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Experimental Infection of Snakes with Ophidiomyces ophiodiicola Causes Pathological Changes That Typify Snake Fungal Disease Jeffrey M. Lorch,a Julia Lankton,a Katrien Werner,a Elizabeth A. Falendysz,a Kevin McCurley,b David S. Bleherta U.S. Geological Survey, National Wildlife Health Center, Madison, Wisconsin, USAa; New England Reptile Distributors, Plaistow, New Hampshire, USAb ABSTRACT Snake fungal disease (SFD) is an emerging skin infection of wild snakes in eastern North America. The fungus Ophidiomyces ophiodiicola is frequently associated with the skin lesions that are characteristic of SFD, but a causal relationship between the fungus and the disease has not been established. We experimentally infected captive-bred corn snakes (Pantherophis guttatus) in the laboratory with pure cultures of O. ophiodiicola. All snakes in the infected group (n ⴝ 8) developed gross and microscopic lesions identical to those observed in wild snakes with SFD; snakes in the control group (n ⴝ 7) did not develop skin infections. Furthermore, the same strain of O. ophiodiicola used to inoculate snakes was recovered from lesions of all animals in the infected group, but no fungi were isolated from individuals in the control group. Monitoring progression of lesions throughout the experiment captured a range of presentations of SFD that have been described in wild snakes. The host response to the infection included marked recruitment of granulocytes to sites of fungal invasion, increased frequency of molting, and abnormal behaviors, such as anorexia and resting in conspicuous areas of enclosures. While these responses may help snakes to fight infection, they could also impact host fitness and may contribute to mortality in wild snakes with chronic O. ophiodiicola infection. This work provides a basis for understanding the pathogenicity of O. ophiodiicola and the ecology of SFD by using a model system that incorporates a host species that is easy to procure and maintain in the laboratory. Skin infections in snakes, referred to as snake fungal disease (SFD), have been reported with increasing frequency in wild snakes in the eastern United States. While most of these infections are associated with the fungus Ophidiomyces ophiodiicola, there has been no conclusive evidence to implicate this fungus as a primary pathogen. Furthermore, it is not understood why the infections affect different host populations differently. Our experiment demonstrates that O. ophiodiicola is the causative agent of SFD and can elicit pathological changes that likely impact fitness of wild snakes. This information, and the laboratory model we describe, will be essential in addressing unresolved questions regarding disease ecology and outcomes of O. ophiodiicola infection and helping to conserve snake populations threatened by the disease. The SFD model of infection also offers utility for exploring larger concepts related to comparative fungal virulence, host response, and host-pathogen evolution. IMPORTANCE Received 10 September 2015 Accepted 26 October 2015 Published 17 November 2015 Citation Lorch JM, Lankton J, Werner K, Falendysz EA, McCurley K, Blehert DS. 2015. Experimental infection of snakes with Ophidiomyces ophiodiicola causes pathological changes that typify snake fungal disease. mBio 6(6):e01534-15. doi:10.1128/mBio.01534-15. Editor Joseph Heitman, Duke University Copyright © 2015 Lorch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Jeffrey M. Lorch, E merging diseases represent significant threats to the conservation of many wildlife species. Over the past several decades, the number of emerging fungal diseases—and the number of species extinctions and extirpations caused by those diseases— has greatly increased (1, 2). The large-scale declines and extinction events caused by the chytrid fungus Batrachochytrium dendrobatidis (3, 4), and the bat pathogen Pseudogymnoascus destructans (the cause of white-nose syndrome [5–7]) highlight the devastating impacts that fungal diseases can have on wild host populations. Due to the ability of many fungi to infect ectothermic hosts whose populations are not well-monitored, fungal diseases in cold-blooded vertebrates may go largely unnoticed until their effects become pronounced. In 2006, the first documented occurrence of population declines associated with skin infections in snakes was reported in New Hampshire (8). Allender et al. (9) subsequently reported fungal infections in a population of massasauga rattlesnakes (Sistru- November/December 2015 Volume 6 Issue 6 e01534-15 rus catenatus) that threatened the viability of the species in the state of Illinois. Conservation concerns over these two imperiled snake populations brought attention to an infection that later became known as snake fungal disease (SFD). While the history of the infections in wild North American snakes remains unclear, mounting evidence indicates that SFD represents an emerging disease that poses a threat to wild snakes in the eastern United States (9–12). Snake fungal disease has been routinely attributed to infection by the fungus Ophidiomyces ophiodiicola (reviewed in reference 12). Ophidiomyces was formerly classified as the Chrysoporium anamorph of Nannizziopsis vriesii species complex (CANV species complex), a group of fungi that are frequently associated with emerging infections in various groups of reptiles (reviewed in reference 13). Recent phylogenetic analyses have demonstrated that CANV represents a species complex that also includes fungi of the genera Nannizziopsis and Paranannizziopsis and that Ophidiomy- ® 1 Downloaded from on January 15, 2016 – Published by crossmark RESEARCH ARTICLE ces is known to occur only on snakes (13–15). However, a clear cause-and-effect relationship between O. ophiodiicola and SFD is lacking. Specifically, it is unclear whether O. ophiodiicola is the cause of the skin lesions that characterize SFD or is simply an opportunistic invader of necrotic tissue. Bohuski et al. (16) reported that when sensitive detection methods were utilized (i.e., real-time PCR), O. ophiodiicola was occasionally found on the skin of snakes without clinical signs of SFD, suggesting that presence of the fungus is not strictly correlated with disease. Direct evidence is needed to establish O. ophiodiicola as a primary pathogen and to demonstrate a causal relationship between O. ophiodiicola and SFD. Along with uncertainty over the cause of SFD, little is understood about progression of the disease and how it influences individual and population fitness. Reported clinical signs associated with purported O. ophiodiicola infections are highly variable and are often recorded at a single time point, providing little information on the affected animal’s history, disease development over time, mechanisms of the host for coping with the infection, and indirect consequences on snake health and survival. Allender et al. (9) reported 100% mortality in Illinois massasauga rattlesnakes that had SFD, and Clark et al. (8) documented over a 50% decline in a population of timber rattlesnakes (Crotalus horridus) following the appearance of clinical signs consistent with SFD. However, the disease has been reported in other areas with apparent resolution of clinical signs and with no obvious impacts to the infected populations (17, 18). Clearly, the ecology of SFD and outcomes of the disease at individual and population levels are complex. Development of a laboratory model will be essential for studying these infections in a controlled manner to better define the intricacies of SFD dynamics. To determine whether O. ophiodiicola is capable of serving as a primary pathogen, we attempted to fulfill Koch’s postulates (19) by exposing captive-bred corn snakes (Pantherophis guttatus) to an isolate of the fungus cultured from a wild snake diagnosed with SFD. In addition to recreating lesions consistent with SFD, we also observed host responses that better elucidate how snakes respond to the infection and how these physiological and behavioral changes may influence disease dynamics in wild populations. Together, this work lays a solid foundation upon which future research directed toward understanding the pathogenicity of O. ophiodiicola and the ecology of SFD can be developed. RESULTS Ophidiomyces ophiodiicola causes SFD. Snakes exposed to O. ophiodiicola developed clinical signs consistent with SFD 4 to 8 days postinoculation. Initial lesions consisted of generalized regional swelling and increased pallor of scales (Fig. 1D). Swelling on the snout often resulted in occlusion of the nares and, in a couple of instances, misalignment of the upper and lower mandibles. Swelling at inoculation sites on the body was less common and more transient than that observed on the snout. Following regional swelling, individual scales became edematous as immune cells infiltrated the specific sites of infection. As the lesions progressed, scales became thickened and yellow (Fig. 1E) and eventually developed rough, brown crusts with hyperpigmentation (Fig. 1F). Lesions often began at the edges of individual scales and frequently coalesced to involve several adjacent scales. Two snakes in the infected group (25%) exhibited bouts of anorexia. Skin lesions on infected snakes became progressively larger and 2 ® more severe, until the animals shed on days 15 to 20. Several days prior to molting, fluid-filled vesicles developed that encompassed, and extended outward from, the inoculation sites (Fig. 1I); on the snout, accumulation of fluid between old and new layers of skin sometimes resulted in severe distortion of the head (Fig. 1H). Molted skins from snakes in the infected group were often bunched up with areas of the shed adhering to one another, and fragments of the molt were occasionally retained on the new skin (dysecdysis). On the molted skin of infected snakes, lesions were clearly visible as brown crusts. Lesions were grossly resolved upon completion of ecdysis; however, some previously affected scales were shrunken, deformed, or slightly depigmented. Reexposure to O. ophiodiicola resulted in recurrence of gross lesions. Throughout the study, snakes in the control group remained healthy (Fig. 1A and B). Control snakes occasionally exhibited minor damage to scales at sham inoculation sites (Fig. 1C). These changes were distinct from the discolored, thickened skin that developed in infected snakes and appeared to be the result of mechanical damage due to abrasion of the skin (see Materials and Methods) or removal of the adhesive portion of the bandage. The shedding process occurred normally in the control group with no abnormalities of the molted skin and no cases of dysecdysis. The presence of lesions indicative of SFD was significantly different between the infected and control groups (P ⬍ 0.001). At necropsy, all eight snakes in the infected group had gross lesions consistent with SFD. Of the 40 sites inoculated (five inoculation sites on eight snakes), 34 had gross lesions. Lesions consistent with SFD occurred more frequently at sites where the skin had been abraded (100%) prior to application of fungal conidia compared to nonabraded inoculation sites (62.5%). As the dose of conidia increased with subsequent inoculations, gross skin lesions appeared to be more likely to develop at nonabraded inoculation sites, but the dose or number of inoculations did not noticeably impact the severity of the lesion. All snakes were in good body condition with adequate fat stores at the time they were euthanized. Microscopically, significant skin lesions in infected snakes included discrete areas of necrosis and granulocytic inflammation in the superficial to midepidermis, with adjacent epidermal granulocytic inflammation and edema (Fig. 2D and E). Necrotic portions of the epidermis often contained a few to many 2- to 5-␮mwide, parallel-walled, occasionally septate, rarely branching, periodic acid-Schiff (PAS) stain-positive and Grocott’s methenamine silver (GMS) stain-positive fungal hyphae. In one animal, rectangular arthroconidia measuring approximately 2 by 5 ␮m were noted on the surface of the stratum corneum (Fig. 2H). Mild to moderate dermal and intramuscular inflammation ranging from granulocytic to mononuclear was consistently present in infected snakes and was typically more severe under areas of necrosis (Fig. 2E). A few snakes exhibited dermal granulomas (Fig. 2G) which occasionally contained fungal hyphae; granulomas were most common on the head but were also noted within the neck and chin of one snake each. The microscopic lesions and morphology of the fungus were consistent with those observed in wild snakes with infections associated with O. ophiodiicola (Fig. 2J, K, and L) (11, 20, 21) and corresponded to the presence of gross lesions observed at necropsy. Small gaps in the stratum corneum of both control and infected animals were almost exclusively present on areas of skin that had been abraded. In snakes exposed to O. ophiodiicola, breaks in the stratum corneum were more com- November/December 2015 Volume 6 Issue 6 e01534-15 Downloaded from on January 15, 2016 – Published by Lorch et al. FIG 1 Clinical signs of SFD in snakes experimentally challenged with Ophidiomyces ophiodiicola. (A to C) Sham-inoculated sites of snakes in the control group did not develop gross lesions characteristic of SFD. However, subtle damage to the scales (arrow) caused by the abrasion process was visible at the dorsal midbody site. In contrast, snakes exposed to O. ophiodiicola developed a range of clinical signs as the disease progressed. (D) Initially, individually infected scales were swollen and whitened (arrow). (E and F) Infected scales later became thickened and turned yellow to brown (E), eventually forming crusts of necrotic skin (F). (G) Infected skin on the snout became similarly thickened and yellow-brown. (H and I) Immediately prior to shedding, fluid accumulated between the old and new layers of skin, causing distortion of the head (H) and vesicle formation at inoculation sites on the body (I). (J to L) The presentations observed in experimentally infected snakes were consistent with those observed in wild snakes diagnosed with SFD at the U.S. Geological Survey National Wildlife Health Center, which often included thickened, yellow-brown areas of skin on the head (J) and ventral scales (K) and edematous scales (arrow) and crusting (asterisk) of the skin (L). November/December 2015 Volume 6 Issue 6 e01534-15 ® 3 Downloaded from on January 15, 2016 – Published by Ophidiomyces ophiodiicola Causes Snake Fungal Disease Downloaded from on January 15, 2016 – Published by Lorch et al. FIG 2 Microscopic lesions of SFD in snakes experimentally challenged with Ophidiomyces ophiodiicola. (A and B) Skin samples from sham-inoculated snakes were within normal limits (PAS stain). Bar, 500 ␮m (A) or 100 ␮m (B). (C) Skin samples from sham-inoculated snakes exhibited focal breaks in the stratum corneum attributed to mechanical damage from abrasion; the underlying epidermis was generally within normal limits (PAS stain). Bar, 100 ␮m. (D and E) Skin samples from snakes exposed to O. ophiodiicola developed multifocal superficial epidermal necrosis with extensive epidermal edema (D) and heterophil infiltration and mononuclear to granulocytic dermal inflammation (E) (PAS stain). Bar, 500 ␮m (D) or 100 ␮m (E). (F) Breaks in the stratum corneum in infected snakes were most common over areas of epidermal necrosis and granulocytic inflammation, suggesting that infection may be facilitated by preexisting damage to the skin surface (PAS stain). Bar, 100 ␮m. (G) Some infected snakes developed granulomas consisting of fungal hyphae (arrow) and epithelioid (Continued) 4 ® November/December 2015 Volume 6 Issue 6 e01534-15 mon over infected areas of skin (Fig. 2F), while breaks in control snakes were equally common over normal and minimally necrotic skin and were attributed to the abrasion process (i.e., not associated with an etiological agent) (Fig. 2C). All control snakes that were undergoing molt at the time of necropsy (n ⫽ 5) also had low numbers of granulocytes in the upper layers of the epidermis in the interscalar space. This finding was considered normal, as heterophils migrate through the epidermis during the molting process (22). Microscopic lesions of internal organs included mild chronic lymphoplasmacytic to lymphohistiocytic inflammation in the liver, lungs, heart, stomach, and colon. Three snakes had granulomas within the coelomic mesentery or spleen; bacteria were noted within the mesenteric granulomas. Two snakes had mild focal to multifocal epithelial necrosis in the esophagus and colon. These lesions were present in both control and infected animals. Six of the eight infected and three of the seven control snakes were PCR positive for one or more of the snake adenoviruses at the end of the trial, and positive detections generally corresponded to mild lesions in internal organs. There was no evidence of disseminated fungal infection, even in snakes that developed SFD. Ophidiomyces ophiodiicola was not detected by real-time PCR on the skin of snakes prior to inoculation. However, O. ophiodiicola was isolated from skin collected at necropsy for all snakes in the infected group. All recovered isolates of O. ophiodiicola for which portions of the intergenic spacer (IGS) region of the rRNA gene complex were sequenced were 100% identical to the isolate used for the initial inoculation. Ophidiomyces ophiodiicola was the only fungus cultured from snakes in the infected group. The ability to culture O. ophiodiicola from snakes in the infected and control groups was significantly different (P ⬍ 0.001), with no fungi being recovered from snakes in the control group. Infection by O. ophiodiicola causes host physiological and behavioral changes. Snakes exposed to O. ophiodiicola molted much more frequently than animals in the control group (t ⫽ 5.627, P ⬍ 0.001). Specifically, mean shed intervals (time between skin molts) prior to experimental treatments were 39.0 (⫾5.3 [standard deviation]) days for the control group and 42.9 (⫾11.9) days for the infected group. Post-sham inoculation, the mean shed interval for the control group was 28.8 (⫾5.0) days. After exposure to O. ophiodiicola, the mean shed interval for the infected group was 15.6 (⫾3.9) days. The two snakes in the infected group that exhibited intermittent periods of anorexia demonstrated 2.0and 2.7-fold reductions in relative growth rate after 5 weeks of exposure to O. ophiodiicola. However, there was no statistical difference in the relative growth rate for snakes in the infected group for the 5 weeks before and the 5 weeks after exposure to the fungus (t ⫽ 0.340, P ⫽ 0.746). There was also no significant difference detected in relative growth rates between the control and infected groups over the 5-week period that snakes were exposed to the fungus or vehicle solution (t ⫽ 1.120, P ⫽ 0.285). Snakes in the infected group were significantly more likely to be encountered in conspicuous areas of their enclosures during daily checks (t ⫽ 3.295, P ⫽ 0.006). Specifically, infected snakes were noted in exposed areas of their cages on 42.9% (⫾10.0%) of checks, while snakes in the control group were found outside hiding areas on only 20.6% (⫾14.8%) of checks. DISCUSSION Ophidiomy

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