Morzaria S P, Dolan T T, Norval R A, Bishop R P, Spooner P R
International Livestock Research Institute, Nairobi, Kenya.
Parasitology. 1995 Jul;111 ( Pt 1):39-49. doi: 10.1017/s0031182000064581.
A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.
开发了一种用于克隆小泰勒虫寄生虫的三步程序。第一步涉及用滴定的子孢子对固定数量的牛淋巴细胞进行体外感染。然后选择使用低于1:100的子孢子/淋巴细胞比率启动感染所获得的细胞系进行克隆，因为这些细胞系含有裂殖体感染的细胞，每个细胞均源自单个子孢子的感染。第二步，通过有限稀释对这些细胞系进行克隆。由于子孢子感染淋巴细胞并转化以诱导克隆增殖，这一步产生了同时含有克隆寄生虫和克隆淋巴细胞的感染细胞系。第三步，使用克隆的细胞系感染牛，并在梨形虫血症期间在蜱中分离寄生虫。最后，从感染的蜱中收获子孢子并用于进一步表征。使用单克隆抗体谱、使用重复和端粒探针检测的DNA限制性片段长度多态性、体内感染性以及在一个案例中的交叉免疫研究，对源自小泰勒虫穆古加、马里凯布尼、博莱尼、乌干达和水牛源7014克隆细胞系的子孢子进行了表征。此外，从穆古加、马里亚卡尼和水牛源7014原种中分离出了几个不同的裂殖体感染淋巴细胞克隆。表征的综合结果表明，克隆程序从已知含有遗传上不同寄生虫群体混合物的亲代原种中选择了小泰勒虫的克隆。所产生的克隆方法和克隆将在寄生虫生物学研究以及阐明牛免疫反应的菌株特异性方面具有价值。
