NOTE Internal Medicine

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

Kazuko NAMITOME1,2), Rui KANO3), Maiko SEKIGUCHI4), Toshiroh IWASAKI1), Takashi KANESHIMA2) and Koji NISHIFUJI1)*

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. Radiographyrevealed osteomyelitis in the distal phalanx bone of the affected limb. Microscopic examination of the claw revealed numerous hyphaein the claw matrix. Fungal DNA fragments coding the ribosomal internal transcribed spacer region (ITS) were detected from the clawmatrix 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 affectedclaw.KEY WORDS: canine, claw, Fusarium, infection, onychomycosis.

J. Vet. Med. Sci. 73(7): 965–969, 2011

Onychomycosis is a term for claw infection caused bydermatophytes, nondermatophyte molds or yeasts. Theincidence 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]. Dermato-phytes such as Trichophyton mentagrophytes andMicrosporum gypseum, nondermatophyte molds such asGeotrichum candidum and Blastomyces dermatitidis andyeasts such as Candida sp. and Malassezia sp. have beenisolated from claws affected with onychomycosis in dogs[1, 24, 25]. Conversely, to date, there have been no reportsdescribing that Fusarium sp. were isolated from onychomy-cotic claws of dogs. This report describes a dog with ony-chomycosis, in which genetic evidence of Fusarium sp. wasdemonstrated in the affected claw as well as in fungal colo-nies of the clinical isolates.

The case was an 8-year-old, intact male GoldenRetriever, presented with a 3-month history of lameness andclaw abnormality in the second digit of the left forelimb.According to the owner’s declaration, the dog has no appar-ent history of trauma on the affected claw prior to develop-ment of the claw abnormality. Simultaneous with thedevelopment of claw changes, a solitary tumor developedon the lumbar area of the skin. The general condition of thedog seemed to be good except for the claw abnormality andthe cutaneous tumor.

On physical examination, the affected claw exhibitedmacronychia and onychorrhexis, and the claw matrixseemed to be dull, brownish and partly brittle (Fig. 1A).

Paronychia and hyperkeratosis of the footpad were also rec-ognized in the affected digit. Besides the affected area,other claws and digits showed no abnormalities. The cuta-neous tumor on the lumbar area consisted of a solitary, wellcircumscribed and firm tumor that was approximately 5 cmin diameter (data not shown).

Radiographic analysis of the affected digit revealed bonedestruction with cortical disruption on the distal phalanxbone (Fig. 1B). Complete blood counts, serum chemistryand thoratic as well as abdominal radiographs yielded unre-markable results. Microscopic examination of the clawsamples revealed numerous hyphae in the claw matrix (Fig.2), suggesting fungal infection of the claw. For fungal cul-ture, the affected claw was initially disinfected with alcoholto avoid contamination of microorganisms present on theclaw surfaces, and a sample was then cultured on a Sab-ouraud's dextrose agar and potato dextrose agar at 27ºC for2 weeks. Fungal colonies with a flat, cinnamon color and acottony texture developed within 1 week. Microscopicexaminations of the isolates revealed branching hyphae, butno microconidia or macroconidia were recognized. There-fore, the fungal species of the clinical isolates was not iden-tified by the morphological characteristics of the culturedisolates.

To confirm the fungal species of the isolates by molecularanalysis, PCR to amplify the ribosomal internal transcribedspacer (ITS) region (5.8 rRNA gene, ITS1 and ITS2) wasperformed. Extraction of genomic DNA samples and PCRanalysis were preformed as previously reported [16]. Thesequences of the primers for the ribosomal ITS region wereconstructed based on sequences reported previously [14];the forward primer was ITS5 (5’-GGA AGT AAA AGTCGT AAC AAG G-3’), and the reverse primer was ITS4(5’-TCC TCC GCT TAT TGA TAT GC-3’). The ampli-

* CORRESPONDENCE TO: NISHIFUJI, K., Laboratory of VeterinaryInternal Medicine, Faculty of Agriculture, Tokyo University ofAgriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo183–8509, Japan.

e-mail: kojimail@cc.tuat.ac.jp

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cons were directly sequenced by the dideoxy chain termina-tion method using an ABI PRISM 310 Genetic Analyzer(Applied Biosystems, Foster City, CA, U.S.A.). Compara-tive sequence analyses with the ITS region in GenBankshowed that the queried sequence of the clinical isolate was> 99% homologous with Fusarium sp. (GenBank accessionnumber, EF611096), with only a single nucleotide beingreplaced (351C to T; Fig. 3), probably due to a single nucle-otide polymorphism in the cultured isolate. PCR amplifica-tion was also performed using DNA extracted directly fromthe affected claw, and nucleotide sequencing revealed thatthe amplicon had 100% homology with Fusarium sp. (Gen-

Bank accession number, EF611096) and Gibberella avena-cea, a teleomorph of Fusarium sp. (GenBank accessionnumber, FJ603000; data not shown). Thus, these findingsprovided genetic evidence of Fusarium sp. in the affectedclaw. Genetic evidence of dermatophytes (e.g. ,Microsporum sp. and Trichophyton sp.) was not identifiedin the affected claw or cultured isolates. Bacterial cultureisolated Escherichia coli from the affected claw.

Under general anesthesia, the cutaneous tumor on lumbararea was removed surgically. As osteomyelitis caused byfungal and/or bacterial infection was considered as a differ-ential diagnosis, amputation of the affected digit was recom-mended. However, as the owner did not agree with theamputation, the affected claw was removed at the proximalend at the time of surgical removal of the lumbar tumor. Todetermine whether or not neoplasia or bullous diseases wererecognized in the distal phalanx of the affected digit, ony-chobiopsy of the affected digit was also performed accord-ing the method reported previously [19]. Histopatholo-gically, the tumor consisted of a subcutaneous and dermalmass comprising spindle and polymorphic cells arranged ina cartwheel pattern with abundant collagen in a part of themass (data not shown). The histopathological findings wereconsistent with those in hemangiopericytoma. Histopathol-ogy of an onychobiopsy sample revealed marked epidermalacanthosis with hyperkeratosis, dilatation of blood vesselswith perivascular-to-diffuse infiltration of mononuclearcells and extravasation of red blood cells in the superficialdermis of the claw fold (Fig. 4). These findings may supportthe clinical finding of paronychia. No neoplasia or bullousdiseases were recognized in the claw fold sample. The clawmatrix was not recognized in multiple histological sections,probably due to complete removal of the claw matrix by

Fig. 1. Clinical and radiographic findings of the affected digit. (A) Macronychiaand onychorrhexis in the second digit of the left forelimb. (B) The radiographreveals bone destruction with cortical disruption on the distal phalanx bone of theaffected digit.

Fig. 2. Microscopic observations of the claw matrix. Note thatnumerous hyphae (arrows) are recognized in the claw matrix.Bar=50 m.

967CANINE ONYCHOMYCHOSIS CAUSED BY FUSARIUM SP.

declawing.Based on these findings, the claw lesion of the present

dog was diagnosed as onychomycosis caused by Fusariuminfection. The dog was treated with oral itraconazole (6.6mg kg–1, q24h) and marbofloxacin (5 mg kg–1, q24h); thelatter was selected according to antibiotic sensitivity tests ofthe isolated E. coli. At 40 days after initiation of therapy,regrowth of a new claw was recognized, although the regen-erated claw did not have a normal shape, probably due toresorption of bone marrow in the distal phalanx bone (Fig.5). Claw scraping at 40 days after initiation of therapyyielded no fungal organisms in the claw matrix (data notshown). The present dog did not exhibit lameness at 40 daysafter initiation of therapy.

Fusarium sp., which are ubiquitous saprophytic fungi andimportant plant pathogens, can also be opportunistic patho-gens causing onychomycosis in mammals [2–7, 9, 10, 12,13, 17, 18, 20–23, 26–28]. In humans, Fusarium sp. havebeen 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 menin-goencephalitis [11], pyelonephritis [8], dermatomycosis[15] and disseminated infection including kidney and sub-cutis infection [16], but no reports have described canineonychomycosis caused by Fusarium sp. to date. In thepresent case, genetic evidence of Fusarium sp. was demon-strated in the affected claw. The present dog was stronglysuspected as having onychomycosis caused by Fusarium sp,as numerous hyphae were present in the claw matrix of theaffected claw, while no genetic evidence of dermatophyteswas found. As the claw is the body site that contacts directlywith soil, it might have greater opportunity to be contami-nated with saprophytes. In addition, as the tip of the claw iscompletely isolated from immune systems, it might be eas-ily infected by saprophytes, even through trivial flaws.

In conclusion, to the authors’ knowledge, this is the firstcase report of canine onychomycosis in which Fusarium sp.was isolated from an affected claw. Our findings also indi-

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 notidentical is indicated by a grey rectangle and an arrow.

K. NAMITOME ET AL.968

cated that the combination of microscopic analysis, fungalculture and PCR would help to identify causative fungi ofinfections in dogs with onychomycosis. Identification ofunderlying diseases affecting immune status is also impor-tant to understand a patient’s condition and manage theinfection.

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