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Intestinal carriage of extended spectrum β-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing, and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over 6 months. We characterized the bacterial clones and plasmids carrying bla ESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [ bla CTX-M-1 (64.7%), bla CTX-M-14 (33.5%), or bla CTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and bla CTX-M -carrying plasmids, few bla CTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone versus plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids, and bacterial clones contribute to optimize fitness in specific environments. IMPORTANCE The gut microbiota is the epicenter of the emergence of resistance. Considerable amount of knowledge on the molecular mechanisms of resistance has been accumulated, but the ecological and evolutionary forces at play in nature are less studied. In this context, we performed a field work on temporal intestinal carriage of extended spectrum β-lactamase (ESBL)-producing Escherichia coli in veal farms. Veal calves are animals with one of the highest levels of ESBL producing E. coli fecal carriage, due to early high antibiotic exposure. We were able to show that calves were colonized successively by different ESBL-producing E. coli clones, and that two main scenarios were at play in the spread of bla CTX-M genes among calves: efficient colonization of several calves by a few bacterial clones and successful plasmid spread in various bacterial clones. Such knowledge should help develop new strategies to fight the emergence of antibiotic-resistance.

Salmonella enterica is a major foodborne pathogen, and contaminated beef products have been identified as one of the primary sources of Salmonella -related outbreaks. Pathogenicity and antibiotic resistance of Salmonella are highly serotype and subpopulation specific, which makes it essential to understand high-resolution Salmonella population dynamics in cattle. Time of year, source of cattle, pen, and sample type (i.e., feces, hide, or lymph nodes) have previously been identified as important factors influencing the serotype distribution of Salmonella (e.g., Anatum, Lubbock, Cerro, Montevideo, Kentucky, Newport, and Norwich) that were isolated from a longitudinal sampling design in a research feedlot. In this study, we performed high-resolution genomic comparisons of Salmonella isolates within each serotype using both single-nucleotide polymorphism-based maximum-likelihood phylogeny and hierarchical clustering of core-genome multilocus sequence typing. The importance of the aforementioned features in clonal Salmonella expansion was further explored using a supervised machine learning algorithm. In addition, we identified and compared the resistance genes, plasmids, and pathogenicity island profiles of the isolates within each subpopulation. Our findings indicate that clonal expansion of Salmonella strains in cattle was mainly influenced by the randomization of block and pen, as well as the origin/source of the cattle, i.e., regardless of sampling time and sample type (i.e., feces, lymph node, or hide). Further research is needed concerning the role of the feedlot pen environment prior to cattle placement to better understand carryover contributions of existing strains of Salmonella and their bacteriophages. IMPORTANCE Salmonella serotypes isolated from outbreaks in humans can also be found in beef cattle and feedlots. Virulence factors and antibiotic resistance are among the primary defense mechanisms of Salmonella , and are often associated with clonal expansion. This makes understanding the subpopulation dynamics of Salmonella in cattle critical for effective mitigation. There remains a gap in the literature concerning subpopulation dynamics within Salmonella serotypes in feedlot cattle from the beginning of feeding up until slaughter. Here, we explore Salmonella population dynamics within each serotype using core-genome phylogeny and hierarchical classifications. We used machine learning to quantitatively parse the relative importance of both hierarchical and longitudinal clustering among cattle host samples. Our results reveal that Salmonella populations in cattle are highly clonal over a 6-month study period and that clonal dissemination of Salmonella in cattle is mainly influenced spatially by experimental block and pen, as well by the geographical origin of the cattle.

Prebiotics are increasingly examined for their ability to modulate the neonate gut microbiota of livestock, and products such as inulin are commonly added to milk replacer used in calving. However, the ability of specific members of the bovine neonate microbiota to respond to inulin remains to be determined, particularly among indigenous lactobacilli and bifidobacteria, beneficial genera commonly enriched by inulin. Screening of Bifidobacterium and Lactobacillus isolates obtained from fresh feces of dairy calves revealed that lactobacilli had a higher prevalence of inulin fermentation capacity (58%) than bifidobacteria (17%). Several Ligilactobacillus agilis (synonym Lactobacillus agilis ) isolates exhibited vigorous growth on, and complete degradation of, inulin; however, the phenotype was strain specific. The most vigorous inulin-fermenting strain, L. agilis YZ050, readily degraded long-chain inulin not consumed by bifidobacterial isolates. Comparative genomic analysis of both L. agilis fermenter and nonfermenter strains indicated that strain YZ050 encodes an inulinase homolog, previously linked to extracellular degradation of long-chain inulin in Lacticaseibacillus paracasei , that was strongly induced during growth on inulin. Inulin catabolism by YZ050 also generates extracellular fructose, which can cross-feed other non-inulin-fermenting lactic acid bacteria isolated from the same bovine feces. The presence of specific inulin-responsive bacterial strains within calf gut microbiome provides a mechanistic rationale for enrichment of specific lactobacilli and creates a foundation for future synbiotic applications in dairy calves aimed at improving health in early life. IMPORTANCE The gut microbiome plays an important role in animal health and is increasingly recognized as a target for diet-based manipulation. Inulin is a common prebiotic routinely added to animal feeds; however, the mechanism of inulin consumption by specific beneficial taxa in livestock is ill defined. In this study, we examined Lactobacillus and Bifidobacterium isolates from calves fed inulin-containing milk replacer and characterized specific strains that robustly consume long-chain inulin. In particular, novel Ligilactobacillus agilis strain YZ050 consumed inulin via an extracellular fructosidase, resulting in complete consumption of all long-chain inulin. Inulin catabolism resulted in temporal release of extracellular fructose, which can promote growth of other non-inulin-consuming strains of lactic acid bacteria. This work provides the mechanistic insight needed to purposely modulate the calf gut microbiome via the establishment of networks of beneficial microbes linked to specific prebiotics., (Copyright © 2020 American Society for Microbiology.)

The commonly used laboratory bacterium Escherichia coli normally does not produce and secrete cellulases due to its complex bilayer membrane structure and poor secretory apparatus. In our previous study, the cellulolytic E. coli strain ZH-4 with extracellular cellulase activity was found in the bovine rumen. In this study, we demonstrate that the secretion of cellulase is a common feature of E. coli isolates from the rumen of animals such as sheep and cattle. Physiological phenotype characterization of these E. coli isolates, together with genome, transcriptome, and comparative genomics analysis, suggests their adaption to the rumen niche. The higher growth rate of the isolated strains under aerobic conditions meets the competitive requirements of the strains in rumen microecosystem, while anaerobic accumulation of reduced H 2 and succinate is hypothesized to be the results of adaptation to the rumen environment. Cellulase secretion increased significantly when the molecular chaperone genes ibpA and ibpB were overexpressed. This was also revealed by the transcriptomic data. A possible mechanism for cellulase secretion by E. coli isolates was proposed based on the transcriptomic data and molecular experiments. IMPORTANCE As an important intestinal microorganism, E. coli is present in the intestinal tract of animals and in many other environments. However, it normally does not produce and secret cellulases due to its complex bilayer membrane structure and poor secretory apparatus. Here, we proved that E. coli is widely present in the rumen of sheep and cattle. Systematic analysis of the isolates indicated that they have adapted to the rumen niche, with phenotypes that include secretion of cellulase and fermentative accumulation of succinate and H 2 The finding that overexpression of small heat shock protein genes ibpA and ibpB could facilitate cellulase BcsZ secretion, which provides a possible insight into the protein secretion mechanism of rumen-colonizing E. coli ., (Copyright © 2020 American Society for Microbiology.)

Analysis of the cow microbiome, as well as host genetic influences on the establishment and colonization of the rumen microbiota, is critical for development of strategies to manipulate ruminal function toward more efficient and environmentally friendly milk production. To this end, the development and validation of noninvasive methods to sample the rumen microbiota at a large scale are required. In this study, we further optimized the analysis of buccal swab samples as a proxy for direct bacterial samples of the rumen of dairy cows. To identify an optimal time for sampling, we collected buccal swab and rumen samples at six different time points relative to animal feeding. We then evaluated several biases in these samples using a machine learning classifier (random forest) to select taxa that discriminate between buccal swab and rumen samples. Differences in the inverse Simpson's diversity, Shannon's evenness, and Bray-Curtis dissimilarities between methods were significantly less apparent when sampling was performed prior to morning feeding ( P  < 0.05), suggesting that this time point was optimal for representative sampling. In addition, the random forest classifier was able to accurately identify nonrumen taxa, including 10 oral and putative feed-associated taxa. Two highly prevalent (>60%) taxa in buccal and rumen samples had significant variance in relative abundances between sampling methods but could be qualitatively assessed via regular buccal swab sampling. This work not only provides new insights into the oral community of ruminants but also further validates and refines buccal swabbing as a method to assess the rumen bacterial in large herds. IMPORTANCE The gastrointestinal tracts of ruminants harbor a diverse microbial community that coevolved symbiotically with the host, influencing its nutrition, health, and performance. While the influence of environmental factors on rumen microbes is well documented, the process by which host genetics influences the establishment and colonization of the rumen microbiota still needs to be elucidated. This knowledge gap is due largely to our inability to easily sample the rumen microbiota. There are three common methods for rumen sampling but all of them present at least one disadvantage, including animal welfare, sample quality, labor, and scalability. The development and validation of noninvasive methods, such as buccal swabbing, for large-scale rumen sampling is needed to support studies that require large sample sizes to generate reliable results. The validation of buccal swabbing will also support the development of molecular tools for the early diagnosis of metabolic disorders associated with microbial changes in large herds.

Staphylococcus agnetis has been previously associated with subclinical or clinically mild cases of mastitis in dairy cattle and is one of several staphylococcal species that have been isolated from the bones and blood of lame broilers. We reported that S. agnetis could be obtained frequently from bacterial chondronecrosis with osteomyelitis (BCO) lesions of lame broilers (A. Al-Rubaye et al., PLoS One 10:e0143336, 2015 [https://doi.org/10.1371/journal.pone.0143336]). A particular isolate, S. agnetis 908, can induce lameness in over 50% of exposed chickens, exceeding normal BCO incidences in broiler operations. We reported the assembly and annotation of the genome of isolate 908. To better understand the relationship between dairy cattle and broiler isolates, we assembled 11 additional genomes for S. agnetis isolates, an additional chicken BCO strain, and ten isolates from cattle milk, mammary gland secretions, or udder skin from the collection at the University of Missouri. To trace phylogenetic relationships, we constructed phylogenetic trees based on multilocus sequence typing and genome-to-genome distance comparisons. Chicken isolate 908 clustered with two of the cattle isolates, along with three isolates from chickens in Denmark and an isolate of S. agnetis we isolated from a BCO lesion on a commercial broiler farm in Arkansas. We used a number of BLAST tools to compare the chicken isolates to those from cattle and identified 98 coding sequences distinguishing isolate 908 from the cattle isolates. None of the identified genes explain the differences in host or tissue tropism. These analyses are critical to understanding how staphylococci colonize and infect different hosts and potentially how they can transition to alternative niches (bone versus dermis). IMPORTANCE Staphylococcus agnetis has been recently recognized as associated with disease in dairy cattle and meat-type chickens. The infections appear to be limited in cattle and systemic in broilers. This report details the molecular relationships between cattle and chicken isolates in order to understand how this recently recognized species infects different hosts with different disease manifestations. The data show that the chicken and cattle isolates are very closely related, but the chicken isolates all cluster together, suggesting a single jump from cattle to chickens., (Copyright © 2020 American Society for Microbiology.)

Survival and growth of the anaerobic gut fungi (AGF; Neocallimastigomycota) in the herbivorous gut necessitate the possession of multiple abilities absent in other fungal lineages. We hypothesized that horizontal gene transfer (HGT) was instrumental in forging the evolution of AGF into a phylogenetically distinct gut-dwelling fungal lineage. The patterns of HGT were evaluated in the transcriptomes of 27 AGF strains, 22 of which were isolated and sequenced in this study, and 4 AGF genomes broadly covering the breadth of AGF diversity. We identified 277 distinct incidents of HGT in AGF transcriptomes, with subsequent gene duplication resulting in an HGT frequency of 2 to 3.5% in AGF genomes. The majority of HGT events were AGF specific (91.7%) and wide (70.8%), indicating their occurrence at early stages of AGF evolution. The acquired genes allowed AGF to expand their substrate utilization range, provided new venues for electron disposal, augmented their biosynthetic capabilities, and facilitated their adaptation to anaerobiosis. The majority of donors were anaerobic fermentative bacteria prevalent in the herbivorous gut. This study strongly indicates that HGT indispensably forged the evolution of AGF as a distinct fungal phylum and provides a unique example of the role of HGT in shaping the evolution of a high-rank taxonomic eukaryotic lineage. IMPORTANCE The anaerobic gut fungi (AGF) represent a distinct basal phylum lineage (Neocallimastigomycota) commonly encountered in the rumen and alimentary tracts of herbivores. Survival and growth of anaerobic gut fungi in these anaerobic, eutrophic, and prokaryote-dominated habitats necessitates the acquisition of several traits absent in other fungal lineages. We assess here the role of horizontal gene transfer as a relatively fast mechanism for trait acquisition by the Neocallimastigomycota postsequestration in the herbivorous gut. Analysis of 27 transcriptomes that represent the broad diversity of Neocallimastigomycota identified 277 distinct HGT events, with subsequent gene duplication resulting in an HGT frequency of 2 to 3.5% in AGF genomes. These HGT events have allowed AGF to survive in the herbivorous gut by expanding their substrate utilization range, augmenting their biosynthetic pathway, providing new routes for electron disposal by expanding fermentative capacities, and facilitating their adaptation to anaerobiosis. HGT in the AGF is also shown to be mainly a cross-kingdom affair, with the majority of donors belonging to the bacteria. This study represents a unique example of the role of HGT in shaping the evolution of a high-rank taxonomic eukaryotic lineage., (Copyright © 2019 American Society for Microbiology.)

Tall fescue, the predominant southeastern United States cool-season forage grass, frequently becomes infected with an ergot alkaloid-producing toxic endophyte, Epichloë coenophialum Consumption of endophyte-infected fescue results in fescue toxicosis (FT), a condition that lowers beef cow productivity. Limited data on the influence of ergot alkaloids on rumen fermentation profiles or ruminal bacteria that could degrade the ergot alkaloids are available, but how FT influences the grazing bovine fecal microbiota or what role fecal microbiota might play in FT etiology and associated production losses has yet to be investigated. Here, we used 16S rRNA gene sequencing of fecal samples from weaned Angus steers grazing toxic endophyte-infected (E+; n  = 6) or nontoxic (Max-Q; n  = 6) tall fescue before and 1, 2, 14, and 28 days after pasture assignment. Bacteria in the Firmicutes and Bacteroidetes phyla comprised 90% of the Max-Q and E+ steer fecal microbiota throughout the trial. Early decreases in the Erysipelotrichaceae family and delayed increases of the Ruminococcaceae and Lachnospiraceae families were among the major effects of E+ grazing. E+ also increased abundances within the Planctomycetes , Chloroflexi , and Proteobacteria phyla and the Clostridiaceae family. Multiple operational taxonomic units classified as Ruminococcaceae and Lachnospiraceae were correlated negatively with weight gains (lower in E+) and positively with respiration rates (increased by E+). These data provide insights into how E+ grazing alters the Angus steer microbiota and the relationship of fecal microbiota dynamics with FT. IMPORTANCE Consumption of E+ tall fescue has an estimated annual $1 billion negative impact on the U.S. beef industry, with one driver of these costs being lowered weight gains. As global agricultural demand continues to grow, mitigating production losses resulting from grazing the predominant southeastern United States forage grass is of great value. Our investigation of the effects of E+ grazing on the fecal microbiota furthers our understanding of bovine fescue toxicosis in a real-world grazing production setting and provides a starting point for identifying easy-to-access fecal bacteria that could serve as potential biomarkers of animal productivity and/or FT severity for tall fescue-grazing livestock., (Copyright © 2019 American Society for Microbiology.)

Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and affect the production of agriculturally relevant products, including milk and meat. Despite this link, interventions to alter the adult microbiota to improve production have proven ineffective, as established microbial communities are resilient to change. In contrast, developing communities in young animals may be more easily altered but are less well studied. Here, we measured the GIT-associated microbiota of 45 Holstein dairy cows from 2 weeks to the first lactation cycle, using Illumina amplicon sequencing of bacterial (16S rRNA V4), archaeal (16S rRNA V6 to V8), and fungal (internal transcribed region 1 [ITS1]) communities. Fecal and ruminal microbiota of cows raised on calf starter grains and/or corn silage were correlated to lifetime growth as well as milk production during the first lactation cycle, in order to determine whether early-life diets have long-term impacts. Significant diet-associated differences in total microbial communities and specific taxa were observed by weaning (8 weeks), but all animals reached an adult-like composition between weaning and 1 year. While some calf-diet-driven differences were apparent in the microbiota of adult cows, these dissimilarities did not correlate with animal growth or milk production. This finding suggests that initial microbial community establishment is affected by early-life diet but postweaning factors have a greater influence on adult communities and production outcomes. IMPORTANCE The gut microbiota is essential for the survival of many organisms, including ruminants that rely on microorganisms for nutrient acquisition from dietary inputs for the production of products such as milk and meat. While alteration of the adult ruminant microbiota to improve production is possible, changes are often unstable and fail to persist. In contrast, the early-life microbiota may be more amenable to sustained modification. However, few studies have determined the impact of early-life interventions on downstream production. Here, we investigated the impact of agriculturally relevant calf diets, including calf starter and corn silage, on gut microbial communities, growth, and production through the first lactation cycle. Thus, this work serves to further our understanding of early-life microbiota acquisition, as well as informing future practices in livestock management., (Copyright © 2019 American Society for Microbiology.)

To assess phenotypic bacterial antimicrobial resistance (AMR) in different strata (e.g., host populations, environmental areas, manure, or sewage effluents) for epidemiological purposes, isolates of target bacteria can be obtained from a stratum using various sample types. Also, different sample processing methods can be applied. The MIC of each target antimicrobial drug for each isolate is measured. Statistical equivalence testing of the MIC data for the isolates allows evaluation of whether different sample types or sample processing methods yield equivalent estimates of the bacterial antimicrobial susceptibility in the stratum. We demonstrate this approach on the antimicrobial susceptibility estimates for (i) nontyphoidal Salmonella spp. from ground or trimmed meat versus cecal content samples of cattle in processing plants in 2013-2014 and (ii) nontyphoidal Salmonella spp. from urine, fecal, and blood human samples in 2015 (U.S. National Antimicrobial Resistance Monitoring System data). We found that the sample types for cattle yielded nonequivalent susceptibility estimates for several antimicrobial drug classes and thus may gauge distinct subpopulations of salmonellae. The quinolone and fluoroquinolone susceptibility estimates for nontyphoidal salmonellae from human blood are nonequivalent to those from urine or feces, conjecturally due to the fluoroquinolone (ciprofloxacin) use to treat infections caused by nontyphoidal salmonellae. We also demonstrate statistical equivalence testing for comparing sample processing methods for fecal samples (culturing one versus multiple aliquots per sample) to assess AMR in fecal Escherichia coli These methods yield equivalent results, except for tetracyclines. Importantly, statistical equivalence testing provides the MIC difference at which the data from two sample types or sample processing methods differ statistically. Data users (e.g., microbiologists and epidemiologists) may then interpret practical relevance of the difference. IMPORTANCE Bacterial antimicrobial resistance (AMR) needs to be assessed in different populations or strata for the purposes of surveillance and determination of the efficacy of interventions to halt AMR dissemination. To assess phenotypic antimicrobial susceptibility, isolates of target bacteria can be obtained from a stratum using different sample types or employing different sample processing methods in the laboratory. The MIC of each target antimicrobial drug for each of the isolates is measured, yielding the MIC distribution across the isolates from each sample type or sample processing method. We describe statistical equivalence testing for the MIC data for evaluating whether two sample types or sample processing methods yield equivalent estimates of the bacterial phenotypic antimicrobial susceptibility in the stratum. This includes estimating the MIC difference at which the data from the two approaches differ statistically. Data users (e.g., microbiologists, epidemiologists, and public health professionals) can then interpret whether that present difference is practically relevant., (Copyright © 2018 Shakeri et al.)

The antibiotic monensin is fed to dairy cows to increase milk production efficiency. A fraction of this monensin is excreted into the cow manure. Previous studies have found that cow manure containing monensin can negatively impact the performance of anaerobic digesters, especially upon first introduction. Few studies have examined whether the anaerobic digester microbiome can adapt to monensin during the operating time. Here, we conducted a long-term time series study of four lab-scale anaerobic digesters fed with cow manure. We examined changes in both the microbiome composition and function of the anaerobic digesters when subjected to the dairy antibiotic monensin. In our digesters, monensin was not rapidly degraded under anaerobic conditions. The two anaerobic digesters that were subjected to manure from monensin feed-dosed cows exhibited relatively small changes in microbiome composition and function due to relatively low monensin concentrations. At higher concentrations of monensin, which we dosed directly to control manure (from dairy cows without monensin), we observed major changes in the microbiome composition and function of two anaerobic digesters. A rapid introduction of monensin to one of these anaerobic digesters led to the impairment of methane production. Conversely, more gradual additions of the same concentrations of monensin to the other anaerobic digester led to the adaptation of the anaerobic digester microbiomes to the relatively high monensin concentrations. A member of the candidate OP11 ( Microgenomates ) phylum arose in this anaerobic digester and appeared to be redundant with certain Bacteroidetes phylum members, which previously were dominating. IMPORTANCE Monensin is a common antibiotic given to dairy cows in the United States and is partly excreted with dairy manure. An improved understanding of how monensin affects the anaerobic digester microbiome composition and function is important to prevent process failure for farm-based anaerobic digesters. This time series study demonstrates how anaerobic digester microbiomes are inert to low monensin concentrations and can adapt to relatively high monensin concentrations by redundancy in an already existing population. Therefore, our work provides further insight into the importance of microbiome redundancy in maintaining the stability of anaerobic digesters., (Copyright © 2018 Spirito et al.)

Microbial communities play critical roles in the gastrointestinal tracts (GIT) of preruminant calves by influencing performance and health. However, little is known about the establishment of microbial communities in the calf GIT or their dynamics during development. In this study, next-generation sequencing was used to assess changes in the bacterial communities of the rumen, jejunum, cecum, and colon in 26 crossbred calves at four developmental stages (7, 28, 49, and 63 days old). Alpha diversity differed among GIT regions with the lowest diversity and evenness in the jejunum, whereas no changes in alpha diversity were observed across developmental stage. Beta diversity analysis showed both region and age effects, with low numbers of operational taxonomic units (OTUs) shared between regions within a given age group or between ages in a given region. Taxonomic analysis revealed that several taxa coexisted in the rumen, jejunum, cecum, and colon but that their abundances differed considerably by GIT region and age. As calves aged, we observed lower abundances of taxa such as Bacteroides , Parabacteroides , and Paraprevotella with higher abundances of Bulleidia and Succiniclasticum in the rumen. The jejunum also displayed taxonomic changes with increases in Clostridiaceae and Turicibacter taxa in older calves. In the lower gut, taxa such as Lactobacillus , Blautia , and Faecalibacterium decreased and S24-7, Paraprevotella , and Prevotella increased as calves aged. These data support a model whereby early and successive colonization by bacteria occurs across the GIT of calves and provides insights into the temporal dynamics of the GIT microbiota of dairy calves during preweaning development. IMPORTANCE The gastrointestinal tracts (GIT) of ruminants, such as dairy cows, house complex microbial communities that contribute to their overall health and support their ability to produce milk. For example, the rumen microbiota converts feed into usable nutrients, while the jejunal microbiota provides access to protein. Thus, establishing a properly functioning GIT microbiota in dairy calves is critical to their productivity as adult cows. However, little is known about the establishment, maintenance, and dynamics of the calf GIT microbiota in early life. In this study, we evaluated the bacterial communities in the rumen, jejunum, cecum, and colon in dairy calves across preweaning development and show that they are highly variable early on in life before transitioning to a stable community. Understanding the dairy calf GIT microbiota has implications for ensuring proper health during early life and will aid in efforts to develop strategies for improving downstream production., (Copyright © 2018 American Society for Microbiology.)

To get insights into the temporal pattern of commensal Escherichia coli populations, we sampled the feces of four healthy cows from the same herd in the Hwange District of Zimbabwe daily over 25 days. The cows had not received antibiotic treatment during the previous 3 months. We performed viable E. coli counts and characterized the 326 isolates originating from the 98 stool samples at a clonal level, screened them for stx and eae genes, and tested them for their antibiotic susceptibilities. We observed that E. coli counts and dominant clones were different among cows, and very few clones were shared. No clone was shared by three or four cows. Clone richness and evenness were not different between cows. Within each host, the variability in the E. coli count was evidenced between days, and no clone was found to be dominant during the entire sampling period, suggesting the existence of clonal interference. Dominant clones tended to persist longer than subdominant ones and were mainly from phylogenetic groups A and B1. Five E. coli clones were found to contain both the stx 1 and stx 2 genes, representing 6.3% of the studied isolates. All cows harbored at least one Shiga toxin-producing E. coli (STEC) strain. Resistance to tetracycline, penicillins, trimethoprim, and sulfonamides was rare and observed in three clones that were shed at low levels in two cows. This study highlights the fact that the commensal E. coli population, including the STEC population, is host specific, is highly dynamic over a short time frame, and rarely carries antibiotic resistance determinants in the absence of antibiotic treatment. IMPORTANCE The literature about the dynamics of commensal Escherichia coli populations is very scarce. Over 25 days, we followed the total E. coli counts daily and characterized the sampled clones in the feces of four cows from the same herd living in the Hwange District of Zimbabwe. This study deals with the day-to-day dynamics of both quantitative and qualitative aspects of E. coli commensal populations, with a focus on both Shiga toxin-producing E. coli and antibiotic-resistant E. coli strains. We show that the structure of these commensal populations was highly specific to the host, even though the cows ate and roamed together, and was highly dynamic between days. Such data are of importance to understand the ecological forces that drive the dynamics of the emergence of E. coli clones of particular interest within the gastrointestinal tract and their transmission between hosts., (Copyright © 2017 American Society for Microbiology.)

Storm water runoff is a major source of pollution, and understanding the components of storm water discharge is essential to remediation efforts and proper assessment of risks to human and ecosystem health. In this study, culturable Escherichia coli and ampicillin-resistant E. coli levels were quantified and microbial source tracking (MST) markers (including markers for general Bacteroidales spp., human, ruminant/cow, gull, and dog) were detected in storm water outfalls and sites along the Humber River in Toronto, Ontario, Canada, and enumerated via endpoint PCR and quantitative PCR (qPCR). Additionally, chemical source tracking (CST) markers specific for human wastewater (caffeine, carbamazepine, codeine, cotinine, acetaminophen, and acesulfame) were quantified. Human and gull fecal sources were detected at all sites, although concentrations of the human fecal marker were higher, particularly in outfalls (mean outfall concentrations of 4.22 log 10 copies, expressed as copy numbers [CN]/100 milliliters for human and 0.46 log 10 CN/100 milliliters for gull). Higher concentrations of caffeine, acetaminophen, acesulfame, E. coli, and the human fecal marker were indicative of greater raw sewage contamination at several sites (maximum concentrations of 34,800 ng/liter, 5,120 ng/liter, 9,720 ng/liter, 5.26 log 10 CFU/100 ml, and 7.65 log 10 CN/100 ml, respectively). These results indicate pervasive sewage contamination at storm water outfalls and throughout the Humber River, with multiple lines of evidence identifying Black Creek and two storm water outfalls with prominent sewage cross-connection problems requiring remediation. Limited data are available on specific sources of pollution in storm water, though our results indicate the value of using both MST and CST methodologies to more reliably assess sewage contamination in impacted watersheds., Importance: Storm water runoff is one of the most prominent non-point sources of biological and chemical contaminants which can potentially degrade water quality and pose risks to human and ecosystem health. Therefore, identifying fecal contamination in storm water runoff and outfalls is essential for remediation efforts to reduce risks to public health. This study employed multiple methods of identifying levels and sources of fecal contamination in both river and storm water outfall sites, evaluating the efficacy of using culture-based enumeration of E. coli, molecular methods of determining the source(s) of contamination, and CST markers as indicators of fecal contamination. The results identified pervasive human sewage contamination in storm water outfalls and throughout an urban watershed and highlight the utility of using both MST and CST to identify raw sewage contamination., (© Crown copyright 2016.)

The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n = 72) collected at 0, 2, and 6 ± 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ± 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ± 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Enterobacteriaceae-associated blaCTX-M genes have become globally widespread within the past 30 years. Among isolates from Washington State cattle, Escherichia coli strains carrying blaCTX-M (CTX-M E. coli strains) were absent from a set of 2008 isolates but present in a set of isolates from 2011. On 30 Washington State dairy farms sampled in 2012, CTX-M E. coli prevalence was significantly higher on eastern than on northwestern Washington farms, on farms with more than 3,000 adult cows, and on farms that recently received new animals. The addition of fresh bedding to calf hutches at least weekly and use of residual fly sprays were associated with lower prevalence of CTX-M E. coli. In Washington State, the occurrence of human pathogens carrying blaCTX-M genes preceded the emergence of blaCTX-M-associated E. coli in cattle, indicating that these resistance determinants and/or their bacterial hosts may have emerged in human populations prior to their dissemination to cattle populations., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Fourteen Holstein cows of similar ages were monitored through their first two lactation cycles, during which ruminal solids and liquids, milk samples, production data, and feed consumption data were collected for each cow during early (76 to 82 days in milk [DIM]), middle (151 to 157 DIM), and late (251 to 257 DIM) lactation periods. The bacterial community of each ruminal sample was determined by sequencing the region from V6 to V8 of the 16S rRNA gene using 454 pyrosequencing. Gross feed efficiency (GFE) for each cow was calculated by dividing her energy-corrected milk by dry matter intake (ECM/DMI) for each period of both lactation cycles. Four pairs of cows were identified that differed in milk production efficiency, as defined by residual feed intake (RFI), at the same level of ECM production. The most abundant phyla detected for all cows were Bacteroidetes (49.42%), Firmicutes (39.32%), Proteobacteria (5.67%), and Tenericutes (2.17%), and the most abundant genera included Prevotella (40.15%), Butyrivibrio (2.38%), Ruminococcus (2.35%), Coprococcus (2.29%), and Succiniclasticum (2.28%). The bacterial microbiota between the first and second lactation cycles were highly similar, but with a significant correlation between total community composition by ruminal phase and specific bacteria whose relative sequence abundances displayed significant positive or negative correlation with GFE or RFI. These data suggest that the ruminal bacterial community is dynamic in terms of membership and diversity and that specific members are associated with high and low milk production efficiency over two lactation cycles., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx1, stx2, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Microbes present in the rumen of dairy cows are essential for degradation of cellulosic and nonstructural carbohydrates of plant origin. The prepartum and postpartum diets of high-producing dairy cows are substantially different, but in what ways the rumen microbiome changes in response and how those changes may influence production traits are not well elucidated. Here, we sequenced the 16S and 18S rRNA genes using the MiSeq platform to characterize the prepartum and postpartum rumen fluid microbiomes in 115 high-producing dairy cows, including both primiparous and multiparous animals. Discriminant analysis identified differences between the microbiomes of prepartum and postpartum samples and between primiparous and multiparous cows. 18S rRNA sequencing revealed an overwhelming dominance of the protozoan class Litostomatea, with over 90% of the eukaryotic microbial population belonging to that group. Additionally, fungi were relatively more prevalent and Litostomatea relatively less prevalent in prepartum samples than in postpartum ones. The core rumen microbiome (common to all samples) consisted of 64 bacterial taxa, of which members of the genus Prevotella were the most prevalent. The Chao1 richness index was greater for prepartum multiparous cows than for postpartum multiparous cows. Multivariable models identified bacterial taxa associated with increased or reduced milk production, and general linear models revealed that a metagenomically based prediction of productivity is highly associated with production of actual milk and milk components. In conclusion, the structure of the rumen fluid microbiome shifts between the prepartum and first-week postpartum periods, and its profile within the context of this study could be used to accurately predict production traits.

The bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for 1% and 0.1% of the total non-rRNAs, respectively. The majority (98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen.

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples.O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P<0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover,simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.

The ability to maintain a dual lifestyle of colonizing the ruminant gut and surviving in nonhost environments once shed is key to the success of Escherichia coli O157:H7 as a zoonotic pathogen. Both physical and biological conditions encountered by the bacteria are likely to change during the transition between host and nonhost environments. In this study, carbon starvation at suboptimal temperatures in nonhost environments was simulated by starving a New Zealand bovine E. coli O157:H7 isolate in phosphate-buffered saline at 4 and 15°C for 84 days. Recovery of starved cells on media with different nutrient availabilities was monitored under aerobic and anaerobic conditions. We found that the New Zealand bovine E. coli O157:H7 isolate was able to maintain membrane integrity and viability over 84 days and that the level of recovery depended on the nutrient level of the recovery medium as well as the starvation temperature. In addition, a significant difference in carbon utilization was observed between starved and nonstarved cells., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047-2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.

Anaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.

The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species.

Shiga toxin-producing Escherichia coli (STEC) O111:NM is an important serotype that has been incriminated in disease outbreaks in the United States. This study characterized cattle STEC O111:NM for virulence factors and markers by PCR. Major conclusions are that STEC O111:NM characterized in this study lacks stx2 and the full spectrum of nle gene markers, and it has an incomplete OI-122.

The aim of this study was to determine if a mixed microbial community from the bovine rumen would respond to excess carbohydrate by accumulating reserve carbohydrate, energy spilling (dissipating excess ATP energy as heat), or both. Mixed microbes from the rumen were washed with N-free buffer and dosed with glucose. Total heat production was measured by calorimetry. Energy spilling was calculated as heat production not accounted by (i) endogenous metabolism (heat production before dosing glucose) and (ii) synthesis of reserve carbohydrate (heat from synthesis itself and reactions yielding ATP for it). For cells dosed with 5 mM glucose, synthesis of reserve carbohydrate and endogenous metabolism accounted for nearly all heat production (93.7%); no spilling was detected (P = 0.226). For cells dosed with 20 mM glucose, energy spilling was not detected immediately after dosing, but it became significant (P < 0.05) by approximately 30 min after dosing with glucose. Energy spilling accounted for as much as 38.7% of heat production in one incubation. Nearly all energy (97.9%) and carbon (99.9%) in glucose were recovered in reserve carbohydrate, fermentation acids, CO2, CH4, and heat. This full recovery indicates that products were measured completely and that spilling was not a methodological artifact. These results should aid future research aiming to mechanistically account for variation in energetic efficiency of mixed microbial communities.

Recent reports in North America and Europe of Clostridium difficile being isolated from livestock and retail meats of bovine origin have raised concerns about the risk to public health. To assess the situation in Australia, we investigated the prevalence and genetic diversity of C. difficile in adult cattle and calves at slaughter. Carcass washings, gastrointestinal contents, and feces were collected from abattoirs across five Australian states. Selective culture, toxin profiling, and PCR ribotyping were performed. The prevalence of C. difficile was 56% (203/360 samples) in feces from <7-day-old calves, 3.8% (1/26) in 2- to 6-month-old calves, and 1.8% (5/280) in adult cattle. Three PCR ribotypes (RTs), RT127, RT033, and RT126, predominated in <7-day-old calves and comprised 77.8% (158/203 samples) of isolates. RT056, which has not been reported in cattle before, was found in 16 <7-day-old calves (7.7%). Surprisingly, RT078 strains, which dominate production animal carriage studies in the Northern Hemisphere, were not isolated.

For exhaustive detection of diarrheagenic Escherichia coli, we previously developed a colony-hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR. To assess the role of domestic animals as the source of atypical enteropathogenic E. coli (aEPEC), a total of 679 samples (333 from foods, fecal samples from 227 domestic animals, and 119 from healthy people) were examined. Combining 48 strains previously isolated from patients and carriers, 159 aEPEC strains were classified by phylogroup, virulence profile, and intimin typing. Phylogroup B1 was significantly more prevalent among aEPEC from patients (50%) and bovine samples (79%) than from healthy carriers (16%) and swine strains (23%), respectively. Intimin type β1 was predominant in phylogroup B1; B1-β1 strains comprised 26% of bovine strains and 25% of patient strains. The virulence profile groups Ia and Ib were also observed more frequently among bovine strains than among porcine strains. Similarly, virulence group Ia was detected more frequently among patient strains than strains of healthy carriers. A total of 85 strains belonged to virulence group I, and 63 of these strains (74%) belonged to phylogroup B1. The present study suggests that the etiologically important aEPEC in diarrheal patients could be distinguished from aEPEC strains indigenous to humans based on type, such as B1, Ia, and β1/γ1, which are shared with bovine strains, while the aEPEC strains in healthy humans are different, and some of these were also present in porcine samples.

Dairy cows have been identified as common carriers of Campylobacter jejuni, which causes many of the human gastroenteritis cases reported worldwide. To design on-farm management practices that control the human infection sourced from dairy cows, the first step is to acquire an understanding of the excretion patterns of the cow reservoir. We monitored the same 35 cows from two dairy farms for C. jejuni excretion fortnightly for up to 12 months. The objective was to examine the concentration of C. jejuni and assess the genetic relationship of the C. jejuni populations excreted by individual cows. Significant differences (P < 0.01) in C. jejuni fecal concentration were observed among the 35 cows, with median concentrations that varied by up to 3.6 log(10) · g(-1) feces. A total of 36 different genotypes were identified from the 514 positive samples by using enterobacterial repetitive intergenic consensus (ERIC)-PCR. Although 22 of these genotypes were excreted by more than one cow, the analysis of frequencies and distribution of the genotypes by model-based statistics revealed a high degree of individuality in the C. jejuni population in each cow. The observed variation in the frequency of excretion of a genotype among cows and the analysis by multilocus sequence typing (MLST) of these genotypes suggest that excretion of C. jejuni in high numbers is due to a successful adaptation of a particular genotype to a particular cow's gut environment, but that animal-related factors render some individual cows resistant to colonization by particular genotypes. The reasons for differences in C. jejuni colonization of animals warrant further investigation.

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.

Staphylococcus aureus is a major agent of bovine mastitis. The concomitant emergence of pig-associated methicillin-resistant S. aureus (MRSA) in human carriage and infection requires a reexamination of the host range and specificity of human- and cow-associated S. aureus strains, something which has not been systematically studied previously. The genetic relatedness of 500 S. aureus isolates from bovine mastitis cases, 57 isolates from nasal carriage of farmers, and 133 isolates from nonfarmers was determined by amplified fragment length polymorphism (AFLP) analysis and spa typing. Multilocus sequence typing (MLST) was conducted on a subset of isolates to match AFLP clusters with MLST clonal complexes (CCs). This data set allowed us to study host range and host specificity and to estimate the extent of bovine-to-human transmission. The genotype compositions of S. aureus isolates from farmers and nonfarmers were very similar, while the mastitis isolates were quite distinct. Overall, transmission was low, but specific genotypes did show increased cow-to-human transmission. Unexpectedly, more than one-third of mastitis isolates belonged to CC8, a lineage which has not been considered to be bovine mastitis associated, but it is well known from human carriage and infection (i.e., USA300). Despite the fact that we did detect some transmission of other genotypes from cows to farmers, no transmission of CC8 isolates to farmers was detected, except for one tentative case. This was despite the close genetic relatedness of mastitis CC8 strains to nonfarmer carriage strains. These results suggest that the emergence of the new bovine-adapted genotype was due to a recent host shift from humans to cows concurrent with a loss of the ability to colonize humans. More broadly, our results indicate that host specificity is a lineage-specific trait that can rapidly evolve.

Bovine Bacillus anthracis isolates from Cameroon were genetically characterized. They showed a strong homogeneity, and they belong, together with strains from Chad, to cluster Aβ, which appears to be predominant in western Africa. However, one strain that belongs to a newly defined clade (D) and cluster (D1) is penicillin resistant and shows certain phenotypes typical of Bacillus cereus.

To longitudinally assess fecal shedding and animal-to-animal transmission of Clostridium difficile among finishing feedlot cattle as a risk for beef carcass contamination, we tested 186 ± 12 steers (mean ± standard deviation; 1,369 samples) in an experimental feedlot facility during the finishing period and at harvest. Clostridium difficile was isolated from 12.9% of steers on arrival (24/186; 0 to 33% among five suppliers). Shedding decreased to undetectable levels a week later (0%; P < 0.001), and remained low (< 3.6%) until immediately prior to shipment for harvest (1.2%). Antimicrobial use did not increase fecal shedding, despite treatment of 53% of animals for signs of respiratory disease. Animals shedding C. difficile on arrival, however, had 4.6 times higher odds of receiving antimicrobials for respiratory signs than nonshedders (95% confidence interval for the odds ratio, 1.4 to 14.8; P = 0.01). Neither the toxin genes nor toxin A or B was detected in most (39/42) isolates based on two complementary multiplex PCRs and enzyme-linked immunosorbent assay testing, respectively. Two linezolid- and clindamycin-resistant PCR ribotype 078 (tcdA+/tcdB+/cdtB+/39-bp-type deletion in tcdC) isolates were identified from two steers (at arrival and week 20), but these ribotypes did not become endemic. The other toxigenic isolate (tcdA+/tcdB+/cdtB+/classic tcdC; PCR ribotype 078-like) was identified in the cecum of one steer at harvest. Spatio-temporal analysis indicated transient shedding with no evidence of animal-to-animal transmission. The association between C. difficile shedding upon arrival and the subsequent need for antimicrobials for respiratory disease might indicate common predisposing factors. The isolation of toxigenic C. difficile from bovine intestines at harvest highlights the potential for food contamination in meat processing plants.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.

Campylobacter jejuni can be isolated from different animal hosts. Various studies have used multilocus sequence typing to look for associations between particular clones of C. jejuni and specific hosts. Here, we describe the isolation of a novel clone (sequence type 3704 [ST-3704]) of C. jejuni associated with the bank vole (Myodes glareolus).

Our objectives were to evaluate the prevalence of Escherichia coli O157:H7 in cattle fed diets supplemented with 20 or 40% dried distillers' grains (DG) (DDG) or wet DG (WDG) and assess whether removing DG from diets before slaughter affected fecal shedding of E. coli O157:H7. Eight hundred forty steers were allocated to 70 pens (12 steers/pen). Treatments were no DG (control), 20% DDG or WDG, and 40% DDG or WDG, and each was replicated in 14 pens. In phase 1, eight floor fecal samples were collected from each pen every 2 weeks for 12 weeks for isolation of E. coli O157:H7 and detection of high shedders. In phase 2, half of the pens with DG were transitioned to the no-DG control diet, and pen floor fecal samples were collected weekly from all pens for 4 weeks. During phase 1, prevalence of E. coli O157:H7 was 20.8% and 3.2% for high shedders. The form of DG had no significant effect on fecal E. coli O157:H7 shedding. The prevalence levels of E. coli O157:H7 and the numbers of high shedders were not different between diets with 0 or 20% DG; however, cattle fed 40% DG had a higher prevalence and more high shedders than cattle fed 0 or 20% DG (P ≤ 0.05). During phase 2, overall and high-shedder prevalence estimates were 3.3% and <0.1%, respectively, and there were no differences between those for different DG forms and inclusion levels or when DG was removed from diets. The form of DG had no impact on E. coli O157:H7; however, fecal shedding was associated with the DG inclusion level.

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10(10) CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10(11) PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.

One clone encoding glycoside hydrolases was identified through functional screening of a rumen bacterial artificial chromosome (BAC) library. Of the 68 open reading frames (ORFs) predicted, one ORF encodes a novel endo-β-1,4-xylanase with two catalytic domains of family GH43 and two cellulose-binding modules (CBMs) of family IV. Partial characterization showed that this endo-xylanase has a greater specific activity than a number of other xylanases over a wide temperature range at neutral pH and could be useful in some industrial applications.

The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P = 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits.

Shiga-toxigenic Escherichia coli (STEC) O157 occurrence was determined along the entire gastrointestinal tract (GIT) of each of four naturally shedding cattle and at three sites in 61 slaughter cattle. STEC O157 was distributed along the entire GIT, though interanimal distribution was variable. Neither feces nor rectoanal-junction samples accurately predicted the STEC O157-negative status of any particular animal.

Following the January 2006 European ban of antibiotics used as growth promoters in the veal calf industry, new feed additives are needed in order to maintain animal health and growth performance. As an alternative to in vivo experiments in the testing of such additives, an in vitro system modeling the intestinal ecosystem of the veal calf was developed. Stabilization of the main cultured microbial groups and their metabolic activity were tracked in an in vitro continuous fermentor operated under anaerobiosis, at pH 6.5, and at a temperature of 38.5 degrees C and supplied with one of three different nutritive media (M1, M2, or M3). These media mainly differed in their concentrations of simple and complex carbohydrates and in their lipid sources. In vitro microbial levels and fermentative metabolite concentrations were compared to in vivo data, and the biochemical composition of the nutritive media was compared to that of the veal calf intestinal content. All three nutritive media were able to stabilize anaerobic and facultative anaerobic microflora, lactate-utilizing bacteria, bifidobacteria, lactobacilli, enterococci, and Bacteroides fragilis group bacteria at levels close to in vivo values. The microbiota was metabolically active, with high concentrations of lactate, ammonia, and short-chain fatty acids found in the fermentative medium. Comparison with in vivo data indicated that M3 outperformed M1 and M2 in simulating the conditions encountered in the veal calf intestine. This in vitro system would be useful in the prescreening of new feed additives by studying their effect on the intestinal microbiota levels and fermentative metabolite production.

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.

Homoacetogens produce acetate from H(2) and CO(2) via the Wood-Ljungdahl pathway. Some homoacetogens have been isolated from the rumen, but these organisms are expected to be only part of the full diversity present. To survey the presence of rumen homoacetogens, we analyzed sequences of formyltetrahydrofolate synthetase (FTHFS), a key enzyme of the Wood-Ljungdahl pathway. A total of 275 partial sequences of genes encoding FTHFS were PCR amplified from rumen contents of a cow, two sheep, and a deer. Phylogenetic trees were constructed using these FTHFS gene sequences and the translated amino acid sequences, together with other sequences from public databases and from novel nonhomoacetogenic bacteria isolated from the rumen. Over 90% of the FTHFS sequences fell into 34 clusters defined with good bootstrap support. Few rumen-derived FTHFS sequences clustered with sequences of known homoacetogens. Conserved residues were identified in the deduced FTHFS amino acid sequences from known homoacetogens, and their presence in the other sequences was used to determine a "homoacetogen similarity" (HS) score. A homoacetogen FTHFS profile hidden Markov model (HoF-HMM) was used to assess the homology of rumen and homoacetogen FTHFS sequences. Many clusters had low HS scores and HoF-HMM matches, raising doubts about whether the sequences originated from homoacetogens. In keeping with these findings, FTHFS sequences from nonhomoacetogenic bacterial isolates grouped in these clusters with low scores. However, sequences that formed 10 clusters containing no known isolates but representing 15% of our FTHFS sequences from rumen samples had high HS scores and HoF-HMM matches and so could represent novel homoacetogens.

The persistence of naturally occurring campylobacteria in aerobic compost constructed of manure from beef cattle that were administered chlortetracycline and sulfamethazine (AS700) or from cattle not administered antibiotics (control) was examined. Although there were no differences in population sizes of heterotrophic bacteria, the temperature of AS700 compost was more variable and did not become as high as that of control compost. There were significant differences in water content, total carbon (C), total nitrogen (N), and electrical conductivity but not in the C/N ratio or pH between the two compost treatments. Campylobacteria were readily isolated from pen manure, for up to day 15 from control compost, and throughout the active phase of AS700 compost. Campylobacter DNA (including Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis, and Campylobacter jejuni) was detected over the ca. 10-month composting period, and no reductions in quantities of C. jejuni DNA were observed over the duration of the active phase. The utilization of centrifugation in combination with ethidium monoazide (EMA) significantly reduced (>90%) the amplification of C. jejuni DNA that did not originate from cells with intact cell membranes. No differences were observed in the frequency of Campylobacter DNA detection between EMA- and non-EMA-treated samples, suggesting that Campylobacter DNA amplified from compost was extracted from cells with intact cell membranes (i.e., from viable cells). The findings of this study indicate that campylobacteria excreted in cattle feces persist for long periods in compost and call into question the common belief that these bacteria do not persist in manure.

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.

The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>10(4) CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (>/=40 CFU/100 cm(2)) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (>/=200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.

We determined the prevalence of Escherichia coli O157:H7 in organically and naturally raised beef cattle at slaughter and compared antibiotic susceptibility profiles of the isolates to those of isolates from conventionally raised beef cattle. The prevalences of E. coli O157:H7 were 14.8 and 14.2% for organically and naturally raised cattle, respectively. No major difference in antibiotic susceptibility patterns among the isolates was observed.

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources.