Veterinary Department of Isfahan Research Center for Agriculture and Natural Resources, Isfahan, Iran.
Vet Res Commun. 2010 Jan;34(1):43-50. doi: 10.1007/s11259-009-9331-3. Epub 2009 Dec 15.
Species of the genus Anaplasma (Rickettsiales: Anaplasmataceae) are obligate intracellular tick borne pathogens. Three species of Anaplasma that infect cattle and sheep (A. marginale, A. centrale and A. ovis) are well recognized. Of these erythrocytic Anaplasma, A. marginale can cause diseases in the livestock with high economical losses. Species-specific PCR based on 16S rRNA gene is commonly used for detection of Anaplasma species but can not differentiate A. marginale, A. centrale and A. ovis because of sequence similarity. In this study DNA extraction was performed on 50 blood samples with presence of Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells was analyzed by PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst1107 I. The restriction endonuclease Bst1107I only recognizes the sequence (GTATAC) in corresponding PCR product of A. marginale and cut it. The nucleotide sequence of the A. marginale 16S rRNA gene was determined and compared with the sequences of A. marginale in GenBank. The 16S rRNA of A. marginale in Iran was completely similar to the related sequence deposited in GenBank at accession number of M60313. In the present study we propose a new PCR-restriction fragment length polymorphism analysis (RFLP) method based on 16S rRNA gene for specific detection of A. marginale.
绵羊无浆体（Rickettsiales: Anaplasmataceae）属的种是专性细胞内蜱传播病原体。感染牛和羊的三种绵羊无浆体（A. marginale、A. centrale 和 A. ovis）已被广泛认识。在这些红细胞内的绵羊无浆体中，A. marginale 可引起牲畜疾病，造成巨大的经济损失。基于 16S rRNA 基因的种特异性 PCR 常用于检测绵羊无浆体，但由于序列相似性，无法区分 A. marginale、A. centrale 和 A. ovis。在本研究中，对 50 份存在于血涂片红细胞边缘点的绵羊无浆体 spp.血液样本进行了 DNA 提取。从血细胞中提取的 DNA 通过 PCR 和使用来自 16S rRNA 基因和限制性内切酶 Bst1107 I 的引物的 PCR-RFLP 进行分析。限制性内切酶 Bst1107I 仅识别 A. marginale 相应 PCR 产物中的序列（GTATAC）并对其进行切割。确定了 A. marginale 16S rRNA 基因的核苷酸序列，并与 GenBank 中 A. marginale 的序列进行了比较。伊朗的 A. marginale 16S rRNA 与 GenBank 中登录号为 M60313 的相关序列完全相似。在本研究中，我们提出了一种基于 16S rRNA 基因的新 PCR-限制性片段长度多态性分析（RFLP）方法，用于特异性检测 A. marginale。
