Frequent transitions in mating-type locus chromosomal organization in Malassezia and early steps in sexual reproduction.

Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining (MAT) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By generating additional chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere-flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight toward the identification of a sexual cycle in Malassezia, with possible implications for pathogenicity.

The population genetics of the causative agent of snake fungal disease indicate recent introductions to the USA.

Snake fungal disease (SFD; ophidiomycosis), caused by the pathogen Ophidiomyces ophiodiicola (Oo), has been documented in wild snakes in North America and Eurasia, and is considered an emerging disease in the eastern United States of America. However, a lack of historical disease data has made it challenging to determine whether Oo is a recent arrival to the USA or whether SFD emergence is due to other factors. Here, we examined the genomes of 82 Oo strains to determine the pathogen's history in the eastern USA. Oo strains from the USA formed a clade (Clade II) distinct from European strains (Clade I), and molecular dating indicated that these clades diverged too recently (approximately 2,000 years ago) for transcontinental dispersal of Oo to have occurred via natural snake movements across Beringia. A lack of nonrecombinant intermediates between clonal lineages in Clade II indicates that Oo has actually been introduced multiple times to North America from an unsampled source population, and molecular dating indicates that several of these introductions occurred within the last few hundred years. Molecular dating also indicated that the most common Clade II clonal lineages have expanded recently in the USA, with time of most recent common ancestor mean estimates ranging from 1985 to 2007 CE. The presence of Clade II in captive snakes worldwide demonstrates a potential mechanism of introduction and highlights that additional incursions are likely unless action is taken to reduce the risk of pathogen translocation and spillover into wild snake populations.

Paranannizziopsis spp. Infection in Wild Vipers, Europe.

We describe the detection of Paranannizziopsis sp. fungus in a wild population of vipers in Europe. Fungal infections were severe, and 1 animal likely died from infection. Surveillance efforts are needed to better understand the threat of this pathogen to snake conservation.

A Cross-Inoculation Experiment Reveals that Ophidiomyces ophiodiicola and Nannizziopsis guarroi Can Each Infect Both Snakes and Lizards.

Host range and specificity are key concepts in the study of infectious diseases. However, both concepts remain largely undefined for many influential pathogens, including many fungi within the Onygenales order. This order encompasses reptile-infecting genera (Nannizziopsis, Ophidiomyces, and Paranannizziopsis) formerly classified as the Chrysosporium anamorph of Nannizziopsis vriesii (CANV). The reported hosts of many of these fungi represent a narrow range of phylogenetically related animals, suggesting that many of these disease-causing fungi are host specific, but the true number of species affected by these pathogens is unknown. For example, to date, Nannizziopsis guarroi (the causative agent of yellow fungus disease) and Ophidiomyces ophiodiicola (the causative agent of snake fungal disease) have been documented only in lizards and snakes, respectively. In a 52-day reciprocal-infection experiment, we tested the ability of these two pathogens to infect currently unreported hosts, inoculating central bearded dragons (Pogona vitticeps) with O. ophiodiicola and corn snakes (Pantherophis guttatus) with N. guarroi. We confirmed infection by documenting both clinical signs and histopathological evidence of fungal infection. Our reciprocity experiment resulted in 100% of corn snakes and 60% of bearded dragons developing infections with N. guarroi and O. ophiodiicola, respectively, demonstrating that these fungal pathogens have a broader host range than previously thought and that hosts with cryptic infections may play a role in pathogen translocation and transmission. IMPORTANCE Our experiment using Ophidiomyces ophiodiicola and Nannizziopsis guarroi is the first to look more critically at these pathogens' host range. We are the first to identify that both fungal pathogens can infect both corn snakes and bearded dragons. Our findings illustrate that both fungal pathogens have a more general host range than previously known. Additionally, there are significant implications concerning the spread of snake fungal disease and yellow fungus disease in popular companion animals and the increased chance of disease spillover into other wild and naive populations.

Prevalence of Ophidiomyces ophidiicola and epizootiology of snake fungal disease in free-ranging Northern Pine Snakes (Pituophis melanoleucus melanoleucus) in New Jersey.

Snake fungal disease, caused by Ophidiomyces ophidiicola, is recognized as a potential concern for North American snakes. We tested skin swabs from Northern Pine Snakes (Pituophis melanoleucus melanoleucus) in the New Jersey pinelands for the presence of O. ophidiicola before emergence from hibernation. We used qPCR to test the collected swabs for the presence of O. ophidiicola, then determined pathogen prevalence as a function of sampling year, sampling location (skin lesion, healthy ventral skin, healthy head skin) sex, and age. There were no temporal trends in O. ophidiicola detection percentages on snakes, which varied from 58 to 83% in different years. Ophidiomyces ophidiicola detection on snakes was highest in swabs of skin lesions (71%) and lowest in head swabs (29%). Males had higher prevalence than females (82% versus 62%). The fungus was not detected in hatchling snakes (age 0) in the fall, but 75% of juveniles tested positive at the end of hibernation (age 1 year). We also screened hibernacula soil samples for the presence of O. ophidiicola. Where snakes hibernated, 69% of soil samples were positive for O. ophidiicola, and 85% of snakes lying on positive soil samples also tested positive for the pathogen. Although a high proportion of snakes (73%) tested positive for O. ophidiicola during our 4-year study, the snakes appeared healthy except for small skin lesions. We conclude that O. ophidiicola prevalence is high on hibernating Northern Pine Snakes and in the hibernacula soil, with a strong association between snakes and positive adjacent soil. This is the first demonstration that snakes likely become infected during hibernation.

Low occurrence of multi-antimicrobial and heavy metal resistance in Salmonella enterica from wild birds in the United States.

Wild birds are common reservoirs of Salmonella enterica. Wild birds carrying resistant S. enterica may pose a risk to public health as they can spread the resistant bacteria across large spatial scales within a short time. Here, we whole-genome sequenced 375 S. enterica strains from wild birds collected in 41 U.S. states during 1978-2019 to examine bacterial resistance to antibiotics and heavy metals. We found that Typhimurium was the dominant S. enterica serovar, accounting for 68.3% (256/375) of the bird isolates. Furthermore, the proportions of the isolates identified as multi-antimicrobial resistant (multi-AMR: resistant to at least three antimicrobial classes) or multi-heavy metal resistant (multi-HMR: resistant to at least three heavy metals) were both 1.87% (7/375). Interestingly, all the multi-resistant S. enterica (n = 12) were isolated from water birds or raptors; none of them was isolated from songbirds. Plasmid profiling demonstrated that 75% (9/12) of the multi-resistant strains carried resistance plasmids. Our study indicates that wild birds do not serve as important reservoirs of multi-resistant S. enterica strains. Nonetheless, continuous surveillance for bacterial resistance in wild birds is necessary because the multi-resistant isolates identified in this study also showed close genetic relatedness with those from humans and domestic animals.

Possibility for reverse zoonotic transmission of SARS-CoV-2 to free-ranging wildlife: A case study of bats.

The COVID-19 pandemic highlights the substantial public health, economic, and societal consequences of virus spillover from a wildlife reservoir. Widespread human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also presents a new set of challenges when considering viral spillover from people to naïve wildlife and other animal populations. The establishment of new wildlife reservoirs for SARS-CoV-2 would further complicate public health control measures and could lead to wildlife health and conservation impacts. Given the likely bat origin of SARS-CoV-2 and related beta-coronaviruses (ß-CoVs), free-ranging bats are a key group of concern for spillover from humans back to wildlife. Here, we review the diversity and natural host range of ß-CoVs in bats and examine the risk of humans inadvertently infecting free-ranging bats with SARS-CoV-2. Our review of the global distribution and host range of ß-CoV evolutionary lineages suggests that 40+ species of temperate-zone North American bats could be immunologically naïve and susceptible to infection by SARS-CoV-2. We highlight an urgent need to proactively connect the wellbeing of human and wildlife health during the current pandemic and to implement new tools to continue wildlife research while avoiding potentially severe health and conservation impacts of SARS-CoV-2 "spilling back" into free-ranging bat populations.

Salmonella enterica Serovar Typhimurium Isolates from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type.

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCE Within-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the United States. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

Confirmed Cases of Ophidiomycosis in Museum Specimens from as Early as 1945, United States.

Ophidiomycosis represents a conservation threat to wild snake populations. The disease was reported in North America early in the 21st century, but the history of ophidiomycosis has not been investigated. We examined museum specimens and confirmed cases of ophidiomycosis >50 years before the disease's reported emergence.

Effects of snake fungal disease on short-term survival, behavior, and movement in free-ranging snakes.

Pathogenic fungi are increasingly associated with epidemics in wildlife populations. Snake fungal disease (SFD, also referred to as Ophidiomycosis) is an emerging threat to snakes, taxa that are elusive and difficult to sample. Thus, assessments of the effects of SFD on populations have rarely occurred. We used a field technique to enhance detection, Passive Integrated Transponder (PIT) telemetry, and a multi-state capture-mark-recapture model to assess SFD effects on short-term (within-season) survival, movement, and surface activity of two wild snake species, Regina septemvittata (Queensnake) and Nerodia sipedon (Common Watersnake). We were unable to detect an effect of disease state on short-term survival for either species. However, we estimated Bayesian posterior probabilities of >0.99 that R. septemvittata with SFD spent more time surface-active and were less likely to permanently emigrate from the study area. We also estimated probabilities of 0.98 and 0.87 that temporary immigration and temporary emigration rates, respectively, were lower in diseased R. septemvittata. We found evidence of elevated surface activity and lower temporary immigration rates in diseased N. sipedon, with estimated probabilities of 0.89, and found considerably less support for differences in permanent or temporary emigration rates. This study is the first to yield estimates for key demographic and behavioral parameters (survival, emigration, surface activity) of snakes in wild populations afflicted with SFD. Given the increase in surface activity of diseased snakes, future surveys of snake populations could benefit from exploring longer-term demographic consequences of SFD and recognize that disease prevalence in surface-active animals may exceed that of the population as a whole.

Experimental infection of bats with Geomyces destructans causes white-nose syndrome.

White-nose syndrome (WNS) has caused recent catastrophic declines among multiple species of bats in eastern North America. The disease's name derives from a visually apparent white growth of the newly discovered fungus Geomyces destructans on the skin (including the muzzle) of hibernating bats. Colonization of skin by this fungus is associated with characteristic cutaneous lesions that are the only consistent pathological finding related to WNS. However, the role of G. destructans in WNS remains controversial because evidence to implicate the fungus as the primary cause of this disease is lacking. The debate is fuelled, in part, by the assumption that fungal infections in mammals are most commonly associated with immune system dysfunction. Additionally, the recent discovery that G. destructans commonly colonizes the skin of bats of Europe, where no unusual bat mortality events have been reported, has generated further speculation that the fungus is an opportunistic pathogen and that other unidentified factors are the primary cause of WNS. Here we demonstrate that exposure of healthy little brown bats (Myotis lucifugus) to pure cultures of G. destructans causes WNS. Live G. destructans was subsequently cultured from diseased bats, successfully fulfilling established criteria for the determination of G. destructans as a primary pathogen. We also confirmed that WNS can be transmitted from infected bats to healthy bats through direct contact. Our results provide the first direct evidence that G. destructans is the causal agent of WNS and that the recent emergence of WNS in North America may represent translocation of the fungus to a region with a naive population of animals. Demonstration of causality is an instrumental step in elucidating the pathogenesis and epidemiology of WNS and in guiding management actions to preserve bat populations against the novel threat posed by this devastating infectious disease.

TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease.

BACKGROUND: Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease. RESULTS: We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. CONCLUSIONS: These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome.

White-nose syndrome (WNS) is an emerging disease of hibernating bats associated with cutaneous infection by the fungus Geomyces destructans (Gd), and responsible for devastating declines of bat populations in eastern North America. Affected bats appear emaciated and one hypothesis is that they spend too much time out of torpor during hibernation, depleting vital fat reserves required to survive the winter. The fungus has also been found at low levels on bats throughout Europe but without mass mortality. This finding suggests that Gd is either native to both continents but has been rendered more pathogenic in North America by mutation or environmental change, or that it recently arrived in North America as an invader from Europe. Thus, a causal link between Gd and mortality has not been established and the reason for its high pathogenicity in North America is unknown. Here we show that experimental inoculation with either North American or European isolates of Gd causes WNS and mortality in the North American bat, Myotis lucifugus. In contrast to control bats, individuals inoculated with either isolate of Gd developed cutaneous infections diagnostic of WNS, exhibited a progressive increase in the frequency of arousals from torpor during hibernation, and were emaciated after 3-4 mo. Our results demonstrate that altered torpor-arousal cycles underlie mortality from WNS and provide direct evidence that Gd is a novel pathogen to North America from Europe.

White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host.

BACKGROUND: The physiological effects of white-nose syndrome (WNS) in hibernating bats and ultimate causes of mortality from infection with Pseudogymnoascus (formerly Geomyces) destructans are not fully understood. Increased frequency of arousal from torpor described among hibernating bats with late-stage WNS is thought to accelerate depletion of fat reserves, but the physiological mechanisms that lead to these alterations in hibernation behavior have not been elucidated. We used the doubly labeled water (DLW) method and clinical chemistry to evaluate energy use, body composition changes, and blood chemistry perturbations in hibernating little brown bats (Myotis lucifugus) experimentally infected with P. destructans to better understand the physiological processes that underlie mortality from WNS. RESULTS: These data indicated that fat energy utilization, as demonstrated by changes in body composition, was two-fold higher for bats with WNS compared to negative controls. These differences were apparent in early stages of infection when torpor-arousal patterns were equivalent between infected and non-infected animals, suggesting that P. destructans has complex physiological impacts on its host prior to onset of clinical signs indicative of late-stage infections. Additionally, bats with mild to moderate skin lesions associated with early-stage WNS demonstrated a chronic respiratory acidosis characterized by significantly elevated dissolved carbon dioxide, acidemia, and elevated bicarbonate. Potassium concentrations were also significantly higher among infected bats, but sodium, chloride, and other hydration parameters were equivalent to controls. CONCLUSIONS: Integrating these novel findings on the physiological changes that occur in early-stage WNS with those previously documented in late-stage infections, we propose a multi-stage disease progression model that mechanistically describes the pathologic and physiologic effects underlying mortality of WNS in hibernating bats. This model identifies testable hypotheses for better understanding this disease, knowledge that will be critical for defining effective disease mitigation strategies aimed at reducing morbidity and mortality that results from WNS.

Contribution of host species and pathogen clade to snake fungal disease hotspots in Europe.

Infectious diseases are influenced by interactions between host and pathogen, and the number of infected hosts is rarely homogenous across the landscape. Areas with elevated pathogen prevalence can maintain a high force of infection and may indicate areas with disease impacts on host populations. However, isolating the ecological processes that result in increases in infection prevalence and intensity remains a challenge. Here we elucidate the contribution of pathogen clade and host species in disease hotspots caused by Ophidiomyces ophidiicola, the pathogen responsible for snake fungal disease, in 21 species of snakes infected with multiple pathogen strains across 10 countries in Europe. We found isolated areas of disease hotspots in a landscape where infections were otherwise low. O. ophidiicola clade had important effects on transmission, and areas with multiple pathogen clades had higher host infection prevalence. Snake species further influenced infection, with most positive detections coming from species within the Natrix genus. Our results suggest that both host and pathogen identity are essential components contributing to increased pathogen prevalence.

Distribution and environmental persistence of the causative agent of white-nose syndrome, Geomyces destructans, in bat hibernacula of the eastern United States.

White-nose syndrome (WNS) is an emerging disease of hibernating bats caused by the recently described fungus Geomyces destructans. First isolated in 2008, the origins of this fungus in North America and its ability to persist in the environment remain undefined. To investigate the correlation between manifestation of WNS and distribution of G. destructans in the United States, we analyzed sediment samples collected from 55 bat hibernacula (caves and mines) both within and outside the known range of WNS using a newly developed real-time PCR assay. Geomyces destructans was detected in 17 of 21 sites within the known range of WNS at the time when the samples were collected; the fungus was not found in 28 sites beyond the known range of the disease at the time when environmental samples were collected. These data indicate that the distribution of G. destructans is correlated with disease in hibernating bats and support the hypothesis that the fungus is likely an exotic species in North America. Additionally, we examined whether G. destructans persists in infested bat hibernacula when bats are absent. Sediment samples were collected from 14 WNS-positive hibernacula, and the samples were screened for viable fungus by using a culture technique. Viable G. destructans was cultivated from 7 of the 14 sites sampled during late summer, when bats were no longer in hibernation, suggesting that the fungus can persist in the environment in the absence of bat hosts for long periods of time.

Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans.

The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was qualified further by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

A culture-based survey of fungi in soil from bat hibernacula in the eastern United States and its implications for detection of Geomyces destructans, the causal agent of bat white-nose syndrome.

The recent emergence of white-nose syndrome (WNS), a fungal disease causing unprecedented mortality among hibernating bats of eastern North America, has revealed a knowledge gap regarding fungal communities associated with bats and their hibernacula. We used culture-based techniques to investigate the diversity of fungi in soil samples collected from 24 bat hibernacula in the eastern United States. Ribosomal RNA regions (internal transcribed spacer and partial intergenic spacer) were sequenced to preliminarily characterize isolates. Geomyces species were one of the most abundant and diverse groups cultured, representing approximately 33% of all isolates. Geomyces destructans was isolated from soil samples from three hibernacula in states where WNS is known to occur, and many of the other cultured Geomyces isolates likely represent undescribed taxa. Further characterization of the diversity of fungi that occur in hibernacula both will facilitate an improved understanding of the ecology of G. destructans within this complex fungal community and provide an opportunity to identify characteristics that differentiate G. destructans from non-pathogenic relatives.

Development and application of a qPCR-based genotyping assay for Ophidiomyces ophidiicola to investigate the epidemiology of ophidiomycosis.

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.

Epistylis spp. Infestation in Two Species of Mud Turtles (Kinosternon spp.) in the American Southwest.

The protistan genus Epistylis contains freshwater colonial species that attach to aquatic organisms in an epibiotic or parasitic relationship. They are known to attach to the epidermis and shells of aquatic turtles, but have not been reported to cause heavy infestations or morbidity in turtles. We documented heavy infestations of Epistylis spp. in several populations of Sonoran mud turtles (Kinosternon sonoriense) inhabiting livestock ponds in Arizona, USA, and rough-footed mud turtles (Kinosternon hirtipes) from livestock ponds in Texas, USA, over the course of several years. Severe Epistylis spp. infestations on mud turtles appeared to alter diving and swimming behavior when compared to uninfested conspecifics. Infestations were cleared in captivity using tap water or a 10% salt solution, and the turtles had no permanent damage to their shell or epidermis upon clearing. While several of the mud turtles we observed had poor body condition, it is possible that the severe infestations we observed were caused by a comorbidity associated with a pathogen, parasite, or poor habitat quality that made the turtles more susceptible to the Epistylis spp. infestation. Further research on causes for these severe infestations are warranted because they contribute to changes in behavior of the heavily infested turtles and may contribute to morbidity in Kinosternon spp. when mud turtles inhabit extremely warm, shallow, eutrophic aquatic habitats, such as livestock ponds.