El-Gazzar Mohamed, Ghanem Mostafa, McDonald Kristina, Ferguson-Noel Naola, Raviv Ziv, Slemons Richard D
A Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
B Poultry Diagnostic and Research Center, 953 College Station Road, The University of Georgia, Athens, GA 30602.
Avian Dis. 2017 Mar;61(1):25-32. doi: 10.1637/11417-040516-Reg.
Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.
滑膜支原体（MS）是一种家禽病原体，在过去几年中其发病率和经济影响不断增加。菌株鉴定对于疫情调查、感染源识别以及促进预防控制和根除工作至关重要。目前，可变脂蛋白血凝素A（vlhA）基因的一个片段（420 bp）是用于MS菌株鉴定的唯一靶点。该检测方法的一个主要局限性是，只有当分型样本的vlhA序列相同时，才能推断其克隆性；然而，如果它们的序列不同，则相关性程度不确定。在本研究中，我们提出了一种多位点序列分型（MLST）检测方法，以进一步完善MS菌株鉴定。在初步筛选了24个管家基因作为潜在靶点后，选择了7个基因用于MLST检测。从58个不同的MS菌株、田间分离株（n = 30）或阳性临床样本（n = 28）中成功扩增并测序了这7个基因各自的一个内部片段（450 - 711 bp）。所有7个基因片段的总体序列（共3960 bp）用于MS序列分型。使用MLST检测方法将58个检测的MS样本分为30种不同的序列类型，巧合的是，使用vlhA检测方法所有样本也被分为30种序列类型。然而，使用MLST数据生成的系统发育树比使用vlhA检测方法生成的树与流行病学信息更一致。我们建议新开发的MLST检测方法和vlhA检测方法可以串联用于MS分型。MLST检测方法将是用于MS序列分型的一种有价值且更可靠的工具，有助于更好地了解MS感染的流行病学。这反过来将有助于疾病的预防、控制和根除工作。
