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

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.

Since 1995, CL cases caused by a hybrid of L. (V.) braziliensis/L. (V.) peruviana have been reported in the eastern inter-Andean valley of Huanuco province in the Department of Huanuco [12], and the hybrid was suggested to increase disease severity when compared to L. (V.) braziliensis and L. (V.) peruviana using an animal model [13]. Extensive sand fly surveillance revealed prevalent sand fly species and Lutzomyia (Lu.) tejadai was identified as a dominant species in endemic areas of Huanuco; however, vector species of hybrid Leishmania parasite have not been determined to date. In our previous study, a method of mass-screening sand fly vectors for Leishmania infections was established and it has become a powerful tool for sand fly research [6,14,15]. In the present study, using the molecular mass-screening method, sand flies from the Department of Huanuco, where CL caused by hybrid L. (V.) braziliensis/L. (V.) peruviana is endemic, were examined for natural Leishmania infections.

Sand fly collection
Sand flies were collected with CDC light traps set inside houses and Shannon traps outside and around houses at 19 localities in Department of Huanuco (Table 1). CDC light traps were operated throughout the night from 18:00-06:00 and Shannon traps from 18:00-20:00 each night. The sand flies were morphologically identified based on measurements of wing veins, the ratio of length of palpus to length of antenna and the color of the thorax [16], and then fixed in 70% ethanol.

DNA extraction
Ethanol-fixed sand flies were placed individually in each well of 96-well plates and lysed in 50 μl of DNA extraction buffer [150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA and 0.1% sodium dodecyl sulfate (SDS)] in the presence of proteinase K (200 μg/ml). The samples were incubated at 37°C overnight and heated for 5 min at 95°C. Each 0.5-μl portion was directly used as a template for mass-screening PCR.
Leishmania species were identified by Leishmania cytochrome b (cyt b) gene sequence analysis. Leishmania cyt b gene fragments were amplified by PCR with a pair of specific primers (L.cyt-S; 5'-GGTGTAGGTTTTAGTYTAGG-3' and L.cyt-R; 5'-CTACAATAAACAAATCAT AATATRCAATT-3') using Ampdirect Plus reagent (Shimadzu Biotech, Tsukuba, Japan), and the products were directly cloned into the plasmid using a pGEM-T Easy Vector System (Promega, Madison, WI). The sequence of the insert was determined by the dideoxy chain termination method using a BigDye Terminator v3.   primer sequences were 5'-GCTCTTCCTGTCGGACAGCGAGC-3' (MPI-S) and 5'-TCACTC TCGAAGGGAGTTCG-3' (MPI-R). PCR was carried out in a volume of 15 μl using the primers (0.4 μM each), Ampdirect Plus reagent (Shimadzu Biotech), and Taq polymerase (NovaTaq Hot Start DNA Polymerase; Novagen, Darmstadt, Germany). After an initial denaturation at 95°C for 10 min, PCR amplification was performed with 35 cycles of denaturation (95°C, 1 min), annealing (55°C, 1 min) and polymerization (72°C, 1 min), followed by a final extension at 72°C for 10 min. Each PCR product was digested with the restriction enzyme, AvaII (Takara Bio) and analyzed by 3% agarose gel electrophoresis. Separately, PCR products were purified and the nucleotide sequences were directly determined.

Phylogenetic analysis
The cyt b gene sequences were aligned with CLUSTAL W software [17] and examined using the MEGA program (Molecular Evolutionary Genetics Analysis) version 5.2 using the Kimura two-parameter [18].

Results
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 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). 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.
In the present collection, only two Lutzomyia species were captured, although three species of sand flies, Lu. tejadai, Lu fischeri and Lu. sallesi have been recorded in the Department of Huanuco [21]. The first species, Lu. tejadai was collected inside, outside and around houses, suggesting its wide range of distributions and activities in the areas [22]. To date, natural infections of Lu. sallesi and Lu. fischeri by L. (L.) infantum and Leishmania (Viannia) species, respectively, have been reported in Brazil [23,24]. However, infection of these sand flies by Leishmania has not been reported in Peru. Furthermore, there is no report on the natural infection of Lu. tejadai by Leishmania species. The present study suggested Lu. tejadai is the vector of a hybrid of L. (V.) braziliensis/L. (V.) peruviana for the first time. A hybrid of Leishmania caused by genetic exchange is experimentally generated in the digestive tract by co-infecting vector sand fly species with two different strains of the same Leishmania species [25][26][27]. In addition, a direct evidence of sexual recombination in natural population was recently provided by whole genome sequencing of Leishmania isolated from sand flies [28]. Midgut molecules of sand fly species are considered to be a major determinant of parasite-vector specificity [29]. The study area, the Department of Huanuco, is located at the mid-eastern region of the Peruvian Andes and is surrounded by seven leishmaniasis-positive departments (San Martin, Ancash, Lima, La Libertad, Loreto, Ucayali, and Pasco). Because of poverty and infertile farm land in the Andean highlands, the inhabitants started to move from the highlands to the lowlands (tropical region) together with their domestic animals (dogs, cats, guinea pigs, cows, sheep, goat, etc.), and some groups migrated to the Department of San Martin, the highest leishmaniasis-endemic area in the country. Around 1975, the number of migrants in these areas increased markedly, and further, in 1983-1984, a massive movement of military personnel from the highlands to the lowlands and vice versa occurred in and around the Department of Huanuco for the purpose of narcotic and guerrilla control in the areas (Personal