Laboratoire d'Epidémiologie Moléculaire Et Pathologie Expérimentale Appliquée Aux Maladies Infectieuses (LR16IPT04), Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia.
Faculté Des Sciences de Tunis, Université Tunis El Manar, Tunis, Tunisia.
Parasit Vectors. 2022 Jan 8;15(1):12. doi: 10.1186/s13071-021-05138-x.
Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia.
Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers.
The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples.
The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.
利什曼病在突尼斯流行，具有不同的临床形式，由利什曼原虫、利什曼原虫和利什曼原虫引起。利什曼原虫的生命周期很复杂，涉及到几种沙蝇媒介和哺乳动物储存宿主。这项工作的目的是开发和评估一种高分辨率熔解 PCR（PCR-HRM）工具，以检测和鉴定突尼斯野生和家养宿主中的利什曼寄生虫，这些宿主被确认为（狗和 Meriones 啮齿动物）或潜在的（刺猬）储存宿主。
使用体外培养的利什曼原虫分离株，针对 7SL RNA 和 HSP70 基因开发了 PCR-HRM 反应。动物在突尼斯西北部的 El Kef 省捕获或取样。从刺猬（Atelerix algirus）（n=3）和啮齿动物（Meriones shawi）（n=7）的肝脏、脾脏、肾脏和心脏以及未出现犬利什曼病症状的狗（n=12）的全血中提取 DNA。共处理了 52 个 DNA 样本，使用两对引物进行 PCR-HRM。
结果显示了能够区分突尼斯存在的三种利什曼原虫的熔解曲线，并通过 Sanger 测序进一步证实。对储存宿主样本的 PCR-HRM 分析显示，在所检查的样本中，共有 45 个样本呈阳性，7 个样本呈阴性，没有利什曼原虫感染。Meriones shawi 感染了 L. major，而狗感染了 L. infantum。然而，在 4 个 Meriones 标本和所有测试的刺猬中都检测到了 L. major/L. infantum 种的合并感染。此外，在一个刺猬标本中发现了三种利什曼原虫的混合感染。PCR-HRM 产物的序列分析证实了分析样本中的利什曼原虫种类。
应用于现场标本的 PCR-HRM 分析结果进一步支持了刺猬作为利什曼原虫储存宿主的可能性。此外，我们还证明了它们在诊断犬利什曼病中的有用性，特别是在无症状的狗中，这将确保更好地评估感染程度，从而改善控制计划的制定。这种 PCR-HRM 方法是一种强大而可靠的利什曼原虫分子检测和鉴定工具，易于在流行地区的流行病学调查中实施。
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