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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:
Received 12 December 2002; Accepted 18 June 2003
2004 ISHAM DOI: 10.1080/13693780410001712061
Medical Mycology August 2004, 42, 355/362
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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
Fema le 3 Incoordination
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
Fema le 7 Incoordination
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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