Summary
Leishmanioses are vector-borne diseases caused by Leishmaniaspp., which are transmitted by phlebotomine sand flies (Diptera, Psychodidae). The recent reports in humans of Leishmania tarentolae , which is primarily found in cold-blooded animals, andLeishmania infantum in Sergentomyia minuta spurred us to develop an internal transcribed spacer 1-based duplex quantitative real-time PCR (dqPCR) assay for the detection and differentiation between these Leishmania spp. The specificity of dqPCR was assessed by processing DNA samples from Phlebotomus spp. (n=188) and Se. minuta (n=171) and from tissues (i.e., heart, liver, muscle, lungs, spleen, kidney, eggs) of Podarcis siculus (n=4) and Tarentola mauritanica (n=3). In the absence of naturally infected and/or co-infected lizards, DNA from cultured L. infantum and L. tarentolae were spiked into tissues of lizards and used as controls. The analytical sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA from both Leishmania spp. and spiked DNA samples from lizards was 2.3 x 10-7ng/2 µl for L. infantum and 2.1 x 10-7 ng/2 µl for L. tarentolae . With the spiked DNA samples, the dqPCR detected up to 2.6 x 10-6 ng/2 µl of L. infantum and up to 2.1 x 10-7 ng/2 µl of L. tarentolae . Of 359 phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph. perniciosus scored positive for L. infantumand L. tarentolae , respectively. Similarly, of 171 Se. minuta , 56 (32.7%) and six (3.5%) scored positive for L. tarentolae and L. infantum, respectively. Co-infection with bothLeishmania spp. was detected in two Se. minuta (1.2%). Out of seven reptiles tested, four P. siculus were positive forL. tarentolae . The newly dqPCR herein described may represent an improvement in the diagnosis of L. infantum and L. tarentolae and may assist in identifying the role of lizards as reservoirs and Se. minuta as vector, for these Leishmaniaspp.
Key words: duplex real-time PCR, ITS1, Leishmania infantum , Leishmania tarentolae , Lizards, Sergentomyia minuta
Introduction
Leishmanioses are vector-borne diseases that cause clinical conditions of varying degrees to different mammal species, including humans. The diseases are caused by Leishmania spp. (Kinetoplastida, Trypanosomatidae), which are transmitted by bites of phlebotomine sand flies of the genera Phlebotomus in the Old World (Maroli et al., 2013). Among the circa 100 sand flies species indicated as proven vectors of Leishmania spp. (Maroli et al., 2013), some are highly specific for certain Leishmania spp. (e.g., Phlebotomus sergenti and Phlebotomus papatasi for Leishmania tropicaand Leishmania major , respectively) (Kamhawi, 2006), whereas others support the development of more than one Leishmania spp. (i.e., Phlebotomus arabicus is susceptible to the development of both L. major and Leishmania infantum ) being considered permissive vectors (Jacobson et al., 2003; Myskova et al., 2007). However, DNA of Leishmania spp. may be retrieved in non-competent vectors such as in the case of L. tropica and L. major inPhlebotomus perniciosus and L. infantum inPhlebotomus mascittii (Vaselek et al., 2017; Gherbi et al., 2020). Meanwhile, phlebotomine sand flies of the genusSergentomyia are known to feed primarily on cold-blooded animals (Lewis, 1987) and are associated to the transmission ofLeishmania (Sauroleishmania ) spp. in lizards (Killick-Kendrick et al., 1986; Lewis, 1987; Noyes et al., 1997; Tuon et al., 2008). These reptiles have also been found molecularly positive for some Leishmania spp. pathogenic to humans (Pombi et al., 2020). For example, DNA of L. major, L. tropica and Leishmania martiniquensis were found in different species of Sergentomyia(e.g., Sergentomyia barraudi , Sergentomyia darling ,Sergentomyia garhami, Sergentomyia clydei , Sergentomyia gemmea , and Sergentomyia iyengari ) (Mutinga et al., 1994; Berdjane-Brouk et al., 2012; Kanjanopas et al., 2013; Nzelu et al., 2014; Ayari et al., 2016; Siripattanapipong et al., 2018). Similarly, the DNA of L. infantum has been detected in severalSergentomyia spp., such as Sergentomyia dubia ,Sergentomyia magna and Sergentomyia schewtzi in Africa (Senghor et al., 2016), and Sergentomyia minuta from areas endemic for canine leishmaniosis in Europe (Tarallo et al., 2010; Campino et al., 2013; Bravo-Barriga et al., 2016; Maia et al., 2015; Latrofa et al., 2018; Gonzales et al., 2020; Pombi et al., 2020).
Sergentomyia spp. female is known to feed mostly on cold-blooded animals, such as lizards, which are, with geckos, the primary hosts ofLeishmania tarentolae (Telford 1995; Sloboda et al., 2007; Halla et al., 2014). This Leishmania species is considered non-pathogenic for mammals, although some strains (e.g., LEM-125) were associated to transient infectious in rodents and humans (Adler, 1962; Breton, 2005; Taylor et al., 2010; Novo et al., 2015). On the contrary, the DNA and/or amastigote forms of pathogenic Leishmania spp. (i.e., L. tropica , Leishmania donovani and L. infantum ) have been detected in reptiles (Wilson and Southgate, 1979; Simpson and Holtz, 1988; Belova, 1971; Zhang et al., 2019; Chen et al., 2019) . Therefore, in addition to mammals and various other hosts such as black rat, hares, horse, cat, red fox and wild carnivores (Criado-Fornelio et al., 2000; Solano-Gallego et al., 2003; Sobrino et al., 2008; Millán et al., 2011; Gramiccia, 2011; Montoya et al., 2016), reptiles have been suggested as potential reservoirs for differentLeishmania spp. (Zhang et al., 2016; Mendoza-Roldan et al., 2020).
The recent reports of L. tarentolae in human blood and ofL. infantum in Se. minuta (Latrofa et al., 2018; Gonzales et al., 2020; Pombi et al., 2020) spurred us to develop a duplex quantitative real-time PCR (dqPCR), with species-specific probes targeting the internal transcribed spacer 1 (ITS1) region, for the detection and differentiation between L. infantum and L. tarentolae . This assay may provide a fundamental contribution for understand the potential ecological role of sand flies and reptiles in spreading these two parasites in endemic and non-endemic geographical areas where they live in sympatry with animals and humans.
Materials and Methods
2.1 Samples
To obtain sequences of Leishmania spp. for primes and probes designing, promastigotes of L. infantum (zymodeme MON-1; 1.6 x 107 promastigotes/ml) and L. tarentolae (strain RTAR/IT/81/ISS21-G.6c; 1.7 x 107 promastigotes/ml) were extracted and their genomic DNA (gDNA) amplified by conventional PCR using primes (L5.8S/LITSR) targeting a fragment of the ITS1 (300-350 bp), as described elsewhere (El Tai et al., 2001). Genomic DNA ofL. major (MHOM/TM/1973/5ASKH), L. aethiopica(MHOM/ET/72/L100), L. donovani (MHOM/IN/00/DEVI) and L. tropica (MHOM/IL/2005/LRC-L1239) were also used for testing the specificity of the newly developed assay (see below).
2.2 Primers, probes, and dqPCR protocol
Generic primers (L.i.t. -ITS1-F 5′-GCAGTAAAAAAAAGGCCG-3′;L.i.t. -ITS1-R 5′- CGGCTCACATAACGTGTCGCG-3′) and two specific hydrolysis TaqMan-MGB probes (L.t. -6-FAM-5’-CACGCCGCGTATACAAAAACAC-3’-non-fluorescent quencher-MGB;L.i.- VIC-5’-TAACGCACCGCCTATACAAAAGCA-3’-non-fluorescent quencher-MGB (Applied Biosystems; Foster City, CA, USA) targeting 150 bp of the ITS1 of L. infantum and L. tarentolae, were designed by alignment of the reference Leishmania spp. sequences using Primer Express 2.0 (Applied Biosystems, Foster City, CA). Sequences of L. major (accession number HG512924), L. tropica (accession number HG512927), L. turanica (accession number AJ272382) and Leishmania aethiopica (accession number HG512923) available from GenBank were included in the alignment. The primer and probe specificity were analysed in silico using the basic local alignment search tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi). dqPCR reactions were carried out in a final volume of 20 µl, consisting of 10 µl of IQ™ Supermix (Bio-Rad Laboratories, Hercules CA, USA), 7 µl of Di-Ethyl Pyro-Carbonate (DEPC) treated pyrogen-free DNase/RNase-free water (Invitrogen, Carlsbad, CA, USA), 2 μl of template DNA and 950 nM of each primer, as well as 200 and 300 nM of FAM and VIC TaqMan-MGB probes, respectively. The thermal cycling conditions consisted of a hot start at 95°C for 3 min, and 40 cycles of denaturation (95°C for 10 sec) and annealing-extension (55°C for 30 sec). All assays were carried out in duplicate and a no-template control (NTC) was included in each run. The dqPCR was performed in a CFX96TM Real-Time System (Bio-Rad Laboratories, Inc., Hercules CA, USA) and the increase in the fluorescent signals were registered during the extension step of the reaction and analysed by the CFX ManagerTM software, version 3.1 (Bio-Rad).
2.3 Specificity and sensitivity of the dqPCR
To investigate the analytical specificity of the assay, gDNA of bothL. infantum and L. tarentolae , alone or in combination, as well as gDNA of L. major , L. aethiopica , L. donovani and L. tropica were tested. In addition, DNA fromPhlebotomus spp. (i.e., five Ph. neglectus , threePh. papatasi , 140 Ph. perniciosus , and 40 Ph. perfiliewi ) and Se. minuta (n=171) previously collected in aL. infantum -endemic area ( Latrofa et al., 2018; Iatta et al., 2020) (Table 1) and DNA from various types of samples (i.e., heart, liver, muscle, lungs, spleen, kidney, and eggs), from four Italian wall lizards (Podarcis siculus ) and three Moorish wall geckos (Tarentola mauritanica ) were tested. In addition, in the absence of naturally infected and/or co-infected lizard samples, DNA fromL. infantum and L. tarentolae were spiked with the DNA from lizard samples and used as controls (Table 2).
The analytical sensitivity of the dqPCR assay was assessed using 10-fold serial dilutions of DNA from L. infantum (ranging from 2.7 × 10-1 to 2.7 × 10-8 ng/2 µl of reaction), L. tarentolae (ranging from 2.1 × 10-1 to 2.1 × 10-8 ng/2 µl of reaction) and from the spiked DNAs of both Leishmania species (ranged from 2.4 × 10-1 to 2.4 × 10-8 ng/2 µl of reaction) and expressed also by threshold cycle values (Cq) and parasite load amount (promastigotes/2 µl of reaction). gDNA was extracted from Leishmania spp. cultures and from lizard tissues using the commercial kit DNeasy Blood & Tissue (Qiagen, GmbH, Hilden, Germany), following the manufacturer’s instructions. The amount of purified DNA from each Leishmaniastrain (L. infantum = 2.6 × 10-1 ng/µl;L. tarentolae = 2.1 × 10-1 ng/µl) was determined using Qubit (Applied Biosystems, Foster City, CA, USA).
Results
Fluorescent signals were recorded for DNA of L. infantum andL. tarentolae , and for the spiked DNA from both Leishmaniaspp. (Figure 1 A, B, C). Though BLASTn analyses did not found sequences identity with other Leishmania spp., fluorescence signals were obtained for L. donovani (Cq=16.5), L. tropica (Cq=16.2) and L. major (Cq=17.7) tested with the VIC-L. infantumprobe, whilst no fluorescence signals were obtained with otherLeishmania spp., or NTC. The performance of the assay was confirmed by the efficiency values (ranging from 106.5 to 115%) of the standard curves obtained with each Leishmania spp. and for spiked DNAs, with R2 ranging from 0.978 to 1.000 and the slope ranging from -3.009 to -3.178 (Figure 1 A, B, C). The analytical sensitivity of the dqPCR was 2.3 x 10-7 ng/2 µl forL. infantum (i.e., 3.3 x 10-3 promastigotes/2 µl, Cq of 37.4) and 2.1 x 10-7 ng/2 µl for L. tarentolae (i.e., 3.3 x 10-3 promastigotes/2 µl, Cq of 36.3). With the spiked DNA, the assay detected up to 2.6 x 10-6 ng/2 µl of L. infantum (i.e., 3.3 x 10-2 promastigotes/2 µl, Cq of 38.0) and up to 2.1 x 10-7 ng/2 µl for L. tarentolae (i.e., 3.3 x 10-3 promastigotes/2 µl, Cq up to 38.6).
Out of 359 phlebotomine sand flies tested, 63 (17.5%) scored positive for Leishmania spp. (Table 1) with five (3.6%) and two (1.4%) Ph. perniciosus specimens, positive for L. infantum and L. tarentolae , respectively (Table 1). Out of 171Se. minuta tested, 56 (32.7%) scored positive for L. tarentolae and six (3.5%) for L. infantum . Co-infection with both Leishmania spp. was detected in two samples of Se. minuta (1.2%). No Ph. papatasi , Ph. neglectus andPh. perfiliewi scored positive for Leishmania spp. (Table 1). The mean DNA load detected in phlebotomine sand flies ranged from 2.2 x 10-5 to 3.0 ng/2 μl for L. infantum (mean Cq up to 35.3) and from 2.1 x 10-6 to 4.1 ng/2 μl forL. tarentolae (mean Cq up to 35.9) (Table 1).
Out of seven reptiles tested, four P. siculus were naturally infected by L. tarentolae . In particular, of the five types of lizard tissue analysed, one each of muscle, heart and spleen (Cq values ranging from 34.1 to 36.9, corresponding to 2.3 x 10-1and 2.9 x 10-2 promastigotes/2 μl) and two each of kidney and egg samples (Cq value ranging from 20.3 to 37.2, corresponding to 3.5 x 103 and 3.1 x 10-2 promastigotes/2 μl) scored positive for L. tarentolae . All DNA samples from lizard spiked with DNA of L. infantum and/or L. tarentolae returned positive signals for one or both species (Table 2). Similar mean Cq values were detected for all tissues spiked with each Leishmania spp. (ranging from 19.0 to 21.4), with the exclusion of lung samples of T. mauritanica , where an increasing Cq values up to 25 were detected (Table 2). A slightly increased Cq values was obtained by testing spiked lizard samples with both Leishmania spp. (Table 2).
Discussion
The dqPCR assay, developed for the simultaneous detection and differentiation of L. infantum and L. tarentolae , proved to be an efficient and sensitive tool for detecting both parasites, alone or in combination. In particular, the smallest amount of DNA detected (2.1 x 10-7 ng/2 µl for L. tarentolaeand 2.6 x 10-6 ng/2 µl for L. infantum ) corresponded to a low parasite load (3.3 x 10-3 and 3.3 x 10-3 promastigotes/2 µl per reaction). However, a slight shift in the Cq values was observed for L. infantum andL. tarentole spiked with DNA samples from lizards. The results obtained may be explained by the copy number (i.e., 200) of the genetic target (ITS1) present in the genome of Leishmania spp. (Van der Auwera and Dujardin 2015; Schonian et al., 2011) and by the high genetic variability of this region, which was pivotal for delineating the two species. Indeed, differently from the minicircle kinetoplast DNA, which exhibits a high sensitivity in detecting low amounts of DNA (equivalent to 0.004 parasites) but low specificity (Kuhls et al., 2011; Galluzzi et al., 2018), the ITS1 has been successfully used to resolve taxonomical and phylogenetic relationships among closely related Leishmaniaspp. (Dávila and Momen, 2000; Parvizi and Amirkhani, 2008; Wang et al., 2010; Hajjaran et al., 2013).
The sensitivity of the dqPCR (100%) was also confirmed by detection of DNA of both Leishmania spp. in all lizard spiked DNA samples, showing approximately the same Cq values (ranging from 19.0 to 22.1). Similarly, the good performance of the dqPCR was also determined by the values of the slope of standard curves, the efficiency and the coefficient of determination obtained with both Leishmania spp. and the lizard-spiked DNA.
The dqPCR assay detected low amounts of Leishmania spp. DNA (mean value of 2.2 x10-5 for L. infantum and 2.1 x 10-6 for L. tarentolae ) in naturally infected phlebotomine sand flies as well as discriminated both species in co-infected Se. minuta. Though a low number of phlebotomine sand flies were tested, the finding of the same prevalence of infection forL. infantum in Ph. perniciosus (3.6%) and in Se. minuta (3.5%) and the co-infection for both Leishmania spp. provide additional circumstantial evidence on the involvement ofSe. minuta in the circulation of L. infantum in some endemic areas (Maia et al., 2015; Maia and Depaquit, 2016; Latrofa et al., 2018; Pombi et al., 2020). Indeed, the role of Se. minuta in spreading Leishmania spp. other than L. tarentolae has been suggested in different countries of the Mediterranean region such as Italy, Portugal, Tunisia and Turkey ( Campino et al., 2013; Jaouadi et al., 2015; Maia et al., 2015; Ayari et al., 2016; Özbel et al., 2016; Latrofa et al., 2018) and also in regions of Africa (Mutinga et al., 1994; Tateng et al., 2018; Nzelu et al., 2014) and Asia (Chusri, et al., 2014; Siripattanapipong et al., 2018). Therefore, given the opportunistic anthropophilic feeding behaviour of this phlebotomine sand fly, susceptible hosts could be infected by L. infantum through bites of Se. minuta females (Maia et al., 2015; Gonzales et al., 2020; Pombi et al., 2020). In this context, the pathogenic role ofL. tarentolae detected in humans should not be ruled out. Indeed, some virulence factors, (i.e., GP63, CPB, LPG3 and amastin) present in the pathogenic L. infantum and L. major is also expressed in L. tarentolae (Azizi et al., 2009; Mizbani et al., 2011; Raymond et al., 2012; Novo et al., 2015) . The detection of the DNA of L. tarentole in P. siculus suggest that more than one lizard species could be infected by this pathogen, besides the previously reported T. mauritanica (Pozio et al., 1983). Finally, even if no lizard samples scored positive for L. infantum in the present investigation, the limited number of samples tested precludes any further inference on this.
In conclusion, the dqPCR herein described may represent an improvement in the discrimination of L. infantum and L. tarentolae in order to identify the role of lizards and Se. minuta as reservoirs and vector, respectively, for these Leishmania spp. Furthermore, this assay will allow large-scale testing of humans (and also dogs, for instance) in areas where Se. minuta occurs, enabling a more robust assessment on how frequent these hosts are exposed to L. tarentolae .