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; Universityof AlbertaMicrofungus Collection,Devonian Botanic Garden, Edmonton, AlbertaT6G 2E1, y Canada; Edward RiverCrocodile Farm, P .O. Pormpuraaw,Queensland 4871,Australia z

The Chrysosporium anamorph of Nannizziopsisvriesii ,recentlyidentiŽ 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 identiŽed 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 identiŽed and it can be difŽcult to evaluate infection by these keratinophilic fungi isdifŽ cult 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 identiŽed as Nannizziopsis vriesii has been identiŽed 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 identiŽed fungi as asigniŽcant 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 fromŽ 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 Žrst 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 roomwere 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 Žrstnoticed. 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 puriŽed where necessary and identiŽed 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 Žve 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, Žve identiŽed 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 electronmicroscopy 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 Žxed 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 the epidermal cells. This tissue appeared necrotic and contained dead inammatory cells. There was loss of the germinal epithelium where the full depth of the epidermis had been destroyed. The dermal melanophore layer was absent or muchreduced beneath areas of epidermal necrosis. Minimal inammation was observed in the hypodermis. The GMSdemonstrated large numbers of hyaline, branching, septate fungal hyphae, Fig. 2 Asectionof the necrotic skin lesion from Figure 1 to demonstratecollections of arthroconidia (arrows). GMS. Scale bar, 2–4 m min width, in the necrotic tissue (Fig. 1). 15 m m. Arthroconidia, measuring 1 5–2 m min width and 2– 3 m min length, occurred in groups in the outer regions of Case 2 the necrotic areas (Fig. 2). The fungus was limited to the Grosspathology. The three juvenile crocodiles had focal necrotic epidermal tissue. In afew sites the fungus had skin lesions 1–2 cmin diameter on their ventral surfaces. penetrated through the dermis and hypodermis up to the The affected skin had adull appearance. When the edge of the muscle tissue. The liver was fatty and there necrotic epidermis was removed, awhite surface was left was some inammation around the portal triads. Other beneath. Lesions were observed on the under surfaces of tissues were not examined. the jaw, the abdomen, the tail, the limbs and the feet.

Histopathology. The epidermal lesions were similar to those in case 1above. However, in some lesions where there was only partial destruction of the epidermal layers, aheavy mononuclear cell inammation was often seen in the hypodermis (Fig. 3). Noother tissues were examined.

Microbiology Case 1 During the Žrstoutbreak, afungus superŽcially resem- bling Chrysosporium species or Trichophyton mentagro- phytes was isolated fromthe plaques removed fromall the 10 crocodiles that died throughout the infection Fig. 1 Asectionof skin showing a heavyinfestation of the period (Table 1). This fungus was relatively fast growing epidermiswith fungal hyphae. Note the lack of inammation in the on SDA with pale yellowish-white powdery colonies that hypodermis(H). GMS. Scalebar, 100 m m. formed small (3 m mdiameter), globose to clavate Ó 2002 ISHAM, Medical Mycology , 40, 143–151 146 Thomas et al.

fungi. No fungus was isolated fromany animal. Aero- monas hydrophila was isolated fromthe heart of four crocodiles; in two of these animals the Chrysosporium fungus was isolated fromskin plaques.

Case 2 The same Chrysosporium fungus was isolated from lesions on the head, abdomen, legs and feet of all three of the sick hatchlings sent tothe laboratory. However, F. solani was recovered also fromplaque samples taken fromtwo of the three hatchlings. No bacteria were cultured fromthe heart, liver or brain of these animals.

Mycology Fig. 3 Asectionof skin to show partial necrosis (N) ofthe epidermis.A heavymononuclear in ammatory cell response Three isolates of the Chrysosporium fungus obtained (arrow)is presentin thehypodermis. H&E. Scale bar, 100 m m. fromthe plaque lesions were referred to the University of Alberta Microfungus Collection and Herbarium (UAMH),Edmonton, Canada, where they were identi- conidia and arthroconidia. Pseudomonas aeruginosa was Žed as the Chrysosporium anamorph of N. vriesii, based isolated fromthe same animals. Different fungi and on comparison with prior isolates obtained fromreptile bacteria were isolated fromthe skin and plaque samples mycoses and with the ex-type culture of N. vriesii [7,19]. of only some hatchlings. No organisms were recovered The isolates were deposited as accession numbers fromthe heart, liver or brain. UAMH9664, 9665, 9666 and examined using methods In the second outbreak, among the 11 crocodiles that previously described [7,19]. They produced yellowish- died during the trial period, only Žve had indications of white, velvety to powdery, sometimes strongly zonate plaques on the head, back or feet. Cultures of these colonies reaching diameters of 3 5–3 8cmon potato lesions yielded afungus similar to that isolated during dextrose agar (PDA, Difco,Becton Dickinson Micro- the Žrstoutbreak (Table 2). Other fungi and bacteria biology Systems, Sparks, Maryland, USA) after 14 days were isolated fromskin samples of 10/11 animals (Table at 30 oC(Fig. 4). Growthwas slightly slower at 25 oC and 2). F. solani was isolated fromthe same Žve animals and no growth occurred at 37 oC.Other growth features 1 fromskin of Žve additional hatchlings, but this fungus, in included tolerance to cycloheximide at 400 m g ml¡ , a conjunction with P. lilacinus,was also isolated fromthe positive urease reaction within 5days, slow growth with backs of hatchlings in anumber of the tanks in various astrong clearing of milk solids and no pHchange after roomswhere the infection did not occur(data not 11 days on Bromcresolpurple– milk solids–glucose agar, shown). Samples of heart were taken fromeach of the and digestion of hairs with perforating bodies within crocodiles and cultured for aerobic bacteria and for 14 days [6,7,19]. Within 2–3 weeks of incubation at

Table 1 Fungaland bacterial isolates from skin plaques on 10 crocodile hatchlings belonging to clutch 89

Crocodile

602617 624 627 629 633 637 651 661 667

Fungi Chrysosporium anamorph of N. vriesii ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ F. solani ‡ ¡ ‡ ¡ ‡ ‡ ¡ ¡ ¡ P. lilacinus ¡ ¡ ¡ ¡ ‡ ¡ ¡ ‡ ‡ ¡ Bacteria Aeromonashydrophila ‡ ‡ ¡ ¡ ¡ ‡ ‡ ¡ ¡ ‡ A. sobria ¡ ¡ ¡ ¡ ¡ ‡ ‡ ¡ ¡ ¡ Pseudomonasaeruginosa ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ Serratiamarcescens ‡ ‡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ‡ Enterobacters ¡ ‡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡

Ó 2002 ISHAM, Medical Mycology , 40, 143–151 Nannizziopsisvriesii incrocodiles 147

Table 2 Bacterialand fungal isolates from skin lesions and heart removed from 11 crocodile hatchlings belonging to clutches 96 (hatchlings 403–461), 99 (hatchlings321– 390) and 102 (hatchling 769)

Crocodile

403413 427 441 461 321 326 343 363 390 769

SSSSSSSHSHSHSSH

Fungi Chrysosporium anamorph of N. vriesii ¡¡‡¡¡‡¡¡‡¡‡¡‡¡¡ F. solani ¡‡‡‡‡‡‡¡‡¡‡¡‡‡¡ Bacteria A. hydrophila ¡¡¡¡¡¡‡‡‡‡‡‡‡‡‡ Ps. aeruginosa ¡‡¡¡‡¡‡‡‡¡¡¡‡‡¡ Enterobacters ¡‡‡‡‡¡‡¡¡¡‡‡‡‡¡ Plaques ¡¡‡¡¡‡¡¡‡¡‡¡‡¡¡

S,skinlesion or skin swab; H, heartsamples yielding positive cultures; no bacteria or fungi were isolated from heart samples of otherhatchlings.

30 oC,colonies on PDAand on oatmeal–salts agar [6] Treatment began to develop globose, buff-coloured ascomata-like Case 1 structures visible to the naked eye (Fig. 5). Microscopic All 12 tanks in the six environmentally controlled rooms examination revealed that these structures, sometimes were receiving formalin in the water at the rate of called pseudogymnothecia, were composed of branched 0 003% to control the growth of Fusarium spp. and P. asperulate hyphae surrounding masses of conidia (Fig. lilacinus which are known to occuras contaminants on 6). No ascospores were produced in cultures held for crocodiles. After the lesions were recognized as aserious several months or when crocodile isolates were mated threat to the hatchlings, the Žrsttreatment was asalt- with each other. Conidia (aleurioconidia) formed on the water bath for 7min; however, this did not have any sides of the hyphae (sessile) or on swollen cells either on great effect and only irritated the crocodiles. The best the vegetative hyphae (Fig. 7) or on the hyphae treatment was acombination of removal of the plaques composing the pseudogymnothecium (Fig. 6). The followed by swabbing of the reddened area with the conidia were subglobose, hyaline, smooth and single- iodine-based antiseptic Betadine (Faulding Pharmaceu- celled. Arthroconidia were also formed, sometimes from ticals, Salisbury, South Australia, Australia). The croco- undulate lateral branches (Fig. 7). diles were allowed to dry out in containers for several hours before placing them back into the tank that had been cleaned out with aracide. Formalin was then added

Fig. 5 Close-upview of the Chrysosporium anamorph of Fig. 4 Colonyof the Chrysosporium anamorph of Nannizziopsis Nannizziopsisvriesii onpotato dextrose agar after 2 monthsgrowth vriesii onpotato dextrose agar after 21 daysat 30 o C (UAMH at 30 oC.Notethe presence of numerous globose infertile ascomata 9664). (pseudogymnothecia)(UAMH 9665). Ó 2002 ISHAM, Medical Mycology , 40, 143–151 148 Thomas et al.

Fig. 6 Apseudogymotheciumof the Chrysosporium anamorph of Fig. 7 The Chrysosporium anamorph of Nannizziopsisvriesii Nannizziopsisvriesii examinedby SEM. (a) A clusterof globose inslide culture preparations examined by light microscopy. conidiaamong asperulate hyphae (UAMH 9665). Scale bar, 7 m m; (a)Globose to pyriform aleurioconidia formed on branched hyphae (b)globose conidia formed sessile (on the sides of thehyphae) or on (UAMH9664). (b) lateral undulate hyphae (9665). (c) divisionof swollencells (9665). Scale bar, 3 m m. hyphaeinto arthroconidia (9664). Scale bar, 20 m m.

Ó 2002 ISHAM, Medical Mycology , 40, 143–151 Nannizziopsisvriesii incrocodiles 149 at the increased rate of 0 013% twice per day. The in the infected tank that did not show signs of lesions healed patches on the survivors remained of adarker hue either visually or by histopathology. F. solani and P. for some time and then faded to the normal coloration of lilacinus are commoncontaminants in the warm,humid the skin. environment of crocodile pens [5] and are especially After the second outbreak, the same disinfection evident in situations where meat and chicken heads are routine was used as for the Žrstoutbreak. However, used as afood source. Fat fromthe food lays on the because of the secondary infections with Aeromonas water and oor of the tank and transfers to the crocodile, hydrophila,one dose of neomycin at 0 2 ml per 100 g thus leaving acovering layer of material suitable for the crocodile followed the next day and then 5days later growth of the fungi. with 0 25 mlper 100 gcrocodile of long-acting tetra- Nannizziopsis vriesii is an ascomycetous fungus cycline were also required to eliminate the problem. classiŽed in the order Onygenales, family Onygenaceae [27,28]. The species was described originally for an Case 2 isolate obtained fromskin and lungs of alizard and is Although the farmhad been notiŽed after the Žrst represented currently only by one other isolate fromsoil outbreak of the successful use of Betadine and formalin in California [27,29]. Both isolates produce the tele- to minimize the number of deaths due to this fungus, no omorph when grown on sporulation media at the o treatment was attempted during the period of time that optimum temperature of 30 C[7]. The gymnothecia deaths were occurring. composed of branched asperulate hyphae are indistin- guishable fromthe pseudogymnothecia produced by the crocodile isolates except that they contain globose ascospores rather than masses of conidia. Additionally, Discussion N. vriesii demonstrates a Chrysosporium anamorph, with Fungi are commoninhabitants of soil and can cause growth habit and physiological proŽle similar to that of opportunistic diseases in reptiles [1–5,8,9,11,12,22]. Fungi the fungus described here frominfected crocodiles, and can enter through wounds, cutsand abrasions and are a fromskin and deep infection in chameleons [7] and particular problem during the Žrststages of growth from brown tree snakes [19]. Itis not uncommon for the egg [23]. Some can easily pass acrossthe egg heterothallic onygenalean fungi to formpseudogym- membrane just after the egg is laid, causing death or nothecia in the absence of acompatible mating partner malformations of the embryos [24–26]. Trapping, hand- [6,27] but matings among reptile isolates have failed, ling, temperature changes, overcrowding and transport thus far, to yield ascomata in vitro (this study, are just some of the stressorsof reptiles that can allow unpublished results). Sexual reproduction in fungi is opportunistic pathogens to cause disease [8,22]. Because inuenced by both genetic (requiring compatible mating different environmental organisms can be cultured from partners among heterothallic species) and physical skin and tissues of reptiles with infections, it can be parameters (culture conditions, medium constituents, difŽcult to establish the cause of infection. temperature, light, etc.). The conditions required for There are several pieces of evidence to suggest that development of the teleomorph among this group of the Chrysosporium anamorph of Nannizziopsis vriesii reptile isolates are still being investigated. In absence of was involved in this disease outbreak. This fungus was the teleomorph, it is difŽcult to conŽrm their identiŽca- the only organism associated with all animals with tion as N. vriesii.For this reason, we are using the plaque-like skin lesions and it was not isolated from anamorphic name pending further investigation of any hatchling in which these lesions were not observed. relationship using molecular methods. The histopathology showed hyaline, septate, branched The source of the outbreaks isnot clear. The infection hyphae that inŽltrated into the epidermis and occasion- occurred on two separate occasions during the Žrstcase. ally into the dermis and hypodermis layers in all infected The Žrstoutbreak was restricted to one clutch: clutch 89. animals. Although these hyphae could not be distin- Itis possible that the fungus occurred in the soil of the guished fromthose of Fusarium or Paecilomyces species, nesting material and contaminated the eggs as they were the presence of arthroconidia (Fig. 2) has been reported being laid. Whether the hatchlings were infected by the previously in infections involving this Chrysosporium fungus crossing the egg membrane, as has occurred with species [19]. The two other fungi that were found to be Fusarium species in reptiles [3,23], or as they discarded commonly present on the backs of the hatchlings in the their shell, isnot known. As there were no isolations of tanks – F. solani and P. lilacinus –were also found on the the fungus frominternal organs of the crocodiles, the backs of hatchlings in other tanks where the infection did latter premise is more likely. Transport of the 2-day-old not occur. These fungi were isolated also fromcrocodiles hatchlings to OVLmay have triggered the infection. Ó 2002 ISHAM, Medical Mycology , 40, 143–151 150 Thomas et al.

However, in the Žrstcase, the electrical failure and the species (Varanus sp. and Egernia bungana ) [16] and a resulting drop in temperature is likely to have exacer- high prevalence of Chrysosporium species has been bated the problem. The treatment regime of Betadine on recorded fromhairs of animals without lesions [16] and the deplaqued areas followed by formalin treatment in fromfeathers of domestic and wild birds [17,18], the water overcame the problem in this clutch and none accounting for 11 1% of fungi isolated frommammals of the surviving members of clutch 89 relapsed during and reptiles, 21 8% of fungi fromnative birds and 84 8% the remainder of the trial. of fungi fromdomestic birds [18]. However, none of The second outbreak (involving crocodiles from these animals or reptiles showed signs of infection. clutches 96, 99 and 102) was more severe and occurred Chrysosporium species have also been isolated fre- in one of the two tanks in the trial held at the lowest quently fromsoils in Queensland and Papua New water temperature of 26 oC.Fungal infections can be Guinea, especially in animal pastures and areas used triggered by lower than ideal temperatures and in these by birds [15,18]. In an attempt toascertain the environ- cases they often involve the skin [30]. Ofthe 11 animals mental source of the Chrysosporium fromthe present submitted for culture fromthis tank, only Žve showed outbreak, cultures were obtained fromcrocodile nests lesions compatible with those observed in the Žrst fromthree farmsin northern Queensland, as well from outbreak. The low-temperature stresshere was enough shells of two crocodile eggs fromdifferent farms.These to also allow abacterial septicaemia due to A. hydro- samples grew Chrysosporium indicum (unpublished phila to cause some of the deaths. Since the second results) [6]. outbreak occurred in adifferent tank to the Žrst Ithas been suggested that the Chrysosporium ana- outbreak and also with crocodiles fromdifferent clutches morph of N. vriesii can readily infect reptilian tissues than the Žrst,it is interesting to speculate on the origin of [7,19]. This could occurthrough cutsand scratches the infection. Only animals in clutches 96 (1/5) and 99 (4/ especially at times of stress. We suggest that the stressof 5) were infected and these animals were transported handling and transport of the hatchlings to the labora- down to OVLwith clutch 89. Clutch 102 that had tory followed by an electrical failure resulting in a animals dying frombacterial septicaemia was trans- marked drop in the water temperature led to infection ported at alater time. The crocodiles in the remaining with this fungus inthe Žrstcase. Crocodiles in captivity tank held at awater temperature of 26 oC were from tend to collect as agroup under ‘hide boards’and can clutches 89, 90 and 103 and showed no signs of fungal stack two to four deep. This is an ideal situation to allow disease during the trial. for the transmission of the arthroconidia that may be Because the nesting data for the farmwas unavailable erumpent through the epidermis of infected animals for the 1994 season, it is impossible to say whether nests (Fig. 2). Overcrowding and environmental factors may 89, 96, 99 and 102 were situated close together and/or have contributed to the second outbreak. As well the had asimilar nest composition. The eggs fromnests 96 fungus appears to be persistent in the environment of and 99 may have come in contact with the fungus in the these farmed animals as evidenced by arecent case soil but it ismore likely the hatchlings were infected observed while this manuscript was in review. Two of six during transport with the hatchlings fromclutch 89. hatchlings sent fromthe same farmdied on route to In the second case, it was impossible to determine to OVLand were examined by culture and histopathology. which clutch an infected crocodile belonged because The Chrysosporium anamorph of N. vriesii was isolated hatchlings fromŽ ve clutches had been penned together in pure culture fromskin plaques of one of these animals during the outbreak. The individual crocodiles were not and hyphae were observed in the necrotic epidermis. identiŽed to clutch. One commonfeature between the 1994 and 1997 outbreaks was that all the affected clutches were laid in Acknowledgements February and hatched in May. The optimal growth of the Chrysosporium anamorph of N. vriesii is 30 oC with a The authors wish to thank R.Jack for his dedication in maximum near 37 oC[7,19, this study]. February is one looking after the crocodiles, L.Kulpa for histology of the hottest and mosthumid of months in northern techniques, M.Chen for assistance with scanning elec- Queensland and therefore the conditions at lay could tron microscopy, and A.Flis for technical assistance. predispose for growth of the fungus. Funding for this work came fromthe Rural and The Chrysosporium anamorph of Nannizziopsis vriesii Industrial Research and Development Council, Austra- has not been reported previously fromAustralia. lia, and fromgrants fromthe Natural Sciences and However, an unidentiŽed Chrysosporium species has Engineering Research Council of Canada and the been reported fromAustralia fromscales of two lizard University of Alberta Small Faculties Fund.

Ó 2002 ISHAM, Medical Mycology , 40, 143–151 Nannizziopsisvriesii incrocodiles 151

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