Shell and Systemic Hyalohyphomycosis in Fly River Turtles, Carettochelys insculpta, caused by Paecilomyces lilacinus

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1 A n e s t h e s i a, A n a l g e s i a, S u r g e r y Shell and Systemic Hyalohyphomycosis in Fly River Turtles, Carettochelys insculpta, caused by Paecilomyces lilacinus Maud Lafortune1, DMV, MSc, DACZM, James F. X. Wellehan1, DVM, MS, Scott P. Terrell2, DVM, DACVP, Elliott R. Jacobson1, DVM, PhD, DACZM, Darryl Heard1, BSc, BVMS, PhD, DACZM, James W. Kimbrough3, PhD 1. Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida , USA 2. Department of Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida , USA 3. Department of Plant Pathology, IFAS, University of Florida, Gainesville, Florida 32611, USA A b s t r a c t : Eleven wild-caught Fly River turtle hatchlings, Carettochelys insculpta, were presented for anorexia and circular shell lesions. One animal died shortly after arrival, and three others within the next month. Necropsy of two animals and one shell biopsy revealed systemic and shell mycoses. A biopsy culture demonstrated infection due to Paecilomyces lilacinus, a ubiquitous fungal pathogen rarely affecting mammals, fish, and reptiles. Malachite green, formaldehyde, and parenteral itraconazole were used to effectively treat the shell lesions. In addition, changes in husbandry were made, and included frequent water changes, increasing the salinity to 7 ppt, increasing the water temperature to 32 C (90 F), and provision of enteral support. K e y W o r d s : Fly River turtle, Carettochelys insculpta, shell dermatitis, Paecilomyces lilacinus, mycosis, hyalohyphomycosis. Introduction Hyalohyphomycosis is a term used for infections caused by hyaline fungal pathogens. Causes of hyalohyphomycosis include species of Acremonium, Beauveria, Fusarium, Scopulariopsis, Penicillium and Paecilomyces. Paecilomyces lilacinus is a ubiquitous mold found in soil and vegetation that is infrequently associated with disease. In humans, infection is rare and generally seen in immunocom promised patients (Carey, et al, 2003). Reports of infection are also rare in dogs, cats and horses (Elliott, et al, 1984, Scott, et al, 1995, Foley, et al, 2002). Case reports have been described in an armadillo, Dasypus novemcinctus, and tilapias, Tilapia aurea and T. mossambica (Gordon, 1984, Rand, et al, 2000). Cutaneous and respiratory systems are the most commonly affected, but other systems have also been involved (Gutierrez-Rodero, et al, 1999, Ono, et al, 1999, Foley, 2002). In reptiles, P. lilacinus has been reported to cause disease in captive individuals kept under suboptim al husbandry conditions, such as overcrowding and low temperatures. Systemic mycoses were reported in hatchling and juvenile saltwater crocodiles, Crocodylus porosus (Maslen, et al, 1988, Hibberd and Harrower, 1993), New Zealand geckos, Hoplodactylus maculatus (Cork and Stockdale, 1994), and an Aldabra tortoise, Geochelone gigantia (Heard, et al, 1986). Finally, dermatitis, bronchopneumonia, and systemic mycosis have been observed in captive sea turtles, Chelonia mydas, Caretta carretta, and Eretmochelys imbricata (Glazebrook, et al, 1993, Posthaus, et al, 1997). This case report describes a shell dermatitis and systemic mycosis due to Paecilomyces lilacinus in Fly River turtles, Carettochelys insculpta, treated with malachite green bath and parenteral itraconazole. Case R eport Initial evaluation Eleven wild-caught Fly River turtle hatchlings were presented to the Veterinary Medical Teaching Hospital of the University of Florida for evaluation following a confiscation of an illegal shipment. On initial physical examination, the turtles were weak and in poor body condition (score 2/5). They weighed between 20 and 25 g. Small white to gray 0.5 to 3 mm diameter circular lesions were present on the carapace, Volume 15, No. 2,2005 Journal of Herpetological Medicine and Surgery 15

2 plastron, head, and legs of six turtles (Figure 1). Initially, the turtles were housed in individual plastic containers (15 x 30 cm, water depth 2-4 cm) and placed into an incuba tor (Animal Intensive Care Unit, Lyon Electric Company Inc, Chula Vista, CA). The water temperature was main tained between C (80-85 F) and monitored twice a day. The turtles were placed outside in a screened area when the ambient temperature and water temperature were above 27 C (80 F). The salinity was set at 1 ppt (0.1%) by adding salt to the water (Instant Ocean, Aquarium System Inc, Mentor, OH) and measuring twice a day with a salini ty meter (Koi Med I, Trans instruments, Singapore). Palm fronds were added for shade and shelter. The containers were cleaned and the water changed daily. The turtles were initially fed separately in their containers, but later placed together to promote feeding. They were offered turtle pel lets (Reptomin, Tetra, Blacksburg, VA), fish pellets (Hikari cichlid gold pellets, Hayward, CA), krill, tomatoes, kiwis, and peppers, and were weighed every day. If the animals were not observed eating or lost weight, they were forcefed (Reptomin pellets and Critical Care Form ula for herbivore, Oxbow company, Murdock, NE; 1% BW PO BID). After feeding, each turtle was replaced into an indi vidual container. Figure 1. Shell lesions on a fly river turtle, Carettochelys insculpta, caused by Paecilomyces lilacinus. Diagnostic and treatment Within a week, all turtles developed lesions on their carapace. The first shell scrap ing performed on two animals was negative for parasites. One week after arrival, the salinity was increased to 5 ppt (0.5%) and dilute iodine solution dip was started (1:50), SID for 3 min at 29 C (84 F) for 10 d. Direct microscopic Jo u rn a l of H erpetological M edicine and Surgery evaluation of a subsequent shell scraping from one animal on day 15 revealed Vorticella spp. and Tetrahymena spp. A D iff Quick stain showed occasional inflammatory cells (heterophils), bacteria and possible fungal hyphae. The salinity was changed to 7 ppt on day 15. Turtles 1, 2, 3, and 4 died on days 2, 22, 23, and 32, respectively, after a short period of extreme lethargy (< 1 day) and abnormal buoyancy. Post-mortem examinations were performed on these four turtles. A biopsy of the shell lesion from turtle five was also taken on day 16. A surgical blade (# 15) was used to harvest the biopsy, containing dermis and epidermis. Results of the histopathology from two turtles and the shell biopsy revealed fiingal infection. Paecilomyces lilacinus was isolated from the shell biopsy and turtle #4. The turtles were treated with malachite green and formaldehyde dips, 15 min BID x 33 d (0.15 mg/l; Rid Ich +, Kordon, Hayward, CA, contains 0.038% mala chite green and 4.26% form aldehyde). Itraconazole therapy was implemented as well on the more severely affected animals (turtles 5, 6 and 7) at 10 mg/kg PO q 48 hr x 20 d (Sporonox, Ortho Biotech, Raritan, NJ). On day 57, all lesions had resolved and the malachite green, formaldehyde dips, and itraconazole treatment were discontinued. On day 104, the turtles were active and eat ing well, and weighed between g. No lesions were visible on any animal. Microbiology (necropsy and biopsy) Samples from turtles 2, 3 and 4 were submitted to the University of Florida Veterinary Medicine Teaching Hospital (VMTH) for fungal culture. Samples were cultured on Mycobiotic agar and Emmons Sabdex agar (Hardy Diagnostics, Santa Maria, CA). The plates were incubated at 30 C ( 86 F) for 30 d and examined under a laminar flow hood once a week. Standard tape preparations were made using lactophenol aniline blue stain and fungi were identified based on spore formation. Fungal growth was observed from the shell of turtle 2 and identified as Penicillium spp. The yolk sac of turtle 3 grew a mixed flora. Finally, Paecilomyces spp. was isolated from the coelomic membrane of turtle 4. No culture was submitted for turtle 1. The shell biopsy was su bm itted to both the VM TH and the M ycology L ab o rato ry o f the D ep artm en t o f P lant Pathology, University of Florida, for identification. The colonies were originally white, but changed to a vinaceous (dark purple) color after sporulating, and then to a drab vinaceous or purple-brown. Microscopically, pm conidiophores were observed on aerial mycelium, each forming verticillate branches with whorls of phialides with swollen bases. Long divergent chains of ellipsoid to fusiform conidia, which m easured x pm, were observed. All cultural and microscopic features conformed to those described for Paecilomyces lilacinus (Thom) Samson (Samson, 1974). H isto p a th o lo g y (n ecrop sy and biopsy) H istopathologic exam ination (hematoxylin and eosin stained slides) of the surgical biopsy o f the shell lesion from turtle five revealed papillary hyperplasia of epidermis with hyperkeratosis and numerous septate branching fun gal hyphae within areas of hyperkeratosis (Figure 2). Post Volume 15, No. 2,2005

3 Figure 2. Numerous dark staining septate branching fungal hyphae are visible within a granuloma, Grocott s Methenamine Silver (GMS) stain, Bar = 50 pm mortem findings from the four turtles that died are summarized as follows: Turtle 1 aspiration pneumonia and severe emaciation, no fungal organisms noted; Turtle 2 -pgranulom atous om phalitis and coelom itis with intralesional septate fungal hyphae (resembling pseudohyphae 3-4 pm in width) fungal colonization of the carapace, bronchopneumonia; Turtle 3 granulomatous omphalitis and severe heterophilic bronchopneumonia; Turtle 4 systemic granulomatous inflammation with intralesional fungal hyphae affecting the yolk sac, liver, kidney, coelom, lung, and carapace. The death of turtle one was unrelated to fungal infection. Although fungal hyphae were not specifically seen in turtle three, it was suspected that an ascending yolk sac infection was responsible for possible systemic inflammation and subsequent severe pneumonia. Definitive yolk sac infection and subsequent systemic fungal infection were documented in turtles two and four. D iscussion Infectious shell and skin lesions in chelonians have been diagnosed with parasitic, bacterial, fungal and viral causes (Rhodin and Anver, 1977, Jacobson, et al, 1980b, Jacobson, et al, 1982, Boyer, 1996, Rossi, 1996, Lackovich, et al, 1999, Harkewicz, 2001, Rose, et al, 2001, Oros, et al, 2003). Non-infectious causes include trauma, neoplasia and nutrition (Boyer, 1996). Finally, a shell disease has been described in the Eastern River cooter, Pseudemys concinna, and the yellow-bellied turtle, Trachemys scripta (Gamer, et al, 1997). The etiology for the dyskeratoses remains unknown. Fungal hyphae were observed in both the shell biopsy of turtle five and the histopathology of two turtles (turtle 2 and 4). Several fungal agents are commonly reported to affect chelonians, including Aspergillus spp., Basidobolus ranarum, Beauvaria bassiana, Candida albicans, Cladosporidium spp., Dermatophyton spp. Fusarium spp., Microsporum spp., Mucor spp., Penicillium spp., and Rhodotolura spp. (Georg, et al, 1962, Jacobson, 1980a, Jacobson, et al, 1980b, Glazebrook and Campbell, 1990, Frye 1991, Glazebrook, et al, 1993, Gonzales, et al, 1995, Cabanes, et al, 1997, Kostka, et al, 1997, Posthaus, et al, 1997, Hernandez-D ivers, 2001, Rose, et al, 2001). However, Paecilomyces spp. infections have only been reported in Aldabra tortoises (Georg, et al, 1962, Heard, et al, 1986) and sea turtles (Glazebrook, et al, 1993, Posthaus, et al, 1997). P. lilacinus is ubiquitous in the environment, and infections are usually, but not always, linked with immunosuppression. In the case of these turtles, the stress of shipment, and most likely the sub optimal husbandry conditions during transport, predisposed these animals to the infection. The immune response of these turtles was not evaluated. It is interesting to note that infected dogs and cats are generally not considered immunosuppressed (Scott, et al, 1995). Also, an increasing number of immunocompetent human patients have recently developed infection with P. liliacinus, suggesting this fungus may be an emerging pathogen in humans (Gutierrez- Rodero, et al, 1999, Carey, et al, 2003). The multiple shell scraping examinations in these animals revealed a mixed infection with parasitic, bacterial and possible fungal agents. It was not possible with the shell scraping to define which of these agents the primary cause of the lesions was, and which secondary invaders were. The shell biopsy, although a more invasive procedure to perform, was very useful for giving a definitive diagnosis of fungal infection (pure culture of Paecilomyces sp.) and permitted appropriate treatment of these animals. Because Paecilomyces is very closely related to Penicillium, it is possible that the fungal growth in turtle 2 was misidentified as Penicilium instead of Paecilomyces (Samson, 1974). The Fly River turtle range includes Australia, southern Irian Jaya, and southern New Guinea (Bargeron, 1997). It is primarily a freshwater turtle, mostly found in rivers, lagoons, lakes, and swamps, but is also found in estuaries, which indicates that it is probably tolerant to brackish water. These turtles tolerated a salinity of 7 ppt without obvious ill effects. Salt baths are routinely, used to treat fungal and parasitic conditions in aquatic animals. Although the initial water temperature at which the turtles were kept was within their recommended range, 26 to 30 C (79-86 F) (Bargeron, 1997), the temperature was raised during treatment to 32 C (90 F) because the animals appeared inactive at the lower temperatures. This seemed to be beneficial to the animals, which became more active and started eating better. Pharmacokinetic studies of itracanazole in Kemp s ridley sea turtles, Lepidochelys kempi, reported that 15 mg/kg q 72 hr or 5 mg/kg SID resulted in therapeutic concentrations (Manire, et al, 2003). The itraconazole dose in this report was empirically determined at 10 mg/kg q 48 hr. In reptiles, 10% iodine in alcohol and malachite green baths have been used to treat fungal cutaneous infections (Jacobson, et al, 1980b, Cabanes, et al, 1997). Systemic antifungals used in reptiles include ketoconazole, itraconazole and fluconazole (Page, et al, 1988, Cabanes, et al, 1997, Mallo, et al, 2002, Manire, et al, 2003). In humans the benzimida- Volume 15, No. 2,2005 Journal of Herpetological Medicine and Surgery 17

4 zoles (especially itraconazole) are usually recommended to treat P. lilacinus infections, although griseofulvin has been used successfully in a few cases. The fungus is usually resistant to amphotericin B and 5-flucytosine (GutierrezRodero, et al, 1999). This report shows the importance of shell biopsy and culture over shell scraping for determination of causative agent of shell lesions. The malachite green, formaldehyde, and itraconazole were successful in treating the shell mycosis. Husbandry measures included force feeding, individual housing, daily water changes, increased salinity (7 ppt) and temperature (32 C). R eferences Bargeron M The Pig-nosed turtle, Carettochelys imculpta. Tortuga Gazette, 33:1-2. Boyer TH In M ader DR (ed): Reptile M edicine and Surgery. 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Vet Rec, 140: Lackovich JK, Brown DR, Homer BL, Garber RL, Mader DR, Moretti RH, Patterson AD, Herbst LH, Oros J, Jacobson ER, Curry SS, Klein PA Association o f herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida. Dis Aquat Organ, 37: M allo KM, H arm s CA, L ew bart GA, P apich MG Pharmacokinetics o f fluconazole in loggerhead sea turtles (icaretta caretta) after single intravenous and subcutaneous injections, and multiple subcutaneous injections. J Zoo Wildl Med, 33: Manire CA, Rhinehart HL, Pennick GJ, Sutton DA, Hunter RP, Rinaldi MG Steady-state plasma concentrations of itra conazole after oral administration in Kemp s Ridley sea turtles, Leidochelys kempi. J Zoo Wild Med, 34: Maslen M, Whitehead J, Forsyth WM, McCraken H, Hocking AD Systemic mycotic disease o f captive crocodile hatchling (Crocodylus porosus) caused by Paecilomyces lilaci nus. 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5 Page CD, M autino M, M eyer JR, M achlinski W Preliminary pharmacokinetics of ketoconazole in gopher tortoises (Gopherus polyphemus). J Vet Pharm, 13: Posthaus H, Krampe M, Pagan O, Gueho E, Suter C, Bacciarini L Systemic paecilom ycosis in a hawksbill turtle (Eretmochelys imbricata). J de Mycologie Medicale, 7: Rand TG, B unkley-w illiam s L, W illiam s EH A hyphomycete fungus, Paecilomyces lilacinus, associated with wasting disease in two species of tilapia from Puerto Rico. J AquatAnim Health, 12: Rhodin ACJ, Anver MR Mycobacteriosis in turtles: cutaneous and hepatosplenic involvement in a Phrynops hilari. J Wild Dis, 13: Rose FL, Koke J, Koehn R, Smith D Identification of the etiological agent for necrotizing scute disease in the Texas tortoise. J Wildl Dis, 37: Rossi JV Dermatology. In M ader DR (ed): Reptile M edicine and Surgery. WB Saunders Co, Philadelphia, PA: Samson RA Paecilomyces and some allied hyphomycetes. Studies in Mycology No. 62, CBS, Baam, The Netherlands. Scott DW, Miller WH, Griffin CE Fungal skin diseases. In Scott DW, Miller WH, Griffin CE (eds): Muller and Kirk s Small Anim al Derm atology, 11 ed., WB Saunders Co, Philadelphia, PA: BIOLOGY, HUSBANDRY, AND MEDICINE OF THE GREEN IGUANA edited by Elliott R. Jacobson Foreword by Thomas Huntington Boyer, DVM r l his multiauthored book spans a range of topics J- relevant to those individuals interested in keeping, breeding, and understanding health problems of the green iguana (Iguana iguana). It offers a unique synthesis of the work and experiences of biologists, nutritionists, and veterinarians who have worked with green iguanas, both in the field and in captivity, and it presents the most current, and in some cases previously unreported, information on iguana biology and medicine. Topics include biology and reproduction in the wild, nutrition in the wild and in captivity, ontogeny of captive iguanas, husbandry, clinical evaluation, diseases, drug dosages and chemotherapeutics, anesthesia and surgery, and diagnostic imaging. Orig. Ed pp. ISBN $46.50 CONTENTS Contributors Foreword by Thomas Huntington Boyer, DVM Preface Introduction 1. Biology and Reproduction in the Wild Gordon H. Rodda, PhD 2. O ntogeny of Captive and Wild Iguanas: From Em ergence to Mating Allison C. Alberts, PhD, Nancy C. Pratt-Hawkes, PhD, and John A. Phillips, PhD 3. Nutrition in the Wild David J. Baer, PhD 4. Nutrition in Captivity Mary E. Allen, PhD and Olav T. Oftedal, PhD 5. Husbandry and Management Juergen Schumacher, DVM, DACZM, Gunther Kdhler, DVM, Lara K. Maxwell, DVM, PhD, Frederick B. Antonio, BS, and Robert W. Ehrig To place your order and obtain shipping costs call or us at: info@krieger-publishing.com 6. Clinical Evaluation and Diagnostic Techniques Elliott R. Jacobson, DVM, PhD, DACZM 7. Infectious and Noninfectious D iseases Lara K. Maxwell, DVM, PhD 8. Drug D osages and Chemotherapeutics Lara K. Maxwell, DVM, PhD, and Kelly E. Helmick, MS, DVM 9. Anesthesia and Surgery Brad Lock, DVM and R. Avery Bennett, MS, DVM, DACVS 10. Diagnostic Imaging Susan M. Newell, DVM, MS, DACVR and Gregory Roberts, DVM, MS, DACVR Index KRIEGER PUBLISHING COMPANY P.O. Box 9542 Melbourne, FL (321) FAX (321) Volume 15, No. 2,2005 Journal of Herpetological Medicine and Surgery 19