A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome

BackgroundLeishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine.Methodology/Principal FindingsA L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine.Conclusions/SignificanceHere we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.背景 巴西利什曼原虫（Leishmania braziliensis）是南美皮肤利什曼病与皮肤黏膜利什曼病患者分离得到的最常见虫株。然而，由于多数小鼠品系对该寄生虫感染具有天然抗性，在体内研究巴西利什曼原虫的致病机制或化疗应答存在诸多困难。本研究旨在开发可用于评估抗巴西利什曼原虫药物疗效的实验模型，并采用米替福辛（miltefosine）对该模型进行了验证。 材料与方法及主要结果 将一株最初从皮肤利什曼病患者体内分离的巴西利什曼原虫虫株，经实验室啮齿动物多次传代后进行基因工程改造，使其表达荧光素酶（luciferase）。从刚由无鞭毛体（amastigotes）转化而来的新鲜寄生虫培养物中收集虫体后，将10^6个野生型或表达荧光素酶的静止期前鞭毛体（promastigotes）皮下接种至年轻BALB/c小鼠或金黄地鼠体内。两组动物的接种部位均出现持续性皮肤损伤，若不进行治疗，接种后4个月内未观察到自发愈合现象。与野生型虫株相比，荧光素酶转基因虫株未表现出显著差异。对感染动物经口服给予5 mg/kg/天或15 mg/kg/天的米替福辛，持续给药15天。治疗结束时，皮损完全消退且未检测到寄生虫，但两种剂量给药组的动物均出现了病情复发。 结论与意义 本研究描述了一种基于表达荧光素酶的巴西利什曼原虫虫株接种的迟发型皮肤利什曼病实验模型，该模型可应用于抗利什曼病药物的开发项目。该实验体系可用于监测短期米替福辛给药的瞬时疗效。