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.

Broad-Scale Assessment of Methylmercury in Adult Amphibians.

Mercury (Hg) is a toxic contaminant that has been mobilized and distributed worldwide and is a threat to many wildlife species. Amphibians are facing unprecedented global declines due to many threats including contaminants. While the biphasic life history of many amphibians creates a potential nexus for methylmercury (MeHg) exposure in aquatic habitats and subsequent health effects, the broad-scale distribution of MeHg exposure in amphibians remains unknown. We used nonlethal sampling to assess MeHg bioaccumulation in 3,241 juvenile and adult amphibians during 2017-2021. We sampled 26 populations (14 species) across 11 states in the United States, including several imperiled species that could not have been sampled by traditional lethal methods. We examined whether life history traits of species and whether the concentration of total mercury in sediment or dragonflies could be used as indicators of MeHg bioaccumulation in amphibians. Methylmercury contamination was widespread, with a 33-fold difference in concentrations across sites. Variation among years and clustered subsites was less than variation across sites. Life history characteristics such as size, sex, and whether the amphibian was a frog, toad, newt, or other salamander were the factors most strongly associated with bioaccumulation. Total Hg in dragonflies was a reliable indicator of bioaccumulation of MeHg in amphibians (R2 ≥ 0.67), whereas total Hg in sediment was not (R2 ≤ 0.04). Our study, the largest broad-scale assessment of MeHg bioaccumulation in amphibians, highlights methodological advances that allow for nonlethal sampling of rare species and reveals immense variation among species, life histories, and sites. Our findings can help identify sensitive populations and provide environmentally relevant concentrations for future studies to better quantify the potential threats of MeHg to amphibians.

Avian-associated Aspergillus fumigatus displays broad phylogenetic distribution, no evidence for host specificity, and multiple genotypes within epizootic events.

Birds are highly susceptible to aspergillosis, which can manifest as a primary infection in both domestic and wild birds. Aspergillosis in wild birds causes mortalities ranging in scale from single animals to large-scale epizootic events. However, pathogenicity factors associated with aspergillosis in wild birds have not been examined. Specifically, it is unknown whether wild bird-infecting strains are host-adapted (i.e. phylogenetically related). Similarly, it is unknown whether epizootics are driven by contact with clonal strains that possess unique pathogenic or virulence properties, or by distinct and equally pathogenic strains. Here, we use a diverse collection of Aspergillus fumigatus isolates taken from aspergillosis-associated avian carcasses, representing 24 bird species from a wide geographic range, and representing individual bird mortalities as well as epizootic events. These isolates were sequenced and analyzed along with 130 phylogenetically diverse human clinical isolates to investigate the genetic diversity and phylogenetic placement of avian-associated A. fumigatus, the geographic and host distribution of avian isolates, evidence for clonal outbreaks among wild birds, and the frequency of azole resistance in avian isolates. We found that avian isolates were phylogenetically diverse, with no clear distinction from human clinical isolates, and no sign of host or geographic specificity. Avian isolates from the same epizootic events were diverse and phylogenetically distant, suggesting that avian aspergillosis is not contagious among wild birds and that outbreaks are likely driven by environmental spore loads or host comorbidities. Finally, all avian isolates were susceptible to Voriconazole and none contained the canonical azole resistance gene variants.

An Opportunistic Survey Reveals an Unexpected Coronavirus Diversity Hotspot in North America.

In summer 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was detected on mink farms in Utah. An interagency One Health response was initiated to assess the extent of the outbreak and included sampling animals from on or near affected mink farms and testing them for SARS-CoV-2 and non-SARS coronaviruses. Among the 365 animals sampled, including domestic cats, mink, rodents, raccoons, and skunks, 261 (72%) of the animals harbored at least one coronavirus. Among the samples that could be further characterized, 127 alphacoronaviruses and 88 betacoronaviruses (including 74 detections of SARS-CoV-2 in mink) were identified. Moreover, at least 10% (n = 27) of the coronavirus-positive animals were found to be co-infected with more than one coronavirus. Our findings indicate an unexpectedly high prevalence of coronavirus among the domestic and wild free-roaming animals tested on mink farms. These results raise the possibility that mink farms could be potential hot spots for future trans-species viral spillover and the emergence of new pandemic coronaviruses.

Soil Reservoir Dynamics of Ophidiomyces ophidiicola, the Causative Agent of Snake Fungal Disease.

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.

Batrachochytrium salamandrivorans (Bsal) not detected in an intensive survey of wild North American amphibians.

The salamander chytrid fungus (Batrachochytrium salamandrivorans [Bsal]) is causing massive mortality of salamanders in Europe. The potential for spread via international trade into North America and the high diversity of salamanders has catalyzed concern about Bsal in the U.S. Surveillance programs for invading pathogens must initially meet challenges that include low rates of occurrence on the landscape, low prevalence at a site, and imperfect detection of the diagnostic tests. We implemented a large-scale survey to determine if Bsal was present in North America designed to target taxa and localities where Bsal was determined highest risk to be present based on species susceptibility and geography. Our analysis included a Bayesian model to estimate the probability of occurrence of Bsal given our prior knowledge of the occurrence and prevalence of the pathogen. We failed to detect Bsal in any of 11,189 samples from 594 sites in 223 counties within 35 U.S. states and one site in Mexico. Our modeling indicates that Bsal is highly unlikely to occur within wild amphibians in the U.S. and suggests that the best proactive response is to continue mitigation efforts against the introduction and establishment of the disease and to develop plans to reduce impacts should Bsal establish.

SURVEY OF AQUATIC TURTLES ON THE SAVANNAH RIVER SITE, SOUTH CAROLINA, USA, FOR PREVALENCE OF RANAVIRUS.

: Ranaviruses have the ability to infect amphibians, fish, and reptiles, and they have caused multiple amphibian die-off events in the US and Europe. Their prevalence in amphibian populations is much more commonly studied than in chelonian populations. We examined blood samples ( n=286) from eight aquatic turtle species collected during 2008-14 on the Savannah River Site, South Carolina, US, as part of long-term mark-recapture efforts. Previous studies in the southeastern US found high prevalence of Ranavirus in amphibians, but we did not detect Ranavirus in any of the turtles sampled, suggesting the absence of the virus or its presence at a very low prevalence in aquatic turtles across the Savannah River Site during the years tested.

Patterns of amphibian infection prevalence across wetlands on the Savannah River Site, South Carolina, USA.

Amphibian diseases, such as chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) and ranaviral disease caused by ranaviruses, are often linked to global amphibian population declines, yet the ecological dynamics of both pathogens are poorly understood. The goal of our study was to determine the baseline prevalence, pathogen loads, and co-infection rate of Bd and ranavirus across the Savannah River Site (SRS) in South Carolina, USA, a region with rich amphibian diversity and a history of amphibian-based research. We tested over 1000 individuals, encompassing 21 amphibian species from 11 wetlands for both Bd and ranavirus. The prevalence of Bd across individuals was 9.7%. Using wetland means, the mean (±SE) Bd prevalence was 7.9 ± 2.9%. Among toad species, Anaxyrus terrestris had 95 and 380% greater odds of being infected with Bd than Scaphiopus holbrookii and Gastrophryne carolinensis, respectively. Odds of Bd infection in adult A. terrestris and Lithobates sphenocephalus were 75 to 77% greater in metal-contaminated sites. The prevalence of ranavirus infections across all individuals was 37.4%. Mean wetland ranavirus prevalence was 29.8 ± 8.8% and was higher in post-metamorphic individuals than in aquatic larvae. Ambystoma tigrinum had 83 to 85% higher odds of ranavirus infection than A. opacum and A. talpoideum. We detected a 4.8% co-infection rate, with individuals positive for ranavirus having a 5% higher occurrence of Bd. In adult Anaxyrus terrestris, odds of Bd infection were 13% higher in ranavirus-positive animals and odds of co-infection were 23% higher in contaminated wetlands. Overall, we found the pathogen prevalence varied by wetland, species, and life stage.

Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser.

The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3 (-)), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F 2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. Lastly, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood.

First case of ranavirus and associated morbidity and mortality in an eastern mud turtle Kinosternon subrubrum in South Carolina.

Ranaviruses are double-stranded DNA viruses that infect amphibians, fish, and reptiles, causing global epidemics in some amphibian populations. It is important to identify new species that may be susceptible to the disease, particularly if they reside in the same habitat as other at-risk species. On the Savannah River Site (SRS) in Aiken, South Carolina, USA, ranaviruses are present in several amphibian populations, but information is lacking on the presence, prevalence, and morbidity of the virus in reptile species. An eastern mud turtle Kinosternon subrubrum captured on the SRS in April 2014 exhibited clinical signs of a ranaviral infection, including oral plaque and conjunctivitis. Quantitative PCR analyses of DNA from liver tissue, ocular, oral, nasal, and cloacal swabs were all positive for ranavirus, and sequencing of the template confirmed infection with a FV3-like ranavirus. Histopathologic examination of postmortem tissue samples revealed ulceration of the oral and tracheal mucosa, intracytoplasmic epithelial inclusions in the oral mucosa and tongue sections, individualized and clusters of melanomacrophages in the liver, and bacterial rods located in the liver, kidney, heart, stomach, and small intestine. This is the first report of morbidity and mortality of a mud turtle with a systemic ranaviral infection.