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  1. IntroductionSpillover events ofMycoplasma ovipneumoniaehave devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread ofM. ovipneumoniaebetween the sheep populations. Most studies involving the typing ofM. ovipneumoniaehave used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing. MethodsOur study aimed to develop and validate an MLST workflow for typing ofM. ovipneumoniaeusing Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes ofM. ovipneumoniaeusing archived DNA samples (N= 68) from nasal swabs. ResultsSequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods. DiscussionOur proposed workflow is a convenient and effective method for strain typing ofM. ovipneumoniaeand can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important. 
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    Free, publicly-accessible full text available July 31, 2025
  2. Abstract The microbiome is a complex community of microorganisms, encompassing prokaryotic (bacterial and archaeal), eukaryotic, and viral entities. This microbial ensemble plays a pivotal role in influencing the health and productivity of diverse ecosystems while shaping the web of life. However, many software suites developed to study microbiomes analyze only the prokaryotic community and provide limited to no support for viruses and microeukaryotes. Previously, we introduced the Viral Eukaryotic Bacterial Archaeal (VEBA) open-source software suite to address this critical gap in microbiome research by extending genome-resolved analysis beyond prokaryotes to encompass the understudied realms of eukaryotes and viruses. Here we present VEBA 2.0 with key updates including a comprehensive clustered microeukaryotic protein database, rapid genome/protein-level clustering, bioprospecting, non-coding/organelle gene modeling, genome-resolved taxonomic/pathway profiling, long-read support, and containerization. We demonstrate VEBA’s versatile application through the analysis of diverse case studies including marine water, Siberian permafrost, and white-tailed deer lung tissues with the latter showcasing how to identify integrated viruses. VEBA represents a crucial advancement in microbiome research, offering a powerful and accessible software suite that bridges the gap between genomics and biotechnological solutions. 
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  3. Chu, Hiutung (Ed.)
    ABSTRACT Crohn’s disease (CD) is a presentation of inflammatory bowel disease (IBD) that manifests in childhood and adolescence and involves chronic and severe enterocolitis, immune and gut microbial dysregulation, and other complications. Diet and gut-microbiota-produced metabolites are sources of anti-inflammatories that could ameliorate symptoms. However, questions remain on how IBD influences biogeographic patterns of microbial location and function in the gut, how early life transitional gut communities are affected by IBD and diet interventions, and how disruption to biogeography alters disease mediation by diet components or microbial metabolites. Many studies on diet and IBD use a chemically induced ulcerative colitis model, despite the availability of an immune-modulated CD model. Interleukin-10-knockout (IL-10-KO) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed a control diet or one containing 10% (wt/wt) raw broccoli sprouts, which was high in the sprout-sourced anti-inflammatory sulforaphane. Diets began 7 days prior to, and for 2 weeks after inoculation withHelicobacter hepaticus,which triggers Crohn’s-like symptoms in these immune-impaired mice. The broccoli sprout diet increased sulforaphane in plasma; decreased weight stagnation, fecal blood, and diarrhea associated; and increased microbiota richness in the gut, especially in younger mice. Sprout diets resulted in some anatomically specific bacteria in younger mice and reduced the prevalence and abundance of pathobiont bacteria which trigger inflammation in the IL-10-KO mouse, for example,Escherichia coliandHelicobacter. Overall, the IL-10-KO mouse model is responsive to a raw broccoli sprout diet and represents an opportunity for more diet-host-microbiome research. IMPORTANCETo our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn’s disease disrupts the lives of patients and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms. 
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  4. Manichanh, Chaysavanh (Ed.)
    ABSTRACT Inflammatory bowel diseases (IBDs) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective and affordable for managing symptoms. Glucosinolate compounds are highly concentrated in broccoli sprouts, especially glucoraphanin (GLR), and can be metabolized by certain mammalian gut bacteria into anti-inflammatory isothiocyanates, such as sulforaphane. Gut microbiota exhibit biogeographic patterns, but it is unknown if colitis alters these or whether the location of glucoraphanin-metabolizing bacteria affects anti-inflammatory benefits. We fed specific pathogen-free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 34-day experiment to simulate chronic, relapsing ulcerative colitis (UC). We monitored body weight, fecal characteristics, lipocalin, serum cytokines, and bacterial communities from the luminal- and mucosal-associated populations in the jejunum, cecum, and colon. Mice fed the broccoli sprout diet with DSS treatment performed better than mice fed the control diet with DSS, and had significantly more weight gain, lower Disease Activity Index scores, lower plasma lipocalin and proinflammatory cytokines, and higher bacterial richness in all gut locations. Bacterial communities were assorted by gut location but were more homogenous across locations in the control diet + DSS mice. Importantly, our results showed that broccoli sprout feeding abrogated the effects of DSS on gut microbiota, as bacterial richness and biogeography were similar between mice receiving broccoli sprouts with and without DSS. Collectively, these results support the protective effect of steamed broccoli sprouts against dysbiosis and colitis induced by DSS. IMPORTANCEEvaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate-induced colitis, that colitis erases biogeographic patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy. 
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  5. Abstract Contaminated drinking water is an important public health consideration in New England where well water is often found to contain arsenic and other metals such as cadmium, lead, and uranium. Chronic or high level exposure to these metals have been associated with multiple acute and chronic diseases, including cancers and impaired neurological development. While individual metal levels are often regulated, adverse health effects of metal mixtures, especially at concentrations considered safe for human consumption remain unclear. Here, we utilized a multivariate analysis that examined behavioral outcomes in the zebrafish model as a function of multiple metal chemical constituents of 92 drinking well water samples, collected in Maine and New Hampshire. To collect these samples, a citizen science approach was used, that engaged local teachers, students, and scientific partners. Our analysis of 4016 metal-mixture combinations shows that changes in zebrafish behavior are highly mixture dependent, and indicate that certain combinations of metals, especially those containing arsenic, cadmium, lead, and uranium, even at levels considered safe in drinking water, are significant drivers of behavioral toxicity. Our data emphasize the need to consider low-level chemical mixture effects and provide a framework for a more in-depth analysis of drinking water samples. We also provide evidence for the efficacy of utilizing citizen science in research, as the broader impact of this work is to empower local communities to advocate for improving their own water quality. 
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  6. Abstract Bacillus anthracis, the causative agent of anthrax, is a considerable global health threat affecting wildlife, livestock, and the general public. In this study, whole‐genome sequence analysis of over 350B. anthracisisolates was used to establish a new high‐resolution global genotyping framework that is both biogeographically informative and compatible with multiple genomic assays. The data presented in this study shed new light on the diverse global dissemination of this species and indicate that many lineages may be uniquely suited to the geographic regions in which they are found. In addition, we demonstrate that plasmid genomic structure for this species is largely consistent with chromosomal population structure, suggesting vertical inheritance in this bacterium has contributed to its evolutionary persistence. This classification methodology is the first based on population genomic structure for this species and has potential use for local and broader institutions seeking to understand both disease outbreak origins and recent introductions. In addition, we provide access to a newly developed genotyping script as well as the full whole‐genome sequence analyses output for this study, allowing future studies to rapidly employ and append their data in the context of this global collection. This framework may act as a powerful tool for public health agencies, wildlife disease laboratories, and researchers seeking to utilize and expand this classification scheme for further investigations intoB. anthracisevolution. 
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  7. Abstract Animals generally benefit from their gastrointestinal microbiome, but the factors that influence the composition and dynamics of their microbiota remain poorly understood. Studies of nonmodel host species can illuminate how microbiota and their hosts interact in natural environments. We investigated the role of migratory behaviour in shaping the gut microbiota of free‐ranging barn swallows (Hirundo rustica) by studying co‐occurring migrant and resident subspecies sampled during the autumn migration at a migratory bottleneck. We found that within‐host microbial richness (α‐diversity) was similar between migrant and resident microbial communities. In contrast, we found that microbial communities (β‐diversity) were significantly different between groups regarding both microbes present and their relative abundances. Compositional differences were found for 36 bacterial genera, with 27 exhibiting greater abundance in migrants and nine exhibiting greater abundance in residents. There was heightened abundance ofMycoplasmaspp. andCorynebacteriumspp. in migrants, a pattern shared by other studies of migratory species. Screens for key regional pathogens revealed that neither residents nor migrants carried avian influenza viruses and Newcastle disease virus, suggesting that the status of these diseases did not underlie observed differences in microbiome composition. Furthermore, the prevalence and abundance ofSalmonellaspp., as determined from microbiome data and cultural assays, were both low and similar across the groups. Overall, our results indicate that microbial composition differs between migratory and resident barn swallows, even when they are conspecific and sympatrically occurring. Differences in host origins (breeding sites) may result in microbial community divergence, and varied behaviours throughout the annual cycle (e.g., migration) could further differentiate compositional structure as it relates to functional needs. 
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  8. Abstract The behavioural ecology of host species is likely to affect their microbial communities, because host sex, diet, physiology, and movement behaviour could all potentially influence their microbiota. We studied a wild population of barn owls (Tyto alba) and collected data on their microbiota, movement, diet, size, coloration, and reproduction. The composition of bacterial species differed by the sex of the host and female owls had more diverse bacterial communities than their male counterparts. The abundance of two families of bacteria, Actinomycetaceae and Lactobacillaceae, also varied between the sexes, potentially as a result of sex differences in hormones and immunological function, as has previously been found with Lactobacillaceae in the microbiota of mice. Male and female owls did not differ in the prey they brought to the nest, which suggests that dietary differences are unlikely to underlie the differences in their microbiota. The movement behaviour of the owls was associated with the host microbiota in both males and females because owls that moved further from their nest each day had more diverse bacterial communities than owls that stayed closer to their nests. This novel result suggests that the movement ecology of hosts can impact their microbiota, potentially on the basis of their differential encounters with new bacterial species as the hosts move and forage across the landscape. Overall, we found that many aspects of the microbial community are correlated with the behavioural ecology of the host and that data on the microbiota can aid in generating new hypotheses about host behaviour. 
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  9. 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, onlyClostridium novyiwas 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. 
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  10. Anthropogenic influences caused depletion and subsequent recovery of marine predators, but ecological consequences of altered predator abundance are not well understood. Although many methods are used to study predator diets, methodological biases and logistical challenges preclude robust sampling schemes. We aimed to compare two non-invasive methods: metabarcoding scat-derived deoxyribonucleic acid and hard parts analysis of scat for the Northwest Atlantic grey seal ( Halichoerus grypus (Fabricius, 1791)), a species that rebounded after near extirpation. We hypothesized that metabarcoding would detect a greater diversity and frequency of prey, and that notable differences in diet will be detected since prior studies. Grey seal scat samples ( N = 247) were collected between 2018 and 2019 from Monomoy Island, Massachusetts, USA. Metabarcoding detected greater prey richness on average, with more frequent detections of clupeids (Clupeidae) and flatfish (Pleuronectiformes), whereas hard parts analysis more frequently detected phycid hakes ( Urophycis spp. Gill, 1863). Combining methods increased detections of 13 prey taxa, with 32 prey taxa identified overall. Skates (Rajidae), flatfish, clupeids, and sand lance ( Ammodytes spp. Linnaeus, 1758) were top-occurring prey. Our study highlights the importance of using multiple methods to characterize generalist predator diets using non-invasive techniques and suggests grey seal diet has changed since the early 2000s. 
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