Laroucau Karine, Vorimore Fabien, Bertin Claire, Mohamad Khalil Yousef, Thierry Simon, Hermann Willems, Maingourd Cyril, Pourcel Christine, Longbottom David, Magnino Simone, Sachse Konrad, Vretou Evangelia, Rodolakis Annie
Unité Zoonoses Bactériennes, Agence Française de Sécurité Sanitaire des Aliments (Lerpaz), 94706 Maisons-Alfort cedex, France.
Vet Microbiol. 2009 Jun 12;137(3-4):335-44. doi: 10.1016/j.vetmic.2009.01.029. Epub 2009 Jan 24.
Chlamydophila (C.) abortus is the causative agent of ovine enzootic abortion with zoonotic potential whose epidemiology has been held back because of the obligate intracellular habitat of the bacterium. In the present study, we report on a molecular typing method termed multiple loci variable number of tandem repeats (VNTR) Analysis (MLVA) for exploring the diversity of C. abortus. An initial analysis performed with 34 selected genetic loci on 34 ruminant strains including the variant Greek strains LLG and POS resulted in the identification of five polymorphic loci, confirming the widely held notion that C. abortus is a very homogeneous species. Analysis of additional 111 samples with the selected five loci resulted in the classification of all strains into six genotypes with distinct molecular patterns termed genotypes [1] through [6]. Interestingly, the classification of the isolates in the six genotypes was partly related to their geographical origin. Direct examination of clinical samples proved the MLVA to be suitable for direct typing. Analysis of the genomic sequences in six C. abortus prototypes of amplicons generated with each of the five selected VNTR primers revealed that variation between genotypes was caused by the presence or absence of coding tandem repeats in three loci. Amplification of Chlamydophila psittaci reference strains with the five selected VNTR primers and of the six C. abortus prototype strains with the eight VNTR primers established for the typing of C. psittaci [Laroucau, K., Thierry, S., Vorimore, F., Blanco, K., Kaleta, E., Hoop, R., Magnino, S., Vanrompay, D., Sachse, K., Myers, G.S., Bavoil, P.M., Vergnaud, G., Pourcel, C., 2008. High resolution typing of Chlamydophila psittaci by multilocus VNTR analysis (MLVA). Infect. Genet. Evol. 8(2), 171-181] showed that both MLVA typing systems were species-specific when all respective VNTR primer sets were used. In conclusion, the newly developed MLVA system provides a highly sensitive, high-resolution and easy-to-perform tool for the differentiation of C. abortus isolates of different origin, which is suitable for molecular epidemiological studies.
流产嗜衣原体是绵羊地方性流产的病原体，具有人畜共患病潜力，由于该细菌为专性胞内寄生菌，其流行病学研究一直受到阻碍。在本研究中，我们报告了一种称为多位点可变数目串联重复序列（VNTR）分析（MLVA）的分子分型方法，用于探索流产嗜衣原体的多样性。对包括希腊变异株LLG和POS在内的34株反刍动物菌株的34个选定基因座进行初步分析，结果鉴定出5个多态性基因座，证实了普遍认为的流产嗜衣原体是一个非常同质化物种的观点。用选定的5个基因座对另外111个样本进行分析，结果将所有菌株分为6种基因型，其具有不同的分子模式，称为基因型[1]至[6]。有趣的是，分离株在这6种基因型中的分类部分与其地理来源有关。对临床样本的直接检测证明MLVA适用于直接分型。对用5个选定的VNTR引物中的每一个产生的流产嗜衣原体6个原型扩增子的基因组序列分析表明，基因型之间的差异是由3个基因座中编码串联重复序列的存在或缺失引起的。用5个选定的VNTR引物对鹦鹉热嗜衣原体参考菌株进行扩增，并用为鹦鹉热嗜衣原体分型建立的8个VNTR引物对6个流产嗜衣原体原型菌株进行扩增[拉鲁考，K.，蒂埃里，S.，沃里莫尔，F.，布兰科，K.，卡莱塔，E.，胡普，R.，马尼诺，S.，范龙佩，D.，萨克塞，K.，迈尔斯，G.S.，巴瓦尔，P.M.，韦尔尼奥，G.，普尔塞尔，C.，2008年。通过多位点VNTR分析（MLVA）对鹦鹉热嗜衣原体进行高分辨率分型。感染。基因。进化。8(2)，171 - 181]表明，当使用所有各自的VNTR引物组时，两种MLVA分型系统均具有种特异性。总之，新开发的MLVA系统为区分不同来源的流产嗜衣原体分离株提供了一种高度灵敏、高分辨率且易于操作的工具，适用于分子流行病学研究。
