2016 Northeastern Naturalist Notes Vol. 23, No. 3 N18 M.J. Ravesi, S.J. Tetzlaff, M.C. Allender, and B.A. Kingsbury Detection of Snake Fungal Disease from a Lampropeltis triangulum (Eastern Milksnake) in Northern Michigan Michael J. Ravesi1,*, Sasha J. Tetzlaff 1, Matthew C. Allender2,3, and Bruce A. Kingsbury1 Abstract - An alarming number of cases of Snake Fungal Disease (SFD), a condition frequently resulting in morbidity and mortality in snakes, have been documented in numerous species across much of the eastern US. We sampled a skin lesion on the face of a free-ranging Lampropeltis triangulum (Eastern Milksnake) from the northern Lower Peninsula of Michigan. The lesion tested positive for Ophidiomyces ophiodiicola, the causative agent of SFD. Our results document the second species from Michigan known to be infected with SFD. This case adds to the growing body of literature detailing the distribution of snake species affected, and further indicates that this pathogen is widespread in the eastern US. We stress the continued need for increased, systematic sampling efforts to determine the species affected by SFD and the potentially deleterious impacts it has on snake populations. Several fungal pathogens have been documented to affect free-ranging wildlife in eastern North America. Pseudogymnoascus destructans (Blehert & Gargas) Minnis & D.L. Lindner, the causative agent of White-nose syndrome (WNS; Lorch et al. 2011), has led to mortality in millions of bats. On a regional scale, for example, WNS caused drastic population declines in New York (Frick et al. 2010). Similarly, Batrachochytrium dendrobatidis Longcore, Pessier & D.K. Nichols, which causes Chytridiomycosis, is associated with mass die-offs and extinction in amphibians globally (Skerratt et al. 2007), including population declines and extirpations in North America (e.g., Vredenburg et al. 2010). Ophidiomyces ophiodiicola Guarro, Deanna A. Sutton, Wickes & Rajeev, a fungal pathogen affecting freeranging North American snakes, is yet another example of an emergent host-specific threat. Ophidiomyces ophiodiicola has received considerable attention and research in recent years (reviewed in Allender et al. 2015a). It is related to other species in the Chrysosporium anamorph Nannizziopsis vriessi (CANV) complex—a group of fungi that are known to cause dermatitis in numerous reptilian taxa (Sigler et al. 2013). Skin conditions associated with CANV were initially reported in captive snakes (Nichols et al. 1999, Vissiennon et al. 1999), though similar symptoms were subsequently reported in 2006 from wild Crotalus horridus (L.) (Timber Rattlesnake) in an imperiled New Hampshire population (Clark et al. 2011). Researchers also noticed skin lesions and facial swelling in Sistrurus catenatus (Rafinesque) (Massasauga Rattlesnake) from a population in Carlyle Lake, IL in 2008, which prompted laboratory analysis confirming the presence of O. ophiodiicola in symptomatic individuals (Allender et al. 2011). In subsequent years, more cases of Snake Fungal Disease (SFD) have been reported in various species in geographically disparate locations throughout eastern North America. Numerous viperids have been documented either with clinical signs of SFD (Cheatwood et al. 2003, Clark et al. 2011) or confirmed O. ophiodiicola infection (Allender et al. 2011, 2015a; Smith et al. 2013; Tetzlaff et al. 2015). Simlarly, 1Department of Biology and Environmental Resources Center, Indiana-Purdue University Fort Wayne, 2101 East Coliseum Boulevard, Fort Wayne, IN 46805. 2Wildlife Epidemiology Lab, Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802. 3Illinois Natural History Survey, Prairie Research Institute, University of Illinois Urbana-Champaign, 1816 South Oak Street, Champaign, IL 61820. *Corresponding author - ravemj01@ipfw.edu. Manuscript Editor: Joseph Milanovich Notes of the Northeastern Naturalist, Issue 23/3, 2016 N19 2016 Northeastern Naturalist Notes Vol. 23, No. 3 M.J. Ravesi, S.J. Tetzlaff, M.C. Allender, and B.A. Kingsbury symptoms of SFD or confirmed O. ophiodiicola have also been documented in several colubrid species (Table 1). Although not the only fungus detected, O. ophiodiicola has been consistently observed in SFD infections, and it was recently confirmed as the causative agent of the disease (Allender et al. 2015b, Lorch et al. 2015). Ophidiomyces causes a range of symptoms including cutaneous ulcers, nodules, hyperkeratosis, and scale deformities; and it can damage deeper tissue in muscle and bone (Allender et al. 2011, Lorch et al. 2015, Sigler et al. 2013, Sleeman 2013). Although O. ophiodiicola infections are nearly always fatal in Massasauga Rattlesnakes, (Allender et al. 2011, Tetzlaff et al. 2015), Timber Rattlesnakes have been documented to recover naturally from the disease (Smith et al. 2013). SFD was first documented in Michigan in 2013 in 2 male Massasauga Rattlesnakes in the northern Lower Peninsula of Michigan (Tetzlaff et al. 2015). In 2014, SFD persisted in snakes at that site and was found at 2 additional Massasauga Rattlesnake research sites in southern Michigan (Allender et al. 2016). Prior to 2015, this species was the only one in Michigan confirmed to have SFD. We found a Lampropeltis triangulum (Lacépède) (Eastern Milksnake; snout-to-vent length = 57.8 cm, tail length = 10.0 cm) on 25 May 2015 in Grayling, MI at 44°40'12.72"N, 84°36'29.46"W, approximately 22 km east of the previously documented cases of SFD in northern Michigan (Tetzlaff et al. 2015). We captured the individual at a private property in sandy habitat dominated by Pinus banksiana (Lamb.) (Jack Pine). The snake had obvious localized thickening of the skin and facial swelling from a discolored, crusty lesion on the right side of the face consistent with clinical signs of SFD (Allender et al. 2011, Tetzlaff et al. 2015). We immediately sampled the lesion using sterile, wooden-handled, micro-tipped Table 1. List of colubrid species in the eastern US either presenting SFD symptoms or confirmed with O. ophiodiicola infection, from published reports. Captive Species State(s) or wild Pantherophis guttatus (L.) (Red Cornsnake) New York (Sigler et al. 2013) Captive Nerodia s. sipedon (L.) (Northern Watersnake) Ohio (Sleeman 2013), Wild Virginia (Guthrie et al. 2016) Thamnophis sirtalis (L.) (Garter Snake) Florida (Cheatwood et al. 2003) Wild Thamnophis s. sauritus (L.) (Common Ribbonsnake) Florida (Cheatwood et al. 2003) Wild Lampropeltis triangulum (Lacépède) (Eastern Milksnake) Wisconsin (Sigler et al. 2013), Wild New York (Sleeman 2013) Nerodia clarkia (Baird and Girard) (Saltmarsh Snake) Florida (Sigler et al. 2013) Wild Coluber constrictor (L.) (Black Racer) Virginia (Guthrie et al. 2016), Wild Florida (Sleeman 2013) Pantheropus obsoletus (Holbrook) (Rat Snake) Georgia (Rajeev et al. 2009), Wild New Jersey (Sleeman 2013) Thamnophis radix (Baird and Girard) (Plains Garter Illinois (Dolinski et al. 2014) Wild Snake) Farancia abacura (Holbrook) (Mud Snake) Georgia (Fenton et al. 2015) Wild Nerodia fasciata confluens (Blanchard) (Broad-banded Louisiana (Glorioso et al. 2016) Wild Water Snake) Farancia erytrograma (Latreille) (Rainbow Snake) Virginia (Guthrie et al. 2016) Wild Nerodia taxispilota (Holbrook) (Brown Watersnake) Virginia (Guthrie et al. 2016) Wild 2016 Northeastern Naturalist Notes Vol. 23, No. 3 N20 M.J. Ravesi, S.J. Tetzlaff, M.C. Allender, and B.A. Kingsbury cotton swabs. We collected 2 swab samples from the lesion, which was the only portion of the snake’s body that we swabbed; the individual was subsequently returned to its original capture location. We transferred the samples to 2-ml Eppendorf tubes and stored them at -20 oC until analysis. We conducted DNA extraction and quantitative PCR amplification (qPCR) tests for the presence of O. ophiodiicola according to Allender et al. (2015c). Briefly, qPCR was performed in triplicate on an ABI 7500 real-time thermocycler targeting a 68-bp segment of the ITS 1 region between the 18S and 5.8S rRNA gene of O. ophiodiicola. The samples were considered positive because all 3 replicates had a lower cycle-threshold value (Ct = 31.277 ± 0.217 SE; 79 fungal copies per reaction) than the lowest detected standard dilution, thus confirming the presence of O. ophiodiicola. Confirmed O. ophiodiicola infection (Sigler et al. 2013) or clinical signs of SFD (Sleeman 2013) have previously been observed in Eastern Milksnakes in other portions of its range. Individual observations such as the one described here continue to be reported for several species from geographically disparate and noncontiguous regions, providing additional evidence that this pathogen is widespread in eastern North America and afflicts a wide breadth of snake species. This observation further emphasizes the need for increased awareness of SFD symptoms by biologists, veterinarians, and resource managers. If coupled with a systematic sampling regime (Allender et al. 2013, 2015c), monitoring efforts will aid in determining the influence of the disease on individual behavior and survivorship, as well as population-level impacts. Acknowledgments. This work was conducted under an approved scientific collector’s permit from the Michigan Department of Natural Resources. Literature Cited Allender, M.C., M. Dreslik, S. Wylie, C. Phillips, D.B. Wylie, C. Maddox, M.A. Delaney, and M.J. Kinsel. 2011. Chrysosporium sp. infection in Eastern Massasauga Rattlesnakes. Emerging Infectious Diseases 17:2383–2385. Allender, M.C., M.J. Dreslik, D.B. Wylie, S.J. Wylie, J.W. Scott, C.A. Phillips. 2013. Ongoing health assessment and prevalence of Chrysosporium in the Eastern Massasauga (Sistrurus catenatus catenatus). Copeia. 2013:97–102. Allender, M.C., E. Hileman, J. Moore, and S. Tetzlaff. 2016. Detection of Ophidiomyces, the causative agent of snake fungal disease, in the Eastern Massasauga (Sistrurus catenatus) in Michigan, USA. Journal of Wildlife Diseases 52:694–698. Allender, M.C., D.B. Raudabaugh, F.H. Gleason, and A.N. Miller. 2015a. The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential impact on free-ranging snake populations. Fungal Ecology 17:187–196. Allender, M.C., S. Baker, D. Wylie, D. Loper, M.J. Dreslik, C.A. Phillips, C. Maddox, and E.A. Driskell. 2015b. Development of snake fungal disease after experimental challenge with Ophidiomyces ophiodiicola in Cottonmouths (Agkistrodon piscivorous). PloS ONE 10:e0140193. Allender, M.C., D. Bunick, E. Dzhaman, L. Burrus, and C. Maddox 2015c. Development and use of a real-time polymerase chain-reaction assay for the detection of Ophidiomyces ophiodiicola in snakes. Journal of Veterinary Diagnostic Investigation 27:217–220. Cheatwood, J.L., E.R. Jacobson, P.G. May, T.M. Farrell, B.L. Homer, D.A. Samuelson, and J.W. Kimbrough. 2003. An outbreak of fungal dermatitis and stomatitis in a free-ranging population of Pigmy Rattlesnakes (Sistrurus miliarius barbouri) in Florida. Journal of Wildlife Diseases 39:329–337. Clark, R.W., M.N. Marchand, B.J. Clifford, R. Stechert, and S. Stephens. 2011. Decline of an isolated Timber Rattlesnake (Crotalus horridus) population: Interactions between climate change, disease, and loss of genetic diversity. Biological Conservation 144:886–891. N21 2016 Northeastern Naturalist Notes Vol. 23, No. 3 M.J. Ravesi, S.J. Tetzlaff, M.C. Allender, and B.A. Kingsbury Dolinski, A.C., M.C. Allender, V. Hsiao, and C.W. Maddox. 2014. Systemic Ophidiomyces ophiodiicola infection in a free-ranging Plains Garter Snake (Thamnophis radix). Journal of Herpetological Medicine and Surgery 24:7–10. Fenton, H., L. Last, and J. Goyner-McGuire. 2015. Snake fungal disease in Georgia. Southeastern Cooperative Wildlife Disease Study, University of Geoergia. SCWDS Briefs 30:4–5. Frick, W.F., J.F. Pollock, A.C. Hicks, K.E. Langwig, D.S. Reynolds, G.G. Turner, C.M. Butchkoski, and T.H. Kunz. 2010. An emerging disease causes regional population collapse of a common North American bat species. Science 329:679–682. Glorioso, B.M., J.H. Waddle, D.E. Green, and J.M. Lorch. 2016. First documented case of snake fungal disease in a free-ranging wild snake in Louisiana. South eastern Naturalist: 15:N4–N6. Guthrie, A.L., S. Knowles, A.E. Ballmann, and J.M. Lorch. 2016. Detection of snake fungal disease due to Ophidiomyces ophidiodiicola in Virginia, USA. Journal of Wildlife Diseases: 52:143–149. Lorch, J.M., C.U. Meteyer, M.J. Behr, J.G. Boyles, P.M. Cryan, A.C. Hicks, A.E. Ballmann, J.T. Coleman, D.N. Redell, and D.M. Reeder. 2011. Experimental infection of bats with Geomyces destructans causes white-nose syndrome. Nature 480:376–378. Lorch, J.M., J. Lankton, K. Werner, E.A. Falendysz, K. McCurley, and D.S. Blehert. 2015. Experimental infection of snakes with Ophidiomyces ophiodiicola causes pathological changes that typify snake fungal disease. mBio 6:e01534–15. Nichols, D.K., R.S. Weyant, E.W. Lamirande, L. Sigler, and R.T. Mason. 1999. Fatal mycotic dermatitis in captive Brown Tree Snakes (Boiga irregularis). Journal of Zoo and Wildlife Medicine 30(1):111–118. Rajeev, S., D.A. Sutton, B.L. Wickes, D.L. Miller, D. Giri, M. Van Meter, E.H. Thompson, M.G. Rinaldi, A.M. Romanelli, and J.F. Cano. 2009. Isolation and characterization of a new fungal species, Chrysosporium ophiodiicola, from a mycotic granuloma of a Black Rat Snake (Elaphe obsoleta obsoleta). Journal of Clinical Microbiology 47:1264–1268. Sigler, L., S. Hambleton, and J.A. Paré. 2013. Molecular characterization of reptile pathogens currently known as members of the Chrysosporium anamorph of Nannizziopsis vriesii complex and relationship with some human-associated isolates. Journal of Clinical Microbiology 51:3338–3357. Skerratt, L.F., L. Berger, R. Speare, S. Cashins, K.R. McDonald, A.D. Phillott, H.B. Hines, and N. Kenyon. 2007. Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. EcoHealth 4:125–134. Sleeman, J. 2013. Snake fungal disease in the United States. Report to Natural Resource/Conservation Managers. Fungal Disease in the United States. US Geological Survey, Reston, VA. Smith, C.E., J.E. Edwards, and L.M. Lorch. 2013. Crotalus horridus (Timber Rattlesnake): Fungal pathogens. Herpetological Review 44:519–520. Tetzlaff, S.J., M. Allender, M. Ravesi, J. Smith, and B. Kingsbury. 2015. First report of snake fungal disease from Michigan, USA, involving Massasaugas, Sistrurus catenatus (Rafinesque 1818). Herpetology Notes 8:31–33. Vissiennon, T.H., O.F. Schüppel, E. Ullrich, E. Kuijpers, and F.A. Angela. 1999. A disseminated infection due to Chrysosporium queenslandicum in a Garter Snake (Thamnophis). Mycoses 42:107–110. Vredenburg, V.T., R.A. Knapp, T.S. Tunstall, and C.J. Briggs. 2010. Dynamics of an emerging disease drive large-scale amphibian population extinctions. Proceedings of the National Academy of Science USA 107:9689–9694.