Case Report: Systemic Candidiasis in a Captured Yellow-Headed Tortoise (Indotestudo elongate)

Transcription

1 台灣獸醫誌 Taiwan VetJ 37 (1): 30-35, 2011 Case Report: Systemic Candidiasis in a Captured Yellow-Headed Tortoise (Indotestudo elongate) 1 Fang-Tse CHAN, 2 Sh ih- Li ng HSUAN, 3 Sh ih -Ch ieh CHANG, 1 Pet-I Ll N, 1 Li ng-m in WANG, 3 Ch ing- Li ng YANG, 2We i-che n g LEE, 2 Maw-Sheng CHIEN, 4Wen-Fa CHANG, 3 Ming-Jang KU O 悶 4 Jiu nn-wang Ll AO 1 Endemic 2 Gradua侶的sfitufe 3 Species Resθarch /nsfitu紗 Chichi, Nanfou, Taiwan of Veferinary Pafhobi%gy, Nafiona/ Chung Hsing Universify, Taichung, Taiwan Deparfmenf of Vefθrinary Mθdicinθ Nafiona/ Chung Hsing University, Taichung, Taiwan 4 Anima/ Disease Diagnosfic Cen的r Nafiona/ Chung Hsing Universify, Taichung, Taiwan (Received: Oecember 29, Accepted: January 19, 2011) ABSTRACT A free-living, fe male yellow-headed tortoise (lndotestudo e/ongate) was rescued and t reated for a laceration wound w ithout improvemen t. and the 叩門oise died after two months of t reatmen t. At nec rop呵 in addition to the skin laceration, multip le w h itish-toγellowish nodules scattered on various visceral organs. The nodular lesions were characterized by coagu lative necrosis and pyogranuloma with intralesionalγeast-like o rganisms under microscopy. Necrotic nodules we re stained using acid-fast staining and screened using po lymerase chain reaction assay for Mycobacterium tuberculosis revea led negative results. The periodic acid Sch iff (PAS) and immunohistochemistry (I HC) staining revea led positive resu lts for Candida albicans. ßased on these analγtical resu lts, a case of rare systemic infection of candidiasis in a tortoise is reported. [Chan FT, Hsuan SL, Chang SC, Lin PI, Wang LM, Yang CL, Lee WC, Chien MS, Chang WF, Kuo MJ, and * Liao JW. Systemic Candid i 泌的lO a Captured Ye llow-headed Tortoise (I ndotestudo elongata). Taiwan Vet J 37 (1): 30-35, * Corresponding author TEL: ex t. 406, FAX : , jwliao@dragon.nchu.edu.tw] Key words: Candidias巧 的dotestudo elongate, pyogranuloma, systemic infection, INTRODUCTION to付oise Usually reptiles colonized with yeasts do not reveal significant symptoms. In a review on the prevalence of Yellow-head tortoises (1ndotestudo elongata) yeasts in repti les, postmortem examinations revealed habitat mainly in the southern Asia, including the for- that intestines of 80.6% of animals carried yeasts [8] ests around the tropic and subtropical areas. The body ln general, virulence in Cand ida is ofyellow-head tortoises is small to middle in size. The hest in C. albicans, but C. tropicalis, C. parapsilosis, o rep 此ed to be hig- suitable temperature and humidity are around C C. krusei and C. glabrata may also occasionally cause and 60-80%, respective ly. However, mycotic diseases of reptiles caused by hyphomycetes and zygomycetes infections in humans and an imals [12]. Deficiencies in ho using, e.g. high humidity, poor water quali ty or have been described mostly as case reports [14]. overcrowding, poor diet and inadequate v itam in

2 Systemic Candidiasis in a Captured Tortoise supplementation, bacterial or paras itic infections and long-term antibiotic treatment are considered predisposing factors for the development of systemic mycosis [15]. Systemic candidiasis results in rapid death of the reptiles, particularly in those with immune depletion [15]. To date, Candida sp. causing systemic mycosis in tortoises has rarely been reported. In this case, we reported system ic mycotic infection in tortoises with chronic pyogranuolomas compared those for suspicion of aspergillosi s or mycobacterial involvemen t. HISTORY. DIAGNOSIS AND TREATMENTS A fema le yellow-headed tortoise of unknown age, 1504 g body weight, was rescued in a field in Nan-Tou County and treated at the Endemic Species 31 Research Institute for anorexia and a laceration wound (5 x 3.5 cm in size) around the cloaca (Fig. la). The to 的oise was then treated with Betadine (Mundipharma GmbH, Limburg, Germany), 5% glucose with 0.9% saline sol ution (Ch i Sheng, Hsinchu County, Taiwan) 20 ml, and Baytril (5% enrofloxacin, Bayer, Leverkusen, Germany) 5 m g/kg SC s.i.d. for three days. The wound was then cleaned and sutured on Day 4 after reception. Same treatment as above was made for two weeks, but patient tortoise still appeared anorexic and interval constipation or diarrhea. A nutritional formula of Ensure (Zwolle, Netherlands) 5 ml was additional supplemented tw ice daily through oral tube. Wound was cleaned; caprofen 4 m g/kg 1M s.i.d. was given, and immersed with antibiotic (gentamicin) and vitamin B 1 for 30 min for two consecutively days. Un司 fortunately, patient tortoise was found dead at the end Fig. 1 Gross findings for a sick tortoise (Indotestudo elongata). The tortoise has a 5 x 3.5 cm skin laceration wound around the cloaca (A, arrow). At necropsy, multiple, whitish-to- yellowish nodules of variable sizes scatter on the lungs (8), kidney (C), and stomach (0)

3 Fang-Tse CHAN et al 32 of 69 lh day's treatmen t. The body was preserved and sent to the Animal Disease Diagnostic Center, National Chung Hsing University, for pathological examinatjon. At necropsy, the tortoise appeared emaciated, and had a 5 x 3.5 cm skin laceration wound around the c1 0aca (Fig. 1A). The wall of gastrointestinal (GI) tract and small intestine were thickened, and a massive ulcerative lesion with a blackish pseudomembrane existed on the mucosa layer in the entire small intestine. Multiple, whitish-to-yellowish nodules varying in sizes at 卜25 mm were found scattered on the oral mucosa and peritoneum, stomach, heart, lungs, liver, kidneys, ovaries and uterus (Fig. lb, C and D). Microscopically, the cloaca had a massive ulceration with Zenker's degeneration of muscles and fibrovascular granulation tissues via hematoxylin and eosin (H&E) staining. Furthermore, severe enteritis with exfoliated necrotic epithelium and inflammatory cells were found in the mucosa of small intestin巳. Many yeast-like organisms, round in shape and measuring 5-10 μm in diameter, with unbranched fungal hyphae were also noted in the mucosa ofthe small intestine. Coagulative necrosis and numerous pyogranulomas varying in sizes were identified in various organs, including the esophagus, heart, kidney, lung, Ii ver, peritoneum, spleen, stomach, ovaries and uterus. The pyogranulomas were mainly infiltrated with heterophils and mononuclear cells with cellular debris and in the central area of crashed round yeast-like organisms were presented. A thin layer of fibrovascular tissue was presented in the peripherally, forming an atypical encapsulation. However, no calcification was found in the pyogranulomas. Numerous brownish pigments of melanomacrophages aggregated in the sple凹 liver and kidneys. In microbiology examination, stomach contents were cultured on blood agar and 恥lacConkey agar, and incubated at in ambient air for h. The numerous grayish and pinkish colonies on cultivated agar plates were identified as a com司 mensally bacterium of E. coli. Organs with pyogranulomas were acid-fast stained to differentiate possible fungal from acid-fast bacteria, such as Mycobacteria, as botb are common pathogens causing similar lesions. The result of acid-fast staining was negative in the pyogranulomas; neverthe- 3rc less, lesions were positive for candidiasis in hyphae and spores on tbe submucosal layers of stomach and small intestine by PAS staining (Fig. 2A and B), which confirmed an intralesional fungal infection was positive for polysaccharides. Some ofthe dead and crashed round yeast-like spores were also PAS positively stained in the central area of pyogranulomas. Tissue sections of organs with pyogranulomas lesions included oral mucosa and the peritoneum, stomach, heart, lun 軒 Iiv 缸" kidneys, ovaries and uterus, were then stained with immunohistochemical kits (IHC kits) for C αlbicans (Biotin, rabbit polyclonal antibody, ab20005, Abcam, Cambridge, UK, 200x dilution), Aspergillus spp. (rabbit polyclonal antibody, ab20419, 200x dilution), and M tubercu/osis (Hsp-65, BD1578, mouse monoclonal antibody, ab20045, 100x dilution) to identi 句 the possible pathogens. Negative reactions for M tuberculosis and Aspergillus spp. were noted ; bowever, a strong positive reaction was found after staining with the C. albicans antibody (Fig. 2C and D). Because gross pathological findings raised suspicion of mycobacterial involvement [1, 11], polymerase chain reaction (PCR) of M tuberculosis was performed. For PCR, DNA was extracted from fixed tortoise tissues using the QIAamp DNA mini elute kit (QIAGEN, Bene lux, The Netherlands). The primer pairs specific to M tuberculosis were 5' -ACC-AAC-GAT-GGT-GTGTCC-AT-3' and 5'-CTτ~GTC - GAA-CCG-CAT-ACC CT-3 ' [11]. A positive control reaction appeared as DNA PCR products of 439 base pairs. However, tissues appeared negative a缸er PCR amplification. These outcomes by acid-fast, IHC, and PCR for M tuberculosis were all negative (data not shown). DISCUSSION C. albicans is a fungus that lives harmlessly in the gastrointestinal (GI) tract of reptiles. A rev iew on the prevalence ofyeasts in reptiles revealed that the intestines of 80.6% of animals carried yeasts. In all, 56 isolates the genera Candida (32), Trichosporon (11), Torulopsis (9) and Rh odotorula (3) were identified [8]. Few reports have addressed mycotic diseases in tortoises compared with those for other reptiles. However, infections have been described in both captive [3,5] and wild sea turtles [9]. Although mycotic granulomas

4 Systemic Candidiasis in a Captured Tortoise 33 Fig. 2 Periodic acid Schiff (PAS) stainings and immunohistochemistry stainings with the Candida albicans antibody. Strong positive reactions of Candida albicans staining on hyphae and spores are noted in the mucosa of the small intestine by PAS (A and ß, pink spores) and IHC stainings (C and 0, brown spores), respectively. can be found in the liver and throughout the coelomic sional fungal elements were strongly stained and iden- cavity of wild and captive sea turtles; contradictory, tified as C. albicans by a polyclonal antibody specific systemic mycotic infections, such as Aspergillosis, occur primarily in the lungs [2]. Intestinal candidiasis in to this organism in histochemical and IHC staining. In a loggerhead sea turtle (Caretta caretta) has been reported [10,1 3]. To date, Candida sp. causing systemic mycotic infection in tortoises has been rarely reported. ln this case, the tortoise was incidentally diagnosed with systemic candidiasis based on severe necrotic enteritis accompanied by many candida organisms growing in the gastro- intestines. Furthermore, coagulative necrosis with various sizes of granulomas was observed in the esophagus, heart, kidney, lungs, liver, peritoneum, spleen, stomach, ovaries and uterus. These lesions closely resemble mycobateriosis in the clinic. However, all tissues examined were negative for mycobacterium by both lh C and PCR assays. Intrale- microbiology examination, E. coli was cultured from stomach contents, and was recognized as a comrnensally bacterium. Unfortunately, the wound culture had not been done for this case that might be lost to identify the microorganisms on the wound. The presumed mechanism for the pathogenesis of C. albicans involves initial mucosal surface colon卜 zation followed by invasion into adjacent tissues and organs [13]. Notab旬 C. albicans typically colonizes the GI tract with subsequent translocation into extraintestinal organs (i.e., mesenteric Iymph nodes, blood stream, liver, and spleen) under chemotherapy-induced neutropenia an djor GI mucosal damage in mice [1 月. Three prima可 mechanisms promoting pathoge-

5 34 Fang-Tse CHAN et al nic microbial (bacterial and fungal) translocation in 4. Candida albicans in a Greek tortoise (Testudo graeca) animal models are disruption of the normal GI micro- and treatment with intrapulmonary amphotericin B. J biologic equilibrium, which facilitates intestinal overgrowth of pathogens, increased permeability ofthe in- Hernadez-Oivers SJ. Pulmonary candidiasis caused by Zoo Wildl Med 32: , Jacobson ER, Gaskin JM, Shields RP, White RH. Mycotic testinal mucosal barrier, and deficiencies in host im- pneumonia in mariculture-reared green sea turtles. J mune defenses [6 月]. AM Vet Med Assoc 175: , For the therapy, intrapulmonary amphotericin at 0.1 m g/kg s.i.d. for B days has proven both safe and effective for pulmonary candidiasis [4]. In nization, and dissemination from the gastrointestinal this case, tract 7. Immun 49: Koh AY, Köhler JR, Coggshall KT, Rooijen NV, Pier GB. Kostka VM, Hoffmann L, Balks E, Eskens U, Wimmershof N. Review of the literature and investigations on the prevalence and consequences of yeasts in reptiles. C. al- ing differential diagnosis of enteritis and pyogranulo- Infect opportunistic infection. However, this report present- bicans, and candidiasis should be considered in mak- antagonism. Candida albicans dissemination. PLoS Pathog 4 : e35, of predisposed factors to cause immunodeficiency. ing a rare case of systemic mycotic infection by bacterial Mucosal damage and neutropenia are required for term antibiotic treatment to this tortoise might be one Therefore, the systenllc candidiasis may be simply an by , 1985 patient tortoise was treated with antibiotics for the laceration wound but it was failed. Ine缸ective and long- Kennedy YMJ, Volz PA. Ecology of Candida albicans gut colonization: Inhibition of Candida adhesion, colo- Vet Rec 140: , Lewbart GA. Medway W. A case of mycotic lung disease in a wild caught juvenile sea turtle. J Sm Exot Anim Med 2: 58-59, mas m tortojses. 10. Mild K, Kostka V, Kaleta EF, Willems H, Jäger C. Multiplex-PCR-based differentiation and characterization ACKNOWLEDGEMENTS of Candida-isolates derived from tortoises (Testudinidae). Vet Microbiol 76: , Authors thank Miss Lee-Jen Chen, Yi-Hsuan Wu and Dr. Wen-Fa Chang for their technical assistances and Dr. Ted Kn oy for his editorial assistance. 11. Morick 0, Kik M, Oe Beer J, Van Oer Zanden AGM J, Houweres O. Isolation of Mycobacterium mageritense from the lung of a harbor porpoise (Phocoena phocoena) with severe granulomatous lesions. J Wildl Ois 44: , Odds FC. Pathogenesis of Candida infections. J Am REFERENCES 1. Acad Oermatol 31: 2-5, Baigrie PJ, ParumasV. Fatal case of disseminated infection with the turtle bacillus Mycobacterium chelonae. J Clin Patho 45: , Oagleish MP, Patterson IAP, Foster G, Reid RJ, Li nton C, Buxton O. Intracranial granuloma caused by asporogenic Aspergillus fumigatus in a harbour porpoise (Phocoena phocoena). Vet Rec 159: , Glazebrook JS, Campbell RSF. A survey of the diseases of marine turtles in northern Australia. turtles. Ois Aquat Org 9: 83-95, Farmed 13. Orós J, Arencibia A, Fernández L, Jensen HE. Intestinal candidiasis in a loggerhead sea turtle (Carelta carelta): an immunohistochemical study. Vet J 167: , Zwart P, Buitelaar MN. Candida tropicalis: enteric infections and their treatment in the chelonians. In : Proceedings of the Annual American Association of Zoo Veterinarians, Washington, 58, Zwart P, Schro自er HO. In: Ippen R, Schr 后der HO, Elze K. eds, Handbuch der Zootierkrankheiten, Reptilien. Akademia Verlage, Berlin, , 1985.

6 Systemic Candidiasis in a Captured Tortoise 35 病例報告 : 野外捕捉黃頭陸龜之全身性念珠菌症 1 詹芳澤 2 宣詩玲 3 張仕杰 1 林佩羿 1 王齡敏 3 楊青玲 2 李維誠 2 簡茂盛 4 張文發 3 郭銘彰 *2,4 廖俊旺 1 特有生物研究保育中心南技縣 2 國立中興大學獸醫病理生物學研究所台中市 3 國立中興大學獸醫學系台中市 4 國立中興大學動物疾病診斷中心台中市 ( 收稿日期 : 99 年 1 2 月 29 日 接受日期 : 10 0 年 1 月 1 9 日 ) 措 i 要病龜為野外捕捉之雌性黃頭陸龜 (lndotestudo e/ongata), 因撕裂傷進行治療, 不幸於 2 個月後發現死亡 剖檢發現病龜全身臟器皆散佈多發性 大小不一之黃白色結節, 組織病理檢查為凝固樣肉芽腫壞死仕並有疑似酵母菌成染, 壞死 1 士經抗酸染色及分枝桿茵 (Mycobacteríum tubercu/osís) 頁頁合酪鍵鎖反應均呈陰性反應, 以化學組織染色法 periodic Acid-Schiff 及念珠菌之組織免疫化學染色法 (Candída a/bícans primary a ntibody ) 染色結果均呈陽性反應 綜合以上結果, 本病例為少見之陸龜全身性念珠菌症 [ 詹芳澤 宣言寄玲 張仕杰 林佩羿 王齡敏 楊青玲 李維 誠 筒茂盛 張文發 享 F 銘彰 * 廖俊旺 病例報告 : 野外捕捉黃頭陸龜之全身性念珠菌症 台灣獸醫言之 37 (1) : 30-35, * 通訊作者 TEL : ext FAX: jwliao@dragon.nchu.edu.tw] - liljilj. 訝 : 念王若 E 症, 黃頭直至重仔 ndo tes 的 do elonga t. 呵, ft!it 衛生戶可穿膺, 全身性