| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of Microbiology and Hygiene (Charité), Humboldt University, Dorotheenstr. 96, D-10117, Berlin, Germany
2 Mycological Research Laboratory, Department of Bioscience, Rani Durgavati University, Jabalpur 482001, MP, India
3 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
Correspondence
Yvonne Gräser
yvonne.graeser{at}charite.de
Received 22 December 2006
Accepted 1 June 2007
Abbreviations: IA, index of association.
 |
INTRODUCTION |
|---|
 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All three species are able to cause human tinea capitis and tinea corporis, especially in prepubescent children. M. audouinii and M. ferrugineum are anthropophilic, generally being transmitted from human to human, and have evolved in Africa and Asia, respectively (Kaszubiak et al., 2004). M. canis has a worldwide distribution and is zoophilic, with humans predominantly only being infected after contact with mammals. The natural habitat of this species is the furred skin of cats, dogs and horses, where it generally resides asymptomatically. The occasional human-to-human infections are self-limiting after a few transmissions.
Epidemiological studies of human and animal infections by contagious strains of M. canis have remained unresolved due to a lack of polymorphic molecular markers. The detection of sources and routes of infection, and identification of contaminated spaces in hospitals, kindergartens, schools and animal nurseries would contribute to optimized therapy, prophylaxis and a hygienic regimen, and thus save financial resources. In addition, human infections due to M. canis tend to be moderately inflammatory, but cases of severe kerion-like tinea capitis or highly inflammatory ringworm infections do occur in similar patient populations (Ernst 1980; Pryce & Verbov, 1992; Stephens et al., 1989; Terragni et al., 1993). The question arises as to whether animal hosts harbour mixed genotypes of M. canis that differ in their degree of virulence to humans. In other words, do all lineages of M. canis have the same potential to infect humans, or is virulence limited to a subset of isolates within a genetically diverse population? Do these genotypes differ in predilection and pathogenicity? Do strains from particular animal host species compose monophyletic groups within M. canis with decreased gene flow? In order to address these questions, there is an urgent need for well-characterized neutral markers to analyse the population structure of M. canis.
In recent studies, several DNA markers (randomly amplified polymorphic DNA, sequencing of internal transcribed spacer and non-transcribed spacer regions of rRNA genes, intergenic spacers of nuclear DNA, and mitochondrial DNA genes) have been applied, but the degree of polymorphism was low within the species (Kaszubiak et al., 2004; Yu et al., 2004). Typing systems based on microsatellite markers have been shown to detect diversity at all levels from species down to individuals in pathogenic fungi such as Histoplasma, Coccidioides and Penicillium (Carter et al., 2001; Fisher et al., 2000, 2004). In the present study, we report on the application of microsatellite markers to a global set of M. canis strains to reveal patterns of genetic variation in this species.
|
METHODS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Of these, 101 strains were identified morphologically as M. canis, 29 were M. audouinii and 7 were M. ferrugineum. Strains of the latter two species were used as outgroups. Thirty-three M. canis strains were isolated from epidemiologically unrelated animals (mainly cats and dogs) in Germany, whilst fifteen strains originated from horses. Fifty-three strains were obtained from epidemiologically unrelated humans (mostly children) at geographically distant locations (Austria, Mexico, Turkey, Korea, The Netherlands, USA, New Zealand and the Dominican Republic). Tinea capitis and tinea corporis was diagnosed in 22 and 18 cases, respectively, in addition to 7 cases of tinea faciei. No clinical data were available on the remaining six patients.
|
Isolation of microsatellites (enrichment methods). Microsatellite sequences were captured using biotinylated (GT)12 and (GA)12 probes and immobilized on avidin-coated beads. The captured DNA was subjected to washing steps, and then eluted, amplified and cloned to produce a library enriched for the target sequence. The method was modified slightly from the one used previously by Ohst et al. (2004). Briefly, genomic DNA was isolated from a clinical isolate of M. canis (H22). Approximately 10 µg DNA was digested with DpnII and cleaned by drop dialysis for 15 min. Linkers (Sau-A, 5'-GCGGTACCCGGGAAGCTTGG-3'; Sau-B, 5'-GATCCCAAGCTTCCCGGGTACCGC-3') were ligated to both ends of the fragments using T4 DNA ligase (New England Biolabs). After purification via columns, pre-hybridization PCR was performed with the Sau-A linker only (annealing temperature of 56 °C, 15 cycles). For enrichment, the PCR product was denatured and hybridized to the biotinylated (CA or GA) probe in a solution of 6x SSC (1x SSC, 0.15 M NaCl, 0.015 M sodium citrate)/0.1 % SDS. The mixture was denatured at 95 °C and cooled slowly (over 15–20 min) to room temperature. The probe was then captured with avidin beads (VECTREX Avidin D; Vector Laboratories) in TBT buffer [100 mM Tris-HCl (pH 7.5), 0.1 % Tween 20] at 50 °C for 30 min and washed three times with TBT plus 150 mM NaCl and three times with 0.2x SSC/0.1 % SDS. The DNA was then denatured from the beads in 10 mM Tris/HCl (pH 8)/0.1 mM EDTA at 95 °C for 5 min and again PCR amplified with Sau-A. The resulting PCR product was cloned and transformed using a TOPO TA cloning kit (Invitrogen). White selection colonies were picked and checked for the repeat insert using M13 primers and (AC)10 or (GA)10 primers. Inserts of 300–600 bp were chosen for sequencing using an M13 primer and an automated sequencing system (3130x Genetic Analyzer). Specific primers were then designed to amplify PCR fragments in the range of 100–300 bp containing more than 12 GT or GA repeats. Amplification of each primer pair was tested on a panel of strains of M. canis, including the genomic DNA of the isolate the library was generated from.
PCR amplification of microsatellite markers using specific primers. Standard PCR conditions were as follows: reactions were performed in 50 µl volumes containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2 (1.5 mM for McGT17), 20 pmol each primer (McGT13 forward, 5'-GATCGGAGCATGCCATACAG-3'; McGT13 reverse, 5'-TCTTCCCACCCTTCTCAATG-3'; McGT17 forward, 5'-GCTCTGGGATAAGGTGTTTG-3'; and McGT17 reverse, 5'-GTAGCAGTAAAGCCAAGAGGG-3'), 50 µM each dNTP, 2.5 U Taq polymerase (Applied Biosystems), and 50 ng template DNA. Samples were amplified through 30 cycles as follows: initial denaturation for 10 min at 95 °C, followed by denaturation for 50 s at 95 °C, annealing for 60 s at 60 °C and extension for 60 s at 72 °C. This was followed by a final extension step of 10 min at 72 °C.
Screening for length polymorphisms. PCR products (15 µl) were loaded onto 12 % polyacrylamide gels (Rotiphorese gel 29 : 1, 40 %; Carl Roth) and the microsatellites were run for 18 h at 12 W (constant power). Gels were silver stained and dried for documentation. Repeat numbers in alleles were calculated visually using a sequenced allele with known repeat number as the reference (EMPL accession nos AM295318 and AM295319).
Data analysis. Different approaches were used to assign strains to populations. Whilst distance-based methods proceed by calculating a pairwise distance matrix whose entries give the distance between every pair of individuals, model-based methods proceed by assuming that observations from each cluster are random draws from some parametric model. In the first case, this matrix is then displayed using a graphical presentation such as a tree. In the second case, inference for the parameters corresponding to each cluster is done jointly with inference for the cluster membership of each individual, using standard statistical methods such as the Bayesian method. The disadvantage of the distance-based method is that the identified clusters may be heavily dependent on the distance measurement and graphical presentation chosen (Pritchard et al., 2000). Therefore, we calculated genetic distances between individuals based on three different measurements, Dc (Cavalli-Sforza & Edwards, 1967), Dm and Ds (Saitou & Nei, 1987) distances, implemented in the software package POPULATIONS version 1.2.28 (http://www.pge.cnrs-gif.fr/bioinfo/populations/index.ph-p?lang=en). Neighbour-joining trees were constructed from the distance matrices and were displayed using TREEVIEW (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). As a model-based method, a Bayesian approach was used in the program STRUCTURE version 2.1 (Pritchard et al., 2000). This method allows the assessment of confidence of the inferred clusters by fine statistical analysis, whilst genetic distance methods are more suited to exploratory data analysis. Various models were used with STRUCTURE, including the no-admixture model, which can deal with clonal reproduction. One million Markov chain Monte Carlo replications and a burn-in period of 100 000 generations were used. The probability of the data, assuming one to five populations (K), was estimated in three replicate analyses. The posterior probability and other values displaying the confidence of the number of populations were recorded.
After structuring the populations, Wright's F statistics were applied to compute the variance in allele frequencies and test for free gene flow versus population differentiation between the inferred populations. Theta (Weir, 1996) was calculated across loci and populations using MULTILOCUS version 1.3 (Agapow & Burt, 2001). Here, the null hypothesis is no population differentiation; 400 000 randomizations were used.
To test for clonality versus recombination in the M. canis sample, the overall and the ‘in population separated’ (based on both cluster methods; see Results) index of association (IA) was calculated using the software MULTILOCUS. In this test, the observed data are compared against the null hypothesis of random mating (random association of alleles from different DNA loci). When the null hypothesis is rejected, a clonal population structure is suggested.
|
RESULTS |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The data for each strain and locus are presented in Table 1
.
|
, Fig. 1). An indication of clonal reproduction within the M. canis sample set was the observation of three multilocus genotypes that were shared by multiple strains from unrelated hosts. Of the 11 multilocus genotypes, 1 was shared by 50 strains, whilst 2 genotypes comprised 14 and 15 strains . This corresponds to a genotypic diversity in this dataset of 0.71. The linkage disequilibrium analysis of the overall sample and the clone-corrected sample rejected the null hypothesis of random mating (IA=0.19, P<0.001). The identity of spatially separated strains of the same genotype demonstrated the high degree of reproducibility of the technique used.
Independent from the underlying assumptions and using several distance measurements – the Bayesian approach and neighbour joining – always revealed trees with three clusters (I–III) within M. canis. Each method generated branches with nearly identical sets of strains, except for three strains that grouped with cluster I (Bayesian approach) or cluster III (distance approach), but with a slight affinity to cluster III (mixed genotype) using the Bayesian approach (Fig. 1). Isolates from cats and dogs were distributed evenly among the three clusters. The 6 multilocus genotypes in cluster III were shared by 13 isolates (87 %) from horses, 8 strains (24 %) from other animals and 5 human isolates (9 %) (Figs 1 and 2). Cluster II was less variable, although the total number of strains was comparable to that in cluster III (26 strains in cluster III and 24 strains in cluster II). Only 4 multilocus genotypes were shared by 15 animal strains (31 %), among which was 1 isolate from a horse, and 9 (17 %) of the human isolates (Figs 1 and 2). Six out of seven (86 %) human strains of Turkish origin were found in group II (Fig. 1). In group I, a single multilocus genotype was shared by most of the human isolates (74 %), independent of their distant geographical origins (Korea, Austria and Mexico), together with 11 animal strains (23 %) from Germany, including one isolate from a horse (Figs 1 and 2).
|
=0.733, P<0.001).
Data on clinical pictures were obtained from 47 of the 53 patients studied. In total, 22, 18 and 7 cases of tinea capitis, tinea corporis and tinea faciei were revealed, respectively. Whilst strains in cluster I were able to cause all three forms of tinea, cases of tinea faciei and tinea corporis were missing in clusters II and III, respectively (Fig. 3).
|
|
DISCUSSION |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
From our results, we could exclude the possibility that geographical differentiation and allopatric speciation played an important role in structuring the populations of M. canis. The strains from Germany (all animal isolates) did not show any monophyletic clustering, and the human isolates from Europe, South America and Asia could be found jointly as a single cluster.
M. canis is a zoophilic fungus that is only isolated rarely from soil. Hence, we anticipated an association of genotypes with particular host species. However, we revealed cat-associated genotypes in all three populations (I–III). Similarly, equine ringworm or colonization of horse fur could not be linked to a monophyletic group of strains. The set of 15 isolates from Germany comprised 5 multilocus genotypes belonging to all 3 populations of M. canis. Of the horse isolates 10 out of 15 had identical alleles at both loci, indicating that a single clone was involved. It is evident that horse isolates have acquired the ability to cause superficial disease more than once in the course of their evolution. Maintenance of horse isolates as a separate species, Microsporum equinum (Delacroix & Bodin, 1896), is therefore not justified. A similar situation has been published with multilocus genotypes of Histoplasma capsulatum (Kasuga et al., 2003). Molecular analysis of four genes of isolates causing equine histoplasmosis, formerly classified as a separate variety, H. capsulatum var. farciminosum, revealed that strains were distributed over three phylogenetic clades. The authors concluded that maintenance of the var. farciminosum was phylogenetically meaningless.
Despite the fact that all of the M. canis strains under study were isolated from epidemiologically unrelated individuals, 74 % of the isolates from human patients but only 23 % of the animal isolates shared a single genotype. The over-representation of this genotype 1 among the human isolates suggests that it may have a higher degree of virulence (possibly a proteinase adapted to human keratin) than the ten remaining genotypes, having a higher potential to infect humans when transmitted from animals. The genotype has a pandemic distribution. Strains harbouring genotype 1 were isolated from patients from three continents (Europe, South America and Asia). This seems to be in conflict with the data presented by Cano et al. (2005) using inter-simple-sequence repeat PCR (ISSR-PCR), who demonstrated that numerous strains from humans have a limited distribution, and may even be restricted to a single patient. The authors found a total of 21 genotypes among 24 mainly human isolates from Spain. With the exception of a single genotype (pattern 1), none of the animal genotypes occurred on humans, and vice versa. The conclusions derived from these data could not be explained by DNA loci located between single repeats, such as the flanking regions of microsatellites. Mutation rates of flanking and repeat regions have been analysed in detail in other fungal species, revealing a 2500-fold higher mutation rate for the latter loci (Dettman & Taylor, 2004). Thus, microsatellite loci are likely to be more variable than the flanking regions analysed using ISSR-PCR. The high variability among strains in the study of Cano et al. (2005) may partly be due to the low reproducibility (93 %) of the technique used. In contrast to Cano et al. (2005), the few other studies performing strain typing in M. canis, e.g. randomly amplified polymorphic DNA, reported a very low variability among epidemiologically unrelated strains from cats, dogs and humans, despite their morphological diversity (Brilhante et al., 2005; Faggi et al., 2001). Such results are in agreement with the extensive developmental work we have done while searching for polymorphic markers: 90 % of the microsatellite markers were unacceptable for the population analysis due to the low variability displayed. This is likely to be caused by the high portion of clonal reproduction within the species. In another clonal species, Trichophyton rubrum, only 30 % of the typable microsatellites were polymorphic, whereas in strongly recombining populations of Microsporum persicolor, almost 100 % of the loci were useful for a population genetic study (R. Sharma, S. de Hoog, W. Presber, Y. Gräser & R. C. Rajak, unpublished data). Although our findings suggest that some M. canis strains have an increased infective potential to humans, there was no indication of an association between genotypes and the type of tinea caused by this genotype, as tinea capitis was caused by genotypes of all three clusters. The missing manifestations of tinea corporis and tinea faciei by strains of clusters II and III, respectively, may have been due to the low number of human cases (nine and five) falling in these population groups.
In conclusion, our data cannot rule out the possibility that there are several asexual, separated lineages within M. canis. Systematic sampling needs to be undertaken, particularly in animal strains from geographically remote locations. Additional microsatellite markers need to be developed to support the indication of the presence of recombination events in this fungus.
In addition to epidemiological studies, the markers developed in this project can be applied to other studies. In addition to virulence traits, as shown here, drug resistance may also be associated with particular genotypes. Although the loci under study are probably not based on resistance genes, the largely clonal reproduction in populations of M. canis keeps genes and their associated traits together. The developed microsatellite markers can also be used as diagnostic tools for the rapid and specific identification of species of the M. canis complex directly from clinical specimens. This has been shown already by Kardjeva et al. (2006) for the detection of T. rubrum. Mc(GT)13 is able to discriminate the species M. canis, M. audouinii and M. ferrugineum by agarose gel electrophoresis.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  T. C. White, B. G. Oliver, Y. Graser, and M. R. Henn Generating and Testing Molecular Hypotheses in the Dermatophytes Eukaryot. Cell, August 1, 2008; 7(8): 1238 - 1245. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
