Cloning of bovine hemoparasites and isolation of genetic markers for the parasite clones

巴贝斯虫和泰勒虫等原生血吸虫引起的疾病仍然是全世界畜牧业的主要威胁。为了方便对这些血寄生虫的实验研究，我们已经建立了一种用牛红细胞替代循环红细胞的SCID小鼠(Bo-RBC-SCID小鼠)的动物模型。在本研究中，我们使用该动物模型系统来获得寄生虫克隆。用卵形芽孢杆菌或瑟氏锥虫感染Bo-RBC-SCID小鼠制备高原虫血症(40%以上)的RBC样品。在显微镜下用微毛细管分离单个红细胞，接种到Bo-RBC-SCID小鼠体内。通过重复这一过程两次，建立了寄生虫克隆。我们能够分离出多个寄生虫克隆，它们的特征似乎彼此不同。为了进一步确定寄生虫的个体克隆，我们从卵形巴贝斯虫的11e号克隆中寻找了具有多态性的遗传标记，克隆了几个基因，包括SPS-7、核糖体小亚基RNA、肌动蛋白、β-微管蛋白、杆状相关蛋白-1、球体蛋白、裂殖子表面蛋白和延伸因子-1。其中许多基因被用作Southern杂交分析的探针时表现出多态性，在一些基因中，寄生虫克隆之间也出现了显著的序列差异。本研究获得的寄生虫克隆和多态遗传标记，将为研究寄生虫种群的多样性和媒介蜱有性阶段可能发生的遗传重组事件提供有用的实验工具。

Diseases caused by hemoparasitic protozoa, such as Babesia and Theileria, still remain to be a major threat to livestock industry worldwide. In order to facilitate the experimental studies on those hemoparasites, we have previously developed an animal model using SCID mice whose circulating red blood cells were substituted with bovine ones (Bo-RBC-SCID mice). In the present study, we used this animal model system to obtain parasite clones. RBC samples containing high parasitemia (more than 40%) were prepared by infection of Bo-RBC-SCID mice with B.ovata or T.sergenti. Single RBCs were individually picked under microscopy with a microcapillary tube, and each of them was inoculated into a Bo-RBC-SCID mouse. Parasite clones were established by repeating this procedure twice. We were able to isolate multiple parasite clones, whose characteristics appeared to be different from each other. To further define the individual parasite clones, we searched genetic markers which show polymorphism among parasite clones From the clone 11E of Babesia ovata, several genes were cloned and sequenced, including SpS-7, small subunit ribosomal RNA, actine, beta-tubulin, roptory associated protein-1, spherical body protein, merozoite surface protein, and elongation factor-1. Many of those genes were found to show polymorphism when they were used as the probe for southern blot analysis.In some genes, significant sequence variations were also seen between parasite clones. We were able to differentiate the individual parasite clones using combinations of several marker genes.The parasite clones and the polymorphic genetic markers obtained in the present study will serve as a useful experimental tool to study the diversified parasite population and possible genetic recombinational event occurring at the sexual stage in the vector ticks.