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Ophidiomycosis (snake fungal disease) is an infectious disease caused by the fungus Ophidiomyces ophidiicola to which all snake species appear to be susceptible. Significant variation has been observed in clinical presentation, progression of disease, and response to treatment, which may be due to genetic variation in the causative agent. Recent phylogenetic analysis based on whole-genome sequencing identified that O . ophidiicola strains from the United States formed a clade distinct from European strains, and that multiple clonal lineages of the clade are present in the United States. The purpose of this study was to design a qPCR-based genotyping assay for O . ophidiicola , then apply that assay to swab-extracted DNA samples to investigate whether the multiple O . ophidiicola clades and clonal lineages in the United States have specific geographic, taxonomic, or temporal predilections. To this end, six full genome sequences of O . ophidiicola representing different clades and clonal lineages were aligned to identify genomic areas shared between subsets of the isolates. Eleven hydrolysis-based Taqman primer-probe sets were designed to amplify selected gene segments and produce unique amplification patterns for each isolate, each with a limit of detection of 10 or fewer copies of the target sequence and an amplification efficiency of 90–110%. The qPCR-based approach was validated using samples from strains known to belong to specific clades and applied to swab-extracted O . ophidiicola DNA samples from multiple snake species, states, and years. When compared to full-genome sequencing, the qPCR-based genotyping assay assigned 75% of samples to the same major clade (Cohen’s kappa = 0.360, 95% Confidence Interval = 0.154–0.567) with 67–77% sensitivity and 88–100% specificity, depending on clade/clonal lineage. Swab-extracted O . ophidiicola DNA samples from across the United States were assigned to six different clonal lineages, including four of the six established lineages and two newly defined groups, which likely represent recombinant strains of O . ophidiicola . Using multinomial logistic regression modeling to predict clade based on snake taxonomic group, state of origin, and year of collection, state was the most significant predictor of clonal lineage. Furthermore, clonal lineage was not associated with disease severity in the most intensely sampled species, the Lake Erie watersnake ( Nerodia sipedon insularum ). Overall, this assay represents a rapid, cost-effective genotyping method for O . ophidiicola that can be used to better understand the epidemiology of ophidiomycosis.

Red-eared sliders (RES; Trachemys scripta elegans ) are a globally invasive species that can impact native chelonian populations through resource competition and disease introduction. In Cook County, Illinois, invasive RES co-occur with, and greatly outnumber, a species of conservation priority in the Great Lakes region: the Blanding’s turtle ( Emydoidea blandingii ). This study sampled free-ranging RES in Cook County during spring, summer, and fall in four unique locations to characterize the possible effect of this non-native species on the health of regional, sympatric, aquatic chelonians. RES (n = 242) were captured and sampled for qPCR pathogen detection, clinical pathology, and necropsy from 2018 to 2022. Multiple pathogens were detected, including Mycoplasma spp., multiple adenoviruses, Trachemys herpesvirus 1 (TrHV-1), frog virus 3, human-pathogenic Leptospira spp., Emydomyces testavorans , and Salmonella typhimurium . Mycoplasma spp. prevalence was significantly higher (p < 0.05) at one isolated site with a suspected greater density of turtles. Detection of TrHV-1 was significantly associated with season (p < 0.001), with detections occurring only in spring and fall. For the 28 hematology, plasma biochemistry, and protein electrophoresis analytes assayed, significant and variable associations occurred based on sample year, season, pathogen detection, age, and sex. Population-based reference intervals were created for hematologic, plasma biochemistry, and plasma protein electrophoresis analytes. Common necropsy findings included mild endoparasitism and related vascular lesions such as endarteritis. Notably, qPCR detection of above pathogens was not associated with any gross or histologic lesions indicative of clinically relevant disease. This study indicates that invasive RES in Cook County may be infected with pathogens of concern for co-occurring turtle species, and absence of associated lesions in the RES suggests they likely serve as carrier species for these pathogens. Continued health monitoring of this species is important as interactions with chelonians of conservation priority increase.

Wildlife disease surveillance and pathogen detection are fundamental for conservation, population sustainability, and public health. Detection of pathogens in snakes is often overlooked despite their essential roles as both predators and prey within their communities. Ophidiomycosis (formerly referred to as Snake Fungal Disease, SFD), an emergent disease on the North American landscape caused by the fungus Ophidiomyces ophiodiicola, poses a threat to snake population health and stability. We tested 657 individual snakes representing 58 species in 31 states from 56 military bases in the continental US and Puerto Rico for O. ophiodiicola. Ophidiomyces ophiodiicola DNA was detected in samples from 113 snakes for a prevalence of 17.2% (95% CI: 14.4-20.3%), representing 25 species from 19 states/territories, including the first reports of the pathogen in snakes in Idaho, Oklahoma, and Puerto Rico. Most animals were ophidiomycosis negative (n = 462), with Ophidiomyces detected by qPCR (n = 64), possible ophidiomycosis (n = 82), and apparent ophidiomycosis (n = 49) occurring less frequently. Adults had 2.38 times greater odds than juveniles of being diagnosed with ophidiomycosis. Snakes from Georgia, Massachusetts, Pennsylvania, and Virginia all had greater odds of ophidiomycosis diagnosis, while snakes from Idaho were less likely to be diagnosed with ophidiomycosis. The results of this survey indicate that this pathogen is endemic in the eastern US and identified new sites that could represent emergence or improved detection of endemic sites. The direct mortality of snakes with ophidiomycosis is unknown from this study, but the presence of numerous individuals with clinical disease warrants further investigation and possible conservation action.

Pathogen-host interactions are important components of epidemiological research, but are scarcely investigated in chelonians. Red-eared sliders (Trachemys scripta elegans), are recognized as a model for frog virus-3 infection (FV3), a ranavirus in the family Iridoviridae that infects multiple classes of ectothermic vertebrates. Previous challenge studies observed differences in disease outcome based on environmental temperature in this species, but the host response was minimally evaluated. We challenged red-eared sliders with an FV3-like ranavirus at both 28°C and 22°C. We monitored several host response variables for 30 days, including: survival (binary outcome and duration), clinical signs, total and differential leukocytes, and select cytokine transcription in the buffy coat (IL-1β, TNFα, IFYg, IL-10). After 30 days, 17% of challenged turtles survived at 28°C (Median survival time [MST]: 15 days, range: 10-30 days) and 50% survived (MST: 28.5 days, range: 23-30 days) at 22°C (range 23-30 days). The most common clinical signs were injection site swelling, palpebral swelling, and lethargy. The heterophil/lymphocyte ratio at 22°C and interleukin-1 beta (IL1β) transcription at both 22°C and 28°C were significantly greater on days 9, 16, and 23 in FV3 challenged groups. Tumor necrosis factor alpha and interleukin-10 were transcribed at detectable levels, but did not display significant differences in mean relative transcription quantity over time. Overall, evidence indicates an over-robust immune response leading to death in the challenged turtles. FV3 remains a risk for captive and free-ranging chelonian populations, and insight to host/pathogen interaction through this model helps to elucidate the timing and intensity of the host response that contribute to mortality.

Snake Fungal Disease (SFD), caused by Ophidiomyces ophiodiicola , is the most recently described fungal disease afflicting wildlife populations across North America and Europe. It has been proposed as a significant conservation threat yielding high mortality and yet much its ecology is unknown. We collected 144 skin swabs from Eastern Massasaugas ( Sistrurus catenatus ) in 2015 and 2016 to determine document ongoing prevalence and assess differences in microbial assemblages between positive and negative individuals. Alpha diversity of fungi was reduced in SFD positive animals, while beta diversity identified distinct assemblages of microbes between SFD–positive and –negative samples. Ophidiomyces was present on the skin of affected animals, even on body sites distant to lesions indicating that the microbiome on entire surface of the skin is altered. Ophidiomyces was not detected in any non-SFD snake. There were smaller, but significant, influences of year sampled. Bacterial genera Janthinobacterium and Serratia were significantly increased in SFD snakes, while Xylanimicrobium , Cellulosimicrobium , and Rhodococcus were the only bacterial taxa significantly reduced. The relative abundance of fungi within the orders Pleosporales and Canopdiales was reduced in SFD-positive samples, though Pyrenochaetopsis pratorum was the only species found to differ significantly. This is the first study to determine the impact that this fungal pathogen has on the skin microbiome.

Wildlife mortality investigations are important for conservation, food safety, and public health; but they are infrequently reported for cryptic chelonian species. Eastern box turtles (Terrapene carolina carolina) are declining due to anthropogenic factors and disease, and while mortality investigations have been reported for captive and translocated individuals, few descriptions exist for free-living populations. We report the results of four natural mortality event investigations conducted during routine health surveillance of three Illinois box turtle populations in 2011, 2013, 2014, and 2015. In April 2011, over 50 box turtles were found dead and a polymicrobial necrotizing bacterial infection was diagnosed in five survivors using histopathology and aerobic/anaerobic culture. This represents the first reported occurrence of necrotizing bacterial infection in box turtles. In August 2013, paired histopathology and qPCR ranavirus detection in nine turtles was significantly associated with occupation of moist microhabitats, identification of oral plaques and nasal discharge on physical exam, and increases in the heterophil count and heterophil to lymphocyte ratio (p < 0.05). In July 2014 and 2015, ranavirus outbreaks reoccurred within a 0.2km radius of highly-disturbed habitat containing ephemeral ponds used by amphibians for breeding. qPCR ranavirus detection in five individuals each year was significantly associated with use of moist microhabitats (p < 0.05). Detection of single and co-pathogens (Terrapene herpesvirus 1, adenovirus, and Mycoplasma sp.) was common before, during, and after mortality events, but improved sample size would be necessary to determine the impacts of these pathogens on the occurrence and outcome of mortality events. This study provides novel information about the causes and predictors of natural box turtle mortality events. Continued investigation of health, disease, and death in free-living box turtles will improve baseline knowledge of morbidity and mortality, identify threats to survival, and promote the formation of effective conservation strategies.

Ophidiomycosis is a prevalent and intermittently pervasive disease of snakes globally caused by the opportunistic fungal pathogen, Ophidiomyces ophidiicola . Host response has yet to be fully explored, including the role of temperature in disease progression and hematologic changes. This study enrolled twelve adult prairie rattlesnakes ( Crotalus viridis ) in an experimental challenge with O . ophidiicola at two temperatures, 26°C (n = 6) and 20°C (n = 6). Each temperature cohort included four inoculated and two control snakes. Assessments involving physical exams, lesion swabbing, and hematology were performed weekly. Differences were observed between inoculated and control snakes in survival, behavior, clinical signs, ultraviolet (UV) fluorescence, hematologic response, and histologic lesions. All inoculated snakes held at 20°C were euthanized prior to study end date due to severity of clinical signs while only one inoculated animal in the 26°C trial met this outcome. In both groups, qPCR positive detection preceded clinical signs with regards to days post inoculation (dpi). However, the earliest appearance of gross lesions occurred later in the 20°C snakes (20 dpi) than the 26°C snakes (13 dpi). Relative leukocytosis was observed in all inoculated snakes and driven by heterophilia in the 20°C snakes, and azurophilia in the 26°C group. Histologically, 20°C snakes had more severe lesions, a lack of appropriate inflammatory response, and unencumbered fungal proliferation and invasion. In contrast, 26°C snakes had marked granulomatous inflammation with encapsulation of fungi and less invasion and dissemination. The results of this study identified that O . ophidiicola -infected rattlesnakes exposed to lower temperatures have decreased survival and more robust hematologic change, though minimal and ineffective inflammatory response at site of infection. Ophidiomycosis is a complex disease with host, pathogen, and environmental factors influencing disease presentation, progression, and ultimately, survival. This study highlighted the importance of temperature as an element impacting the host response to O . ophidiicola .

Wildlife diseases have posed a significant challenge to the conservation of many species in recent years. Diseases have been implicated in population declines over large geographic areas, with severe disease outbreaks leading to either local or complete extinctions of wild populations. Ophidiomycosis, commonly known as snake fungal disease, is caused by the fungus Ophidiomyces ophiodiicola, which has been documented in snake populations across the eastern and southern United States. We collected swab samples from the federally threatened Eastern Indigo Snake (Drymarchon couperi) in populations across the species' Georgia range. We used quantitative PCR to determine the presence of O. ophiodiicola DNA and also recorded skin abnormalities characteristic of ophidiomycosis. From 1 September 2016 to 4 August 2018, Eastern Indigo Snakes tested positive for O. ophiodiicola DNA on 47 of 107 occasions (43.9%) and tested negative for fungal DNA but had skin lesions consistent with ophidiomycosis on 42 occasions (39.3%). Symptomatic and qPCR positive individuals were more likely to be encountered during January and February when compared to November and December. We found no effect of sex (p = 0.517), age-class (p = 0.106), or body size (snout-vent length: p = 0.083; mass: p = 0.206; body condition: p = 0.063) on ophidiomycosis status. Over the two-year study, we encountered individuals in which infection was clearly negatively impacting overall health and also documented individuals in which infection apparently cleared from one year to the next. These results demonstrate that O. ophiodiicola and lesions characteristic of ophidiomycosis are widespread in Georgia's Eastern Indigo Snake populations. However, there are many unanswered questions regarding this disease, including the effects of disease on populations and individuals, the presence of infection vectors, and the change in prevalence over time. More research is needed to address ophidiomycosis and understand its impacts on ongoing conservation efforts.

Coxiella burnetii, the causative bacterium of the zoonotic disease Q fever, has been documented in many different species. We describe documented turtles that were PCR positive for C. burnetii from multiple locations in Illinois and Wisconsin, USA. Assessing the conservation implications, reservoir potential, and zoonotic risk requires further research.

Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.