Medical Mycology 2002, 40, 143–151 Accepted 10July 2001 Chrysosporium anamorph of Nannizziopsis vriesii associated with fatalcutaneous mycoses inthe salt- water crocodile ( Crocodylus porosus ) A.D. THOMAS*, L. SIGLER ,S.PEUCKER*, J. H.NORTON* &A.NIELAN y z *Department ofPrimaryIndustries, Animaland Plant Health Service,Oonoonba Veterinary Laboratory, P .O. Box 1085,Townsville, Queensland 4810,Australia; University ofAlbertaMicrofungus Collection,Devonian Botanic Garden, Edmonton, AlbertaT6G 2E1, y Canada; Edward RiverCrocodile Farm, P .O. Pormpuraaw,Queensland 4871,Australia z The Chrysosporium anamorph of Nannizziopsisvriesii ,recentlyidenti ed as the causeof cutaneous infectionsin chameleonsand brown treesnakes, was associated with skin infectionsand deaths in salt-water crocodile (Crocodylus porosus) hatchlingson two separateoccasions 3 yearsapart. In all,48 animals died from the infection.All hatchlings came from the samefarm in northern Queensland, Australia. Keywords Chrysosporium ,crocodilians,dermatomycosis, reptile infection Introduction disease in mammals[6], but these fungi are rarely implicated in reptile disease [1,7]. Afew reports of Jacobson et al [1] recently reviewed the published mycoticinfection in lacertilians and ophidians have reports of fungal infections in crocodilians. Most reports identied keratinophilic species of soil origin belonging of cutaneous and deep infections have incriminated to the genera Trichophyton and Chrysosporium [8–12] commonenvironmental moulds such as Fusarium or but there have been no prior reports of members of these Paecilomyces species, but causative agents are often genera infecting crocodilians [1,3,5,13,14]. However, incompletely identied and it can be difcult to evaluate infection by these keratinophilic fungi is difcult to whether the isolated fungus is present as acontaminant prove as they are commoninhabitants in the soil [6,15] or involved inapathologic process. There are few prior and may be carried as dormant propagules on the hairs, reports of fungal diseases among crocodiles in Australia skins or feathers of healthy mammals,reptiles and birds and mostconcern farm-reared animals. Paecilomyces [16–18]. Recently, the Chrysosporium anamorph of lilacinus [2] and Fusarium solani [3] were identied as Nannizziopsis vriesii has been identied as the cause of the cause of systemic infection in salt-water crocodile skin infections in three species of chameleons [7] and in hatchlings ( Crocodylus porosus ). Asurvey of farmed captive brown tree snakes [19]. We describe an outbreak salt-water ( C. porosus)and fresh-water ( C. johnsonii) of fatal mycoticdermatitis in saltwater crocodile hatchl- crocodiles identied fungi as asignicant cause of skin ings associated with this fungus [20]. These animals were lesions, but no single fungus was determined to be the part of atrial that investigated the use of pellets as a cause of infection [4,5]. Fusarium sp. was the most nutritional feed for crocodiles. commonfungus isolated, but isolates belonging to the genera Aspergillus, Penicillium , Curvularia, Syncepha- Case report lastrum, Candida and Trichosporon were also obtained. Dermatophytes are the predominant cause of cutaneous History Case 1 The Oonoonba Veterinary Laboratory (OVL),Towns- Correspondence:Annette D. Thomas, Oonoonba Veterinary Laboratory,P.O. Box 1085, Townsville, Queensland 4810, Aus- ville, has an environmentally controlled research facility tralia.Tel.: 617 47222604; fax: 617 47784307; e-mail: for studying the salt-water crocodile, C. porosus. There ‡ ‡ [email protected]. are six roomseach containing two sloping tanks allowing Ó 2002 ISHAM,ISHAM Medical Mycology , 40, 143–151 144 Thomas et al. for both land and water areas. Both air and water died over a23-day period before the infection was temperature can be maintained within set parameters. brought under control. In 1994, 247 hatchlings fromsix clutches (89, 90, 96, 99 hatched on 11 May; 102, 103 hatched on 24 May) were Case 2 transported in two deliveries to OVL.Animals were In 1997, at the farmfrom where the 1994 hatchlings were maintained in separate tanks for 4weeks to acclimatize obtained, 85 hatchlings from ve clutches were placed them to the conditions of water temperature at 32 oC together in the one pen for growth to juvenile stage. and air temperature at 30 oC.After this time, they were These were added to the pen on 9May (28 hatchlings), tagged, weighed and measured. The average weight of 12 May (51) and 19 May (6). Within amonth of the hatchlings was 61 3g. Several of the hatchlings from placement, 30 were dead with plaque-like lesions similar clutch 89 initially had white aky areas on the underside to those registered at OVLin 1994. The mortalities of the body and back of the thighs. During the rst occurred over a16-day period and peaked with 13 deaths month of acclimatisation, there was an electrical on the 12th day. Three live hatchlings with lesions were malfunction that resulted in adrop in the water sent to OVLfor examination. temperature of all tanks down to 22 oC overnight although the air temperature remained at 30 oC. The Necropsyand sampling fault was remedied the next morning. However, within Plaque-like lesions were removed as aseptically as 2–3 days of this problem, acreamy, caseous masswas possible and cultured for both bacteria and fungi. Dead noticed on, and occasionally under, the scales of the crocodiles were also necropsied and skin, heart, liver head, back and feet of several of the 34 crocodiles from and/or brain, were removed for culture and/or histo- clutch 89. Four animals were swabbed for culture. Within pathology. Visual observation by the handlers on the 7–14 days, the animals were more severely affected and farmduring the second case study indicated that the the infection took the formof aleathery, plaque-like lesions on the 33 animals that died were similar to those lesion that spread over the snout, under the jaw and observed in the 1994 outbreak at OVL. along the legs. Most of the animals in this tank (clutch Samples were macerated using aStomacher blender 89) were affected; however the crocodiles in the other (Stomacher Lab-Blender 80, Seward Medical, London, tank situated in the same environmentally controlled UK)and cultured onto blood agar and MacConkey agar room were unaffected. In all, 10 crocodiles died over a (Oxoid Australia Pty Ltd, West Heidelberg, Victoria, 14–16-day period. Twelve samples were collected for Australia), lysine–mannitol– glycerol (LMG)agar [21] culture and histopathology fromthese 10 animals; two and into buffered peptone water (Oxoid) to isolate animals were sampled twice at 3days and 7days after aerobic bacteria including Salmonella spp. The plates lesions were rstnoticed. The nutritional trial was were incubated at 37 oC.After 24 h, adrop of the delayed for 8weeks while treatment was introduced. buffered peptone water was inoculated into Rappaport– When the infection appeared to be under control, the Vassiladis broth (Oxoid) and 24 hlater, adrop of this tanks were cleaned with aracide (Rural West Pty Ltd, medium was inoculated onto another LMGplate. The Bentley, Western Australia, Australia) before re-use. plates were incubated at 37 oCfor 2days. Isolates were The trial recommenced with aweighing, measuring and puried where necessary and identied using conven- sorting of the animals by weight into 11 groups (six tional tests or kit tests such as the Microbact 24E groups containing animals fromclutches 89, 90 and 103 (Medvet Science Pty Ltd, Adelaide, South Australia, and ve groups containing those fromclutches 96, 99 and Australia) or API 20E (bioMe´ rieux, Marcy, l’Etoile, 102). These new groups were then distributed into France) systems. 11 tanks with water temperatures ranging from26 o to Samples were also plated onto Sabouraud dextrose 34 oCat intervals of 2 oC,and air temperatures ranging agar with added gentamicin and chloramphenicol (SDA) from 28 o to 32 oC (also at 2 oCintervals). This was a and mycosel agar with thiamine (MAT) (both Oxoid ) statistical trial and there were two replicates with water and incubated at 28 oCfor 10–14 days in plastic boxes to temperature at 26 oCand air temperature at 28 oC. prevent drying of the agar. Fungal isolates were One month after the trial had commenced, ve identied by microscopicexamination of tease and slide animals in one of the 26 oCtanks (which was adifferent culture preparations. tank fromthat where the original outbreak occurred) developed plaque-like lesions that were more severe Histopathology and electron microscopy than the previous time. Samples were taken for micro- biological culture. The 18 crocodiles in this tank had Tissues for histopathology were stained with either been mixed fromclutches 96, 99 and 102. Eleven animals haematoxylin and eosin (H&E)or Grocott–Gomori’ s Ó 2002 ISHAM, Medical Mycology , 40, 143–151 Nannizziopsisvriesii incrocodiles 145 methenamine silver stain (GMS). For scanning electron microscopy (SEM), aportion of the culture was xed in 2% osmiumtetroxide vapour and dried to the critical point. The material was examined with an S-2500 Hitachi SEM. Pathology Case 1 Grosspathology. The hatchling crocodiles developed leathery plaque-like lesions, mainly on the head, jaw and legs. These lesions progressed over a2-week period. Plaques reached sizes of 1–2 cmin length and these could be peeled off the skin leaving white to reddened areas underneath. Histopathology. The skin lesions consisted of epidermal hypertrophy and hyperplasia, with ballooning of