Vol. 134: 57–64, 2019 DISEASES OF AQUATIC ORGANISMS Published online April 25 https://doi.org/10.3354/dao03358 Dis Aquat Org

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Epidemic nodular facial myxomatous dermatitis in juvenile Cranwell’s horned cranwelli

Kenichi Tamukai1, Junichi Sugiyama2, Yuta Nagata2, Omatsu Tsutomu3, Yukie Katayama3, Tetsuya Mizutani3, Masanobu Kimura4, Yumi Une5,*

1Den-en-chofu Hospital, 2-1-3 Denenchofu, Ota-ku, Tokyo 145-0071, Japan 2Laboratory of Veterinary Pathology, Azabu University,1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan 3Research and Education Center for Global Infectious Diseases of , Tokyo University of Agriculture and Technology, 3-5-8 Fuchu, Tokyo 183-8507, Japan 4Department of Veterinary Science, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan 5Laboratory of Veterinary Pathology, Okayama University of Science, 1-3 Ikoino-oka, Imabari, Ehime 794-8555, Japan

ABSTRACT: In 2017, approximately 40 out of 100 captive Cranwell’s horned frogs Ceratophrys cranwelli from several facilities in Japan exhibited protruding facial lesions. Histopathological examination was performed on 6 specimens with such lesions randomly selected from 2 facilities. Lesions consisted of scattered stellate to spindle-shaped cells without atypia in an abundant myxoid matrix and occasional lymphocytic infiltrates. Maxillary bone was resorbed. No etiological organisms were detected using light microscopy or metagenomic analysis of the lesions. Macro- scopic and histological assessments indicate that the lesions are associated with nodular facial myxomatous dermatitis, which has never been reported in .

KEY WORDS: Myxomatous · Facial lesions · Pacman · · Morphological

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1. INTRODUCTION Myxoid soft tissue dermal masses include neoplas- tic and viral-induced lesions. Myxomas, which origi- The 8 of Ceratophrys, from the family Cer- nate from fibroblasts or multipotent mesenchymal atophryidae, are found in South America and are gen- cells, are benign soft-tissue tumors that have abun- erally called horned frogs or Pacman frogs. Although dant myxoid matrices rich in mucopolysaccharides. some species are very popular pets, few studies have Myxomas are rare in domesticated animals, typically focused on the diseases that affect these species. The occurring in middle-aged or older dogs and cats most common disorders include nutritional disorders (Hendric 2016). Myxomatosis is a generalized, fulmi- (nutritional hyperparathyroidism complex, obesity, and nant, and frequently lethal disease in rabbits caused corneal lipidosis), the impact of substrates, trauma, by the myxoma virus, a poxvirus that is associated atypical bacterial infections, and chytridiomycosis with the appearance of mucoid subcutaneous tumors (Wright 2001, Une et al. 2008). While tu- on the face (Fenner & Fantini 1994). Cunningham et mors are regularly reported, little is known about tu- al. (1993) reported unusual mortality associated with mors in the Ceratophryidae (Asashima et al. 1987, poxvirus-like particles in European common frogs Green & Harshbarger 2001, Stacy & Parker 2004). Rana temporaria.

*Corresponding author: [email protected] © Inter-Research 2019 · www.int-res.com 58 Dis Aquat Org 134: 57–64, 2019

Healthy and blemish-free animals are important facilities A and B. Subjects were less than 6 mo old from an economic point of view given their value as and had vigor and good appetite. Progressive lesions pets. It is also necessary to elucidate the mode of began to appear from 2 mo after metamorphosis and disease progression in captive populations, which were primarily located in the facial region and max- may provide useful insights for the conservation of illa (Fig. 1). However, the lesions were not fatal if wild populations. Juvenile horned frogs with facial feeding was not impeded. deformities caused by bulbous lesions emerged in several distribution facilities in Japan in 2017. Therefore, the aim of this study was to evaluate 2.2. Animals these pathologic findings and elucidate the mecha- nism of pathogenesis in the lesions through mor- Specimens were housed in plastic containers with a phological, microbiological, and molecular biologi- water depth of 0.5 cm. Each facility used a com- cal examination including metagenomic analyses. pletely different source of well water. The daytime ambient temperature ranged from 23 to 28°C. Speci- mens from facility A were fed commercial food (Pac- 2. MATERIALS AND METHODS man Food; Samurai Japan ). The specimens from facility B and pet shop C were fed captive-bred 2.1. Case history house crickets Acheta domesticus, pinky mice, and goldfish. They were also given small quantities of Horned frogs with deformed faces were found in vitamins and mineral supplements. succession from breeding facility A in Saitama pre- Six specimens were randomly selected from 2 facil- fecture, breeding facility B in Ibaraki prefecture, and ities (4 from breeding facility A [Cases 1−4] and 2 pet shop C in Tokyo in 2017. Cranwell’s horned frogs from pet shop C [Cases 5 and 6]; mean body length: Ceratophrys cranwelli, Argentine horned frogs C. 58 mm [range 41−68 mm]; mean body weight: 23.5 g ornata, Surinam horned frogs C. cornuta, and fantasy [range 8.2−31 g]) (Table 1). horned frogs (C. cranwelli × C. cornuta) were housed All animals were euthanized by spinal cord disrup- in the same facilities and given the same diet tion after being anesthetized by a 5 min immersion in and water; however, only captive-bred Cranwell’s a bath of 0.1% eugenol solution, an anesthetic drug horned frogs were affected. All frogs from both facil- for and crustaceans (FA100, Tamura-Seiyaku). ities A and B were reared without the introduction of Consent from the 2 facilities was obtained for all any new frogs, either from the wild or from captivity. specimens, and they were handled and housed in The prevalence of affected individuals reached up accordance with Azabu University’s ethical guide- to 40% (approximately 40 of 100 frogs) at breeding lines for animal experimentation.

Fig. 1. (A) Lateral view of facial lesions in an affected Ceratophrys cranwelli frog (case 5). The face was notably deformed, and multifocal to coalescing nodules of varying sizes extended around the eyes. The inset shows a section of the lesions. Mucoid liquid was emitted. (B) Appearance of an unaffected frog Tamukai et al.: Epidemic myxomatous dermatitis in horned frogs 59

Table 1. Case characteristics of 6 Ceratophrys cranwelli specimens. (+) positive, (−) negative

Case Body Body Sex PCR testing for pathogens Isolation for Brucella weight length Rana- Brucella Facial Liver Femoral (g) (mm) virus Facial Liver Femoral lesion marrow lesion marrow

1 30.94 67 Male − − − − − + − 2 26.92 58 Male − − − + − + − 3 24.94 51 Male − − − + − + − 4 8.15 41 Male − − − − − − − 5 26.64 68 Unidentified − − − + − − + 6 19.37 61 Unidentified − − + + − − −

2.3. Histopathological examination supplement SR0083 (Oxoid), and incubated for 3 d at 37°C in a shaking incubator (BR-23FP, Taitec). Ali - Facial lesions, including the maxilla bone, were col- quots of the cultures were inoculated on brain−heart lected during necropsy, processed routinely, and em- infusion agar plates, and the plates were incubated bedded in paraffin wax. Sections (4 µm) were stained for 1 d at 37°C (Kimura et al. 2017). with hematoxylin and eosin. Selected sections of the facial lesions were stained with colloidal iron stain, Giemsa stain, Ziehl-Neelsen stain, and immunohisto- 2.6. High-throughput sequencing analysis chemical stain for Brucella spp. Primary antibodies for immunohistochemistry staining were obtained from Eight samples, including 6 from facial lesions and atypical Brucella strains originating from White’s tree 2 from eyes, were analyzed with high-throughput frog Litoria caerulea and Denny’s whipping frog Rha- sequencing. Total RNA was extracted from homo - cophorus dennysi (Kimura et al. 2017). For compari- genate tissue samples with a High Pure Viral Nucleic son, we also performed histological examinations on a Acid Kit (Roche), and contaminated ribosomal RNA normal frog (see Figs. 1B & 3C). was depleted with a RiboMinus Eukaryote Kit for RNA-Seq (Thermo Fisher Scientific). To determine the viral RNA genome, cDNA libraries for high- 2.4. PCR testing throughput sequencing were prepared with a NEB- Next® Ultra™ RNA Library Prep Kit for Illumina To test for ranavirus, total DNA was extracted from (New England Biolabs); sequencing was performed fresh kidney and facial lesion samples using a Nucle - using a MiSeq benchtop sequencer (Illumina) with a oSpin Tissue kit (Macherey-Nagel). Three primer MiSeq Reagent Kit v3 (150 cycles). The obtained sets (M68/M154, MCP4/MCP5, and RanaJP556F/ reads were analyzed using CLC Genomics Work- RanaJP772R) were used to amplify the major capsid bench 6.5.1 (https:// www.qiagenbioinformatics. com/). protein gene (MCP) fragments (Mao et al. 1997, Une The obtained raw reads were trimmed based on et al. 2013). sequence quality. Contigs were assembled from To test for Brucella, total DNA was extracted from trimmed reads, using de novo assembly command, facial lesions, liver, and femoral marrow tissue homo- and analyzed with local BLAST analysis based on genates and from isolated bacterial cells using a virus genome data from the National Center for DNA mini kit (Qiagen). Brucella DNA was detected Biotechnology Information. by PCRs specific for bcsP31, which encodes a cell surface protein, and omp2a, omp2b, and omp31 genes, which encode outer membrane proteins (Ima - 3. RESULTS oka et al. 2007). 3.1. Macroscopic findings

2.5. Culture and isolation of Brucella Exophytic and nodular facial lesions were ob - served on all specimens (Fig. 2). Variably sized, mul- Tissue homogenates were cultured on ATCC tifocal to coalescing nodules extended around the medium 488 broth containing Brucella-selective eyes (Fig. 1A). The face was notably deformed and 60 Dis Aquat Org 134: 57–64, 2019

Fig. 2. Macroscopic findings of facial lesions in Ceratophrys cranwelli specimens. (A−F) Cases 1−6, respectively. Nodular lesions on the face were observed in all specimens. Scale bars = 10 mm verrucous (Fig. 2E,F). In Case 3, the left forelimb also less and transparent mucoid liquid was present had a cyst measuring 25 × 17 mm (Fig. 2C). No super- (Fig. 1A). We did not observe significant changes in ficial ulceration was found in the skin lesions. Tumors the internal organs, eyes, or spinal and appendicular were soft, solid, and elastic. On the cut surface, color- skeleton. Tamukai et al.: Epidemic myxomatous dermatitis in horned frogs 61

3.2. Microscopic findings eye samples. We obtained 900, 835, 949, 830, 960, and 701 contigs from each skin sample and 409 and The facial skin was thickened by coalescing nod- 428 contigs from the ocular samples. No known ules (Fig. 3A,B) due to abundant myxoid matrix genomic sequences of virus pathogens, including expanding the subcutis and disrupting resident con- ranavirus, poxvirus, and herpesvirus, were identified nective tissue. Stellate to spindle-shaped mesenchy- using a local BLAST search. mal cells without atypia were dispersed in the mucous substance. These cells had small nuclei and a moderate amount of chromatin, but nucleoli and 4. DISCUSSION mitosis were not observed (Fig. 3E). The myxoid matrix with subcutaneous facial lesions turned blue The histological features of the present case were with Alcian blue staining and was positive for a col- found to be a myxoid matrix of scattered, stellate to loidal iron reaction (Fig. 3F). No inclusion bodies spindle-shaped cells in subcutaneous facial tissues. were observed in the tissues of any lesion. In Case 3, These findings were mostly consistent with myxoma, flattened epithelial cells lined the inner cyst walls. but an infectious etiology cannot be excluded. Diaz- Skin accessory glands were enlarged to varying Figueroa et al. (2004) reported myxoma in an aged degrees. In addition, scattered lymphocytes, as well ; the tumor was evaluated for as focal lymphocytic infiltration around blood ves- proliferative masses, and the morphological findings sels, and some heterophils were observed in the sub- obtained were similar to those of our study. However, cutaneous tissues at the margins of the facial lesions the case pattern of that study differed from ours in its (Fig. 3D). However, these infiltrations of inflamma- unilateral occurrence, as the lesions in their study tory cells were localized and not observed consis- affected only the oral region and not the epidermis. tently throughout the tissue. There are a few reports of spontaneous neoplasia Resorption was prominent in the region of the max- epidemics in wild amphibians. For instance, renal illa bone that was infiltrated by abundant myxoid ma- adenocarcinoma of the northern leopard frog Litho- trix. Osteoclasts had adhered to some of the existing bates pipiens was induced by Lucké tumor her- bone tissue that had been damaged, but there was pesvirus (McKinnell & Carlson 1997, Davison et al. very little osteoblastic activity (Fig. 3A). Furthermore, 1999). Epidermal papillomas are most frequently there were no circulatory disturbances, such as dilata- found in urodele species. Seasonal cutaneous papillo- tion of peripheral vessels, congestion, or thrombosis. mas have been described in Japanese newts Cynops No etiological organisms were found by Giemsa or pyrrhogaster and are suspected to be virally induced Ziehl-Neelsen staining. Immunohistochemistry stain- (Asashima et al. 1982). ing with 2 groups of Brucella antibodies did not In our study, although the histological findings were detect antigens in the facial lesions. similar to myxomatosis in rabbits, inclusion bodies as- sociated with the presence of a poxvirus were not found, and no known viral sequences were observed 3.3. PCR and Brucella isolation using metagenomic analyses. Brucella has been re- ported in many frog species native to Africa, South Ranavirus-specific DNA was not amplified by PCR and Central America, and Australia. However, the with the appropriate primers. Brucella DNA was role of Brucella infection in amphibians is poorly un- detected in the liver and femoral marrow tissues of 4 derstood (Mühldorfer et al. 2017). In some frog spe- of the 6 specimens using PCR. No Brucella DNA was cies, significant pathologic lesions occurred, such as detected in the facial lesions. Four of 6 bacterial cul- subcutaneous abscesses, spinal arthropathy, and bi- tures tested positive for Brucella. Culture and PCR lateral panophthalmitis (Shilton et al. 2008, Fischer et confirmed the presence of Brucella infections in 5 of al. 2012, Mühldorfer et al. 2017). In our study, Brucella the 6 specimens (Table 1). was detected in 83% of the liver and femoral marrow tissues, in 5 out of 6 specimens. In contrast, Brucella antigens and Brucella DNA were not detected in 3.4. High-throughput sequencing analysis facial lesions by immunohistochemistry and PCR. Moreover, no lesions were observed in the eyes or There were 5 640 466, 5 954 826, 6 551 536, 4 916 508, spine of any specimen. Therefore, the presence of 5 461 348, and 3 483 530 raw reads from each lesion Brucella is deemed to be an incidental finding and an sample, and 4 524 722 and 4 628 164 reads from the unlikely cause of the developing facial lesions. 62 Dis Aquat Org 134: 57–64, 2019

Fig. 3. Photomicrographs of facial lesions from Ceratophrys cranwelli specimens. (A−E) Hematoxylin and eosin-stained sec- tions; (F) colloidal iron stain. (A) Facial skin was significantly thickened by a diffuse nodular myxoid matrix in the subcuta- neous tissue and bone resorption of maxilla. (B) Connective tissue architecture in the lesions was disrupted, leaving small is- lands of collagen and appearing as an obliterated structure with indistinct boundaries with the surrounding tissues (asterisk). (C) Normal frog facial tissue; subcutaneous tissue consisting of dense connective tissue was observed. The maxilla was com- posed of a compact bone matrix containing developed bone cavities. (D) Skin accessory glands were dilated to varying de- grees. Scattered lymphocytes, as well as focal lymphocytic infiltration around blood vessels, and some heterophils were ob- served (arrow). (E) Tumor cells consisted of scattered stellate to spindle-shaped cells without atypia against an abundant myxoid matrix. (F) Myxoid matrix with subcutaneous facial tissue stained blue with Alcian blue and showed a positive colloidal iron reaction (Case 6). Scale bars = (A−C) 500 µm, (D) 50 µm, (E,F) 200 µm Tamukai et al.: Epidemic myxomatous dermatitis in horned frogs 63

There are many possible causes for the develop- LITERATURE CITED ment of cutaneous myxomas or tumor formation of Asashima M, Komazaki S, Satou C, Oinuma T (1982) Sea- the face. In humans, Carney complex is a rare, domi- sonal and geographical changes of spontaneous skin nantly inherited syndrome (Bertherat 2006) charac- papillomas in the Japanese newt Cynops pyrrhogaster. terized by spotty skin pigmentation, endocrine over- Cancer Res 42: 3741−3746 activity, and myxomas. Cutaneous myxomas are Asashima M, Oinuma T, Meyer-Rochow VB (1987) Tumors in Amphibia. Zool Sci 4: 411−425 present as non-pigmented subcutaneous nodules. Bertherat J (2006) Carney complex (CNC). Orphanet J Rare Environmental factors, carcinogenic drugs, miscella- Dis 1: 21 neous toxic substances, and genetics are reported to Cunningham AA, Langton TE, Bennett PM, Drury SE, be causative factors for myxoma in dogs (Rani et al. Gough RE, Kirkwood JK (1993) Unusual mortality associ- 2013). The devil facial tumor disease is caused by ated with poxvirus-like particles in frogs (Rana tempo- raria). Vet Rec 133:141−142 clonal transmissible cancers that have led to a cata- Davison AJ, Sauerbier W, Dolan A, Addison C, McKinnell strophic decline in the wild Tasmanian devil Sar- RG (1999) Genomic studies of the Lucké tumor her- cophilus harrisii population (Flies et al. 2016). Tiger pesvirus (RaHV-1). J Cancer Res Clin Oncol 125: salamanders Ambystoma tigrinum inhabiting a sew - 232−238 Diaz-Figueroa O, Mitchell MA, Kim DY, Riggs SR (2004) age sedimentation lagoon developed more neoplas- Oral myxoma in a South American horned frog (Cer- tic skin lesions (e.g. papillomas, fibromas, myxofibro- atophrys ornata, ). Proc Assoc Rep Amph mas, fibrosarcomas, and malignant melano mas) than Vet, Naples, FL, May 8–11, 2004, p 160−161 those in uncontaminated lagoons (Rose & Harsh- Fenner F, Fantini B (1994) Myxomatosis. In: Thompson HV, barger 1977). Myxomas are rarely diagnosed in fish, King CM (eds) The European rabbit. The history and biology of a successful colonizer. Oxford University and the cause of the tumors has not been determined Press, Oxford, p 205–235 (Gjur

management of an uncommon cutaneous myxoma in a Une Y, Kadekaru S, Tamukai K, Goka K, Kuroki T (2008) dog. Int J Adv Vet Sci Tech 2:72−75 First report of spontaneous chytridiomycosis in frogs in Rose FL, Harshbarger JC (1977) Neoplastic and possibly Asia. Dis Aquat Org 82: 157−160 related skin lesions in neotonic tiger salamanders from a Une Y, Kudo T, Tamukai T, Murakami M (2013) Epidemic sewage lagoon. Science 196: 315−317 ranaviral disease in imported captive frogs (Dendrobates Shilton CM, Brown GP, Benedict S, Shine R (2008) Spinal and Phyllobates spp.), Japan, 2012: a first report. JMM arthropathy associated with Ochrobactrum anthropi in Case Rep 2014: jmmcr.0.001198 free-ranging cane toads (Chaunus [Bufo] marinus) in Wright KM (2001) Nutritional disorders. In: Wright KM, Australia. Vet Pathol 45: 85−94 Whitaker BR (eds) Amphibian medicine and captive Stacy BA, Parker JM (2004) Amphibian oncology. Vet Clin husbandry. Krieger Publishing Company, Malabar, FL, North Am Exot Anim Pract 7: 673−695 p 73–87

Editorial responsibility: Stephen Feist, Submitted: June 7, 2018; Accepted: February 10, 2019 Weymouth, UK Proofs received from author(s): April 10, 2019