Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus).

Widespread human SARS-CoV-2 infections combined with human-wildlife interactions create the potential for reverse zoonosis from humans to wildlife. We targeted white-tailed deer (Odocoileus virginianus) for serosurveillance based on evidence these deer have angiotensin-converting enzyme 2 receptors with high affinity for SARS-CoV-2, are permissive to infection, exhibit sustained viral shedding, can transmit to conspecifics, exhibit social behavior, and can be abundant near urban centers. We evaluated 624 prepandemic and postpandemic serum samples from wild deer from four US states for SARS-CoV-2 exposure. Antibodies were detected in 152 samples (40%) from 2021 using a surrogate virus neutralization test. A subset of samples tested with a SARS-CoV-2 virus neutralization test showed high concordance between tests. These data suggest white-tailed deer in the populations assessed have been exposed to SARS-CoV-2.

Plague Exposure in Mammalian Wildlife Across the Western United States.

Plague is caused by a bacterial pathogen (Yersinia pestis) that can infect a wide range of mammal species, but its presence in wildlife is often underappreciated. Using a large-scale data set (n = 44,857) that details the extent of Y. pestis exposure in wildlife, we document exposure in 18 wildlife species, including coyotes (Canis latrans), bobcats (Lynx rufus), and black bears (Ursus americanus). Evidence of plague activity is widespread, with seropositive animals detected in every western state in the contiguous United States. Pathogen monitoring systems in wildlife that are both large scale and long-term are rare, yet they open the door for analyses on potential shifts in distribution that have occurred over time because of climate or land use changes. The data generated by these long-term monitoring programs, combined with recent advances in our understanding of pathogen ecology, offer a clearer picture of zoonotic pathogens and the risks they pose.

Evidence for continental-scale dispersal of antimicrobial resistant bacteria by landfill-foraging gulls.

Anthropogenic inputs into the environment may serve as sources of antimicrobial resistant bacteria and alter the ecology and population dynamics of synanthropic wild animals by providing supplemental forage. In this study, we used a combination of phenotypic and genomic approaches to characterize antimicrobial resistant indicator bacteria, animal telemetry to describe host movement patterns, and a novel modeling approach to combine information from these diverse data streams to investigate the acquisition and long-distance dispersal of antimicrobial resistant bacteria by landfill-foraging gulls. Our results provide evidence that gulls acquire antimicrobial resistant bacteria from anthropogenic sources, which they may subsequently disperse across and between continents via migratory movements. Furthermore, we introduce a flexible modeling framework to estimate the relative dispersal risk of antimicrobial resistant bacteria in western North America and adjacent areas within East Asia, which may be adapted to provide information on the risk of dissemination of other organisms and pathogens maintained by wildlife through space and time.

Author Correction: The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bird-livestock interactions associated with increased cattle fecal shedding of ciprofloxacin-resistant Escherichia coli within feedlots in the United States.

This research study was conducted to determine if bird depredation in feedlots is associated with the prevalence of ciprofloxacin-resistant Escherichia coli in cattle and to determine if removal of invasive bird species could be an effective management strategy to help reduce ciprofloxacin-resistant E. coli in cattle within the United States. European starlings (Sturnus vulgaris) were collected from feedlots within multiple geographic regions within the United States and European starlings within all regions tested positive for ciprofloxacin-resistant E. coli, but prevalence differed by region. Total number of birds on feedlots were positively associated with increased cattle fecal shedding of ciprofloxacin-resistant E. coli. Targeted control of invasive European starlings reduced bird numbers on feedlots by 70.4%, but decreasing populations of European starlings was not associated with corresponding reductions in bovine fecal prevalence of ciprofloxacin-resistant E. coli. These data provide evidence for the role of wild bird depredation in feedlots contributing to fecal shedding of ciprofloxacin-resistant E. coli, but a single month of European starling control in feedlots was not sufficient to impact the fecal carriage of this organism in cattle.

The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations.

Antimicrobial use in livestock production is a driver for the development and proliferation of antimicrobial resistance (AMR). Wildlife interactions with livestock, acquiring associated AMR bacteria and genes, and wildlife's subsequent dispersal across the landscape are hypothesized to play an important role in the ecology of AMR. Here, we examined priority AMR phenotypes and genotypes of Escherichia coli isolated from the gastrointestinal tracts of European starlings (Sturnus vulgaris) found on concentrated animal feeding operations (CAFOs). European starlings may be present in high numbers on CAFOs (>100,000 birds), interact with urban environments, and can migrate distances exceeding 1,500 km in North America. In this study, 1,477 European starlings from 31 feedlots in five U.S. states were sampled for E. coli resistant to third generation cephalosporins (3G-C) and fluoroquinolones. The prevalence of 3G-C and fluoroquinolone-resistant E. coli was 4% and 10%, respectively. Multidrug resistance in the E. coli isolates collected (n = 236) was common, with the majority of isolates displaying resistance to six or more classes of antibiotics. Genetic analyses of a subset of these isolates identified 94 genes putatively contributing to AMR, including seven class A and C ß-lactamases as well as mutations in gyrA and parC recognized to confer resistance to quinolones. Phylogenetic and subtyping assessments showed that highly similar isolates (≥99.4% shared core genome, ≥99.6% shared coding sequence) with priority AMR were found in birds on feedlots separated by distances exceeding 150 km, suggesting that European starlings could be involved in the interstate dissemination of priority AMR bacteria.

Validation of a screening method for the detection of colistin-resistant E. coli containing mcr-1 in feral swine feces.

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 µg/mL) and vancomycin (8 µg/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 100 and 104 CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1-9.99 CFU/g (n = 340), 10-49.99 CFU/g (n = 170), 50-99 CFU/g (n = 255), 100-149 CFU/g (n = 60), and 200-2200 CFU/g (n = 175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment.

Gulls as Sources of Environmental Contamination by Colistin-resistant Bacteria.

In 2015, the mcr-1 gene was discovered in Escherichia coli in domestic swine in China that conferred resistance to colistin, an antibiotic of last resort used in treating multi-drug resistant bacterial infections in humans. Since then, mcr-1 was found in other human and animal populations, including wild gulls. Because gulls could disseminate the mcr-1 gene, we conducted an experiment to assess whether gulls are readily colonized with mcr-1 positive E. coli, their shedding patterns, transmission among conspecifics, and environmental deposition. Shedding of mcr-1 E. coli by small gull flocks followed a lognormal curve and gulls shed one strain >101 log10 CFU/g in their feces for 16.4 days, which persisted in the environment for 29.3 days. Because gulls are mobile and can shed antimicrobial-resistant bacteria for extended periods, gulls may facilitate transmission of mcr-1 positive E. coli to humans and livestock through fecal contamination of water, public areas and agricultural operations.

A model for the prediction of antimicrobial resistance in Escherichia coli based on a comparative evaluation of fatty acid profiles.

Antimicrobial resistance is a threat to agricultural production and public health. In this proof-of-concept study, we investigated predicting antimicrobial sensitive/resistant (S/R) phenotypes and host sources of Escherichia coli (n = 128) based on differential fatty acid abundance. Myristic (14:0), pentadecanoic acid (15:0), palmitic (16:0), elaidic (18:19) and steric acid (18:0) were significantly different (α = 0.05) using a two-way ANOVA for predicting nalidixic acid, ciprofloxacin, aztreonam, cefatoxime, and ceftazidime S/R phenotypes. Additionally, analyses of palmitoleic (16:1), palmitic acid (16:0), methyl palmitate (i-17:0), and cis-9,10-methyleneoctadecanoic acid (19:0Δ) showed these markers were significantly different (α = 0.05) between isolates obtained from cattle and raccoons. S/R phenotype prediction for the above antibiotics or host source, based on linear regression models of fatty acid abundance, were made using a replicated-randomized subsampling and modeling approach. This model predicted S/R phenotype with 79% and 81% accuracy for nalidixic acid and ciprofloxacin, respectively. The isolate host source was predicted with 63% accuracy.

Detection of Viruses from Bioaerosols Using Anion Exchange Resin.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

Short communication: A countrywide survey of antimicrobial-resistant indicator bacteria in Kosovo's dairy farms.

The World Health Organization recently recognized the Republic of Kosovo as one of the highest consumers per capita of antibiotics for human use among non-European Union Eastern European countries; however, data are limited regarding antimicrobial usage and antimicrobial resistance in the livestock sector for this recently formed country. The objective of this study was to conduct the first nationwide survey of antimicrobial resistance phenotypes in indicator bacteria collected from dairy farms in Kosovo. Composite fecal samples were collected from 52 farms located within all 7 administrative districts of Kosovo in the summer of 2014. Isolation and characterization of the indicator bacteria Escherichia coli (n = 165) and Enterococcus spp. (n = 153) from these samples was achieved by culturing on selective/differential media with and without select antibiotics, followed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry-based identification and antimicrobial susceptibility testing using the disk diffusion method. When no selective pressure was applied in culture-based isolation, the majority of E. coli and Enterococcus spp. collected were resistant to ≤1 of 16 and ≤2 of 12 antibiotics tested, respectively. In contrast, E. coli and Enterococcus spp. isolated using sub-minimum inhibitory concentrations of cefoxitin, ciprofloxacin, or erythromycin were typically resistant to at least one and often multiple antibiotic types, which primarily consisted of certain ß-lactams, quinolones, sulfonamides, phenicols, and tetracyclines for E. coli isolates and macrolides, tetracyclines, and rifamycins for enterococci isolates.

A Bead-Based Flow Cytometric Assay for Monitoring Yersinia pestis Exposure in Wildlife.

Yersinia pestis is the causative agent of plague and is considered a category A priority pathogen due to its potential for high transmissibility and the significant morbidity and mortality it causes in humans. Y. pestis is endemic to the western United States and much of the world, necessitating programs to monitor for this pathogen on the landscape. Elevated human risk of plague infection has been spatially correlated with spikes in seropositive wildlife numbers, particularly rodent-eating carnivores, which are frequently in contact with the enzootic hosts and the associated arthropod vectors of Y. pestis In this study, we describe a semiautomated bead-based flow cytometric assay developed for plague monitoring in wildlife called the F1 Luminex plague assay (F1-LPA). Based upon Luminex/Bio-Plex technology, the F1-LPA targets serological responses to the F1 capsular antigen of Y. pestis and was optimized to analyze antibodies eluted from wildlife blood samples preserved on Nobuto filter paper strips. In comparative evaluations with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64× improvement in analytical sensitivity for F1-specific IgG detection and allowed for unambiguous classification of IgG status. The functionality of the F1-LPA was demonstrated for coyotes and other canids, which are the primary sentinels in wildlife plague monitoring, as well as felids and raccoons. Additionally, assay formats that do not require species-specific immunological reagents, which are not routinely available for several wildlife species used in plague monitoring, were determined to be functional in the F1-LPA.

Prevalence and Amounts of Salmonella Found on Raw California Inshell Pistachios.

After harvest, pistachios are hulled with mechanical abrasion and then separated in a float tank containing water; the nuts that float (∼15%; floaters) and those that sink (∼85%; sinkers) are dried and stored separately. To determine the prevalence of Salmonella in pistachios, a total of 3,966 samples (1,032 floaters and 2,934 sinkers) were collected within 4 months of the 2010, 2011, and 2012 harvests from storage silos (12 samples from each silo, in most cases) and were stored at 4°C; 100-g subsamples were enriched for the presence of Salmonella. Twenty-one of the floater samples and 11 of the sinker samples were positive for Salmonella: 2.0% prevalence (95% confidence interval [CI], 1.3 to 3.1%) and 0.37% prevalence (95% CI, 0.21 to 0.67%), respectively, for a weighted average prevalence of 0.61%. Levels of Salmonella were determined for positive samples using a most-probable-number (MPN) method with multiple 50-g, three 5.6-g, and three 0.56-g subsamples. Geometric mean levels of Salmonella in floaters and sinkers were 0.66 MPN/100 g (0.14 to 5.3 MPN/100 g) and 0.18 MPN/100 g (0.10 to 0.62 MPN/100 g), respectively. Seven different serovars were identified among the isolates, with nine pulsed-field gel electrophoresis fingerprints; as many as four serovars were isolated from some samples. Salmonella serovars Montevideo (44%), Enteritidis (19%), Senftenberg (16%), Worthington (12%), and Liverpool (9.4%) were most commonly isolated from the initial 100-g samples. The prevalence and levels of Salmonella in pistachios are within those observed for other tree nuts, but the limited number of serovars isolated suggests a narrow and persistent contamination source.

Francisella tularensis LVS surface and membrane proteins as targets of effective post-exposure immunization for tularemia.

Francisella tularensis causes disease (tularemia) in a large number of mammals, including man. We previously demonstrated enhanced efficacy of conventional antibiotic therapy for tularemia by postexposure passive transfer of immune sera developed against a F. tularensis LVS membrane protein fraction (MPF). However, the protein composition of this immunogenic fraction was not defined. Proteomic approaches were applied to define the protein composition and identify the immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice coupled to liquid chromatography-tandem mass spectrometry identified 24 immunoreactive protein spots containing 45 proteins. A reverse vaccinology approach that applied labeling of F. tularensis LVS surface proteins and bioinformatics was used to reduce the complexity of potential target immunogens. Bioinformatics analyses of the immunoreactive proteins reduced the number of immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted in the identification of 31 surface proteins. However, only 13 of these were reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use of orthogonal proteomic approaches reduced the complexity of potential immunogens in MPF by 96% and allowed for prioritization of target immunogens for antibody-based immunotherapies against tularemia.

Diversity of Salmonella isolates from central Florida surface waters.

Identification of Salmonella serotypes is important for understanding the environmental diversity of the genus Salmonella. This study evaluates the diversity of Salmonella isolates recovered from 165 of 202 Central Florida surface water samples and investigates whether the serotype of the environmental Salmonella isolates can be predicted by a previously published multiplex PCR assay (S. Kim, J. G. Frye, J. Hu, P. J. Fedorka-Cray, R. Gautom, and D. S. Boyle, J. Clin. Microbiol. 44:3608-3615, 2006, http://dx.doi.org/10.1128/JCM.00701-06). Multiplex PCR was performed on 562 Salmonella isolates (as many as 36 isolates per water sample) to predict serotypes. Kauffmann-White serogrouping was used to confirm multiplex PCR pattern groupings before isolates were serotyped, analyzed by pulsed-field gel electrophoresis, and assayed for antimicrobial susceptibility. In 41.2% of the Salmonella-positive water samples, all Salmonella isolates had identical multiplex PCR patterns; in the remaining 58.8%, two or more multiplex PCR patterns were identified. Within each sample, isolates with matching multiplex PCR patterns had matching serogroups. The multiplex patterns of 495 isolates (88.1%) did not match any previously reported pattern. The remaining 68 isolates matched reported patterns but did not match the serotypes for those patterns. The use of the multiplex PCR allowed the number of isolates requiring further analysis to be reduced to 223. Thirty-three Salmonella enterica serotypes were identified; the most frequent included serotypes Muenchen, Rubislaw, Anatum, Gaminara, and IV_50:z4,z23:-. A majority (141/223) of Salmonella isolates clustered into one genotypic group. Salmonella isolates in Central Florida surface waters are serotypically, genotypically, and phenotypically (in terms of antimicrobial susceptibility) diverse. While isolates could be grouped as different or potentially the same using multiplex PCR, the multiplex PCR pattern did not predict the Salmonella serotype.

Colorimetric paper-based detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes of agricultural water.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Post-exposure immunization against Francisella tularensis membrane proteins augments protective efficacy of gentamicin in a mouse model of pneumonic tularemia.

Successful treatment of pneumonic infection with Francisella tularensis, the causative agent of tularemia, requires rapid initiation of antibiotic therapy, yet even then treatment failures may occur. Consequently, new treatments are needed to enhance the effectiveness of antimicrobial therapy for acute pneumonic tularemia. In a prior study, immunization with F. tularensis membrane protein fraction (MPF) antigens 3 days prior to challenge was reported to induce significant protection from inhalational challenge. We therefore hypothesized that MPF immunization might also be effective in enhancing infection control if combined with antibiotic therapy and administered after infection as post-exposure immunotherapy. To address this question, a 24h post-exposure treatment model of acute pulmonary Schu S4 strain of F. tularensis infection in BALB/c mice was used. Following exposure, mice were immunized with MPF and treated with low-dose gentamicin, alone or in combination and the effects on survival, bacterial burden and dissemination were assessed. We found that immunization with MPF significantly increased the effectiveness of subtherapeutic gentamicin for post-exposure treatment of pneumonic tularemia, with 100% of combination-treated mice surviving long-term. Bacterial burdens in the liver and spleen were significantly reduced in combination MPF-gentamicin treated mice at 7 days after challenge. Passively transferred antibodies against MPF antigens also increased the effectiveness of gentamicin therapy. Thus, we concluded that post-exposure immunization with MPF antigens was an effective means of enhancing conventional antimicrobial therapy for pneumonic tularemia.

Differential chitinase activity and production within Francisella species, subspecies, and subpopulations.

Genotyping of Francisella tularensis (A1a, A1b, A2, and type B) and Francisella novicida has identified multiple differences between species and among F. tularensis subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of F. tularensis and F. novicida to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among F. tularensis and F. novicida strains. The activity observed for F. tularensis strains was predominantly associated with whole-cell lysates, while the chitinase activity of F. novicida localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of F. tularensis and between the species. Bioinformatic analyses identified two new putative chitinase genes (chiC and chiD), as well as the previously described chiA and chiB. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between Francisella species and F. tularensis subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of chiA and chiC knockouts in F. tularensis A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of Francisella.