Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal.

BACKGROUND: Zoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe.
METHODS: Phlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence.
RESULTS: Out of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta.
CONCLUSIONS: The anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.

Background

Leishmaniasis caused by Leishmania infantum is the only tropical vector-borne disease that has been endemic in southern Europe for decades [1]. Most of the reported cases are due to zoonotic visceral leishmaniasis (VL), the most dangerous form of Leishmania infection, being lethal when untreated. Dogs are considered the major host for these parasites, and the main reservoir for human infections. In nature, the pathogen transmission occurs via the infective bite of phlebotomine sand flies (Diptera, Psychodidae), for both humans and dogs.

In Portugal, as in other countries in the south of Europe, VL was initially described as a pediatric disease but from the end of the 1980s onwards, the number of cases in children has decreased with a concomitant increase of infection in adults, commonly associated with HIV/AIDS [2]. In the last ten years (2005–2014), 119 new cases of human VL (17 in immunocompetent adults, 36 in children and 66 in immunocompromised patients) and 16 cutaneous leishmaniasis cases were diagnosed at the Leishmaniasis Laboratory at the Institute of Hygiene and Tropical Medicine. Leishmania infantum zymodeme MON-1 is the most common aetiological agent of autochthonous human and canine leishmaniasis cases [3] and Phlebotomus perniciosus and Phlebotomus ariasi have been confirmed as proven vectors [4].

As data regarding Leishmania infection rate and blood meal sources of phlebotomine sand flies in Portugal is still too scarce, this study was implemented so as to allow the (i) detection of Leishmania DNA and (ii) identification of blood meal sources in wild caught female sand flies in Algarve, Portugal/Southwestern Europe.

Methods

Study area

Algarve, located in southern Portugal, has an area of 5,412 Km2 with an estimated number of permanent inhabitants approximating 450,000 [5], which triplicates during summer months. Figs (Ficus carica), almonds (Prunus amygdalus), oranges (Citrus sinensis), carobs (Ceratonia siliqua), strawberries trees (Arbutus unedo) and cork oaks (Quercus suber), are the most common crops in the region [6]. Algarve has a Mediterranean climate with warm weather (annual average temperature of 18°C) and low rainfall almost all year round (annual average of 500 mm). Summer (June-September) is the driest and warmest season with average monthly temperatures between 16° and 28-30°C (www.ipma.pt).

Collection and identification of sand flies

Between May to October from 2011 to 2013, CDC light traps and sticky oil papers were set up in 11 sampling points during three consecutive days per month. Collection places included domestic, peri-domestic and sylvatic environments. In most of the studied biotopes, in addition to humans and dogs, the major vertebrates visible within a 50 m radius of the collection spots were livestock, horses, pigs, rabbits and poultry. Collected sand flies were stored in 70% ethanol for further analysis. A total of 4,971 sand flies (2,584 males and 2,387 females) were collected and identified morphologically. Phlebotomine specimens of both genders were identified by their morphological characteristics to the species level, according to Pires [7]. Female identification was done by microscopic observation of the spermatheca, after dissection and mounting of the three last abdominal segments in Marc-André solution, while males were identified by direct stereomicroscopic observation of the genitalia. In addition, for each female, the presence of eggs (gravid status), and/or blood (engorged: total or partial vs. unfed) in the abdomen was recorded (Table 1).

Table 1

Sand fly specimens collected according to the capture method and their positivity to spp.

	CDC light traps		Sitcky papers		Total			Females
Sand fly species	Females	Males	Females	Males	Females	Males	Females + Males	Blood fed	Gravid	Positive
Phlebotomus ariasi	34	8	0	4	34	12	46	3	2	0
Phlebotomus perniciosus	372	305	59	806	431	1111	1542	49	32	1
Phlebotomus papaptasi	1				1		1		1
Phlebotomus sergenti	27	26	27	94	54	120	174	1	1	0
Sergentomyia minuta	212	149	1655	1192	1867	1341	3208	25	49	2
Total	646	488	1741	2096	2387	2584	4971	78	85	3

DNA extraction, PCR amplification and DNA sequencing

For each female sand fly, the remainder of the body (minus genitalia) was used as the source of DNA, extracted using the Citogene® Cell and Tissue kit (Citomed, Portugal) following the manufacturer’s instructions, with the exception that the maceration of the insect’s tissues was carried out with a piston pellet, and the final elution volume was 30 μl.

The PCR amplification of the internal transcribed spacer 1 (ITS-1) of the ribosomal operon of Leishmania was performed using the LITSR and L5.8S primers generating amplicons with 300–350 bp [8]. A positive control containing L. infantum DNA (MHOM/PT/88/IMT151) and a negative control without DNA template were included. To identify the origin of the blood meal of engorged females, the modified vertebrate-universal specific primers (cytB1-F and cytB2-R) were used to amplify a 350 bp segment of the host mitochondrial cytochrome b gene (cyt-b) [9]. PCR amplifications were performed in a 25 μl final volume containing 12.5 μl of NZYTaq 2× Green Master Mix (Nyztech, Portugal), 1 μl of each primer (10 pmol) and 2 μl of template DNA. The cycling profile used for the amplification of ITS-1 sequences included an initial denaturation step at 95°C for 2 min, followed by 32 repeats of 95°C-20 sec, 53°C-30 sec, 72°C-1 min followed by a final extension step at 72°C-6 min, while the preparation of cyt-b PCR products was carried out starting from 95°C for 5 min, followed by 40 cycles of 94°C-1 min, 55°C-1 min, 72°C-1 min followed by 72°C-7 min. Both amplicons were visualized under UV illumination after their resolution by conventional electrophoresis on 1.5% agarose gels stained with Greensafe premium® (Nzytech, Portugal), using a 100 bp DNA ladder as a molecular weight marker. PCR products were purified with a High Pure PCR Product Purification Kit (Roche® Mannheim, Germany) according to the manufacturer’s instructions. Subsequently, purified products were sent to LIGHTrunTM Sequencing Service (GATC-biotech, Germany) for direct sequencing by Sanger’s method with the same primers used for DNA amplification.

DNA sequence analyses

The identity of the feeding host (species level), carried out on the basis of the analysis of the obtained cyt-b sequences, was determined according to the closest BLASTn match (identity ≥ 99%) to a homologous sequence deposited at GenBank. The sequences obtained in the course of this work were deposited at DNA Data Bank of Japan (DDBJ) (http://www.DDBJ.nig.ac.jp).

Restriction profile was obtained by virtual digestion for ITS-1 sequence by using the Restriction Mapper (version 3 available online at http://www.restrictionmapper.org/).

Phylogenetic relationships were inferred from ITS-1 nucleotide sequence alignments produced with the MAFFT multiple alignment program using a combination of the Q-INS-i and E-INS-i alignment options [10]. Phylogenetic tree construction was carried out using a Maximum Likelihood (ML) approach, and the Kimura’s 2-P (K2P) evolutionary model, also assuming Γ distributed substitution rates among sites, as indicated by Mega6 [11] and as defined by the Akaike information criterion. Alternatively, an empirically defined model (GTR + Γ + I) was also used. The topological robustness of the obtained trees was assessed by bootstrapping, using 1000 resampling of the original alignment data. The final trees were manipulated for display using FigTree v.1.2.2. (available at http://tree.bio.ed.ac.uk/software/figtree/). NeighborNet networks (NNn) were constructed using the same distance matrix using Splits Tree4 software [12]; software available at http://www.splitstree.org/). Mean genetic distance values were calculated with the K2P formula, using Mega6 [11].

Results

Morphological identification of sand flies

S. minuta was the most prevalent species totaling a number of 3,208 specimens (64.53%), followed by P. perniciosus with 1,542 specimens (31.02%). Phlebotomus sergenti (174; 3.50%), P. ariasi (46; 0.93%) and one P. papatasi female (0.02%) were also collected. Eighty five females (2 P. ariasi, 1 P. papatasi, 32 P. perniciosus, 1 P. sergenti and 49 S. minuta) were gravid.

Leishmania DNA detection, sequencing, and phylogenetic inference analysis

Leishmania DNA was detected in three apparently unfed females (0.13%) identified as P. perniciosus (n = 1) and in S. minuta (n = 2). The three positive females were collected in peridomestic biotopes (i.e. P. perniciosus was collected in a horse stable, and S. minuta were collected in a cattle pen and close to a kennel, respectively). The three ITS-1 obtained sequences were submitted to DDBJ (DDBJ accession numbers: LC028233 to LC028235). PCR product obtained from P. perniciosus had a similar size as L. infantum control while the PCRs products from both S. minuta were slightly bigger (data not shown). Furthermore, a HaeIII restriction profile characteristic of L. infantum (184 bp, 72 bp and 55 bp) was obtained after virtual digestion of the ITS-1 sequence obtained from the positive DNA control as well as from P. perniciosus. Finally, sequence homology searches using BLASTn (megablast search option) revealed >99% identity with L. infantum, L. chagasi or L. donovani (E-values = e−154), and a sequence coverage >94%. Curiously, however, species assignment to the ITS-1 sequences amplified from S. minuta could not be carried out on the basis of nucleotide sequence homology search results. In this case the 15 best matches obtained with BLASTn (megablast) revealed >93% sequence identity (>95% sequence coverage and E-values < e−122) with only Leishmania sequences of Chinese origin referred to as Leishmania sp. [13], indicating relatively low identity with any sequence references already deposited in the sequence databases. Virtual HaeIII restriction profiles of the ITS-1 sequences amplified from S. minuta (strains 5277 and 3400) were characterized by three DNA fragments (<193 bp, 89 bp, <54 bp), which were found to be similar, though not identical, to the virtual HaeIII profiles determined for the Chinese Leishmania sp. sequences (<210 bp, 87 bp, <43 bp) mentioned above.

Definition of the species status of the obtained ITS-1 sequences was further pursued on the basis of phylogenetic analyses, along with others directly downloaded from the public database, and used as references (Table 2). The use of the suggested evolutionary model (K2P + Γ) or a more robust one (GTR + Γ + I), empirically defined by the user, resorted in phylogenetic trees with identical topologies as that shown in Figure 1 (data not shown).

Table 2

Nucleotide reference sequences used in this work

Species*	Strain/ isolate/ haplotype	Origin/ host	Accession number
Leishmania donovani	MHOM/KE/83/NLB189	Kenya/Human	AJ634374
Leishmania donovani	MHOM/SD/93/9S	Sudan/Human	AJ634372
Leishmania donovani	MHOM/LK/2002/L60c	Sri Lanka/Human	AM901447
Leishmania donovani	MHOM/LK/2002/L60b	Sri Lanka/Human	AM901448
Leishmania archibaldi	MHOM/SD/93/GE	Sudan/Human	AJ634357
Leishmania archibaldi	MHOM/SD/97/LEM3429	Sudan/Human	AJ634358
Leishmania archibaldi	MHOM/SD/97/LEM3463	Sudan/Human	AJ634359
Leishmania donovani	MHOM/SU/84/LEM0946	Soviet Union/Human	HG512918
Leishmania donovani	MCAN/MA/2002/AD3	Morocco/Canine	AM901453
Leishmania donovani	MHOM/IQ/1981/SUKKAR2	Iraq/Human	AM901452
Leishmania donovani	MHOM/IN/1983/CHANDIGARH	India/Human	AM901449
Leishmania infantum	MCAN/UZ/2007/LRC-L1309	Uzbekistan/Canine	FN398341
Leishmania infantum	MHOM/BR/2007/JFF BM	Brazil/Human	FN398343
Leishmania infantum	MHOM/IT/93/ISS800	Italy/Human	AJ634354
Leishmania infantum	MHOM/PT/00/IMT260	Portugal/Human	AJ634344
Leishmania infantum	MHOM/MT/85/BUCK	Malta/Human	AJ634350
Leishmania infantum	MHOM/SD/93/452BM	Sudan/Human	AJ634371
Leishmania chagasi	MHOM/BR/85/M9702	Brazil/Human	AJ000306
Leishmania chagasi	MHOM/PA/79/WR317	Panama/Human	AJ000305
Leishmania tropica	MHOM/IL/01/LRC-L838	Israel/Human	FN677341
Leishmania tropica	MHOM/EG/90/LPN65	Egypt/Human	HG512927
Leishmania tropica	MHOM/PS/01/ISL590	Palestine*/Human	FN677345
Leishmania tropica	MHOM/YE/86/LEM1015	Yemen/Human	HG512919
Leishmania tropica	MHOM/TN/88/TAT3	Tunisia/Human	AJ300485
Leishmania tropica	IHAM/GH/2007/KLE-18	Ghana/Sergentomyia hamoni	AB787190
Leishmania aethiopica	MHOM/ER/2009/7457	Eritrea/Human	FN252411
Leishmania aethiopica	MHOM/KE/71/KPS-H2	Kenya/Human	HG512908
Leishmania turanica	KD85001	Uzbekistan/Rhombomys opimus	AJ272378
Leishmania turanica	KL3	Kazakhstan/Rhombomys opimus	AJ272382
Leishmania gerbilli	MRHO/UZ/87/KD-87555	Uzbekistan/Rhombomys opimus	AJ300486
Leishmania major	MTAT/KE//NLB089A	Kenya/ND	AJ300482
Leishmania major	MHOM/UZ/02/17h	Uzbekistan/Human	FN677357
Leishmania major	MHOM/BF/2004/REN04-8	Burkina Faso/Human	HG512963
Leishmania major	MHOM/JO/90/JH39	Jordan/Human	HG512945
Leishmania major	MHOM/TN/97/LPN162	Tunisia/Human	FN677342
Leishmania major	MHOM/DZ/89/LIPA228	Algeria/Human	HG512924
Leishmania mexicana	MHOM/PE/02/LH2312	Peru/Human	HG512965
Leishmania mexicana	MHOM/EC/90/LM	Ecuador/Human	HG512934
Leishmania amazonensis	MHOM/BR/73/M2269	Brazil/Human	DQ182536
Leishmania amazonensis	IFLA/BR/67/PH8	Brazil/ND	AF339753
Leishmania braziliensis	MHOM/PE/2003/LH2920	Peru/Human	FN398337
Leishmania braziliensis	MHOM/BR/00/LTB300	Brazil/Human	FN398338
Leishmania peruviana	MHOM/PE/2006/LH3667	Peru/Human	FN398340
Leishmania peruviana	MHOM/PE/1990/HB86	Peru/Human	FN398339
Leishmania guyanensis	MHOM/BR/2002/NMT-RBO013	Brazil/Human	FN398331
Leishmania guyanensis	MHOM/PE/2006/LH3635	Peru/Human	FN398332
Leishmania panamensis	Isolate 18, clone 4	ND/Human	FJ948442
Leishmania sp.	MHOM/CN/80/XJ801	P.R.China/Human	HQ830357
Leishmania sp.	MHOM/CN/89/GS5	P.R.China/Human	HQ830360
Leishmania sp.	MHOM/CN/90/SC10H2	P.R.China/Human	HQ830352
Leishmania sp.	MHOM/CN/86/SC6	P.R.China/Human	HQ830356
Leishmania sp.	MHOM/CN/90/SC10H2	P.R.China/Human	HM130601
Leishmania sp.	MCAN/CN/60/GS1	P.R.China/Canine	HM130600
Leishmania sp.	MHOM/GS6/CHN/SCgq	P.R.China/Human	HM130599
Leishmania sp.	MCAN/CN/86/SC9	P.R.China/Canine	HQ830359
Leishmania sp.	MHOM/CN/83/GS2	P.R.China/Human	HM130603
Leishmania sp.	MHOM/GS5/CHN/SCH2g	P.R.China/Human	HM130602
Leishmania sp.	MHOM/SC11/CHN/SCgz	P.R.China/Human	HM130606
Leishmania sp.	MHOM/CN/84/JS1	P.R.China/Human	HM130605
Leishmania sp.	MHOM/CN/84/SD1	P.R.China/Human	HM130604
Leishmania sp.	MHOM/CN/89/GS6	P.R.China/Human	HQ830355
Leishmania sp.	MHOM/CN/90/SC11	P.R.China/Human	HQ830361

*Species as defined by the depositors; Israel: Occupied Palestinian Territories; P. R. China: People’s Republic of China; ND: not defined.

Figure 1

Maximum likelihood phylogenetic tree (midpoint rooted) of ITS-1 sequences amplified from phlebotomine sand flies collected in Portugal. The percentages of significant (≥77%) bootstrap values of 1000 resamplings of the original data are indicated at specific branch-nodes. The size bar indicates 0.02 substitutions per site.

One of the sequences obtained in this study (strain 1704), amplified from P. perniciosus, was found to segregate in a large monophyletic cluster that included L. infantum, L. donovani, L. archibaldi and L. chagasi (Figure 1), characterized by low genetic variability (average genetic distance of 0.2%). On the other hand, the remainder two ITS-1 sequences (strains 5277 and 3400), amplified from S. minuta, were found to locate in a bootstrap-supported (99%) assemblage of multiple reference sequences of Chinese origin, merely defined as Leishmania sp. [13], and that included a multitude of Leishmania sequences from human and canine origin, with an average genetic distance of 2.6% (ranging from 0% to 8.0%), indicating considerably higher genetic variability than that associated with the L. infantum/L. donovani/L. archibaldi/L. chagasi cluster. Similar conclusions were achieved when, instead of assuming a strict tree-like evolution, the phylogenetic relationships between ITS-1 sequences were represented as a NNn (Figure 2).

Figure 2

NeighborNet network constructed with SplitsTree software employing the matrix of genetic distances (corrected with the K2P formula) between individual ITS-1 sequences amplified from phlebotomine sand flies collected in Portugal, and reference sequences.

Vertebrate DNA detection in female sand flies

A total of 78 engorged female sand flies (3 P. ariasi, 49 P. perniciosus, 1 P. sergenti and 25 S. minuta) were tested to determine the vertebrate host source of the blood meal. A positive PCR amplification result was obtained for 43 of the collected specimens. After DNA sequencing of the amplified partial cyt-b sequences, the origin of 30 (69.77%) blood-meals was identified (Table 3) on the basis of the closest sequence matches, as defined by BLASTn sequence homology searches (>99% identity with deposited at the GenBank/EMBL/DDBJ public databases).

Table 3

Identification of sand fly blood meal sources

Sand fly host	P . ariasi	P. perniciosus	S. minuta	Blast identity for the blood meal	DDBJ accession no.
Horse (Equus caballus)	0	12	0	99-100%	AB985687	AB985693-97	AB985699	AB985703	AB985708	AB985711	AB985714
Chicken (Gallus gallus)	0	5	0	99-100%			AB985704	AB985705	AB985710	AB985713	AB985715
Human (Homo sapiens)	0	0	4	99-100%				AB985688	AB985689	AB985698	AB985712
Rabbit (Oryctolagus cuniculus)	0	3	0	99%					AB985690	AB985700	AB985709
Pig (Sus scrofa)	0	2	0	99						AB985707	AB985716
Cattle (Bos taurus)	1	1	0	99						AB985702	AB985706
Sheep (Ovis aries)	0	1	0	99							AB985701
Lizard (Tarentola mauritanica)	0	0	1	99							AB985692
Total	1	24	5

Discussion

Phlebotomine sand flies are distributed in all countries around the Mediterranean basin, turning both human populations and domestic animals living in these areas into potential targets to sand fly-borne diseases such as leishmaniasis. Therefore, knowledge on the host preferences of sand flies under natural conditions is essential not only to understand their vectorial role, but also as a means to identify potential reservoir hosts. In this work, we detected Leishmania DNA and evaluated blood meal sources of fed females sand flies captured in southern Portugal, where zoonotic leishmaniasis is known to be endemic [2].

Similarly to what has been observed by others [14-19] the blood meal analysis of the engorged P. perniciosus revealed that this species fed on a broad variety of vertebrates hosts (i.e. horses, cattle, sheep, pigs, rabbits and chickens) highlighting its opportunistic feeding behaviour. Interestingly, no dog or human blood was detected in blood-fed P. perniciosus, despite the fact that it has been clearly defined as a proven vector of L. infantum in the Algarve region [19-21]. The apparent absence of P. perniciosus feeding on dogs and humans might indicate that in the sampled biotopes, neither of them were the main blood sources for this sand fly species due to the presence of other larger sized vertebrates (e.g. horses) and/or present in greater numbers (i.e. chicken, rabbits), making them easier targets.

In addition, Leishmania infantum DNA was detected in one unfed P. perniciosus specimen. Assignment of species status for the 1704 ITS-1 sequence could not be clearly carried out solely based on phylogenetic tree analysis due to the low genetic variability of the ITS-1 sequences that define the L. donovani complex [22] (that include L. infantum, L. donovani, L. archibaldi and L. chagasi, precluding a clear resolution of this genetic cluster, as previously observed [13]. Nevertheless, the ITS-1 amplicon size and virtual HaeIII restriction profile obtained for the 1704 sequence amplified from P. perniciosus were compatible with it corresponding to L. infantum, and reinforces the maintenance of this sand fly species as vector of L. infantum in southern Portugal [19-21].

Sand flies of the Sergentomyia genus, which is widely distributed throughout the Old World, are proven vectors of reptile Leishmania species [23]. It is generally accepted that most of Sergentomyia species are not anthropophilic, and as a consequence cannot transmit either Leishmania or any other pathogens to humans. However, in the present study, apart from detecting Tarentola mauritanica (a reptile widely distributed around the Mediterranean area [23,24]) DNA in one engorged S. minuta, human DNA was also amplified in four specimens corroborating that at least some Sergentomyia species disclose sporadic/opportunistic anthropophilic feeding-behaviour [25,26]. Furthermore, Leishmania sp. DNA was detected in two unfed S. minuta females, which unambiguously allocated with references within a cluster of Chinese Leishmania sp. previously isolated from humans and canine leishmaniasis cases [13]. While phylogenetic tree reconstruction and NNn analyses showed that the two ITS-1 sequences amplified from S. minuta (strains 5277 and 3400) clearly segregated away from all the others in a genetically consistent assemblage of Leishmania strains, in this case species assignment was limited by the unavailability of well characterized reference strains. However, despite the inability to clearly define the species of origin of the obtained sequences using phylogenetic analyses, the detection of Leishmania DNA phylogenetically related to those considered pathogenic to humans and dogs in China [13] was somewhat unexpected.

According to Yang et al. [13], the above mentioned Leishmania strains of Chinese origin belonged to an undefined species, that was found to be genetic divergent from any of the known New and Old World Leishmania, on the basis of ITS-1 sequence analysis. Similar results were obtained when kinetoplast cytochrome oxidase II (COII; [27]) or CYT-b coding sequences [28] amplified from these same strains were analysed. Interestingly, both phylogenetic inference reconstruction studies revealed that the Chinese Leishmania sp. isolates were most closely related to the lizard-infecting L. tarentolae. Unfortunately, in the present study it was not possible to evaluate if the two Leishmania sp. detected in S. minuta were genetic related to this reptile Leishmania species, as no ITS-1 sequences of L. tarentolae have yet been deposited in DNA sequence databases for public access. On the other hand, exhaustion of the DNA extracts on which the analysis presented in this report was based ruled out any possibility of generating cyt-b and/or coII sequence data. Nevertheless, and taking into account the results obtained with cyt-b/coII [27,28], in the near future it will be important to analyse more of these Leishmania parasites obtained from both vertebrate (including reptiles) and invertebrate infected hosts for assessment of the parasite species as well as to determine their clinical significance, and estimate the potential risk their endemic establishment in Portugal/Europe. Ideally, should the laboratory settings allow it, further genetic analysis-based studies should be supported, as much as possible, by sequence datasets combining information from multiple genetic loci, so as to tentatively increase the phylogenetic signal, and achieve a better resolution of the observed genetic clusters, including the L. donovani complex [29].

Based upon literature reviews, a consideration of the role of Sergentomyia in the circulation of mammalian leishmaniasis becomes apparent as Leishmania DNA has been identified in several species. These include the molecular detection of L. major in S. sintoni in Iran [30], S. garnhami in Kenya [31], S. darlingi in Mali [25], and S. minuta in Portugal [32]. Furthermore L. donovani has been detected in S. babu in India [33], L. infantum in S. dubia, S. magna and S. schewtzi in Senegal [34], and L. siamennsis in S. gemmea in Thailand [35]. Finally, more recently, L. tropica has been found in S. ingrami and S. hamoni in Ghana [26]. Nevertheless, PCR positivity alone should not be used for incrimination of Sergentomyia sand flies as Leishmania vectors since the detection of DNA does not give any information about the parasites’ viability or its presence as virulent metacyclic promastigotes [36,37]. In fact, and although L. infantum DNA had been detected in S. schwetzi from Senegal [34], the refractoriness of this African species to some Leishmania species infecting humans (including L. donovani, L. infantum and L. major) has also been recently demonstrated [38]. In any case, the refractoriness of this particular Sergentomyia species does not necessarily extend to the whole of the genus. In this line of reasoning, the competence and permissiveness of the different species from Phlebotomus spp. to different Old World Leishmania has also been observed [39]. As L. major DNA had previously been detected in one S. minuta captured in the same region [32], together with the detection in this study of both human and Leishmania sp. DNA in this species, it would be important to determine if S. minuta fulfils the criteria that support its incrimination as vector for this parasite, and that include (i) the isolation of metacyclic promastigotes from the digestive tubes of field-collected specimens; and (ii) the experimental demonstration of its capacity to transmit Old World Leishmania species with medical and veterinarian importance as a result of blood-feeding on mammals.

Conclusion

The apparent anthropophilic behavior of S. minuta together with the detection of Leishmania sp. DNA highlight the need to determine the role played by this sand fly species in the transmission of pathogenic Leishmania to humans. In addition, our data confirms that P. perniciosus is an opportunistic feeder and suggest that is responsible for the maintenance of L. infantum in sourthern Portugal. Altogether, the obtained results reinforce the need for on-going surveillance with systematic epidemiologic surveys on Leishmania vectors so as to investigate the transmission, distribution and spread of infections by Leishmania species.