ABSTRACT 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.

ABSTRACT 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.

Abstract The objective of this study was to determine whether feeding high-concentrate (HC) or high-forage (HF) diets alters the gut microbiome of beef heifers and how these diet-induced microbial changes influence feto-maternal microbial crosstalk at mid-gestation. For this, 20 Angus crossbred heifers (approximately 14 months of age) fed either high forage (HF, 75% forage, 25% concentrate) or high concentrate (HC, 75% concentrate, 25% forage) (n = 10 per group) diets were euthanized for fetal harvesting. Both diets were formulated to meet nutrient requirements and to achieve a targeted daily gain of 0.45 kg per day. Heifers were individually fed using an Insentec feeding system and were fed on their respective diets for 90 days prior to artificial insemination with male-sexed semen from a single sire until euthanization at day 180 of gestation. A total of 263 samples representing 13 distinct sample types were analyzed using 16S rRNA (V4) amplicon sequencing with a host DNA blocking agent. Maternal samples included ruminal fluid, intestinal fluid, vaginal and uterine swabs, and whole blood collected from HF- and HC-fed dams. Fetal-associated samples included fetal ruminal fluid, intestinal tissue, allantoic and amniotic fluid, cord blood, caruncle, cotyledon tissue, and meconium. Multiple environmental (e.g., room air, tap water, and outer uterine swabs) and DNA extraction control samples were included to assess potential contamination. Sequencing of maternal samples revealed significant differences (P < 0.05) in microbial community structure, diversity, and composition in the ruminal and intestinal microbiota of HF- and HC-fed dams, confirming diet-induced modulation of the gut microbiome. However, no significant differences (P > 0.05) were observed in the vaginal, uterine, or blood microbiota between dietary groups. Fetal and environmental control samples were clearly distinct from each other and from maternal samples (PERMANOVA, R² = 0.503, P < 0.00001), confirming minimal environmental contamination. No significant differences (P > 0.05) in community structure, richness, or alpha diversity were observed between fetuses from HF- and HC-fed dams; however, site-specific bacterial communities were evident across fetal sample types. SourceTracker2 analysis is underway to identify potential maternal and environmental contributors to these fetal bacterial profiles. Overall, our findings indicate that microbial exposure of the calf intestine may occur during mid-gestation, but maternal gut microbiome alterations induced by HF or HC diets did not significantly impact the bacterial communities associated with 180-day-old bovine fetuses.

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.

Despite widespread use of antimicrobials and vaccines, the incidence of infectious bovine keratoconjunctivitis (IBK), or pinkeye, continues to increase in North American beef cow-calf operations. Recent research suggests that there is potential for the commensal ocular microbiome to help mitigate IBK. Therefore, this study characterized the ocular microbiome of cattle with and without IBK using culture-based methods and shotgun metagenomic sequencing and assessed the ability of commensal bacteria to inhibit Moraxella spp. in vitro. Ocular swabs (n = 143) were collected from IBK-affected (n = 102) and healthy cattle (n = 41) before antimicrobial treatment from North Dakota herds. Bacteria were cultured aerobically and anaerobically on five different media and the isolates were identified. A subset of swabs (37 IBK-affected; 12 healthy) underwent shotgun metagenomic sequencing. The genomes of 31 isolates, including Moraxella bovoculi, Moraxella bovis, and commensal bacteria, were also sequenced. Fifty-two commensal isolates were screened for inhibition of Moraxella spp. using an agar slab method, with five isolates further tested by qPCR for inhibition in the presence of the culturable ocular microbiome. The 351 bacterial isolates taxonomically identified represented 61 genera from three phyla. The majority of isolates belonged to Bacillus (25.9

ABSTRACT 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.

Abstract Growing evidence from human studies and rodent animal models demonstrate that the maternal gut microbiome during gestation influences immune, metabolic, and neurodevelopmental programming in the offspring from early embryonic stages, supporting its role in the Developmental Origins of Health and Disease (DOHaD) framework. However, whether the bovine maternal microbiome similarly influences calf microbiome development, health, and feed efficiency remains unknown. Therefore, this study evaluated the impact of altering maternal microbiome via feeding high-forage (HF) or high-concentrate (HC) diets during pregnancy on offspring calf gut microbiome development from birth to maturity. For this,119 beef heifers were assigned to one of two treatments and received a diet based on 75% forage (HF group; n = 24) or 75% concentrate (HC group; n = 22) from 15 days pre-breeding through parturition, and immediately after calving a common diet was fed to both groups of dams. All heifers were bred with male-sexed semen from a single sire and managed to gain 0.45 kg/day during early to mid-gestation and 0.79 kg/day in late gestation. Individual feed intake and feeding behavior were monitored, and ruminal fluid, fecal, and vaginal, and deep nasopharyngeal swabs were collected from these heifers at pre-breeding (d-30, d-15 d-2), post-breeding (d28, d56, d91, d180, d238), at calving, and 160 days postpartum. Calves born from HF (n = 22) and HC (n = 19) dams were monitored for growth performance and gut microbiome development from birth to finishing stage. Body weight measurements, ruminal fluid and fecal samples were collected from calves at birth and at 14, 30, 120, 160, 260, 330, and 372 days old. The 16S rRNA gene (V3–V4) sequencing was performed on the dam and calf’s microbiota samples. Average daily gain was not different between HF and HC heifers (P = 0.50), but feed intake and feeding behavior were different between the two groups (P < 0.05). Significant differences in the ruminal, fecal, vaginal, and nasopharyngeal microbiota were observed between HF and HC dams starting from day-2 pre-breeding (13 days after the final treatment diets were fed) and continuing up to calving, with altered ruminal and fecal communities still evident at 160 days post-calving (PERMANOVA: P < 0.05). No differences (P > 0.05) were observed in birth weight or in body weight during suckling, pre-weaning, or post-weaning between HF and HC calves. However, the ruminal microbiota at days 14, 30, 60, and 120, and the fecal microbiota at days 14 and 30, differed between HF and HC calves (P < 0.005), indicating that diet-induced shifts in the maternal microbiome influenced early-life microbial development in the offspring. Overall, our study provides the first evidence in cattle that maternal microbiome alterations contribute to developmental programming and shape offspring gut microbiome trajectory.

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.

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.

The bovine reproductive tract harbors a diverse microbiome that may influence pregnancy outcomes. Recently, we characterized the vaginal and uterine microbiota of virgin yearling heifers and cows at the time of artificial insemination (AI) using both 16S rRNA gene sequencing and culturing approaches. We identified distinct microbial taxa associated with pregnancy success and observed differential abundance between pregnant and non-pregnant groups, however most taxa remained unclassified at the genus level. Therefore, in this study we used shotgun metagenomic sequencing for higher taxonomic resolution and deeper functional insights into the bovine reproductive microbiome. The objective of the present study was to characterize the vaginal and uterine microbiomes of beef cattle that became pregnant compared to those that remained open following AI using shotgun metagenomic sequencing. The vaginal (7 open; 54 pregnant) and uterine (9 open; 41 pregnant) swabs were collected from two different cohorts of Angus-crossbred cattle consisting of mature cows (vaginal and uterine swabs) and heifers (only vaginal swabs) prior to AI. Genomic DNA were extracted from these samples and the microbiomes were profiled using shotgun metagenomic sequencing. We observed that the uterine and vaginal microbiomes had distinct compositions (PERMANOVA: R2 = 0.102 and P < 0.001). The composition (PERMANOVA: R2 = 0.0075, P = 0.7935), as well as the richness and diversity (P > 0.05) of the vaginal microbiome did not differ between open and pregnant cattle. A total of 422 different genera were detected from the vaginal samples, with Negativicutes-UBA1444, Streptococcus, Mycobacterium, and Ureaplasma being the most relatively abundant. Twenty-five of these genera including Aphodosoma, Egerieisoma, Alitiscatomonas, Lentihominibacter, Enterocola, Akkermansia, Ruminococcus, and Faecousia were more abundant (P < 0.05) in the vaginal microbiome of non-pregnant cattle. A significant difference in the composition of the uterine microbiome was observed between pregnant and open cattle (R2 = 0.049 and P = 0.042). Furthermore, microbial richness (P = 0.035) and diversity [(Shannon diversity: P = 0.014), (inverse Simpson diversity: P = 0.011)], as well as evenness (Pielou’s index: P = 0.047) were greater in the uterine microbiome of open than pregnant cattle. Overall, we profiled 329 bacterial genera across uterine samples, with Negativicutes-UBA1444, Cutibacterium, Streptomyces, and Acinetobacter being the most predominant genera. At species level, the vaginal microbiome had 1161 species, including Streptococcus pluranimalium, Ureaplasma diversum, Facklamia hominis, Histophilus somni, and Enterococcus faecalis, whereas the uterine microbiome was dominated by Negativicutes-UBA1444 sp012798135, Cutibacterium acnes, Giesbergeria lacusdiani, Bacillus_J hisashii, Thiopseudomonas sp012518175, and Acinetobacter idrijaensis. While the results of this metagenomic sequencing were negatively impacted by contaminating host DNA and consequently, low microbial sequencing depth, our results suggest that the uterine microbiome may have implications in AI pregnancy success rate.

Maternal gut microbiome has been shown to influence immune, metabolic and neurodevelopmental programming of offspring from the embryonic stage, suggesting a potential role in the Developmental Origins of Health and Disease (DOHaD). Whereas many still support the “sterile-womb hypothesis” that the neonatal microbiome acquisition occurs only during and after birth, very recent studies have provided evidence showing the existence of in utero microbial colonization. Thus, these recent developments in the field of microbiome research of human and vertebrate animals including bovine animals highlight that the maternal gut microbiome during pregnancy should be targeted for harnessing their extended impact on the offspring’s development and health. In this presentation, we will discuss the potential involvement of maternal microbiome and feto-maternal microbial crosstalk in fetal programming, and offspring calf’s health and development. In addition, we will discuss the results from our recently conducted longitudinal study focused on the evaluation of the impact of altering maternal microbiota via high forage or high concentrate diets on offspring microbiome development, energy balance, methane emissions and feedlot performance in beef cattle. For this, 120 beef heifers were assigned to one of two treatments and received a diet based on 75% forage (HF) or 75% concentrate (HC) from 15 days pre-breeding through calving. Heifers were bred using male-sexed semen and fed to target a gain of 0.45kg/d for both groups. Ruminal fluid, fecal and vaginal swabs were collected from both HF (n = 24) and HC (n =22) heifers on pre-breeding (-30, -2), post-breeding (56, 91, 180, and 238 days of gestation) and at calving. Calves born from these heifers were monitored for their animal performance, feed efficiency, gut microbiome development and enteric methane emission (in vitro and in vivo). Body weight measurements, ruminal fluid and fecal samples were collected from the calves at 0, 15, 30, 60, 120, 160, 240, 330 and 340 days old. The 16S rNRA gene sequencing was performed on the dam and calf’s microbiome samples. An in-vitro fermentation assay was performed on the ruminal fluid samples from heifers and their calves for methane and VFA analyses. During finishing stage, a subset of calves born from HF and HC dams (n = 10 each group) were evaluated to examine effects on energy metabolism, nutrient balance and enteric methane emission (using headbox) output between the HF and HC offspring because of the HF or HC diet their dams received during fetal development. The results from this study provide novel insights into the impact of altered maternal gut microbiome during pregnancy on the postnatal animal performance, feed efficiency, microbiome development and enteric methane emission phenotype in cattle.

ABSTRACT 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.

Abstract Increasing evidence suggests that the bovine semen harbors commensal microbiota, and it may be important in male fertility. However, much is unknown regarding the seminal microbiota, its evolution and factors shaping this community in sheep. The objective of this study was to evaluate whether managing the rams on divergent planes of nutrition can influence the seminal microbiota in mature rams and their male offspring. For this, Rambouillet rams (n = 24; 1.5 to 4 yr old) were randomly assigned to one of three groups: 1) a positive plane of nutrition [target 12% gain in their initial body weight (BW; POS, n = 8); 2) a maintenance [maintain the initial BW (MNT, n = 8)]; and 3) a negative [target a 12% reduction in BW (NEG; n = 8)] plane of nutrition over 84 d. The rams were individually housed and fed a common diet, and feed allocations were adjusted weekly. Following the 84-d feeding period, these rams were used to breed 240 mature Rambouillet ewes (1:10 ram:ewe ratio) over 28 d of breeding. After lambing, the male lambs were maintained on a similar diet for 124 d. Semen samples from the mature rams (F0 generation) were collected on d 28, 56, and 84, and semen samples from offspring rams (F1 generation) were collected at 330 d of age via electroejaculation. Genomic DNA was extracted from semen samples and the seminal microbiota characterized using 16S rRNA gene (V3-V4) sequencing. Several environmental and negative extraction controls were also sequenced to assess potential contaminants in these samples. Overall community structure of the seminal microbiota in F0 rams was not affected by the divergent planes of nutrition (PERMANOVA: P ≥ 0.05) or by sampling time (P > 0.05). Microbial richness (observed ASVs) was also not different (P > 0.05) between the three groups of F0 rams at any of the sampling time points; however, microbial diversity (P < 0.05) in the F0 rams was influenced by sire dietary treatment. Microbial community structure, richness and diversity did not differ among the POS, MNT and NEG F1 generation rams (P > 0.05). Actinobacteriota, Firmicutes, Bacteroidota, Proteobacteria and Fusobacteriota were the most abundant phyla. Only Bacteroidota abundance was significantly (P < 0.05) different between three F0 groups, with POS rams had greater (18.6%) of this phylum on d 28 as compared with MNT (2.1%) and NEG (5.1%). The predominant genera identified included Fastidiosipila, Corynebacterium, Trueperella, Arthrobacter, Dietzia, Bifidobacterium, Streptobacillus, Ornithinimicrobium, and Porphyromonas, and their relative abundance was not affected (P > 0.05) by the dietary treatment in F0 rams. Overall, rams managed on positive and negative planes of nutrition harbored similar seminal microbiota to control rams, and divergent nutrition also had no effect on the offspring ram seminal microbiota.

AbstractTrueperella pyogenesis 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 enableT. pyogenesto 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 as well as antimicrobial resistance (AMR) genes inT. pyogenesrecovered from different livestock, companion and wildlife animals. For comparative genomic analysis, 83T. pyogenesgenomes, including 60 isolated in the current study and 23 publicly available genomes were evaluated. These genomes representedT. pyogenesstrains originated from 16 different body sites of 11 different animal hosts (e.g. bovine, swine, ovine, cervid, bison, equine, chamois, feline). Additionally, 49T. pyogenesisolates (bovine, ovine, deer, swine and feline) were evaluated for phenotypic antimicrobial resistance using disk diffusion, and for metabolic profiling using the Biology GENIII MicroPlates. We identified thatT. pyogenesstrains are not host- or body site-specific. The presence of conserved virulence genes (ploandfimA), as well as genotypic and phenotypic AMR may contribute toT. pyogenes’sability 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 contribute toT. pyogenes’ survival, competitive advantage, and pathogenic potential.ImportanceTrueperella pyogenesis 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 characteristics ofT. pyogenesrecovered from 16 different body sites of 11 different animal hosts (livestock, companion, and wild animals). We identified multiple antimicrobial resistance and virulence genes that may enableT. pyogenesfor sustained infection and transmission. Additionally,T. pyogenesstrains displayed metabolic versatility which could also contribute to its ability to thrive in diverse environments. Understanding the genomic and metabolic, and antimicrobial resistance characteristics that enableT. pyogenesto colonize different anatomical sites within a host and its transmission between different animal species is important for the effective control of this pathogen.

Abstract Garlic (Allium sativum) in its dehydrated or distillate (garlic oil) form has been used as a feed additive owing to its insecticidal, antimicrobial, and organoleptic properties. Commercial mineral mixtures have been formulated to contain a low proportion of dehydrated garlic powder as a strategy to deter pest flies and increase mineral intake in cattle. As an antimicrobial, garlic powder has the potential to alter the rumen microbiome, with possible implications for feed digestibility and the growth performance of beef cattle. This study evaluated the effect of short-term feeding of a garlic-mineral mixture in feedlot steers receiving high-grain diets. Four groups of twenty steers [total = 80, ~14 mo old, body weight (BW) = 599 ± 32 kg] were randomly assigned to either a 5% garlic-mineral (5DGP) or plain mineral supplement (MS). The individual feed and mineral intakes of the steers were measured with an automatic feeding system. Feed and mineral supplements were tracked in different bunks for each group. Baseline rumen fluid samples were collected from all steers at the end of an acclimatization period before administering the trial supplements. Rumen samples were later collected from 10 steers in each group with the greatest mineral supplement intake over the trial period. Total DNA and RNA were extracted from all rumen samples and subjected to metagenomic and metatranscriptomic sequencing, respectively. None of the bacterial species with an overall relative abundance of greater than 0.1% were differentially abundant between the two supplement treatments, nor did the rumen microbial community structures differ between the two groups (PERMANOVA: R2 = 0.02, P = 0.43). Regardless of the dietary supplement, the rumen microbiome of the steers was dominated by uncultured Prevotella spp. (in addition to Prevotella lacticifex) as well as the uncharacterized UBA2810 sp900317945 and UBA2810 sp002351705. Overall, these results show that a 5DGP can be effectively administered to cattle without significantly disrupting the rumen microbiome.

The rumen microbiota is important for energy and nutrient acquisition in cattle, and therefore its composition may also affect carcass merit and meat quality attributes. In this study, we examined the associations between archaeal and bacterial taxa in the rumen microbiota of beef cattle and 12 different attributes, including hot carcass weight (HCW), dressing percentage, ribeye area (REA), intramuscular fat content, marbling score, fat thickness, yield grade, moisture content, purge loss, and shear force. There were significant correlations between the relative abundance of certain archaeal and bacterial genera and these attributes. Notably, Selenomonas spp. were positively correlated with live weight and HCW, while also being negatively correlated with purge loss. Members of the Christensenellaceae R-7, Moryella, and Prevotella genera exhibited positive and significant correlations with various attributes, such as dressing percentage and intramuscular fat content. Ruminococcaceae UCG-001 was negatively correlated with live weight, HCW, and dressing percentage, while Acidaminococcus and Succinivibrionaceae UCG-001 were negatively correlated with intramuscular fat content, moisture content, and marbling score. Overall, our findings suggest that specific changes in the rumen microbiota could be a valuable tool to improve beef carcass merit and meat quality attributes. Additional research is required to better understand the relationship between the rumen microbiota and these attributes, with the potential to develop microbiome-targeted strategies for enhancing beef production. KEY POINTS: • Certain rumen bacteria were associated with carcass merit and meat quality • Moryella was positively correlated with intramuscular fat in beef carcasses • Acidaminococcus spp. was negatively correlated with marbling and intramuscular fat.

Maternal diet quality and quantity have significant impacts on both maternal and fetal health and development. The composition and function of the maternal gut microbiome is also significantly influenced by diet; however, little is known about the impact of gestational nutrient restriction on the bovine maternal microbiome during early gestation, which is a critical stage for maternal microbiome-mediated fetal programming to take place. The objective of the present study was to evaluate the impacts of diet restriction and one-carbon metabolite (OCM) supplementation during early gestation on maternal ruminal, vaginal, and blood microbiota in cattle. Thirty-three beef heifers (approx. 14 months old) were used in a 2 × 2 factorial experiment with main factors of target gain (control [CON]; targeted 0.45 kg/d gain vs restricted [RES]; targeted − 0.23 kg/d gain), and OCM supplementation (+ OCM vs − OCM; n = 8/treatment; except n = 9 for RES–OCM). Heifers were individually fed, starting treatment at breeding (d 0) and concluding at d 63 of gestation. Ruminal fluid and vaginal swabs were collected on d − 2, d 35, and d 63 (at necropsy) and whole blood was collected on d 63 (necropsy). Bacterial microbiota was assessed using 16S rRNA gene (V3–V4) sequencing. Overall ruminal microbiota structure was affected by gain, OCM, time, and their interactions. The RES heifers had greater microbial richness (observed ASVs) but neither Shannon nor Inverse Simpson diversity was significantly influenced by gain or OCM supplementation; however, on d 63, 34 bacterial genera showed differential abundance in the ruminal fluid, with 25 genera enriched in RES heifers as compared to CON heifers. In addition, the overall interaction network structure of the ruminal microbiota changed due to diet restriction. The vaginal microbiota community structure was influenced by gain and time. Overall microbial richness and diversity of the vaginal microbiota steadily increased as pregnancy progressed. The vaginal ecological network structure was distinctive between RES and CON heifers with genera-genera interactions being intensified in RES heifers. A relatively diverse bacterial community was detected in blood samples, and the composition of the blood microbiota differed from that of ruminal and vaginal microbiota. Restricted dietary intake during early gestation induced significant alterations in the ruminal microbiota which also extended to the vaginal microbiota. The composition of these two microbial communities was largely unaffected by OCM supplementation. Blood associated microbiota was largely distinctive from the ruminal and vaginal microbiota.

Abstract Pinkeye, clinically known as infectious bovine keratoconjunctivitis (IBK), is one of the most highly contagious diseases impacting cattle at all ages. Despite extensive use of antibiotics and vaccines targeting Moraxella bovis, the primary IBK pathogen, the incidence of pinkeye has been increasing in North American beef cow-calf operations. This highlights the incomplete understanding of the pathogenesis of this disease and the need for novel and alternative approaches to control IBK. The objectives of this study were to: 1) characterize the ocular microbiome of beef cattle with and without IBK using shotgun metagenomic sequencing; 2) investigate the genetic diversity of M. bovis, Moraxella bovoculi, and commensal ocular bacterial isolates; 3) evaluate whether selected ocular probiotic candidate strains inhibit Moraxella pathogen growth in the presence of the ocular microbiome in culture using qPCR. A total of 143 ocular swabs were collected from cattle with (n = 102) or without IBK (n = 42) and before antibiotic treatment from beef herds across North Dakota, USA. Fifty samples from IBK (n = 38) and healthy cattle (n = 12) were also subjected to shotgun metagenomic sequencing. Seven bacterial species were relatively more abundant (P < 0.05) in the healthy cattle ocular microbiome including Bifidobacterium globosum, Bacillus licheniformis, Pasteurella multocida, Ruminococcus sp900316555, and Ruminococcus sp900100595. Arthrobacter luteus was the only bacterial species significantly associated with IBK-affected cattle. From the metagenomes, 37 high-quality dereplicated metagenome-assembled genomes (MAGs) were recovered, nearly a third of which were identified as Mesomycoplasma bovoculi (n = 10). M. bovis (n = 5) and M. bovoculi (n = 13) were also isolated from the ocular swabs of both healthy and IBK-affected cattle. These isolates represented 9 M. bovoculi and 5 M. bovis strains (average nucleotide identity < 99.99%). We also isolated and sequenced several potentially beneficial bacterial species from healthy cattle including Bacillus pumilus, Lactiplantibacillus plantarum, Lentilactobacillus buchneri, Levilactobacillus brevis, and Weizmannia coagulans. Four of these probiotic candidates were also tested for their inhibitory effects against M. bovoculi in co-culture with the ocular microbiome after 24 h incubation using qPCR. The B. pumilus, L. buchneri, L. plantarum, and W. coagulans strains, as well as a mixture of all four isolates, significantly reduced the growth of Moraxella. In summary, our results provide important information on the culturable and non-culturable fraction of the bovine ocular microbiome potentially associated with IBK and suggest that certain bacterial species in the eye may inhibit the growth of Moraxella spp.