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ABSTRACTAlthough circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal (Ribo); linear RNAs degradation (R); linear RNAs and RNAs with structured 3′ ends degradation (RTP); ribosomal RNAs coupled with linear RNAs elimination (Ribo-R); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination (Ribo-RP); and ribosomal RNA, linear RNAs and RNAs with structured 3′ ends elimination (Ribo-RTP), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads (Padj

An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are administered multiple antimicrobial treatments for BRD. A number of factors are responsible for BRD antimicrobial treatment failure, with formation of biofilms possibly being one. It is widely accepted that biofilms play a role in chronic infections in humans and it has been hypothesized that they are the default lifestyle of most bacteria. However, research on bacterial biofilms associated with livestock is scarce and significant knowledge gaps exist in our understanding of their role in AMR of the bacterial BRD complex. The four main bacterial species of the BRD complex, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis are able to form biofilms in vitro and there is evidence that at least H. somni retains this ability in vivo. However, there is a need to elucidate whether their biofilm-forming ability contributes to pathogenicity and antimicrobial treatment failure of BRD. Overall, a better understanding of the possible role of BRD bacterial biofilms in clinical disease and AMR could assist in the prevention and management of respiratory infections in feedlot cattle. We review and discuss the current knowledge of BRD bacteria biofilm biology, study methodologies, and their possible relationship to AMR.

This study evaluated the effects of Pediococcus pentosaceus (PP) and chitinase combinations on the conservation and nutritive value of alfalfa high-moisture hay (HMH). P. pentosaceus [1012 colony forming unit/g fresh forage] combined with (g/tonne of fresh forage) 1.5 (PP + LC), 7.5 (PP + MC), or 15.0 (PP + HC) g of LANiHay01 chitinase (Exp. 1) or with LANiHay02 (PP + Fe; 1.5 g), LANiHay01 (PP + Pe; 1.5 g), or Sigma (PP + Si; 55 mg) chitinase/tonne (Exp. 2) were used in 2016. In 2017, PP was applied alone or in combination with LANiHay01 at 1.5 g (PP + LC) or 7.5 g (PP + MC) chitinase/tonne (Exp. 3 and 4). Deionized water and propionic acid (4.0 L/tonne of fresh forage in Exp. 1 and 2 and at 6.0 L/tonne of fresh forage in Exp. 3 and 4) were applied as neutral (CON) and positive control (CON+), respectively. The maximum temperature (r2 = 0.66) and NH3-N concentration (r2 = 0.80) of the HMH were positively related to total microbial populations. PP + MC had lower (p ≤ 0.05) yeast and mold counts than CON in Exp. 3 and 4 while the neutral detergent fiber degradability was greater (p < 0.01) for PP + MC and PP + LC than CON in Exp. 1 and 3, respectively. P. pentosaceus in combination with chitinase has the potential in conserving the nutrient quality of alfalfa HMH.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one β-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8–16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl β-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Abstract To reduce the transmission risk of bovine spongiform encephalopathy prions (PrPBSE), specified risk materials (SRM) that can harbour PrPBSE are prevented from entering the feed and food chains. As composting is one approach to disposing of SRM, we investigated the inactivation of PrPBSE in lab-scale composters over 28 days and in bin composters over 106–120 days. Lab-scale composting was conducted using 45 kg of feedlot manure with and without chicken feathers. Based on protein misfolding cyclic amplification (PMCA), after 28 days of composting, PrPBSE seeding activity was reduced by 3–4 log10 with feathers and 3 log10 without. Bin composters were constructed using ~ 2200 kg feedlot manure and repeated in 2017 and 2018. PMCA results showed that seeding activity of PrPBSE was reduced by 1–2 log10 in the centre, but only by 1 log10 in the bottom of bin composters. Subsequent assessment by transgenic (Tgbov XV) mouse bioassay confirmed a similar reduction in PrPBSE infectivity. Enrichment for proteolytic microorganisms through the addition of feathers to compost could enhance PrPBSE degradation. In addition to temperature, other factors including varying concentrations of PrPBSE and the nature of proteolytic microbial populations may be responsible for differential degradation of PrPBSE during composting.

ABSTRACT: Ruminant livestock are an important source of anthropogenic methane (CH4). Decreasing the emissions of enteric CH4 from ruminant production is strategic to limit the global temperature increase to 1.5°C by 2050. Research in the area of enteric CH4 mitigation has grown exponentially in the last 2 decades, with various strategies for enteric CH4 abatement being investigated: production intensification, dietary manipulation (including supplementation and processing of concentrates and lipids, and management of forage and pastures), rumen manipulation (supplementation of ionophores, 3-nitrooxypropanol, macroalgae, alternative electron acceptors, and phytochemicals), and selection of low-CH4-producing animals. Other enteric CH4 mitigation strategies are at earlier stages of research but rapidly developing. Herein, we discuss and analyze the current status of available enteric CH4 mitigation strategies with an emphasis on opportunities and barriers to their implementation in confined and partial grazing production systems, and in extensive and fully grazing production systems. For each enteric CH4 mitigation strategy, we discuss its effectiveness to decrease total CH4 emissions and emissions on a per animal product basis, safety issues, impacts on the emissions of other greenhouse gases, as well as other economic, regulatory, and societal aspects that are key to implementation. Most research has been conducted with confined animals, and considerably more research is needed to develop, adapt, and evaluate antimethanogenic strategies for grazing systems. In general, few options are currently available for extensive production systems without feed supplementation. Continuous research and development are needed to develop enteric CH4 mitigation strategies that are locally applicable. Information is needed to calculate carbon footprints of interventions on a regional basis to evaluate the impact of mitigation strategies on net greenhouse gas emissions. Economically affordable enteric CH4 mitigation solutions are urgently needed. Successful implementation of safe and effective antimethanogenic strategies will also require delivery mechanisms and adequate technical support for producers, as well as consumer involvement and acceptance. The most appropriate metrics should be used in quantifying the overall climate outcomes associated with mitigation of enteric CH4 emissions. A holistic approach is required, and buy-in is needed at all levels of the supply chain.

Verotoxigenic Escherichia coli (VTEC), also termed Shiga toxin-producing Escherichia coli (STEC), is a human pathogen transmitted by food, water, animals, and their environment, and from one person to another [...]

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

Antimicrobial use (AMU) in the livestock industry has been associated with increased levels of antimicrobial resistance. Recently, there has been an increase in the number of “natural” feedlots in the beef cattle sector that raise cattle without antibiotics. Shotgun metagenomics was employed to characterize the impact of AMU in feedlot cattle on the microbiome, resistome, and mobilome. Sequenced fecal samples identified a decline (q < 0.01) in the genera Methanobrevibacter and Treponema in the microbiome of naturally vs. conventionally raised feedlot cattle, but this difference was not (q > 0.05) observed in catch basin samples. No differences (q > 0.05) were found in the class-level resistome between feedlot practices. In fecal samples, decreases from conventional to natural (q < 0.05) were noted in reads for the antimicrobial-resistant genes (ARGs) mefA, tet40, tetO, tetQ, and tetW. Plasmid-associated ARGs were more common in feces from conventional than natural feedlot cattle. Interestingly, more chromosomal- than plasmid-associated macrolide resistance genes were observed in both natural and conventional feedlots, suggesting that they were more stably conserved than the predominately plasmid-associated tetracycline resistance genes. This study suggests that generationally selected resistomes through decades of AMU persist even after AMU ceases in natural production systems.

Abstract Background The resistome describes the array of antibiotic resistant genes (ARGs) present within a microbial community. Recent research has documented the resistome in the rumen of ruminants and revealed that the type and abundance of ARGs could be affected by diet and/or antibiotic treatment. However, most of these studies only assessed ARGs using metagenomics, and expression of the resistome and its biological function within the microbiome remains largely unexplored. Results We characterized the pools of ARGs (resistome) and their activities in the rumen of 48 beef cattle belonging to three breeds (Angus, Charolais, Kinsella composite hybrid), using shotgun metagenomics and metatranscriptomics. Sixty (including 20 plasmid-associated) ARGs were expressed which accounted for about 30% of the total number of ARGs (187) identified in metagenomic datasets, with tetW and mefA exhibiting the highest level of expression. In addition, the bacterial hosts of 17 expressed ARGs were identified. The active resistome was less diverse in Kinsella composite hybrid than Angus, however, expression of ARGs did not differ among breeds. Although not associated with feed efficiency, the total abundance of expressed ARGs was positively correlated with metabolic pathways and ‘attenuation values’ (a measurement of stability) of the active rumen microbiome, suggesting that ARGs expression influences the stability and functionality of the rumen microbiome. Moreover, Ruminococcus spp., Prevotella ruminicola, Muribaculaceae spp. and Collinsella aerofaciens were all identified as hosts of expressed ARGs, possibly promoting the dominance of these carbohydrate degraders within the rumen microbiome. Conclusions Findings from this study provide new insight into the active rumen resistome in vivo, which may inform strategies to limit the spread of ubiquitously found ARGs from the rumen to the broader environment without negatively impacting the key functional outcomes of the rumen microbiome.

Seaweeds have received a great deal of attention recently for their potential as methane-suppressing feed additives in ruminants. To date, Asparagopsis taxiformis has proven a potent enteric methane inhibitor, but it is a priority to identify local seaweed varieties that hold similar properties. It is essential that any methane inhibitor does not compromise the function of the rumen microbiome. In this study, we conducted an in vitro experiment using the RUSITEC system to evaluate the impact of three red seaweeds, A. taxiformis, Palmaria mollis, and Mazzaella japonica, on rumen prokaryotic communities. 16S rRNA sequencing showed that A. taxiformis had a profound effect on the microbiome, particularly on methanogens. Weighted Unifrac distances showed significant separation of A. taxiformis samples from the control and other seaweeds (p 0.05). A. taxiformis reduced the abundance of all major archaeal species (p

Abstract Molecular hydrogen (H2) and formate (HCOO−) are metabolic end products of many primary fermenters in the mammalian gut. Both play a vital role in fermentation where they are electron sinks for individual microbes in an anaerobic environment that lacks external electron acceptors. If H2 and/or formate accumulate within the gut ecosystem, the ability of primary fermenters to regenerate electron carriers may be inhibited and microbial metabolism and growth disrupted. Consequently, H2- and/or formate-consuming microbes such as methanogens and homoacetogens play a key role in maintaining the metabolic efficiency of primary fermenters. There is increasing interest in identifying approaches to manipulate mammalian gut environments for the benefit of the host and the environment. As H2 and formate are important mediators of interspecies interactions, an understanding of their production and utilisation could be a significant entry point for the development of successful interventions. Ruminant methane mitigation approaches are discussed as a model to help understand the fate of H2 and formate in gut systems.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5–9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

IntroductionUse of antimicrobial drugs (AMDs) in food producing animals has received increasing scrutiny because of concerns about antimicrobial resistance (AMR) that might affect consumers. Previously, investigations regarding AMR have focused largely on phenotypes of selected pathogens and indicator bacteria, such as Salmonella enterica or Escherichia coli. However, genes conferring AMR are known to be distributed and shared throughout microbial communities. The objectives of this study were to employ target-enriched metagenomic sequencing and 16S rRNA gene amplicon sequencing to investigate the effects of AMD use, in the context of other management and environmental factors, on the resistome and microbiome in beef feedlot cattle.MethodsThis study leveraged samples collected during a previous longitudinal study of cattle at beef feedlots in Canada. This included fecal samples collected from randomly selected individual cattle, as well as composite-fecal samples from randomly selected pens of cattle. All AMD use was recorded and characterized across different drug classes using animal defined daily dose (ADD) metrics.ResultsOverall, fecal resistome composition was dominated by genes conferring resistance to tetracycline and macrolide-lincosamide-streptogramin (MLS) drug classes. The diversity of bacterial phyla was greater early in the feeding period and decreased over time in the feedlot. This decrease in diversity occurred concurrently as the microbiome represented in different individuals and different pens shifted toward a similar composition dominated by Proteobacteria and Firmicutes. Some antimicrobial drug exposures in individuals and groups were associated with explaining a statistically significant proportion of the variance in the resistome, but the amount of variance explained by these important factors was very small (

Abstract Antimicrobials have been widely used to prevent and treat infectious diseases and promote growth in food-production animals. However, the occurrence of antimicrobial resistance poses a huge threat to public and animal health, especially in less developed countries where food-producing animals often intermingle with humans. To limit the spread of antimicrobial resistance from food-production animals to humans and the environment, it is essential to have a comprehensive knowledge of the role of the resistome in antimicrobial resistance (AMR), The resistome refers to the collection of all antimicrobial resistance genes associated with microbiota in a given environment. The dense microbiota in the digestive tract is known to harbour one of the most diverse resistomes in nature. Studies of the resistome in the digestive tract of humans and animals are increasing exponentially as a result of advancements in next-generation sequencing and the expansion of bioinformatic resources/tools to identify and describe the resistome. In this review, we outline the various tools/bioinformatic pipelines currently available to characterize and understand the nature of the intestinal resistome of swine, poultry, and ruminants. We then propose future research directions including analysis of resistome using long-read sequencing, investigation in the role of mobile genetic elements in the expression, function and transmission of AMR. This review outlines the current knowledge and approaches to studying the resistome in food-producing animals and sheds light on future strategies to reduce antimicrobial usage and control the spread of AMR both within and from livestock production systems.

Foodborne illness is exacerbated by novel and emerging pathotypes, persistent contamination, antimicrobial resistance, an ever-changing environment, and the complexity of food production systems. Sporadic and outbreak events of common foodborne pathogens like Shiga toxigenic E. coli (STEC), Salmonella, Campylobacter, and Listeria monocytogenes are increasingly identified. Methods of controlling human infections linked with food products are essential to improve food safety and public health and to avoid economic losses associated with contaminated food product recalls and litigations. Bacteriophages (phages) are an attractive additional weapon in the ongoing search for preventative measures to improve food safety and public health. However, like all other antimicrobial interventions that are being employed in food production systems, phages are not a panacea to all food safety challenges. Therefore, while phage-based biocontrol can be promising in combating foodborne pathogens, their antibacterial spectrum is generally narrower than most antibiotics. The emergence of phage-insensitive single-cell variants and the formulation of effective cocktails are some of the challenges faced by phage-based biocontrol methods. This review examines phage-based applications at critical control points in food production systems with an emphasis on when and where they can be successfully applied at production and processing levels. Shortcomings associated with phage-based control measures are outlined together with strategies that can be applied to improve phage utility for current and future applications in food safety.

Enterococci are commensal bacteria of the gastrointestinal tract of humans, animals, and insects. They are also found in soil, water, and plant ecosystems. The presence of enterococci in human, animal, and environmental settings makes these bacteria ideal candidates to study antimicrobial resistance in the One-Health continuum. This study focused on Enterococcus hirae isolates (n = 4,601) predominantly isolated from beef production systems including bovine feces (n = 4,117, 89.5%), catch-basin water (n = 306, 66.5%), stockpiled bovine manure (n = 24, 0.5%), and natural water sources near feedlots (n = 145, 32%), and a few isolates from urban wastewater (n = 9, 0.2%) denoted as human-associated environmental samples. Antimicrobial susceptibility profiling of a subset (n = 1,319) of E. hirae isolates originating from beef production systems (n = 1,308) showed high resistance to tetracycline (65%) and erythromycin (57%) with 50.4% isolates harboring multi-drug resistance, whereas urban wastewater isolates (n = 9) were resistant to nitrofurantoin (44.5%) and tigecycline (44.5%) followed by linezolid (33.3%). Genes for tetracycline (tetL, M, S/M, and O/32/O) and macrolide resistance erm(B) were frequently found in beef production isolates. Antimicrobial resistance profiles of E. hirae isolates recovered from different environmental settings appeared to reflect the kind of antimicrobial usage in beef and human sectors. Comparative genomic analysis of E. hirae isolates showed an open pan-genome that consisted of 1,427 core genes, 358 soft core genes, 1701 shell genes, and 7,969 cloud genes. Across species comparative genomic analysis conducted on E. hirae, Enterococcus faecalis and Enterococcus faecium genomes revealed that E. hirae had unique genes associated with vitamin production, cellulose, and pectin degradation, traits which may support its adaptation to the bovine digestive tract. E. faecium and E. faecalis more frequently harbored virulence genes associated with biofilm formation, iron transport, and cell adhesion, suggesting niche specificity within these species.

Here, the authors report a large-scale comparative analysis of

Carbapenems are considered a last resort for the treatment of multi-drug-resistant bacterial infections in humans. In this study, we investigated the occurrence of carbapenem-resistant bacteria in feedlots in Alberta, Canada. The presumptive carbapenem-resistant isolates (n = 116) recovered after ertapenem enrichment were subjected to antimicrobial susceptibility testing against 12 different antibiotics, including four carbapenems. Of these, 72% of the isolates (n = 84) showed resistance to ertapenem, while 27% of the isolates (n = 31) were resistant to at least one other carbapenem, with all except one isolate being resistant to at least two other drug classes. Of these 31 isolates, 90% were carbapenemase positive, while a subset of 36 ertapenem-only resistant isolates were carbapenemase negative. The positive isolates belonged to three genera; Pseudomonas, Acinetobacter, and Stenotrophomonas, with the majority being Pseudomonas aeruginosa (n = 20) as identified by 16S rRNA gene sequencing. Whole genome sequencing identified intrinsic carbapenem resistance genes, including blaOXA-50 and its variants (P. aeruginosa), blaOXA-265 (A. haemolyticus), blaOXA-648 (A. lwoffii), blaOXA-278 (A. junii), and blaL1 and blaL2 (S. maltophilia). The acquired carbapenem resistance gene (blaPST-2) was identified in P. saudiphocaensis and P. stutzeri. In a comparative genomic analysis, clinical P. aeruginosa clustered separately from those recovered from bovine feces. In conclusion, despite the use of selective enrichment methods, finding carbapenem-resistant bacteria within a feedlot environment was a rarity.

Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3′)-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade ‘A’ and ‘B’, with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.

Abstract Gut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem. Video abstract

Diversity and compositional analysis are the most common approaches in deciphering microbial community differences. However, these approaches neglect microbial structural differences driven by microbial interactions. In this study, the microbiota data were generated from 12 rectal digesta samples collected from steers in which the Shiga toxin 2 gene (stx2) was not expressed (defined as Stx2− group) in the bacteria, and those with stx2 expressed (defined as Stx2+ group) and used to explore whether microbial networks affect gut microbiota and foodborne pathogen virulence in cattle. Although the Shannon and Chao1 indices of rectal digesta microbial communities did not differ between the two groups (P > 0.05), 24 and 13 taxa were identified to be group-specific genera for Stx2− and Stx2+ microbial communities, respectively. The network analysis indicated 12 and 14 generalists (microbes that were densely connected with other taxa) in microbial communities for Stx2− and Stx2+ groups, and 8 out of 12 generalists and 6 out of 14 generalists were designated to Stx2− and Stx2+ group-specific genera, respectively. However, the 66 core genera were not classified as network generalists. Natural connectivity measurements revealed that the higher stability of the Stx2− microbial network in comparison to the Stx2+ network, suggesting that the structure of each microbial community was inherently different even when their diversity and composition were comparable. Group-specific genera intensely interacted with other taxa in the co-occurrence network, indicating that characterizing microbial networks together with group-specific genera could be an alternative approach to identify variation in microbial communities.

Abstract Background Incorporation of legume species into native North American pastures is considered an effective method to increase native pasture productivity and improve the nutritive value of forage. This study evaluated the effects of inclusion of purple prairie clover (PPC, Dalea purpurea Vent.), a native legume forage, with native cool-season grasses on the in vitro fermentation and in situ digestibility of mixed forages. Methods Whole plant PPC and mixtures of cool-season grasses were harvested when the PPC reached the vegetative (VEG), full flower (FL) and seedpod (SP) stages, and were combined in ratios (DM basis) of 0:100, 25:75, 50:50, 75:25 and 100:0 at each maturity. In vitro ruminal incubations using these mixtures were conducted for 48 h to determine gas production (GP), in vitro DM disappearance (IVDMD), total volatile fatty acids (VFA) and ammonia-N production. Mixtures of forages harvested when the PPC reached the FL stage and 50:50 mixture of forages harvested at VEG, FL and SP stages were incubated in the rumen of three heifers for 0, 2, 6, 12, 24, 48, 72 and 96 h to determine in situ degradabilities of DM, neutral detergent fibre (aNDF) and crude protein (CP). Results Contents of aNDF and ADF increased (P VEG > SP (P FL > SP (P

Abstract A previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P

Abstract Background Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. Results VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p

ABSTRACT Antimicrobial use in food-producing animals has come under increasing scrutiny due to its potential association with antimicrobial resistance (AMR). Monitoring of AMR in indicator microorganisms such as Enterococcus spp. in meat production facilities and retail meat products can provide important information on the dynamics and prevalence of AMR in these environments. In this study, swabs or samples were obtained from various locations in a commercial beef packing operation (n = 600) and from retail ground beef (n = 60) over a 19-month period. All samples/swabs were enriched for Enterococcus spp., and suspected enterococci isolates were identified using species-specific PCR primers. Enterococcus faecalis was the most frequently isolated species, followed by Enterococcus hirae, which was found mostly on post-hide removal carcasses and in ground beef. Enterococcus faecium (n = 9) and E. faecalis (n = 120) isolates were further characterized for AMR. Twenty-one unique AMR profiles were identified, with 90% of isolates resistant to at least two antimicrobials and two that were resistant to nine antimicrobials. Tetracycline resistance was observed most often in E. faecalis (28.8%) and was likely mediated by tet(M). Genomic analysis of selected E. faecalis and E. faecium isolates revealed that many of the isolates in this study clustered with other publicly available genomes from ground beef, suggesting that these strains are well adapted to the beef processing environment. IMPORTANCE Antimicrobial resistance (AMR) is a serious challenge facing the agricultural industry. Understanding the flow of antimicrobial-resistant bacteria through the beef fabrication process and into ground beef is an important step in identifying intervention points for reducing AMR. In this study, we used enterococci as indicator bacteria for monitoring AMR in a commercial beef packaging facility and in retail ground beef over a 19-month period. Although washing of carcasses post-hide removal reduced the isolation frequency of Enterococcus spp., a number of antimicrobial-resistant Enterococcus faecalis isolates were recovered from ground beef produced in the packaging plant. Genome analysis showed that several E. faecalis isolates were genetically similar to publicly available isolates recovered from retail ground beef in the United States.

Current limitations in the understanding and control of antimicrobial resistance (AMR) in Canada are described through a comprehensive review focusing on: (1) treatment optimization; (2) surveillance of antimicrobial use and AMR; and (3) prevention of transmission of AMR. Without addressing gaps in identified areas, sustained progress in AMR mitigation is unlikely. Expert opinions and perspectives contributed to prioritizing identified gaps. Using Canada as an example, this review emphasizes the importance and necessity of a One Health approach for understanding and mitigating AMR. Specifically, antimicrobial use in human, animal, crop, and environmental sectors cannot be regarded as independent; therefore, a One Health approach is needed in AMR research and understanding, current surveillance efforts, and policy. Discussions regarding addressing described knowledge gaps are separated into four categories: (1) further research; (2) increased capacity/resources; (3) increased prescriber/end-user knowledge; and (4) policy development/enforcement. This review highlights the research and increased capacity and resources to generate new knowledge and implement recommendations needed to address all identified gaps, including economic, social, and environmental considerations. More prescriber/end-user knowledge and policy development/enforcement are needed, but must be informed by realistic recommendations, with input from all relevant stakeholders. For most knowledge gaps, important next steps are uncertain. In conclusion, identified knowledge gaps underlined the need for AMR policy decisions to be considered in a One Health framework, while highlighting critical needs to achieve realistic and meaningful progress.

This study aimed to determine the concentration and composition of condensed tannins (CT) in different tissues of purple prairie clover (PPC; Dalea purpurea Vent.) at different maturities and to determine their protein-precipitating capacity. The compositions of CT were elucidated after thiolysis with benzyl mercaptan followed by high-performance liquid-chromatography (HPLC) and 1H–13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The results indicated that PPC flowering heads contained the highest CT concentration. Purple prairie clover CT consisted mainly of epicatechin (EC) and epigallocatechin (EGC) subunits. CT in the leaves were composed of more EC and less EGC than CT in stems and flowering heads at both the early flowering (EF) and late flowering (LF) head stages. The mean degree of polymerization was the highest for CT in stems and increased with maturity. CT isolated from PPC leaves at the early flowering head stage exhibited the greatest biological activity in terms of protein precipitation. Overall, the CT in PPC were predominantly procyanidins and the concentration and composition varied among the plant tissues and with maturity.

A broad, cross-sectional study of beef cattle at entry into Canadian feedlots investigated the prevalence and epidemiology of antimicrobial resistance (AMR) in Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, bacterial members of the bovine respiratory disease (BRD) complex. Upon feedlot arrival and before antimicrobials were administered at the feedlot, deep nasopharyngeal swabs were collected from 2,824 feedlot cattle in southern and central Alberta, Canada. Data on the date of feedlot arrival, cattle type (beef, dairy), sex (heifer, bull, steer), weight (kg), age class (calf, yearling), source (ranch direct, auction barn, backgrounding operations), risk of developing BRD (high, low), and weather conditions at arrival (temperature, precipitation, and estimated wind speed) were obtained. Mannheimia haemolytica, P. multocida, and H. somni isolates with multidrug-resistant (MDR) profiles associated with the presence of integrative and conjugative elements were isolated more often from dairy-type than from beef-type cattle. Our results showed that beef-type cattle from backgrounding operations presented higher odds of AMR bacteria as compared to auction-derived calves. Oxytetracycline resistance was the most frequently observed resistance across all Pasteurellaceae species and cattle types. Mycoplasma bovis exhibited high macrolide minimum inhibitory concentrations in both cattle types. Whether these MDR isolates establish and persist within the feedlot environment, requires further evaluation.

Enterococcus spp. have arisen as important nosocomial pathogens and are ubiquitous in the gastrointestinal tracts of animals and the environment. They carry many intrinsic and acquired antimicrobial resistance genes. Because of this, surveillance of Enterococcus spp. has become important with whole genome sequencing emerging as the preferred method for the characterization of enterococci. A scoping review was designed to determine how the use of whole genome sequencing in the surveillance of Enterococcus spp. adds to our knowledge of antimicrobial resistance in Enterococcus spp. Scoping review design was guided by the PRISMA extension and checklist and JBI Reviewer's Guide for scoping reviews. A total of 72 articles were included in the review. Of the 72 articles included, 48.6% did not state an association with a surveillance program and 87.5% of articles identified Enterococcus faecium. The majority of articles included isolates from human clinical or screening samples. Significant findings from the articles included novel sequence types, the increasing prevalence of vancomycin-resistant enterococci in hospitals, and the importance of surveillance or screening for enterococci. The ability of enterococci to adapt and persist within a wide range of environments was also a key finding. These studies emphasize the importance of ongoing surveillance of enterococci from a One Health perspective. More studies are needed to compare the whole genome sequences of human enterococcal isolates to those from food animals, food products, the environment, and companion animals.

Abstract Background Comparative knowledge of microbiomes and resistomes across environmental interfaces between animal production systems and urban settings is lacking. In this study, we executed a comparative analysis of the microbiota and resistomes of metagenomes from cattle feces, catch basin water, manured agricultural soil and urban sewage. Results Metagenomic DNA from composite fecal samples (FC; n = 12) collected from penned cattle at four feedlots in Alberta, Canada, along with water from adjacent catchment basins (CB; n = 13), soil (n = 4) from fields in the vicinity of one of the feedlots and urban sewage influent (SI; n = 6) from two municipalities were subjected to Illumina HiSeq2000 sequencing. Firmicutes exhibited the highest prevalence (40%) in FC, whereas Proteobacteria were most abundant in CB (64%), soil (60%) and SI (83%). Among sample types, SI had the highest diversity of antimicrobial resistance (AMR), and metal and biocide resistance (MBR) classes (13 & 15) followed by FC (10 & 8), CB (8 & 4), and soil (6 & 1). The highest antimicrobial resistant (AMR) gene (ARG) abundance was harboured by FC, whereas soil samples had a very small, but unique resistome which did not overlap with FC & CB resistomes. In the beef production system, tetracycline resistance predominated followed by macrolide resistance. The SI resistome harboured β-lactam, macrolide, tetracycline, aminoglycoside, fluoroquinolone and fosfomycin resistance determinants. Metal and biocide resistance accounted for 26% of the SI resistome with a predominance of mercury resistance. Conclusions This study demonstrates an increasing divergence in the nature of the microbiome and resistome as the distance from the feedlot increases. Consistent with antimicrobial use, tetracycline and macrolide resistance genes were predominant in the beef production system. One of the feedlots contributed both conventional (raised with antibiotics) and natural (raised without antibiotics) pens samples. Although natural pen samples exhibited a microbiota composition that was similar to samples from conventional pens, their resistome was less complex. Similarly, the SI resistome was indicative of drug classes used in humans and the greater abundance of mercury resistance may be associated with contamination of municipal water with household and industrial products.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90–100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Shiga toxin (stx) is the principal virulence factor of the foodborne pathogen, Shiga toxin-producing Escherichia coli (STEC) O157:H7 and is associated with various lambdoid bacterio (phages). A comparative genomic analysis was performed on STEC O157 isolates from cattle (n = 125) and clinical (n = 127) samples to characterize virulence genes, stx-phage insertion sites and antimicrobial resistance genes that may segregate strains circulating in the same geographic region. In silico analyses revealed that O157 isolates harboured the toxin subtypes stx1a and stx2a. Most cattle (76.0%) and clinical (76.4%) isolates carried the virulence gene combination of stx1, stx2, eae and hlyA. Characterization of stx1 and stx2-carrying phages in assembled contigs revealed that they were associated with mlrA and wrbA insertion sites, respectively. In cattle isolates, mlrA and wrbA insertion sites were occupied more often (77% and 79% isolates respectively) than in clinical isolates (38% and 1.6% isolates, respectively). Profiling of antimicrobial resistance genes (ARGs) in the assembled contigs revealed that 8.8% of cattle (11/125) and 8.7% of clinical (11/127) isolates harboured ARGs. Eight antimicrobial resistance genes cassettes (ARCs) were identified in 14 isolates (cattle, n = 8 and clinical, n = 6) with streptomycin (aadA1, aadA2, ant(3’’)-Ia and aph(3’’)-Ib) being the most prevalent gene in ARCs. The profound disparity between the cattle and clinical strains in occupancy of the wrbA locus suggests that this trait may serve to differentiate cattle from human clinical STEC O157:H7. These findings are important for stx screening and stx-phage insertion site genotyping as well as monitoring ARGs in isolates from cattle and clinical samples.

Shiga toxin (Stx) is the main virulence factor of Shiga toxin-producing Escherichia coli (STEC), and ruminants are the main reservoir of STEC. This study assessed the abundance and expression of Stx genes and the expression of host immune genes, aiming to determine factors affecting these measures and potential gene markers to differentiate Stx gene expression in the recto-anal junction of feedlot beef cattle. Rectal tissue and content samples were collected from 143 feedlot steers of three breeds (Angus, Charolais, and Kinsella Composite) over 2 consecutive years 2014 (n=71) and 2015 (n=72). The abundance and expression of stx1 and stx2 were quantified using qPCR and reverse-transcription-qPCR (RT-qPCR), respectively. Four immune genes (MS4A1, CCL21, CD19, and LTB), previously reported to be down-regulated in super-shedder cattle (i.e., > 104 CFU g-1) were selected, and their expression was evaluated using RT-qPCR. The stx1 gene abundance was only detected in tissue samples collected in year 2 and did not differ among breeds. The stx2 gene was detected in STEC from all samples collected in both years and did not vary among breeds. The abundance of stx1 and stx2 differed (P < 0.001) in content samples collected across breeds (stx1:AN>CH>KC, stx2: AN=CH>KC) in year 1, but not in year 2. Expression of stx2 was detected in 13 RAJ tissue samples (2014: n=6, 2015: n=7), while expression of stx1 was not detected. Correlation analysis showed that the expression of stx2 was negatively correlated with the expression of MS4A1 (R=-0.56, P=0.05) and positively correlated with the expression of LTB (R=0.60, P=0.05). The random forest model and Boruta method revealed that expression of selected immune genes could be predictive indicators of stx2 expression with prediction accuracy of MS4A1 >LTB >CCL21 >CD19. Our results indicate that the abundance of Stx could be affected by cattle breed and sampling year, suggesting that host genetics and environment may influence STEC colonization of the recto-anal junction of feedlot cattle. Additionally, the identified relationship between expressions of host immune genes and stx2 suggests that the host animal may regulate stx2 expression in colonizing STEC through immune functions.

Effectiveness of bacteriophages AKFV33 (Tequintavirus, T5) and AHP24 (Rogunavirus, T1), wV7 (Tequatrovirus, T4), and AHP24S (Vequintavirus, rV5), as well as 11 cocktails of combinations of the four phages, were evaluated in vitro for biocontrol of six common phage types of Escherichia coli O157 (human and bovine origins) at different multiplicities of infection (MOIs; 0.01–1,000), temperatures (37 or 22°C), and exposure times (10–22 h). Phage efficacy against O157 was highest at MOI 1,000 (P < 0.001) and after 14-18 h of exposure at 22°C (P < 0.001). The activity of individual phages against O157 did not predict the activity of a cocktail of these phages even at the same temperature and MOI. Combinations of phages were neutral (no better or worse than the most effective constituent phages acting alone), displayed facilitation (greater efficacy than the most effective constituent phages acting alone), or antagonistic (lower efficacy than the most effective constituent phages acting alone). Across MOIs, temperatures, exposure time, and O157 strains, a cocktail of T1, T4, and rV5 was most effective (P < 0.05) against O157, although T1 and rV5 were less effective (P < 0.001) than other individual phages. T5 was the most effective individual phages (P < 0.05), but was antagonistic to other phages, particularly rV5 and T4 + rV5. Interactions among phages were influenced by phage genera and phage combination, O157 strains, MOIs, incubation temperatures, and times. Based on this study, future development of phage cocktails should, as a minimum, include confirmation of a lack of antagonism among constituent phages and preferably confirmation of facilitation or synergistic effects.

Cattle have been suggested as the primary reservoirs of E. coli O157 mainly as a result of colonization of the recto-anal junction (RAJ) and subsequent shedding into the environment. Although a recent study reported different gene expression at RAJ between super-shedders (SS) and non-shedders (NS), the regulatory mechanisms of altered gene expression is unknown. This study aimed to investigate whether bovine non-coding RNAs play a role in regulating the differentially expressed (DE) genes between SS and NS, thus further influencing E. coli O157 shedding behavior in the animals through studying miRNAomes of the whole gastrointestinal tract including duodenum, proximal jejunum, distal jejunum, cecum, spiral colon, descending colon and rectum. The number of miRNAs detected in each intestinal region ranged from 390 ± 13 (duodenum) to 413 ± 49 (descending colon). Comparison between SS and NS revealed the number of differentially expressed (DE) miRNAs ranged from one (in descending colon) to eight (in distal jejunum), and through the whole gut, seven miRNAs were up-regulated and seven were down-regulated in SS. The distal jejunum and rectum were the regions where the most DE miRNAs were identified (eight and seven, respectively). The miRNAs, bta-miR-378b, bta-miR-2284j, and bta-miR-2284d were down-regulated in both distal jejunum and rectum of SS (log2fold-change: −2.7 to −3.8), bta-miR-2887 was down-regulated in the rectum of SS (log2fold-change: −3.2), and bta-miR-211 and bta-miR-29d-3p were up-regulated in the rectum of SS (log2fold-change: 4.5 and 2.2). Functional analysis of these miRNAs indicated their potential regulatory role in host immune functions, including hematological system development and immune cell trafficking. Our findings suggest that altered expression of miRNA in the gut of SS may lead to differential regulation of immune functions involved in E. coli O157 super-shedding in cattle.

Numerous antimicrobial resistance (AMR) surveillance studies have been conducted in North American feedlot cattle to investigate the major bacterial pathogens of the bovine respiratory disease (BRD) complex, specifically: Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. While most bacterial isolates recovered from healthy cattle are susceptible to a repertoire of antimicrobials, multidrug resistance is common in isolates recovered from cattle suffering from BRD. Integrative and conjugative elements (ICE) have gained increasing notoriety in BRD-Pasteurellaceae as they appear to play a key role in the concentration and dissemination of antimicrobial resistant genes. Likewise, low macrolide susceptibility has been described in feedlot isolates of M. bovis. Horizontal gene transfer has also been implicated in the spread of AMR within mycoplasmas, and in-vitro experiments have shown that exposure to antimicrobials can generate high levels of resistance in mycoplasmas via a single conjugative event. Consequently, antimicrobial use (AMU) could be accelerating AMR horizontal transfer within all members of the bacterial BRD complex. While metagenomics has been applied to the study of AMR in the microbiota of the respiratory tract, the potential role of the respiratory tract microbiome as an AMR reservoir remains uncertain. Current and prospective molecular tools to survey and characterize AMR need to be adapted as point-of-care technologies to enhance prudent AMU in the beef industry.

Multidrug-resistant (MDR; resistance to ≥3 antimicrobial classes) members of the Pasteurellaceae family may compromise the efficacy of therapies used to prevent and treat bovine respiratory disease (BRD) in feedlot cattle. This study examined the prevalence of multidrug resistance in strains of Mannheimia haemolytica and Pasteurella multocida collected from BRD cattle mortalities in North America. Isolates of M. haemolytica (n = 147) and P. multocida (n = 70) spanning 69 Alberta feedlots from 2011 to 2016 and two United States feedlots from 2011 to 2012 were examined for antimicrobial resistance (AMR) in association with integrative and conjugative elements (ICEs). Overall, resistance was high in both bacterial species with an increase in the prevalence of MDR isolates between 2011 and 2016. Resistance to >7 antimicrobial drugs occurred in 31% of M. haemolytica and 83% of P. multocida isolates. Resistance to sulfadimethoxine, trimethoprim/sulfamethoxazole, neomycin, clindamycin oxytetracycline, spectinomycin, tylosin, tilmicosin, and tulathromycin was most common. Although >80% of strains harbored three or more ICE-associated genes, only 12% of M. haemolytica and 77% of P. multocida contained all six, reflecting the diversity of ICEs. There was evidence of clonal spread as P. multocida and M. haemolytica isolates with the same pulsed-field gel electrophoresis profile from the United States in 2011 were isolated in Alberta in 2015–2016. This work highlights that MDR strains of Pasteurellaceae containing ICEs are widespread and may be contributing to BRD therapy failure in feedlot cattle. Given the antimicrobial resistance gene profiles identified, these MDR isolates may be selected for by the use of macrolides, tetracyclines, and/or in-feed supplements containing heavy metals.

Antimicrobial drugs are important tools for maintaining human and animal health. Globally, antimicrobial use (AMU) in food-producing animals is under increasing scrutiny due to its potential to promote antimicrobial resistance (AMR). Historically, comprehensive Canadian data related to the types of antimicrobial drugs used, extent of use, common indicators of use and the demographics of the cattle populations receiving antimicrobial drugs have been limited, in part due to segmentation in the cattle industry and fragmentation of the drug distribution system. Appropriate AMU estimates are required to understand AMU practices, to interpret AMR levels and patterns, to meaningfully assess associated public health risks, and to inform stewardship activities. The Canadian beef cattle industry has a long history of collaboration in AMU and AMR research. Prior research projects identified both opportunities and challenges in the collection of AMU data. Cornerstone projects provided insight into the complexity of collecting AMU data in Canada's feedlot sector. This paper will discuss how the lessons learned from past work have contributed to the formation of a Canadian fed-cattle antimicrobial surveillance program that was initiated in 2019. This important surveillance program will allow feedlot cattle AMU to improve management decisions and support AMU best practices in the evolving Canadian AMR landscape.

A comparative study of microbiota of the respiratory tract and joints of bovine respiratory disease (BRD) cattle mortalities was undertaken. Nasopharynx, trachea, lung and joint samples were collected from 32 cattle that died of BRD, “cases”, and 8 that died of other causes, “controls”. Bacterial diversity was lower (p < 0.05) in the nasopharynx, trachea and lungs of cases as compared to controls. In cases, alpha-diversity (p < 0.05) was lower in the lungs and joints than the nasopharynx. Proteobacteria, Tenericutes, Bacteroidetes, Firmicutes and Actinobacteria were the most abundant phyla in all samples. Relative abundances of Mycoplasma spp. in the lung, Pasteurella spp. in the trachea and lung, and Histophilus spp. in the lung, trachea and nasopharynx of cases were higher (p < 0.001) than controls. Mycoplasma spp. comprised 20.5% of bacterial flora in the joint, 36.0% in the lung, 22.4% in the trachea and 8.8% in the nasopharynx. Mannheimia spp. (21.8%) and Histophilus spp. (10.4%) were more abundant in lungs. Cattle that died of BRD possessed less diverse respiratory microbiomes with a higher abundance of respiratory pathogens. Mycoplasma spp. were prominent members of pneumonic lungs and joints displaying septic arthritis.

Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica are important foodborne pathogens capable of forming both single- and multi-species biofilms. In this study, the mono- and dual-species biofilms were formed by STEC O113:H21 and Salmonella enterica serovar Choleraesuis 10708 on stainless steel in the presence of beef juice over 5 d at 22 °C. The dual-species biofilm mass was substantially (p < 0.05) greater than that produced by STEC O113:H21 or S. Choleraesuis 10708 alone. However, numbers (CFU/mL) of S. Choleraesuis 10708 or STEC O113:H21 cells in the dual-species biofilm were (p < 0.05) lower than their respective counts in single-species biofilms. In multi-species biofilms, the sensitivity of S. Choleraesuis 10708 to the antimicrobial peptide WK2 was reduced, but it was increased for STEC O113:H21. Visualization of the temporal and spatial development of dual-species biofilms using florescent protein labeling confirmed that WK2 reduced cell numbers within biofilms. Collectively, our results highlight the potential risk of cross-contamination by multi-species biofilms to food safety and suggest that WK2 may be developed as a novel antimicrobial or sanitizer for the control of biofilms on stainless steel.

Anaerobic fungi produce extracellular hydrolytic enzymes that facilitate degradation of cellulose and hemicellulose in ruminants. The purpose of this work was to study the impact of three different anaerobic fungal species (Anaeromyces mucronatus YE505, Neocallimastix frontalis 27, and Piromyces rhizinflatus YM600) on hydrolysis of two different lignocellulosic substrates, corn (Zea mays L.) silage and reed (Phragmites australis (Cav.) Trin. ex Steud.). Biomass from each plant species was incubated anaerobically for 11 days either in the presence of live fungal inoculum or with heat-inactivated (control) inoculum. Headspace gas composition, dry matter loss, soluble chemical oxygen demand, concentration of volatile fatty acids, and chemical composition were measured before and after hydrolysis. While some microbial activity was observed, inoculation with anaerobic fungi did not result in any significant difference in the degradation of either type of plant biomass tested, likely due to low fungal activity or survival under the experimental conditions tested. While the premise of utilizing the unique biological activities of anaerobic fungi for biotechnology applications remains promising, further research on optimizing culturing and process conditions is necessary.

Abstract The host mechanisms involved in Escherichia coli O157 super-shedding in cattle is largely unknown. In this study, the comparison of transcriptomes of intestinal tissues between super-shedders (SS) and cattle negative for E. coli O157 (NS) was performed, aiming to identify genes that are potentially associated with super-shedding. In total, 16,846 ± 639 (cecum) to 18,137 ± 696 (distal jejunum) were expressed throughout the intestine, with the expressed genes associated with immune functions more pronounced in the small intestine. In total, 351 differentially expressed (DE) genes were identified throughout the intestine between SS and NS, with 101 being up-regulated and 250 down-regulated in SS. Functional analysis revealed DE genes were involved in increased T-cell responses and cholesterol absorption in the distal jejunum and descending colon, and decreased B-cell maturation in the distal jejunum of SS. RNA-Seq based SNP discovery revealed that the mutations in seven DE genes involved in leukocyte activation and cholesterol transportation were associated with E. coli O157 shedding. Our findings suggest that T-cell responses and cholesterol metabolism in the intestinal tract may be associated with super-shedding phenomenon, and the SNPs in the DE genes are possibly associated with the observed gene expression difference between SS and NS.

Abstract Future growth in demand for meat and milk, and the socioeconomic and environmental challenges that farmers face, represent a “grand challenge for humanity”. Improving the digestibility of crop residues such as straw could enhance the sustainability of ruminant production systems. Here, we investigated if transfer of rumen contents from bison to cattle could alter the rumen microbiome and enhance total tract digestibility of a barley straw-based diet. Beef heifers were adapted to the diet for 28 days prior to the experiment. After 46 days, ~70 percent of rumen contents were removed from each heifer and replaced with mixed rumen contents collected immediately after slaughter from 32 bison. This procedure was repeated 14 days later. Intake, chewing activity, total tract digestibility, ruminal passage rate, ruminal fermentation, and the bacterial and protozoal communities were examined before the first and after the second transfer. Overall, inoculation with bison rumen contents successfully altered the cattle rumen microbiome and metabolism, and increased protein digestibility and nitrogen retention, but did not alter fiber digestibility.

Objective The objectives were to assess the effects of purified lignin from wheat straw (sodium hydroxide dehydrated lignin; SHDL) on in vitro ruminal fermentation and on the growth performance of feedlot cattle. Methods In vitro experiments were conducted by incubating a timothy-alfalfa (50:50) forage mixture (48 h) and barley grain (24 h) with 0, 0.25, 0.5, 1.0, and 2.0 mg/mL of rumen fluid (equivalent to 0, 2, 4, 8, and 16 g SHDL/kg diet). Productions of CH4 and total gas, volatile fatty acids, ammonia, dry matter (DM) disappearance (DMD) and digestion of neutral detergent fiber (NDF) or starch were measured. Sixty Hereford-Angus cross weaned steer calves were individually fed a typical barley silage-barley grain based total mixed ration and supplemented with SHDL at 0, 4, 8, and 16 g/kg DM for 70 (growing), 28 (transition), and 121 d (finishing) period. Cattle were slaughtered at the end of the experiment and carcass traits were assessed. Results With forage, SHDL linearly (p

Horizontal gene transfer of integrative and conjugative elements (ICE) in bacterial pathogens of the bovine respiratory disease (BRD) complex has emerged as a significant cause of antimicrobial resistance (AMR) and therapeutic failure and mortalities in cattle. The aim of this study was to assess an AMR ICE occurring in Pasteurella multocida from a case of BRD, designated ICEMh1PM22 for its structure and host genome insertion site, and to identify consequences for host fitness and antimicrobial therapy. The modular structure of ICEMh1-like elements found in several related livestock pathogens was compared to ICEMh1PM22, and the repertoire of cargo genes in variable ICE modules was functionally categorized. AMR genes were identified as frequent additions to the variable modules of ICEMh1-like elements. Random PCR-based mapping of ICEMh1PM22-genome junctions in transconjugants provided evidence that ICEMh1PM22 integrates into the tRNA-leu for the UUG codon, and not into tRNA-leu for other codons. This was separately confirmed in the genomes of ICEMh1-like-harboring livestock pathogens. Bacterial genera harboring receptive tRNA-leuUUG were identified to establish the potential host range of ICEMh1-like elements. ICEMh1PM22-carrying transconjugants in P. multocida and Mannheimia haemolytica were less fit than isogenic strains without the ICE when grown without antimicrobial selection. This fitness cost was abrogated in the presence of subinhibitory concentrations of antimicrobials. Despite this cost, ICEMh1PM22 was retained in transconjugants in extended culture. To identify possible therapeutic efficiencies, antimicrobial combinations were screened for synergistic interactions against AMR ICEMh1PM22-carrying transconjugants. No antimicrobial combination tested exhibited synergistic interactions against AMR P. multocida or M. haemolytica harboring ICEMh1PM22. In conclusion, this study provided information on the structural variation of ICEMh1-like elements, refined the ICE insertion site and potential host range, and demonstrated the risk and consequences for AMR following horizontal transfer of ICE into BRD pathogens.

The objective of this study was to examine the effect of a pine enhanced biochar (EB) on rumen fermentation, apparent total tract digestibility, methane (CH4) emissions, and the rumen and fecal microbiome of Angus × Hereford heifers fed a barley silage-based diet. The experiment was a replicated 4 × 4 Latin square using 8 ruminally cannulated heifers (565 ± 35 kg initial BW). The basal diet contained 60% barley silage, 35% barley grain and 5% mineral supplement with EB added at 0% (control), 0.5, 1.0, or 2.0% (DM basis). Each period lasted 28 days, consisting of 14 days adaptation and 14 days of measurements. Samples for profiling of the microbiome in rumen liquid, solids and feces were collected on d 15 before feeding. Rumen samples for fermentation characterization were taken at 0, 3, 6, and 12 h post feeding. Total collection of urine and feces was conducted from days 18 to 22. Heifers were housed in open-circuit respiratory chambers on days 26–28 to estimate CH4 emissions. Ruminal pH was recorded at 1-min intervals during CH4 measurements using indwelling pH loggers. Data were analyzed with the fixed effects of dietary treatment and random effects of square, heifer within square and period. Dry matter intake was similar across treatments (P = 0.21). Ammonia N concentration and protozoa counts responded quadratically (P = 0.01) to EB in which both were decreased by EB included at 0.5 and 1.0%, compared to the control and 2.0% EB. Minimum pH was increased (P = 0.04), and variation of pH was decreased (P = 0.03) by 2.0% EB. Total tract digestibility, N balance and CH4 production were not affected (P ≥ 0.17) by EB. Enhanced biochar decreased the relative abundance of Fibrobacter (P = 0.05) and Tenericutes (P = 0.01), and increased the relative abundance of Spirochaetaes (P = 0.01), Verrucomicrobia (P = 0.02), and Elusimicrobia (P = 0.02). Results suggest that at the examined concentrations, EB was ineffective at decreasing enteric CH4 emissions, but did alter specific rumen microbiota.

Deadly outbreaks and illnesses due to Shiga toxin-producing Escherichia coli (STEC) occur worldwide; however, the cultivation methods required for adequate monitoring and traceback investigations are inefficient at best. Detection of STEC relies heavily on enrichment; yet no standard media or protocols exist. Furthermore, whether enrichment may bias detection of multiple STEC serogroups from complex samples is unknown. Thus, 14 STEC strains of serogroups O157 and the top six non-O157s (O26, O45, O103, O111, O121, and O145) were enriched in pairs for 6–78 h in broth and evaluated by quantitative polymerase chain reaction (qPCR). Here we show that a conventional 6-h enrichment protocol did not result in intra-species culture bias for the isolates tested. However, subsequent enrichments often produced biased cultures, with differences in the qPCR gene copy number ≥2 log10 apparent in 12%, 38%, and 52% of competitions after 30, 54, and 78 h of consecutive enrichments, respectively. Some strains were able to prevail and (or) out-compete the opponent strain in 100% of competitions. Our results suggest that culture bias should be considered and (or) evaluated further due to the potential implications during routine pathogen screening and outbreak investigations.