Epidemiology of sarcoptic mange in a geographically constrained insular red fox population.

BACKGROUND: Sarcoptic mange is a skin disease caused by the contagious ectoparasite Sarcoptes scabiei, capable of suppressing and extirpating wild canid populations. Starting in 2015, we observed a multi-year epizootic of sarcoptic mange affecting a red fox (Vulpes vulpes) population on Fire Island, NY, USA. We explored the ecological factors that contributed to the spread of sarcoptic mange and characterized the epizootic in a landscape where red foxes are geographically constrained. METHODS: We tested for the presence of S. scabiei DNA in skin samples collected from deceased red foxes with lesions visibly consistent with sarcoptic mange disease. We deployed 96-100 remote trail camera stations each year to capture red fox occurrences and used generalized linear mixed-effects models to assess the affects of red fox ecology, human and other wildlife activity, and island geography on the frequency of detecting diseased red foxes. We rated the extent of visual lesions in diseased individuals and mapped the severity and variability of the sarcoptic mange disease. RESULTS: Skin samples that we analyzed demonstrated 99.8% similarity to S. scabiei sequences in GenBank. Our top-ranked model (weight = 0.94) showed that diseased red foxes were detected more frequently close to roadways, close to territories of other diseased red foxes, away from human shelters, and in areas with more mammal activity. There was no evidence that detection rates in humans and their dogs or distance to the nearest red fox den explained the detection rates of diseased red foxes. Although detected infrequently, we observed the most severe signs of sarcoptic mange at the periphery of residential villages. The spread of visual signs of the disease was approximately 7.3 ha/week in 2015 and 12.1 ha/week in 2017. CONCLUSIONS: We quantified two separate outbreaks of sarcoptic mange disease that occurred > 40 km apart and were separated by a year. Sarcoptic mange revealed an unfettered spread across the red fox population. The transmission of S. scabiei mites in this system was likely driven by red fox behaviors and contact between individuals, in line with previous studies. Sarcoptic mange is likely an important contributor to red fox population dynamics within barrier island systems.

Assessing avian influenza surveillance intensity in wild birds using a One Health lens.

Wildlife disease surveillance, particularly for pathogens with zoonotic potential such as Highly Pathogenic Avian Influenza Virus (HPAIV), is critical to facilitate situational awareness, inform risk, and guide communication and response efforts within a One Health framework. This study evaluates the intensity of avian influenza virus (AIV) surveillance in Ontario's wild bird population following the 2021 H5N1 incursion into Canada. Analyzing 2562 samples collected between November 1, 2021, and October 31, 2022, in Ontario, Canada, we identify spatial variations in surveillance intensity relative to human population density, poultry facility density, and wild mallard abundance. Using the spatial scan statistic, we pinpoint areas where public engagement, collaborations with Indigenous and non-Indigenous hunter/harvesters, and working with poultry producers, could augment Ontario's AIV wild bird surveillance program. Enhanced surveillance at these human-domestic animal-wildlife interfaces is a crucial element of a One Health approach to AIV surveillance. Ongoing assessment of our wild bird surveillance programs is essential for strategic planning and will allow us to refine approaches and generate results that continue to support the program's overarching objective of safeguarding the health of people, animals, and ecosystems.

A Palearctic view of a bat fungal disease.

The fungal infection causing white-nose disease in hibernating bats in North America has resulted in dramatic population declines of affected species, since the introduction of the causative agent Pseudogymnoascus destructans. The fungus is native to the Palearctic, where it also infects several bat species, yet rarely causes severe pathology or the death of the host. Pseudogymnoascus destructans infects bats during hibernation by invading and digesting the skin tissue, resulting in the disruption of torpor patterns and consequent emaciation. Relations among pathogen, host, and environment are complex, and individuals, populations, and species respond to the fungal pathogen in different ways. For example, the Nearctic Myotis lucifugus responds to infection by mounting a robust immune response, leading to immunopathology often contributing to mortality. In contrast, the Palearctic M. myotis shows no significant immunological response to infection. This lack of a strong response, resulting from the long coevolution between the hosts and the pathogen in the pathogen's native range, likely contributes to survival in tolerant species. After more than 15 years since the initial introduction of the fungus to North America, some of the affected populations are showing signs of recovery, suggesting that the fungus, hosts, or both are undergoing processes that may eventually lead to coexistence. The suggested or implemented management methods of the disease in North America have encompassed, for example, the use of probiotics and fungicides, vaccinations, and modifying the environmental conditions of the hibernation sites to limit the growth of the pathogen, intensity of infection, or the hosts' responses to it. Based on current knowledge from Eurasia, policy makers and conservation managers should refrain from disrupting the ongoing evolutionary processes and adopt a holistic approach to managing the epizootic.

Vista paleártica de una enfermedad fúngica de murciélagos Resumen La enfermedad fúngica que produce el síndrome de nariz blanca en murciélagos en hibernación en Norte América ha resultado en declinaciones poblacionales dramáticas en las especies afectadas desde la introducción del agente causante, Pseudogymnoascus destructans. El hongo es nativo del Paleártico, donde también infecta a varias especies de murciélagos; sin embargo, raramente causa patología severa o la muerte del hospedero. Pseudogymnoascus destructans infecta a los murciélagos durante la hibernación invadiendo y digiriendo el tejido de la piel, lo que resulta en la disrupción de los patrones de torpor y la consecuente emaciación. Las relaciones entre el patógeno, el huésped y el ambiente son complejas, y los individuos, las especies y poblaciones responden al patógeno fúngico de distintas maneras. Por ejemplo, Myotis lucifugus, especie del Neártico, responde a la infección montando una respuesta inmune robusta, produciendo una inmunopatología que a menudo contribuye a la mortalidad. En contraste, M. myotis del Paleártico no presenta respuesta inmunológica significativa a la infección. La falta de una fuerte respuesta, resultado de la larga coevolución entre hospederos y el patógeno en el rango nativo de distribución del patógeno, probablemente contribuye a la supervivencia en especies tolerantes. Después de más de 15 años desde la introducción del hongo en Norte América, algunas de las poblaciones afectadas están mostrando señales recuperación, lo que sugiere que el hongo, hospederos, o ambos, están pasando por procesos que eventualmente pueden conducir a la coexistencia. Los métodos de manejo de la enfermedad sugeridos o implementados en Norte América han abarcado, por ejemplo, el uso de probióticos y fungicidas, vacunaciones y modificación de las condiciones ambientales de los sitios de hibernación para limitar el crecimiento del patógeno, la intensidad de la infección o las respuestas de los hospederos. Con base en conocimiento actual de Eurasia, los formuladores de políticas y los manejadores de la conservación deberían abstenerse de alterar los procesos evolutivos en curso y adoptar un enfoque holístico para gestionar la epizootia.

Metagenomic sequencing sheds light on microbes putatively associated with pneumonia-related fatalities of white-tailed deer (Odocoileus virginianus).

With emerging infectious disease outbreaks in human, domestic and wild animal populations on the rise, improvements in pathogen characterization and surveillance are paramount for the protection of human and animal health, as well as the conservation of ecologically and economically important wildlife. Genomics offers a range of suitable tools to meet these goals, with metagenomic sequencing facilitating the characterization of whole microbial communities associated with emerging and endemic disease outbreaks. Here, we use metagenomic sequencing in a case-control study to identify microbes in lung tissue associated with newly observed pneumonia-related fatalities in 34 white-tailed deer (Odocoileus virginianus) in Wisconsin, USA. We identified 20 bacterial species that occurred in more than a single individual. Of these, only Clostridium novyi was found to substantially differ (in number of detections) between case and control sample groups; however, this difference was not statistically significant. We also detected several bacterial species associated with pneumonia and/or other diseases in ruminants (Mycoplasma ovipneumoniae, Trueperella pyogenes, Pasteurella multocida, Anaplasma phagocytophilum, Fusobacterium necrophorum); however, these species did not substantially differ between case and control sample groups. On average, we detected a larger number of bacterial species in case samples than controls, supporting the potential role of polymicrobial infections in this system. Importantly, we did not detect DNA of viruses or fungi, suggesting that they are not significantly associated with pneumonia in this system. Together, these results highlight the utility of metagenomic sequencing for identifying disease-associated microbes. This preliminary list of microbes will help inform future research on pneumonia-associated fatalities of white-tailed deer.

Evaluation of contingency actions to control the spread of raccoon rabies in Ohio and Virginia.

The raccoon (Procyon lotor) variant of the rabies virus (RRV) is enzootic in the eastern United States and oral rabies vaccination (ORV) is the primary strategy to prevent and control landscape spread. Breaches of ORV management zones occasionally occur, and emergency "contingency" actions may be implemented to enhance local control. Contingency actions are an integral part of landscape-scale wildlife rabies management but can be very costly and routinely involve enhanced rabies surveillance (ERS) around the index case. We investigated two contingency actions in Ohio (2017-2019 and 2018-2021) and one in Virginia (2017-2019) using a dynamic, multi-method occupancy approach to examine relationships between specific management actions and RRV occurrence, including whether ERS was sufficient around the index case. The RRV occupancy was assessed seasonally at 100-km2 grids and we examined relationships across three spatial scales (regional management zone, RRV free regions, and local contingency areas). The location of a grid relative to the ORV management zone was the strongest predictor of RRV occupancy at the regional scale. In RRV free regions, the neighbor effect and temporal variability were most important in influencing RRV occupancy. Parenteral (hand) vaccination of raccoons was important across all three contingency action areas, but more influential in the Ohio contingency action areas where more raccoons were hand vaccinated. In the Virginia contingency action area, ORV strategies were as important in reducing RRV occupancy as a hand vaccination strategy. The management action to trap, euthanize, and test (TET) raccoons was an important method to increase ERS, yet the impacts of TET on RRV occupancy are not clear. The probability of detecting additional cases of RRV was exceptionally high (>0.95) during the season the index case occurred. The probability of detecting RRV through ERS declined in the seasons following initial TET efforts but remained higher after the contingency action compared to the ERS detection probabilities prior to index case incidence. Local RRV cases were contained within one year and eliminated within 2-3 years of each contingency action.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.

Bat white-nose disease fungus diversity in time and space.

White-nose disease (WND), caused by the psychrophilic fungus Pseudogymnoascusdestructans, represents one of the greatest threats for North American hibernating bats. Research on molecular data has significantly advanced our knowledge of various aspects of the disease, yet more studies are needed regarding patterns of P.destructans genetic diversity distribution. In the present study, we investigate three sites within the native range of the fungus in detail: two natural hibernacula (karst caves) in Bulgaria, south-eastern Europe and one artificial hibernaculum (disused cellar) in Germany, northern Europe, where we conducted intensive surveys between 2014 and 2019. Using 18 microsatellite and two mating type markers, we describe how P.destructans genetic diversity is distributed between and within sites, the latter including differentiation across years and seasons of sampling; across sampling locations within the site; and between bats and hibernaculum walls. We found significant genetic differentiation between hibernacula, but we could not detect any significant differentiation within hibernacula, based on the variables examined. This indicates that most of the pathogen's movement occurs within sites. Genotypic richness of P.destructans varied between sites within the same order of magnitude, being approximately two times higher in the natural caves (Bulgaria) compared to the disused cellar (Germany). Within all sites, the pathogen's genotypic richness was higher in samples collected from hibernaculum walls than in samples collected from bats, which corresponds with the hypothesis that hibernacula walls represent the environmental reservoir of the fungus. Multiple pathogen genotypes were commonly isolated from a single bat (i.e. from the same swab sample) in all study sites, which might be important to consider when studying disease progression.

A novel bluetongue virus serotype 2 strain isolated from a farmed Florida white-tailed deer (Odocoileus virginianus) arose from reassortment of gene segments derived from co-circulating serotypes in the Southeastern USA.

Bluetongue disease is a reportable animal disease that affects wild and farmed ruminants, including white-tailed deer (WTD). This report documents the clinical findings, ancillary diagnostics, and genomic characterization of a novel reassortant bluetongue virus serotype 2 (BTV-2) strain isolated from a dead Florida farmed WTD in 2022. Our analyses support that this BTV-2 strain likely stemmed from the acquisition of genome segments from co-circulating BTV strains in Florida and Louisiana. In addition, our analyses also indicate that genetically uncharacterized BTV strains may be circulating in the Southeastern USA; however, the identity and reassortant status of these BTV strains cannot be determined based on the VP2 and VP5 genome sequences. Hence, continued surveillance based on complete genome characterization is needed to understand the genetic diversity of BTV strains in this region and the potential threat they may pose to the health of deer and other ruminants.

Oral Rabies Vaccination of Raccoons (Procyon lotor) across a Development Intensity Gradient in Burlington, Vermont, USA, 2015-2017.

Management of the raccoon rabies virus variant in North America is conducted primarily using oral rabies vaccination (ORV). When a sufficient proportion of the population is vaccinated (∼60%), rabies transmission can be eliminated. To date, ORV programs have successfully controlled and eliminated raccoon rabies in rural areas, but there has been less success in urban areas. We studied the proportions of rabies virus neutralizing antibodies (RVNA) in a raccoon (Procyon lotor) population during a 3-yr ORV trial in developed areas of Burlington, Vermont, US. We used a modified N-mixture model to estimate raccoon abundance, RVNA seroprevalence, and capture rates jointly to examine factors that relate to ORV success to better inform management. We found that raccoon abundance was lower in less-developed areas compared to urban centers. Raccoon RVNA seroprevalence decreased as population abundance increased; it increased as the average age of the population increased. Nontarget opossum (Didelphis virginiana) captures correlated with a decrease in raccoon RVNA seroprevalence in low-development areas, suggesting that they may be competing for baits. The target bait density across the entire study area was 150 baits/km2, but a hand baiting strategy was heavily concentrated on roads, resulting in uneven bait densities within sampling sites (0-484 baits/km2). Uneven bait distribution across the study area may explain low RVNA seroprevalence in some locations. Our results suggest that increases in bait density across the study area may improve RVNA seroprevalence and support annual ORV to account for raccoon population turnover.

A minimally invasive, field-applicable CRISPR/Cas biosensor to aid in the detection of Pseudogymnoascus destructans, the causative fungal agent of white-nose syndrome in bats.

The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd-positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd-positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.

Genetic mechanisms and biological processes underlying host response to ophidiomycosis (snake fungal disease) inferred from tissue-specific transcriptome analyses.

Emerging infectious diseases in wildlife species caused by pathogenic fungi are of growing concern, yet crucial knowledge gaps remain for diseases with potentially large impacts. For example, there is detailed knowledge about host pathology and mechanisms underlying response for chytridiomycosis in amphibians and white-nose syndrome in bats, but such information is lacking for other more recently described fungal infections. One such disease is ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, which has been identified in many species of snakes, yet the biological mechanisms and molecular changes occurring during infection are unknown. To gain this information, we performed a controlled experimental infection in captive Prairie rattlesnakes (Crotalus viridis) with O. ophidiicola at two different temperatures: 20 and 26°C. We then compared liver, kidney, and skin transcriptomes to assess tissue-specific genetic responses to O. ophidiicola infection. Given previous histopathological studies and the fact that snakes are ectotherms, we expected highest fungal activity on skin and a significant impact of temperature on host response. Although we found fungal activity to be localized on skin, most of the differential gene expression occurred in internal tissues. Infected snakes at the lower temperature had the highest host mortality whereas two-thirds of the infected snakes at the higher temperature survived. Our results suggest that ophidiomycosis is likely a systemic disease with long-term effects on host response. Our analysis also identified candidate protein coding genes that are potentially involved in host response, providing genetic tools for studies of host response to ophidiomycosis in natural populations.

Shifts in population density centers of a hibernating mammal driven by conflicting effects of climate change and disease.

Populations wax and wane over time in response to an organism's interactions with abiotic and biotic forces. Numerous studies demonstrate that fluctuations in local populations can lead to shifts in relative population densities across the geographic range of a species over time. Fewer studies attempt to disentangle the causes of such shifts. Over four decades (1983-2022), we monitored populations of hibernating Indiana bats (Myotis sodalis) in two areas separated by ~110 km. The number of bats hibernating in the northern area increased from 1983 to 2011, while populations in the southern area remained relatively constant. We used simulation models and long-term weather data to demonstrate the duration of time bats must rely on stored fat during hibernation has decreased in both areas over that period, but at a faster rate in the northern area. Likewise, increasing autumn and spring temperatures shortened the periods of sporadic prey (flying insect) availability at the beginning and end of hibernation. Climate change thus increased the viability of northern hibernacula for an increasing number of bats by decreasing energetic costs of hibernation. Then in 2011, white-nose syndrome (WNS), a disease of hibernating bats that increases energetic costs of hibernation, was detected in the area. From 2011 to 2022, the population rapidly decreased in the northern area and increased in the southern area, completely reversing the northerly shift in population densities associated with climate change. Energy balance during hibernation is the singular link explaining the northerly shift under a changing climate and the southerly shift in response to a novel disease. Continued population persistence suggests that bats may mitigate many impacts of WNS by hibernating farther south, where insects are available longer each year.

Recovery of multireassortant bluetongue virus serotype 6 sequences from a mule deer (Odocoileus hemionus) and Dorset sheep (Ovis aries) in Colorado.

We report the discovery of two bluetongue virus serotype 6 (BTV-6) reassortants recovered from a domestic sheep and a free-ranging mule deer in northern Colorado. At the time of this publication, whole-genome sequencing of BTV-6 isolates in the Western U.S. have not been undertaken. These findings reflect the incursive movement of geographically distinct BTV serotypes into important agricultural areas of the U.S. and demonstrate reassortment with regionally circulating serotypes.

When the host's away, the pathogen will play: the protective role of the skin microbiome during hibernation.

The skin of animals is enveloped by a symbiotic microscopic ecosystem known as the microbiome. The host and microbiome exhibit a mutualistic relationship, collectively forming a single evolutionary unit sometimes referred to as a holobiont. Although the holobiome theory highlights the importance of the microbiome, little is known about how the skin microbiome contributes to protecting the host. Existing studies focus on humans or captive animals, but research in wild animals is in its infancy. Specifically, the protective role of the skin microbiome in hibernating animals remains almost entirely overlooked. This is surprising, considering the massive population declines in hibernating North American bats caused by the fungal pathogen Pseudogymnoascus destructans, which causes white-nose syndrome. Hibernation offers a unique setting in which to study the function of the microbiome because, during torpor, the host's immune system becomes suppressed, making it susceptible to infection. We conducted a systematic review of peer-reviewed literature on the protective role of the skin microbiome in non-human animals. We selected 230 publications that mentioned pathogen inhibition by microbes residing on the skin of the host animal. We found that the majority of studies were conducted in North America and focused on the bacterial microbiome of amphibians infected by the chytrid fungus. Despite mentioning pathogen inhibition by the skin microbiome, only 30.4% of studies experimentally tested the actual antimicrobial activity of symbionts. Additionally, only 7.8% of all publications studied defensive cutaneous symbionts during hibernation. With this review, we want to highlight the knowledge gap surrounding skin microbiome research in hibernating animals. For instance, research looking to mitigate the effects of white-nose syndrome in bats should focus on the antifungal microbiome of Palearctic bats, as they survive exposure to the Pseudogymnoascus destructans -pathogen during hibernation. We also recommend future studies prioritize lesser-known microbial symbionts, such as fungi, and investigate the effects of a combination of anti-pathogen microbes, as both areas of research show promise as probiotic treatments. By incorporating the protective skin microbiome into disease mitigation strategies, conservation efforts can be made more effective.

A review of sebum in mammals in relation to skin diseases, skin function, and the skin microbiome.

Diseases vary among and within species but the causes of this variation can be unclear. Immune responses are an important driver of disease variation, but mechanisms on how the body resists pathogen establishment before activation of immune responses are understudied. Skin surfaces of mammals are the first line of defense against abiotic stressors and pathogens, and skin attributes such as pH, microbiomes, and lipids influence disease outcomes. Sebaceous glands produce sebum composed of multiple types of lipids with species-specific compositions. Sebum affects skin barrier function by contributing to minimizing water loss, supporting thermoregulation, protecting against pathogens, and preventing UV-induced damage. Sebum also affects skin microbiome composition both via its antimicrobial properties, and by providing potential nutrient sources. Intra- and interspecific variation in sebum composition influences skin disease outcomes in humans and domestic mammal species but is not well-characterized in wildlife. We synthesized knowledge on sebum function in mammals in relation to skin diseases and the skin microbiome. We found that sebum composition was described for only 29 live, wild mammalian species. Sebum is important in dermatophilosis, various forms of dermatitis, demodicosis, and potentially white-nose syndrome. Sebum composition likely affects disease susceptibility, as lipid components can have antimicrobial functions against specific pathogens. It is unclear why sebum composition is species-specific, but both phylogeny and environmental effects may drive differences. Our review illustrates the role of mammal sebum function and influence on skin microbes in the context of skin diseases, providing a baseline for future studies to elucidate mechanisms of disease resistance beyond immune responses.

Paranannizziopsis spp. infections in wild snakes and a qPCR assay for detection of the fungus.

The emergence of ophidiomycosis (or snake fungal disease) in snakes has prompted increased awareness of the potential effects of fungal infections on wild reptile populations. Yet, aside from Ophidiomyces ophidiicola, little is known about other mycoses affecting wild reptiles. The closely related genus Paranannizziopsis has been associated with dermatomycosis in snakes and tuataras in captive collections, and P. australasiensis was recently identified as the cause of skin infections in non-native wild panther chameleons (Furcifer pardalis) in Florida, USA. Here we describe five cases of Paranannizziopsis spp. associated with skin lesions in wild snakes in North America and one additional case from a captive snake from Connecticut, USA. In addition to demonstrating that wild Nearctic snakes can serve as a host for these fungi, we also provide evidence that the genus Paranannizziopsis is widespread in wild snakes, with cases being identified in Louisiana (USA), Minnesota (USA), Virginia (USA), and British Columbia (Canada). Phylogenetic analyses conducted on multiple loci of the fungal strains we isolated identified P. australasiensis in Louisiana and Virginia; the remaining strains from Minnesota and British Columbia did not cluster with any of the described species of Paranannizziopsis, although the strains from British Columbia appear to represent a single lineage. Finally, we designed a pan-Paranannizziopsis real-time PCR assay targeting the internal transcribed spacer region 2. This assay successfully detected DNA of all described species of Paranannizziopsis and the two potentially novel taxa isolated in this study and did not cross-react with closely related fungi or other fungi commonly found on the skin of snakes. The assay was 100% sensitive and specific when screening clinical (skin tissue or skin swab) samples, although full determination of the assay's performance will require additional follow up due to the small number of clinical samples (n = 14 from 11 snakes) available for testing in our study. Nonetheless, the PCR assay can provide an important tool in further investigating the prevalence, distribution, and host range of Paranannizziopsis spp. and facilitate more rapid diagnosis of Paranannizziopsis spp. infections that are otherwise difficult to differentiate from other dermatomycoses.

Contact-independent exposure to Rhodococcus rhodochrous DAP96253 volatiles does not improve the survival rate of Myotis lucifugus (little brown bats) affected by White-nose Syndrome.

Since the emergence of White-nose Syndrome, a fungal disease in bats, caused by Pseudogymnoascus destructans, hibernating populations of little brown bats (Myotis lucifugus) have declined by 70-90% within P. destructans positive hibernacula. To reduce the impact of White-nose Syndrome to North American little brown bat populations we evaluated if exposure to volatile organic compounds produced by induced cells from Rhodococcus rhodochrous strain DAP96253 could improve the overwinter survival of bats infected by P. destructans. Two simultaneous field treatment trials were conducted at natural hibernacula located in Rockcastle and Breckinridge counties, Kentucky, USA. A combined total of 120 little brown bats were randomly divided into control groups (n = 60) which were not exposed to volatile organic compounds and treatment groups (n = 60) which were exposed to volatile organic compounds produced by non-growth, fermented cell paste composed of R. rhodochrous strain DAP96253 cells. Cox proportional hazard models revealed a significant decreased survival at the Rockcastle field trial site but not the Breckinridge field site. At the Breckinridge hibernacula, overwinter survival for both treatment and control groups were 60%. At the Rockcastle hibernacula, Kaplan-Meier survival curves indicated significantly increased overwinter survival of bats in the control group (43% survived) compared to the treatment group (20% survived). Although complete inhibition of P. destructans by volatile organic compounds produced by induced R. rhodochrous strain DAP96253 cells was observed in vitro studies, our results suggest that these volatile organic compounds do not inhibit P. destructans in situ and may promote P. destructans growth.

Conservation genomics of an endangered montane amphibian reveals low population structure, low genomic diversity and selection pressure from disease.

Wildlife diseases are a major global threat to biodiversity. Boreal toads (Anaxyrus [Bufo] boreas) are a state-endangered species in the southern Rocky Mountains of Colorado and New Mexico, and a species of concern in Wyoming, largely due to lethal skin infections caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We performed conservation and landscape genomic analyses using single nucleotide polymorphisms from double-digest, restriction site-associated DNA sequencing in combination with the development of the first boreal toad (and first North American toad) reference genome to investigate population structure, genomic diversity, landscape connectivity and adaptive divergence. Genomic diversity (π = 0.00034-0.00040) and effective population sizes (Ne = 8.9-38.4) were low, likely due to post-Pleistocene founder effects and Bd-related population crashes over the last three decades. Population structure was also low, likely due to formerly high connectivity among a higher density of geographically proximate populations. Boreal toad gene flow was facilitated by low precipitation, cold minimum temperatures, less tree canopy, low heat load and less urbanization. We found >8X more putatively adaptive loci related to Bd intensity than to all other environmental factors combined, and evidence for genes under selection related to immune response, heart development and regulation and skin function. These data suggest boreal toads in habitats with Bd have experienced stronger selection pressure from disease than from other, broad-scale environmental variations. These findings can be used by managers to conserve and recover the species through actions including reintroduction and supplementation of populations that have declined due to Bd.

Total infectome investigation of diphtheritic stomatitis in yellow-eyed penguins (Megadyptes antipodes) reveals a novel and abundant megrivirus.

First identified in 2002, diphtheritic stomatitis (DS) is a devastating disease affecting yellow-eyed penguins (Megadyptes antipodes, or hoiho in te reo Maori). The disease is associated with oral lesions in chicks and has caused significant morbidity and mortality. DS is widespread among yellow-eyed penguin chicks on mainland New Zealand yet appears to be absent from the subantarctic population. Corynebacterium spp. have previously been suspected as causative agents yet, due to inconsistent cultures and inconclusive pathogenicity, their role in DS is unclear. Herein, we used a metatranscriptomic approach to identify potential causative agents of DS by revealing the presence and abundance of all viruses, bacteria, fungi and protozoa - together, the infectome. Oral and cloacal swab samples were collected from presymptomatic, symptomatic and recovered chicks along with a control group of healthy adults. Two novel viruses from the Picornaviridae were identified, one of which - yellow-eyed penguin megrivirus - was highly abundant in chicks irrespective of health status but not detected in healthy adults. Tissue from biopsied oral lesions also tested positive for the novel megrivirus upon PCR. We found no overall clustering among bacteria, protozoa and fungi communities at the genus level across samples, although Paraclostridium bifermentans was significantly more abundant in oral microbiota of symptomatic chicks compared to other groups. The detection of a novel and highly abundant megrivirus has sparked a new line of inquiry to investigate its potential association with DS.

RACCOONS (PROCYON LOTOR) SHOW HIGHER TRYPANOSOMA CRUZI DETECTION RATES THAN VIRGINIA OPOSSUMS (DIDELPHIS VIRGINIANA) IN SOUTH CAROLINA, USA.

Chagas disease, a significant public health concern in the Americas, is caused by a protozoan parasite, Trypanosoma cruzi. The life cycle of T. cruzi involves kissing bugs (Triatoma spp.) functioning as vectors and mammalian species serving as hosts. Raccoons (Procyon lotor) and opossums (Didelphis virginiana) have been identified as important reservoir species in the life cycle of T. cruzi, but prevalence in both species in the southeastern US is currently understudied. We quantified T. cruzi prevalence in these two key reservoir species across our study area in South Carolina, US, and identified factors that may influence parasite detection. We collected whole blood from 183 raccoons and 126 opossums and used PCR to detect the presence of T. cruzi. We then used generalized linear models with parasite detection status as a binary response variable and predictor variables of land cover, distance to water, sex, season, and species. Our analysis indicated that raccoons experienced significantly higher parasite detection rates than Virginia opossums, with T. cruzi prevalence found to be 26.5% (95% confidence interval [CI], 20.0-33.8) in raccoons and 10.5% (95% CI, 5.51-17.5) in opossums. Overall, our results concur with previous studies, in that T. cruzi is established in reservoir host populations in natural areas of the southeastern US.