The MHOM/BR/2006/EFSF isolate, referred herein as EFSF6, was obtained from a patient attending the Anuar Auad Tropical Diseases Hospital in the city of Goiânia, Goiás, Brazil. The patient was infected in Minaçu, a municipality of Goiás State and presented five ulcerated lesions distributed in both arms. The lesions had appeared three months before the diagnosis. The patient was cured after treatment with a 20-day course of pentavalent antimonial. As part of the diagnostic procedure, skin biopsies were taken and the tissue was homogenised and inoculated into Grace's medium (Sigma- Aldrich Chem. Co., St Louis, MO, USA) supplemented with 20% fetal calf serum (FCS) .
The isolate was typed by PCR of ribosomal DNA, glucose-6-phosphate dehydrogenase and META2 gene as described [11–13] and identified as L. (V.) braziliensis. Soon after isolation, the culture was frozen and further tests were performed with freshly recovered cultures with a low passage number (third to seventh).
Atypical promastigotes of the EFSF6 isolate or typical forms of the L. (V.) braziliensis MHOM/BR/75/M2903 reference strain were grown in M199 liquid medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Invitrogen) and 2% sterile male human urine, or in Grace's medium supplemented with 20% FCS and 2 mM L-glutamine (Sigma) and incubated at 22-26°C.
Analysis of the parasite's morphology by optical microscopy was performed by applying and spreading slightly 10 μl of culture onto a slide. The material was left to dry at room temperature and fixed and stained with the Instant Prov kit (Newprov, Pinhais-Paraná, Brazil).
Monoclonal antibody typing
For slide preparation, promastigotes were centrifuged at 1000 × g for 10 min, washed once in PBS pH 7.2 (2.5 mM NaH2PO4, 7.4 mM Na2HPO4, 137 mM NaCl), applied to the slides, dried and fixed with acetone for 15 min.
The following monoclonal antibodies were used: B2, B5, B11, B12, B18, B19, M2, T3, CO2, L1, WIC, W1, W2, N2, N3 and WA2, according to methods described previously [14–16]. The B and N series react with species of the subgenus L. (Viannia); M2, T3, WIC.79.3, W1, W2 and WA2 react with parasites of the subgenus L. (Leishmania). CO2, and L1 are group-specific and react with members of subgenera Viannia and Leishmania, Endotrypanum and some species of the genus Trypanosoma. The monoclonal B18 is specific for L. (V.) braziliensis.
Ribosomal Internal Transcribed Spacer (ITS) amplification and cloning
Genomic DNA was obtained from cultured parasites as described previously . Amplification of the ribosomal DNA internal transcribed spacers 1 and 2 (ITS1, ITS2) and 5.8S ribosomal DNA (5.8S) (approximately 1 kb) was performed with primers IR1 (5' GTA GGT GAA CCT GCA GCA GCT GGA TCA TT 3') and IR2 (5' GCG GGT AGT CCT GCC AAA CAC TCA GGT CTG 3') or 5.8R (5'GGA AGC CAA GTC ATC CAT C 3') as described . Amplified products were cloned into TOPO TA® (Invitrogen). GenBank accession numbers for sequences determined or used in this study are listed in the legend of Additional File 1.
Nucleotide sequences were analyzed using the program BioEdit http://www.mbio.ncsu.edu/BioEdit/bioedit.html and aligned with sequences from GeneBank using ClustalW . The resulting alignments were manually refined. Parsimony and bootstrap analyses were carried out using PAUP* 4.0  with 100 replicates of a random addition sequence followed by branch swapping (RAS-TBR), as previously described .
Infection of macrophages in vitro
Bone marrow-derived macrophages (BMDM) were obtained from BALB/c mice as described by . BMDM were counted and distributed in 24-well plates on round coverslips (3 × 105 macrophages per well) containing RPMI 1640 medium with 10% FCS and 5% L929 cell conditioned medium and allowed to adhere overnight at 37°C, at 5% CO2. Infections of macrophages were performed using ratios of 15 stationary-phase promastigotes per macrophage. Infected macrophage cultures were kept at 33°C, 5% CO2 for 3 h in RPMI 1640 medium with 10% FCS and then washed twice with sterile PBS to remove free promastigotes. After 48 h of incubation, slides were fixed in methanol and stained with the Instant Prov kit (Newprov, Pinhais-Paraná, Brazil) for subsequent examination under light microscopy.
Infection of mice
Female BALB/c mice (n = 5 - 7) were infected in the left ear by intradermal inoculation of 1 × 105 stationary-phase promastigotes grown in M199 liquid medium. Stationary-phase promastigotes (5 × 106/50 μL of PBS) grown in Grace's medium were injected into the hind left footpads of C57BL/6 and BALB/c mice (n = 6). During 12 weeks, the evolution of lesions was evaluated by measuring the thickness of the infected ear or footpad using a calliper.
Logarithmic-phase promastigotes (5 × 107/mL), washed twice with PBS pH 7.2, or tissue fragments, were fixed in a solution containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 24 h at 4°C. After fixation, the material was washed twice in 0.1 M sodium cacodylate buffer, pH 7.2 and post-fixed for 30 min in a solution containing 1% OsO4, 1.25% potassium ferrocyanide and 5 mM CaCl2 in 0.1 M cacodylate buffer, washed in the same buffer, dehydrated in acetone series, and embedded in Epon or Spurr resin. Ultrathin sections were obtained in a Leica Ultramicrotome, stained with uranyl acetate and lead citrate and observed under a Zeiss 900 or a JEOL 100 CX transmission electron microscope operating at 80 kV. Images were recorded with a MegaView III camera (Olympus Soft Imaging Solutions) using the ITem Software. For scanning electron microscopy, cells were fixed as before, adhered to poly-L-lysine-coated coverslips, post-fixed for 30 min in a solution containing 1% OsO4, 1.25% potassium ferrocyanide and 5 mM CaCl2 in 0.1 M cacodylate buffer, washed in the same buffer, dehydrated in ethanol series, critical point dried, and coated with gold in a Balzers gold sputtering system. Cells were observed under a Jeol JSM 6340F field emission scanning electron microscope operating at 5 kV.
Rearing of Sand flies and artificial infection
L. longipalpis were captured at Gruta da Lapinha, Minas Gerais, Brazil, and maintained in an insectary at Instituto Oswaldo Cruz, FIOCRUZ. Capture, maintenance and colonization in laboratory conditions were performed according to Brazil and Brazil . Insects were sugar fed on 30% sucrose solution ad libitum.
For artificial blood-feeding or infection with the EFSF6 isolate or typical forms of the L. (V.) braziliensis MHOM/BR/75/M2903, three-day-old female sand flies were fed through chick skin membrane on hamster blood containing 107 promastigotes/ml.
Quantification of infection
DNA was extracted from individual insects collected at 24, 72 or 96 hours post artificial infection with L. (V.) braziliensis MHOM/BR/75/M2903 or EFSF6 using wizard SV Genomic kit (Promega). Parasite load was assessed as described before . qPCR reactions were performed using the SYBR Green PCR Master Mix (Applied Biosystems) and kDNA  and 18S primers . Standard curves were constructed using serial dilutions of L. braziliensis and L. longipalpis DNA. The relative quantification was calculated using the 2-ΔΔCt method . The Ct values of the 18S reference gene from L. longipalpis were used for data normalization.
This study was approved by the Ethics Committee in Human and Animal Research of the HC/UFG and a consent letter was signed by the patient. All animal procedures were approved by the FIOCRUZ bioethics committee (CEUA - protocol number P0-116-02) and by ICB-USP Ethics Committee on Animal Experimentation.
Results are presented as media ± SEM and data were analyzed by GraphPad Prism 4.0 Software (San Diego, CA, USA), using the Kruskal-Wallis and Mann-Whitney tests (p < 0.05).