Devin B. Holman

Fusobacterium necrophorum is frequently associated with oral cavity infections in livestock and other animal species. Here, we present the draft genome sequence of F. necrophorum subsp. necrophorum FNN105, isolated from the oral cavity of an alpaca ( Vicugna pacos ). The draft genome is 2.49 Mbp and contains 2,237 predicted protein-coding sequences.

Cattle harbor diverse bacterial communities across the gastrointestinal, respiratory, reproductive, mammary and other anatomical systems, but body-wide microbial biogeography remains poorly defined. To address this knowledge gap, we conducted a meta-analysis of publicly available bovine 16S rRNA gene amplicon sequencing data from 5,637 samples from 47 studies across six geographic regions and 27 anatomical sample types. Bacterial community structure differed significantly among sample types (PERMANOVA, R ² = 0.245, P = 0.0001), indicating spatial organization of bacterial communities across the bovine body, although study-level effects also contributed substantially to community variation. Communities were generally more similar within than between anatomical systems. Bacterial richness, diversity, taxonomic composition, and indicator taxa varied among sample types, with gastrointestinal, mammary-associated, ocular, and hoof microbiota exhibiting greater diversity than microbiota from liver, joint, and several reproductive samples. Distinct bacterial communities characterized the gastrointestinal, respiratory, reproductive, and mammary systems, as well as other anatomical sites. Despite these differences, several bacterial taxa were shared across multiple anatomical niches, particularly among male and female reproductive sites and among mammary-associated niches. This study provides a comprehensive body-wide characterization of bacterial biogeography in cattle and establishes a baseline for future studies of bovine microbial ecology and host–microbiome interactions.

Sow colostrum and milk provide essential nutrients, immune protection and one of the earliest microbial exposures for piglets. However, the microbial composition, functional potential and host interactions of these mammary secretions remain poorly characterized. Here, we combined culturomics, metagenomics and proteomics to comprehensively characterize the microbiome and proteome of sow colostrum and milk collected at farrowing and at 7 and 21 days postpartum. We recovered 132 bacterial isolates representing at least 42 species, including 15 putatively novel taxa. These isolates included both potentially pathogenic species, such as Sarcina perfringens and Streptococcus suis, and potentially beneficial bacterial species like Lactobacillus amylovorus and Lactiplantibacillus plantarum . The microbial composition and functional potential shifted significantly as the milk matured, with L. amylovorus , Limosilactobacillus reuteri and Rothia spp . among the most relatively abundant taxa. Several antimicrobial resistance genes, including erm (C), tet(K ), tet (M), lnu(A ), poxtA and fexB , were identified on contigs encoding plasmid replicons in the isolates, indicating potential for horizontal gene transfer. Functional annotation of isolate genomes indicated broad carbohydrate-active enzyme (CAZyme) repertoires, including β-galactosidase-associated families and other CAZyme families consistent with potential milk oligosaccharide utilization. The colostrum and milk proteome also shifted during lactation, reflecting declining immune-related proteins and increasing metabolic and structural proteins. Correlations between specific microbial taxa and host proteins, including Rothia spp . and immune proteins or glycoproteins, suggested potential host–microbe interactions during lactation. Together, these findings provide a multi-omic perspective on how mammary microbiome dynamics and host responses during lactation may influence neonatal microbial colonization and health.

Fusobacterium necrophorum (FN) is an important opportunistic pathogen implicated in necrotizing infections, including liver abscesses, calf diphtheria, metritis, and foot rot in cattle, and tonsillopharyngitis in humans. However, FN also exists as a commensal member of the bovine reproductive microbiota with potential negative, as in metritis, and even positive associations with pregnancy outcomes. The genomic features that enable FN to colonize diverse hosts and anatomical niches as either a commensal or a pathogen is poorly understood. We addressed these knowledge gaps by performing comparative genomic analysis of 137 FN strains (80 newly sequenced, 57 publicly available) from clinical and non-clinical sources across human and bovine hosts. We investigated the pangenome structure, virulence gene repertoire, antimicrobial resistance genes (ARG) prevalence, as well as host-and subspecies-associated genomic signatures of two FN subspecies: subsp. necrophorum (FNN) and subsp. funduliforme (FNF). Comparative genomics revealed an open pangenome with high accessory diversity, and phylogenetic analysis separated the strains into two distinct subspecies clades. Functional profiling revealed substantial metabolic divergence between subspecies, with FNN showing higher prevalence of carbohydrate transport systems and advanced glycation-related pathways, while FNF showed enrichment in threonate metabolism and hemolysin-related systems. Virulence gene analysis identified 84 variants across multiple functional categories with subspecies- and host-specific distributions. Antimicrobial resistance genes, primarily tetracycline resistance genes [tet(O), tet(M), tet(40)] and the macrolide resistance gene erm(B), were detected in 22.6% of strains, with higher prevalence in bovine than human strains. Overall, our results suggest that pathogenic potential of FN appears to be determined by the interplay between an open pangenome, subspecies-specific metabolic and virulence repertoires, host-associated adaptation, and niche specialization. IMPORTANCE Fusobacterium necrophorum, comprising two subspecies, necrophorum (FNN) and funduliforme (FNF), is a major pathogen in cattle and humans, yet it also occurs as a commensal inhabitant in healthy cattle, particularly in the rumen, hindgut, semen, and the female reproductive tract. However, emerging evidence indicates it also occurs as a non-clinical inhabitant, particularly in the bovine reproductive tract, where it may be associated with improved pregnancy outcomes. We conducted a comparative genomic analysis of 137 F. necrophorum strains, including FNN (n = 12) and FNF (n = 125), sourced from humans (n=53) and cattle (n=84) across seven anatomical niches spanning both healthy and diseased sources. We identified subspecies- and host-specific metabolic pathways, antimicrobial resistance profiles, and distinct virulence gene distributions that underpin the ecological versatility of Fusobacterium necrophorum. Overall, these findings provide a genomic framework for understanding its host adaptation and niche specialization across bovine and human hosts.

The female reproductive tract harbors complex microbial communities that may influence reproductive success. In previous work using 16S rRNA gene sequencing, we identified bacterial taxa in the vagina and uterus of beef cattle associated with pregnancy outcomes, but taxonomic resolution and functional inference was limited. Here we used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination (AI) in cows that subsequently became pregnant or remained open. Vaginal (pregnant n = 54; open n = 7) and uterine (pregnant, n = 41; open, n = 9) samples were collected prior to AI. Microbial community structure did not differ between pregnancy outcome groups in either anatomical site (PERMANOVA; P > 0.05). However, cows that remained open showed significantly greater species-level richness and diversity in the vaginal microbiome ( P < 0.05). No diversity differences were observed in the uterine microbiome. In contrast, significant differences were detected between anatomical sites, with distinct dominant taxa and functional profiles. Vaginal microbiomes were enriched in pathways related to genetic information processing, whereas uterine microbiomes exhibited greater representation of metabolic pathways. A total of 105 ARGs spanning 11 antimicrobial classes were identified, with tetracycline resistance genes [ tet (Q), tet (W), and tet (M)] predominating, and bla _TEM-116 more abundant in the uterine microbiome. Overall, while vaginal and uterine microbiomes were compositionally and functionally distinct, no robust pregnancy-associated taxonomic or functional signatures were detected, likely reflecting limited statistical power and challenges inherent to low-biomass metagenomic datasets. IMPORTANCE: Understanding the role of the reproductive tract microbiome in fertility could improve reproductive efficiency in cattle. We used shotgun metagenomic sequencing to characterize the taxonomic composition, functional potential, and antimicrobial resistome of vaginal and uterine microbiomes at the time of artificial insemination in cows that subsequently became pregnant or remained open. Using paired samples from the same animals, we directly compared microbial communities between the upper and lower reproductive tract to identify shared and site-specific features. Although no distinct microbial signatures associated with pregnancy outcomes were detected, this may reflect limited statistical power and low microbial biomass inherent to these samples. Despite these challenges, our study provides high-resolution insights into the composition, functional potential, and resistome of bovine reproductive microbiomes and highlights important technical considerations for studying low-biomass microbial ecosystems.

Sow colostrum and milk provide essential nutrients, immune protection, and one of the earliest microbial exposures for piglets. However, the microbial composition, functional potential, and host interactions of these mammary secretions remain poorly characterized. Here, we combined culturomics, metagenomics, and proteomics to comprehensively characterize the microbiome and proteome of sow colostrum and milk collected at farrowing and at 7 and 21 days postpartum. We recovered 132 bacterial isolates representing at least 42 species, including 15 putative novel taxa. These isolates included both potentially pathogenic species such as Sarcina perfringens and Streptococcus suis and potentially beneficial bacterial species like Lactobacillus amylovorus and Lactiplantibacillus plantarum . The microbial composition and functional potential shifted significantly as the milk matured, with L. amylovorus , Limosilactobacillus reuteri , and Rothia spp. among the most relatively abundant taxa. Several antimicrobial resistance genes, including erm (C), tet(K) , tet (M), lnu(A) , poxtA , and fexB , were identified on contigs encoding plasmid replicons in the isolates, indicating potential for horizontal gene transfer. Functional annotation of isolate genomes indicated broad carbohydrate-active enzyme (CAZyme) repertoires, including those conferring β-galactosidase activity and the capacity to metabolize milk oligosaccharides. The colostrum and milk proteome also shifted during lactation, reflecting declining immune-related proteins and increasing metabolic and structural proteins. Correlations between specific microbial taxa and host proteins, including Rothia spp. and immune proteins or glycoproteins, suggested potential host–microbe interactions during lactation. Together, these findings provide a multi-omic perspective on how mammary microbiome dynamics and host responses during lactation may influence neonatal microbial colonization and health.

The gut microbiome is an important factor in animal health and can be influenced by factors such as age, diet, stress, environmental conditions, and farming practices. Bacterial communities of the gut microbiome in many species have been extensively studied, but research on the fungal microbiota remains limited and underrepresented in the literature. The objective of this study was to characterize the fecal mycobiota of swine raised under two different production systems: outdoor pasture-based or conventional indoor systems. Fecal samples from nursery, growing-finishing, and sow pigs from both farming systems were collected, and the mycobiota was profiled using PCR amplification and sequencing of the universal fungal internal transcribed spacer 1 (ITS1) region. A significant difference in fungal community structure was observed between the conventionally raised and pasture-raised pigs, as well as among all three production phases. Four species, Arthrographis kalrae, Enterocarpus grenotii, Pseudallescheria angusta, and Sagenomella oligospora, were differentially abundant between the two farms, all of which had higher relative abundance in the pasture-raised pigs. Additionally, pasture-raised pigs hosted a more diverse fungal community with higher species richness in their gastrointestinal tract. In summary, farming practices and pig age influenced the pig fecal mycobiota.

Trueperella pyogenes is an important bacterial pathogen implicated in infections such as mastitis, metritis, pneumonia, and liver abscesses in both domestic and wild animals, as well as endocarditis and prosthetic joint infections in humans. Understanding the genomic and metabolic features that enable T. pyogenes to colonize different anatomical sites within a host and its inter-kingdom transmission and survival is important for the effective control of this pathogen. We employed whole-genome sequencing, phenotype microarrays, and antimicrobial susceptibility testing to identify genomic, metabolic and phenotypic features, and antimicrobial resistance (AMR) genes in T. pyogenes recovered from different livestock, companion, and wildlife animals. For comparative genomic analysis, 83 T. pyogenes genomes, including 60 isolated in the current study and 23 publicly available genomes were evaluated. These genomes represented T. pyogenes strains originating from 16 different body sites of 11 different animal hosts (e.g., cattle, swine, ovine, deer, bison, horse, chamois, and cat). Additionally, 49 T. pyogenes isolates (cattle, sheep, deer, swine, and cats) were evaluated for phenotypic AMR using disk diffusion, and for metabolic profiling using the Biology GENIII MicroPlates. The T. pyogenes strains were found not to be host- or body site-specific. The presence of conserved virulence genes ( plo and fimA ), as well as genotypic and phenotypic AMR may contribute to the ability of T. pyogenes to cause infections in livestock, wildlife, and pets. Most of the tested isolates metabolized diverse carbon sources and chemical compounds, suggesting that this metabolic versatility may enhance the survival, competitiveness, and pathogenic potential of T. pyogenes . IMPORTANCE: Trueperella pyogenes is an important animal pathogen with zoonotic potential, posing a significant health concern to both animals and humans due to its ability to cause infections across different animal host species and tissues. Current understanding of this pathogen’s adaptability and survival mechanisms is limited. Here, we evaluated the genomic, virulence, metabolic, and antimicrobial resistance (AMR) characteristics of T. pyogenes recovered from 16 different body sites of 11 different animal hosts (livestock, companion, and wild animals). We identified multiple AMR and virulence genes that may enable T. pyogenes for sustained infection and transmission. Additionally, T. pyogenes strains displayed metabolic versatility which could also contribute to its ability to thrive in diverse environments. Understanding the genomic and metabolic, and AMR characteristics that enable T. pyogenes to colonize different anatomical sites within a host and its transmission between different animal species is important for the effective control of this pathogen.

Evidence suggests that there is a rich and diverse microbial community in the semen of mammals, which may be important in reproductive health and fertility. However, the composition of ram seminal microbiota remains under-characterized, with factors shaping it still largely unknown. The objectives of this study were to; 1) characterize the seminal microbiota of mature rams and their ram lambs using 16S rRNA gene sequencing; 2) evaluate whether managing the mature rams on divergent planes of nutrition can influence their seminal microbiota and that of their male offspring; and 3) compare the seminal microbiota between mature rams and ram lamb cohorts to identify age-related microbiota characteristics. For this, mature rams (n = 24) were assigned to one of the 3 nutritional planes: 1) Positive (POS), to gain 12% of initial body weight (BW) (n = 8), 2) maintenance (MAINT), to maintain BW (n = 8), and 3) negative (NEG), to lose 12% BW (n = 8) over an 84-d period. Semen samples were collected from the mature rams (F0) after 28-d, 56-d, and 84-d from the start of the trial. Following the 84-d period, the 24 rams were used to breed 240 mature ewes over 28-d. After lambing, the ram lambs (F1) sired by POS, MAINT, and NEG rams were maintained on the same diet until 11 months of age, at which semen samples were collected. Genomic DNA was extracted from the semen, and the microbiota was analyzed using 16S rRNA gene (V3-V4) sequencing. Overall, there was a relatively diverse and dynamic bacterial microbial community in the ram semen, mainly dominated by Actinobacteriota, Bacillota, Bacteroidota, and Proteobacteria phyla. The predominant genera identified included Fastidiosipila, Corynebacterium, Trueperella, Arthrobacter, Dietzia, and Bifidobacterium. The seminal microbial community structure, composition, and alpha diversity of F0 rams was influenced by diet during the first 28-d, but these diet influences later diminished. The paternal plane of nutrition did not influence the seminal microbiota of offspring ram lambs. The mature rams and ram lambs had distinct seminal microbiota, with young rams showing greater microbial richness and diversity (P < 0.005). Our results suggest that there is a relatively diverse and dynamic microbial community present in the semen of both mature rams and ram lambs, and that this microbiota is transiently influenced by diet and age. Managing rams on divergent planes of nutrition may not affect their offspring’s seminal microbiota.

Antimicrobial use in food-producing animals such as pigs is a significant issue due to its association with antimicrobial resistance. Florfenicol is a broad-spectrum phenicol antibiotic used in swine for various indications; however, its effect on the swine microbiome and resistome is largely unknown. This study investigated these effects in piglets treated intramuscularly with florfenicol at 1 and 7 days of age. Fecal samples were collected from treated ( n = 30) and untreated ( n = 30) pigs at nine different time points up until 140 days of age, and the fecal metagenomes were sequenced. The fecal microbiomes of the two groups of piglets were most dissimilar in the immediate period following florfenicol administration. These differences were driven in part by an increase in the relative abundance of Clostridium scindens , Enterococcus faecalis , and Escherichia spp. in the florfenicol-treated piglets and Fusobacterium spp., Pauljensenia hyovaginalis , and Ruminococcus gnavus in the control piglets. In addition to selecting for florfenicol resistance genes ( floR , fexA , and fexB ), florfenicol also selected for genes conferring resistance to the aminoglycosides, beta-lactams, or sulfonamides up until weaning at 21 days of age. Florfenicol-resistant Escherichia coli isolated from these piglets were found to carry a plasmid with floR , along with tet (A), aph(6)-Id , aph(3″)-Ib , sul2 , and bla _TEM-1 / bla _CMY-2 . A plasmid carrying fexB and poxtA (phenicols and oxazolidinones) was identified in florfenicol-resistant Enterococcus avium , Enterococcus faecium , and E. faecalis isolates from the treated piglets. This study highlights the potential for co-selection and perturbation of the fecal microbial community in pre-weaned piglets administered florfenicol. IMPORTANCE: Antimicrobial use remains a serious challenge in food-animal production due to its linkage with antimicrobial resistance. Antimicrobial resistance can reduce the efficacy of veterinary treatment and can potentially be transferred to humans through the food chain or direct contact with animals and their environment. In this study, early-life florfenicol treatment in piglets altered the composition of the fecal microbiome and selected for many unrelated antimicrobial resistance genes up until weaning at 21 days of age. Part of this co-selection process appeared to involve an Escherichia coli plasmid carrying a florfenicol resistance gene along with genes conferring resistance to at least four other antimicrobial classes. In addition, florfenicol selected for certain genes that provide resistance to multiple antimicrobial classes, including the oxazolidinones. These results highlight that florfenicol can co-select for multiple antimicrobial resistance genes, and their presence on mobile genetic elements suggests the potential for transfer to other bacteria.