to one of three major groups and nine subgroups. A list of typing phages considered to be valuable for use on Australian poultry strains of S. aureus is given.  相似文献   

18.
The objective was to investigate the spread of S. aureus in seven dairy herds. Milk samples were taken to determine mastitis pathogens and somatic cell count. An approved hygiene programme was established to control the spread of S. aureus from quarter to quarter. S. aureus isolates were differentiated by geno- and phenotyping to trace their spread within a herd.In two herds S. aureus showed a high prevalence, but were eliminated from the herds by the control programme. In these herds and a third herd typing results identified a particular type that was found much more frequently than all other types. The frequent types were repeatedly detectable during the study. In three other herds with a lower S. aureus prevalence and also in one herd with a long history of S. aureus and high prevalence subclinical mastitis was caused by several distinguishable S. aureus types. These types occurred simultaneously in the particular herd and showed little or no tendency to spread from quarter to quarter. They seemed to circumvent the control procedures resulting in a relatively high rate of new infected animals and therefore were hardly eliminated. The typing results and the clinical observations indicated that strains differed in their tendency to spread and their ability to infect udder quarters. In three herds typing methods identified a predominant type with the common epidemiological features of a contagious mastitis pathogen, while in the other herds the S. aureus type patterns were similar to that of environmental pathogens.  相似文献   

19.
Biofilm production by 221 Staphylococcus aureus isolates from 45 dairy herds was evaluated. Isolates were from composite milk of 117 cows, from teat skin of 70 cows, and from 34 milking machine unit liners. Of S. aureus from milk samples, 41.4% were biofilm producers, as compared to 24.7 and 14.7% of the isolates collected from skin and liners. Pulsed field gel electrophoresis (PFGE) best categorized S. aureus biofilm producers as compared to phage typing and binary typing. PFGE types that were significantly associated with isolation from milk as opposed to teat skin or liners, had isolates that were more likely to produce biofilm than PFGE types that were isolated from milk, skin and liners at similar frequencies. By contrast, PFGE type A was significantly associated with isolation from teat skin and had few biofilm producers. PFGE type Q, which is exclusively a milk, isolate produced more biofilm as evidenced by absorbance values. Given S. aureus that are associated with milk are more likely to produce biofilm as compared to extramammary sources (teat skin and milking unit liners), suggests that biofilm production is a risk factor for infection.  相似文献   

20.
Mastitis caused by Staphylococcus aureus is a disease of major economic importance to the dairy industry. Transmission occurs during milking, with chronically-infected cows acting as the major reservoirs of infection. PCR-coagulase gene typing of 151 S. aureus isolates from seven farms generated only six PCR types, with 110 (73%) isolates assigned to PCR type 1 and 23 (15.2%) isolates assigned to type 2. PCR type 1 was the predominant type on five of the seven farms, including farms in geographically separated regions of Victoria, while type 2 predominated on two farms. With the exception of the 41 isolates from one farm, all isolates were resistant to penicillin, but susceptible to other antibiotics that are routinely used to manage mastitis in dairy cattle. Nine of 11 cows with chronic S. aureus infection showed evidence of persistence of a single PCR type for periods of up to 9 months. Two different PCR types of S. aureus were isolated from the other two cows over the same period.  相似文献   

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