Natural infection of the sand fly Phlebotomus kazeruni by Trypanosoma species in Pakistan
© Kato et al; licensee BioMed Central Ltd. 2010
Received: 8 January 2010
Accepted: 25 February 2010
Published: 25 February 2010
The natural infection of phlebotomine sand flies by Leishmania parasites was surveyed in a desert area of Pakistan where cutaneous leishmaniasis is endemic. Out of 220 female sand flies dissected, one sand fly, Phlebotomus kazeruni, was positive for flagellates in the hindgut. Analyses of cytochrome b (cyt b), glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA) gene sequences identified the parasite as a Trypanosoma species of probably a reptile or amphibian. This is the first report of phlebotomine sand flies naturally infected with a Trypanosoma species in Pakistan. The possible infection of sand flies with Trypanosoma species should be taken into consideration in epidemiological studies of vector species in areas where leishmaniasis is endemic.
Phlebotomine sand flies are blood-sucking insects belonging to the family Psychodidae in the order Diptera . The identification of sand fly species is epidemiologically very important because less than 10 percent of over 800 species described are responsible for the transmission of human pathogens such as flagellate protozoa of the genus Leishmania[1–3]. Some sand fly species are reported to transmit non-pathogenic flagellates of Endotrypanum species, originally identified as intraerythrocytic parasites of sloths in the New World . In addition, some Trypanosoma species of mammals, lizards, snakes and toads are transmitted by phlebotomine sand flies [5–11]. Since the flagellated forms of these parasites in the insect gut are morphologically similar to those of Leishmania, careful differentiation is needed for the epidemiological study of the vectors responsible for circulating Leishmania species.
In this study, the natural infection of sand flies by Leishmania was surveyed in a desert area of Pakistan where cutaneous leishmaniasis is endemic. Sand flies were captured at Sono Khan (26° 52'N, 68° 03'E), Sindh Province on the 28-29th of June 2004. Shannon traps with a single compartment (1.0 × 1.0 × 1.3 meters in width, length and height, respectively) were used for the collection. The captured sand flies were dissected and the species identified based on the morphology of their spermathecae. These flies were also examined for Leishmania in the gut microscopically at × 400 magnification. The flagellates detected were inoculated into Difco blood agar (USMARU) biphasic medium containing 20% defibrinated rabbit blood, and later co-cultured with Spodoptera frugiperda Sf 9 insect cells in Grace's medium supplemented with 10% fetal calf serum. The rest of the flagellates in the gut of the sand fly were fixed in absolute ethanol for molecular biological analyses. Genomic DNA was extracted from the ethanol-fixed specimen, and the cytochrome b (cyt b) gene was amplified from the flagellate with primers prepared for the leishmanial cyt b gene (L.cyt-S: GGTGTAGGTTTTAGTYTAGG and L.cyt-R: CTACAATAAACAAATCATAATATRCAATT) . The glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA) genes were also amplified from the parasite using gGAPDH (G3: TTYGCCGYATYGGYCGCATGG and G5: ACMAGRTCCACCACRCGGTG) and SSU rRNA-specific primers (TRY927F: GAAACAAGAAACACGGGAG and TRY927R: CTACTGGGCAGCTTGGA) designed for trypanosomatids [13, 14]. The PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI), and the sequences of the inserts of the plasmids were determined by the dideoxy chain termination method using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The gGAPDH and SSU rRNA gene sequences were aligned with CLUSTAL W software  and examined using the program MEGA (Molecular Evolutionary Genetics Analysis) version 4.0 . Phylogenetic analyses were performed by the neighbor-joining (NJ) and maximum parsimony (MP) methods with the distance algorisms available in the MEGA package .
In the present study, a Trypanosoma species was isolated from P. kazeruni, which is a xerophilic species distributing broadly throughout North Africa and West Asia with little-known behavior [18–20], and the parasite was genetically characterized. To date, trypanosomes in sand flies have been reported on the African and American continents [5–11], but this is the first record of phlebotomine sand flies naturally infected by a Trypanosoma species in Asia. Pakistan is a tropical and subtropical country located in the northwest of South Asia and has areas where leishmaniasis is highly endemic [21, 22]. Although information on the endemicity and spread of cutaneous leishmaniasis is accumulating [23–26], the sand fly species responsible for the transmission is poorly understood. To date, no natural infections of sand flies by flagellates have been reported in Pakistan, and further efforts are ongoing on this matter. Thus, careful identification of the parasites is required for the epidemiological study of sand fly species responsible for the transmission of Leishmania protozoa.
We are indebted to Dr. Juma K. Kakarsulemankhel (Sandflies, Leishmaniasis & Mosquitoes Laboratory, Department of Zoology, Faculty of Scinece, University of Balochistan, Quetta, Balochistan, Pakistan), Hamed A. Chandio and Nuzhat Seema Bhatti (The Leprosy Unit, Chandka Medical College Hospital and the Executive District Office-Health, Larkana, Sindh, Pakistan) for their support during the field phase of the present study. This study was financially supported by the Ministry of Education, Culture and Sports, Science and Technology (MEXT) of Japan (Grant Nos. 14256004, 18256004 and 18780230)
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