Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam
Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam
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DOI：
10.1016/j.meegid.2015.10.029
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发表时间：
2016-01-01
影响因子：
3.2
通讯作者：
Yokoyama, Naoaki
中科院分区：
文献类型：
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作者：
Liyanagunawardena, Nilukshi;Sivakumar, Thillaiampalam;Yokoyama, Naoaki
牛巴贝虫是最致命的巴贝虫生物体，导致牛的高死亡率。B.牛裂殖子表面抗原（MSA），如MSA-1、MSA-2b和MSA-2c，可能与宿主动物中改变的免疫谱有关。本研究旨在建立亚洲msa-1基因型的特异性PCR检测方法，从而重新分析斯里兰卡、蒙古和越南msa-1的遗传多样性。根据GenBank数据库中msa-1基因序列构建的msa-1基因多态性，设计了9个亚洲msa-1基因型的特异性引物。使用含有代表亚洲基因型的msa-1基因片段插入片段的质粒证实了类型特异性PCR检测的特异性。此外，当分析二联巴伯虫、卵形巴伯虫、环形泰勒虫、东方泰勒虫、伊氏锥虫、泰氏锥虫、边缘无浆体和牛无浆体的DNA样品以及未感染的牛血液时，通过这些PCR测定未观察到扩增子。总共有109个B。然后通过类型特异性PCR测定筛选来自斯里兰卡（44头牛）、蒙古（26头牛）和越南（23头牛和16头水牛）的牛阳性血液DNA样品。对来自所有PCR扩增子的序列进行遗传学分析。在109份DNA样本中，23份（20份来自牛，3份来自水牛）至少有一种基因型呈阳性。与以前的研究一致，在来自斯里兰卡和越南的DNA样本中分别检测到五种和四种不同的基因型。与此相反，4个基因型，包括3个新的基因型，从蒙古检测。发现5份DNA样本同时感染多种基因型。PCR扩增子的序列在相应的进化枝内同源地聚簇。这些发现表明本文所述的类型特异性PCR测定可用于确定B的基因型多样性。bovis msa-1基因在亚洲。(C)2015 Elsevier B. V.版权所有。
Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia. (C) 2015 Elsevier B.V. All rights reserved.