Parasitol Res (2012) 111:125–133 DOI 10.1007/s00436-011-2808-3
ORIGINAL PAPER
Zoophilic feeding behaviour of phlebotomine sand flies in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan Saruda Tiwananthagorn & Abdul Manan Bhutto & Javed Hussain Baloch & Farooq Rahman Soomro & Yuta Kawamura & Ryo Nakao & Keisuke Aoshima & Nariaki Nonaka & Yuzaburo Oku & Ken Katakura Received: 7 July 2011 / Accepted: 23 December 2011 / Published online: 14 January 2012 # Springer-Verlag 2012
Abstract Leishmania (Leishmania) major has been identified as the major causative agent of cutaneous leishmaniasis in Sindh Province of southern Pakistan. To make a rational approach for understanding the pathogen transmission cycles, the sand fly species and their natural blood meals in the endemic areas were examined. Total DNA was individually extracted from sand flies collected in four villages in Sindh Province. PCR–RFLP (restriction fragment length polymorphism) and sequence analysis of the 18S ribosomal RNA gene revealed that female sand flies identified were Sergentomyia clydei/Sergentomyia ghesquierei/Sergentomyia magna (68.6%), Sergentomyia dubia (17.1%), S. Tiwananthagorn : Y. Kawamura : R. Nakao : K. Aoshima : N. Nonaka : Y. Oku : K. Katakura (*) Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-ku, Sapporo 060-0818, Japan e-mail:
[email protected] A. M. Bhutto Department of Dermatology, Shaheed Muhtarma Benazir Bhutto Medical University, Larkana, Pakistan J. H. Baloch : F. R. Soomro Leprosy Centre, Chandka Medical College/Hospital, Larkana, Pakistan N. Nonaka Laboratory of Veterinary Parasitology, School of Veterinary Science, University of Miyazaki, Miyazaki 889-2192, Japan Y. Oku Laboratory of Veterinary Parasitology, School of Veterinary Science, Tottori University, Tottori 680-8553, Japan
Phlebotomus papatasi (7.4%), Phlebotomus alexandri-like sand flies (3.4%) and Sergentomyia dentata (3.4%). PCR amplification of leishmanial kinetoplast DNA did not result in positive signals, suggesting that all 175 tested female sand flies were not infected with leishmanial parasites or contained undetectable levels of leishmanial DNA. Amplification and sequencing of the vertebrate cytochrome b gene in 28 blood-fed sand flies revealed that P. papatasi fed on cattle and wild rat whereas P. alexandri-like specimens fed on human, cattle, goat and dog. Although Sergentomyia sand flies are generally known to feed on cold-blooded animals, S. clydei, S. dubia and S. ghesquierei preferred humans, cattle, goat, sheep, buffalo, dog, donkey, wild rat and Indian gerbil. The epidemiological significance of the zoophilic feeding on various host species by Phlebotomus and Sergentomyia sand flies in Pakistan is further required to study for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors.
Introduction Leishmaniasis is one of the neglected tropical diseases. Changing land use, increasing human population and urbanisation are important factors that influence the distribution of leishmaniasis and the sand fly–man relationship (Lewis 1974; Desjeux 2004). These factors have for instance already contributed to the spread of leishmaniasis to South and Southeast Asia (Katakura 2009). Recently, the southern province of Sindh, Pakistan, has been considered as a new endemic area of cutaneous leishmaniasis in which approximately 70% (1,170/1,640) of patients were the residents of Sindh Province without travelling to other provinces (Bhutto et al. 2008). The majority of patients were infected
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with Leishmania (Leishmania) major and only a small proportion of cases were due to L. (L.) tropica infection (Marco et al. 2006; Myint et al. 2008; Bhutto et al. 2009). The spread of leishmaniasis depends on the distribution of vectors and reservoir animals. In Pakistan, at least 29 species of the genus Phlebotomus and 20 species of the genus Sergentomyia have been morphologically identified (Lewis 1982; Alexander 2000; Shakila et al. 2006). However, the natural infection of sand flies as well as of reservoir animals with leishmanial parasites remains to be demonstrated in Sindh Province. Sand fly species are usually identified based on their morphologic characteristics, such as spermatheca, cibarium and pharynx in females and terminal genitalia in males. This method, however, requires refined storage conditions for samples, a highly skilled technique and taxonomic expertise. Recently, various molecular techniques have been used to study the systematics and evolution of sand flies. In particular, PCR amplification of the 18S ribosomal RNA (rRNA) gene followed by restriction enzyme digestion of the PCR product has developed to a powerful tool in sand fly taxonomy (Aransay et al. 1999, 2000; Kato et al. 2005, 2007). Identification of blood meals ingested by haematophagous arthropods is important for determining their host preferences and their probable vectorial capacity for disease agents (Boakye et al. 1999). Because most Leishmania species are zoonotic pathogens, characterising the feeding habits of endemic phlebotomine sand flies is crucial for incriminating putative vectors, elucidating natural transmission cycles and developing efficacious control strategies (Killick-Kendrick 1999; Alexander and Maroli 2003). In the present study, the identification of phlebotomine sand fly species and their blood meals in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan, was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and sequencing of the insect 18S rRNA gene, and by PCR and sequencing of the vertebrate cytochrome b gene. It was revealed that Phlebotomus and Sergentomyia sand flies in this province appear to take multiple blood meals and prefer to feed on humans, domesticated animals and wild rodents.
Materials and methods Study areas The field work of the study was done from late May to early June 2007, during the high season of the sand fly activity, in Sindh Province of Pakistan. Geographically, Sindh is the third largest province of Pakistan, stretching about 579 km from north to south and 442 km (extreme) or 281 km (average) from east to west, with an area of 140,915 km2
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(Fig. 1). Temperatures frequently rise above 46°C between May and August, and the minimum average temperature during December and January is 2°C. Rainfall is highest mainly during July and August, and the average annual rainfall is approximately 178 mm. Intensive sampling of sand flies was conducted in four villages (Sono Khan, Miran Machi, Agani and Thareri Hajira) in Larkana district of Sindh Province. These four villages were selected because of previous reports of a large number of cutaneous leishmaniasis cases (Bhutto et al. 2008, 2009). The inhabitants of these villages are farmers and live together with domestic animals including cattle, goat, sheep, buffaloes, donkeys and/or chickens. Stray or household dogs are very common. Sand fly collection Sand flies were collected using Shannon light traps, insect aspirators and CDC light traps (Model 512; John W. Hock Company, USA). The traps were allocated in settling areas of people and animals, including bedroom, living room, animal shed, animal food storing shed and/or rice polishing shed. Shannon traps were placed for about 1 h between 8:00 and 9:00 PM and CDC traps were placed for about 11 h between 7:00 PM and 6:00 AM of the next day. The collected sand flies were sexed based on the structure of the abdominal terminalia. The flies were individually preserved in 70% ethanol. The presence of a blood meal was determined by visually observing whether the abdomen was enlarged and dark red to slightly brown in colour. DNA extraction The ethanol-fixed whole bodies of individual sand flies were homogenised and lysed in DNA extraction buffer (150 mM NaCl, 10 mM Tris–HCl, 10 mM EDTA and 0.1% sodium dodecyl sulphate, pH 8.0) with 100 μg/ml proteinase K (Qiagen, CA, USA) at 37°C for 12 h. Total DNA of these samples was extracted with phenol/chloroform, precipitated with ethanol, and re-suspended in 10 μl of distilled water. The DNA samples of sand flies were further subjected to whole genome amplification using the REPLI-g Mini Kit (Qiagen). Total DNA was also isolated from the blood samples of vertebrates, such as cattle, goat, sheep, buffaloes, donkeys, dogs, chickens, wild rats, gerbils and geckoes collected in the study areas, using the QIAmp DNA Mini Kit (Qiagen). Identification of sand fly species To perform sand fly identification, PCR–RFLP of the amplified 18S rRNA gene and sequencing of the PCR products were conducted. For amplification of the phlebotomine 18S rRNA gene, a forward primer Lu.18S rRNA-1S (5′-TGC
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Fig. 1 Sand fly collection sites in Sindh Province, Pakistan. The four study villages (Sono Khan, Miran Machi, Agani and Thareri Hajira) and Larkana, the district Capital City, are indicated in the map
CAG TAG TTA TAT GCT TG-3′) and a reverse primer Lu.18S rRNA-1R (5′-TTA CGC GCC TGC TGC CTT CC-3′) were used (Kato et al. 2005). The PCR mixture contained 0.4 μM of each primer, approximately 10 ng/μl template DNA, 0.025 U/μl TaKaRa Ex Taq™ DNA polymerase (Takara Bio Inc., Tokyo, Japan) and 1× Ampdirect® Plus buffer (Shimadzu Corp. Kyoto, Japan) in a volume of 20 μl. After an initial denaturation at 95°C for 5 min, the reaction was carried out with 40 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min with the GeneAmp PCR System 9700 thermal cycler (Applied Biosystems Japan, Tokyo, Japan). PCR products were run on 2% agarose gels, stained with ethidium bromide and photographed under UV light. A total of 21 male sand flies were selected at random and subjected to PCR/sequencing analysis of the 18S rRNA gene. The amplified products were purified using ExoSAPIT (GE Healthcare Japan, Tokyo, Japan) and submitted directly to sequencing using the CEQ™ DTCS-Quick Start Kit and the CEQ 8000 DNA sequencer (Beckman Coulter, CA, USA). The sequences were edited and aligned using BioEdit v7.0.9 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and
compared with available databases of the 18S rRNA gene of Phlebotomus and Sergentomyia species from EMBL/DDBJ/ GenBank. Both the PCR products obtained in this study and 79 corresponding regions of the 18S rRNA gene of Phlebotomus and Sergentomyia species from GenBank databases were examined for restriction enzyme cutting sites discriminating the sand fly species using NEB Cutter version 2.0 (Vincze et al. 2003). Two restriction enzymes, BamHI (Takara Bio) and PaeR7I (New England BioLabs, MA, USA), were selected. The digestion was performed in a volume of 20 μl reaction mixture containing 5 μl of PCR product and 0.025 U/μl of BamHI at 30°C for 2 h or 0.025 U/μl of PaeR7I at 37°C for 2 h. The digested samples were analysed by electrophoresis using 2% or 3% agarose gels and visualised under UV light. Identification of blood source animals of sand flies A total of 28 engorged female sand flies, which appeared to contain blood meals, were subjected to the isolation of total DNA and PCR amplification with primers specific for the vertebrate cytochrome b (cyt b) gene. The forward primer
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used was 5′-CCA TCC AAC ATC TCA GCA TGA TGA AA-3′ (cyt b 1) and the reverse primer was 5′-GCC CCT CAG AAT GAT ATT TGT CCT CA-3′ (cyt b 2) (Steuber et al. 2005). The reaction mixture was prepared as described above. The reaction was started with an initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 30 s. The final extension was carried out at 72°C for 5 min. The resulting PCR products of about 359 bp in size were extracted from agarose gels using a QIAquick Gel Extraction Kit (Qiagen) and cloned into a plasmid using a TOPO TA Cloning® Kit (Invitrogen, CA, USA) and JM109 cells. One to 10 TA clones for each sand fly sample (a total of 112 clones) were sequenced for the insert using a CEQ™ DTCS-Quick Start Kit and the CEQ 8000 DNA sequencer (Beckman Coulter). PCR products of the cyt b gene from mammals and geckoes in the study areas were sequenced directly as described above. Sequences obtained from sand flies were edited using BioEdit v7.0.9 and compared with the sequences obtained from the vertebrates and available sequences in EMBL/DDBJ/GenBank. Detection of Leishmania DNA from sand flies PCR amplification detecting the Leishmania-specific kinetoplast minicircle DNA was carried out in a reaction mixture of 20 μl, containing 1.5 mM MgCl2, 0.2 mM each of four dNTPs, 1 μM each forward (5′-TCG CAG AAC GCC CCT ACC-3′) and reverse (5′-AGG GGT TGG TGT AAA ATA GGC-3′) primers, 10 ng/μl DNA template and 0.025 U/μl Taq DNA polymerase (Qiagen). The primers used were designed for the detection of the genus Leishmania in general and the differentiation between L. (L.) major and L. (L.) tropica in particular (Bhutto et al. 2009; Mahboudi et al. 2001). PCR was started with an initial denaturation at 93°C for 4 min, followed by 35 cycles of denaturation at 93°C for 30 s, annealing at 64°C for 1 min and extension at 72°C for 1.5 min. The final extension was carried out at 72°C for 5 min. The PCR products were run on 2% agarose gels, stained with ethidium bromide and photographed under UV light.
Results Location of sand fly collection In the present limited study, a total of 507 sand flies (168 males and 339 females) were collected in four villages using the insect aspirators, Shannon and CDC light traps located in different places in each village. A large number of sand flies were collected at Sono Khan Village (42.4%, 215/507), the most rural village located at a desert area. Approximately
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half of the sand flies (41.6%, 363/507) were caught using Shannon traps and insect aspirators although this required considerable expertise and patience. The remaining 144 sand flies were captured using CDC traps. In relation to human habitat, 24.2% (82/339) of female sand flies were collected from living rooms and bedrooms. In animal sheds, 29.2% (99/ 339) of females were collected. Identification of sand flies species by PCR/sequencing of the 18S rRNA gene PCR amplification of the 18S rRNA gene was achieved from 21 male sand flies. Sequence analysis of the 21 PCR products revealed seven different nucleotide sequences (sequences A– G). These sequences were analysed using BLAST® against various Sergentomyia spp. and Phlebotomus spp. in GenBank (blastn; http://blast.ncbi.nlm.nih.gov/). Sequence A (eight specimens) was 100% (422/422 bp) identical to Sergentomyia clydei (accession no. AJ391742). Similarly, sequence B (five specimens) was completely identical to Sergentomyia ghesquierei (AJ391743, 421/421), sequence C (three specimens) to Sergentomyia dubia (AJ391738, 421/421), sequence D (one specimen) to Sergentomyia dentata (AJ244425, 421/ 421) and sequence E (one specimen) to Sergentomyia magna (AJ391741, 381/381). Therefore, sequences A to E were identified as Sergentomyia species each. Sequence F (two specimens) was 100% identical to both Phlebotomus papatasi (AJ244414, 431/431) and Phlebotomus perniciosus (AJ391728). Because the distribution of P. perniciosus is restricted to the Mediterranean area (Lewis 1982), sequence F was referred to as P. papatasi. Sequence G (one specimen) was 99.5% (429/431) identical to Phlebotomus alexandri (AJ244401), 99.3% (428/431) to P. papatasi (AJ244414) and Phlebotomus sergenti (AJ391727), and 99.1% (427/431) to P. alexandri (AJ244400) and P. sergenti (AJ244403). Because sequence variations of the 18S rRNA gene among each Phlebotomus spp. have been found in the databases, a phylogenetic tree of the gene was constructed with the neighbourjoining (NJ) method. The resultant phylogenetic tree indicated that sequence G was most related to P. alexandri (data not shown). Thus, sequence G was referred to as P. alexandri-like sand fly in this study. Furthermore, species identification of 28 blood-fed female sand flies was achieved by sequencing of amplified PCR products of the 18S rRNA gene, revealing four S. clydei, one S. dentata, 10 S. dubia, 11 S. ghesquierei, one P. papatasi and one P. alexandri-like species. Identification of Sergentomyia and Phlebotomus species using PCR–RFLP/sequence analysis of the 18S rRNA gene BamHI and PaeR7I were used for PCR–RFLP analysis of seven 18S rRNA gene sequences obtained from Pakistani
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sand flies in this study and for RFLP analysis 79 available sequences of Phlebotomus and Sergentomyia species in GenBank. BamHI digestion of PCR products of 444– 454 bp in size gave two fragments of 171 bp and 273/ 274 bp for S. clydei, S. ghesquierei and S. magna (Figs. 2 and 3a). A PaeR7I digestion of the PCR products not cut with BamHI allowed two fragments of 218 bp and 236 bp for P. papatasi (Figs. 2 and 3b) as well as for P. sergenti, which was not encountered among our sand fly collections. Failure to fragment with these two enzymes indicated that the PCR products were derived from other sand fly species, including S. dentata, S. dubia and P. alexandri-like sand flies (Figs. 2 and 3). Practically, the amplified 18S rRNA fragments from 268 sand flies (121 males and 147 females) were subjected to RFLP analysis. PCR products from 189 samples (70.5%) produced two expected DNA fragments after digestion with BamHI, suggesting one of the sand fly species of S. clydei, S. ghesquierei or S. magna. Although potential restriction enzymes to differentiate among these three Sergentomyia species were suggested from the sequence data, further RFLP analysis and species differentiation were not performed in the present study. Twenty eight samples (10.4%) showed two expected fragments after digestion with PaeR7I, and these samples were subsequently sequenced for confirmation of P. papatasi. The remaining 51 samples (19.0%), which were digested with neither BamHI nor PaeR7I, were sequenced for confirmation of one of the sand fly species of P. alexandri, S. dubia or S. dentata.
PCR products of the 18S rRNA gene of sand fly species (444-454 bp)
Digestion with BamHI YES
NO
Two fragments (171 + 273/274 bp) Sergentomyia clydei
Digestion with PaeR7I
S. ghesqueirei S. magna
YES
Distribution and habitat of Sergentomyia and Phlebotomus sand flies Taking together the results of the sequences of the 21 males, the sequences of the 28 blood-fed females and the PCR– RFLP/sequence analysis of the 121 males and 147 females, a total of 142 males and 175 female sand flies were identified (Table 1). It was revealed that 82.4% (117/142) of the male and 89.1/% (156/175) of the female sand flies identified belonged to the genus Sergentomyia, including 69.0% (98/142) of male and 68.6% (120/175) of female S. clydei/S. ghesquierei/S. magna, 11.3% (16/142) of male and 17.1% (30/175) of female S. dubia, and 2.1% (3/142) of male and 3.4% (6/175) of female S. dentata. The rest of the samples contained 12.7% (18/142) male and 7.4% (13/175) female P. papatasi, and 4.9% (7/142) male and 3.4% (6/175) female P. alexandri-like sand flies. Sergentomyia sand flies were distributed in all four villages, but P. papatasi sand flies were not captured in Agani village and P. alexandri-like specimens were not detected in Agani and Thari Hajira. In relation to the human habitats, 84.6% (11/13) and 66.7% (4/6) of the female P. papatasi and P. alexandri-like specimens, respectively, were collected in living rooms and bedrooms (Table 2). Regarding Sergentomyia sand flies, 16.7% (20/120), 20.0% (6/30) and 33.3% (1/6) of the female S. clydei/S. ghesquierei/S. magna, S. dubia and S. dentata, respectively, were captured in living rooms and bedrooms. With respect to the animal habitats, 39.2% (47/120), 30.0% (9/30) and 50.0% (3/6) of the female S. clydei/S. ghesquierei/S. magna, S. dubia and S. dentata, respectively, were captured in animal sheds. Only one female P. alexandri-like and two female P. papatasi specimens were collected in animal sheds. In rice polishing sheds, 40.0% (48/120), 50.0% (15/30) and 16.7% (1/6) of the female S. clydei/S. ghesquierei/S. magna, S. dubia and S. dentata, respectively, were trapped (Table 2). Identification of host animals from blood meals of sand flies using PCR and sequencing of the vertebrate cyt b gene
NO
Two fragments
S. dubia
(218 + 236 bp) Phlebotomus papatasi P. sergenti
S. dentata P. alexandri
Sequencing
Fig. 2 Flow chart of restriction endonuclease, BamHI and PaeR7I, digestions of the 18S rRNA gene for identification of sand fly species in this study
The DNA recovered from the 28 blood-fed female sand flies which had been identified to species by 18S rRNA gene sequencing was subjected to PCR amplification of the vertebrate cyt b gene, followed by TA cloning and sequencing of the PCR products. Sequences from these sand fly specimens showed the highest homologies (93–100%) to the cyt b gene of human (AY509658), cattle (Bos taurus, EF593094), buffalo (Bubalus bubalis, EF59093), goat (Capra hircus, EU350133), sheep (Ovis aries, AJ971339), dog (Canis familiaris, DQ309764), donkey (Equus asinus, X973337), rat (Rattus sp. EF186492), Indian gerbil (Tatera indica, AJ430563) and gecko (Hemidactylus flaviviridis, EU268388). Some TA clones showed 82% homology to
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Fig. 3 Restriction patterns of PCR products of the 18S rRNA gene from Phlebotomus and Sergentomyia species. DNA fragments after digestion with BamHI (a) or PaeR7I (b). M 0.1–12 kb ladder, 1 S.
clydei, 2 S. ghesquierei, 3 S. dubia, 4 S. magna, 5 S. dentate, 6 P. papatasi, 7 P. alexandri-like. Arrows indicate two fragments after digestion
sequences of Percival’s legless lizard (Acontias percivali, DQ249095) cyt b gene, and others showed 89% homology to the roan antelope (Hippotragus equinus, AF022060) cyt b gene sequence at the maximum level, respectively. Since identification of these sequences would have required further studies, they were referred to as unidentified reptile and unidentified mammal, respectively, in the present study. Blood source animals determined for these sand flies are summarised in Table 3. From a single P. papatasi specimen captured at a living room in Miran Machi, cattle and rat cyt b gene sequences were cloned, indicating that this P. papatasi sand fly fed on two different vertebrate hosts. Similarly, analysis of ten clones of a P. alexandri-like sand fly, captured in an animal shed in Miran Machi, revealed that this fly contained blood meals of five different sources, including human (one clone), cattle (one clone), goat (two clones), dog (three clones) and an unidentified mammal (three clones). One S. dentata, collected in a bedroom in Thareri Hajira, showed an unidentified reptile cyt b gene sequence in all seven clones. Sequence analysis of a total of 42 clones of 10 S. dubia specimens indicated that this species fed on geckoes as well as mammals, including human, cattle, buffalo, goat, sheep, rat and an unidentified mammal host species. Analysis of 13 clones of four S. clydei individuals indicated that this species took blood from five mammalian host
species, namely cattle, human, rat, Indian gerbil and an unidentified mammal. Lastly, S. ghesquierei was found to have fed on at least seven mammalian host species—human, cattle, buffalo, donkey, dog, rat and an unidentified mammal—based on the analysis of a total of 31 clones of 11 sand flies (Table 3). PCR detection of Leishmania DNA from blood-fed sand flies No leishmanial PCR products were obtained from a total of 175 female sand flies using kDNA primers for the identification of L. (L.) major and L. (L.) tropica which are distributed in the study area.
Discussion Blood hosts of sand flies vary according to locality, season and availability of hosts (Lewis 1974). Of the female sand flies collected in this study, 7.4% were P. papatasi, a wellknown vector transmitting L. (L.) major. Although the number of P. papatasi sand flies collected was very small, 11 of 13 females were captured from living rooms and bedrooms, and two were from animal sheds. By PCR and sequencing of the vertebrate cyt b gene, one female was demonstrated to
Table 1 Sand fly species identified in four villages in Sindh Province, Pakistan Village
No. of male and female sand flies identified by PCR–RFLP/sequencing of the 18S rRNA gene P. alexandri-like P. papatasi
Sono Khan Miran Machi Agani Thareri Hajira Total (%)
S. clydei, S. magna or S. S. dubia ghesquierei
S. dentata
Total
♂
♀
♂
♀
♂
♀
♂
♀
♂
♀
♂
♀
2 5 0 0 7 (4.9)
2 4 0 0 6 (3.4)
7 10 0 1 18 (12.7)
7 3 0 3 13 (7.4)
55 24 15 4 98 (69.0)
46 26 42 6 120 (68.6)
4 8 0 4 16 (11.3)
10 11 1 8 30 (17.1)
0 1 0 2 3 (2.1)
0 1 2 3 6 (3.4)
68 48 15 11 142 (100)
65 45 45 20 175 (100)
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Table 2 Location of female sand flies collected in four villages in Sindh Province, Pakistan Location
No. of female sand flies identified P. alexandri-like
P. papatasi
S. clydei, S. magna or S. ghesquierei
S. dubia
S. dentata
Total
Living room
4
9
20
6
1
40
Bedroom Rice polishing shed
0 1
2 0
0 48
0 15
1 1
3 65
Animal shed
1
2
47
9
3
62
Animal food storing shed
0
0
5
0
0
5
Total
6
13
120
30
6
175
contain blood meals from two hosts, cattle and rat. In Iran, double blood meals of human–dog and human–cattle were observed in 0.9% and 3.1% P. papatasi, respectively, by conventional enzyme-linked immunosorbent (ELISA) assay (Yaghoobi-Ershadi et al. 1995). Recently, it was shown in Israel by PCR and reverse line blotting analysis that most P. papatasi sand flies captured inside houses fed on humans, but feeding on two hosts, human–bird and human–mouse (Abbasi et al. 2009). In the same study, P. papatasi captured close to a small farm fed on cattle, avian, cow–avian and cow–human blood sources. Thus, P. papatasi sand flies may be adapted to take blood meals from multiple available vertebrate hosts. This species may take partial blood meals before being disturbed and flying to another host to complete the meal. Recently, PCR amplification and sequencing of the vertebrate cytochrome b gene has been used to identify host blood meals from blood-sucking insects, such as mosquitoes and tsetse flies. This has allowed us to unambiguously identify both avian- and mammalian-derived blood meal sources to the species level. A total of 27 species of birds and seven species of mammals as hosts were identified from
Culex pipiens in the northern United States (Molaei et al. 2006). Similarly, in Japan, Culex pipiens pallens fed on a total of eight avian and seven mammalian species whereas Culex pipiens form molestus fed on three avian and three mammal species (Sawabe et al. 2010). Tsetse flies (Glossina pallidipes) in Zambia fed on eight different vertebrate species, namely human, African elephant, African buffalo, waterbuck, roan antelope, greater kudu, warthog and goat, and sequence analysis of the PCR products revealed that some single tsetse flies were found to harbour blood meals from two to four different host species (Konnai et al. 2008). Thus, blood-sucking insects probably take as many animals as available than we expected. In the present study, a P. alexandri-like sand fly was identified. P. alexandri bites humans in Central Asia and probably in Turkey, where it causes zoonotic dermal leishmaniasis (Lewis 1974). This species is also a proven vector of L. (L.) donovani in China (Guan et al. 1986) and a possible vector of L. (L.) infantum in Iran (Azizi et al. 2006). In Iran, it was shown that 32.5% (39/120) of P. alexandri were positive for human blood by ELISA analysis (Azizi et al. 2006). In this study, a P. alexandri-like female
Table 3 Blood meal source animals of sand flies collected in Sindh Province, Pakistan
Blood meal source animals determined by PCR/sequencing of the vertebrate cyt b gene (sample size)
Species
Phlebotomus (no. collected) P. alexandri-like (1) P. papatasi (1) Sergentomyia (no. collected) S. clydei (4) S. dentata (1) S. dubia (10)
S. ghesquierei (11)
Human + cattle + goat + dog (1) Cattle + rat (1) Human (1), human + cattle + Indian gerbil (1), cattle + rat (1), cattle + rat + unidentified mammal (1) Unidentified reptile (1) Human (1), human + cattle + goat + rat (1), human + cattle + rat (1), human + cattle + rat + unidentified mammal (1), human + cattle + gecko (2), human + cattle + gecko + unidentified mammal (1), cattle + sheep + gecko (1), buffalo (1), rat (1), Human + cattle + rat (2), human + buffalo (1), human + dog + rat (1), cattle (2), cattle + buffalo (1), cattle + donkey (1), cattle + rat (1), rat (1), rat + unidentified mammal (1)
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was shown to contain blood of human, cattle, goat and dog. Thus, it might be possible that P. alexandri-like sand flies participate in the transmission of L. (L.) donovani or L. (L.) infantum to humans and dogs when these parasites are introduced into Sindh Province. On the other hand, the predilection of sand flies for cattle and other domesticated animals may reduce man–sand fly contact, resulting in reduction of patients with leishmaniasis. The role of Sergentomyia sand flies as the vectors of leishmanial parasites is still uncertain. The main reason might be the nature of the blood-feeding habits of Sergentomyia sand flies, which are reported to feed mainly on coldblooded animals, especially small reptiles such as geckoes (Lewis 1982; Lewis and Dyce 1988). Although some Sergentomyia species are known to bite humans and other mammals, few molecular determinations of blood meals have been reported. In this study, it was demonstrated by PCR/sequencing of the vertebrate cyt b gene that at least S. clydei, S. dubia and S. ghesquierei sand flies feed on various mammals, including humans, domesticated animals and wild rodents. Because host DNA in sand fly blood meals can be detected by PCR up to 96 h post-feeding (Abbasi et al. 2009), PCR detection of various hosts’ DNAs in a single sand fly indicates that the sand fly fed on various available host animals in a short time. The presence of Leishmania donovani DNA in Sergentomyia species caught in Indian kala-azar patients’ dwellings has been reported (Mukherjee et al. 1977). Recently, L. (L.) major ITS rDNA was amplified from Sergentomyia sintoni in Iran although sequence data were not presented (Parvizi and Amirkhani 2008). Because Sergentomyia sand flies are widely distributed in the Old World and more abundant than Phlebotomus species in Africa and rainy temperate areas of South and Southeast Asia (Lewis 1974). Recent studies on distribution and population density of Sergentomyia species are reported in some regions, including Saudi Arabia (El-Badry et al. 2008), Nepal (Pandey et al. 2008) and Morocco (Boussaa et al. 2009). Therefore, continuous studies are required to elucidate the role of Sergentomyia species in the transmission of Leishmania parasites. Examination of 175 female sand flies, including 13 P. papatasi and six P. alexandri-like sand flies did not produce any evidence for leishmanial DNA in the present study. A reason for this might be that the sampling size was very small, but on the other hand only few leishmaniasis patients were found in the study area at the time of sand-fly collection. A large-scale and seasonal examination of sand flies will be required for determination of sand fly vectors for leishmanial transmission. The PCR–RFLP method will be a powerful tool for this purpose. With regard to other sand fly transmitted agents, Toscana virus has been detected in Sergentomyia minuta collected in France (Charrel et al. 2006), and Sandfly Sicilian and
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Sandfly Naples viruses have occurred in Pakistan (Amaro et al. 2007). In India, Sergentomyia species were positive for Chandipura virus after an outbreak of human encephalitis (Geevarghese et al. 2005). The demonstrated feeding on various vertebrate host species by Sergentomyia species within a short time therefore raises the possibility of at least mechanical transmission of other sand-fly-borne pathogens. In conclusion, both Phlebotomus and Sergentomyia sand flies were molecularly identified in the endemic areas of cutaneous leishmaniasis in the lowland of Sindh Province, Pakistan. Zoophilic feeding behaviour and variable host preferences were demonstrated in species of both genera sand flies. Sergentomyia flies were more abundant and preferred to feed on humans, domesticated animals and wild rodents. Feeding on various vertebrate host species by these phlebotomine sand flies indicates host preference according to the availability of host animal species. Further studies on the epidemiological significance of the natural blood meal sources are required for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors. Acknowledgements We acknowledge all staff of the Department of Dermatology and Leprosy Centre, Chandka Medical College/Hospital and Regional Health Office of Larkana district, Pakistan, for providing facilities, coordinating field activities and supporting specimen collection. We also thank Dr. Kato of Hokkaido University for valuable suggestion and Ms. Kotera for technical assistance. This work was supported in part by grants from the 21st Century COE program ‘Program of Excellence for Zoonosis Control’, global COE program ‘Establishment of International Collaboration Centres for Zoonosis Control’ and grant no. 183801780 from MEXT, Japan.
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