Medical Mycology, 2015, 53, 369–377 doi: 10.1093/mmy/myu094 Original Article
Original Article
First report of Veronaea botryosa as a causal agent of chromomycosis in frogs Tsuyoshi Hosoya1 , Yasuko Hanafusa2,∗ , Tomoo Kudo3 , Kenichi Tamukai4 and Yumi Une3 1
*To whom correspondence should be addressed. Yasuko Hanafusa, Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, 305-0856, Japan. Tel: +81-29-838-7753; Fax: +81-29-838-7880; E-mail: [email protected] Received 31 August 2014; Revised 17 November 2014; Accepted 8 December 2014
Abstract A dematiaceous hyphomycete, isolated from frogs, was determined as the possible etiologic agent of a case of systemic chromomycosis this cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran. Key words: anuran mycosis, disseminated mycosis, zoonosis, phylogenetic analysis, phaeohyphomycosis.
Introduction Zoonoses are infections in animals caused by various microorganisms and have been gaining increasing attention in clinical research. Fungi are among the common agents of such infectious zoonoses in humans and animals. The most common zoonotic mycoses are dermatomycoses [1–3] and systemic infections are rarely reported, except for blastomycosis [4,5]. In most of these cases, the carrier animals are warm-blooded, and case reports involving cold-blooded animal hosts are limited [6]. Human cutaneous phaeohyphomycosis caused by Veronaea botryosa is extremely rare. To the best of our knowledge, only approximately 10 cases have been
reported in English-language literature to date [7–16] and primarily involve farmers who are exposed to soil or plants [8,11,12,17]. However, there was a single case induced by ear piercing [13,14]. It is suggested that the causal fungus may be a saprophyte or plant pathogen, but the details of the ecology of this fungus have not been elucidated. Despite the fact that frogs are naturally distributed worldwide and are one of the most popular cold-blooded pet animals, there are few reports on diseases affecting them. Since the discovery of chytridiomycosis caused by Batrachochytrium dendrobatidis [18], fungal diseases caused by fungi have been drawing public attention. However, this disease is limited to amphibians and does not infect
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Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 3050005, Japan, 2 Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, 305-0856, Japan, 3 Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan and 4 Den-en-chofu Animal Hospital, 2-1-3 Den-enchofu, Ota-ku, Tokyo 145-0071, Japan
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warm-blooded animals. Although disseminated fungal diseases in frogs have also been reported [6,19–21], very few of these papers provide detailed analyses on the fungal pathology and infection routes [19,20]. The present study describes cases of fatal V. botryosa infection in captive frogs. We detail the isolation of the disease-causing fungus from the frogs and describe its mycological features and molecular analysis. We also confirm the V. botryosa infection by histopathological examination of the organs from infected frogs. This is the first English treatise of the occurrence of a lethal V. botryosa infection in animals other than humans. The frog might play an important role in human V. botryosa infection, as a carrier animal.
Pathogen isolation Isolation of the pathogen was attempted from an individual D. guineti and L. caerulea, respectively, but recovery from B. japonicus formosus was not attempted owing to the decomposition of the bodies. Six small pieces (approximately 1 mm × 1 mm × 1 mm) of tissue were cut out from the liver, lung, kidney, and skin, using sterile injection needles. The tissue pieces were inoculated onto potato dextrose agar (PDA; Nissui, Tokyo, Japan) and Sabouraud agar (Nissui, Tokyo, Japan), both containing 50 μg of chloramphenicol, and incubated at 25◦ C. The hyphae arising from the tissue pieces were cut at the tip using a sterilized needle and transferred to a PDA plate, where it was maintained on PDA slants. The isolates were deposited to the Biological Resource Center, National Institute of Technology and Evaluation (NITE-BRC, Japan) as NBRC 109680–109685.
Animals In September 2012, polypoid lesions with ulceration were found on the body surfaces of seven eastern-Japanese common toads (Bufo japonicus formosus) and seven false tomato frogs (Dyscophus guineti), which had all been maintained in an anuran breeder’s water tank in Tokyo, Japan (water tank A). The symptoms did not improve with oral treatment of the antifungal agent itraconazole (10 mg/ml), administrated at 10 mg/kg/day/for 7 days, and within the following 2 weeks, all 14 infected frogs died or were euthanized as a result of the fatal symptoms. Simultaneously, several goldfish, one barking treefrog (Hyla gratiosa), and one White’s treefrog (Litoria caerulea), which had been held together in another water tank (water tank B), died within a week of each other. In addition, a ruby-eyed treefrog (Leptopelis uluguruensis) and an Orinoco lime treefrog (Sphaenorhynchus lacteus) held together in another water tank (water tank C) also died on the following week. To examine the cause of death, the bodies of B. japonicus formosus, D. guineti, and L. caerulea were sent to our laboratories for examination, while those of the other frogs were disposed of. All procedures in the study were in accordance with the guidelines approved by the Azabu University Animal Experiment Committee.
Necropsy and histopathological examination The dead frogs were chilled and necropsied, with tissues from major organs, fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Tissue sections (approximately 4 μm thick) were obtained using routine histological techniques and stained with hematoxylin and eosin (HE) for histopathological evaluation. To investigate the presence of the fungi, the periodic acid Schiff reaction was performed on the tissue sections.
DNA extraction, sequencing, and phylogenetic analysis The DNA extraction, polymerase chain reaction (PCR), and sequencing procedures followed standard methods [22]. The primer pair ITS1 and ITS4 was used to amplify internal transcribed spacer (ITS1 and ITS2) and 5.8S ribosomal sequences. The sequences obtained (AB98173-109680) were searched using the Basic Local Alignment Search Tool (BLAST) on the GenBank database (http://blast.ncbi.nlm. nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome) and were preserved in GenBank. The extracted DNA samples were preserved in the Center for Molecular Biodiversity Research in the National Museum of Nature and Science (Japan), and are available for future collaborative research. The obtained sequences were aligned with Veronaea spp. sequences in GenBank and with other sequences obtained by BLAST search using BioEdit v. 7.2.0 [23], and were analyzed by the neighbor-joining method using MEGA 6.0 [24]. All the parameters were set to default, except for 1,000 times of bootstrap replicates. Following a previous phylogenetic analysis [25], Rhinocladiella anceps (EU041804) was chosen as the outgroup taxon. The alignments used for the phylogenetic analyses were deposited to TreeBASE (http://purl.org/phylo/treebase/phylows/study/ TB2:S16257).
Cultivation of the fungus and its morphological observation The isolates obtained were inoculated onto the center of PDA plates, Miura’s agar (glucose 1 g, KH2 PO4 1 g, MgSO4 ·7H2 O 0.2 g, KCl 0.2 g, KNO3 2 g, yeast extract
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Materials and methods
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Table 1. Distribution of lesions in examined frogs and toads. Lesion distribution on body surface No.
Species
Status
1 2 3 4 5
Bufo japonics formosus
Dead Dead Euthanized Dead Dead
6 7
Dyscophus guineti
8
Litoria caerulea
by macroscopy
Lesion distribution in major organs by histopahological examination∗ Kidney
Lung
Liver
Spleen
Head, legs Legs Legs Head, legs, back Head, legs, back
+ + + + +
+/− + + + +
– + + + +
– + – – +
Dead Dead
Legs, Head, legs,
+ +
+ +
+ +
– –
Dead
None
+
+
+
–
∗
0.2 g, agar 13 g, and distilled water 1000 ml; pH adjusted to 6.5–7.0), or brain heart infusion agar (BHI; Difco, NJ, USA), and incubated at 20◦ C for 3 weeks. Pantone color codes, adopting the CYMK system that refers to a Pantone color bridge, were used for the color description of the colonies [26].
Results Necropsy, histopathological examination, and treatment attempt According to the gross and histopathological examinations, the distribution of lesions and the response to the pathogen differed in each animal (Table 1). Macroscopically, polypoid skin nodule formation, cutaneous ulceration, and necrosis of the epidermis and skeletal muscle were evident in the head, legs, and back of B. japonicus formosus (Fig. 1A). In these toads, multiple white nodules were observed in the kidneys, lungs, liver, and spleen. In D. guineti, skin lesions were observed in the legs and on the head, the kidneys were highly enlarged and discolored (Fig. 1B) and multiple white nodules were observed in the liver and lungs. On the other hand, in L. caerulea, skin lesions were not observed, although multiple white nodules were apparent in the kidneys, lungs, and liver (Fig. 1C). The histopathological changes were similar in all of the animals examined and were found to be consistent with the macroscopic lesions. Granulomatous inflammation was observed in the dermis and skeletal muscle (Fig. 1D, E). In the kidneys, lungs, liver, and spleen, mainly granulomatous lesions were seen (Fig. 1F) and contained brown fungal hyphae (Fig. 1G). In L. caerulea, the granulomatous reaction consisting of giant cells containing brown fungal hyphae
was remarkable. In the present cases, itraconazole did not have any therapeutic effect at all (Table 1).
Isolation of the possible pathogen Hyphal growth from the tissue sections was observed 4 days after inoculation on all isolation media. Since all isolates were similar in the appearance of their colonies and conidia-producing structures, six representative strains were selected (NBRC 109680-109685; see culture examined in the taxonomy section below) and deposited to the NITE-BRC, Japan. All the isolates obtained showed the same colony morphology on slants and plates. Isolation from the skin failed owing to the heavy bacterial contamination not suppressed by the antibiotic.
Taxonomy of the pathogen Sequence lengths of 562–574 bp were obtained from the isolates (sequences registered as AB981713-981718 for NBRC 109680-109685, respectively) and found to be identical in the compared region and concluded to belong to the same species. When NBRC 109680 was used as a representative for BLAST search, sequences that were identical (e.g., AB369905) or highly similar to that of Veronaea botryosa followed by those of other Veronaea species were obtained (Table 2). The result of phylogenetic analysis (Fig. 2) demonstrated that the query sequence was situated among V. botryosa. The morphology of all the isolates was identical to that of the previously described fungi [25,27] and on the
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Lesion means granulomatous changes. +: Histopathological change + and fungi +. +/−: Histopathological change + and fungi –. –: Histopathological change − and fungi –.
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Figure 1. Pathological findings of chromomycosis in frogs. A. Polypoid skin nodule in Bufo japonicus formosus, no. 5. B. The kidneys are highly enlarged and discolored in Dyscophus guineti, no. 6. C. Multiple white nodules are observed in the liver of Litoria caerulea, no. 8. D. Cutaneous granuloma formation with necrosis of the epidermis and skeletal muscle in B. japonicus formosus, no. 5, HE. E. Granulomatous inflammation is observed in the dermis along with brown fungal hyphae, in B. japonicus formosus, no. 5, HE. F. Mycotic granuloma in the liver and brown fungal hyphae, in D. guineti, no. 6, HE. G. Brown fungal hyphae in granulomas in B. japonicus formosus, no. 5, HE. HE stands for hematoxylin and eosin. Bar, 20 μm. This Figure is reproduced in color in the online version of Medical Mycology.
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Table 2. List of basic local alignment search tool search when NBRC 109680 was used as a query sequence. Total score
Query coverage (%)
Max ident (%)
1 2 3 4 5 6 7 8
Veronaea botryosa IFM 5335 Veronaea botryosa CBS 114829 Veronaea botryosa CBS 126027 Veronaea botryosa CBS 127264 Veronaea botryosa CBS 121.92 Veronaea botryosa CBS 350.65 Veronaea botryosa CBS 254.57 Veronaea botryosa CBS 101462
1199 1194 1194 1222 1144 1101 1094 1074
99 100 100 98 95 91 92 89
9
Veronaea botryosa CBS 121506
1038
10
Veronaea botryosa CBS 102593
11
Acc. No.
Country
100 99 99 100 99 100 99 99
AB369905 KC205972 KC205966 JX566723 EU041815 EU041817 EU041816 JF747141
undisclosed Hong Kong undisclosed undisclosed Australia India Italy Philippines
86
100
JF747140
USA
1038
86
100
JF747142
China
Veronaea botryosa CBS 254.57
1035
87
99
JF747143
Italy
12
Veronaea botryosa CBS 122236
1011
86
99
KC205971
Brazil
13
Veronaea botryosa CBS 122825
1000
83
99
KC205967
Brazil
14
Veronaea botryosa CBS 122823
994
84
99
KC205969
Brazil
15
Veronaea botryosa CBS 122824
994
82
100
KC205968
Brazil
16
Veronaea botryosa CBS 122822
977
81
99
KC205970
Brazil
17
Veronaea botryosa CBS 127016
968
81
99
KC205965
undisclosed
Substrate of Isolate Unknown Wood in fresh water
Dead leaf base Dung of goat Sansa olive slag Skin lesion of 37-year-old male patient Wrist (patient from Japan) Ulcerating subcutaneous lesions, disseminated Phaeohyphomycosis of 12-year-old male Railway tie treated with creosote 20 years ago Railway tie treated with creosote 20 years Railway tie treated with creosote 20 years Railway tie treated with creosote 20 years Railway tie treated with creosote 20 years
The results of up to 17 isolates with E = 0.0 are shown.
basis of the conidia-producing structure, all the isolates were identified as the following fungus. Veronaea botryosa Cif. & Montemart, Atti Ist. bot. Univ. Lab. crittogam. Pavia Ser. 5, 15: 68. Fig. 3, Supplementary data 1. Colony on PDA attaining a diameter of 5.0 cm at 20◦ C, 3wks, low and dense, dark brown (412PC = C53 M56 Y45 K87) to black (C0 M0 Y0 K100) from the surface, concolorous from the reverse; surface velvety, irregularly sulcate; mycelium immersed at the margin, superficial at the center; margin entire. Colony on BHI attaining a diameter of 3.0 cm at 20◦ C, 3 weeks, compact, dark brown (412PC = C53 M56 Y45 K87) from the surface, black (C0 M0 Y0 K100) from the reverse; surface velvety, slightly umbonate at the center; mycelium immersed at the margin, superficial at the center; margin entire. Colony on Miura’s agar attaining a
diameter of 5.0 cm at 20◦ C, 3wks, low and sparse, dark brown (412PC = C53 M56 Y45 K87) from the surface, concolorous from the reverse; surface smooth, slightly floccose due to the development of aerial hyphae at the center; mycelium immersed at the margin, superficial at the center; margin sparsely radiate. Miura’s agar enhanced the conidial production while PDA and BHI were less effective and consequently the following description is based on conidia producing structure on Miura’s agar. Conidiophores macronematous, mononematous, erect, straight or irregularly curved, simple or branched, produced on aerial hyphae 3–4 μm thick tapered at the apex. Conidiogenous cells integrated, polyblastic, terminal or intercalary, sympodial, cicatrized. Conidia mostly two septate, ellipsoid, rounded at the apex, truncate at the base,
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Accession
Code
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two-celled, 6–9 × 4–5 μm (7.7 ± 0.7 × 4.2 ± 0.37 μm on average ± SD, n = 21), dark brown colored, borne solitary on conidiophore. Culture examined. NBRC 109680 (FC-5131), isolated from liver of L. caerulea, dried culture preserved as TNS-F60887; NBRC 109681 (FC-5132), isolated from kidney of L. caerulea; NBRC 109682 (FC-5133), isolated from lung of L. caerulea; NBRC 109683 (FC-5134), isolated from liver of D. guineti; NBRC 109684 (FC-5143), isolated from kidney of D. guineti; NBRC 109685 (FC-5144), isolated from lung of D. guineti. The top 10 candidates resulting from the BLAST search include isolates from various habitats in various countries. Nevertheless, the sequences are very similar in ITS sequences (Table 2). The aligned sequence was composed of 25 sequences with 532 bp, but the sequence of Veronaea musae (GQ184730) was excluded because of its extreme divergence in the alignment, as were short sequences. In the phylogenetic analysis based on small subunit ribosomal RNA sequences [28], V. botryosa was situated in a highly
supported clade, being a sister group of the Exophiala salmonis clade, the majority of which are Exophiala species that are waterborne and invasive in warm-blooded and cold-blooded animals. De Hoog et al. [28] debated that V. botryosa has a strong predilection for human hosts, as supported in various previous case reports [7–10,12– 14,17,29]. However, the facts cumulated do not necessarily suggest the predilection to humans, but instead suggest the versatile ecology of V. botryosa.
Discussion Fungal infection by V. botryosa in frogs Chromomycosis is caused by dematiaceous fungi that usually inhabit soils and plants [9,13,16,28]. Phialophora, Fonsecaea, Scolecobasidium (Scolecosbasidium sensu [6]), and Cladosporium are known to be agents of chromomycosis in amphibians [6,19,21]. In many cases, chromomycosis in frogs and toads is characterized by skin ulcers and multiple white nodules in the liver, spleen, kidneys,
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Figure 2. Neighbor-joining tree based on internal transcribed spacer (ITS1 and ITS2) and 5.8S ribosomal sequences. Veronaea species were outgrouped with Rhinocladiella anceps. Bootstrap values over 80% are shown on the nodes.
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lungs, and other organs [6,20]. Unlike mycotic infection in humans, which is confined to the skin, V. botryosa mycosis in frogs spreads throughout the body, probably the low body temperature allows the rapid growth of the pathogen. Bube et al. [21] reported the transfer of Cladosporium cladosporioides infection to a marine toad (Bufo marinus) from other frogs held in the same water tank. In their paper, the toad had skin lesions, but the frogs showed gross lesions in the brain only. In our case, B. japonicus formosus and D. guineti caused skin lesions but not in L. caerulea. It is difficult to determine the cause of the differences in skin lesions based only on the cases in this report. Such differences might be due to differences in sensitivity in frog species, among other factors. Bube et al. [21] reported different symptoms in the same frog species infected by the same pathogen. We suppose that this is due to differences in the mode of infection, such as an oral infection or percutaneous infection of fungi, rather than the difference of frog species.
In anuran chromomycosis, granulomas including the brown hyphae were the main histopathological changes [6,19–21]. In human V. botryosa infections, the major histological lesions were almost the same as those in anurans, which included granulomas with the brown hyphae [9,13,16]. In the present cases, the histopathological changes were similar in all the animals and were mainly granulomatous lesions, containing brown hyphae. These results are consistent with previously reported cases. We also confirmed the V. botryosa infection by histopathological examination. There has been no report of Veronaea as a pathogen of non-human animals, except for the study by de Hoog et al. [28] in which an isolate of V. botryosa was barely recovered from a frog in Rhode Island Park Zoo, United States. In that report, no information was provided as to the species of frog, the lesions, or was histopathological and mycological examinations provided. To the best of our knowledge, this is the first report of a fatal infection by V. botryosa in anurans, supported by solid morphological and phylogenetic data.
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Figure 3. Veronaea botryosa NBRC 109680. A. Colony on potato dextrose agar (PDA). B. Colony on brain heat infusion (BHI) agar. C. Brown-colored hyphae with septa, constricted at the septa. D–K. Light microscopy of the conidia-producing structure on PDA. D–F. The same conidia-producing structure focused at different optical sections, showing the three-dimensional production of conidia. G. Conidia produced sympodially on the conidiogenous cells. H. Three septate conidia rarely observed on conidiogenous cells. I. Geniculate conidiogenous cells with scars. J. Curved conidiogenous cells with a prominent detachment scar. K. Conidiogenous cell with a detachment scar and pointed apex. This Figure is reproduced in color in the online version of Medical Mycology.
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Possible infection route
Acknowledgments
In spite of its worldwide distribution, the occurrence of V. botryosa in amphibians is limited. In the present case, each water tank (water tanks A, B, and C) employed an independent water circulation system, and they were never exposed to the same farming water. However, the infectivity of this fungus to frogs was very strong, since more than 10 frogs of at least 3 species had died in the same facility in a limited period. We speculate that the spread was mediated by the air or the breeder’s hand, and decontamination may be difficult once the disease is established. Detection of the infection route and a further inoculation study are necessary to evaluate the pathogenicity of V. botryosa. As one of the isolates (CBS 114829) was derived from wood in fresh water, and the occurrence of Veronaea species in watery habitats is known, Czeczuga and Orlowska [30,31] suggested the adaptation of V. botryosa to a watery environment.
This work was partially supported by a research project grant awarded by the Azabu University and Grant-in-Aid for Scientific Research (B) (#23310169), and the integrated research on biodiversity of interspecies relationships in the National Museum of Nature and Science, Japan.
There has been no effective treatment proposed for fungal systemic infections of frogs, except for chytridiomycosis [32]. In the case of the toad infected with Cladosporium cladosporioides, oral treatment with ketoconazole succeeded in the toad regaining its appetite. However, it had to be killed 5 months later, owing to the recurrence of skin lesions [21]. In human, itraconazole [8,11,13], voriconazole [10], and thermotherapy [13] are known to be effective in V. botryosa infections. However, itraconazole- and amphotericin-B-resistant isolates have been reported [11]. In another case study, bathing with povidone-iodine, amphotericin B, or terbinafine did not improve the symptoms during the treatment period [9]. In the present case, the oral administration of itraconazole had no therapeutic effect. Although thermotherapy may be effective against V. botryosa infection in human cases, it is impossible to adopt thermotherapy to frogs because V. botryosa can grow at 35◦ C [10], which is too close to the upper limit survival temperature (36◦ C) for frogs. In addition, in human cases, the lesions are found only in the local and subcutaneous areas whereas systemic and disseminated infections occur in frogs. For the treatment of V. botryosa-infected frogs, voriconazole has been known to be a useful medication for frogs [32] as well as being effective for human V. botryosa infections [10]. In the case of frog mycosis treatment, veterinarians should prepare enough protocols for preventing cross-species contamination to humans.
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
References 1. Takahashi Y, Sano A, Takizawa K et al. The epidemiology and mating behavior of Arthroderma benhamiae var. erinacei in household four-toed hedgehogs (Atelerix albiventris) in Japan. Nihon Ishinkin Gakkai Zasshi 2003; 44(1): 31–38. 2. Ming PX, Ti YL, Bulmer GS. Outbreak of Trichophyton verrucosum in China transmitted from cows to humans. Mycopathologia 2006; 161(4): 225–228. 3. Sitterle E, Frealle E, Foulet F et al. Trichophyton bullosum: a new zoonotic dermatophyte species. Med Mycol 2012; 50(3): 305–309. 4. Weese JS, Peregrine AS, Armstrong J. Occupational health and safety in small animal veterinary practice: Part I - nonparasitic zoonotic diseases. Can Vet J 2002; 43(8): 631–636. 5. Harris JR, Blaney DD, Lindsley MD et al. Blastomycosis in man after kinkajou bite. Emerg Infect Dis 2011; 17(2): 268–270. 6. Anver MR, Pond CL. Biology and diseases of amphibians. In: Fox JG, Cohen BJ, Loew FM, editors. Labortory Animal Medicine. 2nd ed. San Diego, CA: Academic Press, 1984. p. 427–447. 7. Ayadi A, Huerre MR, de Bievre C. Phaehyphomycosis caused by Veronea bothryosa. Lancet 1995; 346(8991–8992): 1703–1704. 8. Foulet F, Duvoux C, de Bievre C et al. Cutaneous phaeohyphomycosis caused by Veronaea bothryosa in a liver transplant recipient successfully treated with itraconazole. Clin Infect Dis 1999; 29(3): 689–690. 9. Matsushita A, Jilong L, Hiruma M et al. Subcutaneous phaeohyphomycosis caused by Veronaea botryosa in the People’s Republic of China. J Clin Microbiol 2003; 41(5): 2219–2222. 10. Sutton DA, Rinaldi MG, Kielhofner M. First US report of subcutaneous phaeohyphomycosis caused by Veronaea botryosa in a heart transplant recipient and review of the literature. J Clin Microbiol 2004; 42(6): 2843–2846. 11. Chen YT, Lin HC, Huang CC et al. Cutaneous phaeohyphomycosis caused by an itraconazole and amphotericin B resistant strain of Veronaeae botryosa. Int J Dermatol 2006; 45(4): 429– 432. 12. Kondo Y, Hiruma M, Matsushita A et al. Cutaneous phaeohyphomycosis caused by Veronaea botryosa observed as sclerotic cells in tissue. Int J Dermatol 2007; 46(6): 625–627. 13. Sang H, Zheng XE, Kong QT et al. A rare complication of ear piercing: a case of subcutaneous phaeohyphomycosis caused by Veronaea botryosa in China. Med Mycol 2011; 49(3): 296–302.
Downloaded from http://mmy.oxfordjournals.org/ at University of Connecticut on June 21, 2015
Possible therapy
Declaration of interest
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23. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1990; 41: 95–98. 24. Tamura K, Stecher G, Peterson D et al. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30(12): 2725–2729. 25. Arzanlou M, Groenewald JZ, Gams W et al. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud Mycol 2007 (58): 57–93. 26. Anonymous. Pantone Color Bridge/Coated. Carlstadt, NJ: Pantone Inc., 2005. 27. Seifert K, Morgan-Jones G, Gams W et al. Genera of Hyphomycetes. Utrecht, The Netherland: CBS-KNAW Fungal Biodiversity Center, 2011. 28. de Hoog GS, Vicente VA, Najafzadeh MJ et al. Waterborne Exophiala species causing disease in cold-blooded animals. Persoonia 2011; 27: 46–72. 29. Nishimura K, Miyaji M, Taguchi H et al. An ecological study on pathogenic dematiaceous fungi in Chiba. In: Miyaji M, Akao M, Kuroda K et al. editors. Proceedings of the Fourth International Symposium of the Research Center for Pathogenic Fungi and Microbial Toxicoses; Tokyo, Japan, February 2–3. Chiba: Research Center for Pathogenic Fungi and Microbial Txicoses, Chiba University, 1989. p. 17–20. 30. Czeczuga B, Orlowska M. Hyphomycetes in the snow from gymnosperm trees. Rocz Akad Med Bialymst 1998; 43: 85–94. 31. Czeczuga B, Orlowska M. Hyphomycetes in the ice of water reservoirs. Rocz Akad Med Bialymst 1999; 44: 64–75. 32. Martel A, Van Rooij P, Vercauteren G et al. Developing a safe antifungal treatment protocol to eliminate Batrachochytrium dendrobatidis from amphibians. Med Mycol 2011; 49(2): 143–149.
Downloaded from http://mmy.oxfordjournals.org/ at University of Connecticut on June 21, 2015
14. Xue YN, Chen H, Hu SQ et al. Cutaneous phaeohyphomycosis on the auricle due to Veronaea botryosa. Eur J Dermatol 2011; 21(3): 418–419. 15. Badali H, Yazdanparast SA, Bonifaz A et al. Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility. Mycopathologia 2013; 175(5–6): 505–513. 16. Bonifaz A, Davoudi MM, de Hoog GS et al. Severe disseminated phaeohyphomycosis in an immunocompetent patient caused by Veronaea botryosa. Mycopathologia 2013; 175(5–6): 497–503. 17. Medina AL, Redondo JAD, Nebrida LM. Two unusual cases of mycoses in the Philippines. In: Society for Mycology, Chinese Society for Microbiology, Japanese Society for Medical Mycology, editors Proceedings of the 4th Chiba Japan International Congress of Mycology; Zhuhai, China, July 17–19. Beijing: International Academic Publishers, 1998: 88. 18. Longcore JE, Pessier AP, Nichols DK. Batrachochytrium dendrobatidis gen et sp nov, a chytrid pathogenic to amphibians. Mycologia 1999; 91(2): 219–227. 19. Cicmanec JL, Ringler DH, Beneke ES. Spontaneous occurrence and experimental transmission of the fungus, Fonsecaea pedrosoi, in the marine toad, Bufo marinus. Lab Anim Sci 1973; 23(1): 43–47. 20. Elkan E, Philpot CM. Mycotic infections in frogs due to a Phialophora-like fungus. Sabouraudia 1973; 11(Jul): 99– 105. 21. Bube A, Burkhardt E, Weiss R. Spontaneous chromomycosis in the marine toad (Bufo marinus). J Comp Pathol 1992; 106(1): 73–77. 22. Hosoya T, Sasagawa R, Hosaka K et al. Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material. Mycoscience 2010; 51(3): 170–181.
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Original Article
First report of Veronaea botryosa as a causal agent of chromomycosis in frogs Tsuyoshi Hosoya1 , Yasuko Hanafusa2,∗ , Tomoo Kudo3 , Kenichi Tamukai4 and Yumi Une3 1
*To whom correspondence should be addressed. Yasuko Hanafusa, Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, 305-0856, Japan. Tel: +81-29-838-7753; Fax: +81-29-838-7880; E-mail: [email protected] Received 31 August 2014; Revised 17 November 2014; Accepted 8 December 2014
Abstract A dematiaceous hyphomycete, isolated from frogs, was determined as the possible etiologic agent of a case of systemic chromomycosis this cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran. Key words: anuran mycosis, disseminated mycosis, zoonosis, phylogenetic analysis, phaeohyphomycosis.
Introduction Zoonoses are infections in animals caused by various microorganisms and have been gaining increasing attention in clinical research. Fungi are among the common agents of such infectious zoonoses in humans and animals. The most common zoonotic mycoses are dermatomycoses [1–3] and systemic infections are rarely reported, except for blastomycosis [4,5]. In most of these cases, the carrier animals are warm-blooded, and case reports involving cold-blooded animal hosts are limited [6]. Human cutaneous phaeohyphomycosis caused by Veronaea botryosa is extremely rare. To the best of our knowledge, only approximately 10 cases have been
reported in English-language literature to date [7–16] and primarily involve farmers who are exposed to soil or plants [8,11,12,17]. However, there was a single case induced by ear piercing [13,14]. It is suggested that the causal fungus may be a saprophyte or plant pathogen, but the details of the ecology of this fungus have not been elucidated. Despite the fact that frogs are naturally distributed worldwide and are one of the most popular cold-blooded pet animals, there are few reports on diseases affecting them. Since the discovery of chytridiomycosis caused by Batrachochytrium dendrobatidis [18], fungal diseases caused by fungi have been drawing public attention. However, this disease is limited to amphibians and does not infect
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Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 3050005, Japan, 2 Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, 305-0856, Japan, 3 Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan and 4 Den-en-chofu Animal Hospital, 2-1-3 Den-enchofu, Ota-ku, Tokyo 145-0071, Japan
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warm-blooded animals. Although disseminated fungal diseases in frogs have also been reported [6,19–21], very few of these papers provide detailed analyses on the fungal pathology and infection routes [19,20]. The present study describes cases of fatal V. botryosa infection in captive frogs. We detail the isolation of the disease-causing fungus from the frogs and describe its mycological features and molecular analysis. We also confirm the V. botryosa infection by histopathological examination of the organs from infected frogs. This is the first English treatise of the occurrence of a lethal V. botryosa infection in animals other than humans. The frog might play an important role in human V. botryosa infection, as a carrier animal.
Pathogen isolation Isolation of the pathogen was attempted from an individual D. guineti and L. caerulea, respectively, but recovery from B. japonicus formosus was not attempted owing to the decomposition of the bodies. Six small pieces (approximately 1 mm × 1 mm × 1 mm) of tissue were cut out from the liver, lung, kidney, and skin, using sterile injection needles. The tissue pieces were inoculated onto potato dextrose agar (PDA; Nissui, Tokyo, Japan) and Sabouraud agar (Nissui, Tokyo, Japan), both containing 50 μg of chloramphenicol, and incubated at 25◦ C. The hyphae arising from the tissue pieces were cut at the tip using a sterilized needle and transferred to a PDA plate, where it was maintained on PDA slants. The isolates were deposited to the Biological Resource Center, National Institute of Technology and Evaluation (NITE-BRC, Japan) as NBRC 109680–109685.
Animals In September 2012, polypoid lesions with ulceration were found on the body surfaces of seven eastern-Japanese common toads (Bufo japonicus formosus) and seven false tomato frogs (Dyscophus guineti), which had all been maintained in an anuran breeder’s water tank in Tokyo, Japan (water tank A). The symptoms did not improve with oral treatment of the antifungal agent itraconazole (10 mg/ml), administrated at 10 mg/kg/day/for 7 days, and within the following 2 weeks, all 14 infected frogs died or were euthanized as a result of the fatal symptoms. Simultaneously, several goldfish, one barking treefrog (Hyla gratiosa), and one White’s treefrog (Litoria caerulea), which had been held together in another water tank (water tank B), died within a week of each other. In addition, a ruby-eyed treefrog (Leptopelis uluguruensis) and an Orinoco lime treefrog (Sphaenorhynchus lacteus) held together in another water tank (water tank C) also died on the following week. To examine the cause of death, the bodies of B. japonicus formosus, D. guineti, and L. caerulea were sent to our laboratories for examination, while those of the other frogs were disposed of. All procedures in the study were in accordance with the guidelines approved by the Azabu University Animal Experiment Committee.
Necropsy and histopathological examination The dead frogs were chilled and necropsied, with tissues from major organs, fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Tissue sections (approximately 4 μm thick) were obtained using routine histological techniques and stained with hematoxylin and eosin (HE) for histopathological evaluation. To investigate the presence of the fungi, the periodic acid Schiff reaction was performed on the tissue sections.
DNA extraction, sequencing, and phylogenetic analysis The DNA extraction, polymerase chain reaction (PCR), and sequencing procedures followed standard methods [22]. The primer pair ITS1 and ITS4 was used to amplify internal transcribed spacer (ITS1 and ITS2) and 5.8S ribosomal sequences. The sequences obtained (AB98173-109680) were searched using the Basic Local Alignment Search Tool (BLAST) on the GenBank database (http://blast.ncbi.nlm. nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome) and were preserved in GenBank. The extracted DNA samples were preserved in the Center for Molecular Biodiversity Research in the National Museum of Nature and Science (Japan), and are available for future collaborative research. The obtained sequences were aligned with Veronaea spp. sequences in GenBank and with other sequences obtained by BLAST search using BioEdit v. 7.2.0 [23], and were analyzed by the neighbor-joining method using MEGA 6.0 [24]. All the parameters were set to default, except for 1,000 times of bootstrap replicates. Following a previous phylogenetic analysis [25], Rhinocladiella anceps (EU041804) was chosen as the outgroup taxon. The alignments used for the phylogenetic analyses were deposited to TreeBASE (http://purl.org/phylo/treebase/phylows/study/ TB2:S16257).
Cultivation of the fungus and its morphological observation The isolates obtained were inoculated onto the center of PDA plates, Miura’s agar (glucose 1 g, KH2 PO4 1 g, MgSO4 ·7H2 O 0.2 g, KCl 0.2 g, KNO3 2 g, yeast extract
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Materials and methods
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Table 1. Distribution of lesions in examined frogs and toads. Lesion distribution on body surface No.
Species
Status
1 2 3 4 5
Bufo japonics formosus
Dead Dead Euthanized Dead Dead
6 7
Dyscophus guineti
8
Litoria caerulea
by macroscopy
Lesion distribution in major organs by histopahological examination∗ Kidney
Lung
Liver
Spleen
Head, legs Legs Legs Head, legs, back Head, legs, back
+ + + + +
+/− + + + +
– + + + +
– + – – +
Dead Dead
Legs, Head, legs,
+ +
+ +
+ +
– –
Dead
None
+
+
+
–
∗
0.2 g, agar 13 g, and distilled water 1000 ml; pH adjusted to 6.5–7.0), or brain heart infusion agar (BHI; Difco, NJ, USA), and incubated at 20◦ C for 3 weeks. Pantone color codes, adopting the CYMK system that refers to a Pantone color bridge, were used for the color description of the colonies [26].
Results Necropsy, histopathological examination, and treatment attempt According to the gross and histopathological examinations, the distribution of lesions and the response to the pathogen differed in each animal (Table 1). Macroscopically, polypoid skin nodule formation, cutaneous ulceration, and necrosis of the epidermis and skeletal muscle were evident in the head, legs, and back of B. japonicus formosus (Fig. 1A). In these toads, multiple white nodules were observed in the kidneys, lungs, liver, and spleen. In D. guineti, skin lesions were observed in the legs and on the head, the kidneys were highly enlarged and discolored (Fig. 1B) and multiple white nodules were observed in the liver and lungs. On the other hand, in L. caerulea, skin lesions were not observed, although multiple white nodules were apparent in the kidneys, lungs, and liver (Fig. 1C). The histopathological changes were similar in all of the animals examined and were found to be consistent with the macroscopic lesions. Granulomatous inflammation was observed in the dermis and skeletal muscle (Fig. 1D, E). In the kidneys, lungs, liver, and spleen, mainly granulomatous lesions were seen (Fig. 1F) and contained brown fungal hyphae (Fig. 1G). In L. caerulea, the granulomatous reaction consisting of giant cells containing brown fungal hyphae
was remarkable. In the present cases, itraconazole did not have any therapeutic effect at all (Table 1).
Isolation of the possible pathogen Hyphal growth from the tissue sections was observed 4 days after inoculation on all isolation media. Since all isolates were similar in the appearance of their colonies and conidia-producing structures, six representative strains were selected (NBRC 109680-109685; see culture examined in the taxonomy section below) and deposited to the NITE-BRC, Japan. All the isolates obtained showed the same colony morphology on slants and plates. Isolation from the skin failed owing to the heavy bacterial contamination not suppressed by the antibiotic.
Taxonomy of the pathogen Sequence lengths of 562–574 bp were obtained from the isolates (sequences registered as AB981713-981718 for NBRC 109680-109685, respectively) and found to be identical in the compared region and concluded to belong to the same species. When NBRC 109680 was used as a representative for BLAST search, sequences that were identical (e.g., AB369905) or highly similar to that of Veronaea botryosa followed by those of other Veronaea species were obtained (Table 2). The result of phylogenetic analysis (Fig. 2) demonstrated that the query sequence was situated among V. botryosa. The morphology of all the isolates was identical to that of the previously described fungi [25,27] and on the
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Lesion means granulomatous changes. +: Histopathological change + and fungi +. +/−: Histopathological change + and fungi –. –: Histopathological change − and fungi –.
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Figure 1. Pathological findings of chromomycosis in frogs. A. Polypoid skin nodule in Bufo japonicus formosus, no. 5. B. The kidneys are highly enlarged and discolored in Dyscophus guineti, no. 6. C. Multiple white nodules are observed in the liver of Litoria caerulea, no. 8. D. Cutaneous granuloma formation with necrosis of the epidermis and skeletal muscle in B. japonicus formosus, no. 5, HE. E. Granulomatous inflammation is observed in the dermis along with brown fungal hyphae, in B. japonicus formosus, no. 5, HE. F. Mycotic granuloma in the liver and brown fungal hyphae, in D. guineti, no. 6, HE. G. Brown fungal hyphae in granulomas in B. japonicus formosus, no. 5, HE. HE stands for hematoxylin and eosin. Bar, 20 μm. This Figure is reproduced in color in the online version of Medical Mycology.
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Table 2. List of basic local alignment search tool search when NBRC 109680 was used as a query sequence. Total score
Query coverage (%)
Max ident (%)
1 2 3 4 5 6 7 8
Veronaea botryosa IFM 5335 Veronaea botryosa CBS 114829 Veronaea botryosa CBS 126027 Veronaea botryosa CBS 127264 Veronaea botryosa CBS 121.92 Veronaea botryosa CBS 350.65 Veronaea botryosa CBS 254.57 Veronaea botryosa CBS 101462
1199 1194 1194 1222 1144 1101 1094 1074
99 100 100 98 95 91 92 89
9
Veronaea botryosa CBS 121506
1038
10
Veronaea botryosa CBS 102593
11
Acc. No.
Country
100 99 99 100 99 100 99 99
AB369905 KC205972 KC205966 JX566723 EU041815 EU041817 EU041816 JF747141
undisclosed Hong Kong undisclosed undisclosed Australia India Italy Philippines
86
100
JF747140
USA
1038
86
100
JF747142
China
Veronaea botryosa CBS 254.57
1035
87
99
JF747143
Italy
12
Veronaea botryosa CBS 122236
1011
86
99
KC205971
Brazil
13
Veronaea botryosa CBS 122825
1000
83
99
KC205967
Brazil
14
Veronaea botryosa CBS 122823
994
84
99
KC205969
Brazil
15
Veronaea botryosa CBS 122824
994
82
100
KC205968
Brazil
16
Veronaea botryosa CBS 122822
977
81
99
KC205970
Brazil
17
Veronaea botryosa CBS 127016
968
81
99
KC205965
undisclosed
Substrate of Isolate Unknown Wood in fresh water
Dead leaf base Dung of goat Sansa olive slag Skin lesion of 37-year-old male patient Wrist (patient from Japan) Ulcerating subcutaneous lesions, disseminated Phaeohyphomycosis of 12-year-old male Railway tie treated with creosote 20 years ago Railway tie treated with creosote 20 years Railway tie treated with creosote 20 years Railway tie treated with creosote 20 years Railway tie treated with creosote 20 years
The results of up to 17 isolates with E = 0.0 are shown.
basis of the conidia-producing structure, all the isolates were identified as the following fungus. Veronaea botryosa Cif. & Montemart, Atti Ist. bot. Univ. Lab. crittogam. Pavia Ser. 5, 15: 68. Fig. 3, Supplementary data 1. Colony on PDA attaining a diameter of 5.0 cm at 20◦ C, 3wks, low and dense, dark brown (412PC = C53 M56 Y45 K87) to black (C0 M0 Y0 K100) from the surface, concolorous from the reverse; surface velvety, irregularly sulcate; mycelium immersed at the margin, superficial at the center; margin entire. Colony on BHI attaining a diameter of 3.0 cm at 20◦ C, 3 weeks, compact, dark brown (412PC = C53 M56 Y45 K87) from the surface, black (C0 M0 Y0 K100) from the reverse; surface velvety, slightly umbonate at the center; mycelium immersed at the margin, superficial at the center; margin entire. Colony on Miura’s agar attaining a
diameter of 5.0 cm at 20◦ C, 3wks, low and sparse, dark brown (412PC = C53 M56 Y45 K87) from the surface, concolorous from the reverse; surface smooth, slightly floccose due to the development of aerial hyphae at the center; mycelium immersed at the margin, superficial at the center; margin sparsely radiate. Miura’s agar enhanced the conidial production while PDA and BHI were less effective and consequently the following description is based on conidia producing structure on Miura’s agar. Conidiophores macronematous, mononematous, erect, straight or irregularly curved, simple or branched, produced on aerial hyphae 3–4 μm thick tapered at the apex. Conidiogenous cells integrated, polyblastic, terminal or intercalary, sympodial, cicatrized. Conidia mostly two septate, ellipsoid, rounded at the apex, truncate at the base,
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Accession
Code
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two-celled, 6–9 × 4–5 μm (7.7 ± 0.7 × 4.2 ± 0.37 μm on average ± SD, n = 21), dark brown colored, borne solitary on conidiophore. Culture examined. NBRC 109680 (FC-5131), isolated from liver of L. caerulea, dried culture preserved as TNS-F60887; NBRC 109681 (FC-5132), isolated from kidney of L. caerulea; NBRC 109682 (FC-5133), isolated from lung of L. caerulea; NBRC 109683 (FC-5134), isolated from liver of D. guineti; NBRC 109684 (FC-5143), isolated from kidney of D. guineti; NBRC 109685 (FC-5144), isolated from lung of D. guineti. The top 10 candidates resulting from the BLAST search include isolates from various habitats in various countries. Nevertheless, the sequences are very similar in ITS sequences (Table 2). The aligned sequence was composed of 25 sequences with 532 bp, but the sequence of Veronaea musae (GQ184730) was excluded because of its extreme divergence in the alignment, as were short sequences. In the phylogenetic analysis based on small subunit ribosomal RNA sequences [28], V. botryosa was situated in a highly
supported clade, being a sister group of the Exophiala salmonis clade, the majority of which are Exophiala species that are waterborne and invasive in warm-blooded and cold-blooded animals. De Hoog et al. [28] debated that V. botryosa has a strong predilection for human hosts, as supported in various previous case reports [7–10,12– 14,17,29]. However, the facts cumulated do not necessarily suggest the predilection to humans, but instead suggest the versatile ecology of V. botryosa.
Discussion Fungal infection by V. botryosa in frogs Chromomycosis is caused by dematiaceous fungi that usually inhabit soils and plants [9,13,16,28]. Phialophora, Fonsecaea, Scolecobasidium (Scolecosbasidium sensu [6]), and Cladosporium are known to be agents of chromomycosis in amphibians [6,19,21]. In many cases, chromomycosis in frogs and toads is characterized by skin ulcers and multiple white nodules in the liver, spleen, kidneys,
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Figure 2. Neighbor-joining tree based on internal transcribed spacer (ITS1 and ITS2) and 5.8S ribosomal sequences. Veronaea species were outgrouped with Rhinocladiella anceps. Bootstrap values over 80% are shown on the nodes.
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lungs, and other organs [6,20]. Unlike mycotic infection in humans, which is confined to the skin, V. botryosa mycosis in frogs spreads throughout the body, probably the low body temperature allows the rapid growth of the pathogen. Bube et al. [21] reported the transfer of Cladosporium cladosporioides infection to a marine toad (Bufo marinus) from other frogs held in the same water tank. In their paper, the toad had skin lesions, but the frogs showed gross lesions in the brain only. In our case, B. japonicus formosus and D. guineti caused skin lesions but not in L. caerulea. It is difficult to determine the cause of the differences in skin lesions based only on the cases in this report. Such differences might be due to differences in sensitivity in frog species, among other factors. Bube et al. [21] reported different symptoms in the same frog species infected by the same pathogen. We suppose that this is due to differences in the mode of infection, such as an oral infection or percutaneous infection of fungi, rather than the difference of frog species.
In anuran chromomycosis, granulomas including the brown hyphae were the main histopathological changes [6,19–21]. In human V. botryosa infections, the major histological lesions were almost the same as those in anurans, which included granulomas with the brown hyphae [9,13,16]. In the present cases, the histopathological changes were similar in all the animals and were mainly granulomatous lesions, containing brown hyphae. These results are consistent with previously reported cases. We also confirmed the V. botryosa infection by histopathological examination. There has been no report of Veronaea as a pathogen of non-human animals, except for the study by de Hoog et al. [28] in which an isolate of V. botryosa was barely recovered from a frog in Rhode Island Park Zoo, United States. In that report, no information was provided as to the species of frog, the lesions, or was histopathological and mycological examinations provided. To the best of our knowledge, this is the first report of a fatal infection by V. botryosa in anurans, supported by solid morphological and phylogenetic data.
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Figure 3. Veronaea botryosa NBRC 109680. A. Colony on potato dextrose agar (PDA). B. Colony on brain heat infusion (BHI) agar. C. Brown-colored hyphae with septa, constricted at the septa. D–K. Light microscopy of the conidia-producing structure on PDA. D–F. The same conidia-producing structure focused at different optical sections, showing the three-dimensional production of conidia. G. Conidia produced sympodially on the conidiogenous cells. H. Three septate conidia rarely observed on conidiogenous cells. I. Geniculate conidiogenous cells with scars. J. Curved conidiogenous cells with a prominent detachment scar. K. Conidiogenous cell with a detachment scar and pointed apex. This Figure is reproduced in color in the online version of Medical Mycology.
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Possible infection route
Acknowledgments
In spite of its worldwide distribution, the occurrence of V. botryosa in amphibians is limited. In the present case, each water tank (water tanks A, B, and C) employed an independent water circulation system, and they were never exposed to the same farming water. However, the infectivity of this fungus to frogs was very strong, since more than 10 frogs of at least 3 species had died in the same facility in a limited period. We speculate that the spread was mediated by the air or the breeder’s hand, and decontamination may be difficult once the disease is established. Detection of the infection route and a further inoculation study are necessary to evaluate the pathogenicity of V. botryosa. As one of the isolates (CBS 114829) was derived from wood in fresh water, and the occurrence of Veronaea species in watery habitats is known, Czeczuga and Orlowska [30,31] suggested the adaptation of V. botryosa to a watery environment.
This work was partially supported by a research project grant awarded by the Azabu University and Grant-in-Aid for Scientific Research (B) (#23310169), and the integrated research on biodiversity of interspecies relationships in the National Museum of Nature and Science, Japan.
There has been no effective treatment proposed for fungal systemic infections of frogs, except for chytridiomycosis [32]. In the case of the toad infected with Cladosporium cladosporioides, oral treatment with ketoconazole succeeded in the toad regaining its appetite. However, it had to be killed 5 months later, owing to the recurrence of skin lesions [21]. In human, itraconazole [8,11,13], voriconazole [10], and thermotherapy [13] are known to be effective in V. botryosa infections. However, itraconazole- and amphotericin-B-resistant isolates have been reported [11]. In another case study, bathing with povidone-iodine, amphotericin B, or terbinafine did not improve the symptoms during the treatment period [9]. In the present case, the oral administration of itraconazole had no therapeutic effect. Although thermotherapy may be effective against V. botryosa infection in human cases, it is impossible to adopt thermotherapy to frogs because V. botryosa can grow at 35◦ C [10], which is too close to the upper limit survival temperature (36◦ C) for frogs. In addition, in human cases, the lesions are found only in the local and subcutaneous areas whereas systemic and disseminated infections occur in frogs. For the treatment of V. botryosa-infected frogs, voriconazole has been known to be a useful medication for frogs [32] as well as being effective for human V. botryosa infections [10]. In the case of frog mycosis treatment, veterinarians should prepare enough protocols for preventing cross-species contamination to humans.
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
References 1. Takahashi Y, Sano A, Takizawa K et al. The epidemiology and mating behavior of Arthroderma benhamiae var. erinacei in household four-toed hedgehogs (Atelerix albiventris) in Japan. Nihon Ishinkin Gakkai Zasshi 2003; 44(1): 31–38. 2. Ming PX, Ti YL, Bulmer GS. Outbreak of Trichophyton verrucosum in China transmitted from cows to humans. Mycopathologia 2006; 161(4): 225–228. 3. Sitterle E, Frealle E, Foulet F et al. Trichophyton bullosum: a new zoonotic dermatophyte species. Med Mycol 2012; 50(3): 305–309. 4. Weese JS, Peregrine AS, Armstrong J. Occupational health and safety in small animal veterinary practice: Part I - nonparasitic zoonotic diseases. Can Vet J 2002; 43(8): 631–636. 5. Harris JR, Blaney DD, Lindsley MD et al. Blastomycosis in man after kinkajou bite. Emerg Infect Dis 2011; 17(2): 268–270. 6. Anver MR, Pond CL. Biology and diseases of amphibians. In: Fox JG, Cohen BJ, Loew FM, editors. Labortory Animal Medicine. 2nd ed. San Diego, CA: Academic Press, 1984. p. 427–447. 7. Ayadi A, Huerre MR, de Bievre C. Phaehyphomycosis caused by Veronea bothryosa. Lancet 1995; 346(8991–8992): 1703–1704. 8. Foulet F, Duvoux C, de Bievre C et al. Cutaneous phaeohyphomycosis caused by Veronaea bothryosa in a liver transplant recipient successfully treated with itraconazole. Clin Infect Dis 1999; 29(3): 689–690. 9. Matsushita A, Jilong L, Hiruma M et al. Subcutaneous phaeohyphomycosis caused by Veronaea botryosa in the People’s Republic of China. J Clin Microbiol 2003; 41(5): 2219–2222. 10. Sutton DA, Rinaldi MG, Kielhofner M. First US report of subcutaneous phaeohyphomycosis caused by Veronaea botryosa in a heart transplant recipient and review of the literature. J Clin Microbiol 2004; 42(6): 2843–2846. 11. Chen YT, Lin HC, Huang CC et al. Cutaneous phaeohyphomycosis caused by an itraconazole and amphotericin B resistant strain of Veronaeae botryosa. Int J Dermatol 2006; 45(4): 429– 432. 12. Kondo Y, Hiruma M, Matsushita A et al. Cutaneous phaeohyphomycosis caused by Veronaea botryosa observed as sclerotic cells in tissue. Int J Dermatol 2007; 46(6): 625–627. 13. Sang H, Zheng XE, Kong QT et al. A rare complication of ear piercing: a case of subcutaneous phaeohyphomycosis caused by Veronaea botryosa in China. Med Mycol 2011; 49(3): 296–302.
Downloaded from http://mmy.oxfordjournals.org/ at University of Connecticut on June 21, 2015
Possible therapy
Declaration of interest
Hosoya et al.
23. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1990; 41: 95–98. 24. Tamura K, Stecher G, Peterson D et al. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30(12): 2725–2729. 25. Arzanlou M, Groenewald JZ, Gams W et al. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud Mycol 2007 (58): 57–93. 26. Anonymous. Pantone Color Bridge/Coated. Carlstadt, NJ: Pantone Inc., 2005. 27. Seifert K, Morgan-Jones G, Gams W et al. Genera of Hyphomycetes. Utrecht, The Netherland: CBS-KNAW Fungal Biodiversity Center, 2011. 28. de Hoog GS, Vicente VA, Najafzadeh MJ et al. Waterborne Exophiala species causing disease in cold-blooded animals. Persoonia 2011; 27: 46–72. 29. Nishimura K, Miyaji M, Taguchi H et al. An ecological study on pathogenic dematiaceous fungi in Chiba. In: Miyaji M, Akao M, Kuroda K et al. editors. Proceedings of the Fourth International Symposium of the Research Center for Pathogenic Fungi and Microbial Toxicoses; Tokyo, Japan, February 2–3. Chiba: Research Center for Pathogenic Fungi and Microbial Txicoses, Chiba University, 1989. p. 17–20. 30. Czeczuga B, Orlowska M. Hyphomycetes in the snow from gymnosperm trees. Rocz Akad Med Bialymst 1998; 43: 85–94. 31. Czeczuga B, Orlowska M. Hyphomycetes in the ice of water reservoirs. Rocz Akad Med Bialymst 1999; 44: 64–75. 32. Martel A, Van Rooij P, Vercauteren G et al. Developing a safe antifungal treatment protocol to eliminate Batrachochytrium dendrobatidis from amphibians. Med Mycol 2011; 49(2): 143–149.
Downloaded from http://mmy.oxfordjournals.org/ at University of Connecticut on June 21, 2015
14. Xue YN, Chen H, Hu SQ et al. Cutaneous phaeohyphomycosis on the auricle due to Veronaea botryosa. Eur J Dermatol 2011; 21(3): 418–419. 15. Badali H, Yazdanparast SA, Bonifaz A et al. Veronaea botryosa: molecular identification with amplified fragment length polymorphism (AFLP) and in vitro antifungal susceptibility. Mycopathologia 2013; 175(5–6): 505–513. 16. Bonifaz A, Davoudi MM, de Hoog GS et al. Severe disseminated phaeohyphomycosis in an immunocompetent patient caused by Veronaea botryosa. Mycopathologia 2013; 175(5–6): 497–503. 17. Medina AL, Redondo JAD, Nebrida LM. Two unusual cases of mycoses in the Philippines. In: Society for Mycology, Chinese Society for Microbiology, Japanese Society for Medical Mycology, editors Proceedings of the 4th Chiba Japan International Congress of Mycology; Zhuhai, China, July 17–19. Beijing: International Academic Publishers, 1998: 88. 18. Longcore JE, Pessier AP, Nichols DK. Batrachochytrium dendrobatidis gen et sp nov, a chytrid pathogenic to amphibians. Mycologia 1999; 91(2): 219–227. 19. Cicmanec JL, Ringler DH, Beneke ES. Spontaneous occurrence and experimental transmission of the fungus, Fonsecaea pedrosoi, in the marine toad, Bufo marinus. Lab Anim Sci 1973; 23(1): 43–47. 20. Elkan E, Philpot CM. Mycotic infections in frogs due to a Phialophora-like fungus. Sabouraudia 1973; 11(Jul): 99– 105. 21. Bube A, Burkhardt E, Weiss R. Spontaneous chromomycosis in the marine toad (Bufo marinus). J Comp Pathol 1992; 106(1): 73–77. 22. Hosoya T, Sasagawa R, Hosaka K et al. Molecular phylogenetic studies of Lachnum and its allies based on the Japanese material. Mycoscience 2010; 51(3): 170–181.
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