2.1 Ethical approval
This study was reviewed and approved by the Ethics Committee of the Department of Biological Science at Shaqra University, according to the ethical principles of animal research (protocol SH 2-2017).
2.2 Study areas
The investigation was conducted from January 2018 to May 2019 in Al-Qaseem province and Riyadh city, Saudi Arabia. Al-Qaseem province is located at the central part of Saudi Arabia (latitude 25°–23° N and longitude 42°–24° E). It has an area of about 58.046 km2 and was reported to be inhabited by approximately 1,423.000 people in 2017 (General Authority for Statistic, 2017). Riyadh city is the capital of Saudi Arabia (latitude 24°–08° N and longitude 47°–18° E), with an area of about 1798 km2 and inhabited by approximately seven million people in 2016  (Figure 1).
2.3 Patients Biopsy tissue collections and gDNA extraction
A total of 27 suspected patients were attended in King Saud Medical City in Riyadh city (n =16) and Buraidah Central Hospital (n =11) in Al-Qaseem province. All samples were diagnosed after clinical and microscopy examination . Briefly, skin biopsies (i.e., 5-10 mm of diameter) were taken under sterile conditions from the border of the ulcer and cutaneous lesions and DNA samples were extracted from all biopsies by MagNaA pure DNA extraction Pure LC DNA Isolation Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instruction (Roche) and the extracted gDNA was checked by Nanodrop spectrophotometer (Thermo, USA), and an aliquot (100 µl of gDNA from each sample) stored at -20 °C prior to nPCR amplification and analysis.
2.4 Sampling of stray dogs
From January 2018 to May 2019, 311 stray dogs were trapped in Al-Qaseem province by bait traps (Havahart®), dogs examined physically for CL in the field and a blood sample of 2-5 ml from the cephalic vein into EDTA vacuum tubes (BD Vacutainer® Tube, Gribbles Pathology, VIC, Australia) and transported to the molecular laboratory, Shaqra University for DNA extraction and further molecular analysis. Seven of the above animals were suspected for CL due to the presence of cutaneous nodules or ulcerated lesions on the skin. Skin biopsies (5 mm in diameter) were taken under sterile conditions from the border of the ulcer and inoculated into medium M199 supplemented (Gibco, Life technologies, Germany) with 25 mmol/L HEPES (pH:7.5) and 20% fetal bovine serum (Gibco, Life technologies, Germany) followed by incubation at 24 °C. Ten days later parasites were harvested and washed with ice-cold phosphate buffered saline (PBS, pH: 7.4) and stored in -20 °C before DNA isolation.
2.5 Leishmania Nested PCR (nPCR)
The specific external CSB2XF primers (5ˊ-ATTTTTCGCGATTTTCGCAGAAACG-3ˊ) and CSB1XR (5ˊ-CGAGTAGCAGAAACTCCCGTTCA-3ˊ) were used initially. In the second step, specific internal 13Z primers (5ˊ-ACTGGGGGTTGGTGTAAAATAG-3ˊ) and LiR (5ˊ-TCGCAGAACGCCCCT-3ˊ) were applied . These primers were able to track the variable part of all forms of the Leishmania kDNA minicircle . The first step of PCR master mix that included CSB2XF and CSB1XR were applied using AccuPower® PCR PreMix kit (Bioneer, Daejeon, Korea). The prepared PCR premix volumes contained KCl 30 mM, MgCl2 1.5mM, Tris-HCL (pH 9.0) 10mM, Taq DNA polymerase, and dNTP were adjusted to 2 µl. In addition, 1 μl of the first step of each initial CSB1XR and CSB2XF primers at concentrations of 10 pmol (Bioneer, Daejeon, Korea) and 3 μl of genomic DNA were added to the complex. Finally, 13 μl of deionized water (ddH2O) were added for a total volume of 20 μl for reaction. The reaction was performed in a thermal cycler (Techne TC-3000, USA) by set up the following conditions; initial denaturation temperature of 94 °C for 5 min; followed by 30 cycles at denaturation 94 °C for 30 s, annealing 55 °C for 60 s, extension 72 °C for 60 s, final extension at 72 °C for 7 min and then the reaction was held at 4 °C. The second step of PCR included 13Z and LiR primers and the same PCR master mix except 3µL of template PCR product. After that, PCR products were electrophoresed on a 1.5% agarose gel containing 1 μL/mL Syber safe (Thermo Scientific™, Nalgene, UK) in Tris-acetate–EDTA buffer at 100 V for 45 min and visualized under UV imaging system (ImageQuant Laz4000, GE Healthcare Life Science, Hammersmith, UK). The size of each sample was estimated by comparison with a 100 bp DNA Ladder Marker (Solis BioDyne OU, Estonia).
2.6 Lesishmania kDNA sequencing and phylogenetic analysis
Positive amplified products of Leishmania species were sent to Macrogen (South Korea) for sequencing, and the results were compared with the sequences available in GenBank database using BLAST (http://blast.ncbi.nlm. nih.gov/). The phylogenetic analysis was performed based on NCBI-Blast alignment identification and maximum composite Likelihood method by phylogenetic tree UPGMA method (MEGA 7.0 version). Bootstrap values were determined with 1,000 replicates of the data sets . The sequences will be deposited onto GenBank (AN will be provided in the R1).