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. 2022 Jan 8;15(1):12.
doi: 10.1186/s13071-021-05138-x.

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

Affiliations

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

Moufida Derghal et al. Parasit Vectors. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 7SL RNA; HSP70; High-resolution melting analysis; Leishmania; Reservoir host; Tunisia.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Gene scanning analysis showing discrimination of 27 strains representing the three Leishmania species L. infantum, L. major, and L. tropica with PCR-HRM using 7SL RNA (a) and HSP70 (b) genes. Leishmania strains used are L. major (n = 8; EMPA10, IL32, Ron 44, EMPA11, EMPA12, Ron155, IL53, and FMH); L. infantum (n = 10; IPT1, LV08, LV10, LV49, LV50, D5, D8, D11, D13, and D16); and L. tropica (n = 9; K27, LA28, L75, Adhanis, Gabai159, Bumm30, Rachnan, Bag9, and Bag17)
Fig. 2
Fig. 2
Gene scanning analysis of PCR-HRM from reservoir samples, using 7SL RNA gene. a Infection of dogs with L. infantum. Tested samples are dog 10, dog 32, dog 37, dog 43, and dog 47. b Identification of Leishmania species infecting hedgehog, Meriones, and dogs. Tested samples are hedgehogs (CEZ4, PES1, and FES1), Meriones (SMZ3, SMZ4, and SMZ1), and dogs (dog 3 and dog 4). Reference isolates correspond to L. infantum, L. major, and L. tropica. See Table 1 for an explanation of samples codes
Fig. 3
Fig. 3
Melting peaks analysis of PCR-HRM from reservoir samples, using HSP70 gene. a Melting peaks of reference isolates L. infantum, L. major, and L. tropica. b Infection of reservoir samples with L. infantum. c Infection of reservoir samples with L. major. Tested samples are dog 47, CED1, SMZ3, CMZ4, SMZ2, SMZ1, SMZ7, RMZ4, and FMZ1. See Table 1 for explanation of sample codes

References

    1. Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, et al. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis. 2016;10:e0004349. - PMC - PubMed
    1. Maia C, Dantas-Torres F, Campino L. Parasite biology: the reservoir hosts. In: Bruschi F, Gradoni L, editors. The Leishmaniases: old neglected tropical diseases. Berlin: Springer; 2018. pp. 79–106.
    1. Aoun K, Jeddi F, Amri F, Ghrab J, Bouratbine A. Current epidemiological data on visceral leishmaniasis in Tunisia. Med Mal Infect. 2009;39:775–779. - PubMed
    1. Fathallah-Mili A, Saghrouni F, BenSaid Z, Saadi-BenAoun Y, Guizani I, BenSaid M. Retrospective analysis of leishmaniasis in Central Tunisia: an update on emerging epidemiological trends. In: Rodriguez-Morales A, editor. Current topics in tropical medicine. New York: InTech; 2012. pp. 227–252.
    1. Chaara D, Bañuls AL, Haouas N, Talignani L, Lami P, Mezhoud H, et al. Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica population structure in Maghreb by microsatellite typing. PLoSNegl Trop Dis. 2015;9:e0004204. - PMC - PubMed
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