Molecular characterization of Leishmania species 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-Qaseem (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-Qaseem 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 leishmaniasis (MCL) 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 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 and wild animals [8][9][10][11][12].
In spite of the number of molecular studies available to reporting the diagnosis and identi cation of 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. El-Beshbishy et al., [17] reported the molecular characterization of CL in patients from Al-Madinah Al-Munawarah province, western Saudi Arabia by internal transcribed spacer 1 (ITS-1) PCR-restriction fragment length polymorphism (RFLP) and kinetoplast DNA (kDNA) PCR established L. major and L. tropica as the causative organisms, with a kDNA PCR sensitivity of 90.7%, whereas ITS-1 PCR had a sensitivity of 70.1%. Rasheed et al., [18] reported the prevalence of Leishmania species among patients with CL in Al-Qaseem province, central Saudi Arabia. They recorded that out of total 206 CL biopsies, 49.5% biopsies were found to be positive for L. major, 28.6% biopsies were positive for L. tropica, 3.9% were found to be positive for L. infantum/donovani. 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-Qaseem 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-Qaseem 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) (Fig. 1).

Patients Biopsy tissue collections and DNA extraction
A total of 27 suspected patients by Leishmania species were attended in both King Saud Medical City in Riyadh city (n =16) and Buraydah Central Hospital (n =11) in Al-Qaseem province. the presence of Leishmania was investigated in all samples collected 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) according to the manufacturer's instructions and the extracted DNA was quanti ed 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 ampli cation and analysis.

Sampling of stray dogs
From January 2018 to May 2019, 311 stray dogs were trapped in Al-Qaseem province by bait traps (Havahart ® ) ( Fig. 1) and 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 collected under sterile conditions from the border of the ulcer and inoculated into M199 medium (Gibco, Life technologies, Germany), supplemented 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 after 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 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 pre-mix 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/uL (Bioneer, Daejeon, Korea) and 3 μl of DNA were added to the reaction mixture. Finally, 13 μl of deionized water (ddH2O) were added for a total volume of 20 μl for reaction. In this study, no negative control was included in the nal nPCR, but all the necessary measures were taken in vitro to nullify unexpected results (i.e; each sample performed separately and individually in isolated PCR room). 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. The obtained 2nd roun, 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 obtained sequences were alignment with other reference sequences in GenBank using CLASTAL W in MEGA software version 7.0 [22]. The phylogenetic tree was constructed using the Maximum-Likelihood method and Tamura-Nei model with 500 bootstrap replicates in MEGA 7.0 software [22,23].

Results
Of the 27 human patients examined, ve from Riyadh and two from Al-Qaseem were positive for L. major and L. tropica respectively ( Fig. 2 and Fig. 3). Of 311 dogs, seven (2.3%) presented with thick cutaneous lesions (i.e., 1.5 × 5 centimeters) in different anatomical sites (e.g., nose, muzzle, abdomen and between ngers) and ve of them were positive for L. tropica. Sequencing analysis of Leishmania kDNA con rmed that the ve positive samples (nos. H1-H5) of the human patients from Riyadh were all L. major with a size ranging from 441 bp to 451 bp yielding a nucleotide identity ranging from 99.34% to 100% with query cover 100% with previous L. major sequences from Iraq (MN313423). Leishmania sequences from two human patients (no. H1 and H3) from Al-Qaseem were most similar 99.66% to 100% with query cover 100%, respectively to L. tropica from Iraq (MF166799) (Fig.4). Sequences for Leishmania kDNA from stray dogs (no. D5, D6 and D7) were closely related (i.e., 99.33% to 99.80%) to kDNA of L. tropica from Iraq (MF166799), whereas two sequences (no. D2 and D4) displayed a close nucleotide identity (i.e., 99.35% and 99,80) to L. tropica kDNA from Iraq (MN334665) and UK (AF308689), respectively (Fig. 4). Phylogenetic tree clustered L. major (no. H1-H5) and L. tropica (no H1 and H3) sequences from human samples to those from Iraq (accession numbers MN313423 and MF166799, respectively), and the phylogenetic tree clustered L. tropica (no. D2 and D4) from Iraq and UK (accession numbers MN334665 and AF308689, respectively) , while (no. D5, D6 and D7) sequences from dog's samples to those from Iraq isolate (accession number MF166799) Fig. 4. In addition, the isolates of L. tropica from human and dogs in present study were closely related (i.e., 98.60% to 99.65% with query cover ranged 98.20% -99.50%) to kDNA of L. tropica (Saudi strain, MHOM/SA/91/WR1063) that recorded on GenBank (accession number X84845.1). Representative sequences of L. major and L. tropica from this study were deposited in the GenBank database under the accession numbers MT787488 -MT787499.

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. has 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., distribution of 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 Iraq (accession number MN313423 and MF166799) as well as of dog L. tropica isolates with Iraq and UK (accession number MN334665, MF166799 and AF308689, respectively) was also con rmed by the phylogenetic dendrogram herein presented. Moreover, this study showed that the isolates of L. tropica from human and dogs were closely related to kDNA of L. tropica (Saudi strain, MHOM/SA/91/WR1063) that recorded on GenBank (accession number X84845.1). This might be due to the distribution of similar sand ies species in the different parts of Saudi Arabia and Middle East, which may act as proper vectors of both Leishmania spp. [24,31]. However, the phylogenetic analysis in this study was limited and performed based on the available sequences on GenBank based on the sequences of L. tropica and L. major in the Middle East countries particularly those from Iraq. In the phylogenetic tree produced, L. tropica and L. major were clustered in separate clades. However, L. tropica were part of the same cluster as members of the L. donovani complex (L. infantum, L. chagasi and L. donovani), although L. tropica sequences presented very limited intra-speci c genetic diversity, unlike sequences classi ed as belonging to the L. donovani complex. It is, thus, only possible to con rm the identity of the L. tropica isolates in this work based on their genetic distances to published L. tropica sequence [32]. Moreover, one limitation of this study is that negative controls were not included in the nal nPCR reactions and, thus it is not possible to assess if positive results were not due to cross-contamination, particularly in the reactions where all samples were positive, but measures have been taken in the lab (i.e; isolated PCR room, each sample handled separately) to prevent it.
Of the 25 species of Phlebotomus (P) reported in Saudi Arabia only ve (i.e., P. papatasi, P. sergenti, P. bergeroti, P. kazeruni, and P. arabicus) have been incriminated as vectors of CL [27,28,33,34]. Of these, P. papatasi is the major, and most predominant vector species for L. major [28,33], while P. sergenti is the natural vector species for L. tropica [27]. Presence of P. papatasi and P. sergenti in Al-Qaseem 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 [35], L. tropica in Iran [26,36] and Israel [37,38] 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, considering that the distribution patterns can easily change through the years in speci c geographic areas [39]. 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.  Figure 1 Map showing the location of the study areas in Saudi Arabia. Kinetoplast DNA based phylogenetic analysis of genotypes identi ed in this study. Phylogenetic tree highlighting the position of Leishmania spp in the present study (Bold) related to other Leishmania spp available in GenBank. The sequences of kDNA were aligned using CLUSTAL W and phylogenetic inferences were constructed in MEGA 7.0 using Maximum Likelihood based on Tamura-Nei Model for nucleotide sequences with 500 bootstrap replicates. There were a total of 587 positions in the nal dataset. The scale bar represents a 2% nucleotide sequence discrepancy.