Introduction

In Spain, leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted by phlebotomine sand flies belonging to the subgenus Larroussius (Diptera: Psychodidae, Phlebotominae); the dog being the main domestic reservoir (WHO 2010).

Leishmaniasis is endemic in rural, peri-urban, and suburban areas of Madrid region (central Spain). Only two sand fly species Phlebotomus perniciosus and Phlebotomus ariasi out of the seven described in this region are proven vectors of the parasite L. infantum, being its main vector P. perniciosus (Conesa Gallego et al. 1999; Gálvez et al. 2010).

Since 2010, a marked and unusual increase of human cases of both visceral and cutaneous leishmaniasis (VL and CL, respectively) is being observed in four municipalities of the southwestern Madrid region, where around 374 cases have been diagnosed until September 2012—of which 36.6 % are cases of visceral leishmaniasis—with an incidence rate of 21.54 cases/100,000 inhabitants. Only in the town of Fuenlabrada, 316 cases have been notified with an incidence rate of 52.99 cases/100,000 inhabitants (Suárez et al. 2012). The creation of a park adjacent to the urban area of the focus was expected to increase the hare population in this open space, and as a result of this, it has increased the population of the sand fly vectors. In fact, we have recently shown by direct xenodiagnosis that hares (Lepus granatensis) infected by L. infantum can transmit the parasites to a competent phlebotomine sand fly (P. perniciosus) suggesting the existence of a sylvatic transmission cycle linked to the urban periphery (Molina et al. 2012). In order to take effective measures to control the situation as soon as possible, the Autonomous Community of Madrid launched in 2011 a survey on human cases, reservoirs, and vectors of the disease in collaboration with the Instituto de Salud Carlos III and other institutions. In the framework of entomological studies that are taking place in the area, a study including the use of CDC light traps was done in 2011 at the end of the Leishmania transmission period with the aim to provide vector epidemiological data.

The usefulness of molecular techniques in epidemiological surveys of leishmaniasis foci has been proved in the determination of blood feeding preferences of sand flies as well as in the detection of Leishmania spp. and vectorial capacity (Rossi et al. 2008; Tiwananthagorn et al. 2012; Azizi et al. 2012; Quaresma et al. 2012; Branco et al. 2013). In this sense, the blood feeding behavior of some arthropods plays a critical role in the transmission and maintenance of vector-borne pathogens in natural systems (Kent 2009). Furthermore, blood meal identification in field-caught sand flies can confirm a strong association between sand flies and hosts in urban areas of Leishmania transmission and also help to improve understanding the role of a particular host or reservoir in urban foci for more effective control strategies (Haouas et al. 2007).

Therefore, the objective of this work was to identify the species of phlebotomine sand flies involved in the leishmaniasis focus. Molecular tools will be used for the first time in the study of the host blood feeding preferences of sand flies together with the detection of L. infantum in a focus of leishmaniasis in southwestern Madrid, Spain.

Materials and methods

Sand fly collection

Sand flies were daily collected using two CDC light traps on the 29th of September and during the first half of October 2011. Traps were situated in two peridomestic stations, named JIC (two sampling points, JIC1 and JIC2) and BOS (one sampling point). The two stations were located very close to the peri-urban green park of around 450 ha and separated each other by 1 km. Sand flies were kept in 70 % ethanol and were sent to the laboratory where they were stored at 4 °C until they were processed.

Sand fly identification and DNA extraction

The 174 sand flies collected were numerically identified according to the code of the sampling stations (JIC1, JIC2, and BOS). Sand flies were washed individually in sterile distilled water in ELISA plates. Females were visually classified as unfed, fed, and gravid. The fed group includes specimens in which a blood meal in different degrees of digestion was observed. Gravid group comprised of females with mature eggs in their abdomen that in some cases can contain traces of undigested blood. Afterwards, the head, wings, and legs of each female sand fly were cut. Moreover, the genitalia were cleared in Marc–André medium during 24 h and mounted the head together on glass slides in Hoyer medium for identification under microscope.

DNA was purified from the thorax and abdomen using the DNeasy® Blood and Tissue Extraction Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Quantification and purity of the DNA samples was determined by spectrophotometry with a NanoDrop ND-1000 spectrophotometer (Nucliber, Madrid, Spain) and the samples stored at −20 °C until use.

Analysis of blood feeding preferences of sand flies

The study was based on the amplification of a fragment of 359 bp of vertebrate cytochrome b (cyt b) gene with universal primers cyto 1: 5'-CCA TCA AAC ATC TCA GCA TGA AA-3' and cyto 2: 5'-CCC CTC AGA ATG ATA TTT GTC CTC-3' (Abassi et al. 2009) following the protocol previously described by Steuber et al. (2005), with a few modifications. PCRs were carried out with 40 ng of DNA in a final volume of 25 μl. To avoid PCR inhibitors such as heme, addition of 1 μl of BSA DNAse Free (20 mg/ml, Roche) was added to the mixture.

The PCR products obtained were removed from the gel under UV exposure and purified by a QIAquick Gel Extraction Kit (QIAGEN). Afterwards, the samples were sequenced with ABI PRISM 3730XL DNA Analyzer (Applied Biosystems®, EEUU).

Electropherograms were manually inspected and corrected using BioEdit v7.0.0.1.program. Nucleotide sequences obtained were analysed with the DNASTAR (Lasergen v7.1®, Madison, WI, USA). Homologies with the available sequences data in GenBank was carried out with the software (http://www.ncbi.nlm.nih.gov/BLAST).

Detection of Leishmania DNA in sand flies

Initially, genomic DNA obtained from individual female sand flies was used to amplify a 120 bp fragment of the conserved region of kDNA as previously described with slight modifications (Kobets et al. 2010). Individual PCRs were performed in a final volume of 20 μl. The amount of DNA in each reaction was 30 ng.

Positive samples by kDNA-PCR were further analysed, in an attempt to identify Leishmania species, following a specific PCR based on the amplification of a fragment of 702 bp of cpb gene from L. infantum as described before (Hide and Bañuls 2006; Oshaghi et al. 2009). This protocol was carried out with a few modifications to improve the sensitivity. The master mix was prepared in a final volume of 50 μl containing: 5 μl of 10× PCR buffer for Ampli-Taq Gold polymerase (Applied Biosystem, UK), 1 μl Ampli-Taq Gold polymerase (5 U/μl) (Applied Biosystem), 0.2 mM of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Sweden), 3 mM of MgCl2 (Applied Biosystem), 2 μl of BSA DNAse Free (20 mg/ml, Roche), and 40 pmol of each primer. Working conditions were 1 cycle at 95 °C for 9 min, then 40 cycles (94 °C for 30 sec, 62 °C for 1 min, 72 °C for 1 min), followed by 1 cycle at 72 °C for 10 min. Sixty to eighty nanogram of DNA was used in each reaction. All PCR products were separated on ethidium bromide 1.5 % agarose gel (Conda, Spain), visualized under UV light, and photographed.

Each sample was tested twice with each set of primers. Negative controls without DNA were included in each PCR. DNA obtained from sand flies reared in the laboratory was used as negative controls. As a positive control, we used DNA obtained from sand flies experimentally infected with L. infantum. To avoid PCR contamination, sample preparation, reactions set-up, and PCR amplifications were carried out in separate rooms, with different lab coats and gloves.

Additionally, amplification of ITS2 region was carried out with DNA obtained from six positive specimens, five from JIC1 station, and one from JIC2. PCR reactions were performed with primers L5.8SR (5'-AAgTgCgATAAgTggTA-3') and LVTSV (5'-ACACTCAggTCTgTAAAC-3') described by El Tai et al. (2000) according to protocols previously defined by Kuhls et al. (2005). Bands of 740 bp were purified and sequenced as explained above.

Results

A total of 174 sand flies (136 females and 38 males) were collected in nine captures completed from the 29th of September to the 14th of October with two CDC traps placed in two stations (JIC and BOS) closest to the urban park, where a significant increase in the number of new cases of human leishmaniasis was detected since 2010.

Of the 174 sand flies captured, 171 were identified as P. perniciosus (36 males and 135 females) and three as Sergentomyia minuta (two males and one female). Among the P. perniciosus females, 121 were unfed, in eight was possible to visualize a blood meal, and six were gravid (Table 1).

Table 1 Number of Phlebotomus perniciosus collected and positive specimens for Leishmania infantum detected by kDNA-PCR and cpb-PCR at each sampling station
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The kDNA-PCR was carried out in genomic DNA obtained from 135 female P. perniciosus and one female S. minuta. The PCR products were visualized and found to be 120 bp (Fig. 1, Panel a, b). kDNA from Leishmania spp. was detected in 79 P. perniciosus samples (58.5 %). Of these, 73 out of 121 unfed (60.33 %), three of eight blood fed (37.5 %), and three of six gravid (50 %) were positive (Table 1). Moreover, bands of 702 bp specific to L. infantum were obtained when the PCR based in the amplification of cpb gene was used with all previously positive samples by kDNA-PCR (n = 79) (Fig. 2, Panel a, b).

Fig. 1
figure 1

Representative kinetoplast DNA (kDNA) PCR products of individual sand flies. (a) From the left to the right lanes; M 100 bp molecular weight marker (Biotools), Lanes 1–3 negative controls (without DNA); Specific band of 120 bp was observed in lanes 4, 5, 6, and 12 (sand flies from JIC1 station) and lane 9 (sand flies from JIC2 station). Absence of specific bands was observed in lanes 8, 10, and lane 13 (female of Sergentomyia minuta), lanes 7 and 11 negative controls (DNA from a male sand fly), lane 14 positive control (30 ng of DNA from a female sand fly experimental infected). (b) From the left to the right lanes; M 100 bp molecular weight marker (Biotools), lane 1 and 2 negative controls (without DNA), lanes 6 and 10 negative control (DNA from males sand flies; lane 13 positive control (30 ng of DNA from a female sand fly experimental infected), M 100 bp molecular weight marker (Biotools); Specific bands of 120 bp was observed in lanes 3, 4, 5, and 12. Absence of specific band was observed in lanes 7, 8, 9, and 11. All DNA samples correspond to sand flies from JIC1 station

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Fig. 2
figure 2

Results of specific Leishmania infantum cpb-PCR products in Phlebotomus perniciosus sand flies. (a) From the left to the right lane M1 100 bp molecular weight marker (Biotools), lane 1 negative control (without DNA); Specific bands of 702 bp was observed in lane 3 to 7 and lanes 10 and 12. Negative samples lane 2, 8, 9, and 11, lane 13 positive control (100 pg of L. infantum gDNA). M2 molecular weight marker IX (Roche). (b) M1 1 kb molecular weight marker (Biotools), lane 1 negative control (without DNA); Specific PCR products of 702 bp are shown in lanes 2, 4, 5, 7, 8, and lanes 10 and 11, Negative samples lanes 3, 6, 9, and 12, lane 13 positive control (60 ng of DNA from a female sand fly experimental infected), M2 100 bp molecular weight marker (Biotools). All DNA samples correspond to sand flies from JIC1 station with the exception of lane 2 in panel B (JIC2)

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Concerning to the feeding preferences, identification of blood meal sources in eight blood-engorged and six gravid females was carried out. In seven blood-fed sand flies all collected in JIC1 station, blood meals identified were mainly from hares (L. granatensis) in six specimens and cat (Felis catus) in the other one. Importantly, in two out of six sand flies with a blood meal from hare, DNA from L. infantum was detected. No homology was found in GenBank with the sequence obtained from the remainder specimen, although a positive result was obtained by kDNA-PCR. In the same way, in the gravid females, a blood source from human was identified in a total of three specimens collected in JIC1, JIC2, and BOS stations, respectively. With the other three samples, the sequencing was unsuccessful. Interestingly, in two out three gravid sand flies, two with a blood source from human and one in which it was impossible to identify the blood source and also a DNA from L. infantum was detected (Table 2).

Table 2 Blood meal identification and Leishmania infantum detection in fed and gravid females from the different stations
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In addition, sequences of the ITS2 products obtained with DNA from positive sand flies from JIC1 (n = 5) and JIC2 (n = 2) were identical to those previously obtained in L. infantum isolates from hares (Molina et al. 2012).

Discussion

In this work, we applied molecular techniques for the detection of L. infantum in P. perniciosus captured in two different stations in the focus of human leishmaniasis of Fuenlabrada, Madrid, Spain, as well as in the study of the host preferences in both fed and gravid female sand flies. PCR based in amplification of kDNA have been applied to the molecular detection of Leishmania spp. in sand flies experimentally infected and have proved to be highly sensitive (Michalsky et al. 2002). In fact, this PCR approach have been applied successfully in the detection of Leishmania spp. in entomological surveys (Aransay et al. 2000; Rossi et al. 2008; Sant’Anna et al. 2008; Torina et al. 2008; Maia et al. 2009). On the other hand, the use of cpb-PCR has revealed the presence of L. donovani/L. infantum in P. perfiliewi in Iran (Oshagi et al. 2009).

In the Iberian Peninsula, the overall infection rate of sand flies obtained from insect dissection and isolation of the parasite ranges between 0.27 and 4.6 % (Rioux et al. 1986; Morillas et al. 1991; Alves-Pires and Ribeiro 1991; Morillas et al. 1996).

On the other hand, there are few studies involved in the molecular detection of Leishmania in entomological surveys. In Spain, pool screen PCR-ELISA technique was used to estimate the prevalence of L. infantum infection in sand flies from Granada region by comparison with dissection. The global prevalence determined by pool screening was situated in 0.45 % (Martín-Sánchez et al. 2006). Recently, in sand flies captured in different counties in the Northeast of Spain (Catalonia), an average rate of 38.7 % of positive sand flies were detected by SSU rDNA in areas with high prevalence of canine leishmaniasis (Alcover et al. 2012). Nevertheless, studies in Algarve region of Portugal and in a new potential zoonotic leishmaniasis focus in Central Region of this country, Portugal reflects an overall infection rate of P. perniciosus of 0.52 and 0.48 %, respectively (Maia et al. 2009; Branco et al. 2013).

In the present work, it should be noted in the first place that the percentage of positive sand flies by kDNA-PCR was noticeably high (58.5 %). Among the positive females, the highest number was found in unfed sand flies (60.33 %), followed by gravid (50 %), and fed sand flies (37.5 %) (Table 1). This could explain the high intensity of transmission of leishmaniasis in the green park, where a high prevalence of over 30 % has been described in the hares studied (Cruz 2012), and there appears to be a close adaptation of the parasite to the sand fly vectors in the area. It is also remarkable that the entomological survey was carried out in October that is considered to be the end of the period in which the parasite transmission occurs and when highest rates of sand fly infections are found. In this sense, it has been shown that the intensity of infection by L. infantum in P. ariasi in natural conditions increases progressively during the second and third ovarian cycles (Killick-Kendrick and Rioux 2002). Also, an increasing degree of blood digestion would result in the presence of more parasites and thus a higher possibility of detecting Leishmania DNA (Alcover et al. 2012). In this way, the use of gravid females in PCR analysis ensures the presence of flies with at least one blood meal almost completely digested and has therefore been proposed for the evaluation of the prevalence of Leishmania in endemic areas where a high proportion of positive has been found among them (Torina et al. 2008). In this study, only six gravid females were collected, half of them are positive to L. infantum. However, among unfed P. perniciosus sand flies, the percentage of positive females found was very high in the PCR analysis (60.33 %), suggesting that the parasites are highly competent to the vector with a great capacity to multiply in the midgut during the blood meal digestion. Among the eight fed females, the percentage of infected sand flies was situated in 37.5 %. This differs slightly from the data of Alcover et al. (2012) who detected a high proportion of P. perniciosus infected (85.7 %) among sand flies with a blood meal in a focus of canine leishmaniasis in Torroja del Priorat (Catalonia), and no sand flies without a blood meal where found infected.

On the other hand, phlebotomine vectors have been observed to feed on a wide range of domestic animals with varying anthropophilic degrees (Guy et al. 1984; Colmenares et al. 1995; Bongiorno et al. 2003; Branco et al. 2013). Even though it was only possible to identify the blood source in 10 out 14 females (the sequencing was unsuccessful, and/or no homology was found in GenBank with four sequences), the highest percentage of host blood feeding preferences in ten sand flies analysed was found in hares (60 %), followed by humans (30 %), and cats (10 %) (Table 2). Interestingly, no dog blood was identified. These results support the existence of a sylvatic transmission cycle of leishmaniasis linked to the urban periphery independent of dogs as we previously proposed (Molina et al. 2012). It is noteworthy that two species of hares, Lepus timidus and L. granantesis, were found in blood meals, based in the analysis of the sequences obtained from cytb. In fact, the mountain hare (L. timidus) type mtDNA was observed in L. granatensis and Lepus europaeus from the Iberian Peninsula, distant from the distributional range of L. timidus. This is because both species of hares, L. granatensis and L. europaeus, contain introgressed L. timidus mtDNA (Alves et al. 2003; Melo-Ferreira et al. 2012).

In conclusion, the usefulness of molecular techniques in the identification of blood feeding preferences in P. perniciosus and the detection of L. infantum was proven for the first time on a preliminary entomological survey in a new focus of leishmaniasis in southwestern Madrid, Spain. In this study, the existence of L. infantum infection in a high proportion of P. perniciosus (58.5 %) in the focus was demonstrated

The data presented here provide significant epidemiological information relative to the spread of human leishmaniasis in the focus. However, additional studies focusing on the transmission dynamics of P. perniciosus need to be done to elucidate the epidemiology of leishmaniasis in this focus. In this sense, we have conducted an entomological survey from May to November of 2012 in the area which data once analyzed could yield more information related to the focus.