High resolution melting analysis as an accurate method for identifying Leishmania infantum in canine serum samples

Leishmania (L.) infantum is the principal agent of visceral leishmaniasis (VL) in the Mediterranean and American regions. So far different molecular methods including high resolution melting (HRM) analysis have been developed for detecting and identifying L. infantum infection. HRM assay is an automted molecular method which detects and identifies different genus and species of infectious agents. This study aimed to diagnose and identify Leishmania infection caused by L. infantum species using real-time PCR coupled with HRM assay in the serum samples in comparison with anti-L. infantum antibodies obtained using direct agglutination test (DAT), in domestic and wild canines of northeastern Iran.

Serum samples of 15 foxes, 14 jackals, seven domestic dogs and three wolves were collected in some villages around Shirvan and Bojnourd districts from the northeast regions of Iran during 2014-15. Initially, all the collected serum samples were tested by DAT for the detection of anti-L. infantum antibodies. Afterwards, genomic DNA was extracted from the samples and tested by real-time PCR-HRM analysis targeting hsp70, ITS1 and gp63 genes. The level of agreement between DAT and HRM assay were analysed statistically.

Out of the 39 serum samples, eight showed anti-L. infantum antibodies at titre 1: 80 while only one of them showed anti-L. infantum antibodies at titre 1 : 160. All the nine seropositive samples showed positive results with HRM analysis. Additionally, three DAT negative serum samples were also found positive in the HRM technique. Altogether, 12 out of the 39 DNA samples showed positive results in HRM analysis. Among the three gene sequences used, gp63 was best for separation and identification of species.

HRM analysis targeting hsp70, ITS1 and gp63 genes can be used as a highly sensitive technique for the screening and early detection of L. infantum infection in the wild and domestic canines. It has higher accuracy than DAT and allows detection and discrimination of different Leishmania species responsible for the Leishmaniases.