CHARACTERIZATION OF TYPICAL AND ATYPICAL MALASSEZIA SPP. FROM CATTLE AND DOG BY RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS

CARACTERIZAÇÃO DE ISOLADOS TÍPICOS E ATÍPICOS DEMALASSEZIA SPP. PROVENIENTES DE BOVINOS E CÃES UTILIZANDO A ANÁLISE DO DNA POLIMÓRFICO AMPLIFICADO ALEATORIAMENTE

E.R. Duarte J.C.P. Resende J.S. Hamdan About the authors

ABSTRACT

There are few numbers of biochemical tests for specie classification in the genus Malassezia and these can to fail in the identification of the atypical isolates. In this study, typical and atypical isolates were analysed by random amplification of polymorphic DNA (RAPD) to compare with biochemical-physiological characteristics of the Malassezia species from bovine and canine ears. RAPD band patterns using OPA4 primer clustered all isolates according its biochemicalphysiological characteristics in the species from cattle and dog. Malassezia nana and M. sympodialis isolates were sub-clustered in separated sub-branches and both were from a different branch of the other species. The DNA pattern of the two atypical lipid-dependent M. pachydermatis strains was similar with of other typical strains but it did not show the one specific band of 200bp. Future studies in the specific RAPD bands of genetic profiles can be important to corroborate the identification of typical and atypical isolates of the genus Malassezia.

KEY WORDS
Malassezia spp.; identification; atypical isolates; RAPD analysis

RESUMO

Existem poucos testes para a identificação das espécies do gênero Malassezia e estes podem ser insuficientes para a correta identificação de isolados atípicos. O objetivo deste estudo foi caracterizar isolados típicos e atípicos dessas leveduras, comparando características bioquímicas e fisiológicas com a análise do DNA polimórfico amplificado aleatoriamente (RAPD). Foram analisados 30 isolados provenientes do ouvido de bovinos e de cão. Utilizando o iniciador OPA4, os padrões de RAPD foram agrupados de acordo com suas características bioquímicas e fisiológicas. Isolados da nova espécie M. nana e de M. sympodialis foram subagrupados em dois diferentes sub-ramos, pertencentes a um grupo distinto das outras espécies. Os padrões de RAPD de duas cepas atípicas lipo-dependentes de M. pachydermatis foram similares com os de outras cepas típicas dessa espécie, entretanto não apresentaram uma banda específica de 200 pb. Em futuros estudos a caracterização e a análise de sequências de bandas específicas podem corroborar na correta identificação de isolados atípicos e típicos pertencentes ao gênero Malassezia.

PALAVRAS-CLAVE
Malassezia spp.; isolados atípicos; identificação; RAPD

INTRODUCTION

The genus Malassezia is composed by thirteen species of yeasts of increasing importance in human and veterinary medicine. In human they have been associated with pityriasis versicolor (PV), seborreic dermatitis (SD), folliculitis, and systemic infection (GUÉHO et al., 1998GUÉHO, E.; BOEKHOUT, T.; ASHBEE, H.R.; GUILLOT, J.; VAN-BELKUM, A.; FAERGEMANN, J. The role of Malassezia species in the ecology of human skin and as pathogens. Medical Mycology, v.36, p.220-229, 1998.; SUGITA et al., 2006SUGITA, T.; TAJIMA, M.; TAKASHIMA, M.; AMAYA, M.; SAITO, M.; TSUBOI, R.; NISHIKAWA, A. A new yeast, Malassezia yamatoensis, isolated from a patient with seborrhoeic dermatitis, and its distribution in patients and healthy subjects. Microbiology and Immunology, v.48, p.755-759, 2006.). M. pachydermatis, M. nana, M. equinaand M. caprae could be considered zoophilic (GUÉHO et al., 1998GUÉHO, E.; BOEKHOUT, T.; ASHBEE, H.R.; GUILLOT, J.; VAN-BELKUM, A.; FAERGEMANN, J. The role of Malassezia species in the ecology of human skin and as pathogens. Medical Mycology, v.36, p.220-229, 1998.; SUGITA et al., 2003SUGITA, T.; TAKASHIMA, M.; KODAMA, M.; TSUBOI, R.; NISHIKAWA, A.; Description of a new yeast species, Malassezia japonica, and its detection in patients with atopic dermatitis and healthy subjects. Journal of Clinical Microbiology, v.41, p.4695-4699, 2003., SUGITA et al., 2004SUGITA, T.; TAJIMA, M.; TAKASHIMA, M. AMAYA, M.; SAITO, M.; TSUBOI, R., NISHIKAWA, A. A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects. Microbiology and Immunology, v.48, p.579-583, 2004.; HIRAI et al., 2004HIRAI, A.; KANO, R.; MAKIMURA, K.; DUARTE, E.R.; HAMDAN, J.S.; LACHANCE, M.A.; YAMAGUCHI, H.; HASEGAWA, A. Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. International Journal of Systematic and Evolutionary Microbiology, v.54, p.623-627, 2004.; CABAÑES et al., 2007CABAÑES, F.J.; THEELEN, B.; CASTELLÁ, G.; BOEKHOUT, T. 2Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands Two new lipid-dependent Malassezia species from domestic animals. FEMS Yeast Research, v.7, p.1064-1076, 2007.). Malassezia pachydermatis is zoophilic and associated with external otitis and dermatitis in dog and cat (BOND et al., 2000BOND, R.; LAMPORT, A.I.; LLOYD, D.H. Colonization status of Malassezia pachydermatis on the hair follicle of healthy beagle dogs. Research Veterinary in Science, v.68, p.291-293, 2000.). The lipid dependent species Malassezia globosa, M. sympodialis, M. furfur and M. slooffiae have been associated with bovine parasitic otitis (DUARTE et al., 2001DUARTE, E.R.; RESENDE, J.C.; ROSA, C.A.; HAMDAN, J.S. Prevalence of yeasts and mycelial fungi in bovine parasitic otitis in the State of Minas Gerais, Brazil. Journal of Veterinary Medicine Series B, v.48, p.631-635, 2001.; DUARTE et al., 2003DUARTE, E.R.; BATISTA, R.D.; HAHN, R.C.; HAMADAN, J.S. Factors associated with the prevalence Malasseziai species in the external ears of cattle from the state of Minas Gerais, Brasil. Medical Mycology, v.41, p.137-142, 2003.).

The random amplification of polymorphic DNA (RAPD) has been employed to epidemiological studies. The intra-species subtypes obtained with RAPD from samples of Malassezia species suggest the presence of genetic population differences that may be an important tool for epidemiological investigation (BOEKHOUT et al., 1998BOEKHOUT, T.; KAMP, M.; GUÉHO E. Molecular typing of Malassezia species with PFG and RAPD. Medical Mycology, v.36, p.365-367, 1998.; THEELEN et al., 2001THEELEN, B.; SILVESTRI, M.; GUÉHO, E.; BELKUM, A.; BOEKHOUT, T. Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP Tm), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE). FEMS Yeast Research, v.1, p.79-86, 2001.). The most important argument to consider the RAPD-PCR as a clearly useful procedure for grouping Malassezia species according to their origin may be attributed to the nosocomial and occasionally fatal infections in neonates as reported by BELKUM et al. (1994)BELKUM, A.; BOEKHOUT, T.; BOSBOOM, R. Monitoring spread of Malasseiza infections in a neonatal intensive care unit by PCR-mediated genetic typing. Journal of Clinical Microbiology, v.32, p.2528-2532, 1994., CHANG et al. (1998)CHANG, H.J.; MILLER, H.L.; WATKINS N.; ARDUINO, M.J.; ASHFORD, D.A.; MIDGLEY, G., AGUERO, S.M., PINTO-POWELL, R., VON REYN, C.F., EDWARDS, W., MCNEIL, M.M., JARVIS, W.R. An epidemic of Malassezia pachydermatis in the intensive care nursery associated with colonization from health care worker’s pet dog. New England Journal of Medicine, v.12, p.706-711, 1998. e CHRYSSANTHOU et al. (2001)CHRYSSANTHOU, E.; BROBERGER, U.; PETRINI, B. Malassezia pachydermatis fungemia in a neonatal intensive care unit. Acta Paediatrica, v.90, p.323-327, 2001..

Malassezia furfur isolates from of patients with PV, SD and SD of the HIV positive patients was characterized by RAPD-PCR analyses. A distinct differentiation between isolates from PV and SD patients with or without AIDS was observed (GANDRA et al., 2006GANDRA, R.F.; SIMÃO, R.C.G.; MATSUMOTO, F.E.; SILVA, B.C.M.; RUIZ, L.S.; SILVA, E.G.; GAMBALE, W.; PAULA, C.R. Genotyping by RAPD-PCR analyses of Malassezia furfur strains from pityriasis versicolor and seborrhoeic dermatitis patients. Mycopathologia, v.162, p.273-280, 2006.). Similar results with isolates obtained from patients presenting different dermatosis or systemic infections were observed by BOEKHOUT et al. (1998)BOEKHOUT, T.; KAMP, M.; GUÉHO E. Molecular typing of Malassezia species with PFG and RAPD. Medical Mycology, v.36, p.365-367, 1998. suggesting that an intra-specific genetic variation determine the presence of the different populations of M. furfur.

In a recent study this technique was applied for genetic typing of Malassezia pachydermatis isolates from different domestic animals. Four different genetic types were distinguished and one predominant genotype was observed in isolates recovered from different anatomical locations in cats, horses, goats and pigs (CASTELLÁ et al., 2005CASTELLÁ, G.; HERNÁNDEZ, J.J.; CABAÑES, J. Genetic typing of Malassezia pachydermatis from different domestic animals. Veterinary Microbiology, v.108, p.291-296, 2005.).

The identification system based on differences in the ability of Malassezia species to grow in the presence of tweens or cremophor EL and to split esculin can fail to identify atypical isolates or new species. Lipiddependent variants of M. pachydermatis and atypical M. furfur have needed complementary tests to achieve a correct identification (DUARTE et al., 2002DUARTE, E.R.; LACHANCE, M.A.; HAMDAN, J.S. Atypical lipid-dependent Malassezia strains from cattle and dog. Canadian Journal of Microbiology, v.48, p.749-752, 2002.). Malassezia species can be distinguished by RAPD patterns. In the study of BOEKHOUT et al. (1998)BOEKHOUT, T.; KAMP, M.; GUÉHO E. Molecular typing of Malassezia species with PFG and RAPD. Medical Mycology, v.36, p.365-367, 1998., Malassezia sympodialis and M. slooffiae showed relative uniform banding patterns with OPA4 and OPA5 respectively.

The objective of the present study was to compare the random amplified polymorphic DNA (RAPD) profiles with specific biochemical-physiological characteristics of typical and atypical Malassezia isolates from bovine and canine ears in Minas Gerais State of Brazil.

MATERIAL AND METHODS

A total of 30 isolates from ear cattle and ear dog in Minas Gerais State of Brazil were identified based on morphological characteristics, growth on Dixon medium at different temperatures, growth on Sabouraud’s medium supplemented with tweens and cremophor EL in diffusion test, esculin hydrolise and catalase screening (MAYSER et al., 1997MAYSER, P.; HAZE, P.; PAPAVASSUKUS, C.; PICKEL, M.; GRUENDER, K.; GUÉHO, E. Differentiation of Malassezia species: selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur. British Journal of Dermatology, v. 137, p.208-213, 1997.; GUEHO et al., 1998).

The typical and atypical isolates of Malassezia spp. and two standard strains used in the RAPD genetic analysis were listed in the Table 1. As controls for the biochemical-physiological tests and genetic analysis the standard strains CBS-1878 (Malassezia furfur) and CBS-1879 (Malassezia pachydermatis) were simultaneously tested with each set of 10 to 18 isolates. The isolates of the novel species M. nana (now standard strains) were identified and characterized by DNA sequence analysis in a previous study (HIRAI et al., 2004HIRAI, A.; KANO, R.; MAKIMURA, K.; DUARTE, E.R.; HAMDAN, J.S.; LACHANCE, M.A.; YAMAGUCHI, H.; HASEGAWA, A. Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. International Journal of Systematic and Evolutionary Microbiology, v.54, p.623-627, 2004.). The identification of the atypical lipiddependent strain of M. pachydermatis (VG Luz 794,) two typical M. furfur strains from cattle with otitis (VG Ig 02 and VG RC 4349) and the atypical strain (C Lerik,) were confirmed by rDNA sequence analysis of D1/D2 domains in DUARTE et al. (2002)DUARTE, E.R.; LACHANCE, M.A.; HAMDAN, J.S. Atypical lipid-dependent Malassezia strains from cattle and dog. Canadian Journal of Microbiology, v.48, p.749-752, 2002..

Following the biochemical-physiological tests of the species identification, the isolates were seeded on solid Dixon medium and incubated for 5 days at 32º C. DNA was prepared as described by BORGES et al. (1990)BORGES, M.J.; AZEVEDO, M.O.; BONATELLI, R.; FELIPE, M.S.S.; ASTOLFI-FILHO, S. A practical method for the preparation of DNA from filamentous fungi. Fungal Genetics Newsletter, v.10, p.7-11, 1990. after enzymatic digestion with glucanase (Glucanex- Novo Nordisk, USA).

The following primers were used: M13 F, M13 R, M13 FR, OPA 1, OPA 2, OPA 4, SOY, 8L AND 10L for RAPD reaction. The RAPD analyses were carried out as described by WILLIAMS et al. (1990)WILLIAMS, J.G.K.; KUBELIK, A.R.; LIVAK, K.J.; FAFALSKI, J.A.; TINGER, S.Y. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, v.18, p.6531-6535, 1990.. Reproducibility was checked by repeating PCR for at least three times. RAPD products were analyzed by electrophoresis on a 8% polyacrilamide gel in Tris borate-EDTA, pH 8.0 buffer and visualized by silver staining as described by Santos et al. (1993)SANTOS, F.R.; PENA, S.D.; EPPLEN, J.T Genetic a population study of a y-linked tetranucleotide repeat DNA polymorphism with a simple nonisotopic technique. Human Genetics, v.90, p.655-656, 1993.. Isolates were considered with similar genetic types varying up to two DNA bands of electrophoresis on polyacrilamide gel.

For RAPD data analysis the relative mobility position of all bands present in each analyzed Malassezia strain were calculated and transformed in a data matrix where the character one means the presence of a specific band and zero represents its absence. We used the Nei & Li algorithm contained in the TREECON computer package program to calculate the genetic distances between the strains (VAN DE PEER; WACHTER, 1994VAN-DE-PEERY; WACHTER R. TREECON for Windows: a software package for the construction and drawing of evolutionary trees in the Microsoft Windows environment. Computer Applications in the Biosciences; v.10, p.569-570, 1994.). The Unrooted phenograms were then constructed by UPGMA (Unweighted Pair Group with Arithmetic Mean) method and the robustness of the tree topology was assessed 1,000 bootstrap resampling.

RESULTS AND DISCUSSION

The primers M13 F (5’-TGACCGGCAGAAAAA TG-3’), OPA 2 (5’-TGCCGAGCTG-3’) and OPA 4 (5-AATCGGGCTG-3’) produced more polymorphic and reproductive profiles. These oligonucleotides were selected based on high-intensity bands, hypervariability and good definition of DNA fragments. The OPA 2 and OPA 4 primers were also used by BOEKHOUT et al. (1998)BOEKHOUT, T.; KAMP, M.; GUÉHO E. Molecular typing of Malassezia species with PFG and RAPD. Medical Mycology, v.36, p.365-367, 1998..

A total of 15 electrophoretic profiles on 8% polyacrilamide gels and their phenograms were selected. Their analysis, supported by bootstrap values higher than 80%, demonstrated that the RAPD band patterns using OPA4 primer clustered all isolates according their biochemical-physiological characteristics. Using the M13 F and OPA2 primers 83.4% of isolates were clustered according to their biochemical-physiological characteristics.

Table 1
Typical and atypical isolates of Malassezia spp. from ear cattle and ear dogs in Minas Gerais State of Brazil and standard strains used in the RAPD genetic analysis.
Fig. 1
Eletrophoretic profiles in 8% polyacrylamide gel and phenogramas of different Malassezia species by RAPDPCR using the OPA2 primer.
Fig. 2
Phenogram constructed by UPGMA method of different Malassezia species derived from RAPD assays generated by using OPA2 primer.
Fig. 3
Phenogram of different Malassezia species based on UPGMA method derived from RAPD assays generated by using OPA4 primer.
Fig. 4
Phenogram constructed by UPGMA method of five Malassezia species derived from RAPD assays generated by using M13 primer.

Better DNA amplification of M. furfur and M. sympodialis isolates were observed using the OPA 2 primer while the DNA polymorphism of M. slooffiae strains was better demonstrated using the primer OPA4. We detected 13.22 +/3.191 (average +/the standard deviation) bands for M. furfur strains varying from 100 to 1200 bp using the OPA2 primer, 22 +/3.823 bands for M. sympodialis strains varying from 100 to 1400 bp using the OPA2 primer. Using the OPA4 primer we detected 17.59 +/3.726 bands for M. slooffiae strains varying from 100 to 1400 bp.

Figure 1 shows the electrophoretic profiles of five different Malassezia species generated by RAPD-PCR with OPA 2 primer. The Figures 2, 3 and 4 show phenograms of Malassezia species based on UPGMA method derived from RAPD assays and generated by using OPA2, OPA4 and M13 primers, respectively. All strains analyzed were demonstrated in these phenograms.

The atypical strains Malassezia werecharacterized in our previous study. The C Lerik strain (M. furfur from dog) did not grow on Sabouraud´s medium supplemented with cremophor EL. The atypical isolate VG Luz 794 of M. pachydermatis never grew on Sabouraud medium without lipids and BM Mc 77 atypical strain showed poor growth (DUARTE et al., 2002DUARTE, E.R.; LACHANCE, M.A.; HAMDAN, J.S. Atypical lipid-dependent Malassezia strains from cattle and dog. Canadian Journal of Microbiology, v.48, p.749-752, 2002.). The DNA pattern of the two atypical lipiddependent M. pachydermatis strains was similar with of other typical strains of this specie (Figs. 2, 3 and 4) but it did not show one band of 200bp present in typical strains. One specific band about 600 bp could be observed to these two atypical strains (Fig. 1). The four M. furfur strains in this figure showed different DNA patterns. However, all these strains had an identical 200 bp band and the atypical strain C Lerik was similar with the typical strain M. furfur VH Duqueza.

In this study, RAPD analysis is shown to be an important complementary procedure for the characterization and identification of atypical strains in the genus Malassezia. It was corroborated by rDNA sequence analysis of D1/D2 domains of these atypical isolates in our preliminary study (DUARTE et al. 2002DUARTE, E.R.; LACHANCE, M.A.; HAMDAN, J.S. Atypical lipid-dependent Malassezia strains from cattle and dog. Canadian Journal of Microbiology, v.48, p.749-752, 2002.).

Using RAPD-PCR and UPGMA methods, the genetic profiles of the new specie M. nana and M. sympodialis strains were sub-clustered in separated sub-branches (Figs. 2, e.1 and e.2, respectively). Both were from a branch (e) different of the other species supported by a bootstrapped value of 88%. All Malasseziasympodialis isolates had a specific band about 400 bp while Mnana isolates showed a band higher than 400 bp (Figs. 1 and 2). In our preliminary study, the physiological and morphological characteristics of the M. nana are similar in many ways to those of M. sympodialis. Phylogenetic trees based on the D1/D2 region of 26S rDNA and ITS1 sequences of M. nana isolatesfromcattleandcatshowed that they were related closely to M. sympodialis (HIRAI et al., 2004HIRAI, A.; KANO, R.; MAKIMURA, K.; DUARTE, E.R.; HAMDAN, J.S.; LACHANCE, M.A.; YAMAGUCHI, H.; HASEGAWA, A. Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. International Journal of Systematic and Evolutionary Microbiology, v.54, p.623-627, 2004.).

Using the OPA4 primer, the genetic profiles of M. slooffiae isolates were clustered in separated in the Figure 3 (cluster a). Some intra-species variations in M. furfur, M. slooffiae and M. sympodialis isolates from cattle have been observed using OP2 primer. In the Figure 2 M. furfur had two sub-types sub-clustered in separated sub-branches (b and d, respectively). The presence of intra-species RAPD subtypes suggesting different populations of M. furfur and M. pachydermatis have been related in some studies (BOEKHOUT et al., 1998BOEKHOUT, T.; KAMP, M.; GUÉHO E. Molecular typing of Malassezia species with PFG and RAPD. Medical Mycology, v.36, p.365-367, 1998.; AIZAWA et al., 2001AIZAWA, T.; KANO, R.; NAKAMURA, Y.; WATANABE, S.; HASEGAWA, A. The genetic diversity of clinical isolates of Malassezia pachydermatis from dogs and cats. Medical Mycology, v.39, p.329-334, 2001.).

Intra-specific polymorphisms have been observed between DNA patterns of Malassezia isolates from different bovine herds in different regions of Brazil (DUARTE et al., 2000DUARTE, E.R.; HAHN, R.C.; HAMDAN, J.S. Molecular characterization of Malassezia species isolated from asymptomatic bovines with otitis using RAPD technique. In: CONGRESS OF THE INTERNATIONAL SOCIETY FOR HUMAN AND ANIMAL MYCOLOGY, 14., 2000, Buenos Aires, Argentina. Resumos. Buenos Aires, 2000. p.130.). The RAPD analysis and phenograms showed the formation of analogous clusters among the isolates from cattle with or without otitis raised in the same herds. These data suggested that genetically similar M. sympodialis and M. furfur strains, found as members of the normal ear microbiota, could become opportunistically active in the inflammatory process in cattle (DUARTE; HAMDAN, 2008DUARTE, E.R.; HAMDAN, J.S. Molecular characterization of Malassezia sympodialis and Malassezia furfur from cattle with and without otitis. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.60, p.779-785, 2008.).

There are few numbers of tested characteristics to species identification in the genus Malassezia and these tests can fail to identify atypical isolates. In this study, the RAPD pattern of typical and atypical Malassezia isolates from bovine and dogs ears was associated with its biochemical-physiological and morphological characteristics, favouring the species identification. Future analyzes in specific bands of genetic profiles from typical and atypical isolates could corroborate the specie identification in the genus Malassezia.

ACKNOWLEDGEMENTS

This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

REFERÊNCIAS

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  • DUARTE, E.R.; LACHANCE, M.A.; HAMDAN, J.S. Atypical lipid-dependent Malassezia strains from cattle and dog. Canadian Journal of Microbiology, v.48, p.749-752, 2002.
  • DUARTE, E.R.; BATISTA, R.D.; HAHN, R.C.; HAMADAN, J.S. Factors associated with the prevalence Malasseziai species in the external ears of cattle from the state of Minas Gerais, Brasil. Medical Mycology, v.41, p.137-142, 2003.
  • DUARTE, E.R.; HAMDAN, J.S. Molecular characterization of Malassezia sympodialis and Malassezia furfur from cattle with and without otitis. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.60, p.779-785, 2008.
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  • MAYSER, P.; HAZE, P.; PAPAVASSUKUS, C.; PICKEL, M.; GRUENDER, K.; GUÉHO, E. Differentiation of Malassezia species: selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur British Journal of Dermatology, v. 137, p.208-213, 1997.
  • SANTOS, F.R.; PENA, S.D.; EPPLEN, J.T Genetic a population study of a y-linked tetranucleotide repeat DNA polymorphism with a simple nonisotopic technique. Human Genetics, v.90, p.655-656, 1993.
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Publication Dates

  • Publication in this collection
    02 June 2021
  • Date of issue
    Apr-Jun 2009

History

  • Received
    02 Apr 2008
  • Accepted
    13 May 2009
Instituto Biológico Av. Conselheiro Rodrigues Alves, 1252 - Vila Mariana - São Paulo - SP, 04014-002 - São Paulo - SP - Brazil
E-mail: arquivos@biologico.sp.gov.br