【作者】 李有全；

【导师】 殷宏； 罗建勋； 白启；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2007， 博士

【摘要】 羊泰勒虫病（ovine and caprine theileriosis）是由媒介蜱传播的由泰勒科泰勒属的原虫寄生于绵羊和山羊巨噬细胞、淋巴细胞和红细胞内所引起的疾病的总称。泰勒虫与巴贝斯虫、无浆体、可厥体可分别引起家畜的泰勒虫病、巴贝斯虫病、无浆体病及心水病，将这些疾病统称为蜱传病（Tick-borne diseases），在全世界每年造成约70亿美元的经济损失。羊泰勒虫病危害严重，可引起羔羊和外地引进羊大量死亡，使慢性发病的山羊和绵羊发育变缓，产肉量和产毛量显著下降，从而给养羊业造成重大经济损失。在我国，羊泰勒虫病是一种重要而常见的蜱传性原虫病，主要分布于我国西部地区，包括甘肃、宁夏、内蒙古、青海、四川和陕西等省。然而，目前国内对羊泰勒虫病的研究尚不全面。本项研究以我国分布的两种羊泰勒虫为主要研究对象，对其生物学特性进行进一步的研究，主要研究结果如下：1．利用传播试验，确定青海血蜱和长角血蜱可以作为我国新定名的吕氏泰勒虫的传播媒介。从感染吕氏泰勒虫的羊体饱血的青海血蜱幼蜱和若蜱发育而来的若蜱和成蜱，能分别传播这种泰勒虫给除脾绵羊；同时，从感染吕氏泰勒虫的羊体饱血的长角血蜱幼蜱和若蜱发育而来的长角血蜱若蜱和成蜱，也能分别传播这种泰勒虫给除脾绵羊。实验结果表明，吕氏泰勒虫至少能被2种硬蜱—青海血蜱和长角血蜱传播，传播方式为阶段性传播。2．利用传播试验，确定青海血蜱和长角血蜱可以作为我国新定名的尤氏泰勒虫的传播媒介。从感染尤氏泰勒虫的羊体饱血的青海血蜱幼蜱和若蜱发育而来的若蜱和成蜱，能分别传播这种泰勒虫给除脾和未除脾绵羊；同时，从感染尤氏泰勒虫的羊体饱血的长角血蜱若蜱发育而来的成蜱，能将这种泰勒虫传播给除脾和未除脾绵羊；但是，从感染尤氏泰勒虫的羊体饱血的长角血蜱幼蜱发育而来的若蜱，对尤氏泰勒虫的传播没有成功。实验表明，尤氏泰勒虫至少能被2种硬蜱—青海血蜱和长角血蜱传播，传播方式为阶段性传播。3．用液氮保存的吕氏泰勒虫和尤氏泰勒虫的虫血分别感染绵羊，待虫体出现后，每日做血液涂片、空气干燥、甲醇固定和姬姆萨染色，进行两种虫体的形态学观察。观察内容包括虫体形态种类、各类虫体出现的先后次序、最高染虫率和染虫率高峰时各类虫体所占比例。结果表明，这两种泰勒虫的虫体胞浆呈浅蓝色，细胞核成紫色，形态类型主要包括圆形、卵圆形、针形、杆状、三叶草形、十字架形、逗点形和不规则等多种形态。在整个病程中，以梨籽形、圆形（卵圆形）、针形3类虫体占优势；而且这2个虫种在形态学上几乎没有明显著异。4．对尤氏泰勒虫人工感染的试验绵羊进行了病理学观察。结果显示：体表少毛部位、眼结膜、皮下脂肪和大网膜轻度黄染；淋巴结和肺脏肿胀，切面湿润多汁；心、肝、肺、肠道利瘤胃表面可见针尖至粟粒大小出血点或出血斑。主要的组织学病变为淋巴结充血、出血及淋巴窦网状内皮细胞增生；肺脏间质、网状细胞增生，肺泡隔增厚；肾小球内皮细胞、血管内皮细胞增生，淋巴网状细胞灶状积累，甚至形成结节；心肌细胞变性坏死，染色不均；肠道肌层、粘膜层和固有膜明显充血、出血；肝细胞颗粒变性和有散在细胞坏死，汇管区和小叶间有淋巴网状细胞增生；瘤胃黏膜下层有散在性出血，肌内层见灶状出血。5．建立了一种从感染有羊泰勒虫的羊血液细胞分离羊泰勒虫裂殖子的快速方法。用嗜水气单孢菌产生的细胞毒素—溶血素Ah-1，裂解寄生有羊泰勒虫的红细胞，继而用Percoll密度梯度离心分离和纯化羊泰勒虫裂殖子。光学显微镜检验表明，从感染羊泰勒虫的红细胞分离的裂殖子仍然保持正常的虫体形态，并从宿主细胞成分中游离出来。6．构建了吕氏泰勒虫cDNA表达文库，文库的重组率为100％，初级文库库容量约为1.0×10⁶PFU，扩增文库滴度约为8.2×10⁸ PFU／mL。本研究用RNAlater RNA Stabilization Reagent保存的吕氏泰勒虫虫体直接提取并纯化mRNA，采用oligo（dT）引物合成双链cDNA，并在其两端加EcoRⅠ／HindⅢ定向接头。将所产生的cDNA分子定向克隆到具有EcoRⅠ／HindⅢ粘性末端的λSCREEN载体的两臂之间。用PhageMaker extract对以上连接产物进行体外包装以形成完整的噬菌体，并用之转染大肠杆菌ER1647，从而构建成吕氏泰勒虫的cDNA表达文库。7．筛选出9个具有免疫反应性的基因。将吕氏泰勒虫cDNA表达文库与宿主菌BL21（DE3）pLysE按一定比例混合孵育并铺板，当噬斑出现后将NC膜覆盖于顶层胶表面并继续培养3.5 h。随后，将此NC膜依次与吕氏泰勒虫阳性血清和碱性磷酸酶标记的兔抗绵羊IgG反应，最后加底物显色，初步筛选得到蓝紫色的阳性克隆，并复筛直至整个膜上均为阳性信号为止。用所得阳性噬菌体转染宿主菌BM25.8使之自动亚克隆为重组质粒，用此亚克隆质粒转化宿主菌JM109并从中提取重组质粒进行PCR、测序和蛋白表达分析，共获得24个新基因，10个基因能成功地诱导表达，9个具有免疫反应性。8．建立了吕氏泰勒虫的PCR检测方法。利用从吕氏泰勒虫筛选的T1401基因，根据其序列设计了其特异性引物，用PCR方法检测吕氏泰勒虫感染血样中虫体基因组DNA。从自然感染吕氏泰勒虫的基因组中，用该引物扩增出368bp大小的DNA片段；而用该引物不能从尤氏泰勒虫，羊巴贝斯虫未定种、绵羊无浆体和健康羊基因组DNA中扩增出368bp大小的DNA片段。PCR检测方法能从1％吕氏泰勒虫虫血稀释到10^-5倍，仍能检测出虫体DNA。该法比光学显微镜检200个视野敏感。为我国吕氏泰勒虫的流行病学调查提供了一种可靠的检测和诊断方法。更多还原

【Abstract】 Ovine and caprine theileriosis is caused by genus Theileria, which is transmitted by ticks and parasitized in macrophage, lymphblast and eryhrocytes of a variety of sheep and goats. Theileria, Babesia, Anaplasma and Cowdria were able to respectively cause serious theileriosis, babesiosis, anaplasmosis and heartwater, and all of them were known as tick-borne diseases. Tick-borne diseases caused a loss of 7 billion $ per year. The disease seriously inflences the sheep industry and results in many exotic sheep （or goats） and local lambs to death each year, and caused a huge economic loss of sheep industry. Theileriosis caused by Theileria luwenshuni and/or Theileria uilenbergi is an important tick-borne protozoan disease, and very common in sheep and goats in China. Investigations carried out over many years indicated that it is mainly distributed in Western China, including Gansu, Ningxia, Inner Mongolia, Qiahai, Sicuan and Shaanxi. However, the disease and causative pathogen have not been systematically studied in China. In this study, Theileria spp. infective to sheep and goats is characterized biologically. The major results were presented below:1. Experimental transmission of a recently identified new Theileria luwenshuni pathogenic for sheep in northern China is described. Haemaphysalis qinghaiensis nymphs and adults developed from larvae and nymph engorged on sheep infected with T. luwenshuni were able to respectively transmit the T. luwenshuni to splenectomized sheep. Meanwhile, H. longicornis nymphs and adults developed from larvae and nymphs engorged on sheep infected with T. luwenshuni were also able to respectively transmit this new T. luwenshuni to splenectomized sheep. This experiment suggested that the T. luwenshuni could be transmitted by at least two species of Haemaphysalis sp. ticks, H. longicornis and H. qinghaiensis, and the mode of transmission is stage to stage.2. Experimental transmission of a recently identified new Theileria uilenbergi pathogenic for sheep in northern China is described. H. qinghaiensis nymphs and adults developed from larvae and nymph engorged on sheep infected with T. uilenbergi were able to respectively transmit the T. uilenbergi to splenectomized and intact sheep. Meanwhile, H. longicornis adults developed from nymphs engorged on sheep infected with T. uilenbergi were also able to respectively transmit this new T. uilenbergi to splenectomized sheep, but H. longicornis nymphs developed from larvae engorged on sheep infected with T. uilenbergi failed to respectively transmit this Theileria sp. to sheep. This experiment suggested that the T. uilenbergi could be transmitted by at least two species of Haemaphysalis sp. ticks, H. longicornis and H. qinghaiensis, and the mode of transmission is stage to stage.3. Description of morphology of T. luwenshuni and T. uilenbergi. The splenectomized sheep were respectively injected jugularly with the cryopreserved blood containing T. luwenshuni and T. uilenbergi. When piroplasms appeared in the blood smear from the ears of two sheep, the morphology and dimensions of parasites was examined from thin blood smears, air-dried, fixed with methanol, and stained with 10% Giemsa solution. The dimensions of the intra-erythrocytic stages of T. luwenshuni and T. uilenbergi were determined from a splenectomized sheep 1246 and sheep 2239 with aid of an ocular micrometer thin blood smears. The parasites selected for measurements were of various morphologies, and 200 parasites were measured. The resulted showed that the protoplasma was slightly blue, and the nucleus was purple by Giemsa staining. The morphology of the two designated strain of Theileria spp. appears mostly as single pyriform, needle, rod, round, oval, cross, amoeboid, etc. forms. In the infected sheep, the pyriform and needle forms were found first, then the rod, round, and oval forms, in the end, three-leaf and cross shaped. The pyriform, the round and the needle almost exist in every stage of the disease and seem to have a dominant shape. Meanwhile, there are no differences in morphology and sizes between T. luwenshui and T. uilenbergi.4. Pathological observation of experimental theileriosis caused by Theileria uilenbergi. Sheep were infected experimentally with T. uilenbergi by subcutaneous injection. The results showed that some tissues, including the less wool part, eye conjunctiva, subcutaneous fat and greater omentum of sheep were slightly yellowish. The heart, liver and lung were generally enlarged, and the cut surfaces of lymph nodes and lung were generally moist and juiceness. Meanwhile, various sizes of haemorrhage were seen in the heart, liver, lung, kidney, especially worst in intestine etc.. The principal microscopic founding was hyperaemia, hemorrhage and proliferation of reticuloendothetial cell in lymph nodes, proliferation of interstitium and reticulocytes in lung, and alveolar septum become wider, proliferation of renal glomerulus intima cells and blood vessel intima cells in ren. Lymph reticulocytes were accumulated into a tubercle in ren. Cardiac muscle cells showed apomorphosis and uneven dyeing. The tunica propria was obvious hyperaemia and hemorrhage in the intestinal tract. Additionally, the hepatocytes showed apomorphosis and necrosis. various sizes of haemorrhage were seen in submucosa and muscle layer of rumen.5. A simple and rapid method for purifying merozoites of Theileria spp. from infected erythrocytes in sheep or goats. Infected erythrocytes were effectively lysed using hemolysin Ah-1 produced from Aeromonas hydrophila, and the lysate was subjected to centrifugation in a Percoll^TM discontinuous density gradient at common speed. Light microscopical study showed that the merozoites purified retained their intact basic structure and were free of erythrocyte membrance and other host-cell components.6. Establishment of cDNA expression library of T. luwenshuni. Purified mRNA was isolated from the T. luwenshuni parasites, which were cryopreserved in RNAlater RNA Stabilization Reagent at-70℃. A library of oligo（dT）-primed cDNA with added directional EcoRⅠ/HindⅢlinkers was constructed and ligated to the EcoRⅠ/HindⅢarms of the screen vector. The recombinant phage DNA was packaged by using PhageMaker packaging extracts, resulting in a primary cDNA library with a size of 1.0×10 PFU and amplified cDNA library with a phage titer of 8.2×10⁸PFU/mL.7. Immunoscreening of nine new genes. Recombinant phages were plated on a host BL21（DE3）pLysE, and when plaques begin to form, the plates were overlaid with nitrocellulose filters and incubated for an additional 3.5 hours. The filters were processed by successive incubations with anti-Theileria luwenshuni serum, rabbit anti- sheep IgG alkaline phosphatase conjugate, and color development substrates and thus positive plaques with intense purple color were found. Pure phage stocks were obtained by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned in BM25.8 were isolated and then transformed into host strain JM109. Twenty-four new genes were obtained after analysis of the plasmids exstracted from JM109 by PCR, sequencing and expression, ten genes of them can be expressed successfully, and nine genes were positive against Theileria luwenshuni sera with Western Blotting.8. Development of PCR for detection of T. luwenshuni. A PCR method was developed with a pair of specific primers, which were designed according to the partial nucleotide sequence of T1401 gene immunoscreened from cDNA expression library of Theileria luwenshuni. A 368-bp DNA fragment was specifically amplified from genomic DNA purified from blood samples from sheep and goats, naturally infected with T. luwenshuni. No PCR products resulted from T. uilenbergi, unidentified Babesia sp., Anaplasma ovis, or normal sheep leucocytes DNA using these specific primers. The PCR was sensitive enough to detect parasite DNA from the dilution of 10^-5 with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. These results indicate that the PCR provides a useful diagnostic tool for the detection of T. luwenshuni infection and epidemiological survey in small ruminants in China.更多还原