Safaee Shirin, Weiser Glen C, Cassirer E Frances, Ramey Rob R, Kelley Scott T
Biology Department, San Diego State University, San Diego, California 92182, USA.
J Wildl Dis. 2006 Jul;42(3):545-55. doi: 10.7589/0090-3558-42.3.545.
We investigated the effectiveness of culture-independent molecular methods for determining host-associated microbial diversity in bighorn sheep (Ovis canadensis). Results from bacterial culture attempts have been the primary source of information on host-associated bacteria, but studies have shown that culture-based results significantly underestimate bacterial diversity in biological samples. To test the effectiveness of culture-independent methods, we extracted DNA from nasal and oropharyngeal swab samples collected from bighorn sheep in four different populations. From these samples, we amplified, cloned, and sequenced small subunit (16S) ribosomal DNA (rDNA) to identify the scope of microbial diversity in bighorn respiratory tracts. Phylogenetic analysis of these rDNA gene sequences revealed organismal diversity an order of magnitude higher than was determined by culture methods. Pasteurellaceae bacteria were the most diverse phylogenetic group in live bighorn sheep, and members of bacterial genera often associated with respiratory disease were found in all the samples. Culture-independent methods were also able to directly detect leukotoxin (lktA) gene sequences in swab and lung tissue samples. Overall, our results show the power of culture-independent molecular methods for identifying microbial diversity in bighorn sheep and the potential for these methods to detect the presence of virulence genes in biological samples.
我们研究了非培养分子方法在确定大角羊（加拿大盘羊）宿主相关微生物多样性方面的有效性。细菌培养尝试的结果一直是宿主相关细菌信息的主要来源，但研究表明，基于培养的结果会显著低估生物样本中的细菌多样性。为了测试非培养方法的有效性，我们从四个不同种群的大角羊采集的鼻腔和口咽拭子样本中提取了DNA。从这些样本中，我们扩增、克隆并测序了小亚基（16S）核糖体DNA（rDNA），以确定大角羊呼吸道中微生物多样性的范围。对这些rDNA基因序列的系统发育分析显示，生物多样性比通过培养方法确定的高出一个数量级。巴斯德氏菌科细菌是活体大角羊中最多样化的系统发育类群，并且在所有样本中都发现了通常与呼吸道疾病相关的细菌属成员。非培养方法还能够直接检测拭子和肺组织样本中的白细胞毒素（lktA）基因序列。总体而言，我们的结果显示了非培养分子方法在识别大角羊微生物多样性方面的强大能力，以及这些方法在检测生物样本中毒力基因存在方面的潜力。
