亚历山大白蛉人工感染不同地区杜氏利什曼原虫的实验

本实验旨在了解新疆砾漠内的亚历山大白蛉对不同地区的杜氏利什曼原虫的适应性,进而推断外来的黑热病患者进入该地带后能否引起传播。 一、材料与方法 (一) 原虫来源新疆的杜氏利什曼原虫(LD)系1977年从巴楚干旱荒漠黑热病疫区内由硕大白蛉吴氏亚种(Phlebotomus major wui)体内分离获得,甘肃的系1988年从文县一例幼儿黑热病患者体内分离获     

杜氏利什曼原虫动基体DNA种林特异片段的克隆

作者在质粒pUC18中克隆了限制性内切酶AIuI消化的Leishmania donovani四川人分离株kDNA片段,筛选后获得能区别杜氏利什曼原虫山丘疫区分离株和平原疫区分离株的的克隆pLK1-10和对杜氏利什曼原虫四川人分离株特异的克隆pLK1-14,对杜氏利什曼原虫种特异的克隆PLK1-1、pLK1-2等。这些克隆将是鉴定杜氏利什曼原虫,区别鉴定山丘及平原疫区黑热病病原体较好的探针。     

杜氏利什曼原虫平原和荒漠疫区分离株LACK基因克隆及序列分析

目的　测定我国平原疫区和荒漠疫区杜氏利什曼原虫分离株活性蛋白激酶 C受体同源物 (L ACK)基因序列 ,并与山丘疫区分离株及国外利什曼原虫分离株进行比较。　方法　应用 RT- PCR扩增 L ACK基因 ,将其克隆入p UC18载体后用双脱氧链末端终止法测序 ,并与 Gen Bank中相关数据进行比较。　结果　用 RT- PCR成功扩增出约95 0 bp的 L ACK基因片段 ,测序结果表明其片段大小均为 94 2 bp,与 Gen Bank中多种利什曼原虫 L ACK基因的核苷酸序列一致性达 97%以上。我国山丘、平原和荒漠 3个不同疫区杜氏利什曼原虫分离株的 L ACK基因序列的一致性达95 %以上。　结论　获得了我国平原和荒漠疫区杜氏利什曼原虫 L ACK基因序列。我国 3个不同疫区杜氏利什曼原虫分离株的 L ACK基因具有高度同源性。     

我国不同地区的杜氏利什曼原虫对亚历山大白蛉感染性的观察

用白蛉人工感染利什曼原虫的方法,观察从新疆荒漠、甘肃山区及河南平原三地自人体成蛉体分离出来的杜氏利什曼原虫对新疆亚历山大白蛉的感染性。以白蛉的感染率、感染程度以及原虫在白蛉消化道内的进展等项指标,来衡量原虫对白蛉的适应性。结果发现,新疆荒漠的原虫对亚历山大白蛉有高度的感染性,甘肃的原虫居次,河南的原虫对白蛉的感染性很差。结合以往用单克隆抗体检测法和K-DNA杂交法对我国一些地区杜氏利什曼原虫的研究,以及我国荒漠、山区和平原地区的黑热病具有不同的流行病学特征等的结果分析,认为我国的杜氏利什曼原虫很可能存在不同的地域株。     

用生物素标记K—DNA鉴定国内不同地区利什曼原虫种株

本文用生物素标记利什曼原虫动基体DNA(K-DNA)以打点杂交和Southern杂交鉴定了我国不同地区杜氏利什曼原虫(Leishmanis donovani,简称L.d.)七个分离株,并在国内首次用K—DNA杂交技术鉴定病灶组织内及白蛉体内利什曼原虫种株。结果表明,我国不同地区类型黑热病病原体间K—DNA同源性存在差异。病灶组织及白蛉体内原虫鉴定获得成功,为黑热病的诊断和流行病学研究提供了一种快速准确可行的新方法。     

陕西省韩城市利什曼原虫SSU rRNA基因系统发育研究

目的 了解陕西省韩城市黑热病区利什曼原虫种类及分子遗传特征。方法 对来自韩城市的利什曼原虫阳性标本,进行SSU rRNA基因序列扩增,扩增产物进行测序,与其它不同种类、不同地区利什曼原虫的SSU rRNA基因序列进行比对,分析突变位点规律,建立系统发育树。结果 有3份标本SSU rRNA基因序列扩增测序成功,其基因序列与杜氏利什曼原虫、夏科氏利什曼原虫、婴儿利什曼原虫的SSU rRNA序列同源性较高,与婴儿利什曼原虫在UQ-II突变区有一个位点的变异,并与新疆荒漠虫株、甘肃省利什曼虫株聚为一类。结论 结合流行病学与SSU rRNA序列分析结果,陕西省韩城市黑热病病原体应为婴儿利什曼原虫。     

新疆喀什地区黑热病预防与控制的思考

黑热病(内脏利什曼病)是由杜氏利什曼原虫引起,通过媒介白蛉传播的一种慢性地方性寄生虫传染病.新疆喀什地区是我国黑热病的重点流行区之一.喀什地区地域广阔,由于独特的地理和生态环境,存在着人源型黑热病和荒漠型黑热病(自然疫源性黑热病)两种.荒漠型黑热病为人兽共患的自然疫源性疾病,流行范围广,传播媒介和保存宿主比较复杂,目前尚无有效的预防控制手段.     

陕西株利什曼原虫ITS-1基因片段序列多态性分析

目的分析陕西株利什曼原虫rRNA内转录间隔区间I(ITS-1)序列多态性,探讨利什曼原虫ITS-1分子遗传规律。方法陕西省韩城市犬、白蛉以及人体利什曼原虫阳性标本7份,提取核酸,PCR法扩增ITS-1片段,双向测序、拼接后与文献发表的代表株进行序列比对,构建系统发育树,进行系统发育分析。结果 7份标本均扩增出约320bp的片段,ITS-1序列之间同源性99%,与国内山丘型疫区虫株高度一致,与荒漠型遗传距离较远。系统发育分析陕西株与婴儿利什曼原虫和杜氏利什曼原虫聚为一类。结论陕西省韩城市分离自病犬、传播媒介白蛉及黑热病患者的利什曼原虫ITS-1序列同源,应为犬源型婴儿利什曼原虫。     

免疫小鼠腹腔巨噬细胞对杜氏利什曼原虫感染的反应

小鼠腹腔巨噬细胞能够支持杜氏利什曼原虫的生长、繁殖和转化。本文进一步观察了免疫小鼠的腹腔巨噬细胞对利什曼感染的反应以及淋巴因子对感染过程的影响。 材料与方法:1.杜氏利什曼原虫785株,系1978年从喀什地区一例黑热病人骨髓中分离,在感染仓鼠中传代保存;2.实验动物及免疫方法:取三周龄     

快速、准确地在现场诊断印度内脏利什曼病

确诊内脏利什曼病(黑热病),需要证明在器官抽吸物中存在利什曼原虫。本文作者进行了前瞻性研究,旨在评定一种浸了利什曼原虫抗原的硝化纤维试纸在检测针对利什曼原虫抗原K39的抗体时,所具有的诊断价值。这是一种无创伤性的、适于现场诊断的试验。K39存在于全世界各种能引起内脏感染的利什曼原虫株的无鞭毛体中。     

新疆利什曼原虫同工酶谱的初步研究：Ⅰ.酶谱型的分布

用５种酶ＭＥ，ＧＰＩ，Ｇ６ＰＤＨ，ＭＤＨ，ＥＳＴ的聚丙烯酰胺凝胶电泳法对新疆不同来源分离的５株利什曼原虫进行了同工酶谱的比较研究。所分析的虫株包括１株杜氏利什曼原虫，２株婴儿利什曼原虫，２株都兰利什曼原虫以及作为参比虫株的杜氏利什曼原虫（ＭＨＯＭ／ＩＮ／８０／ＤＤ８），硕大利什曼原虫（ＭＨＯＭ／ＩＱ／００／ＬＨ３２），沙鼠利什曼原虫（ＲＨＯ／ＣＮ／６２／＃ｌ）和蜥蜴利什曼原虫各１株，共分出９个酶谱型。表明不同来源的２株婴儿利什曼原虫是两个不同的酶谱型，２株都兰利什曼原虫为两个不同的酶谱型，新疆的杜氏利什曼原虫与来源于印度的杜氏利什曼原虫标准株也为两个不同的酶谱型。     

几种利什曼原虫前鞭毛体在不同培养基上生长繁殖的观察

在三恩氏培养基和经过改良的RPMI1640培养基上比较了5株杜氏利什曼,1株沙鼠利什曼和1株蜥蜴利什曼前鞭毛体的繁殖动态。结果表明有四种类型。其中761株杜氏利什曼和蜥蜴利什曼属于一组;杜氏利什曼801株和852株为一组;771株杜氏利什曼与沙鼠利什曼为一组,自1株杜氏利什曼本身为一组。7株利什曼在两种培养基上处于繁殖峰值的原虫数分别为2.8～3.8×10~7和3.9～7.9×10~6。讨论了这四种繁殖动态类型的生物学意义,并认为经过补充的RPMI 1640培养基可供在利什曼原虫的生物化学和免疫学研究中应用。     

An experimental model for canine visceral leishmaniasis

Seven mixed-breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post-infection. Anti-parasite specific antibody levels were measured by enzyme-linked immunosorbence, immunofluorescence, crossed-immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70-2, was also done. Mitogenic and antigen-specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen-specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 1.5 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen.     

新疆不同地区杜氏利什曼原虫抗原制备及应用研究

目的用从新疆不同地区分离得到的利什曼原虫前鞭毛体提取抗原,观察ELISA法检测黑热病病人和疫区健康人血清抗体时的敏感性和特异性,为制备ELISA诊断试剂盒和进一步提高这种检测方法的敏感性和特异性,提高黑热病现场流行病学调查结果的准确性和可靠性提供实验依据。方法(1)抗原提取:收集新疆不同地区分离得到的利什曼原虫前鞭毛体,常规方法提取抗原,滴定出抗原工作浓度。(2)检测:用提取的3株抗原,测定黑热病病人和疫区健康人血清抗体。结果3株抗原检测24份肺结核病人血清,均为阴性;3株抗原分别检测60份非疫区健康人血清801株和951株的检测结果均为阴性,901株检测出1份阳性(临界值);3株抗原分别检测131份黑热病病人血清,阳性率分别为:901株(80.9%)、801株(92.3%)、951株(89.9%)。801株明显高于901株。3株抗原分别检测334份疫区健康人血清,901株(10.5%)明显高于801株(6.0%)和951株(5.1%),有显著差异。按不同地区人群阳性率比较,3株抗原检测巴楚县人群阳性率有显著差异,901株(18.8%)与951株(8.5%)有显著差异,乌什县人群阳性率无显著差异;按不同年龄组的人群阳性率比较,3株抗原在每个年龄组的阳性率无显著差异。结论利什曼原虫前鞭毛体抗原有较好的稳定性,耐热性极好;901株、801株抗原的敏感性、特异性较好,可做为新疆血清流行病学调查所用的抗原株;801株的特异性更好,可用于黑热病的特异性抗体的检测及研究。     

Leishmaniasis in Greece II. Isolation and identification of the parasite causing cutaneous leishmaniasis in man

The serological and biochemical identity of four Greek leishmanial strains isolated from cases of cutaneous leishmaniasis was determined. All four strains were identical and shown to be Leishmania tropica (formerly L. t. minor). The cases are described; two came from the Greek mainland and two from Greek islands, one of the latter being a case of leishmaniasis recidivans. The significance of the results is discussed, in particular the co-existence in Greece of strains of L. tropica and L. donovani infantum.     

新疆家犬体内存在细粒棘球绦虫G1(羊)株和G6(骆驼)株的分子证据

目的收集家犬体内的细粒棘球绦虫成虫以建立新疆棘球蚴病的分子流行病学资料. 方法对家犬体内成虫细胞色素c氧化酶Ⅰ基因序列进行测定以确定其亚株型. 结果所有感染犬体内成虫的基因型为G1型.特别是1条家犬体内发现存在细粒棘球绦虫G1(羊)株和G6(骆驼)株混合感染的情况. 结论在新疆阻断羊犬和羊骆驼循环圈是预防棘球蚴病的重要措施.作为终末宿主的家犬与人类的感染密切相关,对感染犬的管理应该加强.     

我国利什曼原虫RAPD分析

目的　我国不同疫区利什曼原虫分离株的RAPD分析。　方法　用 7种随机引物 ,扩增来自我国 3个疫区 (得自利什曼病患者、病犬及白蛉 )的利什曼原虫分离株和国际标准株 ,并将扩增产物进行聚类分析。　结果　①来自我国山丘疫区及平原疫区的L d .分离株分别聚为两类 ,两者遗传距离较远 ;②来自新疆荒漠疫区、近荒漠疫区及平原地区的L d .XJ771、L d .XJ90 1和L d .XJ80 1聚在一类 ,表明它们的遗传距离较近 ;③山丘疫区虫株中来自病人和病犬的分离株区别不明显 ,两者同源性高 ,表明犬在传播中起着重要作用 ;④印度平原型标准株L d .DD8与我国平原疫区虫株聚在一类 ;⑤L infantum与我国山丘疫区的L d .分离株分属于两类 ;⑥L dJed与平原疫区L d .分离株亲缘关系较近 ;⑦L infantum与L tropica最早聚合 ,遗传距离最近。　结论　我国不同疫区利什曼原虫分离株在基因水平上存有差异。     

单克隆抗体检测砂鼠利什曼原虫的研究

应用淋巴细胞杂交瘤技术,制备出高特异性、高效价的抗砂鼠利什曼原虫单克隆抗体,选择其中2株对我国西北荒漠地区大砂鼠体内的分离物给予检测。从IFA,斑点-ELISA的实验结果,均证明为砂鼠利什曼原虫,同时以抗杜氏利什曼单克隆抗体测定则为阴性。从而进一步阐明砂鼠利什曼原虫为大砂鼠体内特有的一种亲皮肤但对人不致病的原虫。     

Localized cutaneous leishmaniasis due to Leishmania donovani and Leishmania tropica: preliminary findings of the study of 161 new cases from a new endemic focus in himachal pradesh, India

Localized cutaneous leishmaniasis (LCL) in India is due mostly to Leishmania tropica. It is mainly endemic in the deserts of Rajasthan. Recently, Himachal Pradesh has been identified as a new endemic focus for the disease. In the last few years, the number of new cases has been increasing almost to epidemic proportions. This report presents the preliminary findings of clinico-epidemiologic and investigative results of 161 new localized cases of LCL seen between May 2001 and December 2003. The study populaton was composed of 80 males and 81 females between 10 months and 75 years of age. All were indigenous to the sub-alpine valley along the Satluj River in the mountainous region of the Kinnaur District (altitude = 700-2,900 meters). Most patients were seen from April to September and had 1-8 lesions (duration = 1-6 months) that involved mainly the face. Tissue smears were positive for amastigotes in 37% and histopathology showed non-caseating epitheloid cell granuloma in 77% of the cases. Analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ribosomal gene region of 10 biopsy specimens showed amplicons indistinguishable from L. donovani in eight cases and L. tropica in two cases. Leishmania was cultured on modified Nicole-Novy-McNeal (NNN) medium containing RPMI 1640 medium and heat-inactivated fetal bovine serum from 13 of 38 biopsy samples. Three of these isolated strains were identified as L. donovani while a fourth was L. tropica by PCR-RFLP of the ribosomal internal transcribed spacer region. One strain had a gp63 sequence identical to that of east African strains. Another strain had a unique gp63 sequence that has not been found in L. donovani complex strains. Sand flies trapped in the cattle sheds of a few patients were identified as Phlebotomus longiductus (Parrot 1928). Treatment with intralesional sodium stibogluconate was effective in all patients without any major side effects. One patient developed lupoid leishmaniasis that responded to higher dose of sodium stibogluconate. Though rarely reported as a cause of LCL, L. donovani seems to be the predominant pathogen in this new focus of cutaneous leishmaniasis. Phlebotomus longiductus is a possible vector, albeit based on circumstantial evidence.