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Title:Emydomyces testavorans infection in aquatic chelonians
Author(s):Woodburn, Daniel Bond
Director of Research:Terio , Karen A
Doctoral Committee Chair(s):Terio , Karen A
Doctoral Committee Member(s):Allender, Matthew C; Hoyer, Lois L; Maddox, Carol W; Miller, Andrew N
Department / Program:Pathobiology
Discipline:VMS - Pathobiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Pathology, Veterinary
Abstract:Fungi are emerging as significant pathogens of multiple taxa. In addition to the impact of Ophidiomyces ophiodiicola on snakes, Batrachochytrium dendrobatidis (chytrid) has been linked to massive declines in amphibian populations worldwide, and Pseudogymnoascus destructans (white nose syndrome) has been associated with significant declines in North American bat populations. The fungal order Onygenales includes many pathogens of humans and animals, and recent studies have shown some onygenalean fungi to be significant emerging pathogens of reptiles, most notably Nannizziopsis, Paranannizziopsis, and Ophidiomyces. Infections by members of these genera have been previously reported in crocodilians, lizards, snakes, and tuataras with negative impacts on conservation efforts for some populations. Despite the well-documented pathogenicity of these fungi in all other extant reptile lineages, infection has not yet been reported in aquatic turtles. In 2011, however, a previously unreported presentation of shell disease was documented by pathologists at the University of Illinois Zoological Pathology Program (ZPP) from various aquatic turtles in managed care. Shell lesions were not histologically compatible with known causes of shell disease and had the unique and characteristic feature of keratinizing epithelial inclusion cysts within the dermis and shell bone. Additionally, fungi morphologically similar to other onygenalean reptile pathogens were noted at the leading edge of these lesions. Initial molecular testing detected fungal DNA that was similar to these known reptile pathogens, but sequence analysis could not identify the organism to the genus or species level. The research contained within this dissertation was initiated in order to characterize this previously undescribed fungus, describe the shell lesions associated with infection and identify any defining histologic features that would aid in diagnosis, and investigate changes in the shell microflora associated with infection that could provide insight into the pathogenesis of this newly recognized shell disease. In Chapter 1, biology and pathology of the chelonian shell are reviewed in detail, as are known fungal diseases of the other extant reptilian taxa. Disease threats to chelonian conservation efforts are also discussed, with particular attention given to the Western pond turtle (Actinemys marmorata). This species is locally endangered in the state of Washington and has a high (up to 86%) prevalence of fungal shell disease in the few remaining wild populations, already with significant impacts on conservation and research efforts. In Chapter 2, the fungus associated with these unique shell lesions is formally described. The first successful isolation is documented, and the fungus is characterized via standard mycological techniques as well as multilocus phylogenetic analysis. In culture, this fungus shares many features with reptile pathogens in the genera Nannizziopsis, Paranannizziopsis, and Ophidiomyces, though has distinctly larger and more variably sized conidia than these other species. Phylogenetically, all studied isolates (n = 21) of the chelonian fungus are grouped within a highly supported monophyletic clade that is related to, though distinct from, the other described genera of onygenalean reptile pathogens. Together, these findings warranted the designation of a novel species, Emydomyces testavorans (A.N. Mill. & D.B. Woodburn 2018), for these fungi associated with chelonian shell lesions. Chapter 3 investigates the shell lesions associated with Emydomyces infection in freshwater aquatic turtles. Via retrospective histologic review of chelonian submissions (n = 48 individuals) to the University of Illinois Zoological Pathology Program, lesions associated with Emydomyces infection are described, and specific lesional features are compared with those of shell lesions due to other infectious and non-infectious causes. Histologically, Emydomyces-associated shell lesions are characterized by ulceration and squamous metaplasia of the keratinized scute epidermis, necrosis and destruction of the underlying shell bone, and formation of epithelial inclusion cysts within the dermis and bone. These cysts are lined by keratinizing stratified squamous epithelium and are filled with abundant keratin debris and fragments of necrotic bone. Though often undetectable with routine histologic processing and staining, the use of a special stain (Grocott-Gomori’s Methenamine Silver [GMS]) to highlight fungi reveals numerous hyphae morphologically compatible with Emydomyces at the leading edges of these lesions, primarily associated with keratin debris and within the superficial layers of keratinizing cyst epithelium. To confirm infection and provide a rapid diagnostic tool for future cases, a specific quantitative real-time PCR assay was developed to detect minute quantities of Emydomyces DNA. Using this assay in combination with GMS staining, n = 27 cases of Emydomyces infection were confirmed, and lesional features were compared with n = 21 cases of shell lesions resulting from other etiologies. Multivariate logistic regression analysis using manual backward stepwise model selection demonstrated that inclusion cysts are highly associated with Emydomyces infection but not with other etiologies (p < 0.0001), and their presence in histologic section should warrant a thorough search for the fungus. In Chapter 4, a metagenomic approach is utilized to evaluate the shell microflora of healthy and diseased turtles in order to identify common potential bacterial and fungal co-pathogens that may be involved in the development or complicate treatment of shell disease. Standardized shell swab samples were collected from free-ranging WPT from two field sites in the state of Washington. Swabs from ten (n = 10) diseased turtles and ten (n = 10) healthy control turtles, as determined by CT scan and gross evaluation, were analyzed using targeted next-generation sequencing for bacterial (16S) and fungal (ITS) organisms. Significant differences in bacterial diversity and differentially abundant taxa were detected between field sites and locations on the shell (i.e. carapace or plastron). Significant differences in fungal diversity and abundance were only detected between the two field sites. However, no significant differences in bacterial or fungal diversity or differential abundances of taxa were observed to explain the difference between healthy and diseased turtles. This suggests that like infections by related onygenalean fungi in other reptile species, bacterial or fungal co-pathogens may not be required for the development of Emydomyces-associated shell lesions. Chapter 5 concludes this dissertation by summarizing findings of the previous chapters and speculating on potential future studies. This research represents the initial stages of investigation into a newly described fungal pathogen associated with shell disease in freshwater aquatic turtles. There is still much work to be done to more fully understand the pathogenesis of Emydomyces infection. It is the sincere hope of this author that the information presented herein will lay the groundwork for future investigations, with the ultimate goal of minimizing or even eliminating the threat of Emydomyces testavorans to chelonian health and conservation efforts.
Issue Date:2019-07-12
Rights Information:Copyright 2019 Daniel Woodburn
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08

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