Feline Digital Phaeohyphomycosis Due to Exophiala Jeanselmei

NOTE Pathology

Feline Digital Phaeohyphomycosis due to Exophiala jeanselmei

Hirofumi MAEDA1), Hisashi SHIBUYA1), Yoko YAMAGUCHI2), Takuma MIYOSHI2), Mitsuhiro IRIE2) and Tsuneo SATO1)*

1)Laboratory of Veterinary Pathology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–8510 and 2)Irie Animal Hospital, 3308–5 Ikedo Miki-cho, Kitagun, Kagawa 761–0701, Japan

(Received 23 May 2008/Accepted 12 August 2008)

ABSTRACT. The black nodule measuring 1 cm in diameter developed in the base of nail of an 8-year-old Japanese domestic male cat. His- tological examination of the excised nodule revealed a granulomatous lesion extending from the epidermis to adjacent bone. The lesion was consisted of diffuse infiltration of macrophages with epithelioid cells and multinucleated giant cells. These macrophages contained a few to numerous yeast-like brown pigmented fungus cells with a spherical shape and dark thick wall. The PCR amplification with universal primers of the 28S ribosomal RNA gene yielded a 628-bp fragment and the direct sequence confirmed that the diagnosis of the lesion was phaeohyphomycosis caused by the pathogenic dematiaceous fungus, Exophiala jeanselmei. KEY WORDS: feline, granulomatous dermatitis, phaeohyphomycosis. J. Vet. Med. Sci. 70(12): 1395–1397, 2008

Phaeohyphomycosis is used to describe mycotic infec- diameter, at the base of nail in the left 2nd digit. The mass tions caused by various phaeoid fungi that form brown-pig- was easily bleeding when the cat was clawing. The mass mented yeast-like cells and hyphae [2]. Phaeoid fungi was surgically excised and subjected to histological exami- infection has been frequently described in human skin and nation. No recurrence was observed after complete exci- soft tissues in the worldwide including Japan [12]. Fungi sion. inhabiting soil and decomposed plants enter tissues through The mass was fixed in 10% neutral buffered formalin and skin wound. Lesions are usually localized in subcutaneous embedded in paraffin. Sections were stained with hematox- tissues, but the fungus may enter into circulation, especially ylin-eosin (HE) stain, periodic acid-Schiff (PAS) stain, the of immunosuppressed patients and result in systemic dis- potassium permanganate-oxalate (bleaching) method, and semination [1, 15]. There have been several reports of feline Fontana Masson stain. The genomic DNA was extracted cutaneous phaeohyphomycosis, and fungal species were from a paraffin-embedded tissue and used for the PCR successfully determined by culture from a cutaneous granu- amplification. Three to 5 slices of the thin section were loma or abscess [1, 4]. However, culture preparation deparaffinized with xylene and digested in lysis buffer (1 M requires a fresh sample and takes weeks to obtain the iso- Tris-HCl, 0.5 M EDTA, 5 M NaCl, 10% SDS) with protein- lates. This short communication describes histopathologic ase K (20 µg/ml). After phenol/chloroform extraction, the features of the feline case of E. jeanselmei infection in Japan DNA was resuspended with Tris-EDTA buffer and applied as well as molecular identification of fungal species by PCR to PCR. The universal primers for fungal species were con- and sequence technique. structed based on sequences of the internal transcribed An 8-year-old Japanese domestic male cat was presented spacer and 28S ribosomal RNA gene [5, 10]. The following to the private animal hospital because of staggering gait and was the forward and reverse primer sequences respectively; nasal discharge. The symptoms were slightly improved by 5’-GCATATCAATAAGCGGAGGAAAAG-3’ and 5’- antibiotics and forced feeding for a short period. However GGTCCGTGTTTCAAGACG-3’. The PCR amplification the cat developed clinical signs of fever, anorexia, nasal dis- was carried out for 35 cycles of the following conditions; charge, vomiting and depression afterward. A blood exam- 94°C for 30 sec, 53°C for 30 sec, and 72°C for 1 min. The ination revealed aplastic anemia and hyperglobulinemia. PCR product was electrophoresed on 1.5% agarose gel and The cat was negative for FIV and FeLV by SNAP FIV/ then single gel band sized 630 bp approximately was FeLV test (IDEXX Laboratories Inc, Maine, U.S.A.). The excised from the gel. The DNA of gel slice was recovered cat was suspected the FIP infection although it was not con- with the filter cartridge (SUPRECTM-01 TaKaRa, Tokyo, firmed as a definite diagnosis. The cat was treated with per- Japan) and used for sequence analysis with BigDye® Termi- oral medication of antibiotic and subcutaneous infusion nator Cycle Sequencing Kit and ABI 3100 DNA sequencer with corticosteroids. As the cat’s condition improved, the (Applied Biosystems, CA, U.S.A.). The sequence data was dose of steroid medication was reduced gradually. At that subjected to BLAST (The Basic Local Alignment Search time, a veterinarian found a black nodule, the size of 1 cm in Tool) search in the NCBI genebank. Histological examination of the excised nodule revealed a *CORRESPONDENCE TO: SATO, T., Laboratory of Veterinary Pathol- ogy, College of Bioresource Sciences, Nihon University, 1866 granulomatous lesion extending from the epidermis to adja- Kameino, Fujisawa, Kanagawa 252–8510, Japan. cent bone and roughly demarcated from normal tissue. The e-mail: [email protected]. lesion was consisted of diffuse infiltration of macrophages 1396 H. MAEDA ET AL.

from the biopsy sample and a reference strain of E. jeanselmei (Accession number AF050271) except a trans- version of G for T at the nucleotide 975 [16]. These results confirmed that the diagnosis was phaeohyphomycosis caused by the pathogenic dematiaceous fungus, E. jeanselmei. The morphological features of causative agents distin- guish pheohyphomycoses from other brown pigmented fungi: chromoblastomycosis, characterized by spherical fungus cells in tissues (sclerotic cells) and mycetoma, with tissue formation of black grains [1]. The present case showed the fungal cells with a budding body uncharacteris- tic of the sclerotic cells. The observation of distinctive dematiaceous hyphae is important to confirm phaeohyphomycosis. However, the fungi in our case were mostly present as a yeast-like form and no hyphal formation was Fig. 1. Multinucleated giant cells containing numerous yeast-like noted in the lesion. In addition to the morphological confir- brown pigmented fungus cells which had a spherical shape and mation, fungal DNA was successfully extracted from the dark thick wall. Haemotoxylin and eosin stain, bar = 50 µm. formalin-fixed granulomatous lesion in the feline digit and the nucleotide sequence revealed more than 99% homology with E. jeanselmei. This is the first feline case of phaeohyphomycosis diagnosed by molecular analysis. The diagnosis of phaeohyphomycosis has been done obtained by direct examination of an infected tissue with KOH and histological specimens. For identification of the fungal specie, classical culture isolation has been long con- sidered as the only reliable technique. In our case whole excised tissue was fixed in formalin for histological examination and unfortunately it was impossible to perform a classical mycological examination. Like our sample, the several cases in literature were not supported by culture isolation [1]. With the advance of molecular technique, the extraction of genomic DNA recovered from the formalin- fixed samples and the PCR including DNA sequencing may contribute to identify the pathogen, even without a fresh sample. Fig. 2. AS-positive margin of the fungal cells with a budding The listing in the text book cites 60 genera and 109 spe- body (arrow). PAS stain, bar = 10 µm. cies as aetiological agents of phaeohyphomycosis [13]. Phaeohyphomycosis shows a broad spectrum of infections with epithelioid cells, multinucleated giant cells, polymor- ranging from superficial, cutaneous or subcutaneous, to sys- phonuclear neutrophils, fewer lymphocytes and plasma cells temic or disseminated. Most feline cases were reported of accompanied with hemorrhage. These macrophages con- cutaneous phaeohyphomycosis, but there was systemic tained a few to numerous yeast-like brown pigmented fun- involvement caused by Ochroconis gallopava, Cladophialo- gus cells. The fungus cells had a spherical shape and dark phora bantiana and E. jeanselmei [4, 9]. Although we sus- thick wall ranged from 3 to 15 µm in width (Fig. 1). There pected the cat under immunosuppressive condition by a use was a few budding bodies but were no hyphal elements in of corticosteroids, phaeohyphomycosis has been known to the lesion (Fig. 2). The most of fungus cells were intracel- occur in immunocompetent hosts [4, 6]. In the most feline lularly localized. The margin of the fungal cells was posi- cases, diagnostic tests failed to reveal an underlying immu- tive with PAS reaction and Fontana-Masson stain. The nosuppression including a viral infection [3, 7, 8, 11, 14]. brown pigments were eliminated by bleaching method. Little information has been available about the treatment This result indicated the brown pigment was composed by of phaeohyphomycosis. The management of cutaneous and melanin. subcutaneous infection usually involves surgical treatment The amplified product of 628-bp fragment was excised with or without an antifungal agent although antifungal ther- from the gel and used for direct sequence. The obtained apy may result in variable prognosis even within strains of a sequence was subjected to BLAST search in the NCBI Gen- single species [13]. Some phaeoid fungi resist antifungal bank. The nucleotide BLAST showed 99.8% similarity drugs and develop a recrudescent clinical course or an between the nucleotide sequence of 28S ribosomal DNA aggressive course as systemic infection [1, 4, 9]. The FELINE PHAEOHYPHOMYCOSIS 1397 present case had a single lesion and no recurrence after a 702–703. In: Pathology of Domestic Animals, vol. 1, Elsevier, surgical resection, and recovered completely. Philadelphia. 9. Helms, S. R. and McLeod, C. G. 2000. Systemic Exophiala REFERENCES jeanselmei infection in a cat. J. Am. Vet. Med. 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