The basidiomycetous yeast genus Cryptococcus contains two medically important pathogens, Cryptococcus neoformans and C. gattii [1-3]. C. neoformans is one of the common pathogens in acquired immunodeficiency syndrome (AIDS), whereas the most cases of diseases due to C. gattii happened in the healthy individuals [2, 4]. C. gattii has a tendency to affect the respiratory and nervous systems of the humans and domestic animals such as, dogs, cats, and horses . C. gattii is more geographically restricted than C. neoformans and is largely confined to tropical and subtropical regions. Several reports show that C. gattii was isolated from Eucalyptus trees (Eucalyptus tereticornis, E. citriodora and E. camaldulensis) in Australia [4, 6, 7].
These two species are divided into five serotypes including: serotype A (C. neoformans var. grubii), serotype D (C. neoformans), serotype A/D (C. gattii) and serotypes B and C (formerly C. neoformans var. gattii) [1, 3]. C. gattii has two mating types, a and α  and four genotypes designated VGI to VGIV . VGI of C. gattii has a worldwide distribution and is the most customary genotype in Australia. VGII is restricted to the Northern Territory, VGIII reported from Colombia, India and the United States, while VGIV is common in Africa and Central America [4, 9, 10]. The aim of the present study was to evaluate the isolation of C. gattii from Eucalyptus trees in Ahvaz, a capital city of Khuzestan province in south western Iran.
Khuzestan province is located in the southwestern part of Iran with subtropical climatic conditions (Figure 1). June-August temperatures arise more than 52°C. In the present study, a total of 156 samples of flowers (20), fruits (33), leaves (41) and barks (31) of Eucalyptus trees and also soil underneath Eucalyptus trees (31), were collected over a period six months (October-March). Samples were collected from Eucalyptus trees and soils in public gardens and natural reserves in various parts of Ahvaz. In addition, a part of the samples was also collected from the gardens of Ahvaz Jundishapur University of Medical Sciences and Shahid Chamran University of Ahvaz campus. All samples (with exception soils) were cut into small pieces and then 5-10 g of each sample was mixed with 25 ml of sterile distilled water that contained 0.05 mg/ml chloramphenicol (Merck, Germany) in sterile ﬂasks. The ﬂasks were vigorously shaken for few minutes, and then settled for 30 minutes. 0.2 ml of each supernatant sample was inoculated on the Niger seed agar plates and incubated at 30°C for two weeks . Plates were observed daily and all brown and moist colonies suspected to Cryptococcus species, sub-cultured on Sabouraud dextrose agar (Merck, Germany) and incubated at 30°C. Then, all isolates were tested for urease production, growth at 37°C and the presence of capsule around yeasts using India ink preparation.
In the present study, several saprophytic fungi including, Candida species and black yeasts were isolated from samples using Niger seed plates. Although, several isolates were grown as mucoid and brown colonies on selected medium, their urease activity, growth at 37 °C and presence of capsule were negative. As a result, we could not isolate any C. gattii from Eucalyptus trees and soils in Ahvaz.
C. gattii is less virulent than C. neoformans and C. neoformans var. grubii. Thus, most reported cases of disease were among immunocompromised patients. According to Datta et al.,  C. gattii has been increased as a human and animal pathogen in the Pacific Northwest region. Several reports show that most cases of cryptococcosis due to C. gattii were reported from tropical areas .
The successful isolation of C. gattii from E. camaldulensis in Punjab represents the first Indian isolation . However, Australian Eucalyptus trees are the source of C. gattii. Bineshian and Zaini were the only isolated 2 cases of C. gattii from the 600 samples from E. camaldulensis in Northern Iran . Although this study shows that Iranian Eucalyptus trees can be a source of C. gattii, we cannot isolate any C. gattii from Eucalyptus trees in the Southwest of Iran. Direct sunlight sterilizes the sites contaminated by Cryptococcus, in the summer months. With expert studies done in this field, in this research temperature differences in flowering time can be the reason of the unsuccessful isolation of this organism in the environment. In addition, the high temperature in this area probably inhibits the growing of C. gattii on trees.
- Voelz K, May RC. Cryptococcal interactions with the host immune system. Eukaryot Cell. 2010; 9(6):835-46.
- Saul N, Krockenberger M, Carter D. Evidence of recombination in mixed-mating-type and alpha-only populations of Cryptococcus gattii sourced from single Eucalyptus tree hollows. Eukaryot Cell. 2008; 7(4):727-34.
- Datta K, Bartlett KH, Baer R, Byrnes E, Galanis E, Heitman J. Spread of Cryptococcus gattii into pacific northwest region of the United States. Emerg Infect Dis. 2009; 15(8):1185-91.
- Campbell LT, Currie BJ, Krockenberger M, Malik R, Meyer W, Heitman J. Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii. Eukaryot Cell. 2005; 4(8):1403-9.
- Sorrell TC, Brownlee AG, Ruma P, Malik R, Pfeiffer TJ, Ellis DH. Natural environmental sources of Cryptococcus neoformans var gattii. J Clin Microbiol. 1996; 34(5):1261-3.
- Chakrabarti A, Jatana M, Kumar P, Chatha L, Kaushal A, Padhye AA. Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India. J Clin Microbiol. 1997; 35(12):3340-2.
- Werther K, Sousa Ed, Alves Júnior JRF, Ardisson FA, Giannini MJSM. Cryptococcus gattii and Cryptococcus albidus in captive domestic pigeon (Columba livia). Braz J Vet Pathol. 2011; 4(3):247-9.
- Byrnes EJ, Li W, Lewit Y, Perfect JR, Carter DA, Cox GM. First reported case of Cryptococcus gattii in the Southeastern USA: implications for travel-associated acquisition of an emerging pathogen. PLoS One. 2009; 4(6):e5851.
- Ellis D, Marriott D, Hajjeh RA, Warnock D, Meyer W, Barton R. Epidemiology: surveillance of fungal infections. Med Mycol. 2000; 38:173-82.
- Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003; 9(2):189-95.
- Bineshian F, Zaini F. Study of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in some northern regions of Iran. Koomesh. 2002; 3(1):59-67.
- McTaggart L, Richardson SE, Seah C, Hoang L, Fothergill A X, Zhang S. Rapid identification of Cryptococcus neoformans var grubii, C neoformans var neoformans, and C gattii by use of rapid biochemical tests, differential media, and DNA sequencing. J Clin Microbiol. 2011; 49(7):2522-7.