Journal Article

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

Guizani, Ikram, M’Ghirbi, Youmna, Rhim, Adel, Balti, Ghofrane, Bouattour, Ali, Tebai, Abir, Souguir-Omrani, Hajer, Chemkhi, Jomaa, Derghal, Moufida, Guerbouj, Souheila

2022

Overview

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. Graphical Abstract

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: 7SL RNA / Animals / Animals as carriers of disease / Atelerix / Biomedical and Life Sciences / Biomedicine / blood / Control programs / Deoxyribonucleic acid / Diagnosis / Disease Reservoirs - classification / Disease Reservoirs - parasitology / DNA / DNA sequencing / Dogs / Endemic Diseases / Entomology / Epidemiology / Erinaceidae / Evaluation / Gene amplification / Genetic analysis / Genomes / Gerbillinae - parasitology / Heart / Hedgehogs - parasitology / High resolution / High-resolution melting analysis / Hosts / HSP70 / Hsp70 protein / Humans / Identification and classification / In vitro methods and tests / Infections / Infectious Diseases / Kidneys / Leishmania / Leishmania - genetics / Leishmania - growth & development / Leishmania - isolation & purification / Leishmania - pathogenicity / Leishmania infantum / Leishmania major / Leishmania tropica / Leishmaniasis / Leishmaniasis - parasitology / Life cycle / Life cycles / Life Sciences / liver / Mammals / Marsupials / Melting / Meriones / Meriones shawi / Nucleotide sequence / Nucleotide sequencing / Parasites / Parasites of veterinary importance / Parasitic diseases / Parasitology / PCR / Phlebotominae / Polymerase Chain Reaction / Polymorphism / Primers / Reservoir host / Resolution / Risk factors / RNA / Rodentia / Rodents / Species / Spleen / Surveys / Symptoms / Transition Temperature / Tropical Medicine / Tunisia / Vector-borne diseases / Vectors / Veterinary Medicine/Veterinary Science / Virology / Zoonoses