背景:布鲁氏菌病仍然是希腊牲畜和人类的地方病,影响第一产业和整个国民经济。虽然农场动物,特别是反刍动物构成了疾病的自然宿主,传播给人类并不少见,因此也代表了严重的职业病。在这个棱镜下,关于布鲁氏菌在反刍动物中的物种分布的知识被认为是高度优先事项。存在用于布鲁氏菌检测的各种分子方法,然而具有差异判别能力。因此,这项调查的目的是在物种和亚型水平上获得全国布鲁氏菌流行病学基线基因分型数据,以及评估用于检测布鲁氏菌种类的不同分子技术的利弊。30只国内反刍动物的39份组织样本,应用筛选PCR发现阳性,通过四种不同的分子技术进行测试,即16SrRNA的测序,BP26和OMP31地区,和微卫星标记的MLVA分型板1测定。
结果:16SrRNA测序分析仅显示一种单倍型,表明基于该标记的布鲁氏菌的分子鉴定可能仅在属水平上是可行的。BP26测序分析和MLVA的检测完全一致。在11个样本中观察到一个有趣的例外,低质量的提取DNA,其中没有产生所有预期的MLVA扩增子,并且鉴定基于剩余的以及BP26。相反,OMP31未能在任何所检查的样品中提供清晰的条带。
结论:本研究揭示了整个希腊反刍动物中布氏杆菌的持续循环。Further,根据我们的结果,BP26基因代表了MLVA小卫星测定的一个很好的替代方案,特别是低质量的DNA样本。
BACKGROUND: Brucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the
BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers.
RESULTS: Only one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level.
BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on
BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples.
CONCLUSIONS: The present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results,
BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.