Viability and molecular authentication of Coccidioides immitis strains from Culture Collection of the Instituto Oswaldo Cruz , Rio de Janeiro , Brazil

Twenty Coccidioides immitis strains were evaluated. Only 5 of the 20 strains kept under mineral oil maintained their viability while all 5 subcultures preserved in water remained viable and none of the 13 subcultures kept in soil were viable. A 519 bp PCR product from the csa gene confirmed the identity of the strains. Key-words: Coccidioides immitis. Long-term preservation. PCR. RESUMO Vinte cepas de Coccidioides immitis foram avaliadas. Cinco das 20 cepas preservadas sob óleo mineral mantiveram-se viáveis, todas as 5 subculturas preservadas em água permaneceram viáveis e nenhuma das 13 subculturas mantidas em solo foi viável. Um produto de PCR de 519 pb do gene csa confirmou a identidade das cepas. Palavras-chaves: Coccidioides immitis. Preservação por longos períodos. PCR. 1. Serviço de Micologia do Instituto de Pesquisa Clínica Evandro Chagas da Fundação Oswaldo Cruz, Rio de Janeiro, RJ. 2. Departamento de Micologia do Instituto Oswaldo Cruz da Fundação Oswaldo Cruz, Rio de Janeiro, RJ. This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq no 475.219/2006 Address to: Dra. Cintia de Moraes Borba. Depto. de Micologia IOC/FIOCRUZ. Av. Brasil 4365, 21040-900, Rio de Janeiro, RJ, Brasil. Tel: 55 21 3865-8114; Fax: 55 21 3865-8113. e-mail: cborba@ioc.fiocruz.br Recebido para publicação em 1/6/2005 Aceito em 12/4/2006 Coccidioides immitis is a dimorphic, geophilic fungus that causes coccidioidomycosis, a systemic mycosis affecting humans and a wide variety of animals. This mycosis is endemic in many countries of the American continent. The highest prevalence of infection occurs in the Southwestern states of the USA and the Northern Mexican states. However, endemic foci are known in Central and South America. More recently, a large semiarid area in the Northeastern region of Brazil was identified as endemic for this mycosis . Twenty C. immitis strains are maintained in the Culture Collection of the Oswaldo Cruz Institute using different long term storage methods. They were deposited in the collection between 1929 and 1955 and their records show that they came from the USA (9), Venezuela (4), Brazil (1), Netherlands (1), Norway (1) and Argentina (4). Fisher et al described the division of Coccidioides immitis into two species, C. immitis and C. posadasii, respectively the agents of coccidioidomycosis in California and non-California population. Thus, despite the fact that C. posadasii is the agent of mycosis in Brazil, we decided to maintain the older taxonomic status due to the earlier isolation of the strains. Fungi preservation methods have been used in many laboratories with differing results, depending on the species 12 16 . However, some studies have demonstrated that the storage of fungi in vitro for long periods of time may induce morphological changes, alterations in cell wall components, spontaneous mutations and loss or attenuation of virulence. The preservation of fungi which are pathogenic to man and animals is important for research and biotechnology, thus, determining the correct preservation method for each fungal species and periodical monitoring to check their identity has become a requirement. Molecular

Coccidioides immitis is a dimorphic, geophilic fungus that causes coccidioidomycosis, a systemic mycosis affecting humans and a wide variety of animals.This mycosis is endemic in many countries of the American continent.The highest prevalence of infection occurs in the Southwestern states of the USA and the Northern Mexican states.However, endemic foci are known in Central and South America.More recently, a large semiarid area in the Northeastern region of Brazil was identified as endemic for this mycosis 11 23 .
Twenty C. immitis strains are maintained in the Culture Collection of the Oswaldo Cruz Institute using different long term storage methods.They were deposited in the collection between 1929 and 1955 and their records show that they came from the USA (9), Venezuela (4), Brazil (1), Netherlands (1), Norway (1) and Argentina (4).Fisher et al 13 described the division of Coccidioides immitis into two species, C. immitis and C. posadasii, respectively the agents of coccidioidomycosis in California and non-California population.Thus, despite the fact that C. posadasii is the agent of mycosis in Brazil, we decided to maintain the older taxonomic status due to the earlier isolation of the strains.
Fungi preservation methods have been used in many laboratories with differing results, depending on the species 1 12 16 24 .However, some studies have demonstrated that the storage of fungi in vitro for long periods of time may induce morphological changes 22 , alterations in cell wall components 21 , spontaneous mutations 3 and loss or attenuation of virulence 4 .The preservation of fungi which are pathogenic to man and animals is important for research and biotechnology, thus, determining the correct preservation method for each fungal species and periodical monitoring to check their identity has become a requirement.Molecular techniques have proved to be useful for specimen authentication and for obtaining molecular genetic data.
C. immitis produces a specific 19-kDa antigen encoded by the csa gene, with no cross-reaction with other fungi.The primer pair derived from this gene amplify a 519 bp product only when the C. immitis genomic DNA is used as template 17 .
The objective of the present study was to evaluate the viability and morphological characteristics of C. immitis strains maintained for many years under mineral oil, in soil and in water from the Culture Collection of the Oswaldo Cruz Institute (IOC) in Rio de Janeiro, Brazil, and to authenticate them by PCR.

MATERIAL AND METHODS
A total of 20 Coccidioides immitis strains were first preserved at the IOC by successive transplants and maintained at room temperature.In the 1940's the strains were transferred to potato dextrose agar (PDA) medium under mineral oil.Each strain preserved under mineral oil was distributed among a maximum of 5 tubes with different dates, whereas each of the strains preserved in soil and water was represented by only one tube.Samples of the strains subjected to the different preservation methods were grown on PDA (Difco Laboratories, MI, USA) and incubated for 90 days at room temperature to evaluate viability.Figure 1 shows the procedure followed to extract the strains from the preservation methods and their evaluation.
A fragment of the colony grown on PDA was removed and then placed on a slide with one drop of Amann lactophenolcotton blue (20g of phenic acid, 20ml of lactic acid, 20ml of distilled water, 40ml of glycerol and 0.05g of cotton blue) and covered with a coverslip for examination with a Nikon model Labophot microscope.
The viable strains were cultured in 100ml GYE medium (2% glucose, 1% yeast extract, all from Difco) and incubated at 30 o C in a reciprocating shaker at 120 oscillations/min.After 9 days of growth, the strains were subjected to 100 o C in order to kill them.The mycelium mat was separated by vacuum filtration using a sterile filter paper, followed by continuous vacuum until dry.The mycelium mat was lyophilized overnight in a freeze dryer, model L4KR 156 (Edwards, São Paulo, Brazil).Ten milligrams of the dried mycelia were grounded in liquid nitrogen.The resulting mycelium powder was suspended in 400ml of extraction buffer (50mM Tris-HCl pH 8.0; 100mM NaCl; 5mM EDTA and 1% SDS) incubated at 80 o C for 10 min, and incubated again at 40 o C with 10ml of proteinase K (10mg/ml) for 3h.Afterwards, the solution was incubated at 80 o C for 10 min in accordance with Burt et al 7 .The nucleic acids were then immediately extracted with 400ml of phenol: chloroform: isoamyl alcohol (25:24:1) and briefly homogenized.The solution was centrifuged at 10,000 x g for 15min.The upper aqueous phase was removed and the DNA precipitated with 3M sodium acetate and isopropanol.After washing with 70% cold ethanol the DNA was dried by continuous vacuum.The DNA was quantified in a spectrophotometer (GeneQuant™ pro RNA/DNA Calculator, Amersham Pharmacia, Biotech, USA) using 1:1,000 dilutions and visualized on 0.8% agarose gel.
Five nanograms of DNA were used as a template in PCR using specific primers designed on the basis of the csa gene sequence (sense = 5' AAG TTC TCA CTC CTC AGC GCT ATC G 3'; anti-sense = 5' ACA TTA AGG TTC CTC CCC TTC AAC C 3') 17 .The reaction mixture contained 10x buffer PCR (Amersham Pharmacia), 200mM of dNTPs, 10mM of each primer, 1 unit of Taq polymerase (Gibco), DNA template in a final volume of 50ml.Thirty cycles were conducted for amplification in a thermocycler (Gene Amp® PCR System 2,400 -Applied Biosystems).The amplification program was 1 cycle of 94 0 C for 4 min and 30 cycles of 94 0 C for 1 min, 50 0 C for 1 min, 74 0 C for 1 min, followed by 74 0 C for 10 min.After thermal cycling, 20ml of the amplified product was run on a 1.6% (w:v) agarose gel, with 1x TBE as buffer, stained with ethidium bromide and visualized under UV light.

RESULTS AND DISCUSSION
Only 5 (25%) of the 20 C. immitis strains maintained under oil remained viable, whereas all 5 (100%) strains preserved in water were viable and none of the 13 subcultures kept in soil were revealed as viable.Table 1 shows the data concerning the viable strains preserved over different periods of time.The C. immitis strains studied here were kept for long periods of time under mineral oil.This preservation method is based on the reduction of microorganism metabolic rates due to reduced oxygen consumption 6 .However, this decreased metabolic activity over long periods of time may affect the viability, development, spore formation and dimorphic process of many fungal strains 2 14 22 .Other determinants of the fall in viability are the depth of the mineral oil layer and the quality of the product used 10 19 .The small percentage of viable C. immitis strains after preservation under mineral oil detected in this study may be related to their intrinsic characteristics and also to variations in the attributes of the preserved cells and of the technique used.Thus, the low viability of the strains studied clearly shows the high requirements of this fungus for factors related to good development in vitro.
In contrast, the C. immitis cells evaluated here that were preserved in distilled water remained viable for 36 years.Water storage is a simple and inexpensive method of culture preservation described by Castellani 8 , who stored fungi pathogenic to man.Of the fungi studied by Castellani 9 , C immitis grew very well, producing colonies exactly like the original, with the same biochemical characters after 12 months in sterile distilled water.McGinnis et al 15 obtained 93% revival for 12-60 months in cultures preserved in distilled water and recorded reduced pleomorphism in pathogens.
Many of the fungal pathogens of humans accommodated in the Onygenales, like C. immitis, including agents of cutaneous infection (Microsporum and Trichophyton) and human respiratory pathogens (Histoplasma and Blastomyces), showed a rate of viability ranging between 73% and 100% after 6, 12, 18 and 24 months in sterile distilled water 20 .These data are in agreement with our results which show 100% viability for C. immitis strains preserved in distilled water for a long period of time (36 years).According to Onions 16 this method appears to produce very satisfactory results for some isolates.
None of the strains studied survived when preserved in soil, in agreement with Windels 24 , who reported that the use of soil culture for long-term storage may cause mutations with the loss of morpho-and physiological characteristics or cellular death.
The viable strains presented macro and microscopic aspects suggestive of C. immitis as white cream colored velvety colonies formed by hyaline, septate, branched hyphae and only one strain (Ci IOC 2761 preserved in water) presented arthroconidia alternating with empty cells.However, in accordance with Pappagianis 18 the hyphal form is not sufficiently specific for that to yield more than a presumptive diagnosis.In order to avoid the animal inoculation method to demonstrate their virulence, these isolates were authenticated by a molecular tool (PCR).A 519bp PCR product was obtained with the primer pair used and C. immitis genomic DNA was used as the template to confirm the identity of the viable strains (Figure 2).The results of the PCR method for the detection of C. immitis DNA using oligonucleotide primer pair derived from csa gene sequence showed an amplification of identical 519bp product in all seven strains of the pathogen in accordance with Pan and Cole 17 , demonstrating the usefulness of this molecular marker for culture collections.
Culture collections are an important resource for obtaining molecular genetic data and the PCR technique is an effective method for this.The problems of the correct classification of species names, alterations in morphological characteristics, which prevent precise identification and changes in population structure over time, can be examined by analysis of DNA regions.According to Bruns et al 5 , extensive use of preserved specimens for molecular genetic investigations may soon make it desirable for curators to develop guidelines for the sampling of type collections and for methods of storage and retrieval of extracted DNA.

Figure 1 -
Figure 1 -Schematic representation of the evaluation of viability of Coccidioides immitis strains preserved in a culture collection.

Table 1 -C. immitis viable strains preserved using three preservation methods for different periods of time.
u = unknown; ct = contaminated; d = dry; O = mineral oil; W = water; S = soil