-
Loading metrics
Open Access
Peer-reviewed
Research Article
A multifaceted molecular approach to surveillance of leishmaniasis: Identification of sand fly species, Leishmania parasites, and blood meal sources using high-resolution melting analysis
A multifaceted molecular approach to surveillance of leishmaniasis: Identification of sand fly species, Leishmania parasites, and blood meal sources using high-resolution melting analysis
- Liora Studentsky,
- Fouad Akad,
- Debora Diaz,
- Irina Ben Avi,
- Shirly Lea Elbaz,
- Tamar Grossman,
- Maya Davidovich-Cohen,
- Oscar David Kirstein,
- Laor Orshan,
- Gad Baneth
-
- Published: September 24, 2025
- https://doi.org/10.1371/journal.pntd.0013412
Figures
Abstract
Leishmaniasis is a significant public health concern in large parts of the world including Israel, with limited diagnostic tools available for effective surveillance and control. Traditional methods for sand fly species identification, Leishmania detection, and blood meal analysis are time-consuming and prone to errors. To address these challenges, this study aimed to develop PCR-high resolution melt (HRM) assays to accurately determine sand fly species, Leishmania infection and blood meal sources in sand flies. Field-collected sand flies from all regions of Israel were used for the validation of three PCR-HRM assays. These included 254 sand fly males and females identified morphologically for species verification; 1,120 unfed females for Leishmania detection, and 538 engorged females for blood meal identification. PCR products were subjected to HRM curve analysis, and results were compared to nucleotide sequencing and sand fly morphology. Eleven sand fly species, 25 different host species blood meals and four Leishmania species were discerned and each presented a specific HRM pattern. Of the 1,658 analyzed females, 16 (1%) were positive for Leishmania, and the species identified were: Leishmania major, L. tropica, L. infantum and L. donovani. Blood meal source was identified in 520 (96.7%) engorged females. Blood from four animal species (domestic cat, rock hyrax, European hare, cow) accounted for 53% of the sand fly blood meals and the remaining 47% came from 21 other animal species. The sand fly species distribution showed that L. major and L. donovani vectors were mostly prevalent in arid southern Israel while L. tropica and L. infantum vectors were abundant in central and northern Israel. These results present the current knowledge of the different Leishmania species life cycles, vectors, and host species present in Israel and substantiate the utility of the assays developed herein which combine the advantages of PCR and the discriminatory power of HRM.
Author summary
Leishmaniasis is an endemic disease in Israel and four Leishmania species are responsible for cutaneous or visceral leishmaniasis circulation in different zones of the country. This disease affects the health of humans and animals, such as dogs and cats. In this study we presented the development and validation of new analytic methods and conducted an entomological survey using Real-Time PCR and HRM analysis for sand fly and Leishmania species identification, and to determine blood meal sources of field-collected sand flies. The combination of these methods has elucidated the complex prevalence of different sand fly vectors, the most common sources of their blood meal, and the species of Leishmania parasites which they harbor, in Israel. This model would also be useful for epidemiological studies in other areas of the world where leishmaniasis is prevalent.
Citation: Studentsky L, Akad F, Diaz D, Ben Avi I, Elbaz SL, Grossman T, et al. (2025) A multifaceted molecular approach to surveillance of leishmaniasis: Identification of sand fly species, Leishmania parasites, and blood meal sources using high-resolution melting analysis. PLoS Negl Trop Dis 19(9): e0013412. https://doi.org/10.1371/journal.pntd.0013412
Editor: Kyoko Hayashida, Hokkaido University International Institute for Zoonosis Control, JAPAN
Received: February 6, 2025; Accepted: July 30, 2025; Published: September 24, 2025
Copyright: © 2025 Studentsky et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Leishmaniasis comprises a group of zoonotic vector-borne diseases caused by several species of Leishmania parasites. It is endemic in Israel, the Middle East, and many other parts of the world [1]. Leishmania species infect humans and animals and are transmitted by at least 98 confirmed and suspected sand fly vectors within the genera Phlebotomus and Lutzomyia [2]. Leishmaniasis is characterized by several remarkable paradoxes that challenge our understanding of its epidemiology and control. Although different Leishmania species can cause similar clinical symptoms [3–5], some species can induce a variety of clinical manifestations, such as Leishmania infantum causing visceral disease in some patients and cutaneous disease in others [6–8].
At least 70 species of wild and domestic animals have been identified as confirmed or potential reservoirs involved in Leishmania transmission cycles [9,10]. Cutaneous leishmaniasis (CL), alongside visceral leishmaniasis (VL), are notifiable diseases with significant public health implications in Israel. The number of new cases reported annually in Israel is shown in S1 Fig [11–15]. Analysis of disease incidence reveals a concerning trend. Between 2000 and 2004, an average of 58 CL cases were reported annually. This number rose sharply to approximately 200 cases per year between 2005 and 2009. Data from 2010-2014 shows a further surge, with an average of 304 cases reported annually. A peak in the number of cases was observed in 2012–2014, with disease emerging in previously unaffected areas [16,17]. In the following five years, 2015–2019, a decrease was observed in human morbidity, with an average of 237 CL and 5.4 VL annual cases. A further decrease occurred in human morbidity in 2020–2023, with an average 127 CL and one VL cases reported annually [11].
Leishmaniasis in Israel presents complex zoonotic transmission patterns involving four Leishmania species: Leishmania major, Leishmania tropica, L. infantum, and Leishmania donovani, leading to a variety of clinical manifestations in humans and mammals across different ecoregions of the country. All four species cause CL, however, L. infantum is responsible mainly for VL [12–19]. Each Leishmania species present in Israel has a distinct transmission cycle involving specific reservoir hosts. Leishmania major is transmitted by Phlebotomus papatasi with a diverse range of vertebrate reservoir hosts, including the fat sand rats (Psammomys obesus), gerbils (Gerbillus dasyurus), jirds (Meriones crassus and Meriones tristrami), and voles (Microtus guentheri). Leishmania tropica, transmitted by Phlebotomus sergenti and Phlebotomus arabicus, is associated with rock hyraxes (Procavia capensis) as a primary reservoir host [20–23]. Putative vectors for L. infantum include Phlebotomus perfiliewi galilaeus, Phlebotomus syriacus, and Phlebotomus tobbi, with various canid species serving as reservoir hosts [12]. A fourth CL transmission cycle in southern Israel involves L. donovani possibly transmitted by Phlebotomus alexandri and maintained by hares (Lepus europaeus) as reservoir hosts [19,24].
High-resolution melting (HRM) PCR has emerged as a promising approach that offers several advantages over traditional molecular methods. HRM-PCR utilizes the thermal denaturation characteristics of amplicons. The melting temperature (Tm) and typical melting curve shape deduced by the HRM-PCR, are determined by nucleotide sequences, GC content, and amplicon length, and provide valuable information for species-specific genotype identification, making it a relatively cost-effective method compared to traditional PCR techniques [25–28].
This study aimed to develop new HRM-PCR-based assays for identification of sand fly and Leishmania species, and blood meal sources of sand flies as tools for surveillance and control of leishmaniasis.
Materials and methods
Sand fly field collection
Sand flies were collected in entomological surveys conducted by the medical entomology laboratory of the Israeli Ministry of Health between 2022 and 2023 in areas of Israel known for the existence of different Leishmania transmission cycles. The surveys encompassed geographically diverse areas, including the Sea of Galilee region, central Israel, the Jerusalem area, southeastern Israel, and the Arava and Negev deserts. Modified CDC (Centers for Disease Control) traps baited with dry ice, were employed for overnight outdoor collections. The use of CO2 alone, without a light source, follows established national protocols and is based on evidence showing light traps are ineffective for sand fly capture in Israel [29]. The traps were placed on updraft position with their opening placed ~ 1m above the ground, following established protocols [29,30].
Sand fly sample preparation
Collected sand flies were sexed and identified for species level at the medical entomology laboratory at the Israeli Ministry of Health in Jerusalem. Male specimens were morphologically characterized to the species level based on genitalia features. Individual, unfed female sand flies were dissected to examine spermathecal and pharyngeal morphology following established identification keys [31,32]. Individual female sand fly abdomens were separated from the rest of the body using forceps on a chilled plate. Forceps were cleaned with 10% bleach and water between dissections to avoid cross-contamination. Each abdomen was placed in an individual 1.1 ml microcentrifuge tube and stored at -80°C for subsequent DNA extraction and molecular analyses.
DNA extraction
Total genomic DNA was extracted from sand fly samples and from Leishmania parasite promastigote cultures of national and international reference strains obtained from the Kuvin Center cryobank (Hebrew University of Jerusalem) including L. major (MHOM/PS/1967/JerichoII), L. tropica (MHOM/IL/1990/P283), L. infantum (MHOM/SD/62/2S), L. donovani (MHOM/SD/1962/1S-CLD2) and Leishmania aethiopica (MHOM/ET/1972/L102).
The DNA extraction protocol involved homogenization in 50 μL in-house lysis buffer containing DNase- and proteinase-free RNaseA (Thermo Fisher Scientific, Cat. No. EN0531), proteinase K, and ATL tissue lysis buffer (QIAGEN, Cat. No./ID:19076). Sand fly tissues were disrupted using the TissueLyser II (QIAGEN) with 3-mm stainless-steel beads for 5 minutes. Following homogenization, an additional 200 μL of the same lysis buffer was added to each sample and incubated at 56°C for two hours. Subsequently, homogenates were centrifuged, and total DNA was extracted using the QIAsymphony DSP DNA Mini Kit (192) (QIAGEN, Cat. No./ID:937236) in a QIAGEN’s QIAsymphony SP instrument (Cat. No./ID:9001297) following the manufacturer’s instructions. Purified DNA was eluted in 100 μL of kit elution buffer.
Real-time PCR and HRM analysis
Three separate real-time PCR reactions targeting sand fly species identification, Leishmania species detection and identification, and blood meal source analysis, were carried out using a Roche LightCycler 96 instrument (Roche, Mannheim, Germany) with AccuMelt HRM SuperMix (Quanta Bioscience, Gaithersburg, USA, Cat. no. 95103–012) and specific primers (Table 1) following protocols detailed in Studentsky et al. 2023 [19]. HRM analysis was performed following the real-time PCR amplification. Amplicon dissociation analysis was carried out by capturing fluorescence signals in 0.1°C/sec intervals and holding for 60 seconds in each range of the melting curve (between 60°C and 85°C, in the sand fly species and blood meal detection assays, or up to 95°C in the case of Leishmania PCR). Genotype assignment was based on reference controls representing known species, allowing for the recognition of unknown samples based on their melting peak profiles. Normalized melting curves and peak profiles with distinct HRM profiles were categorized into groups based on identical melting peak patterns observed in all individuals of the same species.
HRM-based sand fly species identification
Validation of HRM-PCR was conducted on morphologically identified sand flies (n = 254, 131 males and 123 females). This assay targeted a 368–393-bp fragment of the cytochrome b gene (cytb) for Phlebotomus species identification (Table 1). The established HRM profiles established in this study include all twelve sand fly species documented in Israel to date (Table 2). Following HRM analysis, samples representing distinct melting peak groups were sequenced for confirmation.
Leishmania detection and identification
Leishmania species HRM profiles validation was conducted using two types of controls: (1) parasite promastigote cultures of international reference strains of Leishmania, (L. major MHOM/PS/1967/JerichoII, L. tropica MHOM/IL/1990/P283, L. infantum MHOM/SD/62/2S, L. donovani MHOM/SD/1962/1S-CLD2 and L. aethiopica MHOM/ET/1972/L102) and, (2) Leishmania American Type Culture Collection (ATCC) controls containing genomic DNA from L. major (Cat. no. 30012D), L. tropica (Cat. no. 50129D), L. infantum (strain MHOM/TN/80IPT-1, Cat. no. 50134D) and L. donovani (strain Khartoum, Cat. no. 30030D). Molecular biology-grade water (Bio-Lab, Israel) served as a negative control (NC).
Sand fly blood meal source identification
The HRM method for blood meal source identification was validated using 38 DNA samples from 13 different vertebrate species provided by the Koret School of Veterinary Medicine, Hebrew University (Table 3). A human DNA (Homo sapiens) sample, provided by the Parasitology National Reference Center, Israel Ministry of Health, was included in the tests as a part of their diagnostic procedure. Blood meal identification in field-collected engorged sand flies (n = 538) was performed by amplifying a 500-bp DNA fragment of the host 12S and 16S mitochondrial rRNA genes using universal vertebrate primers [19] (Table 1).
Sample sequencing and sequence alignment
Following HRM analyses, all samples categorized into distinct melting curve groups underwent Sanger sequencing at the Center for Genomic Technologies (Hebrew University) for confirmation. Sequence alignment and correction were conducted using BioNumerics v8.0 software (Applied Maths, Belgium) and compared with sequences in the GenBank database via the NCBI BLASTn algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Leishmania species, blood meal sources, and sand fly species were identified when their sequences were the top BLAST match and exhibited ≥98% identity.
Results
A total of 1,912 sand flies from the entomological survey were used, including 131 males and 1,781 females (1,243 unfed and 538 fed).
1. Development and validation of the HRM assays
Sand fly species identification HRM assay.
To evaluate the reliability of the HRM analysis for sand fly species identification, a total of 254 sand flies (131 males and 123 unfed females) were initially morphologically identified to species level. Subsequently, their DNA was extracted and subjected to HRM analysis targeting a fragment of the cytb gene (368–393 bp). Each sample was analyzed using three methods: morphological identification, HRM, and Sanger sequencing (Tables 4, S1, and S1 File). The HRM analysis generated a unique "fingerprint" pattern for each sand fly species characterized by a specific melting temperature (Tm peak). Furthermore, the HRM results were able to group the tested species into 12 distinct patterns. Each species exhibited a characteristic melting peak pattern, enabling clear differentiation among sand fly species (Fig 1). Notably, 12 (4.7%) out of 254 samples initially misidentified by morphological features were correctly identified by HRM and confirmed through DNA sequencing (Tables 4, S1).
(A) Combined normalized melting peaks (top) and curves (bottom) of sand fly species using the HRM-RT-PCR analysis of the Cyt b gene; (B) The melting peaks of each separate sand fly species Cyt b gene sequences.
HRM assay for detection and differentiation of Leishmania species in sand flies.
For Leishmania species identification, HRM analysis targeting the internal transcribed spacer 1 (ITS1) locus (~265 bp) [33], was validated using DNA from reference Leishmania cultures (L. major, L. tropica, L. infantum, and L. donovani) and ATCC controls to characterize specific HRM profiles for each species based on unique melting peaks (Fig 2). These profiles were then used to identify Leishmania DNA in field-collected sand flies.
(A) Combined normalized melting peaks (top) and curves (bottom) of Leishmania species using the HRM-RT-PCR analysis of ITS1 locus; (B) The melting peaks of separate Leishmania species ITS1 sequences.
The HRM peaks for L. infantum exhibited an HRM curve with two prominent peaks, whereas the other three Leishmania species found in Israel produced three peaks. Leishmania major exhibited three peaks, with the middle peak being the lowest. In contrast, L. donovani had a similar peak pattern but with the middle peak being the highest. Leishmania tropica was characterized by three peaks, with a smaller peak on the right (Figs 2, S1 File).
Sand flies blood meal identification HRM assay.
An HRM assay targeting a fragment of the vertebrate 12S and 16S mitochondrial rRNA genes (~500 bp) was developed and validated to identify blood meal sources of sand flies. DNA from various relevant vertebrate hosts and potential hosts (fat sand rats, European hares, domestic cats, domestic dogs, and humans) was used to generate a reference library of unique HRM profiles for each species (Fig 3). The figure depicts some of the detected blood sources. Each blood source exhibited a distinct HRM pattern. In cases when HRM patterns had similarities, the PCR Tm at the beginning of the peaks was different and enabled to reliably differentiate between the types of blood sources. For example, the golden jackal (Canis aureus) (Fig 3A) and the human (Homo sapiens) exhibited single major peak patterns. However, their Tm differed, with the golden jackal at 78°C, preceded by a small curve, and the human at 81°C. The domestic cat (Felis catus), cow (Bos taurus), and rock hyrax (Procavia capensis) produced curves that had two prominent peaks, but created very different patterns which could not be confused (Fig 3). In addition, although the European hare (Lepus europaeus), fat sand rat (Psammomys obesus) and mountain gazelle (Gazella gazella) produced HRM patterns with three peaks, those patterns were different from each other with the fat sand rat creating an initial low peak and then two higher peaks, the European hare creating three prominent peaks toward the end of the pattern, and the gazelle having three consecutive peaks with similar heights that almost create a plateau space between them (Figs 3, S1 File).
Separate (top) and combined (bottom) normalized melting peaks and curves of blood meal sources of sand flies found in this study using the HRM-RT-PCR analysis of 12S, 16S rRNA genes; (A) Felis catus (domestic cat), Gazella gazella (mountain gazelle), Procavia capensis (rock hyrax), Canis aureus (golden jackal); (B) Psammomys obesus (fat sand rat), Bos taurus (cow), Homo sapiens (human), Lepus europaeus (European hare).
2. HRM analysis of field-collected sand flies
HRM analysis for Leishmania species identification was conducted on 1,120 unfed females sand flies, which were screened for Leishmania DNA and parasite species identification (Table 1) [27,33]. Positive samples were further confirmed by DNA sequencing. Ten unfed sand fly females were found infected with a Leishmania sp. and all positive samples were classified by HRM-RT-PCR and correctly verified by DNA sequencing into four Leishmania species (Table 5). The HRM melting peaks of all 1,120 trapped unfed female sand flies matched the HRM profiles groups of the reference species (Fig 1). Eleven sand fly species were identified and only three of them, Ph. sergenti, Ph. syriacus and Ph. alexandri were found infected with L. tropica, L. infantum, or L. donovani parasites, respectively (Table 5). Phlebotomus canaaniticus, the twelfth species for which the unique HRM profiles was determined in sand fly species validation, was not detected among the analyzed field-collected females.
In addition, ten different sand fly species were identified among the 538 blood-fed sand fly females which were analyzed using the HRM assay to identify sand fly species and Leishmania infection (Table 5). Six females of Ph. papatasi, Ph. sergenti and Ph. alexandri were identified as harboring the following Leishmania parasites: L. major, L. tropica and L. donovani, respectively (Table 5).
The locations in Israel where different sand fly species were detected in the study are shown in Fig 4, where proven vectors and putative vectors of Leishmania species were included, and in S2 Fig where phlebotomine species which are not known vectors are shown. Phlebotomus papatasi, the vector of L. major was found in large quantities in the south and only in smaller numbers in the central parts of Israel. Phlebotomus alexandri, known as a vector of L. donovani in Israel, was found mostly in the Negev desert of southern of Israel and in few locations in other parts of the country. Phlebotomus sergenti, the main vector of L. tropica in Israel was distributed from the Dead Sea to northern Israel, and Ph. arabicus which is also a vector of L. tropica was found only in northern Israel. The putative vectors of L. infantum, the sand fly species Ph. syriacus, Ph. tobbi and Ph. perfiliewi galilaeus, were found predominantly in central and northern Israel (Fig 4).
Map was created using ArcGIS software. Base map source: Natural Earth (https://www.naturalearthdata.com/), public domain.
Additional sand fly species which are not known as Leishmania vectors, were also trapped and identified. Phlebotomus kazeruni, was found in the Negev desert in southern Israel. Phlebotomus jacusieli is a relatively rare species which was trapped near Jerusalem and in other parts of central Israel. Other non-vector species including Phlebotomus halepensis, Phlebotomus simici and Ph. canaaniticus were distributed near the Sea of Galilee and in other parts of northern Israel (Fig 4).
HRM-based blood meal identification was successful in 96.7% (n = 520) of the blood–fed females tested. In addition to known blood meal sources which could be identified by matching to sequences in GenBank, previously unknown blood meal sources were identified through sequencing. Sequence analysis revealed a total of 25 different blood meal sources, with blood from humans, domestic animals, birds, rodents, and other wild animals, including several identified vertebrates known as Leishmania reservoirs (dogs, jackals, rock hyraxes, jirds, hares) (Table 6, Fig 3) [10,19,22,34,35]. Only nine of the eleven field-collected sand fly species included engorged females. No blood-fed Ph. jacusieli and Ph. simici were collected.
The wide variety of blood meals found in the different sand fly species (Tables 6, S2), provides an interesting insight into the feeding habits of sand flies in Israel. The most frequently detected blood meal sources were domestic cat (89/538; 16.5%), rock hyrax (75/538; 13.9%), European hare (63/538; 11.7%), and cow (57/538; 10.6%). Altogether these four animal species accounted for almost 52.9% of all blood meals while 21 other species accounted for 43.8% of the blood meals and an additional 3.3% could not be identified. In addition to this, it is also clear that a variety of sand fly species fed on each of the animal blood source species, with eight different species that have fed on dogs, six on cows, five on hares, and four on cats (Table 6). Despite this, Leishmania species DNA was found only in four of eleven sand fly species. (Tables 5, S2), and in the sand fly species known to be a vector for each of these Leishmania species in Israel and the Middle East. Accordingly, L. major DNA was found in Ph. papatasi fed on Tristram’s jird, L. tropica in Ph. sergenti which fed on rock hyraxes, and L. donovani in Ph. alexandri that fed on European hares [12,19,22].
Discussion
This study describes the development and validation of HRM-PCR assays as a robust tool for sand fly species identification, detection of Leishmania infection, and blood meal sources characterization in field-collected sand flies. The HRM has previously been used to identify different species of Leishmania in human leishmaniasis patients, naturally infected dogs, and other animal hosts [33]. The HRM assay is a rapid, closed-tube system with high sensitivity, eliminating the need for laborious post-PCR processing steps and reducing the risk of contamination inherent in manipulation techniques. The findings demonstrate the efficacy of HRM analysis in overcoming the limitations of traditional morphological identification and Sanger sequencing, providing an accurate, rapid, and cost-effective method for entomological and epidemiological studies related to leishmaniasis and potentially other sand fly-borne diseases. In addition, unlike Sanger sequencing, restriction fragmentation length polymorphism (RFLP), or serological techniques, the HRM method provides results in under two hours without requiring specialized post-PCR steps or advanced bioinformatics, making it particularly suitable for routine diagnostics and large-scale field studies.
We developed, adapted, and tested the surveillance method on 1,781 female sand flies from different regions of Israel, identifying four Leishmania species, each with a unique melting peak pattern. The HRM assay identified twelve sand fly species, each exhibiting a unique "fingerprint" pattern with species-specific melting peaks. The ability of the HRM assay to differentiate species is particularly significant, as 4.7% (9 out of 254) of the sand flies were initially misidentified morphologically but were correctly identified by HRM and confirmed by DNA sequencing. This not only underscores the potential for morphological misidentification but also highlights the reliability of the assay as a diagnostic tool, especially in regions with high sand fly species diversity or cryptic species complexes. In some cases, HRM profiles exhibited multiple melting peaks from a single PCR amplicon. This may result from intra-species sequence variability or the presence of secondary structures especially within the ITS1 region. These observations are consistent with previously reported HRM data and do not affect diagnostic accuracy, as the dominant Tm remains species-specific and stable. The Tm values obtained in our HRM assays (S3 Table) are consistent with those reported in previous studies targeting similar loci. For Leishmania spp., Talmi-Frank et al. [22] described comparable ITS1-based melting profiles, supporting the robustness of this approach for species differentiation. Regarding vertebrate blood meal sources, the HRM profiles are widely used for blood identification observed in our study as well as other studies, substantiating the reliability of the method for host identification [19,36,37]. Although published literature on HRM-based identification of sand fly species remains limited, our findings demonstrate the accuracy, reproducibility, and discriminatory power of this method. These results provide strong support for the utility of HRM-PCR as a reliable tool for sand fly species identification and highlight its potential for broader application in future entomological studies.
While mitochondrial markers such as the cytb gene are widely used for molecular identification, we acknowledge that introgression or retention of ancestral polymorphism may rarely lead to misidentification of closely related species.
Identifying the blood meal sources in hematophagous vectors is crucial for monitoring and managing potential disease transmission foci [38]. Blood meals from 25 different animal host species, as well as human blood, were detected in 538 blood-fed female sand flies. The blood meal could be identified in 520 sand flies and only 18 (3.3%) were not be amplified by HRM PCR and were therefore not be identified molecularly. All three molecular targets (sand fly spp., Leishmania spp., and blood meal sources) can be discerned from a single sand fly specimen. Total DNA extracted from whole individual sand flies is suitable for parallel HRM-PCR assays targeting the cytb (for sand fly), 12S and 16S (for host blood meal) and ITS1 (for Leishmania). This approach maximizes data yield from each specimen and supports integrated surveillance and vector incrimination efforts.
Leishmania amastigotes acquired during a blood meal can develop into promastigotes and multiply within the blood bolus. However, if they are ingested by a non-vector sand fly species, they are typically expelled during blood defecation. For this reason, the mere identification of Leishmania parasites or their DNA in engorged sand fly females (before defecation) is insufficient for vector incrimination [39–41]. However, the detection of L. tropica in unfed Ph. sergenti, L. infantum in unfed Ph. syriacus, and L. donovani in unfed Ph. alexandri, provides further evidence supporting their possible role as active vectors of these Leishmania species.
Although the primary aim of this study was to establish and validate diagnostic PCR techniques for identifying sand fly species, blood meal sources and Leishmania infection, it did not involve random sampling of sand flies across the entire country. However, it did focus on four geographically diverse regions of Israel where both cutaneous and visceral leishmaniasis circulate. The outcomes of this study, identifying the species of sand flies found in each geographical area (Figs 4), determining the most frequent animal hosts for blood meal sources, and detecting the species of Leishmania present in particular sand fly species, provide valuable information regarding the epidemiology of leishmaniasis in Israel. HRM analysis distinguished all the Old World Leishmania species causing disease in Israel and its neighboring countries, including L. infantum and L. donovani as previously reported in other studies [35,42,43]. These two species both belong to the L. donovani complex and can be distinguished based on a short, species-specific DNA sequence in the ITS1 locus [19].
The diversity and distribution of sand fly species in Israel, as demonstrated in this study, are complex and are most likely influenced by the diversity of ecoregions across the country. Israel has three primary climate zones: (1) a Mediterranean region in the central and northern areas, (2) an arid desert zone in the southern and eastern parts, and (3) a semi-arid transition zone situated between the Mediterranean and desert areas [44]. The prevalence of Leishmania infections typically corresponds with these climate zones. Whereas L. infantum infections have been predominantly reported in the Mediterranean region, L. major and L. tropica infections have been mostly documented in arid and semi-arid regions, while L. donovani infections were described only in arid zones [12,19]. The distribution of sand fly species identified in this study corresponds with these infection patterns and supports prior findings about the geographic prevalence of human and animal Leishmania infections. The principal vectors of L. major and L. donovani were primarily found in southern Israel, while the vectors of L. tropica and L. infantum were more commonly detected in central and northern Israel.
Unexpectedly, certain sand fly vectors were identified beyond their historically recognized regions. Phlebotomus sergenti, the principal vector of L. tropica, was identified in southern Israel, whereas Ph. alexandri, a recognized vector of L. donovani, was observed in middle and northern Israel. These findings suggest that sand fly vector distribution in Israel is not strictly confined to expected areas, and that Leishmania transmission could occur beyond traditionally recognized endemic regions. These shifts in distribution underscore the necessity for ongoing surveillance and further studies to determine if these changes are driven by climate change, anthropogenic activities, or ecological shifts, as these factors may contribute to the expansion of sand fly habitats and the transmission of Leishmania to humans and animals.
The detection of 25 different mammalian blood meal sources, including both domestic and wild animals, highlights the complex epidemiology of the leishmaniasis transmission cycle in Israel and the Middle East. The majority of the blood meals derived from just four animal hosts: domestic cats, rock hyraxes, European hares, and cows. This is unexpected, as these animal species are not necessarily the most abundant or largest in terms of body surface area available for sand fly feeding. The composition of blood meals is likely influenced by sand fly host-feeding preferences, which can vary between sand fly species and might be shaped by environmental factors [45,46]. Identifying of blood meal sources and understanding their relationship to Leishmania transmission are essential components devising effective prevention strategies to reduce the spread of leishmaniasis [19,47–49].
In conclusion, the HRM-PCR methods developed in this study enable the simultaneous detection of sand fly species, characterization of blood meals, and identification of Leishmania infection and parasite species discrimination. These methods have provided valuable insights into the distribution of sand fly species in Israel, their predominant blood meal sources, and their infection status with various Leishmania species in Israel. These techniques can be applied in entomological and epidemiological studies, as well as in clinical settings for the rapid diagnosis of Leishmania species, supporting treatment decisions and prognosis. Ultimately, this method holds significant potential to enhance leishmaniasis surveillance and contribute to disease control efforts.
Supporting information
S1 Fig. Human Leishmania morbidity cases number by year (2000–2023) in Israel.
Data reported by the Department of Epidemiology, Ministry of Health of Israel. VL – visceral leishmaniasis, CL – cutaneous leishmaniasis.
https://doi.org/10.1371/journal.pntd.0013412.s001
(TIF)
S2 Fig. Distribution of phlebotomine sand fly species which are not vectors of Leishmania species identified in this study.
Map was created using ArcGIS software. Base map source: Natural Earth (https://www.naturalearthdata.com/), public domain.
https://doi.org/10.1371/journal.pntd.0013412.s002
(TIF)
S1 Table. Complete data of sand fly samples identification by morphology, HRM-RT-PCR for the cytochrome b (cytb) gene, and sequencing.
Species with different morphology identification are marked by red.
https://doi.org/10.1371/journal.pntd.0013412.s003
(DOCX)
S2 Table. Species of engorged sand flies and their blood meal sources detected in the study.
https://doi.org/10.1371/journal.pntd.0013412.s004
(DOCX)
S3 Table. Reference amplicon melting temperatures (Tm) obtained by high-resolution melting (HRM) analysis for the identification of Leishmania species, sand fly vectors, and vertebrate blood meal sources.
https://doi.org/10.1371/journal.pntd.0013412.s005
(DOCX)
S1 File. Multiple sequence alignments for HRM Targets.
https://doi.org/10.1371/journal.pntd.0013412.s006
(DOCX)
Acknowledgments
This study was supported by the public health laboratories of the Ministry of Health in Jerusalem, Israel. We thank Simha Shilo and Anna Sarner from the Laboratory of Entomology, for assisting with the sand flies sorting and sample preparation. We thank Dr. Yaarit Nachum-Biala, Koret School of Veterinary Medicine, Hebrew University, who shared professional knowledge of sequences processing and preparation for GenBank submission. The authors thank the inspectors of the Israeli Ministry of Environmental Protection, for supporting and participating in the fieldwork.
References
- 1. WHO. Fact Sheet: Vector-borne diseases. (2020). https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases
- 2. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27(2):123–47.
- 3. Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, et al. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies. PLoS Negl Trop Dis. 2016;10(3):e0004349. pmid:26937644
- 4. Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol. 2007;37(10):1097–106. pmid:17517415
- 5. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. 2018;392:951–70.
- 6. Cunha J, Carrillo E, Sánchez C, Cruz I, Moreno J, Cordeiro-da-Silva A. Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis. Parasit Vectors. 2013;6:122. pmid:23622683
- 7. Ranasinghe S, Zhang W-W, Wickremasinghe R, Abeygunasekera P, Chandrasekharan V, Athauda S, et al. Leishmania donovani zymodeme MON-37 isolated from an autochthonous visceral leishmaniasis patient in Sri Lanka. Pathog Glob Health. 2012;106(7):421–4. pmid:23265615
- 8. Weiss F, Vogenthaler N, Franco-Paredes C, Parker SRS. Leishmania tropica-induced cutaneous and presumptive concomitant viscerotropic leishmaniasis with prolonged incubation. Arch Dermatol. 2009;145(9):1023–6. pmid:19770442
- 9. Maia C, Dantas-Torres F, Campino L. Parasite Biology: The Reservoir Hosts. The Leishmaniases: Old Neglected Tropical Diseases. Springer International Publishing. 2018. p. 79–106.
- 10. Baneth G, Solano-Callego L. Leishmaniasis. Vet Clin North Am Small Anim Pract. 2022;52(6):1359–75.
- 11. Department of Epidemiology M of H of I s r a e l. Weekly epidemiological reports, 2000-2023.
- 12. Jaffe CL, Baneth G, Abdeen ZA, Schlein Y, Warburg A. Leishmaniasis in Israel and the Palestinian Authority. Trends Parasitol. 2004;20(7):328–32. pmid:15193564
- 13. Azmi K, Krayter L, Nasereddin A, Ereqat S, Schnur LF, Al-Jawabreh A, et al. Increased prevalence of human cutaneous leishmaniasis in Israel and the Palestinian Authority caused by the recent emergence of a population of genetically similar strains of Leishmania tropica. Infect Genet Evol. 2017;50:102–9. pmid:27498421
- 14. Solomon M, Fuchs I, Glazer Y, Schwartz E. Gender and Cutaneous Leishmaniasis in Israel. Trop Med Infect Dis. 2022;7(8):179. pmid:36006271
- 15. Solomon M, Astman N, Warshavsky K, Barzilai A, Meningher T, Avni D, et al. Cutaneous leishmaniasis caused by Leishmania infantum, Israel, 2018–2021. Emerg Infect Dis. 2023;29(5):988–91.
- 16. Gandacu D, Glazer Y, Anis E, Karakis I, Warshavsky B, Slater P. Resurgence of cutaneous leishmaniasis in Israel, 2001–2012. Emerg Infect Dis. 2014;20:1605–11.
- 17. Israel Ministry of Health. Annual report of central laboratories. 2019. https://www.health.gov.il/PublicationsFiles/LAB_JER2019.pdf
- 18. Solomon M, Astman N, Warshavsky K, Barzilai A, Meningher T, Avni D, et al. Cutaneous leishmaniasis caused by Leishmania infantum, Israel, 2018-2021. Emerg Infect Dis. 2023;29(5):988–91.
- 19. Studentsky L, Orshan L, Akad F, Ben Avi I, Diaz D, Elbaz S, et al. Leishmania donovani Transmission Cycle Associated with Human Infection, Phlebotomus alexandri Sand Flies, and Hare Blood Meals, Israel1. Emerg Infect Dis. 2023;29(5):945–55. pmid:37080961
- 20. Wasserberg G, Abramsky Z, Anders G, El-Fari M, Schoenian G, Schnur L, et al. The ecology of cutaneous leishmaniasis in Nizzana, Israel: infection patterns in the reservoir host, and epidemiological implications. Int J Parasitol. 2002;32(2):133–43. pmid:11812490
- 21. Faiman R, Abbasi I, Jaffe C, Motro Y, Nasereddin A, Schnur LF, et al. A newly emerged cutaneous leishmaniasis focus in northern Israel and two new reservoir hosts of Leishmania major. PLoS Negl Trop Dis. 2013;7(2):e2058. pmid:23437408
- 22. Talmi-Frank D, Jaffe CL, Nasereddin A, Warburg A, King R, Svobodova M, et al. Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis. Am J Trop Med Hyg. 2010;82(5):814–8. pmid:20439960
- 23. Schnur LF, Nasereddin A, Eisenberger CL, Jaffe CL, El Fari M, Azmi K, et al. Multifarious characterization of leishmania tropica from a Judean desert focus, exposing intraspecific diversity and incriminating phlebotomus sergenti as its vector. Am J Trop Med Hyg. 2004;70(4):364–72. pmid:15100448
- 24. Ya’ari A, Jaffe CL, Garty BZ. Visceral leishmaniasis in Israel, 1960–2000. Isr Med Assoc J. 2004;6(4):205–8.
- 25. Wittwer CT. High-resolution DNA melting analysis: advancements and limitations. Hum Mutat. 2009;30:857–9.
- 26. Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum Mutat. 2009;30:860–6.
- 27. Slomka M, Sobalska‐Kwapis M, Wachulec M, Bartosz G, Strapagiel D. High Resolution Melting (HRM) for high‐throughput genotyping — limitations and caveats in practical case studies. Int J Mol Sci. 2017;18:2316.
- 28. Tamburro M, Ripabelli G. High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature. Ann Ig. 2017;29(4):293–316. pmid:28569339
- 29. Orshan L, Szekely D, Khalfa Z, Bitton S. Distribution and seasonality of Phlebotomus sand flies in cutaneous leishmaniasis foci, Judean Desert, Israel. J Med Entomol. 2010;47(3):319–28. pmid:20496578
- 30. Orshan L, Elbaz S, Ben-Ari Y, Akad F, Afik O, Ben-Avi I, et al. Distribution and Dispersal of Phlebotomus papatasi (Diptera: Psychodidae) in a Zoonotic Cutaneous Leishmaniasis Focus, the Northern Negev, Israel. PLoS Negl Trop Dis. 2016;10(7):e0004819. pmid:27427959
- 31. Abnnec E. Les phlébotomes de la région éthiopienne (Diptera, Psychodidae). Mém Off Rech Sci Tech Outremer. 1972;55:1–289.
- 32. Lewis DJ. A taxonomic review of the genus Phlebotomus (Diptera: Psychodidae). Bull Br Mus Nat Hist (Ent). 1982;45:121–209.
- 33. Talmi-Frank D, Nasereddin A, Schnur LF, Schönian G, Töz SO, Jaffe CL, et al. Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis. 2010;4(1):e581. pmid:20069036
- 34. Dantas-Torres F, Miró G, Baneth G, Bourdeau P, Breitschwerdt E, Capelli G, et al. Canine Leishmaniasis Control in the Context of One Health. Emerg Infect Dis. 2019;25(12):1–4. pmid:31742505
- 35. Ghawar W, Toumi A, Snoussi M-A, Chlif S, Zâatour A, Boukthir A, et al. Leishmania major infection among Psammomys obesus and Meriones shawi: reservoirs of zoonotic cutaneous leishmaniasis in Sidi Bouzid(central Tunisia). Vector Borne Zoonotic Dis. 2011;11(12):1561–8. pmid:21919726
- 36. Ouso DO, Otiende MY, Jeneby MM, Oundo JW, Bargul JL, Miller SE, et al. Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance. Sci Rep. 2020;10(1):4741. pmid:32179808
- 37. Ogolla KO, Bwana BK, Mang’era CM, Onyango T, Otiende MY, Ochieng B, et al. Putative bloodmeal sources in Glossina austeni tsetse fly of Arabuko Sokoke National Reserve in Kenya. PLoS One. 2024;19(3):e0299243. pmid:38446817
- 38. Pena VH, Fernández GJ, Gómez-Palacio AM, Mejía-Jaramillo AM, Cantillo O, Triana-Chávez O. High-resolution melting (HRM) of the cytochrome b gene: a powerful approach to identify blood-meal sources in Chagas disease vectors. PLoS Negl Trop Dis. 2012;6(2):e1530.
- 39. Sadlova J, Dvorak V, Seblova V, Warburg A, Votypka J, Volf P. Sergentomyia schwetzi is not a competent vector for Leishmania donovani and other Leishmania species pathogenic to humans. Parasit Vectors. 2013;6(1):186. pmid:23786805
- 40. Pruzinova K, Sadlova J, Myskova J, Lestinova T, Janda J, Volf P. Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases. Parasit Vectors. 2018;11(1):37. pmid:29335002
- 41. Sadlova J, Homola M, Myskova J, Jancarova M, Volf P. Refractoriness of Sergentomyia schwetzi to Leishmania spp. is mediated by the peritrophic matrix. PLoS Negl Trop Dis. 2018;12(4):e0006382. pmid:29617364
- 42. Derghal M, Tebai A, Balti G, Souguir-Omrani H, Chemkhi J, Rhim A, et al. High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia. Parasit Vectors. 2022;15(1):12. pmid:34996507
- 43. Bizri NA, Alam W, Khoury M, Musharrafieh U, Ghosn N, Berri A, et al. The Association Between the Syrian Crisis and Cutaneous Leishmaniasis in Lebanon. Acta Parasitol. 2021;66(4):1240–5. pmid:33886042
- 44. Kutiel H. Climate of Israel. Landscapes and Landforms of Israel. 2024. p. 39–47.
- 45. Bongiorno G, Habluetzel A, Khoury C, Maroli M. Host preferences of phlebotomine sand flies at a hypoendemic focus of canine leishmaniasis in central Italy. Acta Trop. 2003;88(2):109–16. pmid:14516922
- 46. Thomaz-Soccol V, Gonçalves AL, Baggio RA, Bisetto A Jr, Celestino A, Hospinal-Santiani M, et al. One piece of the puzzle: Modeling vector presence and environment reveals seasonality, distribution, and prevalence of sandflies and Leishmania in an expansion area. One Health. 2023;17:100581. pmid:37332885
- 47. Baum M, de Castro EA, Pinto MC, Goulart TM, Baura W, Klisiowicz D do R, et al. Molecular detection of the blood meal source of sand flies (Diptera: Psychodidae) in a transmission area of American cutaneous leishmaniasis, Paraná State, Brazil. Acta Trop. 2015;143:8–12. pmid:25530542
- 48. Yared S, Gebresilassie A, Abbasi I, Aklilu E, Kirstein OD, Balkew M, et al. A molecular analysis of sand fly blood meals in a visceral leishmaniasis endemic region of northwestern Ethiopia reveals a complex host-vector system. Heliyon. 2019;5(7):e02132. pmid:31384683
- 49. Remadi L, Chargui N, Jiménez M, Molina R, Haouas N, González E, et al. Molecular detection and identification of Leishmania DNA and blood meal analysis in Phlebotomus (Larroussius) species. PLoS Negl Trop Dis. 2020;14(3):e0008077. pmid:32214313