Isolation of Fusarium sp. from a Claw of a Dog with Onychomycosis

NOTE Internal Medicine Isolation of Fusarium sp. from a Claw of a Dog with Onychomycosis Kazuko NAMITOME 1,2), Rui KANO 3), Maiko SEKIGUCHI 4), Toshiroh IWASAKI 1), Takashi KANESHIMA 2) and Koji NISHIFUJI 1) * 1) Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3 5 8 Saiwai-cho, Fuchu, Tokyo 183 8509, 2) Mizuhodai Animal Hospital, 1 21 5 Nishimizuhodai, Fujimi, Saitama 354 0018, 3) Laboratory of Veterinary Pathobiology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252 0880 and 4) Department of Animal Science, Teikyo University of Science, 2525 Yatsuzawa, Uenohara, Yamanashi 406 0193, Japan (Received 11 January 2011/Accepted 14 March 2011/Published online in J-STAGE 28 March 2011) ABSTRACT. An 8-year-old male Golden Retriever had lameness and claw abnormality in the second digit of the left forelimb. Radiography revealed osteomyelitis in the distal phalanx bone of the affected limb. Microscopic examination of the claw revealed numerous hyphae in the claw matrix. Fungal DNA fragments coding the ribosomal internal transcribed spacer region (ITS) were detected from the claw matrix as well as fungal colonies of the clinical isolates by PCR. Nucleotide sequencing revealed that the amplicons shared > 99% homology with Fusarium sp. Therapy including oral itraconazole resulted in regrowth of a new claw, in which no hyphae were detected. To the authors knowledge, this is the first case report of canine onychomycosis in which Fusarium sp. was isolated from the affected claw. KEY WORDS: canine, claw, Fusarium, infection, onychomycosis. J. Vet. Med. Sci. 73(7): 965 969, 2011 Onychomycosis is a term for claw infection caused by dermatophytes, nondermatophyte molds or yeasts. The incidence of onychomycosis is very rare in dogs. Scott et al. reported that onychomycosis accounted for 7 out of 196 (3.6%) dogs affected with claw diseases [24]. Dermatophytes such as Trichophyton mentagrophytes and Microsporum gypseum, nondermatophyte molds such as Geotrichum candidum and Blastomyces dermatitidis and yeasts such as Candida sp. and Malassezia sp. have been isolated from claws affected with onychomycosis in dogs [1, 24, 25]. Conversely, to date, there have been no reports describing that Fusarium sp. were isolated from onychomycotic claws of dogs. This report describes a dog with onychomycosis, in which genetic evidence of Fusarium sp. was demonstrated in the affected claw as well as in fungal colonies of the clinical isolates. The case was an 8-year-old, intact male Golden Retriever, presented with a 3-month history of lameness and claw abnormality in the second digit of the left forelimb. According to the owner s declaration, the dog has no apparent history of trauma on the affected claw prior to development of the claw abnormality. Simultaneous with the development of claw changes, a solitary tumor developed on the lumbar area of the skin. The general condition of the dog seemed to be good except for the claw abnormality and the cutaneous tumor. On physical examination, the affected claw exhibited macronychia and onychorrhexis, and the claw matrix seemed to be dull, brownish and partly brittle (Fig. 1A). *CORRESPONDENCE TO: NISHIFUJI, K., Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3 5 8 Saiwai-cho, Fuchu, Tokyo 183 8509, Japan. e-mail: kojimail@cc.tuat.ac.jp Paronychia and hyperkeratosis of the footpad were also recognized in the affected digit. Besides the affected area, other claws and digits showed no abnormalities. The cutaneous tumor on the lumbar area consisted of a solitary, well circumscribed and firm tumor that was approximately 5 cm in diameter (data not shown). Radiographic analysis of the affected digit revealed bone destruction with cortical disruption on the distal phalanx bone (Fig. 1B). Complete blood counts, serum chemistry and thoratic as well as abdominal radiographs yielded unremarkable results. Microscopic examination of the claw samples revealed numerous hyphae in the claw matrix (Fig. 2), suggesting fungal infection of the claw. For fungal culture, the affected claw was initially disinfected with alcohol to avoid contamination of microorganisms present on the claw surfaces, and a sample was then cultured on a Sabouraud's dextrose agar and potato dextrose agar at 27ºC for 2 weeks. Fungal colonies with a flat, cinnamon color and a cottony texture developed within 1 week. Microscopic examinations of the isolates revealed branching hyphae, but no microconidia or macroconidia were recognized. Therefore, the fungal species of the clinical isolates was not identified by the morphological characteristics of the cultured isolates. To confirm the fungal species of the isolates by molecular analysis, PCR to amplify the ribosomal internal transcribed spacer (ITS) region (5.8 rrna gene, ITS1 and ITS2) was performed. Extraction of genomic DNA samples and PCR analysis were preformed as previously reported [16]. The sequences of the primers for the ribosomal ITS region were constructed based on sequences reported previously [14]; the forward primer was ITS5 (5 -GGA AGT AAA AGT CGT AAC AAG G-3 ), and the reverse primer was ITS4 (5 -TCC TCC GCT TAT TGA TAT GC-3 ). The ampli-

966 K. NAMITOME ET AL. Fig. 1. Clinical and radiographic findings of the affected digit. (A) Macronychia and onychorrhexis in the second digit of the left forelimb. (B) The radiograph reveals bone destruction with cortical disruption on the distal phalanx bone of the affected digit. Fig. 2. Microscopic observations of the claw matrix. Note that numerous hyphae (arrows) are recognized in the claw matrix. Bar=50 m. cons were directly sequenced by the dideoxy chain termination method using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). Comparative sequence analyses with the ITS region in GenBank showed that the queried sequence of the clinical isolate was > 99% homologous with Fusarium sp. (GenBank accession number, EF611096), with only a single nucleotide being replaced (351C to T; Fig. 3), probably due to a single nucleotide polymorphism in the cultured isolate. PCR amplification was also performed using DNA extracted directly from the affected claw, and nucleotide sequencing revealed that the amplicon had 100% homology with Fusarium sp. (Gen- Bank accession number, EF611096) and Gibberella avenacea, a teleomorph of Fusarium sp. (GenBank accession number, FJ603000; data not shown). Thus, these findings provided genetic evidence of Fusarium sp. in the affected claw. Genetic evidence of dermatophytes (e.g., Microsporum sp. and Trichophyton sp.) was not identified in the affected claw or cultured isolates. Bacterial culture isolated Escherichia coli from the affected claw. Under general anesthesia, the cutaneous tumor on lumbar area was removed surgically. As osteomyelitis caused by fungal and/or bacterial infection was considered as a differential diagnosis, amputation of the affected digit was recommended. However, as the owner did not agree with the amputation, the affected claw was removed at the proximal end at the time of surgical removal of the lumbar tumor. To determine whether or not neoplasia or bullous diseases were recognized in the distal phalanx of the affected digit, onychobiopsy of the affected digit was also performed according the method reported previously [19]. Histopathologically, the tumor consisted of a subcutaneous and dermal mass comprising spindle and polymorphic cells arranged in a cartwheel pattern with abundant collagen in a part of the mass (data not shown). The histopathological findings were consistent with those in hemangiopericytoma. Histopathology of an onychobiopsy sample revealed marked epidermal acanthosis with hyperkeratosis, dilatation of blood vessels with perivascular-to-diffuse infiltration of mononuclear cells and extravasation of red blood cells in the superficial dermis of the claw fold (Fig. 4). These findings may support the clinical finding of paronychia. No neoplasia or bullous diseases were recognized in the claw fold sample. The claw matrix was not recognized in multiple histological sections, probably due to complete removal of the claw matrix by

CANINE ONYCHOMYCHOSIS CAUSED BY FUSARIUM SP. 967 Fig. 3. Comparison of nucleotide sequences between a clinical isolate of the affected claw and Fusarium sp. previously reported (GenBank accession number, EF611096). A single position in which nucleotides are not identical is indicated by a grey rectangle and an arrow. declawing. Based on these findings, the claw lesion of the present dog was diagnosed as onychomycosis caused by Fusarium infection. The dog was treated with oral itraconazole (6.6 mg kg 1, q24h) and marbofloxacin (5 mg kg 1, q24h); the latter was selected according to antibiotic sensitivity tests of the isolated E. coli. At 40 days after initiation of therapy, regrowth of a new claw was recognized, although the regenerated claw did not have a normal shape, probably due to resorption of bone marrow in the distal phalanx bone (Fig. 5). Claw scraping at 40 days after initiation of therapy yielded no fungal organisms in the claw matrix (data not shown). The present dog did not exhibit lameness at 40 days after initiation of therapy. Fusarium sp., which are ubiquitous saprophytic fungi and important plant pathogens, can also be opportunistic pathogens causing onychomycosis in mammals [2 7, 9, 10, 12, 13, 17, 18, 20 23, 26 28]. In humans, Fusarium sp. have been isolated in less than 12.0% of onychomycotic nails by fungal culture or PCR [10, 12, 17, 20 23, 27, 28]. In dogs, Fusarium sp. have been isolated in cases with fungal meningoencephalitis [11], pyelonephritis [8], dermatomycosis [15] and disseminated infection including kidney and subcutis infection [16], but no reports have described canine onychomycosis caused by Fusarium sp. to date. In the present case, genetic evidence of Fusarium sp. was demonstrated in the affected claw. The present dog was strongly suspected as having onychomycosis caused by Fusarium sp, as numerous hyphae were present in the claw matrix of the affected claw, while no genetic evidence of dermatophytes was found. As the claw is the body site that contacts directly with soil, it might have greater opportunity to be contaminated with saprophytes. In addition, as the tip of the claw is completely isolated from immune systems, it might be easily infected by saprophytes, even through trivial flaws. In conclusion, to the authors knowledge, this is the first case report of canine onychomycosis in which Fusarium sp. was isolated from an affected claw. Our findings also indi-

968 K. NAMITOME ET AL. Fig. 5. Radiograph of the affected digit at 40 days after initiation of therapy. Resorption of bone marrow (arrow) was recognized on the distal phalanx bone. Fig. 4. Histopathological findings of the claw fold of the affected digit. (A) Marked epidermal acanthosis with hyperkeratosis is recognized. Bar=200 m. (B) Vascular dilatation and mononuclear cell infiltration are recognized in the superficial dermis. Bar=10 m. cated that the combination of microscopic analysis, fungal culture and PCR would help to identify causative fungi of infections in dogs with onychomycosis. Identification of underlying diseases affecting immune status is also important to understand a patient s condition and manage the infection. REFERENCES 1. Andrino, M., Blanco, J.L., Duran, C., Fernandez Barredo, S., Cruzado, M. and Garcia, M.E. 2003. [Canine onychomycosis produced by Microsporum gypseum. A case report]. Rev. Iberoam. Micol. 20: 169 171 (in Spanish). 2. Arrese, J.E., Pierard Franchimont, C. and Pierard, G.E. 1996. Fatal hyalohyphomycosis following Fusarium onychomycosis in an immunocompromised patient. Am. J. Dermatopathol. 18: 196 198. 3. Baran, R., Faergemann, J. and Hay, R.J. 2007. Superficial white onychomycosis--a syndrome with different fungal causes and paths of infection. J. Am. Acad. Dermatol. 57: 879 882. 4. Baran, R., Tosti, A. and Piraccini, B.M. 1997. Uncommon clinical patterns of Fusarium nail infection: report of three cases. Br. J. Dermatol. 136: 424 427. 5. Bodey, G.P., Boktour, M., Mays, S., Duvic, M., Kontoyiannis, D., Hachem, R. and Raad, I. 2002. Skin lesions associated with Fusarium infection. J. Am. Acad. Dermatol. 47: 659 666. 6. Brasch, J. and Koppl, G. 2009. Persisting onychomycosis caused by Fusarium solani in an immunocompetent patient. Mycoses 52: 285 286. 7. Calado, N.B., Sousa, F. Jr, Gomes, N.O., Cardoso, F.R., Zaror, L.C. and Milan, E.P. 2006. Fusarium nail and skin infection: a report of eight cases from Natal, Brazil. Mycopathologia 161: 27 31. 8. Day, M.J. and Holt, P.E. 1994. Unilateral fungal pyelonephritis in a dog. Vet. Pathol. 31: 250 252. 9. Dignani, M.C. and Anaissie, E. 2004. Human fusariosis. Clin. Microbiol. Infect. 10 (Suppl. 1): 67 75. 10. Ebihara, M., Makimura, K., Sato, K., Abe, S. and Tsuboi, R. 2009. Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences. Br. J. Dermatol. 161: 1038 1044. 11. Evans, J., Levesque, D., de Lahunta, A. and Jensen, H.E. 2004. Intracranial fusariosis: a novel cause of fungal meningoencephalitis in a dog. Vet. Pathol. 41: 510 514. 12. Godoy Martinez, P., Nunes, F.G., Tomimori Yamashita, J., Urrutia, M., Zaror, L., Silva, V. and Fischman, O. 2009. Onychomycosis in Sao Paulo, Brazil. Mycopathologia 168: 111 116. 13. Guilhermetti, E., Takahachi, G., Shinobu, C.S. and Svidzinski, T.I. 2007. Fusarium spp. as agents of onychomycosis in immunocompetent hosts. Int. J. Dermatol. 46: 822 826.

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