Optimization of a Membrane Feeding Assay for Plasmodium vivax Infection in Anopheles albimanus.
Optimization of a Membrane Feeding Assay for Plasmodium vivax Infection in Anopheles albimanus.
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白按蚊间日疟原虫感染膜喂养测定的优化。
DOI：
10.1371/journal.pntd.0004807
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发表时间：
2016-06
影响因子：
3.8
通讯作者：
Arévalo-Herrera M
中科院分区：
文献类型：
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作者：
Vallejo AF;Rubiano K;Amado A;Krystosik AR;Herrera S;Arévalo-Herrera M
长期接触疟疾感染的人会产生免疫反应，能够阻止疟原虫传播到蚊子媒介，从而潜在地限制寄生虫在自然界的传播。开发疟疾结核病疫苗需要更好地了解负责传播阻断(TB)反应的机制和主要效应者。缺乏间日疟原虫的体外培养系统一直是开发评估结核病对这种寄生虫反应的标准化方法的一个重大缺陷。本研究对可能影响按蚊感染的宿主、媒介和寄生虫因素进行了评估，以期建立一种有效、可靠的检测方法来评估结核病的免疫力。在哥伦比亚的两个疟疾流行区(Tierralta和Buenaventura)，共有94名感染间日疟原虫的患者登记为寄生虫捐赠者或直接喂食蚊子的对象。用膜饲法和直接饲养法对实验室饲养的按蚊进行寄生虫感染性评价。在第7天，通过定量聚合酶链式反应和显微镜检查蚊子中肠是否存在卵囊来测量感染情况。以100只/笼饲养4日龄蚊虫的感染力最强。在寄生虫捐献者中，膜饲法比直接饲养法产生显著更好的感染；细胞因子谱显示非感染性个体中干扰素-γ、肿瘤坏死因子和IL-1水平升高。与显微镜相比，聚合酶链式反应能更可靠地评估蚊子感染和寄生虫成熟情况。我们通过人工膜喂养试验评估了可能影响寄生虫传播结果的蚊子、寄生虫和宿主因素。结果表明：1)蚊子的最佳感染力出现在蚊子羽化后4天，笼子密度为100；2)蚊子的感染力最好用聚合酶链式反应来量化，因为显微镜可能低估了它；3)宿主细胞免疫反应似乎没有显著影响蚊子的传染性；4)直接以人为食的蚊子和人工膜喂养的蚊子在传播方面没有显著差异。在这里，我们使用间日疟原虫和白色按蚊以及哥伦比亚流行地区的样本，评估了寄主、媒介和寄生虫因素。这种膜喂养分析方法可以更有效地评估疟疾流行区个人血清以及免疫动物和人的血清中是否存在结核病活性。这种方法有助于更好地评估自然感染引起的结核病免疫反应，以及进一步评估疟疾结核病疫苗的保护效果。
Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity. A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts. Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy. We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays. Here we assessed the host, vector, and parasite factors for membrane feeding assay using Plasmodium vivax and Anopheles albimanus mosquitoes and samples from endemic regions of Colombia. This membrane feeding assay method allowed more efficient assessment of the presence of TB activity in sera of individuals from malaria endemic regions as well as in sera of immunized animals and humans. This method can contribute to better assess the TB immune responses elicited by natural infection as well as further evaluation of protective efficacy of malaria TB vaccines.