Project description:Emerging pathogenic fungi have become a topic of conservation concern due to declines observed in several host taxa. One emerging fungal pathogen, Ophidiomyces ophidiicola, is well documented as the causative agent of ophidiomycosis, otherwise known as snake fungal disease (SFD). O. ophidiicola has been found to cause disease in a variety of snake species across the United States, including the eastern massasauga (Sistrurus catenatus), a federally threatened rattlesnake species. Most work to date has involved detecting O. ophidiicola for diagnosis of infection through direct sampling of snakes, and attempts to detect O. ophidiicola in the abiotic environment to better understand its distribution, seasonality, and habitat associations are lacking. We collected topsoil and groundwater samples from four macrohabitat types across multiple seasons in northern Michigan at a site where Ophidiomyces infection has been confirmed in eastern massasauga. Using a quantitative PCR (qPCR) assay developed for O. ophidiicola, we detected Ophidiomyces DNA in topsoil but observed minimal to no detection in groundwater samples. Detection frequency did not differ between habitats, but samples grouped seasonally showed higher detection during mid-summer. We found no relationships of detection with hypothesized environmental correlates such as soil pH, temperature, or moisture content. Furthermore, the distribution of Ophidiomyces positive samples across the site was not linked to estimated space use of massasaugas. Our data suggests that season has some effect on the presence of Ophidiomyces. Differences in presence between habitats may exist but are likely more dependent on the time of sampling and currently uninvestigated soil or biotic parameters. These findings build on our understanding of Ophidiomyces ecology and epidemiology to help inform where and when snakes may be exposed to the fungus in the environment.
Project description:Wildlife diseases pose an ever-growing threat to global biodiversity. Understanding how wildlife pathogens are distributed in the environment and the ability of pathogens to form environmental reservoirs is critical to understanding and predicting disease dynamics within host populations. Snake fungal disease (SFD) is an emerging conservation threat to North American snake populations. The causative agent, Ophidiomyces ophidiicola (Oo), is detectable in environmentally derived soils. However, little is known about the distribution of Oo in the environment and the persistence and growth of Oo in soils. Here, we use quantitative PCR to detect Oo in soil samples collected from five snake dens. We compare the detection rates between soils collected from within underground snake hibernacula and associated, adjacent topsoil samples. Additionally, we used microcosm growth assays to assess the growth of Oo in soils and investigate whether the detection and growth of Oo are related to abiotic parameters and microbial communities of soil samples. We found that Oo is significantly more likely to be detected in hibernaculum soils compared to topsoils. We also found that Oo was capable of growth in sterile soil, but no growth occurred in soils with an active microbial community. A number of fungal genera were more abundant in soils that did not permit growth of Oo, versus those that did. Our results suggest that soils may display a high degree of both general and specific suppression of Oo in the environment. Harnessing environmental suppression presents opportunities to mitigate the impacts of SFD in wild snake populations.
Project description:The pathogen Ophidiomyces ophidiicola, widely known as the primary cause of snake fungal disease (SFD) has been detected in Texas's naïve snakes. Our team set out to characterize O. ophidiicola's spread in eastern Texas. From December 2018 until November 2021, we sampled and screened with ultraviolet (UV) light, 176 snakes across eastern Texas and detected 27. O. ophidiicola's positive snakes using qPCR and one snake in which SFD was confirmed via additional histological examination. Upon finding the ribbon snake with clear clinical display, we isolated and cultured what we believe to be the first culture from Texas. This cultured O. ophidiicola TX displays a ring halo formation when grown on a solid medium as well as cellular autofluorescence as expected. Imaging reveals individual cells within the septated hyphae branches contain a distinct nucleus separation from neighboring cells. Overall, we have found over 1/10 snakes that may be infected in East Texas, gives credence to the onset of SFD in Texas. These results add to the progress of the disease across the continental United States.
Project description:Dermatophytic pathogens are a source of disturbance to the host microbiome, but the temporal progression of these disturbances is unclear. Here, we determined how Snake Fungal Disease, caused by Ophidiomyces ophidiicola, resulted in disturbance to the host microbiome. To assess disease effects on the microbiome, 22 Common Watersnakes (Nerodia sipedon) were collected and half were inoculated with O. ophidiicola. Epidermal swabs were collected weekly for use in microbiome and pathogen load characterization. For the inoculated treatment only, we found a significant effect of disease progression on microbial richness and Shannon diversity consistent with the intermediate disturbance hypothesis. When explicitly accounting for differences in assemblage richness, we found that β-diversity among snakes was significantly affected by the interaction of time and treatment group, with assemblages becoming more dissimilar across time in the inoculated, but not the control group. Also, differences between treatments in average microbiome composition became greater with time, but this interactive effect was not evident when accounting for assemblage richness. These results suggest that changes in composition of the host microbiome associated with disease largely occur due to changes in microbial richness related to disease progression.
Project description: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.
Project description:Ophidiomyces ophidiicola (Oo) is a fungal pathogen and the causative agent of ophidiomycosis that has affected multiple snake taxa across the United States, Europe, and Asia. Ophidiomycosis has often been referred to as an emerging infectious disease (EID); however, its status as an EID has recently come under debate. Oo infections have been confirmed in wild snake populations in Texas; however, it is unknown if the pathogen is novel (i.e., invasive) or endemic to the state. To address this knowledge gap, we conducted surveys for Oo among preserved Nerodia deposited at three university museums in Texas. First, we visually assessed snakes for signs of infection (SOI), and if SOI were present, we sampled the affected area. We then used quantitative polymerase chain reaction to diagnose the presence of Oo DNA on areas with SOI and used these data to evaluate spatiotemporal patterns of Oo prevalence. We also tested for significant spatial clusters of Oo infenction using a Bernoulli probability model as implemented in the program SatScan. We found that the proportion of snakes exhibiting SOI was constant over time while the prevalence of Oo DNA among those SOI increased across space and time. Within these data, we detected an incidence pattern consistent with an introduction and then spread. We detected six spatial clusters of Oo infection, although only one was significant. Our results support the hypothesis that Oo is an emerging, novel pathogen to Texas snakes. These data narrow the knowledge gap regarding the history of Oo infections in Texas and establish a historical record of confirmed Oo detections in several counties across the state. Thus, our results will guide future research to those areas with evidence of past Oo infections but lacking confirmation in contemporary hosts.