Molecular detection of Trypanosoma sp . and Blastocrithidia sp . ( Trypanosomatidae ) in phlebotomine sand fl ies ( Psychodidae ) in the Federal District of Brazil

Introduction: This study describes the occurrence of trypanosomatids in phlebotomines in Brasília, Brazil. Methods: Two hundred and ten females of 13 sand fl y species were analyzed by polymerase chain reaction (PCR) using different molecular markers (D7 24Sα rRNA, kDNA, and ITS1) and sequencing. Results: PCR revealed trypanosomatid-positive samples from Nyssomyia whitmani and Evandromyia evandroi, which were negative by kDNA and ITS1 Leishmania-specifi c PCRs. DNA sequence analysis of D7 24Sα rRNA amplicons indicated the occurrence of Blastocrithidia sp. and Trypanosoma sp. in Nyssomyia whitmani and Evandromyia evandroi, respectively. Conclusions: Two trypanosomatid species other than Leishmania sp. were found to circulate in sand fl ies in Central Brazil.

In the New World, 58 species of Phlebotominae are proven or suspected vectors for the transmission of Leishmania species to man and other animals (3) .However, the occurrence of trypanosomatids in sand fly species and domestic and wild mammals has yet to be fully investigated in the Cerrado biome, particularly in the Federal District (FD) of Brazil (4) , where autochthonous human cases of visceral and cutaneous leishmaniasis have been recorded (5) (6) .The present study focused on the search for Leishmania and other trypanosomatids in sand fl ies from two localities of FD.Specimens were captured in two areas: 1) a gallery forest located at the Água Limpa Farm owned by the University of Brasília (15°57'41.00''S; 47°56'38.88''W); and 2) a domiciliary unit located in the Boa Vista neighborhood within the administrative region of Fercal (15°37'28.6"S;47°52'23.0"W).At the Água Limpa Farm, the sampling was performed during November, 2013 using light traps: fi ve HP TM and two Shannon.The domiciliary unit was sampled during April, 2014 using two Shannon traps and 10 HP TM traps.The Shannon traps were installed in the forest remnant close to the house and the HP TM traps were installed in animal shelters on the periphery of the house.
The collected female fl ies were dissected to separate the head and the spermathecae; both were cleared and mounted in Canada balsam for species identifi cation (7) .Other body parts (thorax, part of the abdomen, legs, and wings) were placed in an Eppendorf tube (1.5mL) and assigned a code number.A variable number of female specimens (one to ten), belonging to the same species and capture sites, were pooled for total deoxyribonucleic acid (DNA) extraction using the Illustra tissue and cells genomicPrep Mini Spin Kit, according to the manufacturer's instructions (GE Healthcare, New York, USA).Deoxyribonucleic acid integrity of the samples was checked by a PCR designed to amplify the cacophony gene IVS6 region in sand fl ies with specifi c primers (5Llcac 5'-GTG GCC GAA CAT AAT GTT AG-3' and 3Llcac5'-CCA CGA ACA AGT TCA ACA TC-3') (8) , under the following conditions: 95°C for 5 min, 35 cycles of 95°C for 30s, 57°C for 30s, and 72°C for 30s with a fi nal extension at 72°C for 10 min.Tripanosomatid detection was performed by amplifying the polymorphic region of the D7 domain of the 24Sα ribosomal ribonucleic acid (rRNA) gene (primers: D75 5′-GCA GAT CTT GGT TGG CGT AG-3′ and D76 5′-GGT TCT CTG TTG CCC CTT TT-3′) that corresponds to conserved sequences of approximately 225bp found in trypanosome and Leishmania spp.genomes (9) .Polymerase chain reaction was performed in a 25μL volume containing 10ng/μL DNA, 1.0μM MgCl 2 , 0.2μM dNTPs, 0.1μM of each primer, and 1.5U Platinum Taq DNA polymerase (Invitrogen, Life Technologies, Brazil).The amplifi cation conditions were as described by Schijman et al. (10) .The positive controls used in the PCR reactions were obtained from cultures of Trypanosoma cruzi (Berenice strain), Trypanosoma rangeli (SC-58 strain), and Leishmania (Leishmania) infantum (MCER/BR/1979/M6445). Milli-Q water was used as a negative control.All reactions were performed in a thermocycler (MyCycler, Bio-Rad, California, USA).Polymerase chain reaction products were analyzed by electrophoresis on 2% agarose gels.
Internal transcribed spacer 1-PCR was performed using primers LITS1 5′-CTG GAT TTT GCC CAT ATG-3′ and L5.85 5′-TCG TGA TAC CAC TTA CAC TT-3′ as described by Tojal da Silva et al. (11) .The reactions were prepared in a fi nal volume of 25μL containing 10ng/μL DNA, 2.0μM MgCl 2 , 0.2μM dNTPs, 0.1μM each primer, and 1.5U Platinum Taq DNA polymerase, under the following conditions: 95° C for 5 min, 35 cycles at 95°C for 30s, 58°C for 30s, and 72°C for 30s with a fi nal extension step at 72°C for 5 min.To increase the sensitivity of the reaction, PCR products were re-amplifi ed with the same primers and the same conditions.All reactions were performed in a MyCycler thermocycler.Positive and negative controls used in ITS-1 PCR were the same as those used in the kDNA PCR.Amplicons were separated on 1.3% agarose gels.
Amplifi ed fragments were purifi ed using an Illustra GFX PCR DNA & Gel Band Purifi cation Kit (GE Healthcare) and sequenced for identifi cation of trypanosomatids by a commercial provider (Genomic Engenharia Molecular, São Paulo, Brazil).Sequences obtained were edited using Geneious software (Biomatters, New Zealand) and compared with sequences deposited at GenBank using the Basic Local Alignment Search Tool, nucleotides (BLASTn) algorithm at the National Center for Biotechnology Information, USA (http://www.ncbi.nlm.nih.gov/BLAST).
Deoxyribonucleic acid was successfully extracted from 210 females which had been pooled into 24 samples.Fragments that corresponded to the cacophony gene were amplifi ed in all of the samples, demonstrating the integrity of the extracted DNA.Subsequent PCR analyses revealed that three of these samples obtained from the peridomiciliary environment of the domestic unit of Fercal were positive for trypanosomatids (225bp bands).Two were from Ny. whitmani and one was from Ev. evandroi.All samples above tested negative for Leishmania spp.by the kDNA and ITS1 PCRs.DNA sequence analyses of two D7 24Sα rRNA amplicons indicated the presence of Blastocrithidia sp. in Ny. whitmani and Trypanosoma sp. in Ev. evandroi (Table 1).The respective sequences have been deposited in GenBank (accession numbers KR149124 and KR149125).Assuming that only one insect per pool was positive the minimum infection rate was calculated as 1.4% for Ny.whitmani and 12.5% for Ev.evandroi.
To our knowledge, this represents the fi rst registration of the presence of trypanosomatid DNA of the genus Blastocrithidia in Ny. whitmani.Although sand fl ies most commonly serve as hosts for Leishmania species, the occurrence of Trypanosoma, Endotrypanum, Crithidia, Blastocrithidia, Herpetomonas, and Leptomonas have also been reported (1) (2) .For example, Rocha

Ferreira
TS et al. -Trypanosomatids in Brazilian sand fl ies

TABLE 1 -Genera of Trypanosomatids identifi ed in phlebotomine sand fl y species in a domestic unit (peridomicile) of the administrative region of Fercal, Federal District, Brazil.
PCR: polymerase chain reaction; rRNA: ribosomal ribonucleic acid.*BasicLocal Alignment Search Tool.**Number of bases that match the database sequence.