Research Article: First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru

Date Published: January 6, 2016

Publisher: Public Library of Science

Author(s): Hirotomo Kato, Abraham G. Cáceres, Yoshihisa Hashiguchi, Paulo Filemon Pimenta. http://doi.org/10.1371/journal.pntd.0004336

Abstract: The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area.

Partial Text: New World Leishmanias are transmitted by phlebotomine sand flies of the genus Lutzomyia, and around 480 species have been recorded; in Peru, 149 species have been registered [1], and some of which have been implicated as potential vectors of human Leishmanias [2–8]. In Peru, cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) are endemic, and three Leishmania species have been identified as predominant causative agents: Leishmania (Viannia) braziliensis mainly in the tropical rainforest, L. (V.) peruviana mainly in the Andean highland areas, and L. (V.) guyanensis in the northern and central rainforest regions [9–11]. In addition, distribution of Leishmania (Leishmania) mexicana, L. (L.) amazonensis, L. (V.) lainsoni, L, (V.) shawi, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana were reported [9–12]. Concerning sand flies, prevalent species have been extensively researched, especially in Andean areas [2–7]; however, the vector species responsible for transmission of Leishmania have yet to be fully elucidated in most areas because of low infection rates in sand fly populations.

In this study, only two species of the genus Lutzomyia, Lu. tejadai and Lu. fischeri were collected. Namely, a total of 2,997 female sand flies were captured and identified at the species level, of which 2,931 and 66 flies were identified as Lu. tejadai and Lu fischeri, respectively. Of these, Leishmania minicircle DNA was detected from one Lu. tejadai from Chinobamba (13Hua3-1E). The sand fly positive for Leishmania DNA did not contain blood in the gut. The cyt b gene sequence from parasites within the Lu. tejadai 13Hua3-1E was successfully obtained, and the nucleotide sequence was analyzed. The sequence of parasites from 13Hua3-1E had a greater degree of homology with those of L. (V.) braziliensis and L. (V.) peruviana (99.7–100.0%) than with other Leishmania species (88.8–98.8%). The result was supported by a phylogenetic analysis showing that that the specimen from Lu. tejadai was located in the clade of L. (V.) braziliensis and L. (V.) peruviana (Fig 1). To further identify the species infecting the sand fly Lu. tejadai, leishmanial MPI gene sequences were analyzed by PCR-RFLP, since a single nucleotide polymorphism of the gene was reported to be a marker for differentiating between L. (V.) braziliensis and L. (V.) peruviana [6,11,19]. As shown in Fig 2, a restriction enzyme, AvaII, cut the MPI fragment of L. (V.) peruviana completely, but not that of L. (V.) braziliensis. On the other hand, the MPI fragment of Leishmania-positive Lu. tejadai 13Hua3-1E, as well as those of reference strains of the hybrid (LH1099, LC1407, LC1408, LC1418, and LC1419), showed hybrid patterns after digestion by AvaII (Fig 2). The sequences of the MPI fragments were analyzed by direct sequencing, and a single nucleotide polymorphism was confirmed showing “C” in L. (V.) braziliensis, but “G” in L. (V.) peruviana at the corresponding position (Fig 3A and 3B). On the other hand, MPI genes from all reference strains of hybrid (LH1099, LC1407, LC1408, LC1418, and LC1419) and Leishmania-positive Lu. tejadai 13Hua3-1E had both “C” and “G” peaks at the position (Fig 3C and 3D). These results indicated that the parasite species within Lu. tejadai 13Hua3-1E is a hybrid of L. (V.) braziliensis/L. (V.) peruviana.

Despite their importance in the control of leishmaniasis, little is known about vectors involved in disease transmission since the infection ratio among sand flies by Leishmania is generally low (< 1%). CL cases caused by a hybrid of L. (V.) braziliensis/L. (V.) peruviana have been reported in Peru since 1995; however, the vector species remains unidentified. The present study utilized a molecular mass-screening method for analysis of 2,997 female sand flies from the Department of Huanuco, Peru, in which CL caused by L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana is endemic [10,12,20]. As a result, a hybrid of L. (V.) braziliensis/L. (V.) peruviana was detected in one Lu. tejadai which did not contain host blood in the gut, suggesting that Lu. tejadai supports the development of the hybrid Leishmania and is responsible for its transmission in this area. Source: http://doi.org/10.1371/journal.pntd.0004336

 

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