Molecular characterization of Leishmania tropica and Leishmania major from stray dogs and patients in Saudi Arabia

Background: Leishmania major and Leishmania tropica cause cutaneous leishmaniasis in humans and dogs in several parts of the world, with a large number of cases recorded in the Middle East. However, when occurring in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. Methods: To determine the prevalence and to identify the species of Leishmania that infects humans and stray dogs in Riyadh and Al-Qassim (Saudi Arabia), 311 stray dogs and 27 human patients, suspected for Leishmania, were examined for CL by a nested PCR (nPCR). Results: Nested PCR (nPCR) detected seven patients (25.9%) positive for cutaneous leishmaniasis. Five patients from Riyadh were infected by L. major and two from Al-Qassim by L. tropica. In addition, ve dogs (1.6%) were infected by L. tropica. Conclusions: This is one of the rst molecular studies of leishmaniasis from Saudi Arabia. The relationship between the sand y vectors and the reservoirs of both Leishmania spp. is still scarcely known in this region, and further epidemiological investigations of domestic and wild canids infected with L. major and L. tropica are needed towards a control and prevention of the infection in canine and human populations.


Background
Leishmaniases are a complex of sand y transmitted protozoa diseases, listed amongst the neglected tropical diseases affecting millions of people, mainly the world's most vulnerable populations [1]. These diseases are transmitted by phlebotomine sand ies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Leishmaniases include cutaneous (CL), visceral (VL) and mucocutaneous (ML) forms, all of which have been reported in Saudi Arabia [2][3][4]. Moreover, in that Country CL by Leishmania major has the highest prevalence mainly in Riyadh, Qassim, Al-Madinah, Al-Hassa, Hail and Asir [5][6][7] with an estimated number of more than 26,300 cases [2] over the past 10 years (2006-2016). In addition, in Saudi Arabia there are several reports of leishmaniasis by Leishmania infantum, Leishmania major and Leishmania tropica among humans [8][9][10][11][12]. In spite of the number of molecular studies available to diagnose and identify Leishmania species worldwide [13][14][15][16] there is a lack of information on human CL patients as well as dog populations from endemic areas of Saudi Arabia [17,18]. Therefore, the aims of the current study were to use molecular tools to detect and identify the Leishmania species infecting humans and stray dogs in Al-Qassim province and Riyadh city, Saudi Arabia in order to better understand the epidemiology of the infection.

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).

Study areas
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 km 2 with a population of 1,423.000 people in 2017 [19]. 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 km 2 and inhabited by approximately seven million people in 2017 [19]. 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 Buraidah Central Hospital (n =11) in Al-Qassim province. All samples were diagnosed after clinical and microscopy examination [20]. Brie y, 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 quanti ed by Nanodrop spectrophotometer (Thermo, USA), and an aliquot (100 µl of DNA from each sample) stored at -20 °C prior to nPCR ampli cation 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 ® ). Dogs were examined physically for canine leishmaniasis skin lesions in the eld. 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 icecold 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 speci c external CSB2XF primers (5 -ATTTTTCGCGATTTTCGCAGAAACG-3 ) and CSB1XR (5 -CGAGTAGCAGAAACTCCCGTTCA-3 ) were used initially. In the second step, speci c internal 13Z primers (5 -ACTGGGGGTTGGTGTAAAATAG-3 ) and LiR (5 -TCGCAGAACGCCCCT-3 ) were applied [21]. The speci city and sensitivity of this primers is reported to be 92% and 100%, respectively [21]. In addition, these primers were able to track and multiply the variable part of all forms of the Leishmania kDNA. Ampli ed 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 [21]. The rst 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, MgCl 2 1.5mM, Tris-HCL (pH 9.0) 10mM, Taq DNA polymerase, and dNTP were adjusted to 2 µl. In addition, 1 μl of the rst step of each initial CSB1XR and CSB2XF primers at concentrations of 10 pmol (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, nal 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 Scienti c™, 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 ampli ed 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 identi cation 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 [22]. The sequences will be deposited in GenBank (AN will be provided in the R1).

Discussion
This study provides molecular evidence of the circulation of L. major and L. tropica in human and dog populations from the investigated areas. The above Leishmania spp. have already been recorded as agents of cutaneous leishmaniases in Saudi Arabia and Middle Eastern countries [23][24][25][26]. However, L. tropica infection has been herein molecularly diagnosed for the rst time in humans and dogs in the central part of the Saudi Arabia since it was previously reported in the west and southern west of Saudi Arabia in association with the distribution of the sand y species (i.e., Phlebotomus sergenti), which is a proper vector for that species [27]. Conversely, L. major is more prevalent throughout the Country and can be found in the open deserts regions of Saudi Arabia [2,28]. Previous studies in Saudi Arabia have reported the natural infection of L. major in dogs using enzymatic biochemical methods [29,30], though no clinical information was available, nor serology or molecular con rmation were performed. The high nucleotide identity of human L. major and L. tropica isolates with those of Iran (accession number AB678350.1 and LC036307.1) as well as of dog L. tropica isolates with Iraq (accession number MF166799.1 and MF166800.1) was also con rmed by the phylogenetic dendrogram herein presented. This might be due to the distribution of similar sand ies species in the Middle East, which may act as proper vectors of both Leishmania spp. [24,31].
Of the 25 species of Phlebotomus reported in Saudi Arabia only ve (i.e., Phlebotomus papatasi, P. sergenti, Phlebotomus bergeroti, Phlebotomus kazeruni, and Phlebotomus arabicus) have been incriminated as vectors of CL [27,28,32,33]. Of these, P. papatasi is the main and most predominant vector species for L. major [28,32], while P. sergenti is the natural vector species for L. tropica [27]. Presence of P. papatasi and P. sergenti in Al-Qassim province suggests that they could have a potential role in the transmission of human and canine leishmaniasis. Nonetheless, more studies are required to elucidate the role of Phlebotomus spp. in CL disease transmission in Saudi Arabia.
Stray dogs have been often diagnosed in Saudi Arabia, with clinical disease associated with Leishmania species, however previous studies focused on the epidemiology, clinical, histopathological and biochemical aspects [11,29,30]. Conversely, molecular studies have reported the occurrence of dog infection by Leishmania spp. in Qatar [34], L. tropica in Iran [26,35] and Israel [36,37] and by L. major in Iraq [23] and Israel [25], which are in agreement with the current study. Though CL is endemic in many parts of Saudi Arabia, the paucity of data concerning the relationship between the disease, the vectors and reservoirs is a major hindrance to understand the transmission cycles in endemic areas. Data herein provided contribute to ll existing gaps in order to increase the alert by the Ministry of Health in Saudi Arabia in preventing outbreaks and the spread of CL.

Conclusion
This was the rst study that detected and identi ed the causative agent of CL in stray dogs and patients from Saudi Arabia thus con rming that L. major and L. tropica are endemic in Al-Qassim province and Riyadh City. However, it is still unclear the relationship between the sand y vectors and reservoirs and their speci c role in the transmission cycles in endemic areas of Saudi Arabia. Further epidemiological and molecular studies focusing on CL these areas are advocated also in order to elaborate better strategic control plans and assess the risk for human health. Table   Table 1: Data of suspect dogs by canine leishmaniasis that trapped in Al-Qassim province.  Map showing the location of the study areas in Saudi Arabia.

Figure 2
Agarose gel electrophoresis (1.7%) image that show the Nested PCR product analysis of kDNA in Cutaneous L. major from human skin lesion samples from Riyadh city. Where M: marker (3000-100bp) and lane (H1 -H5) positive Human L. major at (560bp) nPCR product.

Figure 4
Phylogenetic tree analysis based on the partial sequence of kinetoplast DNA, non-protein coding region in local L.major and L. tropica human and dogs isolates that used for con rmative genetic detection and genetic relationship analysis. The evolutionary distances were computed using phylogenetic UPGMA tree type (MEGA 7.0 version).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. GraphicalAbstract.pdf