High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

Parasites & Vectors

Hajer Souguir-Omrani¹  Ikram Guizani¹  Souheila Guerbouj¹  Abir Tebai¹  Jomaa Chemkhi¹  Moufida Derghal²  Adel Rhim³  Youmna M’Ghirbi³  Ghofrane Balti³  Ali Bouattour³ 

[1] Laboratoire d’Epidémiologie Moléculaire Et Pathologie Expérimentale Appliquée Aux Maladies Infectieuses (LR16IPT04), Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia;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;Laboratoire d’épidémiologie Et Microbiologie Vétérinaire (LR16IPT03), Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia;Laboratoire Des Virus, Vecteurs Et Hôtes (LR20IPT02), Institut Pasteur de Tunis, Université Tunis El Manar, Tunis, Tunisia;

关键词: Leishmania;    High-resolution melting analysis;    7SL RNA;    HSP70;    Reservoir host;    Tunisia;   

DOI  :  10.1186/s13071-021-05138-x

来源: Springer

【 摘 要 】

BackgroundLeishmaniasis 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.MethodsUsing 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.ResultsThe 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.ConclusionsThe 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.Graphical Abstract

【 授权许可】

CC BY