Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos,... Abstract Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition. Paracoccidioides spp., Onygenales, phylogeny, molecular detection, Dasypus novemcinctus Introduction Paracoccidioides brasiliensis and P. lutzii are the etiological agents of paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America, where Brazil, Colombia, and Venezuela are the leading countries in number of cases.1,2 In Brazil, PCM was already confirmed in 27% of the counties, causing hospitalization in 4.3 per 1.0 million inhabitants, and has been ranked as the eighth most frequent cause of death due to chronic or recurrent infectious and parasitic diseases.3–5 Adult male rural workers presenting the chronic clinical form of the disease are the main group affected, although an acute severe form affecting mainly children or young adults may also occur in nearly 5–10% of cases.6,7 Treatment of PCM still presents enormous challenges, with frequent relapses and debilitating sequelae.8 These pathogens belong to the family Ajellomycetaceae (Onygenales Order) and are typically thermodimorphic, presenting yeast cells when they grow in the animal tissues and mycelia in the environment where they produce the infective propagule.9,10 Molecular phylogenetic studies have indicated two distinct clades among the genus Paracoccidiodes: the brasiliensis clade that harbors five phylogenetic cryptic species (S1a, S1b, PS2, PS3, and PS4) and the lutzii clade containing P. lutzii species.11–13 The cryptic species PS2, PS3, and PS4 were recently reclassified as formal species: P. americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4).14 The geographic distribution of Paracoccidioides spp. is now being revealed, although the final picture is still far from being completed.12P. brasiliensis sensu strictu (S1a and S1b) appears to be widely distributed in Brazil and Argentina, where it has been frequently isolated from humans and armadillos.14P. americana (PS2) seems to occur less frequently, both in human and armadillos in Brazil, although it was already recovered from a Venezuelan patient, and in some uncommon cases/situations, such as from an infected dog in southern Brazil, from dog food contaminated with soil (in the southeast region of Brazil) and in feces of an Antarctic penguin.15–17P. restrepiensis (PS3) occurs mainly in Colombia, both in human and armadillos and in some patients from Brazil.18P. venezuelensis (PS4) is restricted to the single environmental isolate obtained from Venezuelan soil.14,19 The species P. lutzii is thought to occur in the central western and northern regions of Brazil, including Goiás, Mato Grosso, Pará and Rondônia states.20,21 However, the real distribution of P. lutzii has not been fully comprehended until now, since it was already isolated from single cases of patients living in Ecuador and also in southeastern Brazil,22 and in addition, it has been molecularly detected in aerosol and soil samples outside the clinical range of the disease provoked by this species.23,24 Natural infection by P. brasiliensis in the nine-banded armadillo Dasypus novemcinctus has been observed repeatedly in several PCM endemic areas of Brazil and Colombia, where the pathogen was also isolated from the naked-tailed armadillo species Cabassous centralis.25–27 Armadillos have a less active cellular immune system, body temperature slightly lower than other mammals and are constantly in search of food and protection in the soil, which makes them particularly susceptible to infection by soil-dwelling pathogens, including P. brasiliensis. In addition, armadillos present a small home range and no regular migration habits, which makes them ideal for registering the environmental presence of the fungus and for mapping the genotypes that occur in the different endemic areas.26,28 In this work, we aimed at isolating and molecularly detecting Paracoccidioides spp. in armadillo tissues and associated soil samples, which were collected in three well-defined geographic areas, including: (i) a small isolated fluvial island (Cerrito), in São Manuel/SP, (ii) a positive farm (Edgardia) in Botucatu/SP, where P. brasiliensis and the less common P. americana species (former PS2 genotype) were already isolated from armadillos, (iii) a new endemic area (Alta Floresta) from Central region of Brazil, in Alta Floresta and Paranaíta/MT, the probable occurrence area of P. lutzii, whose ecology and geographic distribution is poorly comprehended. Some mycological features of the isolates such as yeast-to-mycelial conversion, growth, and conidial production in soil-related substrates were also evaluated. The data herein obtained present biological and ecological differences in Paracoccidioides spp. that might be relevant for PCM epidemiology. Methods Characteristics of the study areas The collections were carried out in three distinct areas, two located in the southeast and one in the central region of Brazil (Fig. S1). Area 1 (Edgardia Farm) is an experimental farm of São Paulo State University (UNESP), Botucatu, São Paulo state. The S1 genotype of P. brasiliensis and P. americana (former PS2 genotype) were already isolated from nine-banded armadillos (D. novemcinctus), and PCM has been diagnosed in rural workers that have lived and/or worked there. Some of these patients died of PCM. Area 2 is a small fluvial island (Cerrito Island, located in São Manuel County, São Paulo state) that was established in the Tietê River in 1962, when the construction of the Barra Bonita Dam caused a natural population of armadillos to become isolated from the rest of the continent; the location is accessible only by boat and has very little human presence. Area 3 is located in Alta Floresta and Paranaíta Counties, Mato Grosso state, central region of Brazil, under the deforestation arc 29 of the Amazon Biome, where the local economy is mainly based on agriculture and livestock (IBGE, 2016).30 PCM cases have been diagnosed in Alta Floresta, despite its low level of local epidemiological vigilance. Additional ecological data from the sampled areas are provided in Table S1 (Supplementary Material). Animal and soil sampling After obtaining the legal authorization from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA/ICMBio, License number 30585-1) and also from the local Ethics Committee on the Use of Animals (CEUA, protocol number 737), seven armadillos (six males and one female) were captured using traps settled on the animal track or at the entrance of their burrows: two animals from Edgardia Farm (area 1), two from Cerrito Island (area 2), and three from Alta Floresta and Paranaíta (area 3). The armadillos were maintained in appropriate cages for up to 24h and then evaluated. Soil samples (n = 20) were collected in duplicate (four samples from Edgardia Farm, three from Cerrito Island, and 13 from Alta Floresta and Paranaíta) from the armadillo burrows (at 0 and 0.3-m depth) and animal foraging sites located in forest (areas 1 and 2) and also pasture and horticultural fields (area 3). The samples were placed in 50-ml sterile universal bottles, sealed, identified, and stored in an isothermal box at room temperature until their evaluation for fungal molecular detection. Additional soil samples were also collected for determination of their physical and chemical properties, which was carried out at the Soil Fertility Laboratory of the School of Agriculture FCA /UNESP, as well as for preparation of Soil Extract Agar to culture the fungal isolates. Geographic coordinates of the collected sites (armadillos and soil samples, Table S2) were obtained using a Global Positioning System (GPS, Garmin GPS 12) and plotted in the Geographic Information Systems (GIS) by the software QGIS version 2.8. Fungal isolation from the armadillos The animals were anesthetized intramuscularly (Zoletyl® 50 - Virbac, 0.2 ml/kg), euthanized by cardiac puncture and necropsied. The whole liver, spleen, and mesenteric lymph nodes were collected aseptically, as well as feces from two animals. The organs were cleaned in 70% (v/v) alcohol and sterile saline solution (0.9% w/v). One piece of each organ was kept in absolute alcohol and used for molecular detection. The remaining organs were fragmented into small pieces (2–3 mm) and seeded in Mycosel® Agar supplemented with gentamicin (50 μg/ml). Plates were incubated at 35°C and evaluated for fungal growth for up to 60 days. Suspected colonies of Paracoccidioides spp. were screened microscopically by staining with Lacto-phenol cotton blue and subcultured in glucose, peptone, yeast extract and agar (GPYA, 2% glucose, 1% peptone, 0.5% yeast extract and 1% agar) for further mycological and molecular studies. Clinical human isolates were also included herein for molecular and/or mycological comparisons, including four recent isolates (PSM, PbD, 8652 and 3051) of P. brasiliensis obtained from patients assisted at the Dermatology Service of the University Hospital of Botucatu Medical School, São Paulo, Brazil (HC/UNESP), one PS3 isolate (BACR) from Colombia, and three P. lutzii isolates originating from patients (Pb01 and Pb66, from Goiânia-GO, and PbEE, from Cuiabá-MT) in Brazil. Molecular studies DNA extraction For Paracoccidioides spp. isolates, the DNA was extracted from yeast cells, according to the protocol described by McCullough et al.31 with minor modifications (using yeast cells cultured for 7 days on GPYA at 37°C, which were disrupted using the Precellys (Bertin Technologies, MD, USA) equipment). For armadillo tissue samples (spleen, liver, and mesenteric lymph nodes), animal feces and soil samples, the DNA extraction was accomplished by using the commercial kits Macherey-Nagel (MN), QIAamp® DNA Stool (QIAGEN) and Power Soil (MO BIO Laboratories Inc.), respectively. All extracted DNA samples were quantified on the NanoVue (GE Healthcare) spectrophotometer and stored at −20°C until used. Detection in soil, animal tissue, and feces samples The molecular detection was carried out by a nested polymerase chain reaction (PCR) targeting rDNA with primers (ITS1/ITS4 or ITS4/ITS5) in the first PCR reaction, followed by a second amplification with the inner primers (PbITS-E/PbITS-T) (Table 1), supposed to be specific for P. brasiliensis and P. lutzii.23,32 All the PCR products were detected by 1.5% agarose gel electrophoresis stained with SYBR Safe (Invitrogen). The soil environmental amplicons were also purified and sequenced as described below. Table 1. Paracoccidioides spp. isolation by direct culture and molecular detection by nested/PCR in tissue samples (liver, spleen, and mesenteric lymph nodes) of armadillos (Dasypus novemcinctus) captured in two distinct endemic regions of paracoccidioidomycosis in Brazil (Alta Floresta/MT and Botucatu/SP). Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − *Positive growth over the total number of fragments (%). (−) Negative sample (+) Positive sample. MT, Mato Grosso State; SP, São Paulo State, Brazil. View Large Table 1. Paracoccidioides spp. isolation by direct culture and molecular detection by nested/PCR in tissue samples (liver, spleen, and mesenteric lymph nodes) of armadillos (Dasypus novemcinctus) captured in two distinct endemic regions of paracoccidioidomycosis in Brazil (Alta Floresta/MT and Botucatu/SP). Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − *Positive growth over the total number of fragments (%). (−) Negative sample (+) Positive sample. MT, Mato Grosso State; SP, São Paulo State, Brazil. View Large Molecular characterization and phylogenetic analysis of the isolates and environmental amplicons The isolates were characterized by sequencing ITS rDNA and gp43 exon 2 loci. Other DNA regions, such as arf (adenylyl ribosylation factor) gene were sequenced and analyzed for some isolates. In addition, the PCR-RFLP (restriction fragment length polymorphism) of tub1 gene was also carried out to enable differentiation among Paracoccidioides spp. without DNA sequencing18 (details below). All the primers and annealing temperatures employed are listed in Table S3. PCR reactions were performed using a Veriti Thermocycler (Applied Biosystems, Foster City, CA, USA) and GoTaq®Green Master Mix (Promega, Madison, WI, USA), with 25 μl of reaction mixture-containing 3 μl of genomic DNA (300 ng/μl) and 0.5 μM of each primer. For DNA sequencing, the amplicons were purified using ExoProStar (GE Healthcare, Milwaukee, WI, USA) and sequenced in the ABI 3500 DNA Analyzer (Applied Biosystem, Foster City, CA, USA), according to the manufacturer's instructions. The quality of sequences was verified by the software Sequencing Analysis (Applied Biosystems) and only nucleotides with Phred ≥20 were included. Sequencing edition and phylogenetic analysis were accomplished using the software MEGA v6.0.33 Molecular identification was performed by the Basic Local Alignment Search Tool (BLAST)34 from the National Center for Biotechnology Information (NCBI) and International Society for Human Animal Mycology (ISHAM) ITS Databases.35 Alignments of the sequences were accomplished through Clustal W algorithm. Three different phylogenetic constructions were made via the maximum likelihood (ML) method36 by applying Tamura-Nei model37 for ITS1-5.8S-ITS2 and soil environmental amplicons, Kimura 2-parameter model38 for the gp43 exon 2 gene, all with bootstrap of 1000 replicates.37 The PCR-RFLP analysis was performed as described by Roberto et al., 201518 but with the addition of new primers herein designed (Table S3) in order to better amplify the tub1 gene of both Paracoccidioides species. PCR products were visualized on agarose gel (1%) and then subjected to restriction enzyme digestion: 13 μl ultrapure water, 3 μl tub1-PCR, 2 μl 10× of digestion buffer and 1 μl of each endonuclease BclI (10 U/μl) and MspI (10 U/μl), both from Thermo Scientific (Massachusetts, USA). The digestion was incubated at 37°C for 2 hours, and the digested products were visualized on 2.5% agarose gel with electrophoresis at 100 V for 120 min.18 DNA samples from genotypes previously characterized12, namely, Bt84, EPM83, EPM77, Pb18, T16B1, T10B1, and PbDog were also included in this analysis. The DNA sequences from fungal isolates and soil environmental amplicons herein obtained were deposited in the GenBank with accession numbers in parentheses: for ITS sequences of the isolates (KX774393-KX774411), gp43 exon 2 (KY963798-KY963822), arf (KY963821-KY963822), and ITS sequences from environmental amplicons (MF078064-MF078073). Mycological studies Fungal growth, transition from yeast to mycelia, conidia production, and viability in Soil Extract Agar (SEA) Soil extracts (SE) were prepared with soil samples collected at area 1 (Edgardia Farm, Southeast region of Brazil) and Area 3 (Alta Floresta, Central region of Brazil) and performed according to the description of Terçarioli et al.39 Three isolates of P. brasiliensis (T17LM1, T18LM1, 3051) and three of P. lutzii (Pb66, PbEE, Pb01) were evaluated by seeding four yeast fragments in Petri dishes containing SEA, in triplicate, and incubating at 25°C. The isolates were simultaneously evaluated in SEA prepared with SE extracts originating from the two distinct regions. At the 9th day, the isolates were evaluated in relation to the conversion from yeast (Y) to mycelia (M), and classified as: no conversion (0); little conversion, containing few visible mycelia (+); complete conversion with highly visible mycelia (++). Production of conidia was evaluated by using adhesive tape preparations. Fungal mycelial colonies cultured on SEA at 25°C for 35 days were killed by formaldehyde vapor (1–2 ml of 30% formaldehyde added to the bottom plate, for 3–5 h) and employed to prepare the slides with adhesive tapes (Durex, 3M) stained with Lacto-phenol cotton blue. Conidia were counted in at least five fields, at 40× objective magnification, in the photomicrography system Olympus (PM 30), coupled with a Leica digital photomicrography (DMLB) camera and the software Leica Qwin Lite 2.5. Counting data were processed via the software GraphPad Prism version 5.01 (San Diego, CA, USA). The slide culture technique was also applied for isolates (P. brasiliensis and P. lutzii) in SEA,40 with minor modifications, in order to better observe and document conidial production, evaluated on the 40th day and photographed as mentioned above. After 3 months of incubation in SEA (25°C), mycelial forms of Paracoccidioides brasiliensis and P. lutzii isolates were evaluated for the possibility of remaining alive. The mycelia of the isolates were scraped and deposited in test tubes containing saline and glass beads, and vortexed for 20s. After one minute at rest, 100 ul volumes of serial dilutions (10−1 and 10−2) were seeded and spread in Petri dishes (duplicate) containing Mycosel Agar, and incubated at 25°C for up to 60 days. The appearance of typical Paracoccidioides spp. colonies in any dilution indicates positive viability of conidia and/or mycelial fragments. Giant colonies on PDA and preservation/viability after 1 year Yeast inoculums of each armadillo isolate (T17LM1, T18LM1, T19F7-2, T19F33, T20B13-1, T22LM1-1, T23LM1-1 and T23B1-1) were cultivated (triplicate) in a Petri dish containing Potato dextrose agar (PDA, Oxoid) at 25°C for 30 days for evaluating the morphological characteristics (verse and reverse) and diameters of the mycelial colonies. The mycelial plate cultures of T18LM1, T19F7-2, T19F33, T20B13-1, T22LM1-1, T23LM1-1 isolates were also maintained for a period of 1 year under the same conditions (at 25°C) and then evaluated for their viability and capacity to convert to yeast form, by seeding the mycelial/conidial inoculums in GPYA tubes, and incubated at 35°C. Statistical analysis The one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test was used to compare conidial production within isolates of P. brasiliensis and P. lutzii species, in the two soil extract agar conditions (soil from central and southeast regions of Brazil), and also to compare the mycelial growth diameters of giant colonies. Results Fungal isolation from armadillos From January to November 2015, seven armadillos (six male and one female) were evaluated, both for fungal culture and molecular detection (Table 2). The fungi were positively cultured in six animals (85%) that had been collected on Edgardia Farm, area 1 (two animals), Cerrito Island, area 2 (two animals) in the southeast region, and Alta Floresta County, area 3 (two animals) in the central region of Brazil. The fungal growth was detected from the 11th until the 43rd day of organ culture at 35°C. The pathogen was cultured mainly in the fragments of the mesenteric lymph nodes (four animals), followed by spleen (three animals) and liver (one animal) (Table 1). The majority of the isolates were obtained in different fragments of the same organs; however, in two animals the fungus was isolated simultaneously in two distinct organs, namely the liver/spleen in armadillo T19 (area 1) and spleen/mesenteric lymph nodes in T23 (area 3). Whenever possible, more than one isolate from each positive animal, preferably from distinct organs, were selected for additional studies. Table 2. Species and genotypes of Paracoccidioides spp. isolates obtained from armadillos (≠T17 to ≠T23) collected in three distinct geographic areas, as well as human patients from Botucatu PCM endemic area and reference strains. Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 *Edgardia Farm (area 1, Botucatu, SP, Southeast Region); Cerrito Island (area 2, São Manuel, SP, Southeast Region); Alta Floresta (area 3, Alta Floresta, MT, Central Region). MT (Mato Grosso), SP (São Paulo), PR (Paraná) and GO (Goiás) are States of Brazil. ND, Not done. View Large Table 2. Species and genotypes of Paracoccidioides spp. isolates obtained from armadillos (≠T17 to ≠T23) collected in three distinct geographic areas, as well as human patients from Botucatu PCM endemic area and reference strains. Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 *Edgardia Farm (area 1, Botucatu, SP, Southeast Region); Cerrito Island (area 2, São Manuel, SP, Southeast Region); Alta Floresta (area 3, Alta Floresta, MT, Central Region). MT (Mato Grosso), SP (São Paulo), PR (Paraná) and GO (Goiás) are States of Brazil. ND, Not done. View Large Molecular characterization and phylogenetic analysis of the isolates All armadillo isolates were molecularly identified as P. brasiliensis sensu lato, with no occurrence of P. lutzii, according to ITS rDNA sequencing (Table 2 and Fig. 1). The analyses of tub1 gene PCR-RFLP and of gp43 Exon-2 DNA sequencing enabled a better characterization of the isolates and determination of their genotypes or cryptic species. Among the armadillo isolates was observed the presence of S1a (three occurrences) and S1b genotypes (12 occurrences) of P. brasiliensis species and one occurrence of PS2 genotype (P. americana). In the armadillos collected at Edgardia Farm (southeast region), more than one genotype was simultaneously detected in the same animal, namely, the S1a and S1b genotypes of P. brasiliensis in armadillo T17, and S1a (P. brasiliensis) and PS2 (P. americana) in armadillo T18, specifically in the mesenteric lymph nodes. All armadillos collected on Cerrito Island (southeast region) and in Alta Floresta (central region) presented only the genotype S1b of P. brasiliensis. Four recent clinical isolates from patients of the Botucatu PCM endemic area (southeast region, where areas 1 and 2 are located) were also characterized herein as S1a genotypes of P. brasiliensis (Table 2 and Fig. 2). Figure 1. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA sequences from multiple P. brasiliensis isolates from armadillos (marked with T17 to T23) and human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from P. brasiliensis and P. lutzii were also included, and H. capsulatum was used as outgroup. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Figure 1. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA sequences from multiple P. brasiliensis isolates from armadillos (marked with T17 to T23) and human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from P. brasiliensis and P. lutzii were also included, and H. capsulatum was used as outgroup. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Figure 2. View largeDownload slide Phylogenetic analysis of gp43 Exon 2 sequences from multiple isolates of Paracoccidioides spp. obtained from armadillos (marked with T17 to T23), as well as from human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from S1a, S1b, PS2, PS3 and P. lutzii were also included. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Figure 2. View largeDownload slide Phylogenetic analysis of gp43 Exon 2 sequences from multiple isolates of Paracoccidioides spp. obtained from armadillos (marked with T17 to T23), as well as from human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from S1a, S1b, PS2, PS3 and P. lutzii were also included. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The armadillo T18LM3-5 isolate (PS2 genotype) and the clinical Colombian BACR isolate (PS3) were also identified by sequencing the arf gene, confirming their identities with 100% similarity to referenced sequences deposited in the GenBank database. The polymorphic sites of ITS rDNA and gp43 Exon-2 DNA regions detected in P. brasiliensis (S1a and S1b), P. americana (PS2), and P. restrepiensis (PS3) species are shown in Table S4. Molecular detection in animal tissues, feces and soil samples Molecular detection based on nested-PCR indicated positivity in at least one evaluated organ for all animals, including the spleen of armadillo T21 that was negative for fungal culture (Table 2). Using this same technique, we also detected Paracoccidioides spp. in the feces of one armadillo, collected at Edgardia Farm. The pathogens P. brasiliensis sensu lato and P. lutzii were detected in most soil samples from animals’ burrows and foraging areas, collected mainly in forests of the southeast (areas 1 and 2) and central region of Brazil (area 3), where positive samples were also detected in pasture and in a horticultural field (Table S5 and Fig. S2). Eleven environmental soil amplicons generated by the inner primers PbITS-E/PbITS-T (supposed to amplify DNA of all Paracoccidioides species) were sequenced and compared with deposited sequences from GenBank, showing 99% identity with P. lutzii (in 8 out of 11 amplicons) and 100% with P. brasiliensis sensu lato (in 3 out of 11 amplicons), indicating that both species groups occur in soils of both regions. The soil amplicons phylogenetically associated with P. lutzii have a greater genetic variability compared to the amplicons associated with P. brasiliensis clade (Fig. 3). Figure 3. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA partial sequences, obtained by amplification with the inner primers PbITSE/PbITST (Arantes et al., 2012) in soil samples collected in Central/MT and Southeast/SP regions of Brazil, and grouping pattern in relation to P. brasiliensis (accession number AY374337) and P. lutzii (accession number XR001551846) species. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site positions in the final dataset. Figure 3. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA partial sequences, obtained by amplification with the inner primers PbITSE/PbITST (Arantes et al., 2012) in soil samples collected in Central/MT and Southeast/SP regions of Brazil, and grouping pattern in relation to P. brasiliensis (accession number AY374337) and P. lutzii (accession number XR001551846) species. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site positions in the final dataset. P. brasiliensis and P. lutzii conversion to mycelial and conidial production in SEA Three isolates of P. brasiliensis and three of P. lutzii were evaluated in relation to mycelial conversion and conidial production in soil extract agar (SEA), using SEA prepared with soil samples from the southeast (SP) and central (MT) regions of Brazil. All isolates of P. brasiliensis from both regions converted well, beginning their transition on the 4th day in SEA, while in P. lutzii species, the conversion started only at the 9th day in two of three isolates in SEA prepared with soil from the southeast region—in SEA from the central region the mycelial conversion was less effective, since it occurred in just one isolate of P. lutzii (Table 3). Conidial production appeared to be higher in P. brasiliensis than in P. luzii isolates, whereas SEA from the southeast region seems to be more favorable both for mycelial growth and conidial production (Fig. 4). Figure 4. View largeDownload slide Conidia production of P. brasiliensis and P. lutzii isolates seeded in soil extract Agar (SEA), prepared with soil samples from Central (MT) and the Southeast (SP) regions of Brazil (25ºC). Counting carried out at 35th day in the 40× magnification. Figure 4. View largeDownload slide Conidia production of P. brasiliensis and P. lutzii isolates seeded in soil extract Agar (SEA), prepared with soil samples from Central (MT) and the Southeast (SP) regions of Brazil (25ºC). Counting carried out at 35th day in the 40× magnification. Table 3. Conversion from yeast (Y) to mycelium (M) of P. brasiliensis and P. lutzii isolates, at 9th day of growing in Soil Extract Agar (SEA), prepared with soil samples collected in Central and Southeast Regions, Brazil. Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ No Y to M conversion (0); moderate conversion (+); plenty conversion (++). View Large Table 3. Conversion from yeast (Y) to mycelium (M) of P. brasiliensis and P. lutzii isolates, at 9th day of growing in Soil Extract Agar (SEA), prepared with soil samples collected in Central and Southeast Regions, Brazil. Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ No Y to M conversion (0); moderate conversion (+); plenty conversion (++). View Large Giant colonies on PDA and preservation/viability after 1 year All P. brasiliensis isolates obtained from armadillos presented typical mycelial colonies, such as white cotton and crater-like aspects on the verse, and the presence of brown pigments with some cracks and/or folds on the reverse. The growths were relatively slow, since the colony diameters at the 30th day were less than 2.0 cm in all isolates, except T17LM1 (originating from Botucatu, SP) that differs statistically from T19B7-2 and T20B13-1 (both originated from Alta Floresta, MT), as shown in Table 4. Table 4. Colony morphology of Paracoccidioides brasiliensis isolates from armadillos, evaluated at 30th day on Potato Dextrose Agar (PDA) at 25°C, and fungal viability after one year by direct culture and conversion to yeast form in GPYA at 35°C. Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated *T17LM1 differs statistically from T19B7-2 and T20B13-1, by one way ANOVA and Tukey's Test. View Large Table 4. Colony morphology of Paracoccidioides brasiliensis isolates from armadillos, evaluated at 30th day on Potato Dextrose Agar (PDA) at 25°C, and fungal viability after one year by direct culture and conversion to yeast form in GPYA at 35°C. Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated *T17LM1 differs statistically from T19B7-2 and T20B13-1, by one way ANOVA and Tukey's Test. View Large Six isolates growing on sealed plates containing PDA at 25°C were maintained for 1 year under this condition, when they produced a sparse radial growth. Mycelial fragments, containing conidia (observed microscopically, not shown here), were then collected and seeded on GPYA at 35°C, which subsequently produced typical yeast colonies in three out of the six isolates evaluated (Table 4). Yeast cells in cotton blue preparation present evidence of excellent vigorous growth, such as yeasts with intense multibudding daughter cells, well-defined walls and homogenous blue-colored cytoplasm. Discussion This work took advantage of using armadillos as a natural animal model for studying biological aspects of PCM agents. Despite intense advances in the knowledge of fungal genomics and phylogeny, and the emergence of new important endemic areas of PCM, mostly in the central and northern regions of Brazil,41 the ecology of Paracoccidioides species remains poorly comprehended. Herein we confirm the high frequency of P. brasiliensis infection in armadillos in three distinct areas, two located in the southeast and one in the central region of Brazil. The mesenteric lymph nodes were the organ with the highest positivity of isolation, followed by the spleen and liver. The recovery of P. brasiliensis in armadillos from the southeast region occurred exclusively in the mesenteric lymph nodes, whereas in those from the country's central region, the fungus was distributed among the three organs (spleen, liver, and mesenteric lymph nodes). It was possible to detect the presence of the pathogen, and at the same time define its genotypes, in the three well-defined geographic areas, which is particularly welcome for further ecological and epidemiological studies. All armadillo isolates, as well as some recent human clinical isolates from the Botucatu endemic area (São Paulo state), were genotyped in order to define species, according to the latest phylogenetical and taxonomical proposal for Paracoccidioides genus.13,14 Despite persistent efforts, we have not succeeded in obtaining human clinical isolates from the Alta Floresta (MT) region, which was recently characterized as an important endemic area for PCM.42,43 According to Volpato et al. 201643, who evaluated oral manifestation in patients from Mato Grosso state, PCM occurs in 37% of counties in a heterogeneous manner, with most cases occurring in the north of the state, where Alta Floresta County ranks first in the number of cases. Although P. lutzii is supposed to be prevalent in this endemic area,20 and it has been isolated from patients of this region,44 the two positive armadillos from Alta Floresta harbored only P. brasiliensis and no P. lutzii species. In fact, P. lutzii has never been isolated from armadillos.24 Curiously, we observe herein that all armadillos collected in Alta Floresta (central region) and on Cerrito Island (southeast region) presented the same S1b genotype, which was the most frequent genotype among armadillo isolates (12 occurrences). The S1a genotype was the second most frequent in armadillos (three occurrences) and also the genotype found in the four recent human isolates from the Botucatu endemic area. The PS2 genotype (P. americana species) presented only one occurrence in the armadillos, confirming previous findings that this species is relatively rare both in human and armadillos, when comparing with P. brasiliensis (S1a and S1b genotypes).12 In the armadillos collected at Edgardia Farm (Southeast region), it was possible to determine the occurrence of multiple infection by distinct genotypes in the two evaluated animals—S1a and S1b genotypes of P. brasiliensis species in the armadillo T17 and S1a and PS2 genotypes (P. brasiliensis and P. americana) in armadillo T18—confirming what had already been observed by Sano et al.45 It is also important to mention that the PS2 genotype (P. americana), which is phylogenetically highly distinct from other P. brasiliensis cryptic species,11,13 was herein isolated in the armadillo T18, which was collected in the same exact area (Edgardia Farm) where this genotype had already been isolated in a previous animal (T10), evaluated 15 years ago.11,26 The occurrence of three distinct fungal groups of Paracoccidioides (S1a, S1b, and PS2) on Edgardia Farm might be associated with the local environmental conditions that comprise the simultaneous presence of diversified natural vegetation, such as semideciduous and savanna forest, as well as small rivers, channels, and flooded paddy-field agricultural areas.46 The presence of these pathogens was also detected by molecular methods in tissues of all animals, in feces of one animal, and in soil samples collected in armadillos’ burrows and foraging sites in the three environmental areas. The sequencing of these environmental amplicons in an attempt to define the species suggested that P. brasiliensis sensu lato and P. lutzii species occur simultaneously in both the central and southeastern regions of Brazil, as reported previously.23,24 The samples “Burrow Pasture MT05 and MT04” collected in Alta Floresta (MT) clustered in a different branch from the clinical isolate Pb01 (XR00155186) and the other P. lutzii amplicons. Arantes et al. (2016)24 performed phylogenetic analysis on environmental sequences obtained from soil and aerosols and observed that sequences of P. lutzii were grouped into two distinct clades, clade I of the Central-West (GO) and clade II of the Northern Regions (RO). These findings reinforce the genetic diversity of Paracoccidioides spp. in the environment and also suggest that cryptic species may exist within P. lutzii. Paracoccidioides brasiliensis sensu lato was also detected by molecular methods in soil samples collected in a horticultural field of Alta Floresta (MT), whose owner presents PCM and was under treatment when the samples were collected. Horticultural fields present abundant organic matter, high humidity, since they are regularly irrigated, and the addition of fertilizers, which might comprise favorable environmental conditions for development of the fungus in its mycelial phase, as already suggested.9,32,39,47,48 In addition, the nine-banded armadillo frequently visits these areas in search of food, such as earthworms. Horticultural workers presenting intense daily activities in such endemic areas should be monitored for PCM infection. The reason why P. lutzii has not been already isolated from armadillos is an open question. It has been argued that P. lutzii is less virulent or produces fewer infective conidia than P. brasiliensis or might occupy a distinct ecological niche, since they diverged from each other around 22.5 million years ago.12,13 In addition, P. lutzii may have developed different evolutionary strategies and may have hosts other than armadillos. The mycological findings herein observed seem to support the existence of ecological or other biological differences between P. brasiliensis and P. lutzii species, although additional experimental data must be obtained for confirmation. For instance, when these species were cultured on Soil Extract Agar (SEA), P. brasiliensis generally presents a faster conversion from yeast to mycelium, as well as larger mycelial colonies and more conidial production than P. lutzii. It is worth noting that P. brasiliensis and P. lutzii differed in their ability to survive in vitro over a long period (5 months, 3 in SEA and 2 in Mycosel agar) under stress conditions (water deficit). This suggests that P. brasiliensis may be more tolerant of adverse conditions in the environment, thereby augmenting its chance of causing infection in their hosts in comparison with P. lutzii. Lastly, another curious mycological observation was the ability of P. brasiliensis to maintain its viability when growing as a mycelial form in PDA at 25°C for 1year and, most importantly, with the capacity for easily converting to yeast form when seeded directly in a relatively richer medium such as GPYA and cultured at 35°C. The yeast cells herein obtained, since the first-round growth, present evidence of good viability and vigor, presenting typical well-rounded mother cells with a large number of buddings. These findings indicate that the fungus might survive for a longer period in soil in a state of discrete mycelial form and easily convert to yeast when exposed to a richer substrate and higher temperature (35°C) or when introduced into a mammalian host. Acknowledgments We thank the owners of Cerrito Island—Edson Gruppi, Edson Luiz Gruppi and Silvio José Gruppi—for allowing our studies in this private area. We also thank the Foundation for Research Support of the State of Mato Grosso (FAPEMAT) for granting a scholarship and IBAMA for the environmental license, as well as the forestry engineer Gabriel Moraes Gasparoto for the services rendered in Geoprocessing and map elaboration. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Shikanai-Yasuda MA , Telles Filho F de Q , Mendes RP , Colombo AL , Moretti ML . Guideliness in paracoccidioidomycosis . Rev Soc Bras Med Trop . 2006 ; 39 : 297 – 310 . Google Scholar Crossref Search ADS PubMed 2. Martinez R . Epidemiology of Paracoccidioidomycosis . Rev Inst Med Trop Sao Paulo . 2015 ; 57 : 11 – 20 . Google Scholar Crossref Search ADS PubMed 3. Coutinho ZF , Silva D , Lazéra M et al. Paracoccidioidomycosis mortality in Brazil (1980–1995) . Cad Saúde Pública . 2002 ; 18 : 1441 – 1454 . Google Scholar Crossref Search ADS PubMed 4. Coutinho ZF , Wanke B , Travassos C , Oliveira RM , Xavier DR , Coimbra CEA . Hospital morbidity due to paracoccidioidomycosis in Brazil (1998–2006) . Trop Med Int Heal . 2015 ; 20 : 673 – 680 . Google Scholar Crossref Search ADS 5. Colombo AL , Tobón A , Restrepo A , Queiroz-Telles F , Nucci M . Epidemiology of endemic systemic fungal infections in Latin America. Med Mycol . 2011 ; 49 : 785 – 798 . Google Scholar PubMed 6. Lacaz C da S , Porto E , Martins JEC . Medical Mycology, Actinomycetes and Algae of Medical Importance, 8th edn. Sarvier, São Paulo , 1991 , 695 p. [in Portuguese] . 7. Paniago AM , Ivan J , Aguiar A , Aguiar ES . Paracoccidioidomycosis: a clinical and epidemiological study of 422 cases observed in Mato Grosso do Sul . Rev Soc Bras Med Trop . 2003 ; 36 : 455 – 459 [in Galician] . Google Scholar Crossref Search ADS PubMed 8. Shikanai-Yasuda MA . Paracoccidioidomycosis Treatment . Rev Inst Med Trop Sao Paulo . 2015 ; 57 : 31 – 37 . Google Scholar Crossref Search ADS PubMed 9. Restrepo A , McEwen JG , Castañeda E . The habitat of Paracoccidioides brasiliensis: how far from solving the riddle? Med Mycol . 2001 ; 39 : 233 – 241 . Google Scholar Crossref Search ADS PubMed 10. Untereiner WA , Scott JA , Sigler L , Canada TG . The Ajellomycetaceae, a new family of vertebrate-associated Onygenales. Mycologia . 2004 ; 96 : 812 – 821 . Google Scholar Crossref Search ADS PubMed 11. Matute DR , McEwen JG , Puccia R et al. Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies . Mol Biol Evol . 2006 ; 23 : 65 – 73 . Google Scholar Crossref Search ADS PubMed 12. Theodoro RC , Teixeira M , de M , Felipe MSS et al. Genus Paracoccidioides: species recognition and biogeographic aspects . PLoS One . 2012 ; 7 : e37694 . Google Scholar Crossref Search ADS PubMed 13. Muñoz JF , Farrer RA , Desjardins CA et al. Genome diversity, recombination, and virulence across the major lineages of Paracoccidioides . mSphere . 2016 ; 1 : 1 – 18 . Google Scholar Crossref Search ADS 14. Turissini DA , Gomez OM , Teixeira MM , McEwen JG , Matute DR . Species boundaries in the human pathogen Paracoccidioides . Fungal Genet Biol . 2017 ; 106 : 9 – 25 . Google Scholar Crossref Search ADS PubMed 15. Ferreira MS , Freitas LH , Lacaz C da S et al. Isolation and characterization of a Paracoccidioides brasiliensis strain from a dogfood probably contaminated with soil in Uberlândia, Brazil. Med Mycol . 1990 ; 28 : 253—256 . Google Scholar Crossref Search ADS 16. Garcia NM , Del Negro GM , Heins-Vaccari EM , de Melo NT , de Assis CM , Lacaz C da S . Paracoccidioides brasiliensis, a new sample isolated from feces of a penguin (Pygoscelis adeliae) . Rev Inst Med Trop Sao Paulo . 1993 ; 35 : 227 – 235 . 17. de Farias MR , Zeni Condas LA , Ribeiro MG et al. Paracoccidioidomycosis in a dog: case report of generalized lymphadenomegaly . Mycopathologia . 2011 ; 172 : 147 – 152 . Google Scholar Crossref Search ADS PubMed 18. Roberto TN , Rodrigues AM , Hahn RC , de Camargo ZP . Identifying Paracoccidioides phylogenetic species by PCR-RFLP of the alpha-tubulin gene . Med Mycol . 2016 ; 54 : 240 – 247 . Google Scholar Crossref Search ADS PubMed 19. De Albornoz MB . Isolation of Paracoccidioides brasiliensis from rural soil in Venezuela . Sabouraudia . 1971 ; 9 : 248 – 253 . Google Scholar Crossref Search ADS PubMed 20. Teixeira MM , Theodoro RC , de Carvalho MJ A et al. Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus . Mol Phylogenet Evol . 2009 ; 52 : 273 – 283 . Google Scholar Crossref Search ADS PubMed 21. Teixeira Mde M , Theodoro RC , Derengowski Lda S , Nicola AM , Bagagli E , Felipe MS . Molecular and morphological data support the existence of a sexual cycle in species of the genus Paracoccidioides . Eukaryot Cell . 2013 ; 12 : 380 – 389 . Google Scholar Crossref Search ADS PubMed 22. Takayama A , Itano EN , Sano A , Ono MA , Kamei K . An atypical Paracoccidioides brasiliensis clinical isolate based on multiple gene analysis . Med Mycol . 2010 ; 48 : 64 – 72 . Google Scholar Crossref Search ADS PubMed 23. Arantes TD , Theodoro RC , Da Graça Macoris SA , Bagagli E . Detection of Paracoccidioides spp. in environmental aerosol samples . Med Mycol . 2013 ; 51 : 83 – 92 . Google Scholar Crossref Search ADS PubMed 24. Arantes TD , Theodoro RC , Teixeira M , de M , Bosco S , de MG , Bagagli E . Environmental mapping of Paracoccidioides spp. in Brazil reveals new clues into genetic diversity, biogeography and wild host association . PLoS Negl Trop Dis . 2016 ; 10 : 1 – 18 . 25. Bagagli E , Sano A . Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus novemcinctus) capured in an endemic area of paracoccidioidomycosis . Am J Trop Med Hyg . 1998 ; 58 : 505 – 512 . Google Scholar Crossref Search ADS PubMed 26. Bagagli E , Franco M , Bosco SDMG , Hebeler-Barbosa F , Trinca LA , Montenegro MR . High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study . Med Mycol . 2003 ; 41 : 217 – 223 . Google Scholar Crossref Search ADS PubMed 27. Corredor GG , Peralta LA , Castãno JH , Zuluaga JS , Henao B , Restrepo A . The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis. Molecular identification of the isolate . Med Mycol . 2005 ; 43 : 275 – 280 . Google Scholar Crossref Search ADS PubMed 28. McDonough CM. , Loughry WJ. Behavioral ecology of armadillos . In: Vizcaíno SF , Loughry WJ , eds. The Biology of the Xenarthra . Gainesville, FL : University Press of Florida ; 2008 : 281 – 293 . 29. INPE . National Institute of Spacial Researches . 2016 . Available at: http://www.inpe.br/ [in Portuguese] . 30. IBGE . Brazilian Institute of Geography and Statistics . 2016 . Available at: http://www.ibge.gov.br [in Portuguese] . 31. McCullough M , DiSalvo A , Clemons K , Park P , Stevens D . Molecular epidemiology of Blastomyces dermatitidis . Clin Infect Dis . 2000 ; 30 : 328 – 335 . Google Scholar Crossref Search ADS PubMed 32. Theodoro RC , Candeias JMG , Araújo JP et al. Molecular detection of Paracoccidioides brasiliensis in soil . Med Mycol . 2005 ; 43 : 725 – 729 . Google Scholar Crossref Search ADS PubMed 33. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . MEGA6: Molecular evolutionary genetics analysis version 6.0 . Mol Biol Evol . 2013 ; 30 : 2725 – 2729 . Google Scholar Crossref Search ADS PubMed 34. NCBI . Basic Local Alignment Search Tool . 2015 . Available at: http://www.blast.ncbi.nlm.nih.gov/blast . 35. Irinyi L , Serena C , Garcia-Hermoso D et al. International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database—the quality controlled standard tool for routine identification of human and animal pathogenic fungi . Med Mycol . 2015 ; 53 : 313 – 337 . Google Scholar Crossref Search ADS PubMed 36. Stocker HJ . Of Mathematics and Computational Science . New York : Springer ; 1998 . 37. Tamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases . Mol Biol Evol . 1992 ; 9 : 678 – 687 . Google Scholar PubMed 38. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences . J Mol Evol . 1980 ; 16 : 111 – 120 . Google Scholar Crossref Search ADS PubMed 39. Terçarioli GR , Bagagli E , Reis GM et al. Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection . BMC Microbiol . 2007 ; 7 : 92 . Google Scholar Crossref Search ADS PubMed 40. Larone Davise H. Medically Important Fungi: A Guide to Identification . 5th ed. Washington, DC : ASM Press , 2011 . 41. Vieira GD , da Cunha Alves T , Dias de Lima SM , Aranha Camargo LM , de Sousa CM . Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the desease . Rev Soc Bras Med Trop . 2014 ; 47 : 63 – 68 . Google Scholar Crossref Search ADS PubMed 42. Carvalhosa AA , Borges FT , França DCC , Queiroz RR , Moimaz SAS GC . Paracoccidioidomycosis prevalence in a public laboratory of the Brazilian unified health system . J Oral Diag . 2016 ; 1 : 31 – 35 . 43. Volpato MCPF , Volpato LER , Guedes OA , Musis CR , Estrela CRA , Carvalhosa AA . Spatial distribution of cases of paracoccidioidomycosis with oral manifestations in the state of Mato Grosso, Brazil. Rev Odontol Bras Cent . 2016 ; 25 : 84 – 87 [in Portuguese] . 44. Hahn RC , Rodrigues AM , Fontes CJF et al. Fatal fungemia due to Paracoccidioides lutzii . Am J Trop Med Hyg . 2014 ; 91 : 394 – 398 . Google Scholar Crossref Search ADS PubMed 45. Sano A , Defaveri J , Tanaka R et al. Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo (Dasypus novemcinctus). Mycopathologia . 1998 ; 144 : 61 – 65 . Google Scholar Crossref Search ADS PubMed 46. Jorge LAB , Sartori MS . Land use and temporal analysis of natural vegetation at Edgardia experimental farm Botucatu-SP. Rev Árvore . 2002 ; 26 : 585 – 592 . Google Scholar Crossref Search ADS 47. Salazar ME , Restrepo A . Morphogenesis of the mycelium-to-yeast transformation in Paracoccidioides brasiliensis . Sabouraudia . 1985 ; 23 : 7 – 11 . Google Scholar Crossref Search ADS PubMed 48. Barrozo LV , Benard G , Silva MES , Bagagli E , Marques SA , Mendes RP . First description of a cluster of acute/subacute paracoccidioidomycosis cases and its association with a climatic anomaly . PLoS Negl Trop Dis . 2010 ; 4 : e643 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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1369-3786
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1460-2709
D.O.I.
10.1093/mmy/myx142
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Abstract

Abstract Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition. Paracoccidioides spp., Onygenales, phylogeny, molecular detection, Dasypus novemcinctus Introduction Paracoccidioides brasiliensis and P. lutzii are the etiological agents of paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America, where Brazil, Colombia, and Venezuela are the leading countries in number of cases.1,2 In Brazil, PCM was already confirmed in 27% of the counties, causing hospitalization in 4.3 per 1.0 million inhabitants, and has been ranked as the eighth most frequent cause of death due to chronic or recurrent infectious and parasitic diseases.3–5 Adult male rural workers presenting the chronic clinical form of the disease are the main group affected, although an acute severe form affecting mainly children or young adults may also occur in nearly 5–10% of cases.6,7 Treatment of PCM still presents enormous challenges, with frequent relapses and debilitating sequelae.8 These pathogens belong to the family Ajellomycetaceae (Onygenales Order) and are typically thermodimorphic, presenting yeast cells when they grow in the animal tissues and mycelia in the environment where they produce the infective propagule.9,10 Molecular phylogenetic studies have indicated two distinct clades among the genus Paracoccidiodes: the brasiliensis clade that harbors five phylogenetic cryptic species (S1a, S1b, PS2, PS3, and PS4) and the lutzii clade containing P. lutzii species.11–13 The cryptic species PS2, PS3, and PS4 were recently reclassified as formal species: P. americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4).14 The geographic distribution of Paracoccidioides spp. is now being revealed, although the final picture is still far from being completed.12P. brasiliensis sensu strictu (S1a and S1b) appears to be widely distributed in Brazil and Argentina, where it has been frequently isolated from humans and armadillos.14P. americana (PS2) seems to occur less frequently, both in human and armadillos in Brazil, although it was already recovered from a Venezuelan patient, and in some uncommon cases/situations, such as from an infected dog in southern Brazil, from dog food contaminated with soil (in the southeast region of Brazil) and in feces of an Antarctic penguin.15–17P. restrepiensis (PS3) occurs mainly in Colombia, both in human and armadillos and in some patients from Brazil.18P. venezuelensis (PS4) is restricted to the single environmental isolate obtained from Venezuelan soil.14,19 The species P. lutzii is thought to occur in the central western and northern regions of Brazil, including Goiás, Mato Grosso, Pará and Rondônia states.20,21 However, the real distribution of P. lutzii has not been fully comprehended until now, since it was already isolated from single cases of patients living in Ecuador and also in southeastern Brazil,22 and in addition, it has been molecularly detected in aerosol and soil samples outside the clinical range of the disease provoked by this species.23,24 Natural infection by P. brasiliensis in the nine-banded armadillo Dasypus novemcinctus has been observed repeatedly in several PCM endemic areas of Brazil and Colombia, where the pathogen was also isolated from the naked-tailed armadillo species Cabassous centralis.25–27 Armadillos have a less active cellular immune system, body temperature slightly lower than other mammals and are constantly in search of food and protection in the soil, which makes them particularly susceptible to infection by soil-dwelling pathogens, including P. brasiliensis. In addition, armadillos present a small home range and no regular migration habits, which makes them ideal for registering the environmental presence of the fungus and for mapping the genotypes that occur in the different endemic areas.26,28 In this work, we aimed at isolating and molecularly detecting Paracoccidioides spp. in armadillo tissues and associated soil samples, which were collected in three well-defined geographic areas, including: (i) a small isolated fluvial island (Cerrito), in São Manuel/SP, (ii) a positive farm (Edgardia) in Botucatu/SP, where P. brasiliensis and the less common P. americana species (former PS2 genotype) were already isolated from armadillos, (iii) a new endemic area (Alta Floresta) from Central region of Brazil, in Alta Floresta and Paranaíta/MT, the probable occurrence area of P. lutzii, whose ecology and geographic distribution is poorly comprehended. Some mycological features of the isolates such as yeast-to-mycelial conversion, growth, and conidial production in soil-related substrates were also evaluated. The data herein obtained present biological and ecological differences in Paracoccidioides spp. that might be relevant for PCM epidemiology. Methods Characteristics of the study areas The collections were carried out in three distinct areas, two located in the southeast and one in the central region of Brazil (Fig. S1). Area 1 (Edgardia Farm) is an experimental farm of São Paulo State University (UNESP), Botucatu, São Paulo state. The S1 genotype of P. brasiliensis and P. americana (former PS2 genotype) were already isolated from nine-banded armadillos (D. novemcinctus), and PCM has been diagnosed in rural workers that have lived and/or worked there. Some of these patients died of PCM. Area 2 is a small fluvial island (Cerrito Island, located in São Manuel County, São Paulo state) that was established in the Tietê River in 1962, when the construction of the Barra Bonita Dam caused a natural population of armadillos to become isolated from the rest of the continent; the location is accessible only by boat and has very little human presence. Area 3 is located in Alta Floresta and Paranaíta Counties, Mato Grosso state, central region of Brazil, under the deforestation arc 29 of the Amazon Biome, where the local economy is mainly based on agriculture and livestock (IBGE, 2016).30 PCM cases have been diagnosed in Alta Floresta, despite its low level of local epidemiological vigilance. Additional ecological data from the sampled areas are provided in Table S1 (Supplementary Material). Animal and soil sampling After obtaining the legal authorization from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA/ICMBio, License number 30585-1) and also from the local Ethics Committee on the Use of Animals (CEUA, protocol number 737), seven armadillos (six males and one female) were captured using traps settled on the animal track or at the entrance of their burrows: two animals from Edgardia Farm (area 1), two from Cerrito Island (area 2), and three from Alta Floresta and Paranaíta (area 3). The armadillos were maintained in appropriate cages for up to 24h and then evaluated. Soil samples (n = 20) were collected in duplicate (four samples from Edgardia Farm, three from Cerrito Island, and 13 from Alta Floresta and Paranaíta) from the armadillo burrows (at 0 and 0.3-m depth) and animal foraging sites located in forest (areas 1 and 2) and also pasture and horticultural fields (area 3). The samples were placed in 50-ml sterile universal bottles, sealed, identified, and stored in an isothermal box at room temperature until their evaluation for fungal molecular detection. Additional soil samples were also collected for determination of their physical and chemical properties, which was carried out at the Soil Fertility Laboratory of the School of Agriculture FCA /UNESP, as well as for preparation of Soil Extract Agar to culture the fungal isolates. Geographic coordinates of the collected sites (armadillos and soil samples, Table S2) were obtained using a Global Positioning System (GPS, Garmin GPS 12) and plotted in the Geographic Information Systems (GIS) by the software QGIS version 2.8. Fungal isolation from the armadillos The animals were anesthetized intramuscularly (Zoletyl® 50 - Virbac, 0.2 ml/kg), euthanized by cardiac puncture and necropsied. The whole liver, spleen, and mesenteric lymph nodes were collected aseptically, as well as feces from two animals. The organs were cleaned in 70% (v/v) alcohol and sterile saline solution (0.9% w/v). One piece of each organ was kept in absolute alcohol and used for molecular detection. The remaining organs were fragmented into small pieces (2–3 mm) and seeded in Mycosel® Agar supplemented with gentamicin (50 μg/ml). Plates were incubated at 35°C and evaluated for fungal growth for up to 60 days. Suspected colonies of Paracoccidioides spp. were screened microscopically by staining with Lacto-phenol cotton blue and subcultured in glucose, peptone, yeast extract and agar (GPYA, 2% glucose, 1% peptone, 0.5% yeast extract and 1% agar) for further mycological and molecular studies. Clinical human isolates were also included herein for molecular and/or mycological comparisons, including four recent isolates (PSM, PbD, 8652 and 3051) of P. brasiliensis obtained from patients assisted at the Dermatology Service of the University Hospital of Botucatu Medical School, São Paulo, Brazil (HC/UNESP), one PS3 isolate (BACR) from Colombia, and three P. lutzii isolates originating from patients (Pb01 and Pb66, from Goiânia-GO, and PbEE, from Cuiabá-MT) in Brazil. Molecular studies DNA extraction For Paracoccidioides spp. isolates, the DNA was extracted from yeast cells, according to the protocol described by McCullough et al.31 with minor modifications (using yeast cells cultured for 7 days on GPYA at 37°C, which were disrupted using the Precellys (Bertin Technologies, MD, USA) equipment). For armadillo tissue samples (spleen, liver, and mesenteric lymph nodes), animal feces and soil samples, the DNA extraction was accomplished by using the commercial kits Macherey-Nagel (MN), QIAamp® DNA Stool (QIAGEN) and Power Soil (MO BIO Laboratories Inc.), respectively. All extracted DNA samples were quantified on the NanoVue (GE Healthcare) spectrophotometer and stored at −20°C until used. Detection in soil, animal tissue, and feces samples The molecular detection was carried out by a nested polymerase chain reaction (PCR) targeting rDNA with primers (ITS1/ITS4 or ITS4/ITS5) in the first PCR reaction, followed by a second amplification with the inner primers (PbITS-E/PbITS-T) (Table 1), supposed to be specific for P. brasiliensis and P. lutzii.23,32 All the PCR products were detected by 1.5% agarose gel electrophoresis stained with SYBR Safe (Invitrogen). The soil environmental amplicons were also purified and sequenced as described below. Table 1. Paracoccidioides spp. isolation by direct culture and molecular detection by nested/PCR in tissue samples (liver, spleen, and mesenteric lymph nodes) of armadillos (Dasypus novemcinctus) captured in two distinct endemic regions of paracoccidioidomycosis in Brazil (Alta Floresta/MT and Botucatu/SP). Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − *Positive growth over the total number of fragments (%). (−) Negative sample (+) Positive sample. MT, Mato Grosso State; SP, São Paulo State, Brazil. View Large Table 1. Paracoccidioides spp. isolation by direct culture and molecular detection by nested/PCR in tissue samples (liver, spleen, and mesenteric lymph nodes) of armadillos (Dasypus novemcinctus) captured in two distinct endemic regions of paracoccidioidomycosis in Brazil (Alta Floresta/MT and Botucatu/SP). Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − Liver Spleen Mesenteric lymph nodes Weight Geographic *Fungal Molecular *Fungal Molecular *Fungal Molecular Armadillo Sex (kg) region (area) culture Detection culture Detection culture Detection T17 Male 4.7 Southeast, SP (Edgardia Farm) 0 − 0 − 4/147 (2.72) + T18 Male 3.9 Southeast, SP (Edgardia Farm) 0 − 0 − 6/59 (10.16) − T19 Male 6.0 Central/MT (Alta Floresta) 7/910 (0.76) + 29/230 (12.60) + 0 − T20 Male 6.0 Central/MT (Alta Floresta) 0 + 2/270 (0.74) + 0 + T21 Male 5.0 Central/MT (Alta Floresta) 0 − 0 + 0 − T22 Female 3.9 Southeast/SP (Cerrito Island) 0 + 0 − 8/68 (11.76) − T23 Male 4.3 Southeast/SP (Cerrito Island) 0 + 6/395 (1.51) + 19/84 (22.61) − *Positive growth over the total number of fragments (%). (−) Negative sample (+) Positive sample. MT, Mato Grosso State; SP, São Paulo State, Brazil. View Large Molecular characterization and phylogenetic analysis of the isolates and environmental amplicons The isolates were characterized by sequencing ITS rDNA and gp43 exon 2 loci. Other DNA regions, such as arf (adenylyl ribosylation factor) gene were sequenced and analyzed for some isolates. In addition, the PCR-RFLP (restriction fragment length polymorphism) of tub1 gene was also carried out to enable differentiation among Paracoccidioides spp. without DNA sequencing18 (details below). All the primers and annealing temperatures employed are listed in Table S3. PCR reactions were performed using a Veriti Thermocycler (Applied Biosystems, Foster City, CA, USA) and GoTaq®Green Master Mix (Promega, Madison, WI, USA), with 25 μl of reaction mixture-containing 3 μl of genomic DNA (300 ng/μl) and 0.5 μM of each primer. For DNA sequencing, the amplicons were purified using ExoProStar (GE Healthcare, Milwaukee, WI, USA) and sequenced in the ABI 3500 DNA Analyzer (Applied Biosystem, Foster City, CA, USA), according to the manufacturer's instructions. The quality of sequences was verified by the software Sequencing Analysis (Applied Biosystems) and only nucleotides with Phred ≥20 were included. Sequencing edition and phylogenetic analysis were accomplished using the software MEGA v6.0.33 Molecular identification was performed by the Basic Local Alignment Search Tool (BLAST)34 from the National Center for Biotechnology Information (NCBI) and International Society for Human Animal Mycology (ISHAM) ITS Databases.35 Alignments of the sequences were accomplished through Clustal W algorithm. Three different phylogenetic constructions were made via the maximum likelihood (ML) method36 by applying Tamura-Nei model37 for ITS1-5.8S-ITS2 and soil environmental amplicons, Kimura 2-parameter model38 for the gp43 exon 2 gene, all with bootstrap of 1000 replicates.37 The PCR-RFLP analysis was performed as described by Roberto et al., 201518 but with the addition of new primers herein designed (Table S3) in order to better amplify the tub1 gene of both Paracoccidioides species. PCR products were visualized on agarose gel (1%) and then subjected to restriction enzyme digestion: 13 μl ultrapure water, 3 μl tub1-PCR, 2 μl 10× of digestion buffer and 1 μl of each endonuclease BclI (10 U/μl) and MspI (10 U/μl), both from Thermo Scientific (Massachusetts, USA). The digestion was incubated at 37°C for 2 hours, and the digested products were visualized on 2.5% agarose gel with electrophoresis at 100 V for 120 min.18 DNA samples from genotypes previously characterized12, namely, Bt84, EPM83, EPM77, Pb18, T16B1, T10B1, and PbDog were also included in this analysis. The DNA sequences from fungal isolates and soil environmental amplicons herein obtained were deposited in the GenBank with accession numbers in parentheses: for ITS sequences of the isolates (KX774393-KX774411), gp43 exon 2 (KY963798-KY963822), arf (KY963821-KY963822), and ITS sequences from environmental amplicons (MF078064-MF078073). Mycological studies Fungal growth, transition from yeast to mycelia, conidia production, and viability in Soil Extract Agar (SEA) Soil extracts (SE) were prepared with soil samples collected at area 1 (Edgardia Farm, Southeast region of Brazil) and Area 3 (Alta Floresta, Central region of Brazil) and performed according to the description of Terçarioli et al.39 Three isolates of P. brasiliensis (T17LM1, T18LM1, 3051) and three of P. lutzii (Pb66, PbEE, Pb01) were evaluated by seeding four yeast fragments in Petri dishes containing SEA, in triplicate, and incubating at 25°C. The isolates were simultaneously evaluated in SEA prepared with SE extracts originating from the two distinct regions. At the 9th day, the isolates were evaluated in relation to the conversion from yeast (Y) to mycelia (M), and classified as: no conversion (0); little conversion, containing few visible mycelia (+); complete conversion with highly visible mycelia (++). Production of conidia was evaluated by using adhesive tape preparations. Fungal mycelial colonies cultured on SEA at 25°C for 35 days were killed by formaldehyde vapor (1–2 ml of 30% formaldehyde added to the bottom plate, for 3–5 h) and employed to prepare the slides with adhesive tapes (Durex, 3M) stained with Lacto-phenol cotton blue. Conidia were counted in at least five fields, at 40× objective magnification, in the photomicrography system Olympus (PM 30), coupled with a Leica digital photomicrography (DMLB) camera and the software Leica Qwin Lite 2.5. Counting data were processed via the software GraphPad Prism version 5.01 (San Diego, CA, USA). The slide culture technique was also applied for isolates (P. brasiliensis and P. lutzii) in SEA,40 with minor modifications, in order to better observe and document conidial production, evaluated on the 40th day and photographed as mentioned above. After 3 months of incubation in SEA (25°C), mycelial forms of Paracoccidioides brasiliensis and P. lutzii isolates were evaluated for the possibility of remaining alive. The mycelia of the isolates were scraped and deposited in test tubes containing saline and glass beads, and vortexed for 20s. After one minute at rest, 100 ul volumes of serial dilutions (10−1 and 10−2) were seeded and spread in Petri dishes (duplicate) containing Mycosel Agar, and incubated at 25°C for up to 60 days. The appearance of typical Paracoccidioides spp. colonies in any dilution indicates positive viability of conidia and/or mycelial fragments. Giant colonies on PDA and preservation/viability after 1 year Yeast inoculums of each armadillo isolate (T17LM1, T18LM1, T19F7-2, T19F33, T20B13-1, T22LM1-1, T23LM1-1 and T23B1-1) were cultivated (triplicate) in a Petri dish containing Potato dextrose agar (PDA, Oxoid) at 25°C for 30 days for evaluating the morphological characteristics (verse and reverse) and diameters of the mycelial colonies. The mycelial plate cultures of T18LM1, T19F7-2, T19F33, T20B13-1, T22LM1-1, T23LM1-1 isolates were also maintained for a period of 1 year under the same conditions (at 25°C) and then evaluated for their viability and capacity to convert to yeast form, by seeding the mycelial/conidial inoculums in GPYA tubes, and incubated at 35°C. Statistical analysis The one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test was used to compare conidial production within isolates of P. brasiliensis and P. lutzii species, in the two soil extract agar conditions (soil from central and southeast regions of Brazil), and also to compare the mycelial growth diameters of giant colonies. Results Fungal isolation from armadillos From January to November 2015, seven armadillos (six male and one female) were evaluated, both for fungal culture and molecular detection (Table 2). The fungi were positively cultured in six animals (85%) that had been collected on Edgardia Farm, area 1 (two animals), Cerrito Island, area 2 (two animals) in the southeast region, and Alta Floresta County, area 3 (two animals) in the central region of Brazil. The fungal growth was detected from the 11th until the 43rd day of organ culture at 35°C. The pathogen was cultured mainly in the fragments of the mesenteric lymph nodes (four animals), followed by spleen (three animals) and liver (one animal) (Table 1). The majority of the isolates were obtained in different fragments of the same organs; however, in two animals the fungus was isolated simultaneously in two distinct organs, namely the liver/spleen in armadillo T19 (area 1) and spleen/mesenteric lymph nodes in T23 (area 3). Whenever possible, more than one isolate from each positive animal, preferably from distinct organs, were selected for additional studies. Table 2. Species and genotypes of Paracoccidioides spp. isolates obtained from armadillos (≠T17 to ≠T23) collected in three distinct geographic areas, as well as human patients from Botucatu PCM endemic area and reference strains. Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 *Edgardia Farm (area 1, Botucatu, SP, Southeast Region); Cerrito Island (area 2, São Manuel, SP, Southeast Region); Alta Floresta (area 3, Alta Floresta, MT, Central Region). MT (Mato Grosso), SP (São Paulo), PR (Paraná) and GO (Goiás) are States of Brazil. ND, Not done. View Large Table 2. Species and genotypes of Paracoccidioides spp. isolates obtained from armadillos (≠T17 to ≠T23) collected in three distinct geographic areas, as well as human patients from Botucatu PCM endemic area and reference strains. Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 Molecular Approach Geographic ITS1/ITS2 rDNA PCR/RFLP gp43 EXON-2 Isolate Host Origin* rDNA sequencing tub1 sequencing Reference T17LM1 Armadillo Edgardia Farm P. brasiliensis ND ND This study T17LM2 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T17LM3 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T17LM4 Armadillo Edgardia Farm P. brasiliensis S1 S1b This study T18LM1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM2 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-1 Armadillo Edgardia Farm P. brasiliensis S1 S1a This study T18LM3-3 Armadillo Edgardia Farm P. brasiliensis S1 ND This study T18LM3-4 Armadillo Edgardia Farm P. brasiliensis ND ND This study T18LM3-5 Armadillo Edgardia Farm P. brasiliensis PS2 PS2 This study T19B7-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19F24-1 Armadillo Alta Floresta P. brasiliensis S1 ND This study T19F33-1 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T19B15-2 Armadillo Alta Floresta P. brasiliensis S1 S1b This study T20B13-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T20B15-1 Armadillo Alta Floresta P. brasiliensis ND S1b This study T22LM1-1 Armadillo Cerrito Island P. brasiliensis S1 S1b This study T22LM2-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM1-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23LM2-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23LM3-4 Armadillo Cerrito Island P. brasiliensis ND S1b This study T23B3-1 Armadillo Cerrito Island P. brasiliensis S1 ND This study T23B9-1 Armadillo Cerrito Island P. brasiliensis ND S1b This study PbD Human Botucatu, SP P. brasiliensis ND S1a This study PSM Human Botucatu, SP P. brasiliensis S1 S1a This study 8652 Human Botucatu, SP P. brasiliensis S1 S1a This study 3051 Human Botucatu, SP P. brasiliensis S1 S1a This study Pb18 Human São Paulo, SP P. brasiliensis S1 S1b Matute et al., 2006 T16B1 Armadillo Botucatu, SP P. brasiliensis S1 S1a Aranteset al., 2013 BACR Human Colombia P. brasiliensis PS3 PS3 Almeida et al., 2015 BT84 Human Botucatu, SP P. brasiliensis PS2 ND Theodoroet al., 2012 EPM83 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 EPM77 Human Colombia P. brasiliensis PS3 ND Theodoro et al., 2012 T10B1 Armadillo Botucatu, SP P. brasiliensis PS2 ND Matute et al., 2006 PbDog Dog Curitiba, PR P. brasiliensis PS2 ND Theodoro et al., 2012 PbEE Human Cuiabá, MT P. lutzii ND ND Theodoro et al., 2012 Pb66 Human Goiânia, GO P.lutzii ND ND Theodoro et al., 2012 *Edgardia Farm (area 1, Botucatu, SP, Southeast Region); Cerrito Island (area 2, São Manuel, SP, Southeast Region); Alta Floresta (area 3, Alta Floresta, MT, Central Region). MT (Mato Grosso), SP (São Paulo), PR (Paraná) and GO (Goiás) are States of Brazil. ND, Not done. View Large Molecular characterization and phylogenetic analysis of the isolates All armadillo isolates were molecularly identified as P. brasiliensis sensu lato, with no occurrence of P. lutzii, according to ITS rDNA sequencing (Table 2 and Fig. 1). The analyses of tub1 gene PCR-RFLP and of gp43 Exon-2 DNA sequencing enabled a better characterization of the isolates and determination of their genotypes or cryptic species. Among the armadillo isolates was observed the presence of S1a (three occurrences) and S1b genotypes (12 occurrences) of P. brasiliensis species and one occurrence of PS2 genotype (P. americana). In the armadillos collected at Edgardia Farm (southeast region), more than one genotype was simultaneously detected in the same animal, namely, the S1a and S1b genotypes of P. brasiliensis in armadillo T17, and S1a (P. brasiliensis) and PS2 (P. americana) in armadillo T18, specifically in the mesenteric lymph nodes. All armadillos collected on Cerrito Island (southeast region) and in Alta Floresta (central region) presented only the genotype S1b of P. brasiliensis. Four recent clinical isolates from patients of the Botucatu PCM endemic area (southeast region, where areas 1 and 2 are located) were also characterized herein as S1a genotypes of P. brasiliensis (Table 2 and Fig. 2). Figure 1. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA sequences from multiple P. brasiliensis isolates from armadillos (marked with T17 to T23) and human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from P. brasiliensis and P. lutzii were also included, and H. capsulatum was used as outgroup. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Figure 1. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA sequences from multiple P. brasiliensis isolates from armadillos (marked with T17 to T23) and human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from P. brasiliensis and P. lutzii were also included, and H. capsulatum was used as outgroup. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Figure 2. View largeDownload slide Phylogenetic analysis of gp43 Exon 2 sequences from multiple isolates of Paracoccidioides spp. obtained from armadillos (marked with T17 to T23), as well as from human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from S1a, S1b, PS2, PS3 and P. lutzii were also included. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Figure 2. View largeDownload slide Phylogenetic analysis of gp43 Exon 2 sequences from multiple isolates of Paracoccidioides spp. obtained from armadillos (marked with T17 to T23), as well as from human clinical isolates obtained from Botucatu (PSM, PbD, 8652, 3051) and Colombia (BACR). Reference sequences from S1a, S1b, PS2, PS3 and P. lutzii were also included. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. The tree with the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The armadillo T18LM3-5 isolate (PS2 genotype) and the clinical Colombian BACR isolate (PS3) were also identified by sequencing the arf gene, confirming their identities with 100% similarity to referenced sequences deposited in the GenBank database. The polymorphic sites of ITS rDNA and gp43 Exon-2 DNA regions detected in P. brasiliensis (S1a and S1b), P. americana (PS2), and P. restrepiensis (PS3) species are shown in Table S4. Molecular detection in animal tissues, feces and soil samples Molecular detection based on nested-PCR indicated positivity in at least one evaluated organ for all animals, including the spleen of armadillo T21 that was negative for fungal culture (Table 2). Using this same technique, we also detected Paracoccidioides spp. in the feces of one armadillo, collected at Edgardia Farm. The pathogens P. brasiliensis sensu lato and P. lutzii were detected in most soil samples from animals’ burrows and foraging areas, collected mainly in forests of the southeast (areas 1 and 2) and central region of Brazil (area 3), where positive samples were also detected in pasture and in a horticultural field (Table S5 and Fig. S2). Eleven environmental soil amplicons generated by the inner primers PbITS-E/PbITS-T (supposed to amplify DNA of all Paracoccidioides species) were sequenced and compared with deposited sequences from GenBank, showing 99% identity with P. lutzii (in 8 out of 11 amplicons) and 100% with P. brasiliensis sensu lato (in 3 out of 11 amplicons), indicating that both species groups occur in soils of both regions. The soil amplicons phylogenetically associated with P. lutzii have a greater genetic variability compared to the amplicons associated with P. brasiliensis clade (Fig. 3). Figure 3. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA partial sequences, obtained by amplification with the inner primers PbITSE/PbITST (Arantes et al., 2012) in soil samples collected in Central/MT and Southeast/SP regions of Brazil, and grouping pattern in relation to P. brasiliensis (accession number AY374337) and P. lutzii (accession number XR001551846) species. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site positions in the final dataset. Figure 3. View largeDownload slide Phylogenetic analysis of ITS1/ITS2 rDNA partial sequences, obtained by amplification with the inner primers PbITSE/PbITST (Arantes et al., 2012) in soil samples collected in Central/MT and Southeast/SP regions of Brazil, and grouping pattern in relation to P. brasiliensis (accession number AY374337) and P. lutzii (accession number XR001551846) species. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model. The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the boostrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site positions in the final dataset. P. brasiliensis and P. lutzii conversion to mycelial and conidial production in SEA Three isolates of P. brasiliensis and three of P. lutzii were evaluated in relation to mycelial conversion and conidial production in soil extract agar (SEA), using SEA prepared with soil samples from the southeast (SP) and central (MT) regions of Brazil. All isolates of P. brasiliensis from both regions converted well, beginning their transition on the 4th day in SEA, while in P. lutzii species, the conversion started only at the 9th day in two of three isolates in SEA prepared with soil from the southeast region—in SEA from the central region the mycelial conversion was less effective, since it occurred in just one isolate of P. lutzii (Table 3). Conidial production appeared to be higher in P. brasiliensis than in P. luzii isolates, whereas SEA from the southeast region seems to be more favorable both for mycelial growth and conidial production (Fig. 4). Figure 4. View largeDownload slide Conidia production of P. brasiliensis and P. lutzii isolates seeded in soil extract Agar (SEA), prepared with soil samples from Central (MT) and the Southeast (SP) regions of Brazil (25ºC). Counting carried out at 35th day in the 40× magnification. Figure 4. View largeDownload slide Conidia production of P. brasiliensis and P. lutzii isolates seeded in soil extract Agar (SEA), prepared with soil samples from Central (MT) and the Southeast (SP) regions of Brazil (25ºC). Counting carried out at 35th day in the 40× magnification. Table 3. Conversion from yeast (Y) to mycelium (M) of P. brasiliensis and P. lutzii isolates, at 9th day of growing in Soil Extract Agar (SEA), prepared with soil samples collected in Central and Southeast Regions, Brazil. Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ No Y to M conversion (0); moderate conversion (+); plenty conversion (++). View Large Table 3. Conversion from yeast (Y) to mycelium (M) of P. brasiliensis and P. lutzii isolates, at 9th day of growing in Soil Extract Agar (SEA), prepared with soil samples collected in Central and Southeast Regions, Brazil. Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ Soil Extract Agar Isolate Species Central Region Southeast Region Pb01 P. lutzii 0 0 Pb66 P. lutzii 0 + PbEE P. lutzii + ++ T17LM1 P. brasiliensis + ++ T18LM1 P. brasiliensis ++ ++ 3051 P. brasiliensis ++ ++ No Y to M conversion (0); moderate conversion (+); plenty conversion (++). View Large Giant colonies on PDA and preservation/viability after 1 year All P. brasiliensis isolates obtained from armadillos presented typical mycelial colonies, such as white cotton and crater-like aspects on the verse, and the presence of brown pigments with some cracks and/or folds on the reverse. The growths were relatively slow, since the colony diameters at the 30th day were less than 2.0 cm in all isolates, except T17LM1 (originating from Botucatu, SP) that differs statistically from T19B7-2 and T20B13-1 (both originated from Alta Floresta, MT), as shown in Table 4. Table 4. Colony morphology of Paracoccidioides brasiliensis isolates from armadillos, evaluated at 30th day on Potato Dextrose Agar (PDA) at 25°C, and fungal viability after one year by direct culture and conversion to yeast form in GPYA at 35°C. Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated *T17LM1 differs statistically from T19B7-2 and T20B13-1, by one way ANOVA and Tukey's Test. View Large Table 4. Colony morphology of Paracoccidioides brasiliensis isolates from armadillos, evaluated at 30th day on Potato Dextrose Agar (PDA) at 25°C, and fungal viability after one year by direct culture and conversion to yeast form in GPYA at 35°C. Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated Colony Features Isolate Geographical origin Diameter (cm) Verse Reverse One year mycelia/conidia viability T17LM1 Botucatu, SP 2.20 ± 0.08* White cotton-like surface, well-defined crater in the center Beige with folds Not evaluated T18LM1 Botucatu, SP 1.73 ± 0.09 White cotton-like surface; well-defined protrusion in the center Without cracks and folds; Brown pigment. Not evaluated T19B7-2 Alta Floresta, MT 1.33 ± 0.04 White cotton-like surface Without cracks and folded; dark brown Positive T19F33 Alta Floresta, MT 1.53 ± 0.20 White cotton-like surface; with depression in the center Without cracks and folded; brownish Positive T20B13-1 Alta Floresta, MT 1.16 ±0.28 White cotton-like surface; point of depression in the center Without cracks and folds; brownish with white borders. Positive T22LM1-1 São Manuel, SP 1.6 ± 0.10 White cotton-like surface; with ripples. A “ring” surrounding the center in a yellowish tone Without cracks and folds; brownish in the center with white borders Negative T23LM1-1 São Manuel, SP 1.6 ± 0.16 White cotton-like surface; with ripples Without cracks and folded; dark brown in the center and beige in the borders Not evaluated T23B1-1 São Manuel, SP 1.5 ± 0.28 White to yellowish cotton-like surface; with crater in the center. Few cracks, without folds; dark brown in the center and beige in the borders Not evaluated *T17LM1 differs statistically from T19B7-2 and T20B13-1, by one way ANOVA and Tukey's Test. View Large Six isolates growing on sealed plates containing PDA at 25°C were maintained for 1 year under this condition, when they produced a sparse radial growth. Mycelial fragments, containing conidia (observed microscopically, not shown here), were then collected and seeded on GPYA at 35°C, which subsequently produced typical yeast colonies in three out of the six isolates evaluated (Table 4). Yeast cells in cotton blue preparation present evidence of excellent vigorous growth, such as yeasts with intense multibudding daughter cells, well-defined walls and homogenous blue-colored cytoplasm. Discussion This work took advantage of using armadillos as a natural animal model for studying biological aspects of PCM agents. Despite intense advances in the knowledge of fungal genomics and phylogeny, and the emergence of new important endemic areas of PCM, mostly in the central and northern regions of Brazil,41 the ecology of Paracoccidioides species remains poorly comprehended. Herein we confirm the high frequency of P. brasiliensis infection in armadillos in three distinct areas, two located in the southeast and one in the central region of Brazil. The mesenteric lymph nodes were the organ with the highest positivity of isolation, followed by the spleen and liver. The recovery of P. brasiliensis in armadillos from the southeast region occurred exclusively in the mesenteric lymph nodes, whereas in those from the country's central region, the fungus was distributed among the three organs (spleen, liver, and mesenteric lymph nodes). It was possible to detect the presence of the pathogen, and at the same time define its genotypes, in the three well-defined geographic areas, which is particularly welcome for further ecological and epidemiological studies. All armadillo isolates, as well as some recent human clinical isolates from the Botucatu endemic area (São Paulo state), were genotyped in order to define species, according to the latest phylogenetical and taxonomical proposal for Paracoccidioides genus.13,14 Despite persistent efforts, we have not succeeded in obtaining human clinical isolates from the Alta Floresta (MT) region, which was recently characterized as an important endemic area for PCM.42,43 According to Volpato et al. 201643, who evaluated oral manifestation in patients from Mato Grosso state, PCM occurs in 37% of counties in a heterogeneous manner, with most cases occurring in the north of the state, where Alta Floresta County ranks first in the number of cases. Although P. lutzii is supposed to be prevalent in this endemic area,20 and it has been isolated from patients of this region,44 the two positive armadillos from Alta Floresta harbored only P. brasiliensis and no P. lutzii species. In fact, P. lutzii has never been isolated from armadillos.24 Curiously, we observe herein that all armadillos collected in Alta Floresta (central region) and on Cerrito Island (southeast region) presented the same S1b genotype, which was the most frequent genotype among armadillo isolates (12 occurrences). The S1a genotype was the second most frequent in armadillos (three occurrences) and also the genotype found in the four recent human isolates from the Botucatu endemic area. The PS2 genotype (P. americana species) presented only one occurrence in the armadillos, confirming previous findings that this species is relatively rare both in human and armadillos, when comparing with P. brasiliensis (S1a and S1b genotypes).12 In the armadillos collected at Edgardia Farm (Southeast region), it was possible to determine the occurrence of multiple infection by distinct genotypes in the two evaluated animals—S1a and S1b genotypes of P. brasiliensis species in the armadillo T17 and S1a and PS2 genotypes (P. brasiliensis and P. americana) in armadillo T18—confirming what had already been observed by Sano et al.45 It is also important to mention that the PS2 genotype (P. americana), which is phylogenetically highly distinct from other P. brasiliensis cryptic species,11,13 was herein isolated in the armadillo T18, which was collected in the same exact area (Edgardia Farm) where this genotype had already been isolated in a previous animal (T10), evaluated 15 years ago.11,26 The occurrence of three distinct fungal groups of Paracoccidioides (S1a, S1b, and PS2) on Edgardia Farm might be associated with the local environmental conditions that comprise the simultaneous presence of diversified natural vegetation, such as semideciduous and savanna forest, as well as small rivers, channels, and flooded paddy-field agricultural areas.46 The presence of these pathogens was also detected by molecular methods in tissues of all animals, in feces of one animal, and in soil samples collected in armadillos’ burrows and foraging sites in the three environmental areas. The sequencing of these environmental amplicons in an attempt to define the species suggested that P. brasiliensis sensu lato and P. lutzii species occur simultaneously in both the central and southeastern regions of Brazil, as reported previously.23,24 The samples “Burrow Pasture MT05 and MT04” collected in Alta Floresta (MT) clustered in a different branch from the clinical isolate Pb01 (XR00155186) and the other P. lutzii amplicons. Arantes et al. (2016)24 performed phylogenetic analysis on environmental sequences obtained from soil and aerosols and observed that sequences of P. lutzii were grouped into two distinct clades, clade I of the Central-West (GO) and clade II of the Northern Regions (RO). These findings reinforce the genetic diversity of Paracoccidioides spp. in the environment and also suggest that cryptic species may exist within P. lutzii. Paracoccidioides brasiliensis sensu lato was also detected by molecular methods in soil samples collected in a horticultural field of Alta Floresta (MT), whose owner presents PCM and was under treatment when the samples were collected. Horticultural fields present abundant organic matter, high humidity, since they are regularly irrigated, and the addition of fertilizers, which might comprise favorable environmental conditions for development of the fungus in its mycelial phase, as already suggested.9,32,39,47,48 In addition, the nine-banded armadillo frequently visits these areas in search of food, such as earthworms. Horticultural workers presenting intense daily activities in such endemic areas should be monitored for PCM infection. The reason why P. lutzii has not been already isolated from armadillos is an open question. It has been argued that P. lutzii is less virulent or produces fewer infective conidia than P. brasiliensis or might occupy a distinct ecological niche, since they diverged from each other around 22.5 million years ago.12,13 In addition, P. lutzii may have developed different evolutionary strategies and may have hosts other than armadillos. The mycological findings herein observed seem to support the existence of ecological or other biological differences between P. brasiliensis and P. lutzii species, although additional experimental data must be obtained for confirmation. For instance, when these species were cultured on Soil Extract Agar (SEA), P. brasiliensis generally presents a faster conversion from yeast to mycelium, as well as larger mycelial colonies and more conidial production than P. lutzii. It is worth noting that P. brasiliensis and P. lutzii differed in their ability to survive in vitro over a long period (5 months, 3 in SEA and 2 in Mycosel agar) under stress conditions (water deficit). This suggests that P. brasiliensis may be more tolerant of adverse conditions in the environment, thereby augmenting its chance of causing infection in their hosts in comparison with P. lutzii. Lastly, another curious mycological observation was the ability of P. brasiliensis to maintain its viability when growing as a mycelial form in PDA at 25°C for 1year and, most importantly, with the capacity for easily converting to yeast form when seeded directly in a relatively richer medium such as GPYA and cultured at 35°C. The yeast cells herein obtained, since the first-round growth, present evidence of good viability and vigor, presenting typical well-rounded mother cells with a large number of buddings. These findings indicate that the fungus might survive for a longer period in soil in a state of discrete mycelial form and easily convert to yeast when exposed to a richer substrate and higher temperature (35°C) or when introduced into a mammalian host. Acknowledgments We thank the owners of Cerrito Island—Edson Gruppi, Edson Luiz Gruppi and Silvio José Gruppi—for allowing our studies in this private area. We also thank the Foundation for Research Support of the State of Mato Grosso (FAPEMAT) for granting a scholarship and IBAMA for the environmental license, as well as the forestry engineer Gabriel Moraes Gasparoto for the services rendered in Geoprocessing and map elaboration. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Shikanai-Yasuda MA , Telles Filho F de Q , Mendes RP , Colombo AL , Moretti ML . Guideliness in paracoccidioidomycosis . Rev Soc Bras Med Trop . 2006 ; 39 : 297 – 310 . Google Scholar Crossref Search ADS PubMed 2. Martinez R . Epidemiology of Paracoccidioidomycosis . Rev Inst Med Trop Sao Paulo . 2015 ; 57 : 11 – 20 . Google Scholar Crossref Search ADS PubMed 3. Coutinho ZF , Silva D , Lazéra M et al. Paracoccidioidomycosis mortality in Brazil (1980–1995) . Cad Saúde Pública . 2002 ; 18 : 1441 – 1454 . Google Scholar Crossref Search ADS PubMed 4. Coutinho ZF , Wanke B , Travassos C , Oliveira RM , Xavier DR , Coimbra CEA . Hospital morbidity due to paracoccidioidomycosis in Brazil (1998–2006) . Trop Med Int Heal . 2015 ; 20 : 673 – 680 . Google Scholar Crossref Search ADS 5. Colombo AL , Tobón A , Restrepo A , Queiroz-Telles F , Nucci M . Epidemiology of endemic systemic fungal infections in Latin America. Med Mycol . 2011 ; 49 : 785 – 798 . Google Scholar PubMed 6. Lacaz C da S , Porto E , Martins JEC . Medical Mycology, Actinomycetes and Algae of Medical Importance, 8th edn. Sarvier, São Paulo , 1991 , 695 p. [in Portuguese] . 7. Paniago AM , Ivan J , Aguiar A , Aguiar ES . Paracoccidioidomycosis: a clinical and epidemiological study of 422 cases observed in Mato Grosso do Sul . Rev Soc Bras Med Trop . 2003 ; 36 : 455 – 459 [in Galician] . Google Scholar Crossref Search ADS PubMed 8. Shikanai-Yasuda MA . Paracoccidioidomycosis Treatment . Rev Inst Med Trop Sao Paulo . 2015 ; 57 : 31 – 37 . Google Scholar Crossref Search ADS PubMed 9. Restrepo A , McEwen JG , Castañeda E . The habitat of Paracoccidioides brasiliensis: how far from solving the riddle? Med Mycol . 2001 ; 39 : 233 – 241 . Google Scholar Crossref Search ADS PubMed 10. Untereiner WA , Scott JA , Sigler L , Canada TG . The Ajellomycetaceae, a new family of vertebrate-associated Onygenales. Mycologia . 2004 ; 96 : 812 – 821 . Google Scholar Crossref Search ADS PubMed 11. Matute DR , McEwen JG , Puccia R et al. Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies . Mol Biol Evol . 2006 ; 23 : 65 – 73 . Google Scholar Crossref Search ADS PubMed 12. Theodoro RC , Teixeira M , de M , Felipe MSS et al. Genus Paracoccidioides: species recognition and biogeographic aspects . PLoS One . 2012 ; 7 : e37694 . Google Scholar Crossref Search ADS PubMed 13. Muñoz JF , Farrer RA , Desjardins CA et al. Genome diversity, recombination, and virulence across the major lineages of Paracoccidioides . mSphere . 2016 ; 1 : 1 – 18 . Google Scholar Crossref Search ADS 14. Turissini DA , Gomez OM , Teixeira MM , McEwen JG , Matute DR . Species boundaries in the human pathogen Paracoccidioides . Fungal Genet Biol . 2017 ; 106 : 9 – 25 . Google Scholar Crossref Search ADS PubMed 15. Ferreira MS , Freitas LH , Lacaz C da S et al. Isolation and characterization of a Paracoccidioides brasiliensis strain from a dogfood probably contaminated with soil in Uberlândia, Brazil. Med Mycol . 1990 ; 28 : 253—256 . Google Scholar Crossref Search ADS 16. Garcia NM , Del Negro GM , Heins-Vaccari EM , de Melo NT , de Assis CM , Lacaz C da S . Paracoccidioides brasiliensis, a new sample isolated from feces of a penguin (Pygoscelis adeliae) . Rev Inst Med Trop Sao Paulo . 1993 ; 35 : 227 – 235 . 17. de Farias MR , Zeni Condas LA , Ribeiro MG et al. Paracoccidioidomycosis in a dog: case report of generalized lymphadenomegaly . Mycopathologia . 2011 ; 172 : 147 – 152 . Google Scholar Crossref Search ADS PubMed 18. Roberto TN , Rodrigues AM , Hahn RC , de Camargo ZP . Identifying Paracoccidioides phylogenetic species by PCR-RFLP of the alpha-tubulin gene . Med Mycol . 2016 ; 54 : 240 – 247 . Google Scholar Crossref Search ADS PubMed 19. De Albornoz MB . Isolation of Paracoccidioides brasiliensis from rural soil in Venezuela . Sabouraudia . 1971 ; 9 : 248 – 253 . Google Scholar Crossref Search ADS PubMed 20. Teixeira MM , Theodoro RC , de Carvalho MJ A et al. Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus . Mol Phylogenet Evol . 2009 ; 52 : 273 – 283 . Google Scholar Crossref Search ADS PubMed 21. Teixeira Mde M , Theodoro RC , Derengowski Lda S , Nicola AM , Bagagli E , Felipe MS . Molecular and morphological data support the existence of a sexual cycle in species of the genus Paracoccidioides . Eukaryot Cell . 2013 ; 12 : 380 – 389 . Google Scholar Crossref Search ADS PubMed 22. Takayama A , Itano EN , Sano A , Ono MA , Kamei K . An atypical Paracoccidioides brasiliensis clinical isolate based on multiple gene analysis . Med Mycol . 2010 ; 48 : 64 – 72 . Google Scholar Crossref Search ADS PubMed 23. Arantes TD , Theodoro RC , Da Graça Macoris SA , Bagagli E . Detection of Paracoccidioides spp. in environmental aerosol samples . Med Mycol . 2013 ; 51 : 83 – 92 . Google Scholar Crossref Search ADS PubMed 24. Arantes TD , Theodoro RC , Teixeira M , de M , Bosco S , de MG , Bagagli E . Environmental mapping of Paracoccidioides spp. in Brazil reveals new clues into genetic diversity, biogeography and wild host association . PLoS Negl Trop Dis . 2016 ; 10 : 1 – 18 . 25. Bagagli E , Sano A . Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus novemcinctus) capured in an endemic area of paracoccidioidomycosis . Am J Trop Med Hyg . 1998 ; 58 : 505 – 512 . Google Scholar Crossref Search ADS PubMed 26. Bagagli E , Franco M , Bosco SDMG , Hebeler-Barbosa F , Trinca LA , Montenegro MR . High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study . Med Mycol . 2003 ; 41 : 217 – 223 . Google Scholar Crossref Search ADS PubMed 27. Corredor GG , Peralta LA , Castãno JH , Zuluaga JS , Henao B , Restrepo A . The naked-tailed armadillo Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis. Molecular identification of the isolate . Med Mycol . 2005 ; 43 : 275 – 280 . Google Scholar Crossref Search ADS PubMed 28. McDonough CM. , Loughry WJ. Behavioral ecology of armadillos . In: Vizcaíno SF , Loughry WJ , eds. The Biology of the Xenarthra . Gainesville, FL : University Press of Florida ; 2008 : 281 – 293 . 29. INPE . National Institute of Spacial Researches . 2016 . Available at: http://www.inpe.br/ [in Portuguese] . 30. IBGE . Brazilian Institute of Geography and Statistics . 2016 . Available at: http://www.ibge.gov.br [in Portuguese] . 31. McCullough M , DiSalvo A , Clemons K , Park P , Stevens D . Molecular epidemiology of Blastomyces dermatitidis . Clin Infect Dis . 2000 ; 30 : 328 – 335 . Google Scholar Crossref Search ADS PubMed 32. Theodoro RC , Candeias JMG , Araújo JP et al. Molecular detection of Paracoccidioides brasiliensis in soil . Med Mycol . 2005 ; 43 : 725 – 729 . Google Scholar Crossref Search ADS PubMed 33. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . MEGA6: Molecular evolutionary genetics analysis version 6.0 . Mol Biol Evol . 2013 ; 30 : 2725 – 2729 . Google Scholar Crossref Search ADS PubMed 34. NCBI . Basic Local Alignment Search Tool . 2015 . Available at: http://www.blast.ncbi.nlm.nih.gov/blast . 35. Irinyi L , Serena C , Garcia-Hermoso D et al. International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database—the quality controlled standard tool for routine identification of human and animal pathogenic fungi . Med Mycol . 2015 ; 53 : 313 – 337 . Google Scholar Crossref Search ADS PubMed 36. Stocker HJ . Of Mathematics and Computational Science . New York : Springer ; 1998 . 37. Tamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases . Mol Biol Evol . 1992 ; 9 : 678 – 687 . Google Scholar PubMed 38. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences . J Mol Evol . 1980 ; 16 : 111 – 120 . Google Scholar Crossref Search ADS PubMed 39. Terçarioli GR , Bagagli E , Reis GM et al. Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection . BMC Microbiol . 2007 ; 7 : 92 . Google Scholar Crossref Search ADS PubMed 40. Larone Davise H. Medically Important Fungi: A Guide to Identification . 5th ed. Washington, DC : ASM Press , 2011 . 41. Vieira GD , da Cunha Alves T , Dias de Lima SM , Aranha Camargo LM , de Sousa CM . Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the desease . Rev Soc Bras Med Trop . 2014 ; 47 : 63 – 68 . Google Scholar Crossref Search ADS PubMed 42. Carvalhosa AA , Borges FT , França DCC , Queiroz RR , Moimaz SAS GC . Paracoccidioidomycosis prevalence in a public laboratory of the Brazilian unified health system . J Oral Diag . 2016 ; 1 : 31 – 35 . 43. Volpato MCPF , Volpato LER , Guedes OA , Musis CR , Estrela CRA , Carvalhosa AA . Spatial distribution of cases of paracoccidioidomycosis with oral manifestations in the state of Mato Grosso, Brazil. Rev Odontol Bras Cent . 2016 ; 25 : 84 – 87 [in Portuguese] . 44. Hahn RC , Rodrigues AM , Fontes CJF et al. Fatal fungemia due to Paracoccidioides lutzii . Am J Trop Med Hyg . 2014 ; 91 : 394 – 398 . Google Scholar Crossref Search ADS PubMed 45. Sano A , Defaveri J , Tanaka R et al. Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo (Dasypus novemcinctus). Mycopathologia . 1998 ; 144 : 61 – 65 . Google Scholar Crossref Search ADS PubMed 46. Jorge LAB , Sartori MS . Land use and temporal analysis of natural vegetation at Edgardia experimental farm Botucatu-SP. Rev Árvore . 2002 ; 26 : 585 – 592 . Google Scholar Crossref Search ADS 47. Salazar ME , Restrepo A . Morphogenesis of the mycelium-to-yeast transformation in Paracoccidioides brasiliensis . Sabouraudia . 1985 ; 23 : 7 – 11 . Google Scholar Crossref Search ADS PubMed 48. Barrozo LV , Benard G , Silva MES , Bagagli E , Marques SA , Mendes RP . First description of a cluster of acute/subacute paracoccidioidomycosis cases and its association with a climatic anomaly . PLoS Negl Trop Dis . 2010 ; 4 : e643 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Medical MycologyOxford University Press

Published: Nov 1, 2018

References

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