mycoses

Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Original article

Molecular typing of Sporothrix schenckii isolates from cats in Malaysia Rui Kano,1 Miki Okubo,1 Han Hock Siew,2 Hiroshi Kamata1 and Atsuhiko Hasegawa3 1 Department of Veterinary Pathobiology, Nihon University College of Bioresource Sciences, Fujisawa, Kanagawa, Japan, 2Han Veterinary Surgery, Johor Bahru, Johor, Malaysia and 3Teikyo University Institute of Medical Mycology, Hachioji, Tokyo, Japan

Summary

Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia.

Key words: Feline sporotrichosis, molecular typing, mating type, MAT 1-1-1, Sporothrix schenckii.

Introduction Sporothrix schenckii complex, a fungus able to grow on living and dead vegetation, comprises at least six sibling species: S. brasiliensis, S. schenckii sensu stricto, S. globosa, S. mexicana, S. luriei and S. pallida (formerly S. albicans).1–3 Sporothrix brasiliensis, S. globosa, S. schenckii sensu stricto and S. luriei also have been detected as human and animal infectious agents, with transmission apparently occurring by subdermal inoculation in tropical and sub-tropical environments.3,4 The long-lasting feline sporotrichosis outbreaks, such Correspondence: R. Kano, Department of Veterinary Pathobiology, Nihon University College of Bioresource Sciences, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. Tel.: +81 466 84 3649. Fax: +81 466 84 3649. E-mail: [email protected] Submitted for publication 10 October 2014 Revised 13 January 2015 Accepted for publication 13 January 2015

doi:10.1111/myc.12302

as those that are ongoing in South and Southeast Brazil, have highlighted the importance of zoonotic transmission to humans.5 Sporothrix brasiliensis is the main species involved in the feline sporotrichosis outbreaks in Brazil, with a low frequency of S. schenckii sensu stricto.5–7 As demonstrated by Zhou et al. [3], S. schenckii sensu stricto can be divided into 2 clinical clades (C and D) based on ITS (internal transcribed spacer) region sequence analysis and geographic distribution. These authors reported that the pathogens most commonly implicated in human sporotrichosis in Asia were S. globosa and clinical clade C of S. schenckii sensu stricto; however, predominance in individual countries remained unknown.3 Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in this country has been reported to be transmitted primarily via cat scratch.8 In the present work, we characterised Sporothrix clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the

© 2015 Blackwell Verlag GmbH Mycoses, 2015, 58, 220–224

Sporothrix schenckii from Malaysia

rDNA ITS region, and selective PCR of mating type (MAT) loci. To the best of our knowledge, our work represents the first report of the molecular epidemiology of S. schenckii isolates from feline sporotrichosis in Malaysia.

Materials and methods Isolates

The patient cats included both domestic and feral cats that harboured cutaneous sporotrichosis. These cats resided primarily in urban areas, but some of the subjects were from rural areas of various states of peninsular Malaysia (Selangor, Melaka, Johor, Kelantan, Perak, Penang, and Kuala Lumpur). The cotton swab smear samples were collected in 2014 from skin lesions and inoculated on Saboraud’s dextrose agar9 containing 0.1 mg ml1 of chloramphenicol (OXOID, Hampshire, UK). Eighteen isolates were confirmed as S. schenckii complex by morphological examination.1,9 Phylogenetic analysis of the ITS region and calmodulin gene

Sequence analyses of the ITS region of rDNA and calmodulin were performed using gene-specific primers as described in previous reports.10,11 The amplicons from the ITS region and calmodulin genes of clinical isolates were purified with the ExoSAP-ITâ kit (USB Corporation, Cleveland, OH, USA) and sequenced on an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The resultant nucleotide sequences were edited using GENETYX-MAC software (ver. 7; GENETYX Corporation, Tokyo, Japan). The sequences of the ITS region were compared with those from the reference strains of S. brasiliensis (GenBank accession number JQ070115), S. globosa (FN549904), clinical clade C of S. schenckii sensu stricto (KC113215),3 clinical clade D of S. schenckii sensu stricto (KC113221 and FJ545232),3 and S. luriei (AB128012) using ClustalW software.12 The phylogenetic trees were constructed using the TREEVIEW program to display phylogenies.13 Bootstrap analysis was performed on 1000 random samples and analysed using ClustalW. PCR of MAT1-1-1 and MAT1-2-1

Primers used for amplification of the MAT1-1-1 fragment were 50 -CGGCTTGGATGCAGACTTAA-30 and 50 -

© 2015 Blackwell Verlag GmbH Mycoses, 2015, 58, 220–224

TCGTGTTCGCTGTTGTCTCG-30 (primers SpalphaSP1S and SpalphaSP1R (respectively), corresponding to nucleotides (nt) 195–214 and 540–559 of the S. schenckii MAT1-1-1 sequence of Genbank accession no. AB898676).14 The primers used for amplification of the MAT1-2-1 fragment were 50 -GTCAAATTCGTGATTGATGA-30 and 50 -GCCTCCTTTACAGCTCTGTG-30 (primers SpHMGEPS and SpHMGEPR (respectively), corresponding to nt 570–589 and 840–859 of the S. schenckii MAT1-2-1 sequence of DDBJ accession no. AB693152).15 The MAT1-1-1-specific and MAT1-2-1-specific primers were used to identify MAT in each of the 18 isolates (Table 1). Genomic DNA samples (100 ng) were amplified by PCR in a volume of 30 ll, in a reaction mixture containing 10 mmol l1 Tris-HCl (pH 8.3), 50 mmol l1 KCl, 1.5 mmol l1 MgCl2, 0.001% gelatin, 200 mmol l1 each deoxynucleoside triphosphate, 1.0 unit of Taq polymerase (Takara, Kyoto, Japan) and 30 lmol of each primer pair. PCR amplification was carried out for 30 cycles consisting of denaturation for 1 min at 94 °C, primer annealing for 2 min at 60 °C and extension for 2 min at 72 °C. A final extension was performed at 72 °C for 5 min. PCR products were analysed on a 2% agarose gel.

Results Eighteen isolates were confirmed as S. schenckii sensu stricto by sequence analyses of the ITS region of rDNA and the calmodulin gene (Table 1). Phylogenetic analysis revealed that the sequences of the ITS region of the 18 isolates clustered with clinical clade D of S. schenckii sensu stricto, as a branch distinct from clinical clade C of S. schenckii sensu stricto and from isolates of S. globosa, S. brasiliensis and S. luriei (Fig. 1). The phylogenetic analysis also revealed that the sequences of the calmodulin genes from the 18 isolates clustered with S. schenckii sensu stricto (AM117438), genetically distinct from isolates of S. globosa, S. brasiliensis and S. luriei (Fig. 2). The ratio of MAT1-1 : 1-2 in our isolates was found to be 1 : 0, since PCR detected MAT1-1-1 (and not MAT1-2-1) in all 18 isolates (Table 1).

Discussion Human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch,8 a route similar to that reported in Brazil.5 However, Rodrigues et al. [6] reported that predominant aetiological agent in feline sporotrichosis in Brazil is S. brasiliensis, which is

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Table 1 Isolates of Sporothrix schenckii from feline sporotrichosis in Malaysia used in this study. Strain

ITS identity

ITS number2

CAL identity

CAL number3

MAT1-14

MAT1-25

NUBS1 14001 NUBS 14002 NUBS 14003 NUBS 14004 NUBS 14005 NUBS 14007 NUBS 14008 NUBS 14009 NUBS 14010 NUBS 14011 NUBS 14013 NUBS 14014 NUBS 14015 NUBS 14016 NUBS 14017 NUBS 14018 NUBS 14019 NUBS 14022

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

LC012514 LC012515 LC012512 LC012516 LC012513 LC012518 LC012519 LC012520 LC012521 LC012598 LC012599 LC012600 LC012601 LC012602 LC012603 LC012604 LC012605 LC012606

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

LC013252 LC014618 LC014619 LC014624 LC014620 LC014623 LC014621 LC014625 LC014627 LC014626 LC014628 LC014629 LC014630 LC014631 LC014632 LC014633 LC014634 LC014636

+ + + + + + + + + + + + + + + + + +

                 

schenckii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii schenkii

schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii schenckii

ITS, internal transcribed spacer. 1

NUBS: Nihon University College of Bioresource Sciences.

2

GenBank accession numbers of the ITS regions of S. schenckii sensu stricto.

3

GenBank accession numbers of the calmodulin gene of S. schenckii sensu stricto. MAT1-1: MAT1-1-1 specific PCR, +: positive, : negative.

4

MAT1-2: MAT1-2-1 specific PCR, +: positive, : negative.

5

Figure 1 Phylogenetic trees, derived by

neighbour-joining methods, showing the molecular taxonomy of the internal transcribed spacer (ITS) region fragments. The numbers at the branch points represent the bootstrap values. GenBank accession numbers of the ITS regions of Sporothrix species are indicated in parentheses.

geographically restricted to the South and Southeast regions of Brazil. Rodrigues et al. [6] indicated that the zoonotic transmission of Sporothrix through cats clearly differentiates Brazil from other outbreaks worldwide, where the source and vector of infection are primarily soil and decomposing organic matter. We suspect the epidemic feline sporotrichosis in

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Malaysia is caused by cat fights and contact with the environment, because the patient animals included both domestic and feral cats and the diseases did not occur in a seasonal pattern. Therefore, in Malaysia, as in Brazil, feline sporotrichosis appears to be the predominant aetiological agent for transmission to humans.

© 2015 Blackwell Verlag GmbH Mycoses, 2015, 58, 220–224

Sporothrix schenckii from Malaysia

Figure 2 Phylogenetic trees, derived by

neighbour-joining methods, showing the molecular taxonomy of the calmodulin gene fragments. The numbers at the branch points represent the bootstrap values. GenBank accession numbers of the calmodulin genes of Sporothrix species are indicated in parentheses.

In the present study, isolates from cats with sporotrichosis were identified as S. schenckii sensu stricto by sequence analyses of the calmodulin gene and of the ITS region. Phylogenetic analysis of ITS region demonstrated that these isolates sorted with clinical clade D of this species. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be most prevalent in feline sporotrichosis in Malaysia. Our results contrasted with the those of Zhou et al. [3], which indicated (using isolates from Japanese patients) that S. globosa and S. schenckii sensu stricto clinical clade C were the most common human sporotrichosis pathogens in Asia. Clearly, further molecular epidemiological studies are required to characterise sporotrichosis in Asia. In our previous study,15 we observed a 1 : 3 ratio of MAT1-1-1:MAT1-2-1 among isolates of S. globosa obtained from multiple regions of Japan. In contrast, a MAT1-1-1:MAT1-2-1 ratio of 1 : 0 was observed in the present study among 18 isolates of S. schenckii sensu stricto obtained in Malaysia. We speculate that a clonal strain of S. schenckii may serve as the causative agent of feline sporotrichosis in Malaysia. Teixeira et al. [16] reported that the MAT11 : MAT1-2 ratio was not significantly different from 1 : 1 among S. brasiliensis and S. schenckii in Brazil, but the population of S. brasiliensis in the outbreaks from S~ao Paulo originated from a single MAT. Therefore, we are fearful of an ongoing epidemic of feline sporotrichosis in Malaysia. Kwon-Chung et al. [17] studied the MAT distribution for isolates of histoplasmosis outbreaks and reported a ratio of + and  types of 1 : 2.5. In Cryptococcus neoformans, a MAT strains outnumber a strains by almost 45 : 1 in environmental isolates and by

© 2015 Blackwell Verlag GmbH Mycoses, 2015, 58, 220–224

almost 30 : 1 in clinical isolates.18 Moreover, the MAT (+)-specific gene of the high-mobility group (HMG) DNA-binding domain in Trichophyton interdigitale, an anthropophilic dermatophyte, is ordinarily detected in isolates from human tinea pedis.19 Thus, pathogenic fungi often appear to be biased towards the prevalence of a single MAT. However, it is unclear how this phenomenon (single MAT) arose when pathogenic fungi evolved from saprophytes. Further investigation of the evolution of virulence and the selection of MAT in isolates of Sporothrix species will be necessary to address this question.

Acknowledgments This study was supported by a grant (‘International joint research and training of young researchers for zoonosis control in the globalized world’) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan, and by a Nihon University Multidisciplinary Research Grant (2013-2014).

Conflicts of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is ...
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