羊梨形虫核糖体RNA内转录间隔区序列的测定及反向线状印迹技术的建立
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摘要
羊梨形虫病(旧称羊焦虫病)是由顶复门、梨形虫纲、梨形虫目中的巴贝斯科(Babesiidae)巴贝斯属(Babesia)和泰勒科(Theileriidae)泰勒属(Theileria)的多种原虫寄生于羊的红细胞或网状内皮细胞内所引起的一类血液原虫病的统称,临床以高热、贫血、黄疸、血红蛋白尿或体表淋巴结肿大为主要特征,严重时可引起动物死亡。本病在我国分布广、危害严重,常常给疫区的养羊业造成巨大损失。
本研究利用实验室液氮保存的羊梨形虫病病原,包括2种(6株)羊的巴贝斯虫和2种(2株)羊的泰勒虫,采用核糖体RNA的两个内转录间隔区(ITS1和ITS2)基因组区域和5.8S rRNA基因序列分析方法,对我国的新疆喀什地区、河北承德以及甘肃省的临潭、宁县、天祝、麻当六个不同地区羊的巴贝斯虫和甘肃省的渭源、宁夏的隆德两个地区羊泰勒虫进行分子分类学及分子诊断研究。其主要研究结果包括两个方面:
1.利用实验室保存的8株羊梨形虫,提取基因组DNA,测得ITS1-5.8S-ITS2、ITS1、5.8S和ITS2序列,分别构建系统发生树,并进行同源性比较。结果显示我国羊的梨形虫可明显分为巴贝斯虫和泰勒虫两枝;其中,莫氏巴贝斯虫株之间的同源性明显高于新疆喀什株未定种巴贝斯虫。巴贝斯虫的ITS1和ITS2核苷酸区段变异较大,种间同源性分别在19.7%-99.4%,河北株、宁县株、临潭株、天祝株和麻当株的巴贝斯虫都是由血蜱属传播的,在遗传演化过程中的亲缘关系较近,同源性比较高,在ITS1-5.8S-ITS2、ITS1和ITS2序列的系统发生树上,共处一枝。新疆喀什株是由璃眼蜱属传播的,被其它的一些巴贝斯虫隔开,独立占有一枝,可以认为是一个独立的种。5.8S rRNA较保守,种间同源性在87.1%-100%之间,系统发生树上各种巴贝斯虫的分布与ITS1-5.8S-ITS2、ITS1和ITS2系统发生树基本一致。两种泰勒虫之间的同源性比较高,同已经测得的18S rRNA基因区段相似,其ITS1和ITS2区段种间变异较小,ITS1-5.8S-ITS2、ITS1、5.8S和ITS2序列的同源性分别为96.8%、96.4%、100%和95.2%。该结果与18S rRNA基因序列分类方法基本符合。
2.在已经确定的18S rRNA基因V4高变区段的基础上,利用反向线状印迹杂交技术(RLB)高特异性和高敏感性的特点,设计了梨形虫通用探针和属种特异性探针,用RLB技术检测以上8株羊梨形虫。所有种特异性探针都仅仅和它们各自的靶序列发生反应,无论是在不同虫种,还是不同种群之间都没有交叉反应,与绵羊/山羊基因组对照试验没有杂交信号。将两种羊巴贝斯虫(莫氏巴贝斯虫和巴贝斯虫未定种喀什株)和两种羊泰勒虫(吕氏泰勒虫渭源株,尤氏泰勒虫隆德株)基因组经分光光度计定量在100ng/ul,连续10倍稀释10-1-10-12,用RLB方法检测,结果显示泰勒虫较羊巴贝斯虫的敏感性高,莫氏巴贝斯虫、巴贝斯虫未定种喀什株、吕氏泰勒虫和尤氏巴贝斯虫的敏感性分别是0.01pg、0.1ng、0.0001pg、0.1pg,吕氏泰勒虫的敏感性明显高于尤氏泰勒虫。该方法还可以用来鉴别诊断吕氏泰勒虫和尤氏泰勒虫感染。对117份野外样品检测,并与ELISA血清检测和PCR结果相比较,发现反向线状印迹的检出率明显高于ELISA和PCR。所建立的方法可用于羊梨形虫病的诊断和流行病学调查。
Ovine piroplasmosis is caused by Apicomplexa, Piroplasmia, Babesiidae Babesia and Theileriidae Theileria. The disease is usually characterized by fever, hemolytic anemia, hemoglobinuria, and even death in severe case. It is widely distributed in China, and considered to be the most frequent and important tick-born disease of small ruminants and responsible for vast economic loss.
In this study, genomic DNA of the etiological agents (6 isolates of 2 Babesia species and 2 isolates of 2 Theileria species) of ovine piroplasmosis was extracted. The agents of Babesia were originated from Chengde County of Hebei Province, Kashi region of Xinjiang Province and Lintan, Ningxian, Tianzhu and Madang regions of Gansu Province, while agents of Theileria were from Weiyuan in Gansu province and Longde regions in Ningxia province. The molecular taxonomy was carried out based on the gene sequencing of ITS1, ITS2 and 5.8SrRNA of these isolates. The major results were presented as following: 1. The sequencing of ITS1-5.8S-ITS2 gene was sequenced, and then alignment of ITS1-5.8S-ITS2, ITS1, 5.8S and ITS2 was conducted by Clustal W method respectively and the corresponding phylogenetic trees were inferred. The method was successfully discriminating Babesia from Theileria. The phylogenetic trees illustrated that Babesia isolates from Hebei, Ningxian, Lintan, Tianzhu and Madang considered as Babesia motasi were placed onto one branch, whereas the isolate from Kashi remained onto another. The homology among Babesia motasi was obviously higher than that of Babesia sp. Kashi indicating the later is a new species of ovine Babesia. Moreover, B. motasi is transmitted by ticks of genus of Haemaphysalis, whereas B. sp Kashi is transmitted by ticks of Hyalomma genus. This supports that B. sp Kashi could be an independent species. B. motasi and Babesia sp. Kashi possessed unique size(s) of ITS1 and ITS2 and species specific nucleotide sequences. The 5.8S gene is conserved; the homology among the species was 87.1%-100%. The homology between two Theileria was high, which of ITS1-5.8S-ITS2、ITS1、5.8S and ITS2 was 96.8%、96.4%、100% and 95.2% respectively.
2. Based on the V4 hypervariable region of 18S rRNA sequences of ovine piroplasma, the general piroplasma probes and species-specific probes were synthesized for RLB technique. Eight isolates of ovine piroplasma were detected by applying this method, the species-specific probes could recognize only their corresponding piroplasma genomic DNA, while there was no hybridization signal with the genome of sheep/goat as the control. The sensitivity of RLB was detected by 10-fold dilution of genome of two ovine Babesia spp (B. motasi and B. sp Kashi strain ) and two ovine Theileria(T. luwenshuni Weiyuan strain and T. uilenbergi strain )from 100ng/ul on. The results indicated that the sensitivity of ovine Babesia spp is lower than the ovine Theileria, and the sensitivity of T. luwenshuni is higher than T. uilenbergi obviously. We found that the detection ability of RLB is higher than that of ELISA and PCR through detected 117 samples from field by these methods. The established RLB can be used to detect, diagnose and perform epidemiologic survey.
本研究利用实验室液氮保存的羊梨形虫病病原,包括2种(6株)羊的巴贝斯虫和2种(2株)羊的泰勒虫,采用核糖体RNA的两个内转录间隔区(ITS1和ITS2)基因组区域和5.8S rRNA基因序列分析方法,对我国的新疆喀什地区、河北承德以及甘肃省的临潭、宁县、天祝、麻当六个不同地区羊的巴贝斯虫和甘肃省的渭源、宁夏的隆德两个地区羊泰勒虫进行分子分类学及分子诊断研究。其主要研究结果包括两个方面:
1.利用实验室保存的8株羊梨形虫,提取基因组DNA,测得ITS1-5.8S-ITS2、ITS1、5.8S和ITS2序列,分别构建系统发生树,并进行同源性比较。结果显示我国羊的梨形虫可明显分为巴贝斯虫和泰勒虫两枝;其中,莫氏巴贝斯虫株之间的同源性明显高于新疆喀什株未定种巴贝斯虫。巴贝斯虫的ITS1和ITS2核苷酸区段变异较大,种间同源性分别在19.7%-99.4%,河北株、宁县株、临潭株、天祝株和麻当株的巴贝斯虫都是由血蜱属传播的,在遗传演化过程中的亲缘关系较近,同源性比较高,在ITS1-5.8S-ITS2、ITS1和ITS2序列的系统发生树上,共处一枝。新疆喀什株是由璃眼蜱属传播的,被其它的一些巴贝斯虫隔开,独立占有一枝,可以认为是一个独立的种。5.8S rRNA较保守,种间同源性在87.1%-100%之间,系统发生树上各种巴贝斯虫的分布与ITS1-5.8S-ITS2、ITS1和ITS2系统发生树基本一致。两种泰勒虫之间的同源性比较高,同已经测得的18S rRNA基因区段相似,其ITS1和ITS2区段种间变异较小,ITS1-5.8S-ITS2、ITS1、5.8S和ITS2序列的同源性分别为96.8%、96.4%、100%和95.2%。该结果与18S rRNA基因序列分类方法基本符合。
2.在已经确定的18S rRNA基因V4高变区段的基础上,利用反向线状印迹杂交技术(RLB)高特异性和高敏感性的特点,设计了梨形虫通用探针和属种特异性探针,用RLB技术检测以上8株羊梨形虫。所有种特异性探针都仅仅和它们各自的靶序列发生反应,无论是在不同虫种,还是不同种群之间都没有交叉反应,与绵羊/山羊基因组对照试验没有杂交信号。将两种羊巴贝斯虫(莫氏巴贝斯虫和巴贝斯虫未定种喀什株)和两种羊泰勒虫(吕氏泰勒虫渭源株,尤氏泰勒虫隆德株)基因组经分光光度计定量在100ng/ul,连续10倍稀释10-1-10-12,用RLB方法检测,结果显示泰勒虫较羊巴贝斯虫的敏感性高,莫氏巴贝斯虫、巴贝斯虫未定种喀什株、吕氏泰勒虫和尤氏巴贝斯虫的敏感性分别是0.01pg、0.1ng、0.0001pg、0.1pg,吕氏泰勒虫的敏感性明显高于尤氏泰勒虫。该方法还可以用来鉴别诊断吕氏泰勒虫和尤氏泰勒虫感染。对117份野外样品检测,并与ELISA血清检测和PCR结果相比较,发现反向线状印迹的检出率明显高于ELISA和PCR。所建立的方法可用于羊梨形虫病的诊断和流行病学调查。
Ovine piroplasmosis is caused by Apicomplexa, Piroplasmia, Babesiidae Babesia and Theileriidae Theileria. The disease is usually characterized by fever, hemolytic anemia, hemoglobinuria, and even death in severe case. It is widely distributed in China, and considered to be the most frequent and important tick-born disease of small ruminants and responsible for vast economic loss.
In this study, genomic DNA of the etiological agents (6 isolates of 2 Babesia species and 2 isolates of 2 Theileria species) of ovine piroplasmosis was extracted. The agents of Babesia were originated from Chengde County of Hebei Province, Kashi region of Xinjiang Province and Lintan, Ningxian, Tianzhu and Madang regions of Gansu Province, while agents of Theileria were from Weiyuan in Gansu province and Longde regions in Ningxia province. The molecular taxonomy was carried out based on the gene sequencing of ITS1, ITS2 and 5.8SrRNA of these isolates. The major results were presented as following: 1. The sequencing of ITS1-5.8S-ITS2 gene was sequenced, and then alignment of ITS1-5.8S-ITS2, ITS1, 5.8S and ITS2 was conducted by Clustal W method respectively and the corresponding phylogenetic trees were inferred. The method was successfully discriminating Babesia from Theileria. The phylogenetic trees illustrated that Babesia isolates from Hebei, Ningxian, Lintan, Tianzhu and Madang considered as Babesia motasi were placed onto one branch, whereas the isolate from Kashi remained onto another. The homology among Babesia motasi was obviously higher than that of Babesia sp. Kashi indicating the later is a new species of ovine Babesia. Moreover, B. motasi is transmitted by ticks of genus of Haemaphysalis, whereas B. sp Kashi is transmitted by ticks of Hyalomma genus. This supports that B. sp Kashi could be an independent species. B. motasi and Babesia sp. Kashi possessed unique size(s) of ITS1 and ITS2 and species specific nucleotide sequences. The 5.8S gene is conserved; the homology among the species was 87.1%-100%. The homology between two Theileria was high, which of ITS1-5.8S-ITS2、ITS1、5.8S and ITS2 was 96.8%、96.4%、100% and 95.2% respectively.
2. Based on the V4 hypervariable region of 18S rRNA sequences of ovine piroplasma, the general piroplasma probes and species-specific probes were synthesized for RLB technique. Eight isolates of ovine piroplasma were detected by applying this method, the species-specific probes could recognize only their corresponding piroplasma genomic DNA, while there was no hybridization signal with the genome of sheep/goat as the control. The sensitivity of RLB was detected by 10-fold dilution of genome of two ovine Babesia spp (B. motasi and B. sp Kashi strain ) and two ovine Theileria(T. luwenshuni Weiyuan strain and T. uilenbergi strain )from 100ng/ul on. The results indicated that the sensitivity of ovine Babesia spp is lower than the ovine Theileria, and the sensitivity of T. luwenshuni is higher than T. uilenbergi obviously. We found that the detection ability of RLB is higher than that of ELISA and PCR through detected 117 samples from field by these methods. The established RLB can be used to detect, diagnose and perform epidemiologic survey.
引文
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