Molecular tools for the identification of Phlebotomine sand flies and detection of Leishmania spp. parasites in Misiones province, Argentina.

MOYA, S.L.; GIULIANI, M.G.; MANTECA ACOSTA, M.; SALOMÓN, O.D.; LIOTTA, D.J.

Reims

Congreso; International Symposium On Phlebotomine Sandflies IX (ISOPS IX); 2016

University of Reims Champagne Ardenne

In Argentina, Tegumentary Leishmaniasis (TL) is endemic in 9 provinces, while the number of Visceral Leishmaniasis (VL) cases have increased sharply in the last years, accompanied by an expansion to new areas, being Misiones the province with the highest incidence. Because the disease spread depends largely on the geographical distribution of the vectors, simultaneously characterization of vectors and parasites through molecular tools will contribute to the eco-epidemiological knowledge of this disease through a simplified experimental approach. The procedures for phlebotomine identification by the observation of its morphology alters the nature of the sample, and inhibits its posterior analysis through molecular biology, such as the ones requires for detection and identification of parasites. In this context, the aim of this work was the evaluation of both protocols: PCR-Sequencing of cacophony-IVS6, as a complementary method in the taxonomic identification of phlebotomine sand flies, and PCR-RFLP of ITS-1 for detection of presence and identification of Leishmania spp. Female sand flies were capture with light traps and classified at species level through dissection and observation of the spermathecae according to taxonomic keys. Sandfly identification assay was carried out through DNA extraction of the remaining sandfly body, amplification and sequencing of the 220pb DNA fragment obtained by cacophony-IVS6 protocol (Lins, et. al., 2002). Sequences were evaluated in purity and identity with Codon Code Aligner? software and Basic Local Alignment Search Tool-MEGA (BLASTm), respectively. Datasets were analyzed by Neighbor-Joining (NJ) (software MEGA) using Kimura 2 parameters model (Bootstrap of 1000 pseudoreplicates). Regarding to the detection and identification of Leishmania parasites, it was done according to the ITS-1 PCR-RFLP protocol (Schönian et. al., 2003) in 39 of the blood fed captured females.Amplification and sequencing of cac-IVS6 was achieved in 107 female sand flies. The multiple alignment showed a conserved exon and the polymorphism present in an intragenic region of the gen. NJ consensus tree shows that sequences belonging to sand flies of different genus (Lutzomyia, Nyssomyia, Migonemyia and Evandromyia) grouped in different clusters, showing that the intron polymorphism is enough for its differentiation. Further, the protocol of cac-IVS6 allowed the identification of 4 sand flies whose identification could not be achieved by morphological means due to the condition of the specimens. Despite the promising results, sequences belonging to the same genus, like the ones from Ny. whitmani and Ny. neivai (both proven to be vectors of L. braziliensis) clustered together making unable the differentiation between them. Regarding to the detection of Leishmania parasites, in 15% of the samples (6/39) was possible the amplification of ITS-1, but the RFLP assay for species identification resulteddifferent than expected. Because of this, ITS-1 products were purified, sequenced and analyzedfor identity with BLASTm, resulting in 6 sequences of L. infantum (detected in Ny. whitmani and Migonemyia migonei sand flies). In conclusion, even though a sequencing protocol was required for Leishmania species identification, both protocols were applied in the same biological sample with positives results,allowing a simultaneously identification of sandfly and the parasite present. It is important to remark that the detection of Leishmania DNA itself is not enough to determine a sandfly specie as a vector of a Leishmania species, although these findings may serve as a first step to define the direction of future eco-epidemiological studies in Misiones province.