Vaccine and Infectious Disease Organization (VIDO) University of Saskatchewan, 120 Veterinary Rd, Saskatoon, SK S7N 5E3, Canada.
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology, Huazhong Agricultural University, Wuhan 430070, China.
Vet Microbiol. 2022 Oct;273:109532. doi: 10.1016/j.vetmic.2022.109532. Epub 2022 Aug 4.
Mycoplasma bovis (M. bovis) is an emerging major bovine pathogen, causing economic losses worldwide in the dairy and beef industry. Whole-genome sequencing (WGS) now allows high resolution for tracing clonal populations. Based on WGS, we developed the core genome multilocus sequence typing (cgMLST) scheme and applied it onto 151 genomes of clonal and non-clonal strains of M. bovis isolated from China, Australia, Israel, Denmark, Canada, and the USA. We used the complete genome of M. bovis PG45 as the reference genome. The pairwise genome comparison of these 151 genome sequences resulted in 478 cgMLST gene targets present in > 99.0 % clonal and non-clonal isolates with 100 % overlap and > 90 % sequence similarity. A total of 478 core genes were retained as cgMLST target genes of which an average of 90.4-99 % were present in 151 M. bovis genomes, while M. agalactiae (PG2) had 17.0 % and M. mycoides subsp. capri (PG3), M. ovipneumoniae (Y98), and M. arginine resulted in 0.0 % of good targets. When tested against the clonal and non-clonal strains, we found cgMLST clusters were congruent with the MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish between clonal and epidemiologically unrelated strains of the same clonal group, which could not be achieved using traditional MLST schemes. Our results showed that ninety-two M. bovis genomes from clonal group isolates had > 10 allele differences and unambiguously differentiated from unrelated outgroup strains. Additionally, cgMLST revealed that there might be several sub-clones of the emerging ST-52 clone. The cgMLST phylogenetic analysis results showed substantial agreement with geographical and temporal information. cgMLST enables the use of next-generation sequencing technology to bovine mycoplasma epidemiology at both the local and global levels. In conclusion, the novel cgMLST scheme not only showed discrimination resolution highly as compared with MLST and SNP cgMLST in sub-typing but also indicated the capability to reveal more population structure characteristics than MLST.
牛支原体（M. bovis）是一种新兴的主要牛病原体，在全球乳制品和牛肉行业造成了经济损失。全基因组测序（WGS）现在可以实现对克隆种群的高分辨率追踪。基于 WGS，我们开发了核心基因组多位点序列分型（cgMLST）方案，并将其应用于从中国、澳大利亚、以色列、丹麦、加拿大和美国分离的克隆和非克隆牛支原体 151 株。我们使用牛支原体 PG45 的完整基因组作为参考基因组。对这 151 个基因组序列的两两基因组比较，导致了 478 个 cgMLST 基因靶标，这些靶标在> 99.0%的克隆和非克隆分离株中存在，且重叠率为 100%，序列相似度> 90%。共保留了 478 个核心基因作为 cgMLST 靶基因，其中 151 个牛支原体基因组中存在 90.4-99%的平均靶基因，而无乳链球菌（PG2）有 17.0%，牛支原体亚种。山羊（PG3）、绵羊肺炎支原体（Y98）和精氨酸支原体则完全不存在好的靶基因。当用该方案对克隆和非克隆菌株进行测试时，我们发现 cgMLST 聚类与 MLST 定义的克隆群一致，这些克隆群在全基因组水平上具有不同程度的多样性。值得注意的是，cgMLST 可以区分同一克隆群的克隆和流行病学上无关的菌株，而传统的 MLST 方案则无法做到这一点。我们的结果表明，来自克隆群分离株的 92 个牛支原体基因组有> 10 个等位基因差异，与无关的外群菌株明显不同。此外，cgMLST 显示新兴 ST-52 克隆可能存在几个亚克隆。cgMLST 系统发育分析结果与地理和时间信息具有很大的一致性。cgMLST 使我们能够使用下一代测序技术在本地和全球范围内研究牛支原体的流行病学。总之，与 MLST 和 SNP-cgMLST 相比，新的 cgMLST 方案不仅在亚分型方面具有更高的分辨力，而且还具有揭示比 MLST 更多的种群结构特征的能力。
