用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定
Summary
标准膜喂养测定（SMFA）被视为评估和鉴定潜在抗疟化合物的金标准。这种人工喂养系统用于感染蚊子，以进一步评估这些化合物对 恶性疟原 虫寄生虫强度和患病率的影响。
Abstract
疟疾仍然是全世界最具破坏性的疾病之一，迄今为止，非洲区域仍占全世界所有病例的94%。这种寄生虫病需要原生动物寄生虫、 按蚊 媒介和脊椎动物宿主。 按蚊 属包括500多种，其中60种被称为寄生虫的载体。 疟原虫 属由250种组成，其中48种参与疾病传播。此外， 近年来，恶性疟原 虫已导致撒哈拉以南非洲约99.7%的疟疾病例。
配子细胞是寄生虫性阶段的一部分，在以受感染的人类宿主为食时被雌性蚊子摄入。蚊子中肠的有利环境条件促进了蚊子体内寄生虫的进一步发展。在这里，雌性和雄性配子发生融合，并且运动卵动子起源。卵动体进入蚊子的中肠上皮，成熟的卵泡形成卵囊，卵囊又产生活动孢子体。这些孢子体迁移到蚊子的唾液腺，并在蚊子取血时注射。
出于药物发现目的，在标准膜喂养测定（SMFA）中，蚊子被配子细胞感染的血液人工感染。为了检测蚊子内的感染和/或评估抗疟化合物的功效，雌性蚊子的中肠在感染后被移除并用汞铬染色。该方法用于增强显微镜下卵囊的视觉检测，以准确测定卵囊患病率和强度。
Introduction
疟疾被称为全球最具破坏性的疾病之一，它仍然对一些国家，特别是非洲地区的国家构成巨大威胁，并占全球约95%的病例1。这种疾病是由原生动物寄生虫引起的，连同其按蚊媒介，这些罪魁祸首会对人类宿主造成巨大伤害2。更具体地说，疟原虫属的恶性疟原虫物种估计占撒哈拉以南非洲疟疾病例的99%1。除此之外，几种主要的按蚊媒介（包括An. gambiae Giles，An. arabiensis Patton，An. coluzzii Coetzee & Wilkerson sp.n.和An. funestus Giles）可能被归咎于全球95%以上的寄生虫传播3，4，5，6，7，8.为了建立理想的寄生虫-媒介陪伴，蚊媒应该对寄生虫敏感并能够传播它9.此外，媒介和寄生虫都应该克服物理障碍，形成完美的感染组合——蚊媒应该能够维持寄生虫的发育，寄生虫应该有能力克服宿主的防御机制10，11。
配子细胞是 恶性疟 原虫的性阶段，在连接载体和寄生虫伙伴方面起着至关重要的作用12。性发育发生在 体内，配子细胞发生描述了成熟配子细胞分化为活动雄性微配子和雌性大配子的过程13。蚊子体内发生的另一个过程是鞭打 – 雄性配子细胞转化为配子并从血餐中摄取的红细胞中出来的过程11。进一步建议通过蚊子中肠环境的有利变化来增强释放过程14。驭毛后，由雄配子和雌配子融合形成受精卵13。从受精卵中，产生一个运动的卵动体，并从血粉移动到蚊子中肠的上皮13。在这里，卵泡成熟，形成卵囊，进而产生运动孢子体13，15。然后，孢子体迁移到蚊子唾液腺，当蚊子从宿主那里获取血粉时，这些孢子体被注射到宿主的血液中15。
疟疾控制干预措施，将病媒控制战略与使用有效的抗疟药物相结合，已成为防治这一疾病的关键15。随着寄生虫和蚊子耐药性的增加，鉴定新型抗疟化合物的紧迫性正在增加16。因此，传递阻断化合物的体内评价很重要16。在开发出这种有效的传播阻断药物后，SMFA已被用于评估这些化合物是否抑制恶性疟原虫在按蚊中的性发育17，18，19。自 1970-1980 年代以来，该测定已获得认可，成为评估传播阻断20，21 的金标准。该检测方法比其他检测方法（如需要专用设备的RT-qPCR）提供了更便宜的替代方案。此外，不需要患者来执行实验。该测定还涉及向雌性蚊子提供配子细胞诱导的血液，然后解剖这些血液以评估是否存在卵囊发育21。这允许配子细胞定量和检测由于化合物22而变形的卵囊。对于被归类为有效的化合物，必须评估患病率（在中肠中至少包含一个卵囊的蚊子比例）和蚊子中肠中的卵囊数量（强度）以评估感染抑制17，21，22。
Protocol
有关协议的图示，请参阅 图 1 。比勒陀利亚大学健康科学伦理委员会（506/2018）获得了提取和使用人类血液的伦理许可。 1. 配子细胞培养 注意：在建立SMFA之前，比勒陀利亚大学准备了配子细胞培养物（有关完整方案，请参阅Reader et al.22 ）。 制备由来自 NF54 寄生虫菌株的 V 期配子细胞组成的配…
Representative Results
解剖的对照标本总数为47个，平均患病率为89%，强度为每中肠9.5个卵囊（表1，如之前发表的22）。对于化合物MMV1581558，样本量共达到42个标本，卵囊患病率为36%，平均强度为1.5个卵囊。这表明在所有三个生物学重复中，卵囊患病率降低了58%，TRA降低了82%（表1）。 MMV1581558的%TRA和%TBA均高于50%;因此，这种化合物可被视为阻断和减少传播…
Discussion
为了成功执行此协议，应注意每个步骤，即使它可能是一个乏味而费力的过程。最重要的步骤之一是在开始 SMFA23，24 之前，确保配子细胞培养物质量良好，并且由成熟的配子细胞组成，具有正确的男女比例。在SMFA期间，将配子细胞培养物保持在正确的温度以防止雄配子在进入蚊子之前鞭打也很重要。在建立成功的人工感染中起关键作用的另一个因素是…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢教授。Lyn-Mari Birkholtz和比勒陀利亚大学可持续疟疾控制研究所生物化学，遗传学和微生物学系的Janette Reader博士培养和供应配子细胞培养物。寄生虫菌株是从后一个部门获得的（不是本出版物的一部分）。科学与创新部（DSI）和国家研究基金会（NRF）;南非研究教席倡议（UID 64763 至 LK 和 UID 84627 至 LMB）;NRF 实践社区（LMB 和 LK 的 UID 110666）;南非医学研究理事会战略健康创新伙伴关系（SHIP）也获得了DSI的资金认可。
Materials
| Bovine intestine/ | Butchery | ||
| Compound MMV1581558 | MMV | Pandemic response box | |
| Dissecting needles | WRIM | Custom made | |
| falcon tube | Lasec | ||
| Glass feeders | Glastechniek Peter Coelen B.V. | ||
| Graphpad Prism (8.3.0) | Graphpad | ||
| Mercurochrome | Merck (Sigma-Aldrich) | 129-16-8 | |
| Microscope slides | Merch (Sigma-Aldrich) | S8902 | |
| Parafilm | Cleansafe | ||
| PBS tablets | ThermoFisher Scientific | BP2944 | |
| Perspex biosafety cabinet | Wits University | Made by the contractors at Wits | |
| Plastic cups (350 mL) | Plastic Land |
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Cite This Article
Erlank, E., Venter, N., Koekemoer, L. L. Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors. J. Vis. Exp. (183), e63546, doi:10.3791/63546 (2022).