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Cryptococcosis outbreak in psittacine birds in Brazil

T. F. RASO*, K. WERTHER*, E. T. MIRANDA$ & M. J. S. MENDES-GIANNINI$ ria (FCAV/UNESP), Jaboticabal and $Departamento de Ana lises Cl *Departamento de Patologia Veterina nicas, Faculdade de ncias Farmace uticas (UNESP), Araraquara, SP, Brazil Cie

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An outbreak of cryptococcosis occurred in a breeding aviary in Sa o Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou , Lorius lory, Trichoglossus goldiei , Psittacula krameri and Psittacus erithacus ) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 mg/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil. Keywords avian, Brazil, Cryptococcus neoformans var. gattii , cryptococcosis, psittacine birds

Introduction
Cryptococcus neoformans is widespread in natural environments, mainly in plant debris, soil and avian droppings [1]. On the basis of physiological, ecological and serological differences, C. neoformans has been subdivided in two varieties: C. neoformans var. neoformans (serotypes A, D and AD) and C. n. var. gattii (serotypes B and C) [2]. Recently, C. neoformans var. grubii has been described and comprises isolates of serotype A [3]. In Brazil, the two long-established varieties of the fungus are recognized as aetiological agents of human cryptococcosis [4,5].

Received 12 December 2002; Accepted 18 June 2003 Correspondence: Dr Karin Werther, Departamento de Patologia Veterina ria, Faculdade de Cie ncias Agra rias e Veterina rias, Universidade Estadual Paulista, FCAV/UNESP, Via de acesso Prof. Paulo Donato Castellane, s/n8, Jaboticabal, Sa o Paulo, 14884-900, Brazil. Tel: '/55 16 3209 2664; Fax: '/55 16 3202 4275; E-mail: werther@fcav.unesp.br

In birds, clinical disease is rare, although the presence of Cryptococcus spp. in the faeces has frequently been demonstrated [6,7]. Ante-mortem diagnosis of cryptococcosis is difficult because clinical signs are not pathognomonic. Signs include weakness, depression, dyspnoea, anorexia, weight loss, diarrhoea, oral masses, blindness, incoordination, progressive paralysis and eventually death [8,9]. The most common lesion seen in post-mortem examinations is a gelatinous myxomatous material in the respiratory tract, abdominal cavity, sinuses and brain. C. neoformans may be seen in specimens stained with Gram or India ink as spherical cells surrounded by a thick mucopolysaccharide capsule that does not stain and is seen as a clear halo [10]. The species can be easily isolated, thus providing the means for studies on its sexuality and its antigenic and genetic structures [2]. In psittacine birds, at the time of this writing, cryptococcosis has been reported only in a moluccan cockatoo (Cacatua moluccenis ) [11], a Major Mitchells cockatoo (Cacatua leadbeateri) [12], a thick-billed parrot (Rhynchopsitta pachyrhyncha ), an African grey
DOI: 10.1080/13693780410001712061

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parrot (Psittacus erithacus ) [13] and a green-winged macaw (Ara chloroptera ) [14]. The present report describes an outbreak of cryptococcosis in psittacine birds in Brazil.

Materials and methods

Clinical cases
A breeding aviary in Sa o Paulo, Brazil, imported many psittacine species from the Netherlands, Belgium and Germany in 1996 and 1997. Couples of birds were kept in 1.5 )/3.0-m enclosures with sand floors and separated by metal fencing (Fig. 1a). Perches were made with twigs taken from nearby Eucalyptus spp. and were spread throughout the enclosure (Fig. 1a,b). Each enclosure was equipped with a stainless steel cage, a wood nest and stainless steel water and food containers, which were cleaned daily. Birds were fed fresh fruits and a commercial diet (Lorinectar; Aves Products, Deventer, the Netherlands). Seven birds died of cryptococcosis during a period of 3 years in this aviary; six between August 1999 and May 2000 and one in 2002. Birds 1 and 2 were blackcapped Lories (Lorius lory ), bird 3 was a Goldies lorikeet (Trichoglossus goldiei ), bird 4 was a ring necked parrot (Psittacula krameri ) and bird 5 was an African grey parrot (Psittacus erithacus ). These five birds died a few hours after showing incoordination and paralysis, and two birds had alterations related with the respiratory tract and were submitted for treatment.

A mass (approximately 2 )/2 cm) was seen involving the upper beak and the infraorbital sinus (Fig. 2) of the sixth bird, a Goldies lorikeet. Structures suggestive of cryptococccal and inflammatory cells were detected by aspirative biopsy. Fluconazole (Zoltec; Pfizer, New York, NY) was prescribed for 30 days, at 8 mg/kg per os (p.o.)/single in day (SID) [15]. After that period, a second aspirative biopsy was done and fluconazole was injected directly into the mass at the same dosage. Faeces were collected for smears throughout the treatment. This bird died 10 days after the beginning of the second period of treatment. Bird 7 was a Papua lori (Charmosyna papou ) that had difficulty in closing the beak due to a gelatinous mass at the choanas. Biopsy of the mass was performed by aspiration and the faecal smears were examined. A

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Fig. 2 A Lorikeet (Trichoglossus goldiei ) with cryptococcosis. Note a mass involving the upper beak (white arrow) and infraorbital sinus (black arrow).

Fig. 1 (A) Couples of birds were kept in enclosures with sand oor and separated by metal fence. Perches were made with Eucalyptus sp. twigs. (B) At prominence, perches chewed by the birds.

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treatment consisting of an intralesional injection of fluconazole (8 mg/kg SID) into the gelatinous mass was prescribed for 30 days. After 15 days of treatment, a biotherapeutic drug (Cryptococcus 30CH, Jaboticabal, Sa o Paulo, Brazil) was also administered in the food (10 drops SID), prepared using the yeast aspirated from the mass [16]. After 45 days of treatment, the gelatinous mass had disappeared. Serum of this bird was assayed by latex agglutination test and high titres of cryptococcal antigen were detected. Oral fluconazole was prescribed, but the bird died 21 days later.

104 c.f.u./ml [20]. One set of microplates was wrapped with film sealer to prevent medium evaporation, attached to an electrically driven wheel inside the incubator, and agitated at 350 r.p.m. The minimum inhibitory concentration (MIC) endpoint was defined as the lowest drug concentration exhibiting a reduction in growth of 50% or more compared with the growth of the control. C. neoformans ATCC 90012 (American Type Culture Collection, Manassas, VA) was included on each test as quality control strain.

Pathology
All birds were necropsied and organs collected for microscopic examination. Samples were fixed in buffered 10% formalin, processed and embedded in paraffin. Sections of 4 mm were stained with haematoxylin and eosin (H&E) and with periodic acid-Schiff (PAS).

Results
Structures of Cryptococcus spp. and inflammatory cells were seen in biopsy material from bird 6 before and after the treatment. Faecal smears from the same bird stained with India ink demonstrated the presence of Cryptococcus spp. The first aspiration biopsy of bird 7 also showed Cryptococcus spp. structures and inflammatory cells. Nevertheless, the second aspiration biopsy taken after 30 days of treatment with fluconazole was negative. All faecal smears stained with India ink were negative for Cryptococcus spp. Serum latex agglutination titres were 1:512 during treatment and 1:1024 when the bird died. Clinical, macroscopic and microscopic findings from all birds are shown in Table 1. Macroscopic and microscopic lesions were seen predominantly in the liver, respiratory system, spleen, kidneys and intestines. In investigation of the seven cases, the liver displayed hepatomegaly in four, hydropic and lipid degeneration in three, multifocal hepatitis with polymorphic inflammatory cells in three, yellow areas in the capsule and parenchyma in three, loss of cellular architecture and massive destruction of parenchyma in two, congestion in one and thickened capsule and hyperplasia of biliary ducts in one. The lungs were congested and haemorrhagic in four cases. A yellow gelatinous material replaced the pulmonary parenchyma in three. Oedema and inflammatory cell infiltration were seen in three cases, necrotic foci and calcification in two and emphysema in one. Air sacs were thickened with an adherent mass in one case and sinuses were filled with yellow material in one. Alterations in the kidneys included nephromegaly in three cases, tubular and glomerular degeneration with sclerosis in one and congestion in one. Macroscopic alterations observed in the spleen were splenomegaly in three cases, paleness in one and a presence of multiple white foci in one. Microscopic examination of spleen disclosed splenitis with heterophils in one case. One bird had congested intestines, with black material inside the lumen. Some additional alterations were seen only in single cases:

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Mycological and serological tests

Faeces, organ fragments and aspirated biopsy materials from birds 6 and 7 were collected in sterile vials. After maceration, samples were stained with India ink, prepared with 20% KOH and plated on Sabouraud agar and Niger seed agar medium (manipulated by the laboratory of the university). Plates were kept at room temperature and monitored for 2 /7 days. Brown smooth colonies on Niger seed agar were transferred to Sabouraud glucose agar and C. neoformans was identified by the API 20 C Aux System (Biome rieux, Rio de Janeiro, Brazil). The canavanine /glycine /bromothymol blue (CGB) medium test was used to determine the variety of C. neoformans [17]. Isolates were serotyped using the Crypto Check Iatron (Iatron Laboratories, Tokyo, Japan). Serum samples from bird 7 were analysed by the latex agglutination test for cryptococcal antigen (IMMY; Immuno-mycologics, Norman, OK) [18]. All isolates were submitted to the broth microdilution test, which was performed according to the National Committee for Clinical Laboratory Standards (NCCLS) [19] with modifications [20], for in-vitro susceptibility testing of fluconazole (FLC). Broth microdilution testing was performed with RPMI 1640 medium (Gibco, NY; Biome rieux) with L-glutamine, without bicarbonate and buffered with morpholinepropanesulfonic acid (MOPS) at pH 7.0 with addition of 2% glucose. An inoculum of 1 )/106 to 5 )/106 colonyforming units (c.f.u.) per ml was made as recommended by NCCLS and then diluted 1:10 with sterile distilled water. The final inoculum contained 0.5 )/104 to 2.5 )/
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Table 1 Clinical, pathological and histopathological ndings from seven psittacine birds with cryptococcosis caused by Cryptococcus neoformans var. gattii Number of bird/species Sex Age (years) Clinical signs Pathology Histopathology Alterations Histopathology Presence of Cryptococcus neoformans (Cn ) Cn in parenchyma and pulmonary vessels with inflammatory cells (heterophils) Cn in hepatic capsule and vessels. Heart with cn between the endocardial fibres Cn in kidney and intestinal lumen Cn in the vessels, the pulmonary parenchyma and the gelatinous mass Cn in the kidneys, brain (with heterophils), meninges, and spinal cord Faecal smears at necropsy showed cn Cn in pulmonary parenchyma with inflammatory cells (heterophils) and liver Cn in renal parenchyma/capsule with inflammatory cells (heterophils)

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Bird 1 Lorius lory

Female 3

Incoordination and paralysis

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Congestion and haemorrhage in left lung, yellow gelatinous mass (4 )/ 3 cm) in right lung Hepatomegaly with yellow areas at the capsule and parenchyma Splenomegaly with pale colour Congestion in the intestines and presence of black material in the lumen

Lungs with congestion, necrotic foci and calcification Liver enlarged with hydropic and lipid degeneration Spleen pale and enlarged, lymphoid tissue reduced Loss of the epithelial intestine and congestion

Bird 2 Lorius lory

Female 3

Incoordination and paralysis

Loss of the hepatic cell architecture and massive Yellow gelatinous mass instead of pulmonary parenchyma between the destruction of the parenchyma two lungs and adherent to spin Spleen unaltered Hepatomegaly

Bird 3 Trichoglossus goldiei

Female 2

Incoordination and paralysis

Yellow areas at the capsule and parenchyma of the liver Nephromegaly

Bird 4 Psittacula krameri

Female 7

Incoordination and paralysis

Yellow areas in the pulmonary parenchyma, congestion and oedema Thickened air sacs with adherent mass near ovary Thickened pericardial sac Hepatomegaly, ascites Presence of multiple white foci in spleen Nephromegaly Brain congestion, haemorrhage at the skull

Lungs with oedema and discrete inflammatory cell infiltration Liver with hydropic and lipid degeneration, congestion, loss of the cellular architecture and massive destruction of the parenchyma Spleen without alteration Kidneys with diffuse tubular and glomerular degeneration, sclerosis glomerular, oedema, and congestion Lungs with congestion and haemorrhage Airsaculits Pericarditis with inflammatory cells Multifocal hepatitis with polymorphic inflammatory cells, thickened capsule and hyperplasia of the biliary ducts Splenitis Kidneys congested

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Cn in lungs, bronchium, air sacs, kidneys, and liver. Cn in spleen around heterophils

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Table 1 (Continued ) Number of bird/species Sex Age (years) Clinical signs Pathology Histopathology Alterations Histopathology Presence of Cryptococcus neoformans (Cn )

Bird 5 Psittacus erithacus

Male

Difficulty in flying

Yellow gelatinous mass in the pulmonary parenchyma Liver congested. Splenomegaly Kidneys congested Lungs with white foci, haemorrhagic areas, emphysema and oedema Hepatomegaly Splenomegaly Sinus filled with liquid yellow material

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Bird 6 Trichoglossus goldiei

Male

Mass in the upper beak, difficulty in closing the beak and dyspnoea Intraorbital sinus was affected

Bird 7 Charmosyna papou

Female 3

Gelatinous mass in the choana with difficulty in closing the beak

lungs with haemorrhage, oedema and emphysema Hepatomegaly Splenomegaly

Cn in lungs (areas with inflammatory cells and areas without inflammatory cells), liver, intestines and central nervous system Lungs without inflammation Lungs with inflammatory cell infiltration reaction near to cn (heterophils) near to bacterial foci and Cn in choana, beak, nasal mass, congestion Choana, beak and nasal mass with necrosis Air liver and air sacs (with heterophils) sacs with inflammatory reaction Cn in vessels with inflammatory (heterophils) reaction (heterophils) in elastic Multifocal hepatitis with polymorphic fibres inflammatory cells (heterophils), lipidic Aspiration biopsy reveals cn degeneration, thick capsule, hyperplasia of the and biliary ducts Multiple white foci in spleen with heterophils and inflammatory cells (twice) Faecal smears showed cn hyperplasic tissue Choana with destruction of the tissue with Cn in choana discrete inflammatory cells. Pulmonary Aspiration biopsy revealed cn necrosis and Multifocal hepatitis with heterophils and diffuse inflammatory cells lipidic degeneration Latex agglutination titres: 1/512 Spleen with hyperplasic of the lymphoid tissue and 1/1024

Necrosis of parenchyma of lungs Spleen with lymphoid tissue hyperplasia

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pericarditis and thickening of the pericardial sac, ascites, and congested brain with skull haemorrhage. Histopathological examination (Table 1) evidenced Cryptococcus spp. in the sinus, choana, beak, air sacs, lungs, liver, spleen, kidneys, intestines, cardiac fibres, brain, meninges and bone marrow (Figs. 3 /7). Such cells were also present in the various gelatinous masses seen. Strains were identified as C. neoformans because of the characteristic patterns of sugar assimilation, cycloheximide intolerance, urease production and growth at 378C. Strains produced brown pigment in the Niger seed medium. The CGB test was positive, indicating that the strains all belonged to C. neoformans var. gattii . Agglutination tests with Crypto Check demonstrated that all strains were serotype B. All strains were resistant to fluconazole (minimum inhibitory concentration (MIC), 64 mg/ml).

Fig. 4 Photomicrography of the heart showing C. neoformans between the muscle bres without inammatory reaction (PAS, 40x). Bird 1.

Discussion
This paper provides the first report of C. n. var. gattii infection in three species in the family Loriidae and two species in the family Psittacidae in Brazil. There are few reports in literature concerning cryptococcosis in psittacine birds [11 /14]. In the seven birds described here, only two cases of superficial lesions were seen; the other birds had discrete neurological signs with no clinical evidence of disseminated cryptococcosis. Clinical diagnosis of cryptococcosis in birds is difficult due to non-specific signs. In our cases, primary infection probably occurred in the upper respiratory system and then spread haematogeneously to other organs. Also coelomic dissemination may have occurred between organs within close proximity. In most previous reports, initial colonization was thought to have occurred within the

Fig. 5 Photomicrography of the medullar space, showing Criptococcus neoformans at the meninges and around the spinal cord without inammatory reaction (HE, 4x). Bird 2.

Fig. 3 Photomicrography of the lungs showing C. neoformans in the lumen of the blood vessels (PAS, 20x). Bird 1.

respiratory tract. It has been suggested that the upper respiratory tract may be particularly susceptible to initial colonization because of its lower temperature [8,10]. In the cases presented here, however, disseminated infection was predominant, indicating that the body temperature of the birds was not a barrier to further ingress. The superficial lesion seen in birds 5 and 6 was a gelatinous mass occupying the choanas, sinus and upper beak. To date only one such case of avian superficial infection has been reported. In this case, cryptococcosis was localized at the infraorbital sinus of a Beccariss crowned pigeon [21]. Gelatinous material was also seen in the lung lesions of three of our birds. In the present report, some birds showed inflammatory cells, predominantly heterophils, close to the yeast cells. Peripheral inflammatory response in cryptococ 2004 ISHAM, Medical Mycology, 42, 355 /362

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Fig. 6 Photomicrography of the kidney showing a massive inltration of C. neoformans at the parenchyma (HE, 20x). Bird 3.

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cosis is usually minimal, consisting of epithelioid macrophages or multinucleated giant cell and heterophils [10]. An interesting feature of these infections with C. neoformans was that sometimes there was no inflammatory reaction and in other cases we were able to observe a reaction only in some organs. However, all birds experienced aggressive dissemination to internal organs. Confirmation of cryptococcal infection can be made by culture or by demonstrating a positive titre for cryptococcal antigen in the serum. Analysis of serum samples from bird 7 showed that serology could be useful in ante-mortem diagnosis of avian cryptococcosis. It may also be useful in monitoring the efficacy of treatment. Though fluconazole is generally effective against C . n. var. gattii [22] we found that titres of cryptococcal antigen in serum evidenced an apparently poor response to antifungal therapy, a finding that corresponded well with the in-vitro resistance to fluconazole seen. This predictive value of such titres has previously been reported by Aller et al . [23].

Fig. 7 Photomicrography of the lungs showing C. neoformans with inammatory cells (HE, 40x). Bird 5.

The diagnosis of cryptococcosis when birds were alive permitted a tentative treatment in two cases. Bird 6, which had a large mass involving the upper beak, was treated for 30 days with fluconazole alone, but no improvement was observed. In bird 7, the infected mass at the choana was treated with fluconazole in combination with a biotherapeutic drug. The mass disappeared completely after 45 days, but the use of two drugs concomitantly does not allow defining which one was effective. Even though the mass had disappeared, the presence of high antigen titres in the serum showed that the animal was still infected. The progressive increase in cryptococcal antigen titre in bird 7 and the eventual fatal outcome showed that the fluconazole therapy had failed. Continued infection was confirmed at necropsy. Therapeutic levels of antifungal drugs that are suggested for humans or other animals are not appropriate for these birds, due to resistance problems. Further studies are needed to determine appropriate therapy for avian cryptococcosis, as well as the best use of antigen serology, paralleling the use made in human cases. Cryptococcus n. var. neoformans has a cosmopolitan distribution and is usually associated with bird excreta, especially that of pigeons. On the other hand, C. n. var. gattii (the infectious agent in our cases), is frequently associated with blooming Eucalyptus spp. in tropical regions [1,24,25]. The yeast might have come from eucalyptus trees that are sometimes used as perches in bird enclosures. Nevertheless, attempts to isolate C. neoformans from the trees were not successful, perhaps because the trees were not blooming. In a similar case, an unsuccessful attempt to isolate this yeast from eucalypt trees was made after a kiwi died of cryptococcosis [26]. In a park in Sa o Paulo, Brazil, material from 12 eucalypt trees was collected monthly and C. n. var. gattii was isolated for 2 consecutive years from only one tree and C. n. var. neoformans was isolated from another tree, indicating that these microorganisms exist in urban areas [27]. The authors believe that the twigs of eucalypt used as perches for the birds were the source of infection. Psittacine birds have the habit of chewing wooden objects, such as the perches (Fig. 1b). Possibly the birds had extensive contact with cryptococcal inoculum. The idea that these birds might have been infected prior to importation seems unlikely because of the time elapsed between importation and the death of these birds. Also, bird 5 was born in the breeding aviary. No difference in infection rates nor types was observed related to the sex or the age of the birds. Human cryptococcosis frequently develops after exposure to dust derived from contaminated avian

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droppings [8,25]. One immunosuppressed patient is known to have developed cryptococcal meningitis as a result of contact with a pet cockatoo [28]. Control of this disease is best accomplished by adequate ventilation and frequent removal of bird droppings and organic debris. After cryptococcosis was diagnosed in our birds, hygienic measures were implemented in the breeding aviary. All the nests and perches were removed and incinerated, and the upper layer of sand was removed from the enclosures. As another infection control measure, birds undergoing therapy were kept in isolation. Although we treated birds in this study, we suggest that the value of such therapy be carefully considered given the zoonotic potential of this organism and the poor prognosis associated with disseminated cryptococcosis infection in birds.

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