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Keywords:

  • Infected bats;
  • Histoplasma capsulatum;
  • RAPD-patterns;
  • DNA sequences

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

The pathogenic fungus Histoplasma capsulatum var. capsulatum-Darling 1906 is frequently found in the guano accumulated underneath bat colonies in confined spaces, such as caves, abandoned mines, and uninhabited houses [1]. Nutrients from guano together with soil and environmental conditions constitute the ecological niche of this microorganism [2–8]. By raising the temperature and humidity, bat colonies may also influence the physical parameters necessary for fungal survival and reproduction. The role of bats in spreading H. capsulatum was first proposed by Hoff and Bigler [9]; however, the influence of bats on the ecology of H. capsulatum remains unclear, especially their potential to disperse the pathogenic agent in nature.

The high risk of natural bat infection with H. capsulatum in Mexican cave environments has been well-documented [7,10]; and the first molecular approaches involving H. capsulatum isolates from infected bats were reported by Chávez-Tapia et al. [10], Taylor et al. [11] and Kasuga et al. [12]. In consequence, the ability to distinguish isolates of the fungus in infected bats, based on genetic patterns, could provide a biological marker to better define the distribution of H. capsulatum polymorphism in specific geographic areas.

Histoplasma capsulatum is distributed worldwide, but its presence is relevant in those geographic areas where histoplasmosis is endemic or epidemic. This systemic mycosis is caused by the inhalation of infective propagules of fungal saprobe mycelial-phase developed in natural environments. The best-known endemic areas of histoplasmosis are the Ohio and Mississippi Valleys in the USA, but areas associated with high frequency of outbreaks are found in Latin America. In Mexico, histoplasmosis has been shown to be variable in prevalence in endemic areas and is widely distributed throughout the country [13–15]. Epidemiological studies in Mexico have demonstrated that individuals with occupations associated with bat shelters, such as mining, geological exploration and, particularly, the collection of bat guano to be used as fertilizer, have a high risk of infection and develop a mild to severe clinical epidemic form [16–20].

In the past years, H. capsulatum isolates from different geographic origins have been classified by their DNA polymorphism using molecular assays. These assays have revealed variation in genetic polymorphism depending on fungal sources or origins [21–25]. Diversity in population structures of H. capsulatum isolates has been suggested [26–29], including distinct fungal geographic populations [27]. Kasuga et al. [12,30] and Taylor et al. [31] studied the phylogenetic relationships of several H. capsulatum isolates, representing the varieties capsulatum, duboisii, and farciminosum. Partial DNA sequences of four protein-coding genes (arf-ADP ribosylation factor; H-anti-H antigen precursor; ole-delta-9 fatty acid desaturase; and tub1-alpha-tubulin), used to analyze H. capsulatum isolates of different geographic origins, suggest that Histoplasma form at least eight genetically distinct geographical populations, of which seven are considered phylogenetic species [12]. Furthermore, a strong differentiation between H. capsulatum population structures from North (USA) and South (Colombia) America was found by using microsatellite markers, supporting the separation of both H. capsulatum populations into different species, based on the phylogenetic species concept [28].

Molecular findings based on DNA polymorphism patterns of H. capsulatum should be explored further by defining the fungal distribution in nature. According to this idea, the present paper provides support for the use of molecular markers as tools to detect the distribution of this fungus in geographical areas from Mexico.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

2.1Source of H. capsulatum

Isolates were taken from the Fungal Immunology Laboratory Culture Collection of the Department of Microbiology and Parasitology, School of Medicine-UNAM. H. capsulatum Culture Collection can be accessed at: http://www.histoplas-mex.unam.mx, and is registered in the database of the World Data Centre on Microorganisms (WDCM) of the World Federation for Culture Collections (WFCC) under the acronym and number LIH-UNAM WDCM817. Isolates were maintained in brain heart infusion-agar (Bioxón, Becton-Dickinson, Mexico City) at 26 °C and preserved in Sabouraud-agar added with sterile mineral oil at 4 °C. They were originally obtained from naturally infected adult male bats, captured by chance in caves located in three states of the subtropical semi-arid region of central Mexico [7,10]. EH-372 to 377 isolates were recovered from Artibeus hirsutus (non-migratory) from “El Salitre” cave and EH-383I and 383P from Leptonycteris nivalis (migratory) from “El Diablo” cave, both in Morelos (MS); EH-384I and 384P from Tadarida brasiliensis (long-distance migratory) and EH-393 and EH-394 from L. curasoae (migratory) from “La Claraboya” cave, in Oaxaca (OC); EH-408H and 408P from L. nivalis from “Tzicanostoc” cave, in Puebla (PL). EH-317 from MS and G-217B from USA (a gift from Dr. G.S. Kobayashi, Washington University), both isolated from human histoplasmosis clinical cases, one from the subtropical region studied and the other from a non-related region, were used in the assays as reference strains.

2.2DNA isolation

Procedures for isolation of H. capsulatum whole-cell DNA were performed as described elsewhere [32]. The DNA was quantified fluorometrically and checked against standard concentrations by agarose gel electrophoresis. Finally, it was frozen at −20 °C until required.

2.3Two-primer random amplification of polymorphic DNA (RAPD)-PCR assay

This assay was performed as described by Hu et al. [33] in a 20 μl reaction, using 10 ng H. capsulatum DNA, 2.5 mM MgCl2, 200 μM of each dATP, dTTP, dCTP, and dGTP (Applied Biosystems Inc., Foster City, CA), 15 pmol of each primer 1281 (5′-AACGCGCAAC-3′) and 1253 (5′-GTTTCCGCCC-3′) supplied by Operon Technologies Inc. (Alameda, CA), and 1 U Taq DNA polymerase (Applied Biosystems). Controls without DNA were also set up and run with each set of reactions. Optimal PCR amplification was performed in a Perkin–Elmer Cetus DNA thermal cycler, programmed as follows: one cycle of 7 min at 94 °C followed by 45 cycles of 1 min at 92 °C (denaturation); 1 min at 35 °C (annealing); and 1 min at 72 °C (extension). A final 5-min cycle at 72 °C ensured full extension of all amplified products, and samples were maintained at 4 °C. The polymorphic amplified patterns containing DNA fragments revealed by RAPD-PCR were analyzed. Digital images of ethidium bromide (0.5 μg ml−1)-stained agarose gels (1.5%) were captured with a documentation system (GeneCam; Syngene, Cambridge, MA) and printed on a digital graphic printer (Sony Electronics Inc.; Park Ridge, NJ).

The RAPD-PCR products were compared and bands were scored as either present (1) or absent (0). A pair-wise genetic similarity matrix of H. capsulatum isolates was produced using the simple matching coefficient (SM) [34]. The SM coefficient was defined as: (nw +nz)/(nw +nx +ny +nz), where n= the number of band positions; nw 11 = the number of positions where isolate 1 and 2 show band presence; nx 10 = the number of positions where isolate 1 exhibits the band and isolate 2 exhibits no band; ny 01 = the number of positions where isolate 1 shows no band and isolate 2 shows the band; nz 00 = the number of positions where isolate 1 and 2 exhibit band absence.

A dendrogram based on simple matching coefficient using unweighted pair group method analysis (UPGMA) was generated. The cophenetic correlation coefficient (r), between the similarity matrix and the matrix generated by UPGMA to construct the dendrogram, was estimated by means of Mantel's non-parametric test [35] to measure distortion of the dendrogram.

2.4PCR for nuclear genes of H. capsulatum

DNA fragments of four H. capsulatum nuclear genes were obtained from each fungal isolate studied and were PCR-amplified as described by Kasuga et al. [30]. The primers (Operon Technologies) were selected as follows: For arf gene, arf1 (5′-AGAATATGGGGCAAAAAGGA-3′) and arf2 (5′-CGCAATTCATCTTCGTTGAG-3′); for H-anti gene, H-anti3 (5′-CGCAGTCACCTCCATACTATC-3′) and H-anti4 (5′-GCGCCGACATTAACCC-3′); for ole gene, ole3 (5′-TTTAAACGAAGCCCCCACGG-3′) and ole4 (5′-CACCACCTCCAACAGCAGCA-3′); and for tub1 gene, tub1 (5′-GGTGGCCAAATCGCAAACTC-3′) and tub2 (5′-GGCAGCTTTCCGTTCCTCAGT-3′). The PCR products were sent to the Cellular Physiology Institute, UNAM-Mexico, for sequencing in an ABI-automated DNA sequencer (Applied Biosystems).

2.5Sequencing analysis

Sequences were generated for a single strand. They were edited, aligned, and compared by means of the BLAST program, version 2.0 (National Center for Biotechnology Information-NCBI-Databases) [36] with sequences available from strain G-217B (USA reference strain) for arf gene (GenBank Accession No. L25117), for H-anti gene (GenBank Accession No. U20346), for ole gene (GenBank Accession No. X85962), and for tub1 gene (GenBank Accession No. M28358). Sequences of the four gene regions studied were also aligned using Clustal X version 1.7 program (NCBI) [37], and the online alignments can be accessed at http://www.histoplas-mex.unam.mx. The following set was used: slow/accurate; gap opening penalty – 15.00; gap extension penalty – 6.66; delay divergent sequences 30%; DNA transitions weight – 0.50; DNA weight matrix – IUB; negative matrix – OFF.

The nucleotide diversity (π) and divergence for the four gene regions were estimated among the H. capsulatum isolates studied, according to Nei and Li [38]. Distance and maximum-parsimony (MP) analyses were performed, for each gene region, using PHYLIP version 3.5 [39] and PAUP version 4.0 [40]. Parsimony analysis was based on a heuristic search of data set, with the tree bisection-reconnection branch-swapping option on. Pair-wise distance was calculated by means of Kimura's 2-parameter [41], and grouped using the neighbor-joining (NJ) algorithm. Bootstrap consensus trees by majority rule were constructed using heuristic search on 1000 bootstrap resampled data set [42]. The sequences in the GenBank for the four genes studied from G-217B strain were considered as outgroups in all analyses.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

Kunz [43] compiled all the reports of H. capsulatum isolated from bats in the world and most of them were from North America. In the last 12 years, H. capsulatum has been recovered from gut, lung, liver, and spleen of some short-distance migratory and non-migratory infected bats captured in Mexico by our team [6,7,10]. A serendipitous Histoplasma infection in the long-distance migratory bat, T. brasiliensis, has also been recorded in one of the selected states of Mexico for this study (Taylor, unpublished data).

3.1Two-primer RAPD-PCR patterns

The first genetic polymorphic studies of H. capsulatum isolated from non-migratory and short-distance migratory infected bats, captured in central Mexico, revealed a common DNA banding pattern that was shared by several isolates, when testing with different primers with one- or two-primer RAPD-PCR assays [10,11,19].

An interesting new finding represented by a different DNA banding pattern was registered when two H. capsulatum isolates recovered from the gut (EH-384I) and lung (EH-384P) of one specimen of T. brasiliensis (long-distance migratory) were studied. An identical molecular pattern was revealed in both isolates through the two-primer RAPD-PCR assay, by using 1281 and 1253 primers combination (Fig. 1). To ease comparisons with a previous report by Taylor et al. [11], in Fig. 1 the DNA pattern of H. capsulatum isolates from central Mexico is represented by EH-373 (non-migratory A. hirsutus bat) and EH-317 (human clinical case) both from Morelos, as well as by EH-393 from a short-distance migratory bat (L. curasoae) from Oaxaca, the same state where the long-distance migratory T. brasiliensis specimen was captured. The DNA banding pattern of EH-384I and 384P isolates also differed from the pattern of the G-217B reference strain, originally isolated from an infected human in USA. The dendrogram from Fig. 1 was developed to estimate relationships among the H. capsulatum isolates analyzed. A good cophenetic correlation (r= 0.990, P= 0.0002) was achieved and three groups, according to RAPD-patterns, were identified. Group A formed by EH-384I and 384P isolates recovered from T. brasiliensis at 1.0 similarity. Group B formed by EH-393, EH-373, and EH-317 isolates also showed 1.0 similarity. The G-217B strain from USA formed group C. Group A was linked to B and C clusters at a similarity of 0.55, whereas B and C clusters were linked at 0.75. Consequently, the RAPD-pattern of H. capsulatum isolated from T. brasiliensis could represent another H. capsulatum geographic pattern.

image

Figure 1. Two-primer RAPD-PCR patterns of H. capsulatum isolated from the long-distance migratory bat T. brasiliensis captured in the Mexican tropical central zone. For this assay, the primers tested were 1281 and 1253; and the molecular marker was 123 bp DNA Ladder. The RAPD-PCR assay was performed as described under Section 2. The similarity matrix generated by simple matching coefficient was plotted by UPGMA. The H. capsulatum isolates analyzed were: EH-384I and 384P from T. brasiliensis, EH-393 from L. curasoae, EH-373 from A. hirsutus. Clinical isolates, EH-317 from Mexico and G-217B from the USA, were used as human reference strains. Abbreviations: Oaxaca (OC); Morelos (MS); United States of America (USA).

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3.2DNA polymorphism and phylogenetic analyses of H. capsulatum based on DNA sequences of four protein-coding genes

The DNA from 14 H. capsulatum isolates and two human reference strains was amplified in appropriate and standardized conditions; the generated sequences of arf, H-anti, ole, and tub1 H. capsulatum genes were deposited in the GenBank nucleotide sequence databases (Accession Nos. AF495591–AF495654). BLAST 2 program [36] showed high similarity, ranging from 88% to 99%, for the four genes tested in all isolates compared with strain G-217B and few insertions and/or deletions were observed, revealing no critical genome changes in the partial sequences of the four genes studied.

Nucleotide diversity (π) for each H. capsulatum gene studied was low: π= 0.01066 for the arf gene; π= 0.01668 for the H-anti gene; π= 0.00739 for the ole gene; and π= 0.02062 for the tub1 gene. Nucleotide divergence among sequences of H. capsulatum isolates was also low: the arf gene showed 21 polymorphic sites from 463; the H-anti gene 26 polymorphic sites from 404; the ole gene 13 polymorphic sites from 426; and the tub1 gene 25 polymorphic sites from 300 sites analyzed. According to nucleotide diversity and divergence values, a highly homogeneous H. capsulatum population was revealed among most isolates from the central zone of Mexico. Besides, the best homology among the group I isolates was associated with the ole gene and the lowest nucleotide diversity for this gene (π= 0.00739) confirms its high homology. Nevertheless, the greatest π values (ranging from 0.01 to 0.03) found for the four genes in isolates EH-384I and 384P from the long-distance migratory bat T. brasiliensis suggest that these isolates could represent a different H. capsulatum population.

Three groups were revealed by the phylogenetic trees of the four genes analyzed either by NJ (Fig. 2) or MP (Fig. 3). Group I clustered most H. capsulatum isolates from infected bats captured in Morelos, Puebla, and Oaxaca (EH-372-377, EH-383I and 383P, EH-393, EH-394, EH-408H and 408P) and one clinical isolate from Morelos (EH-317). Group II was represented by the two isolates recovered from T. brasiliensis (EH-384I and 384P). Taking into account that the four genes studied were characterized in the G-217B strain from the USA, this strain was found closely related to these outgroups for original gene sequences; hence, it was separated into group III, which was independent from the other two groups. Concordance was observed among trees elaborated through NJ and MP, and topologies were congruent with the nucleotide diversity observed among the isolates studied. Low π values were found among H. capsulatum isolates from group I. In contrast, higher π values were detected between group I and II or group II and III, suggesting another genetic isolation. No critical phylogenetic differences were found among H. capsulatum isolates from group I, suggesting that gene flow could be occurring among the states of Morelos, Puebla, and Oaxaca, where group I isolates were originated. Besides, Kasuga et al. [12] in a previous report using sequence analyses of the same four protein-coding genes classified the H. capsulatum isolates from group I into a Latin American group A population.

image

Figure 2. Neighbor-joining consensus trees of all H. capsulatum isolates studied. Partial sequences from arf, H-anti, ole, and tub1 H. capsulatum genes were analyzed through Kimura's 2-parameter distance. Sequences of these four protein-coding genes from strain G-217B, reported in GenBank, were used as outgroups. Bootstrap values lower than 70% are not shown. Abbreviations: Oaxaca (OC); Morelos (MS); Puebla (PL); United States of America (USA).

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image

Figure 3. Maximum-parsimony consensus trees of all H. capsulatum isolates studied. Partial sequences from arf, H-anti, ole, and tub1 H. capsulatum genes were analyzed based on a heuristic search of data set, with the tree bisection-reconnection branch-swapping option on. Outgroup, bootstrap and abbreviation details are the same as in Fig. 2.

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In the present paper, EH-384I and 384P isolates (group II) showed diversity in relation to group I and III, and may have originated from a different phylogenetic group. In order to establish whether group II behaved as a different group, since it contained only two isolates from the same infected bat, we compared the sequences of their four gene fragments studied with the sequences of H. capsulatum isolates from different sources and origins reported by Kasuga et al. [30]. Results showed that group II remained independent from the other isolates studied by Kasuga et al. [30] (data not shown). This last finding suggests that group II could represent a new phylogenetic species. Therefore, it is possible that the genetic pattern of H. capsulatum isolated from T. brasiliensis may represent another H. capsulatum geographic pattern, associated with bat shelters along its migratory route, where these bats were probably infected. This suggestion is quite feasible since T. brasiliensis' migratory streams include the south of Brazil, continuing through Argentina and Chile, crossing Mexican territory and arriving to the USA mainly across Texas [44].

Hence, appropriate genetic markers associated with fungal distribution in a central zone of Mexico (Morelos, Puebla, and Oaxaca) could be proposed, based on a common RAPD-pattern formerly described by Taylor et al. [11], corroborated by the new RAPD results now reported, and supported by DNA sequence similarity in four independent genes of H. capsulatum. However, the usefulness of these markers to discriminate isolates from other geographic origins cannot be generalized yet.

3.3Genetic polymorphism of H. capsulatum as a regional biological marker

Histoplasma capsulatum propagules can be dispersed across varying distances. In general, infected mammals appear to be ideal candidates for spreading H. capsulatum either along short or long distances. The colonial behavior of bats in roosting caves, the size of colonies, the ability to fly, and the high fidelity to roosts are all important factors that explain the dynamics of fungal dispersion in nature. For instance, diseased bats could act as parasite dispersers by incorporating the fungus into new favorable environments, possibly through their carcasses once dead. The opportunity of having specific fungus markers associated with migratory and resident bats in a well-defined area is potentially valuable. Seasonal and climatic changes together with food availability may modulate bat movement [45]. Furthermore, geographic barriers do not represent important obstacles for some bat species in developing long-distance migratory routes. However, some non-migratory species, such as A. hirsutus, from which the fungus has been isolated, have a low-rate of regional movement and share shelters with other bat species. Other species living in caves but with important regional movements, such as L. curasoae and L. nivalis, are probably involved in the spread of H. capsulatum isolates with particular polymorphic patterns in Mexico. It has been proposed that the nectarivorous bat, L. curasoae, migrates between the southern part of the USA and the tropical regions of Mexico; evidence of this migration is circumstantial and, a resident and non-migratory population of L. curasoae was monitored in Mexico [46,47]. Besides, inter-tropical movements between caves of the semi-arid region of central Mexico (Guerrero–Hidalgo, Hidalgo–Morelos, and Morelos–Oaxaca) have also been documented [48]. According to bat distribution data referred in Medellín et al. [49], a map was created to show that these three bat species coexist in central Mexico (Fig. 4). Interestingly, in this same region, a widespread polymorphic DNA pattern of H. capsulatum isolated from these bats has been described [11,19]; this finding is now strongly supported by our present data.

image

Figure 4. Location of sampled caves in central Mexico. The four caves sampled are depicted in the map (©): two in Morelos (MS), one in Puebla (PL) and another in Oaxaca (OC) (cave details are under Section 2). Map shows also the distribution of L. curasoae, L. nivalis and A. hirsutus. Horizontal lines –L. curasoae area; gray zone –L. nivalis area; vertical lines –A. hirsutus area; black spot – the geographic area shared by the three bat species, where a widespread H. capsulatum molecular pattern was identified in isolates recovered from several specimens of these short-distance migratory and non-migratory bats.

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Finally, there are important facts about the behavior of T. brasiliensis that are remarkable and could explain why H. capsulatum isolates from group II molecular pattern was scarcely detected in the Mexican caves. First, their migratory route is unstable and sometimes indefinable; second, frequently, they are not quite faithful to their shelters and they remain for a short time in their transitory roosts, and third, quite often, they roost in other places different from caves.

In conclusion, based on studies of genetic polymorphism under standardized conditions, we propose that the molecular geographic-pattern of H. capsulatum isolated from infected bats could be used mainly as a tool to monitor the distribution of the fungus in nature.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

This research was supported mainly by Dirección General de Asuntos del Personal Académico (DGAPA) DGAPA-UNAM-IN203197 and by CONACYT 1018-PM. The authors thank Alice Piaget and Ingrid Mascher for their editorial assistance, as well as Frank Mallory and David S. Gernandt for their constructive criticism.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References
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