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).
The investigation was conducted from January 2018 to May 2019 in Al-Qassim province and Riyadh city, Saudi Arabia. Al-Qassim 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 with a population of 1,423.000 people in 2017 . Al-Qassim province is known as an agricultural region and it has a typical desert climate, with an average temperature of 13˚C and hot summer (an average temperature of 35˚C), with few annual rainfalls (214 mm) and low humidity ranging from 25% to 76% (http://www.pme.gov.sa). Conversely, 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 2017 . Riyadh is characterized by very hot summers with an average temperature of 45˚C in July, whereas winters are cold. The overall climate is arid, with scarce annual precipitations (21.4 mm), with a relative humidity ranging from 10% to 47% throughout the year. Riyadh is also known to have many dust storms (http://www.pme.gov.sa) (Figure 1).
Patients Biopsy tissue collections and DNA extraction
A total of 27 suspected patients were attended in both King Saud Medical City in Riyadh city (n =16) and Buraydah Central Hospital (n =11) in Al-Qassim 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 instructions and the extracted DNA was quantified by Nanodrop spectrophotometer (Thermo, USA), DNA concentration differed from sample to another, but it ranged from 18ng/ul to 33ng/ul, and an aliquot (100 µl of DNA from each sample) was stored at -20 °C prior to nPCR amplification and analysis.
Sampling of stray dogs
From January 2018 to May 2019, 311 stray dogs were trapped in Al-Qassim province by bait traps (Havahart®) (Figure 1).Dogs were examined physically for canine leishmaniasis skin lesions in the field. Seven dogs were suspected for canine leishmaniasis due to the presence of cutaneous nodules or ulcerated lesions on the skin (Table 1). 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. DNA from parasites cultures was isolated by using the ReliaPrep™ gDNA Tissue Miniprep System Kit (Promega, Madison, United States), following the manufacturer’s instructions.
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 . The specificity and sensitivity of this primers is reported to be 92% and 100%, respectively . In addition, these primers were able to track and multiply the variable part of all forms of the Leishmania kDNA. Amplified fragments of L. infantum were 680 bp in length and fragments of L. tropica and L. major were 750 and 560 bp in length, respectively . 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 pre-mix 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/uL (Bioneer, Daejeon, Korea) and 3 μl of 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 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).
Leishmania kDNA sequencing and BLAST 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 BLAST analysis was performed based on NCBI-BLAST alignment identification and an UPGMA tree performed based on distances calculated using a composite likelihood model (MEGA 7.0 version). Bootstrap values were determined with 1,000 replicates of the data sets .