Cryptococcus neoformans var. gattii in the koala (Phascolarctos cinereus): a review of 43 cases of cryptococcosis.

Details of 11 previously reported cases and 32 new cases of cryptococcosis in captive and wild koalas were analysed. Cryptococcus neoformans var. gattii accounted for all 29 cases in which varietal status was determined. No age or sex predisposition was observed. The respiratory tract was the primary focus of disease in 77% of cases. Although the lower respiratory tract was affected most commonly (60% of cases), 30% of cases had upper respiratory tract lesions and 14% had both. Dissemination was common, especially to the central nervous system (37% cases). Local extension to surrounding tissues was a feature of upper respiratory tract disease. Other tissues showing cryptococcal invasion included lymph nodes (19%), gastrointestinal tract (12%), kidneys (12%), spleen (9%) and skin (7%). Only three cases (7%) had no respiratory tract or central nervous system involvement, two cases of primary skin inoculation and one case of primary lymphadenopathy. Late presentation was a likely factor in the high proportion of cases with disseminated disease (40%). The proportion of koala cases with involvement of the central nervous system, lower respiratory tract and skin, parallels what has been reported for immunocompetent people. Cryptococcosis in the koala appears to be an excellent naturally occurring model for examination of the cryptococcal host-parasite relationship in all species.     

Cryptococcus neoformans in the koala (Phascolarctos cinereus): colonization by C n. var. gattii and investigation of environmental sources.

This study is the one in a series looking at the relationship among Cryptococcus neoformans var. gattii, koalas and the environment. The koala was used as a natural biological sampler in an attempt to understand the dynamics of C. neoformans var. gattii in Australian environments. Evidence of asymptomatic nasal and skin colonization for extended periods by large numbers of C. n. var. gattii was obtained and geographical factors assessed. The key finding was the ability of koalas to amplify numbers of C. n. var. gattii in certain environments. Koalas were not found to be obligatory for the survival of the organism in all environments. Geographical factors alone could not explain differing rates of nasal and skin colonization in koalas in different environments. A strong association between healthy koalas and C. n. var. gattii was confirmed and C n. var. gattii was isolated from novel sources, including the turpentine gum tree (Syncarpia glomulifera), tallowwood (Eucalyptus microcorys) and flooded gum (E. grandis). It seems likely that as yet undiscovered environmental sources of C. n. var. gattii exist in eastern Australia. Further investigation of host, environmental and organism factors integral to the hostpathogen relationship will assist an understanding of the progression from colonization to tissue invasion and cryptococcosis in all species.     

Asymptomatic carriage of Cryptococcus neoformans in the nasal cavity of the koala (Phascolarctos cinereus).

Over a 22-month period, sequential nasal and skin swabs were obtained from 52 healthy captive koalas (Phascolarctos cinereus) from the Sydney region. Cryptococcus neoformans was isolated in 17 koalas from 64 of 262 (24%) nasal swabs and from nine of 262 (3%) skin swabs. Prevalence of nasal colonization varied seasonally from 12% (3/25) to 38% (10/26). Cryptococcus neoformans var. gattii alone was cultured from 37, var. neoformans alone from 22 and both varieties from five nasal swabs. Of 33 koalas sampled on three or more occasions, organisms were isolated persistently from six, occasionally from eight and never from 19. Two koalas were persistently and heavily (>/=100 colonies/plate) colonized by C. neoformans var. gattii and two with var. neoformans. Isolation of C. neoformans var. gattii from the skin was low grade and sporadic. No koalas from which C. neoformans was persistently isolated showed clinical signs of cryptococcosis and all except one had a negative latex cryptococcal antigen test, therefore the nasal cavity was presumed to be colonized by, rather than infected with, C. neoformans. Preliminary observations of koalas from Coffs Harbour indicated a much higher prevalence of colonization by C. neoformans, suggesting that environmental factors influenced the extent of carriage by C. neoformans.     

Cryptococcus neoformans var. gattii in Australia.

An examination of 45 clinical isolates of Cryptococcus neoformans revealed an unusually high incidence of C. neoformans var. gattii in South Australia (65%) and in the Northern Territory (95%). In assessing all the available data from Australian isolates of C. neoformans, there appeared to be an endemic focus for the incidence of C. neoformans var. gattii in the rural aboriginal population of the Northern Territory.     

First case of human cryptococcosis due to Cryptococcus neoformans var. gattii in Spain

We report the first case of human cryptococcosis due to Cryptococcus neoformans var. gattii described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patient's environment was carried out to find the source of infection.     

Cryptococcus neoformans var. gattii meningitis in Singapore.

We describe here two cases of Cryptococcus neoformans var. gattii meningitis that occurred in Singapore. This variety of Cryptococcus neoformans differs from the more widely known C. n. var. neoformans in its ecology and epidemiology. Infections with this variety are still rare in Singapore.     

Intraorbital anatomy of the koala (Phascolarctos cinereus)

This is the first documented study of the anatomical details of the contents of the normal koala orbit, excluding the bulbus oculi. Baseline data were established which are necessary for understanding and treating ocular disease in the koala (Phascolarctos cinereus). The anatomy of the orbital contents of the koala were examined and described from animals that presented dead or were euthanized for humane reasons. Dissections of the orbital cavity were performed under magnification. Polymethyl methacrylate (PMMA) casts of the nasolacrimal system and the vascular supply of the orbit were also made in order to study these systems. The superficial lymphatic drainage of the conjunctival tissues was studied by subcutaneous injection of Evan's Blue into the palpebral conjunctiva of a freshly deceased animal, and by Microfil casts of the efferent lymphatics. In general, the orbital contents of the koala are consistent with those of other carnivorous polyprotodont and herbivorous diprotodont marsupials.     

Natural habitat of Cryptococcus neoformans var. gattii.

Environmental isolations have established that Cryptococcus neoformans var. gattii appears to have a specific ecological association with Eucalyptus camaldulensis. So far, we have isolated C. neoformans var. gattii on 35 separate occasions, all from samples associated with E. camaldulensis. The global distribution of E. camaldulensis appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattii. No other environmental source for the fungus has yet been detected, and no other eucalypt has the distribution pattern corresponding to reported cases caused by this fungus. These findings may provided an explanation for the high incidence of infections caused by C. neoformans var. gattii in Australian aborigines living in the Northern Territory and for its low worldwide incidence in acquired immunodeficiency syndrome patients.     

Bone marrow examination in the koala (Phascolarctos cinereus)

A technique for bone marrow aspiration from the iliac crest in the koala (Phascolarctos cinereus) is described. Bone marrow was obtained from ten healthy koalas under general anaesthesia using the combination of tiletamine HCL and zolazepam HCL. Reference ranges were wide. The mean value for M:E was 1.7 (range 0.8–2.7), the mean percentage proliferating erythroid was 11% (range 5%–16%), and the mean percentage proliferating myeloid was 25% (range 17%–35%). In addition, bone marrow aspiration was performed on 14 koalas with various haematological diseases. Results showed aspiration to be useful in the diagnosis of leukaemia, and the investigation of regenerative anaemia and dysplastic anaemia. It was of varying use in the investigation of non-regenerative anaemia.     

Spontaneous lymphoid neoplasia in the koala (Phascolarctos cinereus)

Thirteen cases of lymphoid neoplasia were examined in koalas from New South Wales and Queensland. Haematological and biochemical data were collected from 2 cases while all were subjected to detailed necropsies. Affected koalas were adults of both sexes and the disease had a variable time course. The 2 koalas examined haematologically were leukaemic. On a morphological basis, 5 cases were classified as multicentric, 7 cases as abdominal (but not necessarily with alimentary tract involvement) and one case as solitary. In most cases, the neoplastic cells appeared to be either lymphoblasts or prolymphocytes.     

Natural environmental sources of Cryptococcus neoformans var. gattii.

We sought evidence for new environmental sources of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA (RAPD) analysis of isolates from 29 animals with a restricted territorial range in five Australian states. Twenty-three of the 29 isolates and 45 of 45 eucalypt isolates tested previously exhibited one RAPD profile, VGI. RAPD profile VGII was identified in 6 of 17 isolates from domesticated species but in none of 12 native species. Four VGII isolates originated from an area of Western Australia with no natural stands of known eucalypt host, indicating the existence of at least one unrecognized natural source of C. neoformans var. gattii.     

Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C).

A simple new agar medium containing L-canavanine, glycine, and bromthymol blue was found to give a clearer and more accurate distinction between serotype A or D (Cryptococcus neoformans var. neoformans) and serotype B or C (C. neoformans var. gattii) than creatinine-dextrose-bromthymol blue or glycine-cycloheximide-phenol red media. Among 143 isolates of serotype A or D and 70 isolates of serotype B or C, the new medium correlated completely with the serotype, whereas nearly 11% of these isolates gave discrepant reactions with creatinine-dextrose-bromthymol blue and glycine-cycloheximide-phenol red media.     

Chronic peritonitis and pancreatitis in a koala (Phascolarctos cinereus)

The gross, light microscopic and ultrastructural appearance of a nodular peritonitis accompanied by pancreatic changes in a Koala (Phascolarctos cinereus) is described. Its pathogenesis in relation to pancreatitis is discussed.     

Cryptococcus neoformans var. gattii can exploit Acanthamoeba castellanii for growth.

It has recently been proposed that the origin and maintenance of virulence in certain environmental fungi is influenced by their interactions with non-vertebrate hosts such as amoebae and nematodes. In prior studies we have shown that the interactions of the soil amoebae Acanthamoeba castellanii with Cryptococcus neoformans varieties neoformans and grubii resemble those with macrophages. Here we extend those studies to C. neoformans variety gattii and describe quantitative differences in the type and outcome of the interactions observed relative to the other varieties. C. neoformans var. gattii proliferated in the presence of A. castellanii but the interaction was primarily extracellular with a paucity of phagocytic events. Experiments with acapsular cells coated with polysaccharide suggest that differences in the capsule structure may be responsible for the different interactions between cells of varieties neoformans, grubii, and gattii with amoebae. The ability of C. neoformans var. gattii to exploit amoebae indicates that despite major biological differences between C. neoformans varieties, all retain the ability to be pathogenic for A. castellanii.     

The stomach gland patch of the koala (Phascolarctos cinereus)

A large gland patch is found in the submucosa of the lesser curvature of the koala stomach near the entrance of the esophagus. It measures approximately 3 cm in diameter and is comprised of numerous invaginations continuous with the surrounding mucosa. The glandular invaginations drain into the lumen of the stomach by 25 to 30 large orifices. The mucosa of the gland patch invaginations differs from the mucosal lining of the stomach chiefly in thickness. The elongate gastric glands that comprise the invaginations exhibit an increased number of parietal and chief cells. Ultrastructural examination of the gland patch illustrates that the parietal, chief, and mucous neck cells are similar in structure to those of other mammalian species.     

Cryptococcosis in a bottlenose dolphin (Tursiops truncatus) caused by Cryptococcus neoformans var. gattii

We describe the first case of cryptococcosis caused by Cryptococcus neoformans var. gattii in a male Atlantic bottlenose dolphin (Tursiops truncatus). The dolphin showed clinical signs of tachypnea, transient dyspnea, and mild tachycardia and developed multiple hyperechoic nodules, parenchymal consolidation, and thickening of pleura. A diagnosis of bronchopneumonia with pleuritis was made. Itraconazole therapy was implemented for 120 days, and trough levels in serum were within or above the suggested therapeutic range. Titers of cryptococcal antigen in serum increased eightfold during therapy, and the case had a fatal outcome. Necropsy examination findings included enlarged pulmonary lymph nodes and extensive coalescing granulomatous lesions throughout both lungs. Histologic examination revealed numerous, spherical to ellipsoidal, mucicarmine-positive, 3- to 14-microm, encapsulated, budding cells consistent with C. neoformans. Culture of the lung tissue yielded colonies of C. neoformans. The isolate was urease positive and nitrate negative and exhibited phenoloxidase activity. It was positive on canavanine-glycine-bromothymol blue agar. When tested by the Iatron serodiagnostic reagent kit (Iatron Laboratories, Inc.), it was shown to belong to serotype B.     

Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India.

Cryptococcus neoformans var. gattii has an ecological association with five Eucalyptus species: E. blakelyi, E. camaldulensis, E. gomphocephala, E. rudis, and E. tereticornis. After human infections due to C. neoformans var. gattii were diagnosed in the states of Punjab, Himachal Pradesh, and Karnataka, India, a study was undertaken to investigate the association of C. neoformans var. gattii with Indian eucalypts, especially in the state of Punjab. A total of 696 specimens collected from E. camaldulensis, E. citriodora and E. tereticornis (hybrid) trees were examined for the presence of C. neoformans var. gattii. Flowers from two trees of E. camaldulensis in the Chak Sarkar forest and one from the village of Periana near the Ferozepur area yielded five isolates of C. neoformans var. gattii. The origin of the trees could be traced to Australia, thus providing evidence that the distribution of E. camaldulensis correlated with the distribution of human cryptococcosis cases caused by C. neoformans var. gattii in northern India.     

Development of a Singleplex PCR Assay for Rapid Identification and Differentiation of Cryptococcus neoformans var. grubii,Cryptococcus neoformans var. neoformans,Cryptococcus gattii,and Hybrids

A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and large-scale epidemiologic studies.     

Pathogenicity of Cryptococcus neoformans var. gattii in an immunocompetent mouse model.

The pathogenicity of two different genomic profiles of Cryptococcus neoformans var. gattii serotype B isolated from goats that died from cryptococcal pneumonia was assessed in an experimental model of immunocompetent mice. One strain of each randomly amplified polymorphic DNA (RAPD) profile (GR52 and GR56) and three reference C. neoformans isolates representing serotypes B, D and C were used. BALB/c male mice were inoculated by the intraperitoneal route with each strain. After 4 weeks of follow-up, the animals were sacrificed and autopsy specimens of testes, liver, spleen, kidney, lungs and brain were cultured and stained for histopathology. Although spontaneous mortality was only 2% (one animal), all mice except for those inoculated with serotype C showed positive cultures in almost one organ. The strain GR52 isolated from goat showed the highest rate of positive cultures (80%) followed by serotype D (77%). Serotype B reference strain and second goat strain GR56 were both isolated from 70% of samples. Serotype C was recovered in only 33% of organs, and never from brain or lung specimens. GR52 grew abundantly from all lung cultures, and yeast cells with large capsules were seen in histopathology inside the alveoli, peribronchial vessels and interalveolar spaces. They appeared to elicit no inflammatory response. We conclude that intraperitoneally inoculated C. neoformans var. gattii shows high virulence in this immunocompetent mouse model. Strain GR52 was highest in pathogenicity and had marked lung tropism. In contrast, the serotype C reference strain showed the lowest pathogenicity and seemed not to spread outside the abdominal viscera.