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Identification of attenuation markers of a Theileria lestoquardi cell line to be used for the development of live vaccine against malignant ovine theileriosis

机译:鉴定用于开发抗恶性绵羊黑胫病活疫苗的泰勒虫(Theileria lestoquardi)细胞系的减毒标记物

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摘要

Theileria lestoquardi is a tick-borne protozoan parasite and highly pathogenic for sheep. The disease caused by the pathogen is known as malignant ovine theileriosis (MOT) and is transmitted by Hyalomma ticks. Control of the disease can be achieved by immunization of sheep with attenuated T. lestoquardi schizont-infected ovine cells that provides the animal with solid immunity. The approach of using the attenuated vaccine against malignant ovine theileriosis has been carried out successfully in Iraq and Iran. Better characterization of attenuated cell lines could result in the identification of markers that would allow more rapid selection of attenuated vaccine and reduce the cost of vaccine production. Since no work has been reported regarding attenuation mechanisms in T. lestoquardi, the following study investigated potential attenuation markers of T. annulata infected cells in a T. lestoquardi cell line at different passages. Two markers associated with attenuation in T. annulata vaccine strains were analyzed, matrix metalloproteinase activity and TNF-alpha mRNA expression. Furthermore, differentially expressed genesin higher passage and lower passage were analyzed using suppression subtractive hybridization in order to identify genes whose expression correlates with subculturing and thus potentially with attenuation. The expression of matrix metalloproteinase-9 (MMP9) and matrix metalloproteinase-2(MMP2) in the investigated cell line was confirmed by using specific inhibitors. The results showed gradual reduction in the activity of matrix metalloproteinase-9 (MMP9)with increasing passage number. Following the mRNA expression of TNF-alpha in different passages revealed down regulation of this cytokine from the low passage compared with high passage. Analysis of randomly selected clones in the suppression subtractive hybridization libraries identified nine differentially expressed genes, one from the parasite and eight from the host. Transcripts of retinoblastoma binding protein 7, Enolase-a (ENO 1), Ki-67 antigen and H2A histone from the host and vacuolar H+ATPase from the parasite were more plentiful in low passage culture. RAB14, a member of the RAS oncogene family, glucose transporter type 3, creatine kinase B, and cytochrome C oxidase transcripts from the host were more abundant in high passage culture. Quantitative real time-PCR confirmed mRNA expression of the parasite vacuolar H+ATPase to be downregulated at higher passages. The expression of the Ki-67 protein was clearly decreased with increasing passage number in western blot using specific antibody. Moreover, assessment of thymidine incorporation as a measure for the proliferation rate clearly showed that with increasing passage number, the proliferation rate of the T. lestoquardi infected cells decreases. This study revealed that the matrix metalloproteinase enzymes (9 and 2) and TNF-alpha could be potential molecular markers for identification of attenuation in the Theileria lestoquardi (Atbara) cell line. Also the down regulated parasite gene, vacuolar H+- ATPase could be considered as a molecular marker for attenuation. Immunization trials in sheep with different passages are required to provide in vivo evidence to support these findings.
机译:Theileria lestoquardi是a传播的原生动物寄生虫,对绵羊具有高致病性。由病原体引起的疾病被称为恶性羊脂化病(MOT),并由透明质酸tick传播。该疾病的控制可通过用减毒的T. lestoquardi schizont感染的绵羊细胞免疫绵羊来实现,该绵羊细胞为动物提供了坚实的免疫力。在伊拉克和伊朗已经成功地使用了减毒疫苗来对抗恶性羊脂化病。减毒细胞系的更好表征可导致鉴定标记,这将允许更快速地选择减毒疫苗并降低疫苗生产成本。由于尚无关于T. lestoquardi衰减机制的报道,以下研究调查了T. lestoquardi细胞系中不同传代时间的被T. lesnquarata感染细胞的潜在衰减标记。分析了与无环线虫疫苗株减毒有关的两个标志物,基质金属蛋白酶活性和TNF-αmRNA表达。此外,使用抑制消减杂交法分析了较高传代和较低传代的差异表达基因,以鉴定其表达与继代培养有关并因此与减毒相关的基因。基质金属蛋白酶9(MMP9)和基质金属蛋白酶-2(MMP2)在所研究的细胞系中的表达已通过使用特异性抑制剂得以证实。结果表明,随着传代次数的增加,基质金属蛋白酶9(MMP9)的活性逐渐降低。继不同传代中TNF-α的mRNA表达后,与高传代相比,该细胞因子从低传代下调。在抑制消减杂交文库中随机选择的克隆的分析鉴定出九个差异表达的基因,一个来自寄生虫,八个来自宿主。在低传代培养中,来自宿主的视网膜母细胞瘤结合蛋白7,烯醇酶-a(ENO 1),Ki-67抗原和H2A组蛋白以及来自寄生虫的液泡H + ATPase的转录本更为丰富。在高传代培养中,来自宿主的RAS癌基因家族的成员RAB14、3型葡萄糖转运蛋白,肌酸激酶B和细胞色素C氧化酶转录物更为丰富。实时定量PCR证实,寄生虫液泡H + ATPase的mRNA表达在较高传代时被下调。在使用特异性抗体的蛋白质印迹法中,随着传代次数的增加,Ki-67蛋白的表达明显降低。而且,评估胸腺嘧啶核苷掺入作为增殖速率的指标清楚地表明,随着传代次数的增加,莱氏梭菌感染细胞的增殖速率降低。这项研究表明,基质金属蛋白酶(9和2)和TNF-α可能是鉴定Theileria lestoquardi(Atbara)细胞系减毒的潜在分子标记。另外,下调的寄生虫基因液泡H + -ATPase也可以被认为是减毒的分子标记。需要在不同传代的绵羊中进行免疫试验,以提供体内证据来支持这些发现。

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