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Case Report

Canine paracoccidioidomycosis

G. RICCI*, F. T. MOTA$, A. WAKAMATSU%, R. C. SERAFIM, R. C. BORRA* & M. FRANCO*

*Department of Pathology, Federal University of Sao Paulo (UNIFESP-EPM), $Laboratory of Pathology, Mogi-Guacu, %Adolfo

Lutz Institute, and Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo, Brazil

Paracoccidioidomycosis (PCM) is a severe disease caused by the dimorphic fungus

Paracoccidioides brasiliensis, which is characterized by granulomatous pulmonary

and systemic lesions, affecting mainly men between 20 and 60 years of age. Reports

of PCM disease in animals are rare, but the disease has been described in

armadillos. On the other hand, PCM infection of domestic and wild animals

detected by serological or cutaneous tests in the absence of apparent disease has

been frequently reported. We present here the case of a female adult Doberman

that developed cervical lymphadenomegaly. Histopathological examination of a

cervical biopsy specimen revealed active PCM, with an epithelioid, granulomatousinflammation containing numerous yeast-like, multiple budding fungal forms. The

diagnosis of PCM was confirmed by immunohistochemistry using a specific

antibody anti-gp43 and by nested PCR using primers for the amplification of the

gp43 gene region. This is the first report of PCM disease occurring in a dog, an

animal that has been shown to play an important role in the natural history of

North American blastomycosis.

Keywords animals, fungus, paracoccidioidomycosis, PCR

Introduction

Paracoccidioidomycosis (PCM) is a systemic mycosis

almost exclusively found in man, whose etiologic agent

is Paracoccidioides brasiliensis, a dimorphic thermo-

dependent fungus which exists in two forms:

(i) mycelial, the saprophytic infectious form growing

at an ambient temperature of 22/288C, and (ii) yeast-

like, the parasitic form found in host tissue which grows

at a temperature of 35/378C. The main clinical

characteristic of the mycosis is the development of

granulomatous lesions in the lungs, reticuloendothelial

system and integument [1,2]. The route of infection is

through of the inhalation of conidia, the infectious

propagules of the parasite [1,3]. Although its natural

habitat is still unknown, there is consensus that the

fungus inhabits the soil of endemic areas, from which itwas isolated in Brazil, Argentina and Venezuela [4/7].

The gp43 glycoprotein is an immune-dominant antigen

in patients with PCM. It is protective against murine

PCM and is a putative virulence factor [8,9].

Paracoccidioides brasiliensis has been rarely recov-

ered from animals; even the detection of the fungus in

bats and armadillos has been always in the absence of

apparent disease [10,11]. In only two reports, four

infected armadillos presented pathological lesions of

the disease at autopsy, characterizing subclinical PCM

[12,13]. In addition, numerous serological studies or

surveys using paracoccidioidin have demonstrated thatdomestic or wild animals had contact with the fungus

but did not develop signs or symptoms of the disease

[14/19]. The fungus has also been isolated from soil-

contaminated ration, although the dog did not develop

the mycosis [20].

In contrast to PCM, other systemic granulomatous

mycoses caused by dimorphic fungi, such as North

Correspondence: Professor Marcello Franco, Department of

Pathology, Rua: Botucatu 740, Sao Paulo 04023-900, SP/Brazil.

Tel: '/5 5 1 1 5 57 6 4 26 6; Fa x: '/55 11 5571 9295; E-mail:

mfranco@patologia.epm.br

Received 22 June 2003; Accepted 7 August 2003

2004 ISHAM DOI: 10.1080/1369378032000141417

Medical Mycology August 2004, 42, 379/383

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American blastomycosis and histoplasmosis, frequently

affect animals [21,22]. We present here the first case of a

dog with PCM disease.

Case report

A female adult Doberman presented nodules in the

neck and a decline in general health condition. Based

on the diagnosis of cervical lymphadenomegaly, one of

the lymph nodes was biopsied. Histopathological

examination revealed active PCM with numerous

pathognomonic yeast-like forms of P. brasiliensis.

Identification of the agent was confirmed by

Grocott/Gomori staining, immunohistochemistry and

PCR. General clinical examination did not show

involvement of other organs or systems. The dog was

from the town of Mogi Guacu, interior of the State of

Sao Paulo, a region endemic for the disease. The animal

shared the space with another dog that did not develop

the disease, nor did the human inhabitants of the house.The owner of the infected dog and the other dog that

shared the same house were submitted to anti-

P. brasiliensis antibody serological testing, by immu-

nodiffusion, and the results were negative.

It is important to add that the house only had a small

garden and that the animal rarely went out to the

surrounding rural areas. Treatment was instituted with

ketoconazole, leading to total regression of the lym-

phadenomegaly. However, clinical recurrence was

observed after 18 months, and the dog was euthanized

without being submitted to autopsy.

Materials and methods

Histopathology

After resection, the cervical lymph node was fixed in

10% formalin (Sigma, St Louis, MO, USA), embedded

in paraffin (Sigma) and processed for histology. The

sections were stained with standard hematoxylin-eosin,

Grocott/Gomori and periodic acid Schiff.

Immunohistochemistry

Paraffin sections on 3-aminopropyl-triethoxysilane

(Sigma) coated microslides were deparaffinized

and stained overnight at 48C with a polyclonal anti-

gp43 antibody diluted 1/40 000 (provided by Dr

Mendes-Giannini), followed by biotinylated goat anti-

mouse/rabbit Ig (DAKO, Carpinteria, CA, USA)

diluted 1/200. The reaction was amplified with the

avidin/biotin/peroxidase complex (DAKO) diluted

1/200. The signal was developed using hydrogen

peroxidase as substrate and 3,3?,5,5?-diaminobenzidine

tetrachloride (Sigma) enhanced with 0.006% H2O2. All

reactions were incubated for 30 min and the sections

were washed in phosphate-buffered saline (PBS) at pH

7.4 and lightly counterstained with Harris hematoxylin

(Sigma). Substitution of the primary antibody with

PBS applied to the same sample resulted in a consistent

absence of immunostaining [23,24].

PCR

DNA extraction. DNA was extracted according to the

method of Shibata [25], with minor changes. Histolo-

gical sections of the lymph node (5/6-mm thick) were

placed in heated (808C) xylene solution and incubated

at 378C for 30 min. After centrifugation, the procedure

was repeated twice. The sample was washed twice in

absolute ethanol and centrifuged. The supernatant was

decanted and the sample dried. TE buffer (445 ml) and

5 ml 10 mg/ml proteinase K (Qiagen, Valencia, CA,

USA) were added, and the sample was incubatedovernight at 378C. The sample was submitted to

thermal shock in liquid nitrogen and incubated at

968C to inactivate proteinase K. Two extractions with

saturated phenol-chloroform-isoamyl alcohol were

carried out and the sample was centrifuged and 40 ml

3 mol/l sodium acetate, pH 4.0, was added to the

supernatant. After 30 min at /208C, the precipitated

DNA was dried and resuspended in autoclaved MilliQ

water. The total DNA concentration was determined

by spectrophotometry.

Primers. The primer design was based in 196-bp

specific region of gp43 gene sequence [26]. For nested

PCR, the following primers. Outer: I / 5? AAC TAGAAT ATC TCA CTC CCA GTC C 3?, and II / 5? TGT

AGA CGT TCT TGC ATG TCT TGG G 3?. Inner:

III / 5? GAT CGC CAT CCA TAC TCT CGC AAT C

3?, and IV / 5? GGG CAG AGA AGC ATC CGA

AAT TGC G 3?, were synthesized by Invitrogen (Life

Technologies, Carlsbad, CA, USA).

Conditions. The PCR mix consisted of 20 mmol/l

Tris-HCl buffer, pH 8.0, containing 50 mmol/l KCl,

2 mmol/l MgCl2, 1 mmol/l deoxynucleoside tripho-

sphate and Taq DNA polymerase (5 U/ml), all products

from Invitrogen. The primers were added to a final

concentration of 1 ng per 50 ml reaction. One tube

containing template-free DNA and another containing

Histoplasma capsulatum DNA were added as negative

controls. The nested PCR was amplified in a thermo-

cycler (GeneAmp PCR System 9700; Applied Biosys-

tems, Foster City, CA, USA). Initial denaturation was

achieved by heating the samples at 948C for 3 min,

followed by 30 cycles at 948C for 30 s, 658C for 30 s and

728C for 30 s, and a final extension at 728C for 5 min.

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For the second PCR, 1 ml of the product of the first

PCR was submitted to the following conditions: 5 min

at 948C, 30 s at 948C, 30 s at 508C, 1 min at 728C,

and a final extension at 728C for 10 min. The PCR

products (10 ml) were separated by electrophoresis on

ethidium bromide-stained 2% agarose gels and the

band intensities were analyzed using the Kodak Digital

Science EDAS 120 system (Invitrogen). The molecular

Fig. 1 Lymph node histopathology. (A, B) Epithelioid granuloma with the presence of numerous yeast-like forms of Paracoccidioides

brasiliensis inside of the giant cells (H&E; )/100). (C,D) Granulomas with numerous single or multiple budding yeast-like forms of

P. brasiliensis (Grocott/Gomori; )/100). (E,F) The location of yeast-like forms of P. brasiliensis is shown with an antibody directed against the

protein gp43 of fungus cells (immunohistochemistry; )/100).

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weight standard used was the 100-bp DNA Ladder

(Invitrogen).

Results

The histopathological findings of the lymph node werecharacterized by a granulomatous, epithelioid lympha-

denitis, with compact and loose areas accompanied by

necrosis. Numerous yeast-like forms of P. brasiliensis

were identified, with a predominance of viable forms

showing multiple exosporulation (Fig. 1A/D). The

immunohistochemistry shows various fungus cells

stained in brown, spread throughout the tissue

(Fig. 1E,F). Agarose gel (2%) electrophoresis of the

PCR products revealed a band indicating the presence

of P. brasiliensis (Fig. 2).

DiscussionThis is, to the best of our knowledge, the first case

published in the literature of a natural canine infection

with PCM. The dog developed the lymphadenomegalic

acute form of the mycosis and initially responded to

treatment, but later relapsed as frequently observed in

the human disease.

PCM infection in animals, although relatively rare

compared to the incidence of this condition in inhabi-

tants of endemic areas, has particularly been detected

in animals such as armadillos, bats and penguins. These

animals are believed to serve as probable wild reser-

voirs. Among primates, Cebus apella has been shown

to be most susceptible to infection [10/13,18,27,28].

Epidemiological surveys using paracoccidioidin con-

ducted on dogs from endemic areas have demonstrated

an elevated frequency of positive tests; however,

reactive animals submitted to clinical examination did

not show signs or symptoms of the disease. The same

author later used dogs as an experimental model of

PCM. The inoculated animals developed a self-limited

disease which resolved spontaneously within one

month [15/17].

Contrariwise, canine North American blastomycosis

in endemic area, has been considered as a important

epidemiologic marker, alerting physicians to the possi-

ble presence for concomitant blastomycosis in humans.

Nevertheless, the innate or acquired defense mechan-

isms that confer resistance to the development of

disease on wild or domestic animals from PCM-

endemic regions are still unknown. Identification of

these mechanisms will eventually result in clinical and

therapeutic benefits to the population at risk of

contracting the mycosis.

Further surveys involving domestic animals from

hyperendemic areas of the mycosis are necessary to

demonstrate that the dog plays a significant role in the

natural history of PCM.

Acknowledgements

The study was supported by a FAPESP grant (no.

01/07563-3). The authors would like to thank

Dr Ismael D. C. G. da Silva for molecular biology

support, Dr Zoilo Pires de Camargo for providing the

H. capsulatum DNA samples, Mr Antonio Carlos de

Souza and Mr Joaquim Soares de Almeida for

technical assistance and Ms Maria C. Aparecida do

Nascimento for secretarial assistance.

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