Medical Mycology August 2004, 42, 379 /383

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 Sa o Paulo (UNIFESP-EPM), $Laboratory of Pathology, Mogi-Guac u, %Adolfo Lutz Institute, and Department of Biochemistry, Federal University of Sa o Paulo, Sa o Paulo, Brazil

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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, granulomatous inflammation 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 thermodependent fungus which exists in two forms: (i) mycelial, the saprophytic infectious form growing at an ambient temperature of 22 /288C, and (ii) yeastlike, 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

Received 22 June 2003; Accepted 7 August 2003 Correspondence: Professor Marcello Franco, Department of Pathology, Rua: Botucatu 740, Sa o Paulo 04023-900, SP /Brazil. Tel: '/ 55 11 5576 4266; Fax: '/ 55 11 5571 9295; E-mail: mfranco@patologia.epm.br

habitat is still unknown, there is consensus that the fungus inhabits the soil of endemic areas, from which it was 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 recovered 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 that domestic 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 soilcontaminated 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
DOI: 10.1080/1369378032000141417

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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 Guac u, interior of the State of Sa o 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 antiP. brasiliensis antibody serological testing, by immunodiffusion, 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 lymphadenomegaly. However, clinical recurrence was observed after 18 months, and the dog was euthanized without being submitted to autopsy.

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. Histological 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 incubated overnight 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 TAG AAT 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 triphosphate 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 thermocycler (GeneAmp PCR System 9700; Applied Biosystems, 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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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 antigp43 antibody diluted 1/40 000 (provided by Dr Mendes-Giannini), followed by biotinylated goat antimouse/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

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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

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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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Fig. 2 PCR. Lane 1, 100-bp molecular-weight marker; lane 2, template-free DNA, lane 3, negative control (H. capsulatum ); lane 4, P. brasiliensis gp43 amplicom (195 bp) from a human biopsy; lane 5, P. brasiliensis gp43 amplicom (196 bp) from the canine biopsy.

weight standard used was the 100-bp DNA Ladder (Invitrogen).

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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 possible presence for concomitant blastomycosis in humans. Nevertheless, the innate or acquired defense mechanisms that confer resistance to the development of disease on wild or domestic animals from PCMendemic 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.

Results
The histopathological findings of the lymph node were characterized by a granulomatous, epithelioid lymphadenitis, 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).

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.

References
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Discussion
This 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 inhabitants of endemic areas, has particularly been detected in animals such as armadillos, bats and penguins. These animals are believed to serve as probable wild reservoirs. Among primates, Cebus apella has been shown to be most susceptible to infection [10 /13,18,27,28]. Epidemiological surveys using paracoccidioidin conducted 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

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