FULL PAPER Internal Medicine

Treatment of Spontaneous Chytridiomycosis in Captive Using Itraconazole

Kenichi TAMUKAI1,2), Yumi UNE2)*, Atsushi TOMINAGA3), Kazutaka SUZUKI4) and Koichi GOKA4)

1)Den-en-chofu Hospital, 2–1–3 Denenchofu, Ota-ku, Tokyo 145–0071, 2)Laboratory of Veterinary Pathology, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252–5201, 3)Faculty of Education Lifelong Education Program, University of the Ryukyu, 1 Senbaru, Nishihara, Okinawa 901–0213 and 4)National Institute for Environmental Studies, 16–2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan

(Received 14 June 2010/Accepted 31 August 2010/Published online in J-STAGE 14 September 2010)

ABSTRACT. We performed this study in order to establish an effective, simple and safe treatment for chytridiomycosis. The subjects were 12 amphibians (11 anurans of 4 different species and 1 urodela) diagnosed with chytridiomycosis by clinical signs and a PCR test. A 0.01% aqueous solution of the antifungal agent itraconazole was used to treat the subjects, and we evaluated the efficacy of treatment by 3 methods: clinical signs, direct microscopy and a nested PCR test. A 10-min immersion in a 0.01% aqueous solution of itraconazole every other day for a total of 7 treatments resulted in an improvement of clinical signs in 11 of the 12 cases. Specifically, we observed an abatement of increased sloughing and disappearance of zoosporangia by direct microscopy. DNA fragments of Batrachochytrium dendrobatidis were not detected by a PCR test at the end of treatment, nor were they detected after treatment (20–57 days following treatment; average, 34.4 days). No recurrence was observed 12 months after the end of treatment, nor did we observe any obvious side effects from itraconazole. Therefore, we recommend this as a treatment method for chytridiomycosis and as an elimination technique for use in captive amphibians. KEY WORDS: , Batrachochytrium dendrobatidis, chytridiomycosis, itraconazole. J. Vet. Med. Sci. 73(2): 155–159, 2011

Chytridiomycosis is an emerging fungal disease that treatment and elimination techniques is particularly impor- infects amphibians and is caused by Batrachochytrium den- tant for conservation of species such as those typified in the drobatidis. It is an important disease from the standpoint of Amphibian Ark project created by the International Union conservation medicine for wild animal and is listed in the for Conservation of Nature and Natural Resources (IUCN) obligatory Aquatic Animal Health Code published by The [4]. In this study, we investigated treatments targeting clin- World Organization for Animal Health (OIE), as it contin- ically symptomatic spontaneous chytridiomycosis using the ues to devastate wild amphibian populations [1, 14, 23]. antifungal agent itraconazole and established an effective B. dendrobatidis is a fungus first described in 1999 that protocol. belongs to the Chytridiomycetes class of the Chytridiales order of fungi [16]. The primary life stages of B. dendroba- MATERIALS AND METHODS tidis include 2 phases, a flask-shaped zoosporangia 6–15 μm in diameter, and a flagellated zoospore that resides in aque- The research were amphibians admitted to a vet- ous environments [16]. B. dendrobatidis only infects the erinary hospital with clinical symptoms consistent with keratinous layer of the stratum corneum in amphibians and chytridiomycosis such as increased sloughing, miosis and causes skin hyperplasia and hyperkeratosis [1, 4]. Amphib- anorexia (Table 1, Figs. 1 and 2). They included 11 anurans ian skin regulates transport for water, electrolytes and respi- (hereafter, ) of 4 different species (4 ratory gases [7]. As a result, when B. dendrobatidis infects laevis, 3 Ceratophrys spp., 3 Chacophrys pierotti and 1 Lep- and multiplies in the epidermal stratum corneum, it causes todactylus pentadactylus) and 1 urodela (Ambystoma tigri- extensive damage to the skin, reducing skin function and num) for a total of 12 animals of 5 different species. The causing electrolyte imbalances that can lead to death [25, ages of the animals were not known; their body weights are 26]. shown in Table 1. All had been bred in captivity and indi- The first cases of chytridiomycosis in Asia were com- vidually housed. We definitively diagnosed the disease as firmed in captive amphibians in Japan in 2006, with some of chytridiomycosis based on detection of DNA fragments the cases resulting in death [24]. Although there have been specific to B. dendrobatidis by a polymerase chain reaction many reports of chytridiomycosis in wild amphibians [1, 5, (PCR) test and observation of fungal zoospores by direct 11, 15], clinical information regarding symptoms and treat- microscopy. PCR tests for B. dendrobatidis were performed ment in captive amphibians is limited. Establishment of using samples attained by swabbing the animal’s ventral surface, inner thigh and toepads with cotton swabs (Men-tip *CORRESPONDENCE TO: UNE, Y., Laboratory of Veterinary Pathol- ogy, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagami- 1P1501, Nihon-Menbo Co., Tokyo, Japan). The PCR tests hara, Kanagawa 252–5201, Japan. were performed at 3 time points: at the time of initial medi- e-mail: [email protected] cal examination, on completion of treatment and during a 156 K. TAMUKAI ET AL.

Table 1. Clinical signs and examination data for the 12 amphibians diagnosed with chytridiomycosis Test at Body Clinical signs Pretreatment tests end of Retest Case Species weight treatment no. (g) Increased Direct PCR test Miosis Anorexia Other PCR test PCR test sloughing method (dayb)) posterior 1 Lepidobatrachus laevis 142 + PNDN N (29) paresis 2 Lepidobatrachus laevis 129 + P P N N (30) 3 Lepidobatrachus laevis 120 + P ND N N (29) 4 Lepidobatrachus laevis 86 + P P N N (30) 5 Ceratophrys ornata 68 + + P P N N (30) 6 Ceratophrys sp. 55 + + P ND N N (29) 7 Ceratophrys ornataa) 15 + + + Lethargy P P N Generalized 8 Chacophrys pierotti 80 + + PP NN (31) edema 9 Chacophrys pierotti 78 + + + P ND N N (29) 10 Chacophrys pierotti 68 + + P N N N (30) Increased 11 Leptodactylus pentadactylus 31 + + + body surface P ND N N (20) sheen Dark 12 Ambystoma tigrinum 111 + + discoloration P N N N(57) of the skin +: Present. P: Positive. N: Negative, ND: Not determined. a): Died on the 2nd day of treatment. b): Number of days from end of treatment until retesting. retest at a later point. Swab samples were stored in micro- following treatment. Treatment efficacy was determined tubes at –28°C until use in PCR tests. B. dendrobatidis based on improvement of clinical signs (principally a DNA was extracted by soaking and agitating swab samples reduced rate of sloughing) and by the absence of B. dendro- in lysis buffer (1 mg/ml proteinase K, 0.01 M NaCl, 0.1 M batidis DNA fragments on PCR testing and fungal zoospo- EDTA, 0.01 M Tris-HCl, 0.5% Nonidet P-40). Samples rangia on direct microscopy. were allowed to react for 120 min at 50°C and at 95°C for 20 min and were then diluted in TE buffer (10 mM Tris-HCl, 1 RESULTS mM EDTA). For PCR testing, we performed a nested-PCR test using a primer from the B. dendrobatidis ITS gene Before treatment, the nucleotide sequences of the PCR region, as described by Goka et al. [10]. PCR products were products in all 12 cases were consistent with the ITS1-5.8S- separated on 6% polyacrylamide gels, and bands of DNA ITS2 regions already registered in the DNA Data Bank of fragments were visualized by means of ethidium bromide Japan (A haplotype, accession number AY997031) [10]. staining under UV light. In addition, we performed direct Eleven frogs of the 12 amphibians examined presented sequencing on the PCR products. with abnormally increased sloughing. At the beginning of For direct microscopy, we stretched pieces of unstained treatment, large amounts of sloughed skin were found float- sloughed skin over a glass slide, covered them with a cover ing in their cages (Table 1, Fig. 1), and when sloughed skin glass and confirmed the presence of zoosporangia in the was directly examined under a microscope, zoosporangia sloughed skin (Fig. 3) [12, 28]. were observed in botryoidal clusters (Fig. 3). One , We prepared a 0.01% aqueous solution of itraconazole as Case 7, which had the lowest body weight and serious clini- a treatment for chytridiomycosis by diluting itraconazole cal signs, died 2 days after the beginning of treatment. (Itrizole Oral Solution 1%, Janssen Pharmaceutical K.K., However, after the 5th day (3rd round) of treatment, the Tokyo, Japan) with purified water. The solution was poured amount of sloughed skin in the habitats of the remaining 10 into a 230 × 155 × 150-mm plastic container to a depth of 2 frogs was decreased and improvements were seen in clinical cm. The animals were manually moved from their cages, signs such as miosis. In addition, a decrease in zoosporan- using gloves, to the plastic container, and the solution was gia infection density concurrent with the increase in number applied to their entire bodies using a ladle. They were then of treatments was observed via direct microscopy. No left briefly in the medicinal bath. In the treatment protocol zoosporangia were found in any of the frogs at the comple- used, treatments were administered every other day for a tion of treatment. There was also no evidence of itracona- total of 7 treatments, each lasting 10 min. We confirmed zole-related side effects such as depigmentation. Only one changes in the clinical signs of the animals both during and urodela, Ambystoma tigrinum (Case 12), presented without TREATMENT OF SPONTANEOUS CHYTRIDIOMYCOSIS 157

Fig. 1. Large amounts of sloughed skin in the water of an affected frog (Case 5. Ceratophrys ornata). Fig. 2. Mitosis and generalized edema in an affected frog (Case 8. Chacophrys pierotti). Fig. 3. Botryoidal clusters of B. dendrobatidis zoosporangia in unstained sloughed skin under direct microscopy. Bar=10 μm (Case 5. Ceratophrys ornata). Fig. 4. Dark discoloration of the skin before treatment (Case 12. Ambystoma tigrinum ). Fig. 5. Normal skin pigmentation after treatment (13th day; Case 12. Ambystoma tigrinum). Note the yellow spots on the skin.

increased sloughing; however, dark discoloration of the skin animals are currently in good health with no evidence of was observed (Fig. 4). This animal recovered its normal chytridiomycosis recurrence. pigmentation following completion of treatment (Fig. 5). PCR tests performed on all 11 surviving animals on comple- DISCUSSION tion of treatment and again after completion of treatment (20–57 days following treatment; average, 34.4 days) iden- B. dendrobatidis is believed to be involved in the tified no B. dendrobatidis genes in any of the animals (Table decrease in the number of species and populations of wild 1). Of course, no zoosporangia were observed by the direct amphibians in areas around the world. As a result, research method either. At the time of writing, over 12 months had on B. dendrobatidis in wild amphibians and its ecology as a passed since completion of treatment; all of the surviving pathogen is being actively pursued [13, 17, 18], though very 158 K. TAMUKAI ET AL. few studies have targeted captive amphibians. Recently, ment), we concluded that the method used was extremely activities for protection and breeding of endangered effective. Itraconazole is a lipid-soluble triazole compound amphibian species in captivity have become more common. used in veterinary medicine to treat fungal infections such as The present study was conducted in order to establish an blastomycosis, histoplasmosis, aspergillosis, candidosis, effective, simple and safe treatment targeting clinical cases cryptococcal infection and fungal keratitis [16]. It inhibits of chytridiomycosis in various amphibian species. the cytochrome P450 enzyme system, which produces There are several reported methods for B. dendrobatidis ergosterol, a main component of fungal cell membranes elimination during quarantine of newly obtained individu- [22]. Because the principle sterol in the genus Batra- als. These methods include elimination of B. dendrobatidis chochytrium is also ergosterol [6], we surmised that itracon- by placing Litoria chloris in a 37°C environment [27], as B. azole, by the same mechanism of action, would have a dendrobatidis ceases propagation and is inactivated at tem- similar antifungal effect on B. dendrobatidis . peratures over 28°C [21], and the use of formalin and mala- Side effects of itraconazole treatment reported in amphib- chite green, which are reportedly effective on Xenopus ians include skin depigmentation in Alytes muletensis tad- tropicalis [20]. However, these methods are difficult to use poles; the possibility of hepatotoxicity has also been with amphibians that are not suited to high-temperature suggested [9]. In the present study, there was no evidence of environments, particularly urodeles, and malachite green is depigmentation in any of the animals, and physical exami- considered to be carcinogenic. A treatment method using nation did not reveal any apparent side effects. However, chloramphenicol for Leiopelma archeyi found to be positive we did not perform detailed clinical examinations such as for chytridiomycosis on PCR testing has recently been blood tests, and because potential toxicities are not well reported [3]. However, the mechanism by which chloram- understood, we believe that sufficient observation is phenicol affects B. dendrobatidis is not clear, and its effi- required when using this medication. cacy on amphibians that have already become symptomatic Case 7 died on the 2nd day of treatment. As we were and worsened under standard conditions remains uncertain. unable to perform pathological testing, the exact cause of Itraconazole is effective against B. dendrobatidis in vitro death was not confirmed, but we conclude that it was [2]. In both a study by Nichols et al. [19] using poison dart chytridiomycosis based on the fact that no depigmentation frogs experimentally infected with B. dendrobatidis and a following treatment with itraconazole was observed; it had study by Forzán et al. [8] examining quarantine methods for the lowest body weight of all the test animals (15 g), sug- amphibians in an aquarium following a widespread out- gesting that it was a juvenile; and its clinical signs closely break of chytridiomycosis, treatment consisting of 5 min- resembled end-stage chytridiomycosis with severely utes immersion in a 0.6% saline bath containing 0.01% increased sloughing and lethargy. itraconazole for 11 consecutive days succeeded in eliminat- In the present study, we proposed a treatment and elimi- ing B. dendrobatidis. In addition, treatment with flucona- nation protocol targeting spontaneously occurring chytridio- zole, another azole antifungal agent similar to itraconazole, mycosis in 5 different species of amphibians (including 1 in frogs experimentally infected with B. dendrobatidis urodela) and confirmed the efficacy of the protocol using a resulted in death in all cases, although survival was pro- highly sensitive B. dendrobatidis detection method and longed [2]. The method used in the present study was based long-term follow-up. To our knowledge, no such method on the methods used by Nichols et al. and Forzán et al. [8, has been reported previously. We believe that this treatment 19], in which a 0.01% aqueous solution of itraconazole method will be extremely useful in the preservation of without NaCl was used as a bath. In addition, to reduce endangered amphibian species. stress induced by the treatment and the effect of the medica- tion, we developed a protocol with a reduced number of ACKNOWLEDGMENTS. We are especially grateful to treatments. Although efficacy of elimination was confirmed Yuko Ichimaru for providing valuable specimens. This histopathologically in the studies by Nichols et al. and study was supported by the Global Environmental Research Forzán et al. [8, 19], we decided to adopt a nested PCR test Fund (F-3 and F-081, 2008, Leader: K. Goka) of the Minis- with a higher detection sensitivity in order to definitively try of the Environment, Japan, by the Promotion and Mutual determine the elimination efficacy of this treatment. The Aid Corporation for Private Schools of Japan, Grant-in-Aid nested PCR tests were 10 to 100 times more sensitive than a for Matching Fund Subsidy for Private University, and by a single-round PCR test and real-time TaqMan PCR assay, Grant-in-Aid for Scientific Research C (20580347). and could detect specific fragments with as little as 0.001 pg (=1 fg) of fungal DNA in the second amplification [10]. REFERENCES The method in the present study resulted in successful treatment and elimination in all 11 subjects completing the 1. Berger, L., Speare, R., Daszak, P., Green, D. E., Andrew, A. protocol. Efficacy was confirmed by post-treatment retest- C., Goggin, L., Slocombe, R., Mark, R. A., Hyatt, A. D., Keith, ing (average of 34.4 days post-treatment), and no recurrence R. M., Hines, H. B., Lips, K. R., Marantelli, G. and Parkes, H. was observed, even after 12 months. 1998. 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