2.4 Laboratorial analyses
2.4.1 Trypanosoma sp, : The presence of T. cruziwas evaluated by the hemoculture previously performed in the field. Cultures were analyzed every 15 days for three to five months and when at least one flagellate parasite was observed in optical microscopy, samples were classified as positive. From positive samples, parasites were grown in LIT medium, cryopreserved and deposited in theTrypanosoma collection from wild and domestic animals and vectors-COLTRYP (Oswaldo Cruz Foundation). Genomic DNA was extracted using the standard phenol-chloroform protocol (Sambrook et al., 1989). The obtained DNA was first characterized by multiplex PCR amplification of the mini-exon intergenic region (Fernandes et al., 2001); and secondly using the PCR-RFLP protocol targeting the histone 3 gene and digestion with AluI (Westenberger et al., 2005). Moreover, a Nested PCR targeting the 18S rRNA gene (∼600 bp) was performed as described by Noyes et al. (1999) and dos Santos et al. (2018). From this latter, amplified PCR products were purified using Illustra GFX PCR DNA and a gel band purification kit (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). The purified PCR products were sequenced with ABI 3730 BigDye Terminator (v3·1) Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystem DNA Analyzer on the PDTIS/FIOCRUZ sequencing platform. Nucleotide sequences were manually edited using the DNASTAR’s Lasergene Sequence Analysis Software (Burland, 2000) and aligned using CLUSTALW. The obtained sequences were compared with nucleotide sequences deposited in GenBank using the NCBI BLAST (Basic Local Alignment Search Tool) algorithm to identifyTrypanosoma species and/or T. cruzi DTUs.
2.4.2 Toxoplasma gondii: For the serodiagnosis ofT. gondii , the Modified Agglutination Test (MAT) was applied with the use of tachyzoites inactivated by formalin as an antigen, as described by Dubey and Desmonts (1987). As a cut-off point, a 1:25 dilution (Dubey, 2010) was used, with concomitant tests being performed with positive and negative controls previously known to mice. Positive samples were diluted until the final reaction titer. The antigen for carrying out the MAT was provided by Dr. Jitender P. Dubey of the United States Department of Agriculture (USDA), Agriculture Research Service, Animal and Natural Resources Animal Parasitic Diseases Laboratory, Beltsville, Maryland (USA)
2.4.3 Leishmania sp. : Nucleic acids contained in samples of armadillo ear tissue were extracted using the DNeasy Blood & Tissue kit (Qiagen), according to manufacturers. Each extraction procedure included duplicate extraction controls containing 25mg of bovine liver. PCR targeting a conserved region of the kinetoplast DNA ofLeishmania sp. was conducted using the pureTaq Ready-To-Go PCR beads (Amersham Biosciences, Buckinghamshire, UK) and primers forward 5’-GGGAGGGGCGTTCTGCGAA-3’ and reverse 5’GGCCCACTATATTACACCAACCCC-3’ (Cássia-Pires et al., 2014). The PCR products were visualized after electrophoresis on 8% polyacrylamide gel and silver staining using a specific kit (DNA Silver Staining, GE Healthcare). Positive and negative controls were derived from fragments of liver from noninfected and infected (Leishmania braziliensis – IOC-L2483) hamsters.
2.4.4 Mycobacterium leprae : The DNA samples extracted for Leishmania sp. analysis were also used to detect M. leprae . Specific sequences of 16 Variable Number Tandem Repeats (VNTRs) were applied for four separate multiplex PCR assays, as described by Kimura et al., (2009), with a volume of 2µL of DNA used in each multiplex reaction. The sensitivity of the multiplex assay, incorporating the AC8b, GTA9, GGT5, AT17, 6-3 VNTR targets, was determined using nude mouse-derived M. leprae cells as follows: a total of 2x105 cells was combined with duplicate samples of 25mg of bovine liver and DNA extracted using the same protocol described for the armadillo samples, with elution performed in 100µL of AE.
The mouse-derived samples (denominated S0), were considered to contain a quantity of DNA equivalent to 2x103 cells/µL (assuming 100% extraction efficiency). The S0 samples were submitted to 10-fold serial dilution in AE buffer to generate samples containing quantities of DNA ranging from equivalent to 2x103 cells/µL to 2 cells/µL. The sensitivity of the multiplex assay was determined based on the last dilution that produced clearly amplified products, suitable for genotyping based on fluorescent fragment size analysis (Kimura et al., 2009).
The M. leprae specific repetitive element (RLEP) PCR was amplified in a nested PCR reaction. The primers for RLEP2-1 (5’-ATATCGATGCAGGCGTGAG-3’) and RLEP2-2 (5’-GGATCATCGATGCACTGTTC-3’) amplified a 282-bp sequence of the RLEP element. The second set of inner primers, RLEP2-3 (5’- GGGTAGGGGCGTTTTAGTGT-3’) and RLEP2-2, amplified a 238-bp product. A 1µL aliquot of the isolated DNA was added to 24µL of PCR mix, which contained 15mM Tris-HCl (pH 8.0), 50mM KCl, 1.5mM MgCl2, 0.2mM dNTP, 5% DMSO, 1.25 units of Taq DNA Polymerase and 0.2µM of each primer.
The mixture was denatured at 94ºC for 4 min, followed by 35 PCR cycles (30s at 94ºC, 30s at 59.6ºC and 1min at 72ºC), with a final extension at 72ºC for 10min. Each run included negative and positive controls. For the nested PCR, 0.5µL of product was used as the DNA template. The amplification reactions were visualized on a 1.5% agarose gel. When the results for the same sample were different, a third PCR was performed for confirmation.
Different amounts of purified DNA from M. leprae were added to all negative PCR samples to assess the presence of inhibitory substances. A standard curve was constructed by serial dilution of purified M. leprae DNA ranging from 10fg to 1µg. PurifiedM. leprae DNA was also used as a positive control for the amplifications.
2.4.5 Paracoccidioides sp.: PCR analyses and sequencing were performed to detect the infection by Paracoccidioides sp. (Hrycyck et al., 2018) in distinct tissues. The DNA extraction from ear, lung, liver and spleen samples was performed using the commercial kit Macherey-Nagel (MN) and the DNA was quantified on NanoVue (GE Healthcare) and stored at ˗20°C before use. A Nested PCR technique was applied aiming to amplify the ITS1-5.8S-ITS2 region with primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCGTAGGTGAACCTGCGG-3’). Later, a second amplification using the specific primers PbITS-E (5’-GAGCTTTGACGTCTGAGACC-3’) and PbITS-T (GTATCCCTACCTGATCCGAG) was applied. Both amplifications were performed by employing GoTaq MasterMix (Promega). Cycling conditions were stablished according to the Taq polymerase manufacturer requirement with annealing temperature of 55°C for ITS1/ITS4 and 62°C for PbITS-E/ PbITS-T primers. The PCR products were detected by 1.5% agarose gel electrophoresis.