2.12. Molecular Detection of Leishmania spp.

Detection of Leishmania spp. was solely attempted in rodents, the only mammalian host for which tissue samples were available. Identification of this kinetoplastida parasite was carried out by a nested PCR protocol to amplify a partial fragment (358 bp) of the ssu rRNA gene of the parasite [38]. The primary PCR reaction (50 µL) contained 10 µL of template DNA and 15 pmol of the primer pair R221/R332 (Table S2). Conditions of PCR for ssu rRNA amplification were initial denaturation for 5 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, and terminal elongation for 10 min at 72 °C. In the secondary PCR reaction (25 µL), 10 µL of a 1:40 dilution of the primary PCR product was re-amplified using 7.5 pmol of the primer pair R223/R333 (Table S2). Cycling conditions were as described above except that the annealing temperature was set at 65 °C.

All the direct, semi-nested, and nested PCR protocols described above were conducted on a 2720 Thermal Cycler (Applied Biosystems). Reaction mixes always included 2.5 units of MyTAQ^TM DNA polymerase (Bioline GmbH, Luckenwalde, Germany), and 5× MyTAQ^TM Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl₂, except for the amplification of Leishmania spp., for which 0.7–1.4 units of Tth DNA polymerase (Biotools B&M Laboratories, S.A., Madrid, Spain) were used. Laboratory-confirmed positive and negative DNA samples of human and animal origin for each parasitic species investigated were routinely used as controls and included in each round of PCR. PCR amplicons were visualized on 1.5–2% D5 agarose gels (Conda, Madrid, Spain) stained with Pronasafe (Conda) or Gel Red (Biotium, Fremont, CA, USA) nucleic acid staining solutions. A 100 bp DNA ladder (Boehringer Mannheim GmbH, Baden-Wurttemberg, Germany) was used for the sizing of obtained amplicons. Positive-PCR products were directly sequenced in both directions using appropriate internal primer sets (Table S2). DNA sequencing was conducted by capillary electrophoresis using the BigDye^® Terminator chemistry (Applied Biosystems) on an on ABI PRISM 3130 automated DNA sequencer.

The sequences obtained in this study have been deposited in GenBank under accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"MW417420-MW417422","start_term":"MW417420","end_term":"MW417422","start_term_id":"2020706927","end_term_id":"2020706931"}}MW417420-MW417422 (G. duodenalis), {"type":"entrez-nucleotide-range","attrs":{"text":"MW414634-MW414644","start_term":"MW414634","end_term":"MW414644","start_term_id":"1950848784","end_term_id":"1950848804"}}MW414634-MW414644 and {"type":"entrez-nucleotide","attrs":{"text":"MW581486","term_id":"1977684660","term_text":"MW581486"}}MW581486 (Blastocystis sp.), {"type":"entrez-nucleotide-range","attrs":{"text":"MW406908-MW406921","start_term":"MW406908","end_term":"MW406921","start_term_id":"1950033975","end_term_id":"1950033988"}}MW406908-MW406921 (Cryptosporidium spp.) and {"type":"entrez-nucleotide","attrs":{"text":"MW414645","term_id":"1950848806","term_text":"MW414645"}}MW414645 (E. bieneusi).