Canine samples
This study included domestic dogs collected by the Diretoria de Vigilância Ambiental (DIVAL) of the FD during the survey of the CVL control program in the region. Dogs were either voluntarily supplied by their owners to DIVAL officers during the CVL survey, or stray dogs collected by routine DIVAL activities in the FD were included in the study. For convenience, the sample consisted of dogs collected in the period ranging from 4 July to 30 September in 2011 after approval was granted by the Animal Ethics Committee of the Faculty of Medicine at University of Brasilia (Process No. 57444/2011). To be included in the study, dogs must have presented at least one positive result after three serological tests used for detection of Leishmania spp. infection (see below). Dogs vaccinated against leishmaniasis, under suspicion of and/or confirmed with rabies and those using collars impregnated with insecticides were excluded from this study.
Blood samples from each dog (3 mL) were collected by venipuncture of the cephalic, femoral or jugular vein, and transferred to tubes with an anticoagulant (Vacuette® K3E K3 EDTA). Two tubes were collected from each dog: one to undergo serologic testing by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT) and the other to undergo DNA extraction and the rapid immunoassay dual-path platform test (DPP). The plasma samples were frozen at −20 °C until testing.
Tick samples
A clinical examination was performed in anesthetized dogs, with preferred places, such as the ears, interdigital spaces, and the areas around the orbits, being explored for ticks. The ticks were collected with tweezers, stored in properly identified tubes and kept at room temperature for approximately 2 h until the identification and dissection procedure. The ectoparasites were separated by sex and morphologically identified prior to their dissection based on taxonomic keys [17, 18].
The dissection was performed while the ticks were still alive according to the procedure described by Edwards et al. [19] After the internal contents were exposed, the salivary glands (SGs) were removed and then the intestine. The SGs and intestine samples were placed in separate Eppendorf tubes containing 0.2 mL of 0.9 % saline and 5-fluorocytosine (100 μg/mL) and stored at −20 °C for later DNA extraction.
The ticks taken from each dog were separated into pools (of up to 10 ticks) of males, females and nymphs. The processing flow of these samples is shown in Fig. 1. After isolation of the salivary glands and of the intestine of each tick, the remaining extravasated content was transferred to an Eppendorf tube containing 0.2 mL of 0.9 % saline and 5-fluorocytosine (100 μg/mL) and kept cool at 8 °C for 30 min to one hour. Then, the material was inoculated into the culture media [20]. These cultures were evaluated every 2 days until the 30th day.
Serological diagnosis
The blood samples intended for the ELISA (EIE-LVC kit Bio-Manguinhos/FIOCRUZ, Rio de Janeiro, Brazil) and the IFAT (IFI-LVC kit Bio-Manguinhos/FIOCRUZ, Rio de Janeiro, Brazil) tests were stored and processed by DIVAL. Only samples with positive ELISA results were subjected to IFAT for confirmation of positivity. The IFAT cut-off was 1:40.
The rapid immunochromatographic test, DPP® Canine Visceral Leishmaniasis (Bio-Manguinhos, Brazil), was performed according to the manufacturer’s instructions on plasma samples stored at −20 °C that had been thawed at room temperature.
DNA extraction and PCR
DNA obtained from blood samples was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Salivary glands and intestine samples were processed using Illustra Tissue Cells & Genomic Prep Mini Spin kit (GE Healthcare, New York, USA). DNA extraction of the parasites isolated in the culture medium was performed by the phenol-chloroform technique. Primers used to amplify 120 bp of the conserved kDNA region of Leishmania spp. were as follows: BW-B: 5 'CCG CCC CTA TTT TAC CCC ACC ACC 3'; FW: 5 'GGG GAG GGG CGT TCT GCG AA 3'; BW-CA: 5 'GGC CCA CTA TAT TAC ACC AAC CCC 3' [15]. The amplification conditions included a final volume of 10 μL, with the following standard mixture for each reaction: 1 μL Buffer (5x), 1 μL dNTPs (2 mM), 1 μL MgCl2 (25 mM), 1 μL FW primer (1.2 mM), 1 μL BW-B/BW-CA primer (0.6 mM), 0.1 μL Taq DNA polymerase (5 μ/μL), 3.9 μL of H2O and 1 μL of DNA template. The thermocycling conditions were 95 °C for 5 min for denaturation; then 39 cycles of 30 s at each temperature of 95 °C, 66 °C and 72 °C; and 5 min of final extension at 72 °C.
The primers used to amplify a 234-bp target of hsp70 of Leishmania spp. were FW: 5 'GAT CGA CGA GGA GGC CAT GGT 3' and BW: 5 'GAC TTC TCC GCC TCC TGG TTG 3'. The amplification yielded a final volume of 25 μL, and the standard mix for each reaction was as follows: 5 μL Buffer (5x), 2 μL of dNTPs (25 mM), 1.5 μL MgCl2 (25 mM) 0.25 μL FW primer (20 pm), 0.25 BW μL of primer (20 pm), 0.15 μL of Taq DNA polymerase (5 μ/μL), 14.85 μL of H2O and 1 μL of DNA. The thermocycling conditions were: 94 °C for 5 min for denaturation; 30 cycles of 94 °C for 30 s, 63 °C for 1 min and 72 °C for 10 min; and final extension at 72 °C for 10 min.
All PCR products were examined by electrophoresis in a polyacrylamide gel at 7.5 %, 150 V, and 75 Amp for 90 min. The molecular weight marker used in all gels was the DNA Molecular Weight Marker V (Roche Applied Science, Germany).
Enzymatic digestion with restriction endonucleases
The PCR-RFLP test with the HaeIII restriction enzyme required 5 μL of PCR products plus enzyme, which were incubated in a water bath at 37 °C. BstUI digestion was performed with 10 μL of PCR products plus enzyme, which were incubated in a water bath at 60 °C. The sample digested by the HaeIII and the sample digested by the BstUI enzymes were allowed to incubate for 1 h, and then they were subjected to electrophoresis in a polyacrylamide gel at 7.5 %.
DNA sequencing
PCR products were purified with an Illustra GFX PCR DNA & Gel Band Purification Kit (GE Healthcare, New York, USA), according to the manufacturer’s instructions. Sequencing was performed by the company Genomic Engenharia Molecular with the BigDye® Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems. Obtained sequences were edited by the DNAMAN software (Lynnon Corporation, Canada). Later on, these sequences were compared with the sequences of Leishmania species available in GenBank through the BLASTn algorithm (Basic Local Alignment Search Tool) from the National Center for Biotechnology Information of the United States of America.
Multilocus analysis (MLEE - multilocus enzyme electrophoresis and MLMT – multilocus microsatellite typing)
The Leishmania culture was maintained on a semi-solid medium and is on deposit in the Leishmania Collection at the Oswaldo Cruz Institute. This culture was typed by MLEE, as described [21]. The DNA obtained from this culture were subjected to microsatellite analysis, using a previously described protocol [22]. The MLEE profiles obtained were compared with a reference strain for L. infantum (MHOM/BR/1974/PP75). The microsatellite data were compared with the panel presented in Ferreira et al. [22].