Eur. J. Epidemiol.0392-2990


Vol. 8, No. 3


May 1992,p. 321-325



THE ECOLOGYOF CRYPTOCOCCUS NEOFORMA S D. ELLIS 1 and T. PFEIFFER Mycology Unit- Adelaide Children's Hospital- North Adelaide 5006- Australia.

Key words: Cryptococcus neoformans vat. gattii - Ecology Environmental isolations have established that Cryptococcus neoformans var. gattii serotype B appears to have a specific ecological association with Eucalyptus camaldulensis. The global distribution of the tree appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattiL The epidemiology of cryptococcosis can primarily be explained by exposure to an infective aerosolized inoculum, such as basidiospores released from specific host plants and/or desiccated blastoconidia (yeast cells) disseminated from accumulations of dried pigeon dung. The ecology of C. neoformans still remains largely unresolved, studies on the hostparasite interaction between serotype B and E. camaldulensis are still in progress, and extensive environmental searches are now underway to determine the natural habitats of serotypes A, C and D.


Cryptococcosis is a cosmopolitan infectious disease of humans and animals caused by a yeast-like fungus called Cryptococcus neoformans. Humans are believed to acquire infection by the inhalation of infectious airborne particles, however the identity of the infectious unit remains unknown. In general terms two types of disease may develop. Primary lung infections which show no diagnostic symptoms and are usually asymptomatic. These generally occur in otherwise healthy individuals, sometimes showing flulike symptoms, which resolve spontaneously without the need for treatment. Unfortunately, we have no way of determining the incidence of this form of cryptococcosis in the general population. However, it has been described as the "sleeping giant" among infections caused by fungi and many thousands of individuals are probably infected every year (1). In 'some cases the fungus may disseminate from the lungs and incite systemic disease, in particular meningitis. However, systemic disease is more often associated with immunosuppressed patients suffering i Corresponding author.


from other serious diseases, such as cancer and AIDS. Once again, the fungus usually shows a predilection for the central nervous system, however skin, bones and other visceral organs may also become involved. This form of cryptococcosis is a serious life threatening infection and patients require hospitalization and prolonged antifungal chemotherapy. AIDS is now the most common detectable predisposing factor for cryptococcosis and between 10-20% of all AIDS patients suffer from serious infections caused by C. neoformans. Overall, cryptococcosis is a serious, world-wide infectious disease of great medical importance in terms of morbidity and mortality (19). C. neoformans is an encapsulated basidiomycetous yeast-like fungus which has been divided into two varieties; C. neoformans var. neoformans (teleomorph Filobasidiella neoformans var. neoformans) and C. neoformans var. gattii (teleomorph Filobasidiella neoformans var. bacillispora) (11). The teleomorph is the sexual state of a fungus, which in this case is characterized by the production of basidiospores (sexual propagules). Asexual reproduction in C. neoformans is the anamorph state which is characterized by the production of budding yeast-like cells (asexual propagules) that typically

Ellis D. and Pfeiffer T.

Eur. J. Epidemiol.

develop a wide polysaccharide capsule (Fig. 1). Canavanine-glycine-bromthymol blue (CGB) agar (12) is now the media of choice to determine the varietal status of C. neoformans isolates. This simple biotype test is based on the ability of C. neoformans var. gattii isolates to grow in the presence of Lcanavanine and to assimilate glycine as a sole carbon source (6). The cryptococcal polysaccharide capsule also exhibits antigenic variability, and five serotype groups (A, B, C, D and AD) can now be distinguished by agglutination of the organism with specific antisera (10). Serotypes A, D and AD are synonymous with C. neoformans vat. neoformans whereas serotypes B and C belong with C. neoformans var. gattiL

climate (13). A recent assessment of the available data from Australian patient isolates indicated that rural areas of South Australia and the Northern Territory are endemic regions for the occurrence of this pathogenic fungus (7). Environmental isolations, initially from the Barossa Valley in South Australia (8), have now established that C. neoformans vat. gattii has a specific ecological association with Eucalyptus camaldulensis (the river red gum) (Fig. 2, and Fig. 3).

Figure 1. - Encapsulated yeast cells of C. neoformansvar. gattii (X1750).

The cryptococcal polysaccharide capsule is also an important virulence factor both for its ability to inhibit phagocytosis of cryptococci and by its innate antiphagocytic and immunosuppressive activity (17). Although, antibody to this capsular material neutralizes its protective effect, it may not be formed in time to prevent dissemination of the disease. Cell mediated immunity develops after weeks to months and probably acts to contain cryptococci at all body sites. This is why AIDS patients who have a defective T-lymphocyte cell mediated immunity are more susceptible to infections by C. neoformans (19, 21). C. neoformans var. neoformans (A, D and AD serotypes) has a world-wide distribution. It has been isolated from various sources in nature and is noted for its association with accumulations of avian guano, especially with pigeon excreta. The fungus has also been isolated from the dung of caged birds including canaries, parrots and budgerigars. Other environmental isolations of C. neoformans var. neoformans have been from rotting vegetables, fruits and fruit juices, wood, dairy products and soil (16,19). On the other hand, C. neoformans var. gattii (serotypes B and C) has a more restricted global distribution corresponding to a subtropical to tropical

Figure 2. - Eucalyptus camaldulensis growing on the banks of the Murrumbidgee River at Balranald, positive for C. neoformans vat. gattiL

Culture characteristics of these C. neoformans var. gattii isolates included the following: very mucoid


growth on Sabouraud dextrose agar, intense brown colour effect on Staib's bird seed agar (22), strong assimilation of creatinine, assimilation of D-proline, high virulence for white mice and a positive reaction on CGB media within 5 days. In addition, 30 environmental isolates of C. neoformans var. gattii were randomly selected for drug sensitivity testing and all were found to be fully sensitive to the following 6

Vol. 8, 1992

Ecology of C. neoformans

(the forest red gum). Although this species is closely related to Eucalyptus camaldulensis further work remains to be done. It is noteworthy that E. tereticornis has also been extensively exported overseas, especially to California (23). Dispersal of infectious propagules of C. neoformans vat. gattii occurs from November to March, concomitant with the flowering of E. camaldulensis (8). Direct microscopy of water suspensions (9) that were strongly positive on culture for the fungus demonstrated the presence of numerous smooth-walled encapsulated bacillary-like structures (1.5-2 x 6-8 urn), similar in shape and size to that described for basidiospores belonging to the teleomorph F. neoformans var. bacillispora (Fig. 4), (Table 1).

Figure 3. - Primary isolation plate from E. camaldulens& showing a heavy growth of C. neoformans vat. gattii.

antifungal drugs; Amphotericin B, Miconazole, Ketoconazole, 5-Fluorocytosine, Itraconazole and Fluconazole. Thus, it would appear that there is no inherent resistance associated with isolates of this fungus from E. camaldulensis. The global distribution ofE. camaldulensis appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattii (8). In Australia, endemic loci occur around Darwin, Alice Springs, rural South Australia, New South Wales, Victoria, Queensland and Western Australia (7). E. camaldulensis does not occur in Tasmania and to our knowledge nor do C. neoformans var. gattii infections (8). No other Eucalyptus species has this distribution pattern in Australia (3). The trees have also been exported extensively from Australia to other regions; especially Hawaii, California, Mexico, Brazil, parts of Africa and South East Asia (23) from where C. neoformans var. gattii infections have been reported (13). We have now recovered environmental isolates of the fungus from E. camaldulensis trees growing at a site near Fort Point, San Francisco, California (18). Thus, it would appear that this important medical pathogen has been exported from Australia along with the river red gum. It is of great interest that so far, all Australian patient and environmental isolates of C. neoformans var. gattii from E. camaldulensis have proved to be serotype B. We have not yet recovered an isolate of C. neoformans vat. gattii serotype C from any source in Australia and the natural habitat of this serotype remains unknown. Recent environmental collections from southern Queensland have also yielded C. neoformans vat. gattii serotype B from wood debris ofEucalfptus tereticornis 323

Figure 4. - Encapsulated basidiospores of C. neoformans var. gattii (X600).

The significance of the difference in size of the potential infectious propagules of C. neoformans has been debated in the literature for some time (2, 5, 20). Many authors favour the desiccated yeast cell as the infectious unit for the following reasons (1) their small size, (2) the environmental source has been identified as pigeon dung and (3) compatible mating types are not usually found in the pigeon dung - thus excluding the formation of basidiospores. However, in view of our findings with C. neoformans vat. gattii it would appear that both yeast cells and basidiospores may act as infectious propagules, and that size differences may help explain some of the varietal differences in the virulence and/or pathogenicity of C. neoformans. It would appear that basidiospores not landing on susceptible E. camaldulensis host tissue synthesize

Ellis D. and Pfeiffer T.

Eur. J. Epidemiol.

TABLE 1. - Relative size and shape of potential infectious propagules o f Cryptococcus neoformans (14). Propagule



encapsulated yeast cells (both varieties)

globose or oval

desiccated yeast cells ex pigeon dung

globose or oval

basidiospores of var. neoformans

spherical,oblong elliptical or cylindrical with finely rough walls

1.8-3 x 2-5 um

bacillary smooth walled

1-1.8 x 3-8 um

basidiospores of var. gattii

serotypes A, C and D. Finally, we submit this brief review o f our recent work on Cryptococcus neoformans as a tribute to Libero AjeUo, and to encourage others to also re-evaluate the ecology of C. neoformans and other medically important fungi.

3-8 (15) um Acknowledgements < 2 um

This work has been supported by grants from the National Health and Medical Research Council and the Adelaide Children's Hospital Foundation. REFERENCES

protective capsular material and remain dormant until they are either stimulated to replicate as budding yeast cells, by a suitable growth medium, or cleared from the environment by the action o f sunlight and other microorganisms. The transformation o f basidiospores into encapsulated yeast cells is well documented (14). Both are haploid cells, but yeast cells are the only vegetative units that grow readily on artificial media. Further studies on the host-parasite interaction are in progress to determine where the formation o f dikaryotic hyphal elements and basidia o f the fungus occur in the host trees. In conclusion, we believe that C. neoformans is either an endophytic or epiphytic yeast-like fungus and that the epidemiology o f cryptococcosis can primarily be explained by exposure to an infective aerosolized inoculum (9). For C. neoformans var. gattii we have postulated that the principle infectious propagule is the basidiospore and that exposure to E. camaMulensis or other host eucalypt trees, is required to initiate infection in man and animals (9). For C. neoformans vat. neoformans we have postulated that both the basidiospore and desiccated encapsulated yeast cells act as infectious propagules. The basidiospores showing a seasonal distribution in association with an as yet unidentified host plant, while the latter are dispersed from accumulations of dried pigeon/bird dung which acts as a year round vector (9). The need for exposure to flowering E. camaldulensis trees in order to acquire an infection by C. neoformans vat. gattii serotype B also provides an explanation for the high incidence of infections caused by this fungus in Australian Aborigines living in the Northern Territory (8) and possibly for its low world-wide incidence in AIDS patients (4, 15). The ecology o f C. neoformans still remains largely unresolved, studies on the host-parasite interaction between serotype B and E. camaldulensis are still in progress, and extensive environmental searches are now underway to determine the natural habitats of 324

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6. Dimech W.J. (1991): Diagnosis, identification and epidemiology of Cryptococcus neoformans infection Australian Journal of Medical Laboratory Science 12: 13-21. 7. Ellis D.H. (1987): Cryptococcus neoformans var. gattii in Australia - J. Clin. Microbiol. 25: 430-431. 8. Ellis D.H. and Pfeiffer T.J. (1990): Natural habitat of Cryptococcus neoformans vat. gattii J. Clin. Microbiol. 28: 430-431. 9.

Ellis D.H. and Pfeiffer T.J. (1990): Ecology, life cycle, and infectious propagule of Crvptococcus neoformans - The Lancet 366: 923-925.

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12. Kwon-Chung K.J., Polacheck I. and Bennett J.E. (1982): Improved diagnostic medium for separation of Cryptococcus neoformans vat. neoformans (Serotypes A and D) and Cryptococcus neoformans var. gattii (Serotypes B and C) - Journal of Clinical Microbiology •5(3): 535-537. 13. Kwon-Chung K.Z and Bennett J.E. (1984): High prevalence of Cryptococcus neoformans var. gattii in tropical and subtropical regions - Zbl. Bakt. Hyg. A 257: 213-218.

17. Murphy J.W. (1989): Cryptococcosis. In: Immunology of the Fungal Diseases - edited by R.A. Cox - CRC Press - Inc. 18. Pfeiffer T.J. and Ellis D.H. (1990): Environmental isolation of Cryptococcus neoformans var. gattii from California - J. Infect. Dis. 163: 929-930. 19. Rippon J.W. (1988): Medical Mycology: The pathogenic fungi and the pathogenic actinomycetes 3rd edition, W.B. Saunders Co. W.B. Saunders Co Philadelphia.

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23. Zacharin R.F. (1978): Emigrant eucalypts: gum trees as exotics - Melbourne University Press.

The ecology of Cryptococcus neoformans.

Environmental isolations have established that Cryptococcus neoformans var. gattii serotype B appears to have a specific ecological association with E...
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