Molecular Characterisation 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 they occur in sympatry, the role of each species of Leishmania in the epidemiology of cutaneous leishmaniasis (CL) is not clear. Methods: To determine the frequency of occurrence and to identify the species of Leishmania that infect humans and stray dogs in Riyadh and Al-Qaseem (Saudi Arabia), 311 stray dogs and 27 human patients who were suspected for Leishmania were examined for CL by a nested polymerase chain reaction (nPCR). Results: The use of nPCR detected seven patients (25.9%) who were 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 to be conducted in Saudi Arabia. The relationship between the sand-y vectors and the reservoirs of both Leishmania spp. order and canine


Background
Leishmaniases are a complex group of protozoa diseases that are transmitted by sand ies. They are listed among a group of neglected tropical diseases that affect 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 the forms known as cutaneous (CL), visceral (VL) and mucocutaneous leishmaniasis (MCL), all of which have been reported in Saudi Arabia [2][3][4]. Moreover, in that country, CL that is caused by Leishmania major has the highest prevalence in the Riyadh, Qassim, Al-Madinah, Al-Hassa, Hail and Asir regions [5][6][7], where more than 26,300 cases are estimated to have occurred [2] over a 10 years period (from 2006 to 2016). In addition, in Saudi Arabia there are several reports of leishmaniasis by Leishmania infantum, Leishmania major and Leishmania tropica in humans and wild animals [8][9][10][11][12].
In spite of the availability of several molecular studies that report the diagnosis and identi cation of Leishmania species worldwide [13][14][15][16], there is a lack of information on human CL patients or on dog populations from endemic areas of Saudi Arabia.
In western Saudi Arabia (Al-Madinah Al-Munawarah province) CL was diagnosed in human patients by internal transcribed spacer 1 (ITS-1) polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) and kinetoplast DNA (kDNA) ampli cation [17]. PCR established L. major and L. tropica as the causative organisms for the above infection, 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, of a total of 206 CL biopsies, 49.5% were found to be positive for L. major, 28.6% for L. tropica, and 3.9% for L. infantum/donovani, respectively. Therefore, the aims of the current study were to use molecular tools to detect and identify the Leishmania spp. that infected humans and stray dogs in Al-Qaseem province and in Riyadh city, Saudi Arabia, in order to understand better the epidemiology of the infection.

Ethical approval
This study was reviewed and approved by the Ethics Committee of the Department of Biological Sciences 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 in 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 and in 2017, it had a population of 1,423,000 people [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 a hot summer (an average temperature of 35˚C). It has low annual rainfall (214 mm) and low humidity that ranges 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). It covers an area of about 1,798 km 2 and in 2017, was inhabited by approximately seven million people [19]. Riyadh is characterised 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 rainfall (21.4 mm) and a relative humidity that ranges from 10% to 47% throughout the year. Riyadh also has many dust storms (http://www.pme.gov.sa) (Fig. 1).

Patient biopsy tissue collection and DNA extraction
A total of 27 patients who were suspected to be infected by Leishmania species were seen in either King Saud Medical City in Riyadh city (n =16) or Buraydah Central Hospital (n =11) in Al-Qaseem province. The presence of Leishmania was investigated in all samples, which were collected after clinical and microscopy examination [20]. Brie y, skin biopsies (i.e., 5-10 mm in diameter) were taken under sterile conditions from the border of the ulcerous and cutaneous lesions and DNA samples were extracted from all biopsies by MagNaA pure DNA extraction through use of a Pure LC DNA Isolation Kit (Roche Applied Science, Germany) according to the manufacturer's instructions. The extracted DNA was quanti ed by Nanodrop spectrophotometer (Thermo, USA). The DNA concentration differed from sample to sample, but it ranged from 18ng/ul to 33ng/ul. An aliquot (100µl 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 of infection with canine leishmaniasis due to the presence of cutaneous nodules or ulcerated lesions on the skin (Table 1). Skin biopsies (5mm in diameter) were collected under sterile conditions from the borders of the ulcers and were inoculated into M199 medium (Gibco, Life Technologies, Germany), which was supplemented with 25 mmol/l of 4-(2-hydroxyethyl)-1piperazineethanesulphonic acid (HEPES) (pH:7.5) and 20% fetal bovine serum (Gibco, Life Technologies, Germany). These samples were then incubated at 24 °C. Ten days after sample incubation, parasites were harvested and washed with ice-cold phosphate-buffered saline (10X PBS, pH: 7.4) and stored at -20°C before DNA isolation. DNA from parasite cultures was isolated by use of the ReliaPrep™ gDNA Tissue Miniprep System Kit (Promega, Madison, United States), following the manufacturer's instructions.

Leishmania nested PCR
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 these primers are reported to be 92% and 100%, respectively [21]. 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 680bp in length and fragments of L. tropica and L. major were 750bp and 560bp in length, respectively [21].
The rst step of the addition of the PCR master mix, which included CSB2XF and CSB1XR, was performed using the AccuPower ® PCR PreMix kit (Bioneer, Daejeon, Korea). The prepared PCR pre-mix volumes contained potassium chloride (KCl) at a concentration of 30mM, magnesium chloride (MgCl 2 ) at 1.5mM, tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCL at pH 9.0) at 10mM, Taq DNA polymerase, and deoxynucleoside triphosphate (dNTP) were adjusted to 2µl. In addition, 1μl of each initial CSB1XR and CSB2XF primer at concentrations of 10pmol/ul (Bioneer, Daejeon, Korea) and 3μl of DNA were added to the reaction mixture. Finally, 13μl of deionised water (ddH 2 O) were added up to a total volume of 20μl for reaction. Negative control was included in the nal nPCR. The reaction was performed in a thermal cycler (Techne TC-3000, US) according to the following conditions: initial denaturation temperature of 94°C for 5min; 30 cycles of denaturation at 94°C for 30s, annealing at 55°C for 60s and extension at 72°C for 60s; nal extension at 72°C for 7min; and then the reaction was held at 4°C. The second step of PCR involved 13Z and LiR primers and the same PCR master mix except that 3µl of template PCR product were used. The second round, PCR products that were obtained were electrophoresed on a 1.5% agarose gel that contained 1μl Syber safe (Thermo Scienti c™, Nalgene, UK) in tris-acetate-ethylenediaminetetraacetic acid (EDTA) buffer (50X) at 100V for 45min and visualised under a UV imaging system (ImageQuant Laz4000, GE Healthcare Life Science, Hammersmith, UK). The size of each sample was estimated by comparison with a 100bp 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 that were available in the GenBank database through use of BLAST (http://blast.ncbi.nlm. nih.gov/). The obtained sequences were aligned with a set of reference sequences that were available in GenBank using CLUSTALW in MEGA software version 7.0 [22]. The phylogenetic tree was constructed using the maximum-likelihood method and with the Hasegawa-Kishino-Yano (HKY) model with 2000 bootstrap replicates in MEGA 7.0 software, using Trypanosoma cruzi (AJ748063) as outgroup [22,23].

Results
Of the 27 human patients who were examined, ve from Riyadh and two from Al-Qaseem were positive for L. major and L. tropica, respectively (Figs. 2 and 3). Of 311 dogs, seven (2.3%) presented with thick cutaneous lesions (i.e., 1.5 × 5 cm) in different anatomical sites (e.g., nose, muzzle, abdomen and interdigital spaces) and ve of them were positive for L. tropica. Sequencing analysis of the Leishmania kDNA con rmed that the ve positive samples (nos. H1-H5) that were taken from the human patients from Riyadh were all L. major of sizes that ranged from 441 bp to 451 bp. These yielded nucleotide identities that ranged from 99.34% to 100% with query cover of 100% with previous L. major sequences from Iraq (MN313423). The Leishmania sequences that were collected from two human patients (nos. 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). The sequences for Leishmania kDNA that were obtained from stray dogs (nos. D4, D5, D6 and D7) were closely related (i.e., 99.33% to 99.80%) to kDNA of L. tropica from Iraq (MF166800), whereas one sequence (nos. D2) displayed close nucleotide identity (i.e., 99.35% and 99.80%) with L. tropica kDNA from Iraq (MN334661) and with that from the UK (AF308689), respectively (Fig. 4). The phylogenetic tree for L. major sequences (nos. H1-H5) and L. tropica sequences (nos. H1 and H3) from human samples clustered these with those from Iraq (accession numbers MN313423 and MF166799, respectively), and the phylogenetic tree for L. tropica (nos. D2 and D4) clustered these with samples from Iraq and UK (accession numbers MN334665 and AF308689, respectively). The sequences from dogs (nos. D5, D6 and D7) matched with those from Iraq isolate (accession number MF166799) (Fig. 4). In addition, the isolates of L. tropica that were taken from humans and dogs in the present study were closely related (i.e., 98.60% to 99.65% with query cover that ranged from 98.20% -99.50%) to kDNA of L. tropica (Saudi strain, MHOM/SA/91/WR1063) that were recorded on GenBank (accession number X84845.1). Representative sequences of L. major and L. tropica that were retrieved in the present 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. have already been recorded as agents of cutaneous leishmaniases in Saudi Arabia and Middle Eastern countries [23][24][25][26]. However, in this study L. tropica infection has been molecularly diagnosed for the rst time in humans and dogs in the central part of Saudi Arabia. This infection has been previously reported in the west and south west of Saudi Arabia in association with the distribution of Phlebotomus sergenti, a proper vector for that species [27]. Conversely, L. major is more prevalent throughout the country and can be found in the open desert regions of Saudi Arabia [2,28]. Previous studies that have been performed in Saudi Arabia have reported the natural infection by L. major in dogs through use of enzymatic biochemical methods [29,30], though in these studies no clinical information was available, nor were serology or molecular con rmation studies performed.
The high similarities of the nucleotides of human L. major and L. tropica isolates with those of Iraq (accession number MN313423 and MF166799) and of the dog L. tropica isolates of Iraq and UK (accession number MN334665, MF166799 and AF308689, respectively) were con rmed by the phylogenetic analysis. Moreover, this study showed that L. tropica from humans and dogs was closely related with the kDNA of L. tropica. This might be due to the distribution of similar sand y species in the different parts of Saudi Arabia and the Middle East, which may act as proper vectors of both Leishmania spp. [24,31]. However, the phylogenetic analysis in this study was performed based on the sequences available on GenBank, which were based on the sequences of L. tropica and L. major from Middle East countries, particularly Iraq. In the phylogenetic tree that was inferred, L. tropica and L. major clustered in separate clades, distinct from the L. donovani complex (i.e., L. infantum and L. donovani). Moreover, L. tropica sequences presented very limited intra-speci c genetic diversity, unlike the sequences that were classi ed as belonging to the L. donovani complex [32].
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] with P. papatasi as the major and most predominant vector species for L. major [28,33], and P. sergenti for L. tropica [27]. The 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 spp. infection. However, previous studies have 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], by L. tropica in Iran [26,36] and Israel [37,38] and by L. major in Iraq [23] and Israel [25], and these ndings 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 the comprehension of the transmission cycles, particularly given that the distribution patterns can easily change through the years in speci c geographic areas [39]. Data that have been provided in this study contribute to the lling of existing gaps in knowledge in order to increase the awareness of the Ministry of Health in Saudi Arabia to prevent outbreaks and the spread of CL.

Conclusion
This is the rst study that has detected and identi ed the causative agent of CL in stray dogs and patients in Saudi Arabia and has con rmed that L. major and L. tropica are endemic in Al-Qaseem province and Riyadh City. However, the relationship between sand y vectors and reservoirs of disease remains unclear and their speci c role in transmission cycles in endemic areas of Saudi Arabia is unknown. Further epidemiological and molecular studies that focus on CL in these areas are advocated in order to draw up better strategic control plans and assess the risk for human health.