Ocampo-Sosa Alain A, Agüero-Balbín Jesús, García-Lobo Juan M
Departamento de Biología Molecular, Facultad de Medicina Universidad de Cantabria, Unidad asociada al CIB, CSIC, C/Cardenal Herrera Oria s/n, 39011 Santander, Spain.
Vet Microbiol. 2005 Sep 30;110(1-2):41-51. doi: 10.1016/j.vetmic.2005.06.007.
One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.
1999年3月至2003年2月间从西班牙坎塔布里亚地区牛群中分离出129株布鲁氏菌野毒株，采用AMOS-ERY PCR检测法并使用插入序列IS711的探针进行Southern印迹杂交分析。大多数野毒株在AMOS-ERY检测中仅产生ery条带，其杂交模式与流产布鲁氏菌生物变种5、6和9的参考菌株相同，但与属于流产布鲁氏菌生物变种3的图利亚菌株不同。然而，通过标准方法对这些菌株进行分型表明它们属于流产布鲁氏菌生物变种3。这些结果表明，流产布鲁氏菌生物变种3在基因上并非同质，至少可分为两类。在一类中，即我们所称的生物变种3a，是图利亚菌株，而当地野毒株则属于生物变种3b。对包含一个IS711拷贝的DNA片段进行克隆和核苷酸测序，该片段为生物变种3b的流产布鲁氏菌野毒株及生物变种5、6和9的参考菌株所特有，结果显示在一个IS711拷贝附近存在一个5.4 kb的缺失。基于这些数据，我们设计了一种新引物，该引物与IS711 AMOS引物一起仅从流产布鲁氏菌生物变种3b、5、6和9的分离株中产生一个1.7 kb的PCR片段。使用这些引物对布鲁氏菌其他种和生物变种的菌株以及本研究中分析的与布鲁氏菌系统发育关系相近的细菌进行扩增时，未产生扩增产物。将该引物添加到AMOS-ERY PCR引物混合物中，可将流产布鲁氏菌生物变种3b、5、6和9与布鲁氏菌其他种和生物变种进行阳性区分。
