Motiwala Alifiya S, Amonsin Alongkorn, Strother Megan, Manning Elizabeth J B, Kapur Vivek, Sreevatsan Srinand
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Ohio State University, Wooster, Ohio 44691, USA.
J Clin Microbiol. 2004 Apr;42(4):1703-12. doi: 10.1128/JCM.42.4.1703-1712.2004.
Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.
分枝杆菌分离株是通过放射性培养从美国六个地理区域的33种圈养或自由放养动物（n = 106）和环境来源（n = 3）中获得的。使用对分枝杆菌素J的依赖性以及五个明确的分子标记（包括IS900的两个整合位点（位点L1和L9）、一个鸟分枝杆菌副结核亚种（副结核分枝杆菌）特异性序列（位点251）和一个鸟分枝杆菌鸟亚种特异性标记（IS1245））以及hsp65和IS1311限制性内切酶分析，确认了所有109株分离株的身份。76株抗酸分离株被鉴定为副结核分枝杆菌，15株被鉴定为属于鸟分枝杆菌-胞内分枝杆菌复合群（但不是副结核分枝杆菌），其余18株被鉴定为鸟分枝杆菌-胞内分枝杆菌复合群之外的分枝杆菌。针对IS900整合位点的多重PCR指纹图谱将76株副结核分枝杆菌菌株中的67株聚为一个单一进化枝（指定为进化枝A18），辛普森多样性指数（D）为0.53。相比之下，基于序列的最近鉴定的副结核分枝杆菌短序列重复（SSR）区域的特征分析能够将进化枝A18中的副结核分枝杆菌分离株分为七个不同的等位基因（D = 0.75）。分析揭示了33种动物中的八个亚型，表明特定菌株存在种间传播。综上所述，我们的分析结果表明，SSR分析能够对来自不同宿主物种的副结核分枝杆菌分离株进行遗传特征分析，并为一些副结核分枝杆菌菌株的宿主特异性以及野生和家养动物物种之间菌株的共享提供了证据。
