Sergentomyia (Neophlebotomus) gemmea, a potential vector of Leishmania siamensis in southern Thailand

Background Leishmaniasis, caused by Leishmania siamensis, is an emerging disease in Thailand. Although reported cases have been increasing, epidemiological information of the disease including host and vector aspects is not clearly known. This study was a preliminary survey of the potential vector of L. siamensis in an affected area of leishmaniasis, Trang Province, southern Thailand. Methods The collection of sandflies was performed around the area where a case of leishmaniasis was reported using CDC light traps. Species of sandfly were identified based on morphological characteristics according to Lewis’s key. PCR amplification and sequencing of the heat shock protein 70 gene (hsp70) was used to identify L. siamensis DNA in sandflies. Results A total of 146 male and female sandflies were collected in the affected areas. Of 71 female sandflies, four species were identified, i.e., Sergentomyia (Neophlebotomus) gemmea, S. (Neophlebotomus) iyengari, S. (Parrotomyia) barraudi and Phlebotomus (Anaphlebotomus) stantoni. Among these species, S. (Neophlebotomus) gemmea was the most predominant species in all areas. DNA of L. siamensis was identified in S. (Neophlebotomus) gemmea. Nucleotide sequences of PCR products using DNA extracted from S. (Neophlebotomus) gemmea showed 99.8% identity to L. siamensis. Conclusion S. (Neophlebotomus) gemmea might be a potential vector of L. siamensis in an affected area, Trang Province, southern Thailand. However further studies are needed to prove whether these sandflies can be natural vectors of leishmaniasis.


Background
Autochthonous visceral leishmaniasis (VL) caused by Leishmania siamensis, firstly described from southern Thailand in 2008, is now considered an emerging disease in Thailand [1]. Previously, only two autochthonous leishmaniasis cases were reported in Thailand, i.e., in 1999 and 2007; however, the causative species were not identified [2,3]. After 2008, more cases of autochthonous leishmaniasis were reported [4][5][6]. Characterization of the causative species, L. siamensis was performed and showed that L. siamensis is a novel species [4,7]. HIV/ AIDS patients are a high risk group to acquire the infection which can cause not only VL but also disseminated dermal leishmaniasis [4][5][6]. Until now, the disease has been reported from six provinces in the South, two provinces in the North as well as one province in the East of Thailand [4,6]. Zoonotic transmission of L. siamensis could possibly occur since animal cutaneous leishmaniasis caused by L. siamensis was recently reported from Switzerland, Germany and the USA [8][9][10]. Identification of the vectors is crucial for the prevention and control of the disease. Surveys of sandfly species have been conducted in the Central, West, North, Northeast and South of Thailand where Sergentomyia is the most predominant genus reported in all study areas [1,[11][12][13][14], of which S. (Neophlebotomus) gemmea is the most prevalent in the South [14]. However, the particular species of sandfly that could serve as the vector for leishmaniasis has not yet been demonstrated.

Methods
In 2011, a sentinel surveillance of sandfly species distribution and their potential vectors was conducted in an affected area, Hat Samran District, Trang Province, southern Thailand where a case of VL-HIV co-infection was reported. The altitude of the affected area is 18 m above the sea level where palm trees were grown in the area. In August 2011, the average temperature was 27.9°C with a humidity of 85.5%. The average wind speed was 1.3 knots and the rainfall was 10.0 mm. Outdoors, a 200-m perimeter surrounding the house included animal enclosures, i.e., cows, goats, pigs, dogs, and Persian cats was surveyed. Neighborhood houses were about 10 m apart. A palm tree plantation was located about 50 m away from the house.
To capture sandflies, CDC light traps were placed indoors and outdoors of the patient's house in August, 2011 from 18.00-06.00 h. Outdoors, areas of the 200-m perimeter surrounded house included areas behind the house, spaces under the kitchen, animal enclosures, stacks of bricks and wood in the back yard, banana plants, palm trees, a store house for wood and rice, a thatched roof hut and a nearby abandoned house.
Species of female sandflies were identified by welltrained entomologists based on morphological characteristics [15,16] at the Vector-borne Disease Control Center 11.3, Suratthani Province, Thailand. These female sandflies were pooled in the same microcentrifuge tube containing 70% alcohol if they shared the same characteristics, i.e., species and collected place. The samples were then sent to the Department of Parasitology, Phramongkutklao College of Medicine for molecular studies. DNA from each pool was extracted using the Genomic DNA Mini Kit (Tissue) (Geneaid, Taiwan) and evaluated for natural infections with L. siamensis using PCR amplification of the heat shock protein 70 gene (hsp70) and the internal transcribed spacer 1 region (ITS1) of the small subunit ribosomal RNA (SSU-rRNA) gene. For the hsp70, PCR was performed using primers HSP70sen and HSP70ant with the conditions previously described [17]. For the ITS1, the method of El Tai et al. (2001) was performed [18]. DNA sequencing was then conducted by 1st Base Pte. Ltd., Singapore. All PCR products positive for hsp70 gene were ligated with pGEM-T Easy vector (Promega, WI, USA) and cloned into E.coli JM109. Three clones of each positive pool were selected and sequenced.
The 18s rRNA region of each pool sandfly sample was amplified using primers Lu.18S rRNA-1S and Lu.18S rRNA-1R with conditions previously described by Kato et al. [19]. The PCR products from each pool were then directly sequenced.
The minimum infection rate (MIR) was calculated using the standard formula: [number of positive pools / total specimens tested] x 1000), with the data representing a single sandfly species, collected over a time period and geographic area.
All sequencing results from the cloning of positive hsp70 amplification showed identical amplicon sizes at

Discussion
In the present study, we reported S.  [21]. Recently L. major DNA was also detected in S. (Spelaeomyia) darlingi collected from zoonotic cutaneous leishmaniasis affected areas in Mali [22]. Moreover, L. major was also isolated from S. garnhami and successfully cultured in NNN medium [23].
Since Sergentomyia sandflies are zoophilic, they might have a role in the transmission of zoonotic leishmaniasis. Leishmaniasis caused by L. siamensis might be also zoonotic since phylogenetic analysis showed that L. siamensis was closely related to L. enrietti, a Leishmania species infecting Brazilian guinea pigs [6]. In addition, L. siamensis DNA was identified in animals such as rats and dogs (personal communication). The distribution of sandfly species in the present study was similar to our previous report showing that S. (Neophlebotomus) gemmea was predominant in southern Thailand, the affected area of leishmaniasis caused by L. siamensis [14]. Unfortunately, we could not detect L. siamensis by PCR in any samples of the previous survey [14]. Of the 26 species of sandflies described in different regions of Thailand [11], the distribution of species in the South was different from other parts of Thailand [11,14]. All captured S. (Neophlebotomus) gemmea were found outdoors where the cow corral was the most common habitat. Habitat preference could be related to their blood meal feeding. In this study, we identified S. (Neophlebotomus) gemmea based on morphological characteristics [15,16]. The 18s rRNA of L. siamensispositive S. (Neophlebotomus) gemmea was amplified and sequenced. The nucleotide sequence was then submitted to GenBank. Unfortunately no other 18s rRNA nucleotide sequence of S. (Neophlebotomus) gemmea is available to be compared in GenBank. Since the chromatogram of nucleotide sequences showed a completed consensus, we postulated that each pool contained only one species.

Conclusion
Our study showed that S. (Neophlebotomus) gemmea might be a vector of L. siamensis, the causative agent of leishmaniasis in Thailand. Further studies with larger samples are urgently needed to determine the potential sandfly vectors, their infection rate and behaviors such as host-preference behavior, blood meal feeding and breeding habitat in all affected areas of leishmaniasis. In addition, the natural reservoir host should be identified. The awareness of the disease by public health personnel and community health workers as well as having an effective vector control program are important keys to prevent the disease from spreading.