INVESTIGATION OF KINETOPLASTID CHITINASE FOR THE DIAGNOSIS AND PHYLOGENY OF LEISHMANIA
Leishmaniasis is an infectious disease that affects humans, domestic and wild animals, caused by 20 of the 53 known Leishmania species. Leishmania genus, belonging to the Trypanosomatidae Family and Kinetoplastida Order, are grouped in five subgroups according to its evolutive and biogeographical histories, which ends up in incongruencies and paraphyly. Leishmania GH18 chitinase, encoded by a single copy species-specific gene, conserved in basal taxa of trypanosomatids and absent in Trypanosoma genus, was evaluated as a phylogenetic marker and target for diagnosis. This study was divided into two parts. For the first part, primers were designed to detect Leishmania in biological samples of hosts, to obtain gene sequences non available in public data banks. GH18 chitinase gene and its genomic context was phylogenetic evaluated. A PCR protocol to differentiate the Leishmania subgenus was developed by using in silico tools. A PCR method to detect a partial fragment of 953 bp of the GH18 chitinase presented high sensitivity and specificity in parasite isolate DNA. The preservation of the chitinase locus demonstrated a possible marine origin of the GH18 chitinase gene and the phylogenetically reconstruction corroborates de Supercontinent Origin of Leishmania. For the second part of this study, DNA was extracted from the liver of anurans collected at the Parque Natural Municipal Nascentes de Paranapiacaba (Paranapiacaba - SP). Detection of Trypanosomatidae from anurans samples was made through PCR that amplified the chitinase encoding gene and the markers gGAPDH and 18SrRNA. The positive results were sequenced and demonstrated that the anurans were infected by the same parasite that showed higher homology to Leishmania lainsoni. The results for the chitinase encoding gene had more sensitivity than the other markers and corroborated the marine origin hypothesis presented in the first part of this work.