Development of a Targeted NGS Assay for the Detection of Respiratory Pathogens including SARS-CoV-2 in Felines.

Acute respiratory diseases in felines can be attributed to a diverse range of pathogens. The recent emergence of novel viruses, particularly SARS-CoV-2 and its variants, has also been associated with respiratory ailments in cats and other pets, underscoring the need for a highly sensitive diagnostic assay capable of concurrently detecting multiple respiratory pathogens. In this study, we developed a targeted next generation sequencing panel using Ion Torrent Ampliseq technology to detect multiple respiratory pathogens, including recent SARS-CoV-2 variants and Feline herpesvirus-1, Feline calicivirus, Bordetella bronchiseptica, Mycoplasmopsis (previously Mycoplasma) felis, and Chlamydia felis. A PCR amplification-based library preparation, employing primers designed for pathogen target regions, was synthesized and divided into two pools, followed by sequencing and assembly to a repertoire of target pathogen genomes. Analytical sensitivity was assessed based on Ct values from real-time PCR for the corresponding pathogens, indicating an equivalent detection limit. Most of the pathogens under study were positively identified to a limit of approximately Ct 36, whereas for Feline herpesvirus-1 and SARS-CoV-2, positive reads were observed in samples with a Ct of 37. Based on a limited number of samples, the diagnostic sensitivity values for the SARS-CoV-2, Feline herpesvirus-1, and M. felis samples were 100% with no false negative results. The diagnostic specificity of SARS-CoV-2, Feline herpesvirus-1, Feline calicivirus, and C. felis were 100%. Importantly, none of the target primers exhibited non-specific amplification, ensuring the absence of false positive results for other pathogens within the study. Additionally, the assay's specificity was validated by cross-referencing the raw sequencing data with established databases like BLAST, affirming the high specificity of the targeted Next-Generation Sequencing (tNGS) assay. Variations in the sequencing reads of different pathogens were observed when subjected to diverse extraction methods. Rigorous assessment of the assay's reliability involved reproducibility across testing personnel and repeated runs. The developed assay's clinical applicability was tested using samples submitted to the diagnostic laboratory from cat shelters and suspected cases. The developed targeted next-generation sequencing methodology empowers the detection of multiple respiratory pathogens manifesting similar clinical symptoms while offering confirmation of results through genome sequencing.

Investigating the rise of Omicron variant through genomic surveillance of SARS-CoV-2 infections in a highly vaccinated university population.

Novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge as the coronavirus disease 2019 (COVID-19) pandemic extends into its fourth year. Understanding SARS-CoV-2 circulation in university populations is vital for effective interventions in higher education settings and will inform public health policy during pandemics. In this study, we generated 793 whole-genome sequences collected over an entire academic year in a university population in Indiana, USA. We clearly captured the rapidity with which Delta variant was wholly replaced by Omicron variant across the West Lafayette campus over the length of two academic semesters in a community with high vaccination rates. This mirrored the emergence of Omicron throughout the state of Indiana and the USA. Further, phylogenetic analyses demonstrated that there was a more diverse set of potential geographic origins for Omicron viruses introduction into campus when compared to Delta. Lastly, statistics indicated that there was a more significant role for international and out-of-state migration in the establishment of Omicron variants at Purdue. This surveillance workflow, coupled with viral genomic sequencing and phylogeographic analyses, provided critical insights into SARS-CoV-2 transmission dynamics and variant arrival.

Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis.

A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

Phylogenomic assessment of 23 equid alphaherpesvirus 1 isolates obtained from USA-based equids.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is a global viral pathogen of domestic equids which causes reproductive, respiratory and neurological disease. Few isolates acquired from naturally infected USA-based hosts have been fully sequenced and analyzed to date. An ORF 30 (DNA polymerase) variant (A2254G) has previously been associated with neurological disease in host animals. The purpose of this study was to perform phylogenomic analysis of EHV-1 isolates acquired from USA-based hosts and compare these isolates to previously sequenced global isolates. METHODS: EHV-1 was isolated from 23 naturally infected USA-based equids (6 different states, 15 disease outbreaks) with reproductive (22/23) or neurological disease (1/23). Following virus isolation, EHV-1 DNA was extracted for sequencing using Illumina MiSeq. Following reference-based assembly, whole viral genomes were annotated and assessed. Previously sequenced EHV-1 isolates (n = 114) obtained from global host equids were included in phylogenomic analyses. RESULTS: The overall average genomic distance was 0.0828% (SE 0.004%) for the 23 newly sequenced USA isolates and 0.0705% (SE 0.003%) when all 137 isolates were included. Clade structure was predominantly based on geographic origin. Numerous nucleotide substitutions (mean [range], 179 [114-297] synonymous and 81 [38-120] non-synonymous substitutions per isolate) were identified throughout the genome of the newly sequenced USA isolates. The previously described ORF 30 A2254G substitution (associated with neurological disease) was found in only one isolate obtained from a host with non-neurological clinical signs (reproductive disease), six additional, unique, non-synonymous ORF 30 substitutions were detected in 22/23 USA isolates. Evidence of recombination was present in most (22/23) of the newly sequenced USA isolates. CONCLUSIONS: Overall, the genomes of the 23 newly sequenced EHV-1 isolates obtained from USA-based hosts were broadly similar to global isolates. The previously described ORF 30 A2254G neurological substitution was infrequently detected in the newly sequenced USA isolates, most of which were obtained from host animals with reproductive disease. Recombination was likely to be partially responsible for genomic diversity in the newly sequenced USA isolates.

Evaluation of RapidChek® SELECT™ for the detection of Salmonella spp. in environmental samples from a veterinary hospital.

Nosocomial salmonellosis in hospitalized animals is a recognized hazard, especially in large animal clinics. A standardized culture protocol (SCP) for detecting Salmonella spp. in environmental samples using a 48-h enrichment step results in a 5-day turnaround time for negative results. The RapidChek® SELECT™ Salmonella (RCSS) test system offers detection of organisms in 22-44 h through double enrichment followed by a lateral flow immunoassay. Negative results are reported within 48 h. To determine the most sensitive and rapid method for detecting Salmonella spp. from environmental samples collected at the large animal Purdue Veterinary Hospital (LA-PVH), a preliminary study compared the performance of RCSS and a SCP when testing artificially spiked and naturally contaminated samples. An expanded study analyzed results obtained using the RCSS method to test 872 environmental samples over a 12-month period. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was chosen as the confirmation method for RCSS-presumptive positive samples. A randomly selected subset of samples received additional confirmation by real-time PCR. Here, we reported the performance data of RCSS in terms of sensitivity, specificity, and positive predictive value using MALDI-TOF results as reference for comparison. We also provide guidelines for reporting results obtained using this system.

Adherent-invasive Escherichia coli associated with granulomatous colitis and extraintestinal dissemination in a Sphynx cat.

This case report describes a case of granulomatous colitis (GC) associated with adherent-invasive Escherichia coli (AIEC) with extension to cecum and ileum and dissemination to multiple lymph nodes, the spleen, and brain in a 10-year-old, male Sphynx cat. The cat had an episode of diarrhea 4 months prior to consultation due to sudden blindness. Signs rapidly progressed to ataxia, seizures, and death. Gross and histologic findings were consistent with granulomatous inflammation in all affected organs. In situ hybridization confirmed the presence of intracellular E. coli within enterocytes and infiltrating macrophages, and whole genome sequencing identified virulence traits commonly linked to AIEC strain. This is the first characterization of GC in a cat associated to AIEC resembling the metastatic form of Crohn's disease in humans and GC of dogs. Extraintestinal involvement might provide evidence of the ability of AIEC to promote granulomatous inflammation beyond the gut.

Next-Generation Diagnostics for Pathogens.

Next-generation sequencing (NGS) was initially developed to aid sequencing of the human genome. This molecular method is cost effective for sequencing and characterizing genomes, not only those of humans or animals but also those of bacteria and other pathogens. However, rather than sequencing a single organism, a targeted NGS method can be used to specifically amplify pathogens of interest in a clinical sample for detection and characterization by sequencing. Targeted NGS is an ideal method for ruminant syndromic testing due to its ability to detect a variety of pathogens in a sample with a single test.

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism.

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

Hyperkeratotic erythema multiforme variant in 17 dogs.

BACKGROUND: A new canine subgroup defined as 'old-dog' or 'hyperkeratotic' erythema multiforme (HKEM) with marked hyperkeratosis and parakeratosis has been proposed without any detailed description of larger case series. OBJECTIVES: We report herein the signalment, clinical signs, treatment outcome, and histopathological and immunological findings in 17 dogs with HKEM. ANIMALS: Inclusion criteria were the presence of (i) scaly skin lesions with or without crusting; and (ii) microscopic lesions typical of EM (i.e. a panepidermal cytotoxic lymphocytic dermatitis with or without basal keratinocyte apoptosis); and (iii) microscopic ortho- and/or parakeratotic hyperkeratosis affecting the interfollicular epidermis. MATERIALS AND METHODS: Clinical questionnaires and skin biopsies were reviewed. Polymerase chain reactions for epidermotropic viruses and direct immunofluorescence were performed. RESULTS: Various breeds were affected with an over-representation of males in their mid-to-late adulthood (median age 9 years). Generalised skin lesions included multifocal-to-coalescing, linear and annular macules and plaques with erythema and adherent firm crusting. Microscopic lesions were specific for EM and featured prominent superficial epidermal apoptosis with lymphocytic satellitosis and parakeratosis. No drug triggers were identified. Polymerase chain reactions for canine herpesvirus polymerase gene, canine parvovirus and canine distemper virus were negative in all HKEM and canine erosive EM (15 dogs) biopsies. Lesions failed to respond to oral and/or topical antimicrobials. Complete remission of signs was achieved in 9 of 17 dogs (53%) using immunosuppressive regimens. CONCLUSIONS AND CLINICAL RELEVANCE: Hyperkeratotic erythema multiforme (HKEM) is a chronic, persistent and clinically distinctive erythema multiforme (EM) variant that differs from 'classic' vesiculobullous erosive-to-ulcerative EM in dogs.

Targeted detection and molecular epidemiology of turkey coronavirus spike gene variants in turkeys and chickens.

Turkey coronavirus (TCoV) is a member of the Avian coronavirus species with infectious bronchitis virus (IBV), which is considered to be the source of TCoV. These 2 viruses are highly similar in all regions of their genomes, except for the spike gene, which is necessary for virus attachment. Although TCoV causes severe enteric disease in turkey poults, it does not cause clinical disease in chickens. However, considering that TCoV can infect chickens, it is important to distinguish TCoV from IBV in chickens. This is particularly true for chickens that are housed near turkeys and thus might be infected with TCoV and serve as a silent source of TCoV for turkeys. We developed and validated a real-time PCR assay to detect the spike gene of TCoV and sequenced a portion of this gene to evaluate the molecular epidemiology of TCoV infections associated with a commercial turkey premises in the United States in 2020-2021. We identified natural infections of TCoV in chickens, and based on the molecular epidemiology of the viruses detected, these chickens may have served as a source of infection for the commercial turkey premises located nearby.

Targeted Next-Generation Sequencing for Comprehensive Testing for Selected Vector-Borne Pathogens in Canines.

The standard for detecting vector-borne pathogens is real-time PCR (rtPCR). However, this requires many individual tests to obtain an accurate diagnosis. The purpose of this study was to develop and validate a targeted next-generation sequencing (NGS) assay for vector-borne pathogens. Pathogen target regions were amplified via PCR using two primer pools that were developed in conjunction with ThermoFisher Scientific, and barcoded DNA libraries were prepared and sequenced with the Ion Torrent S5 system. Data were assembled using SPAdes and mapped to a reference file containing sequences from the pathogens. The raw reads were analyzed to confirm the results. Test feasibility and analytical specificity were evaluated with type strains or validated positive clinical samples from dogs. The analytical sensitivity of the method was compared to Ct values obtained by rtPCR testing. Diagnostic sensitivity and specificity were assessed with a set of known positive and negative clinical samples based on rtPCR testing. Positive and negative percent agreements and Cohen's kappa were calculated. The primer sets were specific for the intended targets, based on sequence analysis of the amplified products, and the method detected 17 different pathogens. Analytical sensitivity was equivalent to an rtPCR Ct value of approximately 35-36. The positive percent agreement was 92%, and the negative percent agreement was 88%. Cohen's kappa was 0.804, which indicates almost perfect agreement between the rtPCR assays and the targeted NGS assay. Using a targeted method reduces the costs associated with NGS sequencing and allows for a 2-3 day turn-around time, making this a viable method for detection of vector-borne pathogens in canine whole blood samples.

Genomic Surveillance of SARS-CoV-2 in a University Community: Insights Into Tracking Variants, Transmission, and Spread of Gamma (P.1) Variant.

Background: Using a combination of data from routine surveillance, genomic sequencing, and phylogeographic analysis, we tracked the spread and introduction events of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants focusing on a large university community. Methods: Here, we sequenced and analyzed 677 high-quality SARS-CoV-2 genomes from positive RNA samples collected from Purdue University students, faculty, and staff who tested positive for the virus between January 2021 and May 2021, comprising an average of 32% of weekly cases across the time frame. Results: Our analysis of circulating SARS-CoV-2 variants over time revealed periods when variants of concern (VOC) Alpha (B.1.1.7) and Iota (B.1.526) reached rapid dominance and documented that VOC Gamma (P.1) was increasing in frequency as campus surveillance was ending. Phylodynamic analysis of Gamma genomes from campus alongside a subsampling of >20 000 previously published P.1 genomes revealed 10 independent introductions of this variant into the Purdue community, predominantly from elsewhere in the United States, with introductions from within the state of Indiana and from Illinois, and possibly Washington and New York, suggesting a degree of domestic spread. Conclusions: We conclude that a robust and sustained active and passive surveillance program coupled with genomic sequencing during a pandemic offers important insights into the dynamics of pathogen arrival and spread in a campus community and can help guide mitigation measures.

Effects of Phosphonate Herbicides on the Secretions of Plant-Beneficial Compounds by Two Plant Growth-Promoting Soil Bacteria: A Metabolomics Investigation.

Plant growth-promoting rhizobacteria (PGPR) that colonize plant roots produce a variety of plant-beneficial compounds, including plant-growth regulators, metal-scavenging compounds, and antibiotics against plant pathogens. Adverse effects of phosphonate herbicides, the most extensively used herbicides, on the growth and metabolism of PGPR species have been widely reported. However, the potential consequence of these effects on the biosynthesis and secretion of PGPR-derived beneficial compounds still remains to be investigated. Here, using high-resolution mass spectrometry and a metabolomics approach, we investigated both the intracellular metabolome and the extracellular secretions of biomass-normalized metabolite levels in two PGPR species (Pseudomonas protegens Pf-5, a Gram-negative bacterium; Priestia megaterium QM B1551, a Gram-positive bacterium) exposed to three common phosphonate herbicides (glyphosate, glufosinate, and fosamine; 0.1-1 mM) in either iron (Fe)-replete or Fe-deficient nutrient media. We quantified secreted auxin-type plant hormone compounds (phenylacetic acid and indole-3-acetic acid), iron-scavenging compounds or siderophores (pyoverdine and schizokinen), and antibiotics (2,4-diacetylphloroglucinol and pyoluteorin) produced by these PGPR species. The Fe-replete cells exposed to the phosphonate herbicides yielded up to a 25-fold increase in the production of both auxin and antibiotic compounds, indicating that herbicide exposure under Fe-replete conditions triggered metabolite secretions. However, the herbicide-exposed Fe-deficient cells exhibited a near 2-fold depletion in the secretion of these auxin and antibiotic compounds as well as a 77% decrease in siderophore production. Intracellular metabolomics analysis of the Fe-deficient cells further revealed metabolic perturbations in biosynthetic pathways consistent with the impaired production of the plant-beneficial compounds. Our findings implied that compromised cellular metabolism during nutrient deficiency may exacerbate the adverse effects of phosphonate herbicides on PGPR species.

Canine Distemper Virus in Endangered Species: Species Jump, Clinical Variations, and Vaccination.

Canine morbillivirus (Canine distemper virus, CDV) is the cause of distemper in a large number of different species, some of which are endangered. The clinical outcome associated with infection is variable and based on many factors, including the host species, the immune response of the individual animal to the infection, and variation in virus tropism and virulence. Unfortunately, the viral characteristics associated with virulence versus attenuation are not fully characterized, nor are the specific mutations that allow this virus to easily move and adapt from one species to another. Due to its wide host range, this virus is difficult to manage in ecosystems that are home to endangered species. Vaccination of the domestic dog, historically considered the reservoir species for this virus, at dog-wildlife interfaces has failed to control virus spread. CDV appears to be maintained by a metareservoir rather than a single species, requiring the need to vaccinate the wildlife species at risk. This is controversial, and there is a lack of a safe, effective vaccine for nondomestic species. This review focuses on topics that are paramount to protecting endangered species from a stochastic event, such as a CDV outbreak, that could lead to extinction.

Analogous Metabolic Decoupling in Pseudomonas putida and Comamonas testosteroni Implies Energetic Bypass to Facilitate Gluconeogenic Growth.

Gluconeogenic carbon metabolism is not well understood, especially within the context of flux partitioning between energy generation and biomass production, despite the importance of gluconeogenic carbon substrates in natural and engineered carbon processing. Here, using multiple omics approaches, we elucidate the metabolic mechanisms that facilitate gluconeogenic fast-growth phenotypes in Pseudomonas putida and Comamonas testosteroni, two Proteobacteria species with distinct metabolic networks. In contrast to the genetic constraint of C. testosteroni, which lacks the enzymes required for both sugar uptake and a complete oxidative pentose phosphate (PP) pathway, sugar metabolism in P. putida is known to generate surplus NADPH by relying on the oxidative PP pathway within its characteristic cyclic connection between the Entner-Doudoroff (ED) and Embden-Meyerhoff-Parnas (EMP) pathways. Remarkably, similar to the genome-based metabolic decoupling in C. testosteroni, our 13C-fluxomics reveals an inactive oxidative PP pathway and disconnected EMP and ED pathways in P. putida during gluconeogenic feeding, thus requiring transhydrogenase reactions to supply NADPH for anabolism in both species by leveraging the high tricarboxylic acid cycle flux during gluconeogenic growth. Furthermore, metabolomics and proteomics analyses of both species during gluconeogenic feeding, relative to glycolytic feeding, demonstrate a 5-fold depletion in phosphorylated metabolites and the absence of or up to a 17-fold decrease in proteins of the PP and ED pathways. Such metabolic remodeling, which is reportedly lacking in Escherichia coli exhibiting a gluconeogenic slow-growth phenotype, may serve to minimize futile carbon cycling while favoring the gluconeogenic metabolic regime in relevant proteobacterial species. IMPORTANCE Glycolytic metabolism of sugars is extensively studied in the Proteobacteria, but gluconeogenic carbon sources (e.g., organic acids, amino acids, aromatics) that feed into the tricarboxylic acid (TCA) cycle are widely reported to produce a fast-growth phenotype, particularly in species with biotechnological relevance. Much remains unknown about the importance of glycolysis-associated pathways in the metabolism of gluconeogenic carbon substrates. Here, we demonstrate that two distinct proteobacterial species, through genetic constraints or metabolic regulation at specific metabolic nodes, bypass the oxidative PP pathway during gluconeogenic growth and avoid unnecessary carbon fluxes by depleting protein investment into connected glycolysis pathways. Both species can leverage instead the high TCA cycle flux during gluconeogenic feeding to meet NADPH demand. Importantly, lack of a complete oxidative pentose phosphate pathway is a widespread metabolic trait in Proteobacteria with a gluconeogenic carbon preference, thus highlighting the important relevance of our findings toward elucidating the metabolic architecture in these bacteria.

Malakoplakia in the Urinary Bladder of 4 Puppies.

Malakoplakia in humans most often affects the urinary bladder and is characterized by inflammation with von Hansemann-type macrophages, with or without Michaelis-Gutmann bodies, and is frequently associated with Escherichia coli infection. We describe the microscopic features of malakoplakia in the urinary bladder of 4 puppies. In all cases, the lamina propria of the urinary bladder was markedly expanded by sheets of large, round to polygonal macrophages with intracytoplasmic, periodic acid-Schiff-positive granules and granular inclusions, and rare Prussian blue-positive inclusions. Macrophages were positive for CD18 and Iba1. In 2 cases, Michaelis-Gutmann bodies were detected with hematoxylin and eosin stain and were best demonstrated with von Kossa stain. E. coli infection was confirmed in 2 cases with bacterial culture or polymerase chain reaction (PCR) and sequencing of the bacterial 16S ribosomal RNA gene. Transmission electron microscopy of one case demonstrated macrophages with abundant lysosomes, phagolysosomes, and rod-shaped bacteria. Microscopic features were similar to human cases of malakoplakia. In dogs, the light microscopic characteristics of malakoplakia closely resemble granular cell tumors and histiocytic ulcerative colitis.

Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.