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Cryptococcosis outbreak in psittacine birds in Brazil

T. F. RASO*, K. WERTHER*, E. T. MIRANDA$ & M. J. S. MENDES-GIANNINI$

*Departamento de Patologia Veterinaria (FCAV/UNESP), Jaboticabal and$Departamento de Analises Clnicas, Faculdade de

Ciencias Farmaceuticas (UNESP), Araraquara, SP, Brazil

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 crypto-

coccosis. 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 naturalenvironments, 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. neofor-

mans (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].

In birds, clinical disease is rare, although the

presence of Cryptococcus spp. in the faeces hasfrequently been demonstrated [6,7]. Ante-mortem diag-

nosis of cryptococcosis is difficult because clinical signs

are not pathognomonic. Signs include weakness, de-

pression, 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, abdom-

inal cavity, sinuses and brain. C. neoformans may be

seen in specimens stained with Gram or India ink as

spherical cells surrounded by a thick mucopolysacchar-

ide capsule that does not stain and is seen as a clear

halo [10]. The species can be easily isolated, thusproviding 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

Correspondence: Dr Karin Werther, Departamento de Patologia

Veterinaria, Faculdade de Ciencias Agrarias e Veterinarias,

Universidade Estadual Paulista, FCAV/UNESP, Via de acesso Prof.

Paulo Donato Castellane, s/n8, Jaboticabal, Sao Paulo, 14884-900,

Brazil. Tel: '/55 16 3209 2664; Fax: '/55 16 3202 4275; E-mail:

[email protected]

Received 12 December 2002; Accepted 18 June 2003

2004 ISHAM 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 methodsClinical cases

A breeding aviary in Sao 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 sepa-

rated 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, De-venter, 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 black-

capped 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 respira-

tory 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, asecond 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

Fig. 1 (A) Couples of birds were kept in enclosures with sand floor and separated by metal fence. Perches were made with Eucalyptus sp. twigs.

(B) At prominence, perches chewed by the birds.

Fig. 2 A Lorikeet (Trichoglossus goldiei) with cryptococcosis. Note

a mass involving the upper beak (white arrow) and infraorbital sinus

(black arrow).

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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,

Sao 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 crypto-

coccal antigen were detected. Oral fluconazole was

prescribed, but the bird died 21 days later.

Pathology

All birds were necropsied and organs collected for

microscopic examination. Samples were fixed in buf-

fered 10% formalin, processed and embedded in

paraffin. Sections of 4 mm were stained with haematox-

ylin and eosin (H&E) and with periodic acid-Schiff

(PAS).

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 (Biomerieux,

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 microdilu-

tion 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; Biomerieux) with L-glutamine,

without bicarbonate and buffered with morpholinepro-

panesulfonic acid (MOPS) at pH 7.0 with addition of

2% glucose. An inoculum of 1)/106 to 5)/106 colony-

forming 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)/

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.

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 inflam-

matory cells in three, yellow areas in the capsule andparenchyma 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 haemor-

rhagic 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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Table 1 Clinical, pathological and histopathological findings from seven psittacine birds with cryptococcosis caused by Cryptococcus neofor

Number of

bird/species

Sex Age

(years)

Clinical signs Pathology Histopathology Alterations

Bird 1

Lorius lory

Fema le 3 Incoordination

and paralysis

Congestion and haemorrhage in left

lung, yellow gelatinous mass (4 )/ 3

cm) in right lung

Hepatomegaly with yellow areas atthe capsule and parenchyma

Splenomegaly with pale colour

Congestion in the intestines and

presence of black material in the

lumen

Lungs with congestion, necrotic foci an

calcification

Liver enlarged with hydropic and lipid

degenerationSpleen pale and enlarged, lymphoid tiss

Loss of the epithelial intestine and con

Bird 2

Lorius lory


and paralysis

Yellow gelatinous mass instead of

pulmonary parenchyma between the

two lungs and adherent to spin

Hepatomegaly

Loss of the hepatic cell architecture an

destruction of the parenchyma

Spleen unaltered

Bird 3Trichoglossus

goldiei

Fema le 2 Incoordinationand paralysis

Yellow areas at the capsule andparenchyma of the liver

Nephromegaly

Lungs with oedema and discrete inflamcell infiltration

Liver with hydropic and lipid degenera

congestion, loss of the cellular architec

massive destruction of the parenchyma

without alteration

Kidneys with diffuse tubular and glom

degeneration, sclerosis glomerular, oed

congestion

Bird 4

Psittacula

krameri


and paralysis

Yellow areas in the pulmonary

parenchyma, congestion and oede-

ma

Thickened air sacs with adherent

mass near ovary

Thickened pericardial sac

Hepatomegaly, ascitesPresence of multiple white foci in

spleen

Nephromegaly

Brain congestion, haemorrhage at

the skull

Lungs with congestion and haemorrha

Airsaculits

Pericarditis with inflammatory cells

Multifocal hepatitis with polymorphic

inflammatory cells, thickened capsule a

hyperplasia of the biliary ducts

SplenitisKidneys congested

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Table 1 (Continued)

Number of

bird/species

Sex Age

(years)

Clinical signs Pathology Histopathology Alterations

Bird 5

Psittacus

erithacus

M ale 1 Difficulty in flying Yellow gelatinous ma ss in the

pulmonary parenchyma

Liver congested. Splenomegaly

Kidneys congested

Necrosis of parenchyma of lungs

Spleen with lymphoid tissue hyperplas

Bird 6

Trichoglossus

goldiei

Male 5 Mass in the upper beak,

difficulty in

closing the beak and

dyspnoea

Intraorbital sinus was

affected

Lungs with white foci,

haemorrhagic areas, emphysema

and oedema

Hepatomegaly

Splenomegaly

Sinus filled with liquid yellow

material

Lungs with inflammatory cell infiltrati

(heterophils) near to bacterial foci and

congestion

Choana, beak and nasal mass with nec

sacs with inflammatory reaction

(heterophils)

Multifocal hepatitis with polymorphic

inflammatory cells (heterophils), lipidic

degeneration, thick capsule, hyperplasi

biliary ducts

Multiple white foci in spleen with heter

hyperplasic tissue

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

Choana with destruction of the tissue

discrete inflammatory cells. Pulmonary

necrosis

Multifocal hepatitis with heterophils an

lipidic degeneration

Spleen with hyperplasic of the lympho

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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, cyclo-

heximide 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 demon-

strated that all strains were serotype B. All strains were

resistant to fluconazole (minimum inhibitory concen-

tration (MIC), 64 mg/ml).

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

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, dissemi-

nated 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 aviansuperficial 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 inflamma-

tory cells, predominantly heterophils, close to the yeast

cells. Peripheral inflammatory response in cryptococ-Fig. 3 Photomicrography of the lungs showing C. neoformans in the

lumen of the blood vessels (PAS, 20x). Bird 1.

Fig. 4 Photomicrography of the heart showing C. neoformans

between the muscle fibres without inflammatory reaction (PAS,

40x). Bird 1.

Fig. 5 Photomicrography of the medullar space, showing Cripto-

coccus neoformans at the meninges and around the spinal cord

without inflammatory reaction (HE, 4x). Bird 2.


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cosis is usually minimal, consisting of epithelioid

macrophages or multinucleated giant cell and hetero-

phils [10]. An interesting feature of these infections with

C. neoformans was that sometimes there was noinflammatory 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 cryptococco-

sis. 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 apparentlypoor 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].

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 combina-

tion 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 appro-

priate for these birds, due to resistance problems.

Further studies are needed to determine appropriate

therapy for avian cryptococcosis, as well as the best useof 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 fromeucalypt trees was made after a kiwi died of crypto-

coccosis [26]. In a park in Sao 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 avianFig. 7 Photomicrography of the lungs showing C. neoformans with

inflammatory cells (HE, 40x). Bird 5.

Fig. 6 Photomicrography of the kidney showing a massive infiltra-

tion of C. neoformans at the parenchyma (HE, 20x). Bird 3.


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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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