for conducting the layer study and formulating and preparing the layer diets. Our gratitude to Dr. Jason Keegan (Alltech Bioscience Centre (Ireland)) for project administration (animal science component) and initial data analysis (animal science component). Author contributions' section now is as follows: RL formatted all data for downstream analyses, performed all statistical, computational biology, and bioinformatic analyses, applied computational approaches for measuring uniformity, devised the ϙ metric for measuring relative dispersion in productivity observations, produced all figures, formatted all tables, wrote the draft papers, and applied corrections as advised by other authors. AC coordinated sample collection, sequencing, and productivity observations. RM and FW provided research supervision and project direction. All authors provided input to the corrected draft paper. The graphical abstract was produced under a paid licence with BioRender.com (2213-9170). Author contributions' section should be as follows: RL formatted all data for downstream analyses, performed all statistical, computational biology, and bioinformatic analyses, applied computational approaches for measuring uniformity, devised the ϙ metric for measuring relative dispersion in productivity observations, produced all figures, formatted all tables, wrote the draft papers, and applied corrections as advised by other authors. AC coordinated sample collection, sequencing,

BackgroundTreponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock.ResultsThe bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci.ConclusionsThe bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations.

BackgroundThe gastrointestinal microbiome and metabolome vary greatly throughout the different segments of the gastrointestinal tract, however current knowledge of gastrointestinal microbiome and metabolome in health and disease is limited to fecal samples due to ease of sampling. The engineered Small Intestinal MicroBiome Aspiration (SIMBA™) capsule allows specific sampling of the small intestine in humans. We aimed to determine whether administration of SIMBA™ capsules to healthy beagle dogs could reliably and safely sample the small intestinal microbiome and metabolome when compared to their fecal microbiome and metabolome.ResultsEleven beagle dogs were used for the study. Median transit time of capsules was 29.93 h (range: 23.83–77.88). Alpha diversity, as measured by the Simpson diversity, was significantly different (P = 0.048). Shannon diversity was not different (P = 0.114). Beta diversity results showed a significant difference between capsule and fecal samples regarding Bray–Curtis, weighted and unweighted unifrac (P = 0.002) and ANOSIM distance metric s (R = 0.59, P = 0.002). In addition to observing a statistically significant difference in the microbial composition of capsules and feces, distinct variation in the metabolite profiles was seen between the sample types. Heat map analysis showed 16 compounds that were significantly different between the 2 sampling modes (adj-P value ranged between 0.004 and 0.036) with 10 metabolites more abundant in the capsule than in the feces and 6 metabolites more abundant in the feces compared to the capsules.ConclusionsThe engineered Small Intestinal MicroBiome Aspiration (SIMBA™) capsule was easy and safe to administer to dogs. Microbiome and metabolome analysis from the capsule samples were significantly different than that of the fecal samples and were like previously published small intestinal microbiome and metabolome composition.Graphical abstract

BackgroundInfectious bovine keratoconjunctivitis (IBK) is a common cause of morbidity in cattle, resulting in significant economic losses. This study aimed to characterize the bovine bacterial ocular surface microbiome (OSM) through conjunctival swab samples from Normal eyes and eyes with naturally acquired, active IBK across populations of cattle using a three-part approach, including bacterial culture, relative abundance (RA, 16 S rRNA gene sequencing), and semi-quantitative random forest modeling (real-time polymerase chain reaction (RT-PCR)).ResultsConjunctival swab samples were obtained from eyes individually classified as Normal (n = 376) or IBK (n = 228) based on clinical signs. Cattle unaffected by IBK and the unaffected eye in cattle with contralateral IBK were used to obtain Normal eye samples. Moraxella bovis was cultured from similar proportions of IBK (7/228, 3.07%) and Normal eyes (1/159, 0.63%) (p = 0.1481). Moraxella bovoculi was cultured more frequently (p < 0.0001) in IBK (59/228, 25.88%) than Normal (7/159, 4.40%) eyes. RA (via 16 S rRNA gene sequencing) of Actinobacteriota was significantly higher in Normal eyes (p = 0.0045). Corynebacterium variabile and Corynebacterium stationis (Actinobacteriota) were detected at significantly higher RA (p = 0.0008, p = 0.0025 respectively) in Normal eyes. Rothia nasimurium (Actinobacteriota) was detected at significantly higher RA in IBK eyes (p < 0.0001). Alpha-diversity index was not significantly different between IBK and Normal eyes (p > 0.05). Alpha-diversity indices for geographic location (p < 0.001), age (p < 0.0001), sex (p < 0.05) and breed (p < 0.01) and beta-diversity indices for geographic location (p < 0.001), disease status (p < 0.01), age (p < 0.001), sex (p < 0.001) and breed (p < 0.001) were significantly different between groups. Modeling of RT-PCR values reliably categorized the microbiome of IBK and Normal eyes; primers for Moraxella bovoculi, Moraxella bovis, and Staphylococcus spp. were consistently the most significant canonical variables in these models.ConclusionsThe results provide further evidence that multiple elements of the bovine bacterial OSM are altered in the context of IBK, indicating the involvement of a variety of bacteria in addition to Moraxella bovis, including Moraxella bovoculi and R. nasimurium, among others. Actinobacteriota RA is altered in IBK, providing possible opportunities for novel therapeutic interventions. While RT-PCR modeling provided limited further support for the involvement of Moraxella bovis in IBK, this was not overtly reflected in culture or RA results. Results also highlight the influence of geographic location and breed type (dairy or beef) on the bovine bacterial OSM. RT-PCR modeling reliably categorized samples as IBK or Normal.

BackgroundThe dissemination of antibiotic resistance genes (ARGs) poses a substantial threat to environmental safety and human health. Herein, we present a longitudinal paired study across the swine lifetime from birth to market, coupled with metagenomic sequencing to explore the dynamics of ARGs and their health risk in the swine fecal microbiome.ResultsWe systematically characterized the composition and distribution of ARGs among the different growth stages. In total, 829 ARG subtypes belonging to 21 different ARG types were detected, in which tetracycline, aminoglycoside, and MLS were the most abundant types. Indeed, 134 core ARG subtypes were shared in all stages and displayed a growth stage-associated pattern. Furthermore, the correlation between ARGs, gut microbiota and mobile genetic elements (MGEs) revealed Escherichia coli represented the main carrier of ARGs. We also found that in most cases, the dominant ARGs could be transmitted to progeny piglets, suggesting the potential ARGs generation transmission. Finally, the evaluation of the antibiotic resistance threats provides us some early warning of those high health risk ARGs.ConclusionsCollectively, this relatively more comprehensive study provides a primary overview of ARG profile in swine microbiome across the lifetime and highlights the health risk and the intergenerational spread of ARGs in pig farm.Graphical

BackgroundThe nasal microbiota of the piglet is a reservoir for opportunistic pathogens that can cause polyserositis, such as Glaesserella parasuis, Mycoplasma hyorhinis or Streptococcus suis. Antibiotic treatment is a strategy to control these diseases, but it has a detrimental effect on the microbiota. We followed the piglets of 60 sows from birth to 8 weeks of age, to study the effect of ceftiofur on the nasal microbiota and the colonization by pathogens when the treatment was administered to sows or their litters. We also aimed to revert the effect of the antibiotic on the nasal microbiota by the inoculation at birth of nasal colonizers selected from healthy piglets. Nasal swabs were collected at birth, and at 7, 15, 21 and 49 days of age, and were used for pathogen detection by PCR and bacterial culture, 16S rRNA amplicon sequencing and whole shotgun metagenomics. Weights, clinical signs and production parameters were also recorded during the study.ResultsThe composition of the nasal microbiota of piglets changed over time, with a clear increment of Clostridiales at the end of nursery. The administration of ceftiofur induced an unexpected temporary increase in alpha diversity at day 7 mainly due to colonization by environmental taxa. Ceftiofur had a longer impact on the nasal microbiota of piglets when administered to their sows before farrowing than directly to them. This effect was partially reverted by the inoculation of nasal colonizers to newborn piglets and was accompanied by a reduction in the number of animals showing clinical signs (mainly lameness). Both interventions altered the colonization pattern of different strains of the above pathogens. In addition, the prevalence of resistance genes increased over time in all the groups but was significantly higher at weaning when the antibiotic was administered to the sows. Also, ceftiofur treatment induced the selection of more beta-lactams resistance genes when it was administered directly to the piglets.ConclusionsThis study shed light on the effect of the ceftiofur treatment on the piglet nasal microbiota over time and demonstrated for the first time the possibility of modifying the piglets’ nasal microbiota by inoculating natural colonizers of the upper respiratory tract.

BackgroundThe importance of the gut microbiota for physiological processes in mammals is well established, but the knowledge of their functional roles in fish is still limited. The aims of this study were to investigate associations between variation in taxonomical composition of the gut microbiota and gut health status in Atlantic salmon and to explore possible modulatory effects of dietary prebiotics in one net-pen farm in open water. The fish with initial mean body weight of around 240 g were fed diets based on the same basal composition, either without (Ref diet) or with (Test diet) yeast cell wall based-prebiotics, during the marine production phase from December to September the following year. Sampling was conducted at three sampling time points: January, April, and September, with average water temperature of 3.9 ℃, 3.4 ℃ and 9.6 ℃, respectively.ResultsAs the fish progressed towards September, growth, brush border membrane enzyme activities, and the expression in the gut of most of the observed genes involved in immune (e.g., il8, cd4a, myd88, il1b, gilt, tgfb, cd8b and cd3), barrier (e.g., zo1, occludin, ecad, claudin25b and claudin15), and metabolism increased significantly. Lipid accumulation in pyloric enterocytes decreased remarkably, suggesting improvement of gut health condition. The growth of the fish did not differ between dietary treatments. Further, dietary prebiotics affected the gut health only marginally regardless of duration of administration. Regarding gut microbiota composition, a decrease in alpha diversity (Observed species, Pielou and Shannon) over time was observed, which was significantly associated with an increase in the relative abundance of genus Mycoplasma and decrease in 32 different taxa in genus level including lactic acid bacteria (LAB), such as Lactobacillus, Leuconostoc, and Lactococcus. This indicates that developmental stage of Atlantic salmon is a determinant for microbial composition. Multivariate association analysis revealed that the relative abundance of Mycoplasma was positively correlated with gut barrier gene expression, negatively correlated with plasma glucose levels, and that its relative abundance slightly increased by exposure to prebiotics. Furthermore, certain LAB (e.g., Leuconostoc), belonging to the core microbiota, showed a negative development with time, and significant associations with plasma nutrients levels (e.g., triglyceride and cholesterol) and gene expression related to gut immune and barrier function.ConclusionsAs Atlantic salmon grew older under large-scale, commercial farm settings, the Mycoplasma became more prominent with a concomitant decline in LAB. Mycoplasma abundance correlated positively with time and gut barrier genes, while LAB abundance negatively correlated to time. Dietary prebiotics affected gut health status only marginally.

BackgroundDespite the long-established importance of zebrafish (Danio rerio) as a model organism and their increasing use in microbiome-targeted studies, relatively little is known about how husbandry practices involving diet impact the zebrafish gut microbiome. Given the microbiome’s important role in mediating host physiology and the potential for diet to drive variation in microbiome composition, we sought to clarify how three different dietary formulations that are commonly used in zebrafish facilities impact the gut microbiome. We compared the composition of gut microbiomes in approximately 60 AB line adult (129- and 214-day-old) zebrafish fed each diet throughout their lifespan.ResultsOur analysis finds that diet has a substantial impact on the composition of the gut microbiome in adult fish, and that diet also impacts the developmental variation in the gut microbiome. We further evaluated how 214-day-old fish microbiome compositions respond to exposure of a common laboratory pathogen, Mycobacterium chelonae, and whether these responses differ as a function of diet. Our analysis finds that diet determines the manner in which the zebrafish gut microbiome responds to M. chelonae exposure, especially for moderate and low abundance taxa. Moreover, histopathological analysis finds that male fish fed different diets are differentially infected by M. chelonae.ConclusionsOverall, our results indicate that diet drives the successional development of the gut microbiome as well as its sensitivity to exogenous exposure. Consequently, investigators should carefully consider the role of diet in their microbiome zebrafish investigations, especially when integrating results across studies that vary by diet.

BackgroundHuman encroachment into nature and the accompanying environmental changes are a big concern for wildlife biodiversity and health. While changes on the macroecological scale, i.e. species community and abundance pattern, are well documented, impacts on the microecological scale, such as the host’s microbial community, remain understudied. Particularly, it is unclear if impacts of anthropogenic landscape modification on wildlife gut microbiomes are species-specific. Of special interest are sympatric, generalist species, assumed to be more resilient to environmental changes and which often are well-known pathogen reservoirs and drivers of spill-over events. Here, we analyzed the gut microbiome of three such sympatric, generalist species, one rodent (Proechimys semispinosus) and two marsupials (Didelphis marsupialis and Philander opossum), captured in 28 study sites in four different landscapes in Panama characterized by different degrees of anthropogenic disturbance.ResultsOur results show species-specific gut microbial responses to the same landscape disturbances. The gut microbiome of P. semispinosus was less diverse and more heterogeneous in landscapes with close contact with humans, where it contained bacterial taxa associated with humans, their domesticated animals, and potential pathogens. The gut microbiome of D. marsupialis showed similar patterns, but only in the most disturbed landscape. P. opossum, in contrast, showed little gut microbial changes, however, this species’ absence in the most fragmented landscapes indicates its sensitivity to long-term isolation.ConclusionThese results demonstrate that wildlife gut microbiomes even in generalist species with a large ecological plasticity are impacted by human encroachment into nature, but differ in resilience which can have critical implications on conservation efforts and One Health strategies.

BackgroundExposure to microbes early in life has long-lasting effects on microbial community structure and function of the microbiome. However, in commercial poultry settings chicks are reared as a single-age cohort with no exposure to adult birds which can have profound effects on microbiota development and subsequent pathogen challenge. Microbiota manipulation is a proven and promising strategy to help reduce pathogen load and transmission within broiler flocks. However, administration of microbiota transplant products in a hatchery setting may prove challenging. Effective administration strategies are dependent on key factors, such as; the age of chicks receiving interventions and mode of delivery. This study aimed to assess these two aspects to provide supporting evidence towards microbiome manipulation strategies for use in commercial hatcheries.ResultsManipulation of the microbiota between 4 and 72 h of hatch markedly reduced faecal shedding and colonisation with the foodborne pathogen Salmonella enterica serovar Typhimurium (ST4/74). Administration of transplant material via spray or gel drop delivery systems had minimal effect on the protection conferred with fewer birds in transplant groups shown to shed ST4/74 in the faeces compared to PBS-gavaged control birds. Analysis of the microbiome following transplantation demonstrated that all transplant groups had higher diversity and species richness than non-transplant groups during the first week of life and the early stages of infection with ST47/4.The relative abundance of the bacterium Faecalibacterium prausnitzii was significantly higher in CMT groups compared to PBS controls. The presence of F. prausnitzii was also shown to increase in PBS-challenged birds compared to unchallenged birds potentially indicating a role of this bacterium in limiting Salmonella infections.ConclusionsThis study demonstrated that administration of microbiome transplants, using methods that would align with hatchery practices, effectively reduced colonisation and shedding of Salmonella in chickens. Age of chicks at microbiome administration had limited effect on the diversity and composition of the microbiome and conferred protection against Salmonella infections. Traditional hatchery delivery systems, such as spray or gel-drop, are sufficient to transfer donor material, alter the microbiome and confer protection against Salmonella. This study helps highlight the opportunity for use of microbiome modification methods within the hatchery.