Allender Et Al., 2015)

fungal ecology 17 (2015) 187e196

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The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential impact on free-ranging snake populations

Matthew C. ALLENDERa,1, Daniel B. RAUDABAUGHb,d,1, Frank H. GLEASONc,*, Andrew N. MILLERd aDepartment of Comparative Biosciences, University of Illinois, Urbana, IL, USA bDepartment of Plant Biology, University of Illinois, Urbana, IL, USA cSchool of Biological Sciences, A12, University of Sydney, NSW 2006, Australia dIllinois Natural History Survey, University of Illinois, Champaign, IL, USA article info abstract

Article history: Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, is a serious emerging Received 17 December 2014 fungal pathogen of North American-endemic and captive snakes. We provide a detailed Revision received 4 April 2015 literature review, introduce new ecological and biological information and consider aspects Accepted 7 May 2015 of O. ophiodiicola that need further investigation. The current biological evidence suggests Available online 18 June 2015 that this fungus can persist as an environmental saprobe in soil, as well as colonizing living Corresponding editor: hosts. Not unlike other emerging fungal pathogens, many fundamental questions such as Matt Fisher the origin of O. ophiodiicola, mode of transmission, environmental inﬂuences, and effective treatment options still need to be investigated. Keywords: ª 2015 Elsevier Ltd and The British Mycological Society. All rights reserved. Emerging fungal pathogen Herpetology Massasaugas Pitvipers Snake fungal disease Wildlife diseases

Introduction Pseudogymnoascus destructans, causing white-nose syndrome in North American bat populations (Blehert et al., 2009). One of True fungal pathogens have been increasingly associated with the latest emerging fungal pathogens of vertebrate animal free-ranging wildlife epidemics. Two recently studied exam- populations, Ophidiomyces ophiodiicola, was ﬁrst observed in ples include the effects of chytridriomycosis, caused by 2006 affecting populations of pitvipers in the eastern United Batrachochytrium dendrobatidis, on global frog populations States (Clark et al., 2011), and later conﬁrmed in Eastern (Skerratt et al., 2007) and the asexual ascomycete, Massasaugas (Sistrurus catenatus) from Illinois in 2008

* Corresponding author. Tel.: þ61 2 9971 2071. E-mail address: [email protected] (F.H. Gleason). 1 This text was written initially by Matthew C. Allender and Daniel B. Raudabaugh with equal participation and subsequently edited by Frank H. Gleason and Andrew N. Miller. http://dx.doi.org/10.1016/j.funeco.2015.05.003 1754-5048/ª 2015 Elsevier Ltd and The British Mycological Society. All rights reserved. 188 M.C. Allender et al.

(Allender et al., 2011). With the current rate of decline in global biodiversity, it is apparent that wildlife diseases similar to those described above are serving as threats to population declines potentially resulting in species extinctions. Fisher et al. (2012) described a model or concept for analysis of emerging infectious fungal diseases in animal and plant populations and ecosystem health in general. This model is useful for the study of newly emerging highly viru- lent agents of disease such as the snake fungal pathogen, O. ophiodiicola (see Table 1). Ophidiomyces ophiodiicola (formerly Chrysosporium ophiodiicola) is currently placed in the order Onygenales (Ascomycota) within the family Onygenaceae(Sigler et al., 2013). This fungus is closely related to other species of Onygenaceae within the Fig 1 e Current known US distribution of O. ophiodiicola. Chrysosporium anamorph Nannizziopsis vriesii (CANV) complex States where O. ophiodiicola is known to occur are grey, that causes dermal lesions in reptiles (Sigler et al., 2013). The based on previous reports (Allender et al., 2011; Sleeman genus Ophidiomyces currently contains only one species, O. et al., 2014) and unpublished data (MCA). ophiodiicola, and it is known to infect only snakes leading to the syndrome Snake Fungal Disease (SFD) which causes wide- spread morbidity and mortality across the eastern United Since 2008, Eastern Massasaugas from the Carlyle Lake States (Fig 1)(Allender et al., 2013; Sigler et al., 2013; Sleeman, population in Illinois have been diagnosed with SFD 2013). (Fig 5AeD) (Allender et al., 2011, 2015). Free-range infected Historically, several case reports of skin disease in snakes massasaugas display skin lesions, which vary from pustules were confirmed (Pare and Jacobson, 2007) or suspected to be or nodules to severe swelling of the skin (Allender et al., 2011). CANV or CANV-like. However, since publication of the Infections in this species have been known to invade deep description of the genus Ophidiomyces in 2013 and with the use muscle tissue and infect muscle and bone (Allender et al., of advanced molecular techniques, it is now thought that 2011). Prior to 2014, lesions were thought to only affect the many of these infections may have been caused by O. ophio- head and neck of snakes, but lesions have now been observed diicola. An outbreak of fungal mycosis with clinical signs in the skin of the entire body in massasaugas in Michigan consistent with SFD was seen in pigmy rattlesnakes (S. mil- (Tetzlaff et al., 2015). In 2006, a population of timber rattle- iarius) in Florida in the early 2000’s, but neither Ophidiomyces snakes (Crotalus horridus) in New Hampshire was observed nor CANV were identified in that case (Cheatwood et al., 2003). with lesions on the head, neck, and body consistent with SFD This outbreak included 42 pigmy rattlesnakes, one garter (Clark et al., 2011). The authors hypothesized that environ- snake (Thamnophis sp.), and a ribbon snake (Thamnophis sau- mental conditions (increased rainfall) were likely the explan- ritus) within a 2 yr period, and retrospectively the authors ation for the increased prevalence and/or severity as no other identified an additional 59 pigmy rattlesnakes with signs lesions were noted in their follow-up study in 2009 (Clark consistent with mycotic disease (Cheatwood et al., 2003). et al., 2011). To date, all reports related to pitvipers have While these were not confirmed to be SFD at that time, it been consistently associated with dermatitis. highlights the need to utilize advanced molecular techniques While infections have been observed with great frequency for accurate identification of the etiological agent during in pitvipers (Cheatwood et al., 2003; Allender et al., 2011; Clark mycotic outbreaks. et al., 2011; Smith et al., 2013; Tetzlaff et al., 2015), there are numerous reports of CANV or SFD in nonvenomous colubrid snakes (Jacobson, 1980; Vissiennon et al., 1999; Nichols et al., 1999; Bertelsen et al., 2005; Rajeev et al., 2009; Dolinski et al., 2014). The manifestation of SFD in North American colubrids Table 1 e Properties of emerging infectious diseases of Ophidiomyces which fit the Fisher model snakes is variable and has included pneumonia, ocular infections, and subcutaneous nodules (Rajeev et al., 2009; Properties of emerging Ophidiomyces ophiodiicola infectious diseases Sleeman, 2013; Dolinski et al., 2014). A case of O. ophiodiicola was observed in a captive black rat snake (Elaphe obsoleta Propagules for rapid transmission Conidia obsoleta) with a subcutaneous nodule (Rajeev et al., 2009). In Threshold host population size Unknown addition, mycosis was observed in the skin as well as a more for disease persistence Host species resistant to disease Some host species systemic invasion involving the lungs and eye (one case) and Ecotypes with high virulence Yes lungs and liver (one case) of garter snakes (Vissiennon et al., Long-lived environmental stages Conidia and hyphae 1999; Dolinski et al., 2014). growing on detritus While the presence of the fungus causing infections in Generalist and opportunist Yes individuals is concerning, the role that it might play in pop- pathogens ulation declines is more alarming. A timber rattlesnake pop- Alternative hosts Yes ulation in New Hampshire consisted of 40 individuals pre- Sexual life cycle Not known in this species Transport of infected species International Animal Trade SFD; however, by the conclusion of that particular study, the entire population was reduced to 19 individuals (Clark et al., History, ecology, and epidemiology of O. ophiodiicola 189

Fig 2 e Morphology and carbon utilization of O. ophiodiicola. (A) Ophidiomyces ophiodiicola isolate on PDA at 25 d; left is the upper surface of the colony, right is the bottom of the plate. (B) Ophidiomyces ophiodiicola asexual reproduction via arthroconidia demonstrating schizolytic dehiscence and (C) stalked aleurioconidia. Ophidiomyces ophiodiicola growth at after 25 d on (D) autoclaved Carassius sp., (E) autoclaved Locusta migratoria and (F) autoclaved Lentinula edodes Growth of O. ophiodiicola after 20 d (G) demineralized Pleoticus muelleri exoskeletons and (H) demineralized and deproteinated exoskeletons. All size bars [ 5 mm.

2011). While many factors are affecting the conservation of this population, the occurrence of this pathogen may serve as Methods and materials an additional threat to its conservation. In this report we describe the ecology of O. ophiodiicola in Four Illinois O. ophiodiicola isolates (isolated in 2014) from the laboratory and consider aspects of this disease that various infection sites located on free range snakes were require further investigation. While the true global dis- examined: three from infected massasaugas (12-34933: iso- tribution of this fungus is unknown, it is possible that snake lated from facial region, 13-42282: isolated from skin/bone, fungal disease, if present only in North America, could be and 13-40265: isolated from caudal body) and one isolate (12- spread to other ecosystems around the world through the 33400: isolated from lung tissue) from an infected plains garter animal trade. A thorough understanding of this disease is of snake (T. radix). Isolates were maintained on Difco Sabour- paramount importance in managing free ranging snake pop- aud’s Dextrose agar (SDA) at room temperature. Unless noted, ulations especially with rapid global climate change. all media were sterilized at 121 C for 15 min, and wrapped 190 M.C. Allender et al.

Fig 3 e In vitro growth of Ophidiomyces ophiodiicola. (A) O. ophiodiicola demonstrating gelatinase activity. (B) Carbon enzymatic assays: PDA (control, bottom), Mn-dependent peroxidase (negative, left), chitinase (negative, top) and b-glucosidase (positive, right). (C) Carbon enzymatic assays: PDA (control, bottom), lipase using olive oil (left, positive), lipase using lard (top, positive) and lipase/esterase using Tween 80 (right, positive). (D) Keratinase assay: control left (negative) and inoculated right (positive). (E) Sole nitrogen source assays: left to right, columns 1e2 [ nitrate, columns 3e4 [ nitrite, columns 5e6 [ ammonium, columns 7e8 [ L-asparagine, columns 9e10 [ control with no nitrogen source, columns 11e12 [ uric acid. Rows A-D (12-34933), Rows E-H (12-33400), rows A, E (pH 5), rows B, F (pH 6), rows C, G (pH 7), rows D, H (pH 8). (F) Colony diameter of O. ophiodiicola isolates over the pH range of 5e11. Isolate colony diameters vary but growth pattern was consistent. Solid line represents mean values; dotted lines represent standard error of the mean.

with Paraﬁlm after inoculation. All assays were inoculated Analysis with actively growing mycelium, maintained in an upright position, and replicated twice at different times using two Matric potential was adjusted with polyethylene glycol 8 000 replicates each time unless otherwise noted. All carbon and [PEG] following the equation J ¼ 1.29[PEG]2T140[PEG]24.0 nitrogen assays were incubated under 24 hr darkness at room [PEG] where [PEG] ¼ gram of PEG 8 000 per gram of water and temperature and all agar based assays contained approx- T ¼ temperature (C) (Michel, 1983), spore counts were con- imately 20 ml of media. Unless noted, all chemicals utilized ducted with an improved Neubauer hemocytometer, pH within this experiment were purchased through Sigma measurements were conducted with a Milwaukee MW102 pH/ Aldrich or Fischer Scientiﬁc. temperature meter, and microscopic evaluations were History, ecology, and epidemiology of O. ophiodiicola 191

Fig 4 e Growth response of Ophidiomyces ophiodiicola to sulfur compounds and temperature. (A) O. ophiodiicola growth response to elevated levels of different sulfur compounds. (B) Effects of temperature on the growth of O. ophiodiicola and (C) representative colony from each temperature in Fig 3B. Left to right: 7 C, 14 C, 20 C, 25 C, 35 C. (A, B) Bars represent mean values; error bars indicate standard error of the mean.

conducted using an Olympus SZX12 stereo microscope. Data tolerance to sulfur compounds and environmental pH was driven charts were generated using GraphPad Prism version determined 25 d after inoculation by measuring the diameter of 5.03 for Windows, and the evaluation of colony growth at var- each colony twice at 90 angles and averaging the two values. ious temperatures was conducted in a Blue M dry type bacter- All isolate identities were veriﬁed using rDNA sequence iological incubator and a Lab-Line Ambi-Hi-Lo-chamber. Fungal comparison of the internal transcribed spacer (ITS) region. 192 M.C. Allender et al.

Fig 5 e Ophidiomyces ophiodiicola in vipers. (A) Active infection in the ocular region in a cottonmouth (Agkistrodon piscivorous). (B) Crusts within the scales in a massasauga (Sistrurus catenatus). (C, D) More advanced infection of the face and tail in a massasauga. Arrows indicate location of infection.

DNA was extracted from frozen mycelium by adding 200 ml SV Gel and PCR Clean-Up System (Promega)]. A BigDye Ter- 0.5 M NaOH. The mycelium was ground, centrifuged (14 000 minator 3.1 cycle sequencing kit (Applied Biosystems Inc.) was RPM for 2 min), and 5 ml of the resulting supernatant was used to sequence the ITS in one direction using the ITS5 pri- added to 495 ml 100 mM TriseHCl (pH 8.5e8.9) (Osmundson mer on an Applied Biosystems 3730XL high-throughput et al., 2013). PCR was completed on a Bio-Rad PTC 200 ther- capillary sequencer. Identity was confirmed through nBLAST mal cycler. The total PCR reaction volume was 25 ml (12.5 ml analysis. GoTaq Green Master Mix, (with 1 ml of each 10 mM primer ITS4 and ITSIF, 3 ml of the Tris-HCl-DNA extraction solution and Assays 7.5 ml DNA free water). The thermal cycle parameters were: (1) initial denaturation at 94 C for 2 min, (2) 94 C for 30 s, 55 C Whole carbon source assays were conducted on autoclaved for 45 s, 72 C for 1 min (30 cycles), and (3) a final extension insect (freeze-dried Locusta migratoria), fish (fresh Poecilia sp.) step of 72 C for 10 min. Gel electrophoresis (1 % TBE agarose and mushroom (Lentinula edodes) biomass. A representative gel stained with ethidium bromide) was used to verify the sample of each was placed into glass Petri plates and inocu- presence of a PCR product prior to PCR purification [Wizard lated with an autoclaved cotton swab that was pre-moistened History, ecology, and epidemiology of O. ophiodiicola 193

in a 1 % NaCl solution and rubbed over ca. 5 mm 5 mm area and 100 mg, respectively) to liquid medium cooled to 55 C of a sporulating culture. In addition, a portion of fresh exo- and poured into Petri plates. Colloidal chitin was added to skeleton of Pleoticus muelleri was subjected to a deminerali- another portion of the colloidal chitin basal medium to zation step and another portion was subjected to a produce a 2 % colloidal chitin overlay medium of which 2 ml demineralization and deproteination step (Rødde et al., 2007). was pipetted on top of the solidified basal medium. The The demineralization step consisted of two, 300 ml ice cold overlay was inoculated with a 5 mm agar plug, and exam- 0.25 M HCl treatments (one for 5 min and the second for ined twice per week for the presence of a clearing zone 35 min) followed by neutralization with distilled water and the around the colony. deproteination step incorporated three separate 100 ml (1.0 M Cellulase and manganese-dependent peroxidase activities NaOH) treatments at 95 C; the first and second treatments were assayed with esculin plus iron agar and Difco Potato 1 were 2 hr, and the third was for 1 hr followed by neutralization Dextrose Agar (PDA) supplemented with MnSO4 l , respec- with distilled water. The demineralized and deproteinated tively (Pointing, 1999; Overton et al., 2006). Esculin plus iron exoskeletons were placed in glass Petri plates, autoclaved, and agar consisted of 5 g L-tartaric acid diammonium salt, 0.5 g $ inoculated as above. All plates were visually monitored twice MgSO4 7H2O,1gK2HPO4, 0.1 g yeast extract, 0.001 g CaCl2, a week for the presence of mycelial growth and conidiation. 2.5 g esculin sesquihydrate and 8 g agar l 1 distilled water. The Proteinase activity was determined using gelatin as the medium was adjusted to pH 7.0 0.1 with 3 % KOH prior to sole carbon and nitrogen source. The medium consisted of sterilization. Filter sterilized (0.20 mm) aqueous ferric sulphate distilled water containing 3 % (pH 6.0 0.1) or 6 % (adjusted to (2 %) was added to 55 C medium at a ratio of 1 ml per 100 ml of pH 7.0 0.1 with KOH) gelatin (Knox original unflavored) and media. The medium was then poured into Petri plates, ino- 0.002 % phenol red as the pH indicator (Raudabaugh and culated with a 5 mm diameter agar plug and evaluated twice Miller, 2013). Each sterilized gelatin medium (150 ml) was per week for the appearance of a brownish black precipitate. pipetted into a separate UV-sterilized 96-well standard Manganese-dependent peroxidase was assayed by supple- 1 1 microplate and each test well was inoculated with a BB size menting 39 g PDA with 100 mg l and 200 mg l MnSO4. The pellet of scraped mycelium. The 96-well plates were covered medium was sterilized, inoculated as above, and examined with microtiter plate sealing film, incubated, and monitored twice per week for a black precipitate. daily for gelatin degradation (liquification) and pH change Lipase and esterase activity were assayed using Rhod- (color change from yellow below pH 6.8 to red/pink above pH amine B agar (8 g tryptone, 4 g NaCl, 0.001 % wt./vol. rhod- 8.2). amine B, 31.25 ml of sterile lipid (lard or olive oil) and 10 g agar Keratinase activity was determined using the following l 1 distilled water) and Victoria blue B agar (10 g tryptone, 5 g medium: 0.1 % glucose, 0.2 % yeast extract, 0.1 % KH2PO4, NaCl, 0.1 g CaCl2, 0.01 % wt./vol. Victoria blue B, 10 g sterile $ 1 0.02 % MgSO4 7H2O, 1 % Na2CO3 (autoclaved separately and Tween 80 and 15 g agar l distilled water) (Carissimi et al., added to achieve pH 8) and 1.5 % agar l 1 distilled water (Ito 2007; Rajan et al., 2011). Both media were adjusted to pH et al., 2005). Keratin azure was cut into small ca. 3 mm 7.0 0.1 with 3 % KOH prior to sterilization. The dye rhod- length sections, autoclaved separately in a dry beaker and a amine B was filter sterilized (0.20 mm) separately, while all portion of the cooled (55 C) basal medium was added to the lipid sources (lard, olive oil and Tween 80) were autoclaved keratin azure to produce a 1 % keratin azure overlay medium. separately, and both dye and lipid source were added to the Using sterile techniques, 5 ml of basal medium was added to appropriate basal medium cooled to 55 C. Each medium was 10 ml autoclaved glass test tubes. After the basal medium shaken vigorously for 1 min to emulsify the lipid, poured into solidified, 1 ml of the 1 % azure keratin overlay medium was Petri plates, inoculated with a 5 mm diameter agar plug, and pipetted into the glass test tubes. Each test tube was inocu- monitored twice a week for a positive reaction. A positive lated with a BB size pellet of scraped mycelium. Test tubes reaction for Rhodamine B agar was the appearance of orange were sealed with Parafilm and visually monitored weekly for fluorescence when exposed to UV light at 365 nm, while a dye release. positive reaction for Victoria blue B agar was the presence of Chitinase activity was determined using colloidal chitin insoluble long chain fatty acid calcium soap crystals. obtained from crab shell (Murthy and Bleakley, 2012). Col- Assays for sole nitrogen source were conducted for loidal chitin was obtained by grinding 20 g of crab shells into ammonium, L-asparagine (amino acid), nitrate, nitrite, urea a powder followed by acid digestion for 1 hr, at room tem- and uric acid. Stuart’s urea broth base (0.1 g yeast extract, 9.1 g m 1 perature, in 150 l of 12 M HCl under constant stirring. The KH2PO4, 9.5 g Na2HPO4, 0.012 g phenol red l distilled water chitin was precipitated in ice cold water followed by vacuum (pH 6.8 0.2)) and modified Christensen’s urea broth base (1.0

filtration (coffee filter: two layers). Neutralization was con- Tryptone, 2 g KH2PO4, 1 g dextrose, 5 g NaCl, 0.012 g phenol red ducted by wrapping the precipitate in multiple layers of l 1 distilled water (pH 6.8 0.2)) without urea were used as coffee filters and floating the wrapped precipitate in two control media. Filter sterilized (0.22 mm) urea (20 g l 1) was changes of distilled water (500 ml) at room temperature for a added to a portion of each cooled medium above to test for total of 24 hr. The colloidal chitin was unwrapped, auto- urease activity. An aliquot of each medium (150 ml) with and claved, and stored in a sealed container at 10 Cuntilnee- without urea was pipetted into a separate UV-sterilized well in ded. Colloidal chitin basal medium consisted of 0.7 g a 96-well standard microplate. For control purposes, only half $ K2HPO4, 0.3 g KH2PO4, 0.5 g MgSO4 7H2O, 0.5 g NaCl, 0.5 g of each of the medium types were inoculated with a BB size 1 FeSO4, 0.2 mg ZnSO4, 0.1 mg MnSO4 and 2 % agar l distilled pellet of scraped mycelium. The 96-well plate was covered water. Filter sterilized (0.20 mm) thiamine chloride and biotin with microtiter plate sealing film and was monitored daily solution was added (providing a final concentration of 5 mg (visually) for a change in color (yellow to pink) in both the 194 M.C. Allender et al.

inoculated and control wells. Ammonium, L-asparagine, accumulative linear growth of all the replicates at 15 d post nitrate, nitrite, and uric acid assays were conducted using the inoculation was reported. following basal media (Hacskaylo et al., 1954): 20 g D-glucose, $ 2gKH2PO4, 0.5 g MgSO4 7H2O, 0.5 g FeSO4, 0.2 mg ZnSO4, m m 0.1 mg MnSO4,5 g thiamine chloride, and 100 g biotin Results (0.20 mm ﬁlter sterilized and added to 55 C media) l 1 distilled water and one of the following ﬁve nitrogen sources: (1) 3.07 g In vitro, O. ophiodiicola isolates produced powdery to zonate

KNO3, (2) 2.0 g (NH4)2SO4, (3) 2.58 g KNO2, (4) 2.28 g asparagine yellowish-white colonies, with a reverse side that appeared 1 monohydrate, or (5) 1.275 g C5H4N4O3)l . After sterilization white to pale yellow (Fig 2A). All isolates of O. ophiodiicola and addition of the micronutrient solution, each medium pH reproduced asexually by arthroconidia (schizolytic dehis- was adjusted using a saturated solution of NaOH and 12 M cence) via sessile to stalked, hyaline to pale yellow aleur- HCl. Assays were conducted at pH 5, 6, 7, and 8 for basal media ioconidia with rhexolytic dehiscence (Fig 2BeC respectively). containing nitrate, nitrite, L-asparagine, ammonium, or no In vitro, O. ophiodiicola demonstrated robust growth on dead added nitrogen source (control) and at pH 8 for uric acid. An ﬁsh, dead insect, dead mushroom tissue (Fig 2DeF) and aliquot of each medium was pipetted into a separate UV- demineralized shrimp exoskeletons (Fig 2G), while sparse sterilized 96-well standard microplate well and inoculated as growth occurred on demineralized-deproteinated exoskel- stated above. The 96-well plate was covered with microtiter etons (Fig 2H). Ophidiomyces ophiodiicola isolates were positive plate sealing ﬁlm and monitored daily for mycelial growth. for gelatinase activity (with subsequent medium alkaliniza- Three tolerance assays were utilized to determine the tol- tion), b-glucosidase, lipase, lipase/esterase, and keratinase erance of O. ophiodiicola to pH, water availability (matric activity (Fig 3AeD respectively), while no positive results were potential) and environmental sulfur compounds. The pH tol- obtained for Mn-dependent peroxidase and chitinase activity erance assay consisted of 9 g Malt broth, 2.25 g tryptone, and (Fig 3B). Assays for sole nitrogen source demonstrated that O. 18 g agar per 900 ml of distilled water (Nagai et al., 1998). One ophiodiicola produced robust growth on ammonium sulfate of the following buffer solutions (100 ml) was added after and L-asparagine monohydrate under neutral to alkaline sterilization to achieve the following pH levels: pH 5 (6.9 g conditions (pH7 and pH8) compared to growth under the same $ $ NaH2PO4 H2O), pH 7 (3.904 g NaH2PO4 H2O and 3.105 g conditions on nitrate or nitrite (Fig 3E). The urease assay was

Na2HPO4), pH 9 (0.318 g Na2CO3 and 3.948 g NaHCO3), and pH positive for all isolates, while mycelial growth was sparse on

11 (5.3 g Na2CO3). After sterilization and addition of buffer, the uric acid (Fig 3E). Ophidiomyces ophiodiicola isolates grew over Petri plates were inoculated with one 5 mm diameter agar the entire pH range 5e11, with the largest colony diameter at plug and assayed for tolerance as described above. The matric pH 9 (Fig 3F), was tolerant of matric induced water stress potential assay medium consisted of 0.2 g sucrose, 0.2 g D- (5 MPa), and all isolates grew at elevated levels of L-cysteine, $ glucose, 1 L-asparagine, 1 g KH2PO4, 0.5 g MgSO4 7H2O, 0.5 g sulfate, sulfite and thiosulfate (Fig 4A). For all isolates, growth NaCl l 1 distilled water and adjusted to pH 7 with NaOH inhibition occurred at 7 C, significant growth reduction (Raudabaugh and Miller, 2013). The medium was amended occurred at 14 C, greatest growth occurred at 25 C, followed with PEG 8 000 as previously stated to generate the following by a significant reduction of growth at 35 C(Fig 4B and C). matric potentials at 20 C: 0.07 MPa, 1 MPa, 2.5 MPa, and 5 MPa. After sterilization, 40 ml of each medium was inoculated with a 500 ml spore suspension in water (average spore Discussion count ¼ 4.0 106 per 500 ml), sealed with parafilm, and placed on shake culture at 100 rpm for 25 d at 20 C. Tolerance was The effects of infectious diseases on wildlife populations are qualitatively assayed as either visible growth or no visible of increasing concern, especially for endangered species growth after 25 d. The nitrogen source (KNO3) from already persisting at small population sizes (Daszak et al., Raudabaugh and Miller (2013) was replaced with L-asparagine 2000). Furthermore, fungal pathogens of at least one species, because O. ophiodiicola lacked robust growth when nitrate was the sharp-snouted day frog (Taudactylus acutirostris), have led the sole nitrogen source (see Results). The environmental to an extinction event due to chytridiomycosis (Schloegel sulfur compound tolerance assays consisted of 39 g PDA et al., 2006). The impact Ophidiomyces has on snake pop- medium supplemented with 0.2 g FeSO4 and one of the fol- ulations is unknown, but understanding the ecology is the first $ lowing: Na2S2O3 5H2O, Na2SO3,or97%L-cysteine at 100 mg, step to determining the approach to management and char- 300 mg, 500 mg, and 700 mg l 1 distilled water. After steri- acterizing the epidemiology. lization, Petri plates were inoculated with one 5 mm diameter While the sexual stage of the life cycle has not yet been agar plug and assayed for tolerance as described above. encountered (Rajeev et al., 2009), O. ophiodiicola has been To determine the temperature range in which O. ophiodii- shown to reproduce asexually by arthroconidia with schizo- cola could grow, each isolate was grown on SDA at 7 C, 14 C, lytic dehiscence (Sigler et al., 2013) and asexually via sessile to 20 C, 25 C, 30 C and 35 C. Each isolate was assayed in sets of stalked, hyaline to pale yellow aleurioconidia with rhexolytic four replicates, with three temporal replications. The diame- dehiscence (Rajeev et al., 2009)(Fig 2BeC respectively). Several ter of each colony was measured 3 d post inoculation (spore factors support O. ophiodiicola occurring as an environmental inoculation) and the linear extension of each colony was saprobe: (1) its ability to utilize multiple complex carbon and measured every 2e3 d until 15 d post inoculation. The linear nitrogen sources, (2) its ability to tolerate pH and most natu- growth for each set of replicates was averaged to determine rally occurring sulfur compounds, and (3) its ability to tolerate the average growth of each isolate, and the average low matric potentials occurring in soil. In vitro, O. ophiodiicola History, ecology, and epidemiology of O. ophiodiicola 195

grew on a variety of dead substrata and had a broad comple- Tetzlaff et al., 2015), so the continued search for an effective ment of enzymes for saprotrophically utilizing many envi- treatment is of paramount importance. In addition, the mode ronmental carbon and nitrogen sources. Also, it tested of transmission and the influence of environmental triggers positive for urease activity, which has been proposed as a dual on prevalence of this disease are not understood. Further use virulence factor in other fungal pathogens (Casadevall research in these areas is necessary for a more thorough et al., 2003). Taken together, the data suggest that O. ophiodii- understanding of the dynamics of this disease. cola most likely infects snakes in an opportunistic manner. The data also suggest that the environmental presence of ammonium would be more beneficial for growth of O. ophio- Conclusions diicola than the presence of nitrate or nitrite. All isolates grew over a wide pH range (pH 5e11), and ele- The fungus O. ophiodiicola is acting alone or in conjunction vated levels of sulfur compounds found in soils and reptile with other pathogens to cause snake fungal disease (Allender skin did not inhibit the growth of O. ophiodiicola. Substratum et al., 2015; Sleeman, 2013). The threat to free-ranging snakes pH and the presence of elevated sulfur compounds are needs to be determined. Ophidiomyces was observed to be unlikely to inhibit its persistence in the environment. Sur- active at a range of temperatures and pH, in addition to its prisingly, O. ophiodiicola demonstrated tolerance to matric ability to utilize complex carbon, nitrogen, and sulfur induced water stress, which is one of the most limiting factors resources. These characteristics indicate this pathogen may for soil fungi (Marın et al., 1995). In general, soil fungi are be present in numerous ecosystems, and thus many snakes typically intolerant of matric induced water stress may be exposed to it. Future and current epidemiological below 3 MPa (Magan and Lynch, 1986; Deacon, 2006); how- surveys and ecological experiments that determine the extent ever O. ophiodiicola demonstrated growth down to 5 MPa of disease, species range, fungal characteristics, prevention indicating that soil matric potential will also be unlikely to strategies, and therapeutic options are greatly needed. inhibit its persistence in most soils. Several studies have indicated that temperature is a sig- references nificant factor affecting the growth of O. ophiodiicola in natural ecosystems, with inhibition occurring at 37 C(Rajeev et al., 2009; Sigler et al., 2013). Our data suggest that snake populations that hibernate in the lower part of the thermal range Allender, M.C., Dreslik, M., Wylie, S., Phillips, C., Wylie, D., of 0 Ce10 C(Macartney et al., 1989) should have less infec- Maddox, C., Delaney, M.A., Kinsel, M.J., 2011. An unusual mortality event associated with Chrysosporium in eastern tion during the spring than snake populations that hibernate massasauga rattlesnakes (Sistrurus catenatus catenatus). e in the upper part of the thermal range of 0 C 10 C. In addi- Emerging Infectious Disease 17, 2383e2384. tion, our data also suggest that with increasing global tem- Allender, M.C., Dreslik, M.J., Wylie, D.B., Wylie, S.J., Phillips, C.A., peratures, snake populations will be more vulnerable to O. 2013. Ongoing health assessment and prevalence of ophiodiicola infection in regions experiencing frequent mild Chrysosporium in the eastern massasauga (Sistrurus catenatus e winter conditions. catenatus). Copeia,97 102. 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