不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况,为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中,22 ℃温箱中无菌静置培养,显微镜下连续观察计数8 d,绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖,在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05）,在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异,同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

我国不同流行区婴儿利什曼原虫前鞭毛体比较蛋白质组学分析

目的 应用比较蛋白质组学方法分析两株来自我国内脏利什曼病不同流行区的婴儿利什曼原虫前鞭毛体蛋白表达差异,为筛选和鉴定我国不同流行区利什曼原虫致病差异相关分子奠定基础。方法 分别培养两株分离于我国四川九寨沟县（SC6株）和新疆伽师县内脏利什曼病流行区（JIASHI?5株）的婴儿利什曼原虫前鞭毛体,经酶解后进行液相色谱串联质谱（Liquid chromatography?tandem mass spectrometry, LC?MS/MS）非标记（Label?free）定量分析,利用MaxQuant软件（版本号1.3.0.5）查库,进行非标定量（Label?free quantitation,LFQ）分析。结果 成功鉴定蛋白4 274个,筛选出差异蛋白1 219个（差异倍数＞2.0 或 ＜0.5,P < 0.05）,其中JIASHI?5株前鞭毛体特有蛋白550个,SC6 株前鞭毛体特有蛋白174个。SC6 株和 JIASHI?5 株间差异表达蛋白495个,其中SC6株上调蛋白（高表达）167个,JIASHI?5株上调蛋白328个。这些差异表达蛋白主要涉及能量代谢、应激反应、延长感染宿主细胞的寿命以及利什曼原虫存活与增殖。结论　来自我国内脏利什曼病不同流行区的婴儿利什曼原虫前鞭毛体蛋白质表达存在差异。     

利什曼原虫前鞭毛体体外生长动力学研究

目的观察不同种株利什曼原虫前鞭毛体在体外培养条件下的生长增殖情况，确定其最适体外培养条件。方法分别使用RPMI1640和M199复合培养液，观察温度、pH及新生小牛血清对硕大利什曼原虫、热带利什曼原虫、婴儿利什曼原虫、墨西哥利什曼原虫、杜氏利什曼原虫前鞭毛体体外增殖速度与增殖周期的影响。结果当培养温度为26℃时，杜氏利什曼原虫前鞭毛体在含和不含新生小牛血清、pH中性、偏碱和偏酸的PMI1640或M199复合培养基中，早期均能生长，且生长周期相对较长，其他种株利什曼原虫前鞭毛体在无血清或偏碱培养基中生长缓慢，增殖周期缩短；培养温度为37℃时，各种株利什曼原虫前鞭毛体均发生沉积，增殖停滞，不同程度地向无鞭毛体转化并发生死亡。结论使用复合培养液培养利什曼原虫前鞭毛体，温度、pH和新生小牛血清均可显著影响增殖速度和生长周期。各种株利什曼原虫前鞭毛体在相同培养条件下增殖速度和生长周期存在差异，可能与其遗传背景不同有关。     

利什曼原虫培养方法的改进

利什曼原虫的培养,对于利什曼病的诊断、虫种鉴定以及抗原制备和开展免疫学等方面的研究是一项不可缺少的技术.国内的实验室常用的培养基有NNN培养基(NOVY-MC Neal-Nicollemedium三恩氏培养基)和199培养基(medium199)两种.经多年的实践证明,将国内的几种利什曼原虫的前鞭毛体转种入NNN培养基内并放置22℃～24℃内培养,需要放置10～12d左右才有显著的增长,故难以在短期内收集大量的前鞭毛体.199培养基虽有成品购买,随时可供配置使用,但当前鞭毛体直接转种入该培养基内后,原虫繁殖不佳,1w后虫体变圆,呈衰残型,因此,仅可供短期保存虫种之用.     

田鼠巨噬细胞的体外粘附试验不能区分杜氏利什曼前鞭毛体有无感染性

作者用有感染性和无感染性的杜氏利什曼原虫前鞭毛体与田鼠的脾、淋巴结和腹膜渗出物中的巨噬细胞,在体外进行粘附试验。杜氏利什曼原虫IS株按照Stanber法用田鼠保种。前鞭毛体在肝浸胰胨(LIT)培养基或含20%灭活胎牛血清的Schueider培养基中置于27℃培养,当它们静止6～8天后收集。每ml培养基接种10~6个脾的无鞭     

免疫血清对同种和异种利什曼原虫的影响

利什曼原虫的种类颇多,它们的形态和结构等颇为相似,难于相互鉴别。因此我们用NNN培养基加抗血清对同种及异种利什曼原虫生长影响作了光学显微镜的观察。 一、材料和方法 1.利什曼前鞭毛体抗血清的制备: 抗原:将利什曼原虫置NNN培养基内培养10～12天,收集前鞭毛体用生理盐水离心洗涤4～5次,取有原虫部分。最后按前鞭毛体的压积量加9倍的pH7.2PBS制成15     

斑点酶联免疫吸附试验检测人内脏利什曼病的标准化

斑点酶联免疫吸附试验(Dot-ELISA)是一种经济,简易,可微量目测,适于现场应用的检测抗体的方法。本文对在检测人内脏利什曼病时应用的实验参数进行了观察比较。血清来自查见原虫的内脏利什曼病人,共5份;5份北美正常人血清作对照。杜氏利什曼原虫前鞭毛体培养于含20%灭活胎牛血清等的RPMI1640培养基内,培养至对数生长期时收虫,活原虫约占≥97%;     

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。     

两例输入性皮肤利什曼病的诊断与病原体鉴定

目的对两例疑似皮肤利什曼病进行病原学诊断,并应用分子生物学方法对虫种进行鉴定。方法两例皮肤病患者,分别曾在阿尔及利亚(病例1)和沙特阿拉伯(病例2)务工,表皮均有多个面积较大的溃疡。取皮损处组织涂片、染色、镜检,皮损处组织液置NNN培养基培养,查找原虫。取含利什曼原虫的培养液,离心收集原虫,用2对利什曼原虫种特异性引物ITS1-ITS2和K13A-K13B分别扩增利什曼原虫核糖体DNA内转录间隔区和动基体DNA的基因片段,扩增产物进行测序和Blast序列分析。结果病例1患者的皮损组织涂片镜检未查见利什曼原虫,皮损处组织液经NNN培养基培养10 d后查见前鞭毛体;病例2患者的皮损组织涂片镜检发现利什曼原虫无鞭毛体,皮损处组织液培养8 d后查见前鞭毛体。引物ITS1-ITS2从分离于2例患者的利什曼原虫均扩增出约330 bp的片段,与硕大利什曼原虫(Leishmania major)相应序列同源性均为100%;引物K13A-K13B均扩增出约120 bp的片段,与硕大利什曼原虫相应序列同源性均为96%。4个序列GenBank登录号为JF831924~JF831927。结论两例皮肤病患者均确诊为输入性皮肤利什曼病,...     

克拉玛依地区的利什曼病XIV．硕大白蛉吴氏亚种自然感染前鞭毛体的鉴定

硕大白蛉吴氏亚种是新疆克拉玛依地区的主要蛉种之一，具有强的亲人性，在野外和居民点内常能查见该蛉有前鞭毛体的自然感染。本文结果表明，白蛉自然感染的前鞭毛体能使仓鼠及ＢＡＬＢ／ｃ小鼠发生内脏利什曼病；在感染仓鼠内脏涂片上的无鞭毛体，由蛉体而来的明显较由大沙鼠而来的都兰利什曼原虫为小；白蛉自然感染的前鞭毛体在ＮＮＮ培养基内生长不良；用￣（３２）Ｐ标记的ｇｐ￣（６３）基因为探针，与婴儿利什曼原虫、都兰利什曼原虫及白蛉自然感染的前鞭毛体的ＤＮＡ进行杂交，证实蚌体自然感染的原虫与婴儿利什曼原虫同源。克拉玛依无内脏利什曼病人，但人群中有皮肤利什曼病流行。关于硕大白蛉吴氏亚种自然感染的来源以及当地的皮肤利什曼病究竟是由都兰利什曼原虫抑或婴儿利什曼原虫所致，尚待阐明。     

Use of modified Grace's insect medium for the primary isolation of Leishmania donovani donovani in Bihar, India

Grace's insect medium, supplemented with 20% (v/v) heat-inactivated fetal bovine serum (FBS) and 15% defibrinated pooled rabbit blood is statistically shown to be more sensitive than modified NNN medium (P less than 0.05), Grace's insect medium with 20% (v/v) and Grace's insect medium with 30% (v/v) FBS (P less than 0.01) for in vitro primary isolation of promastigotes of Leishmania donovani donovani from cultures of bone marrow aspirates. This medium has been found to be especially useful for parasitological diagnosis of untreated cases of visceral leishmaniasis and for cases where microscopic examination of bone marrow aspirates has been negative for Leishman-Donovan bodies.     

Comparison of microscopy and culture in the detection of Leishmania donovani from splenic aspirates

Three culture media were compared with Giemsa-stained smears for the detection of Leishmania in splenic aspirates from Kenyan patients with visceral leishmaniasis. Ninety-nine splenic aspirates obtained from 26 patients at various times before, during, and after treatment were cultured in Schneider's Drosophila medium and RPMI medium 1640 (each supplemented with 20% fetal bovine serum) and McConnell's modification of Senekje's medium overlayed with 0.9% saline. From 13 splenic aspirates obtained before treatment, amastigotes were identified microscopically in all and promastigotes were cultured in 12. During and after treatment, Schneider's medium was the most sensitive method for detecting parasites, followed by microscopic examination of stained smears which was more sensitive than either of the other two media tested. Results indicate that, for initial diagnosis, both culture and direct microscopy of aspirates should be employed.     

Successful replacement of fetal calf serum with human urine for in vitro culture of Leishmania donovani.

Leishmania donovani causes visceral leishmaniasis claiming several thousand lives every year in Indian sub-continent. The etiological agent is grown in cell free media supplemented with fetal calf serum (FCS). Urine from human beings and other mammalian species has also been reported to stimulate growth of Leishmania species No study has been carried out Leishmania donovani. Therefore, we studied the feasibility of culturing Leishmania donovani promastigotes in M199 medium supplemented with 10% human urine and compared the phenotypic and genotypic characters under supplementation with urine vis-a-vis FCS. The growth curve showed no significant difference in the promastigote counts in urine vs. FCS supplementation. The best growth was observed in cultures supplemented with the post-menopausal urine. No difference in antigenic bands and RFLP pattern was seen indicating that no alteration occurred in strain specific characters of the parasites cultured with human urine.     

Destruction of Leishmania mexicana amazonensis promastigotes by normal human serum

Fresh normal human serum was observed to have a lethal effect on Leishmania mexicana amazonensis promastigotes obtained from laboratory-bred Lutzomyia longipalpis or on promastigotes grown in liquid culture medium, inoculated with the same isolates. Heat inactivation abolished the Leishmania lytic activity from the sera. Resistance of culture promastigotes to lysis by normal human serum was investigated in three isolates of L. m. amazonensis. Development of resistance (up to 7%) was found in only one isolate, obtained from the bone marrow in a human case of visceral leishmaniasis.     

Diagnosis of cutaneous leishmaniasis by in vitro cultivation of saline aspirates in Schneider's Drosophila Medium

A culture system was developed and evaluated as an improved diagnostic procedure for human infections of New and Old World cutaneous leishmaniasis. Needle aspirates of 55 suspect lesions from 40 human cases were made by injecting and withdrawing sterile saline from the outer margin of a lesion. Aspirates were inoculated and cultured in an insect cell culture medium, Schneider's Drosophila Medium, supplemented with 30% fetal bovine serum. Paralleled cultures in a blood-based medium, NNN, were used as a basis of comparison for sensitivity. Promastigotes were observed in 37 of 55 (67%) of the Schneider's Medium cultures on an average of 6.0 days, as compared to only 8 of 55 of the NNN cultures on an average of 11.6 days.     

Use of non-human plasma for in vitro cultivation and antimalarial drug susceptibility testing of Plasmodium falciparum

Viability, growth rate, and chemotherapeutic susceptibility of the CDC/Indochina III, CDC/Sierra Leone I, and FCR-3 (Subline F-86) isolates of Plasmodium falciparum grown continuously in RPMI 1640 medium supplemented with goat, horse, porcine, bovine, or ovine plasma were evaluated. Results were compared to those obtained from parallel cultures maintained in medium supplemented with non-immune human plasma. Only media supplemented with goat or horse plasma supported significant continuous multiplication of the isolates. Medium supplemented with either ovine or porcine plasma supported continuous multiplication of the CDC/Indochina III isolate, but not the FCR-3 isolate. Medium supplemented with bovine plasma did not support continuous growth of any of the isolates tested. The light microscopic appearance of the isolates during and after continuous culture in medium supplemented with either goat or horse plasma was identical to that of the control parasites maintained in medium supplemented with human plasma. There were no statistically significant differences in the susceptibility to antimalarial drugs of the culture lines maintained in medium supplemented with either human or goat plasma.     

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.     

First molecular identification of Leishmania species in a new endemic area of cutaneous leishmaniasis in Lorestan,Iran

ObjectiveTo identify Leishmania using PCR.MethodsThis study was conducted from April 2009 to March 2011 in order to identify Leishmania species in a new endemic area of CL in Lorestan, Iran. Samples were taken from 62 patients that referred to the health centers in different cities of Lorestan province, the presence of Leishmania was confirmed using direct smear and then grown in NNN media and mass cultured in RPMI 1 640 medium supplemented with 10% heat-inactivated fetal bovine serum. DNA was extracted from cultured promastigotes and used in ITS-PCR.Results45(72.6%) samples out of 62 showed a band in the range of 485 bp and 17 (27.4%) with a band in the range of 626 bp which were similar to standard strains of Leishmania tropica(L. tropica) and Leishmania major(L. major), respectively. 50 (65.80%) of samples were collected from people with no history of travel in at least a year prior to the onset which shows that indigenous source of infection.ConclusionsSince the vector and reservoir of the two species are different, so precise and extensive control and prevention methods should be designed and carried out.