Case report Pathology

A Suspected Case of Ceti in a Male Aquarium-maintained Pacific White-sided Dolphin（Lagenorhynchus obliquidens）in Japan

Tomoko MINAKAWA1）, Keiichi UEDA2）, Ayako SANO3）, Haruka KAMISAKO2）, Mikuya IWANAGA1）, Takeshi KOMINE1） and Shinpei WADA1 ）＊

1） Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-Cho, Musashino, Tokyo 180-8602, Japan 2） Okinawa Churashima Foundation, 888 Aza Ishikawa, Motobu-Cho, Kunigami-Gun, Okinawa 905-0206, Japan 3） Department of Animal Sciences, Faculty of Agriculture, University of the Ryukyus, 1 Sembaru, Nishihara-Cho, Nakagusuku-Gun, Okinawa 903-0213, Japan

［Received 21 March 2017; accepted 1 June 2017］

ABSTRACT A Pacific white-sided dolphin diagnosed with suspected paracoccidioidomycosis ceti has suffered clinical manifestations since September 2008. Skin biopsy samples were examined microbiologically, pathologically, and with molecular biology. However, we detected no clear evidence of infection other than multiple budding- cells in a skin stamp smear. The lesion improved after the oral administration of and topical treatment with an ointment containing amphotericin B powder. These results imply that some fungal agents might be involved in the pathogenesis of the present case. Key words: Pacific white-sided dolphin, paracoccidioidomycosis ceti － Jpn. J. Zoo. Wildl. Med. 23（2）：45-50，2018

Japan is an endemic area for paracoccidioidomycosis covering most of the area of the tail fluke, the right side of the ceti (PCM-C), with three confirmed cases, two in bottlenose body, and the ventral side of the keel by August 2016 (Fig. dolphins (BDs, Tursiops truncatus) [1] and one in a Pacific 1a, b). The individual foci were 1–2 cm in diameter, although white-sided dolphin (PWSD, Lagenorhynchus obliquidens) [2]. the major axes of some fused lesions were up to 15 cm. The Molecular biological data are essential for the diagnosis of dolphin was captured off the Japan Sea coast in 1996, and PCM-C, so its diagnosis is sometimes difficult. In the present was estimated to have been more than 25 years old at the end case, a PWSD with suspected PCM-C developed clinical of August 2016. Its bodyweight was 121.0 kg in April 2016. manifestations and cytological features similar to those of Topical treatments with ointments containing antimicrobial PCM-C. The male aquarium-maintained PWSD had multiple agents, including gentamicin sulfate (Takeda Pharmaceutical lesions, consisting of whitish-gray dermal tubercles, on the tip Co. Ltd., Osaka, Japan), oxytetracycline hydrochloride (Yoshindo of the right side of its tail fluke, since September 2008. This Inc., Toyama, Japan), and polymyxin B sulfate (Yoshindo Inc.), was similar to a previous case of PCM-C in a PWSD [2] and and steroids, such as hydrocortisone (Yoshindo Inc.) and to a nontuberculous mycobacterial infection in a white whale betamethasone valerate (Shionogi & Co. Ltd., Osaka, Japan), (Delphinopterus leucas) [3]. The lesions gradually increased, were administered at least a few days. Povidone iodine (Meiji fusing with one another to give a keloidal appearance, and Seika Pharma Co. Ltd., Tokyo, Japan), gentian violet (Kishida Chemical Co. Ltd., Osaka, Japan), and hydrogen peroxide (Showa Seiyaku Co. Ltd., Toyohashi, Japan) were also applied to the * Corresponding author：Shinpei WADA（E-mail: [email protected]） lesions as disinfectants. Although these clinical treatments

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TM on potato dextrose agar (potato dextrose agar, Difco , Becton 㻌a Dickinson and Company, Sparks, MD, USA) supplemented

with 100 mg/L of chloramphenicol (Wako Chemicals Co. Ltd., Osaka, Japan), Mycosel agar (BBLTM Mycosel agarTM, Becton TM Dickinson and Company), and 1 % yeast extract (Difco , Becton Dickinson and Company), and 1 % glucose (Wako

Chemicals Co. Ltd.) added brain heart infusion agar (DifcoTM, Becton Dickinson and Company). The agar plates were incubated at 25 and 35℃ for 4 weeks. Then, stamp smears were performed with the residue from the fresh skin samples,

and these samples were stained with Giemsa, periodic acid- Schiff reaction (PAS), and/or Gomori methenamine silver nitrate stain (GMS) for fungi. b㻌 After these procedures, each sample was divided into four

small blocks. One was fixed in 10 % phosphate-buffered formalin solution, one in 70 % ethanol, and one in absolute ethanol, all immediately after sampling. The other block was stored in a freezer ( −20℃ ) for up to 48 h, and transported to

the laboratory. The frozen skin tissues were thawed and treated with 1N HCl according to Eddyani et al. [4] and/or 1N NaOH according to Yajko et al. [5] for decontamination. Then, each sample

was inoculated on 2 % Ogawa slant (Kyokuto, Tokyo, Japan), Middlebrook 7H11 agar with oleic acid-albumin-dextrose- Fig. 1 Granulomatous skin lesions (arrows) on the tail fluke catalase complex (OADC, DifcoTM, Becton Dickinson and (a) and ventral side of the keel (b) in July 2016. Company) enrichment and Middlebrook 7H9 broth (DifcoTM, Becton Dickinson and Company) for NTM cultivation. All of were continued for several months, the skin lesions did not these media were incubated at 30℃. improve. For molecular analysis of NTM, genomic DNA was extracted Two punch biopsies (9 mm in diameter) of the dermal from each skin sample fixed with absolute ethanol using lesions were collected from the tail flukes, and three excisions QIAamp DNA Mini kit (QIAGEN, Venlo, Netherlands) after were made with a surgical blade to obtain small tissue blocks performing the freezing-boiling method according to Fukano (around 0.25 cm2 and 1.0 cm in depth) from the right body et al. [6]. PCR analysis was performed targeting the 16S rRNA surface and the ventral side of the keel, under infiltration gene, the 65-kDa heat shock protein (hsp65) gene, and the anesthesia induced with lidocaine hydrochloride supplemented RNA polymerase β-subunit (rpoB) gene. The primers used with 2 % adrenaline (AstraZeneca K.K., Osaka, Japan) in August for this analysis [6] are listed in Table 1. PCR analysis was and October, 2016. The biopsied tissue samples were divided conducted with GoTaq DNA polymerase (Promega, Madison, into small pieces to culture any fungi or nontuberculous WI, USA) with the following amplification conditions: 95℃ for mycobacteria (NTM), and for cytological studies of stamp 10 min, 35 cycles of 95℃ for 40 sec, 55℃ for 40 sec, 72℃ for smears on glass slides, histopathology, and molecular biological 90 sec, and a final cycle of 72℃ for 3 min. studies. The operative wounds were treated with an ointment For molecular analysis of Paracoccidioides sp., detection of including gentamicin sulfate. the 43-kDa glycoprotein coding gene (gp43) was carried out For mycological examinations, smaller pieces of dermal on the biopsied samples fixed with 70 % ethanol, according to tissue were removed from the fresh samples, and inoculated Ueda et al. [1]. After purification by an ethanol precipitation

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Dermatitis in a Pacific White-sided Dolphin

Table 1 PCR primer sets for detection of nontuberculous mycobacteria used in this study. Genes Primers Sequences (5′–3′) 16S rRNA 8F AGAGTTTGATCCTGGCTCAG 1047R TGCACACAGGCCACAAGGGA

830F GTGTGGGTTTCCTTCCTTGG 1542R AAGGAGGTGATCCAGCCGCA

hsp65 Tb11 ACCAACGATGGTGTGTCCAT Tb12 CTTGTCGAACCGCATACCCT rpoB MycoF CGCCACTTCGGCAACCG 㻌 MycoR TCGATCGGGCACATCCGG 20 µm

Fig. 2 Chains of spherical budding-yeast-like structures method, the DNA solutions were subjected to nested PCR to (arrows) stained with Gomori methenamine silver nitrate stain amplify the gp43 gene using the primer set MAE (5′ -TGC TGC observed in the stamp smears of dermal lesions. Bar=20µm. GGC GGG GTT AAA CCA TGT C-3′) and ATO (5′ -GTT GTG GTA TGT GTC GAT GTA GAC G-3′) for the first round of PCR, and the primer set SUM F1 (5 ′ -GTC ATC GAT CTC CAT GGT GTT a AAG-3′ ) and SUM R2 (5′ -GGC AGA RAA GCA TCC GAA A -3′ ) for the second round, using the same protocol as our previous report [2]. The fixed samples with 10 % phosphate-buffered formalin solution were processed routinely to prepare paraffin sections 4 to 5 µm thick for histopathology. The sections were stained with hematoxylin and eosin (H&E). Some other histological staining methods including the Ziehl-Neelsen (ZN) stain for acid-fast bacteria, PAS, and/or GMS, were also performed with the sections. No microorganisms were detected with culture, histopathology, or molecular biology. However, the cytological analysis revealed typical fungal elements consisting of chains b of spherical budding-yeast-like structures, 15–25 µm in diameter, which stained positively with PAS and GMS (Fig. 2). The morphological features of these structures were quite similar to those described in the previous cases of Paracoccidioides brasiliensis infection diagnosed by molecular analyses of the gp43 gene [2, 7]. The characteristics of PCM-C, formerly called “lacaziosis,” “Jorge Lobo’ s disease,” or “lobomycosis” [8], include chronic keloidal skin lesions with or without visceral involvement [1, 2, 8-11]. Taborda et al. [12] classified the causative agent of lacaziosis as loboi in 1999, and the name “lacaziosis” Fig. 3 Improved skin lesions (arrows) on the tail fluke has been used preferentially for both human and cetacean (a) and ventral side of the keel (b) in January 2017, after administration of agents. cases [13]. In the latest report of Vilela et al. [7], the name

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Tomoko MINAKAWA et al.

“PCM-C” is proposed for cetacean lacaziosis caused by an Bristol Myers, Squibb, Tokyo, Japan), three times daily, uncultured Paracoccidioides sp., based on a phylogenetic referring to Murata et al. [21]. We applied these dosages of the analysis of multiple genes and some partial gene sequences: antifungal agents to a previous case of PCM-C in PWSD [2], kexin coding gene (Kex), chitin synthase 4 (CHS4), a partial and remarkable improvement of the skin lesions was observed sequence of the 43-kDa glycoprotein coding gene (gp43), and (data not shown). the internal transcribed spacer region of ribosomal RNA (ITS After several months on this regimen, the skin lesions rDNA). These sequences showed that the causative agent of became smaller and flatter than the original keloidal skin PCM-C shares highest identities with P. brasiliensis [9], one lesions (Fig. 3a, b). From these data, we conclude that some of the causative agents of paracoccidioidomycosis, which is fungal agents might be involved in the pathogenesis of the globally endemic, including Japanese offshore areas [1, 2]. present case. We plan to examine this case further to clarify Therefore, at present, molecular biological data are essential the pathogenesis of the disease in detail. for a diagnosis of PCM-C. From these data, we conclude that ACKNOWLEDGMENT PCM-C is globally endemic in cetacean species, and is caused by uncultivated P. brasiliensis [2, 7] or Paracoccidioides sp. We thank Janine Miller, PhD, and Whitney Bagge, PhD, for [1, 9, 10]. However, according to the definition of “lacaziosis,” editing a draft of this manuscript. it is caused by L. loboi [12], which is endemic to the Amazon REFERENCES area and causes an infection that is limited to humans [7]. Based on the new concept of PCM-C, the confirmed hosts are 1. Ueda K, Sano A, Yamate J, Itano EN, Kuwamura M, Izawa T, BD [1, 7, 9, 10] and PWSD [2], although the Indian Ocean Tanaka M, Hasegawa Y, Chibana H, Izumisawa Y, Miyahara bottlenose dolphin (T. aduncus) [11, 14], costero estuarine H, Uchida S. 2013. Two cases of lacaziosis in bottlenose dolphin (Sotalia guianensis) [8], and Indian Ocean humpback dolphins (Tursiops truncatus) in Japan. Case Report Vet Med, dolphin (Sousa plumbea), are suspected hosts [14]. Suspected 318548. doi:10.1155/2013/318548. cases have also been recorded in an Australian snubfin dolphin 2. Minakawa T, Ueda K, Tanaka M, Tanaka N, Kuwamura (Orcaella heinsohni) [15] and in Irrawaddy dolphins (Orcaella M, Izawa, Konno T, Yamate J, Itano EN, Sano A, Wada S. brevirostris) based on photographic images [16]. 2016. Detection of multiple budding yeast cells and a The gross skin lesions of the present case are similar to those partial sequence of 43-kDa glycoprotein coding gene of of our previous PCM-C cases in PWSD [2], a Paracoccidioides brasiliensis from a case of lacaziosis in asteroids infection in a BD [17], and an NTM infection in a a female Pacific white-sided dolphin (Lagenorhynchus white whale [3]. However, we detected no clear evidence of obliquidens). Mycopathologia 181: 523–529. fungal or mycobacterial infection in the biopsied skin samples, 3. Bowenkamp KE, Fraska S Jr, Draghi A II, Tsongalis GJ, other than typical chains of spherical budding-yeast-like cells, Koerting C, Hinckley L, De Guise S, Montali RJ, Goertz CEC, a cytological characteristic of PCM-C [2, 7]. In addition, some Aubin DJ St, Dunn JL. 2001. Mycobacterium marinum previous cases of “lobomycosis-like disease” in cetaceans were dermatitis and panniculitis with chronic pleuritis in a captive described without proof of fungal elements, and the diagnostic white whale (Delphinapterus leucas) with aortic rupture. J basis for each was only the gross features of skin lesions [11, Vet Diagn Invest 13: 524–530. 18-20]. 4. Eddyani M, Debacker M, Martin A, Aguiar J, Johnson CR, We began oral administration of itraconazole (Kaken Uwizeye C, Fissette K, Portaels F. 2008. Primary culture of Pharmaceutical Co., Ltd, Tokyo, Japan) at a dose of 300 mg Mycobacterium ulcerans from human tissue specimens after (2.4 mg/kg) twice a week after skin biopsies were performed. storage in semisolid transport medium. J Clinic Microbiol 46: In combination with systemic administration of itraconazole, 69-72. we also applied topical treatments of the skin lesions with an 5. Yajko DM, Nassos PS, Sanders CA, Gonzalez PC, Reingold AL, ointment composed of 10 g of white petrolatum (Maruishi Horsburgh, Jr CR, Hopewell PC, Chin DP, Hadley WK. 1993. Pharmaceutical, Osaka, Japan) and 50 mg of amphotericin B Comparison of four decontamination methods for recovery powder (fungizone [50 mg/vial] for systemic administration, of Mycobacterium avium complex from stools. J Clinic

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Microbiol 31: 302-306. humpback (Sousa plumbea) dolphins incidentally caught in 6. Fukano H, Wada S, Kurata O, Katayama K, Fujiwara N, shark nets off the KwaZulu-Natal Coast, South Africa. PLoS Hoshino Y. 2017. Mycobacterium stephanolepiae sp. One 9: e107038. nov., a rapid-growing nonchromogenic species related to 15. Palmer C, Peterson A. 2014. First report of a lacaziosis- Mycobacterium chelonae, isolated from marine teleost fish. like disease (LLD) observed in the Australian snubfin dolphin Int J Syst Evol Microbiol 67: 2811-2817. (‘Orcaella heinsohni’) in Darwin Harbour, Northern Territory, 7. Vilela R, Bossart GD, St. Leger JA, Dalton LM, Reif JA, Australia. North Territ Nat 25: 3–6 Schaefer AM, McCarthy PJ, Fair PA, Mendoza L. 2016. 16. Van Bressem MF, Minton G, Sutaria D, Kelkar N, Peter C, Cutaneous granulomas in dolphins caused by novel Zulkarnaen M, Mansur RM, Porter L, Vargas LH, Rajamani uncultivated Paracoccidioides brasiliensis. Emerg Infect Dis L. 2014. Cutaneous nodules in Irrawaddy dolphins: an 22: 2063–2069. emerging disease in vulnerable populations. Dis Aquat Organ 8. Horner KL, Raugi GJ. 2017. Lobomycosis on MedScap. 107: 181–189 http://emedicine.medscape.com/article/1092451-overview. 17. Ueda K, Nakamura I, Itano EN, Takemura K, Nakazato Updated April 17. Y, Sano A. 2017. Trichosporon asteroides isolated 9. Esperón F, García-Párraga D, Bellière EN, Sánchez-Vizcaíno from cutaneous lesions of a suspected case of JM. 2012. Molecular diagnosis of lobomycosis-like disease in "paracoccidioidomycosis ceti" in a bottlenose dolphin a bottlenose dolphin in captivity. Med Mycol 50: 106–109. (Tursiops truncatus). Mycopathologia. doi: 10.1007/s11046- 10. Rotstein DS, Burdett LG, McLellan W, Schwacke L, Rowles 017-0147-3. [Epub ahead of print] T, Terio KA, Schltz S, Pabst A. 2009. Lobomycosis in offshore 18. Bermudez L, Van Bressum MF, Reyes-Jaimes O, Sayegh bottlenose dolphins (Tursiops truncatus), North Carolina. AJ, Paniz-Mondolfi AE. 2009. Lobomycosis in man and Emerg Infect Dis 15: 588–590. lobomycosis-like disease in bottlenose dolphin, Venezuela. 11. Kiszka J, Van Bressem MF, Pusineri C. 2009. Lobomycosis- Emerg Infect Dis 15: 1301-1303. like disease and other skin conditions in Indo-Pacific 19. Tajima Y, Sasaki K, Kashiwagi N, Yamada TK. 2015. A bottlenose dolphins, Tursiops aduncus, from the Indian case of stranded Indo-Pacific bottlenose dolphin (Tursiops Ocean. Dis Aquat Org 84: 151–157. aduncus) with lobomycosis-like skin lesions in Kinko-wan, 12. Taborda PR, Taborda VA, McGinnis MR. 1999. Lacazia loboi Kagoshoma, Japan. J. Vet. Med. Sci. 77: 989-992. gen. nov., comb. nov., the etiologic agent of lobomycosis. J 20. Van Bressem MF, Van Waerebeek J, Reyes J, Felix F, Clin Microbiol 37: 2031–2033. Echegaray SS. 2007. A preliminary overview of skin and 13. Vilela R, Mendoza L, Rosa PS, Belone AF, Madeira S, skeletal diseases and traumata in small cetaceans from South Opromolla DV, Resenda MM. 2005. Molecular model for American waters. Latin Am J of Aqua Mam 6: 7-42. studying the uncultivated fungal pathogen Lacazia loboi. J 21. Murata Y, Sano A, Ueda Y, Inomata T, Takayama A, Clin Microbiol 43: 3657–3661. Poonwan N, Nanthawan M, Mikami Y, Miyaji M, Nishimura 14. Lane EP, de Wet M, Thompson P, Siebert U, Wohlsein P, K, Kamei K. 2007. Molecular epidemiology of canine Plön S. 2014. A systematic health assessment of Indian in Japan. Medical Mycology 45: 233–247. ocean bottlenose (Tursiops aduncus) and Indo-Pacific

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症例報告 病理学

日本における水族館飼育下の雄のカマイルカにみられた クジラ型パラコクシジオイデス症（paracoccidioidomycosis ceti）を疑う一例

皆川智子 1），植田啓一 2），佐野文子 3），上迫春香 2），岩永海空也 1），小峰壮史 1），和田新平 1 ）＊

1） 日本獣医生命科学大学水族医学研究室 〒180-8602 東京都武蔵野市境南町 1-7-1 2） 沖縄美ら島財団 〒905-0206 沖縄県国頭郡本部町字石川 888 3） 琉球大学農学部家畜衛生学研究室 〒903-0213 沖縄県中頭郡西原町字千原 1

［2017 年 9 月 22 日受領，2018 年 3 月 8 採択］

要 約 水族館飼育下のカマイルカの体表にクジラ型パラコクシジオイデス症を疑う多発性の肉芽腫性結節患部を観察した。生検を実施 して各種検査を行ったところ，渡銀染色下にて多極性出芽を示した球形の酵母様細胞を認めた以外は何らかの感染症を示唆する所 見は得られなかったが，抗真菌剤を用いた治療により患部が緩解しているため，本症例の病態に何らかの真菌が関与している可能 性が考えられた。 キーワード：カマイルカ，クジラ型パラコクシジオイデス症，paracoccidioidomycosis ceti －日本野生動物医学会誌 23（2）：45-50，2018

＊ 責任著者：和田新平（ E-mail: [email protected] ）

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