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In ruminants, the rumen is a specialized stomach that is adapted to the breakdown of plant-derived complex polysaccharides through the coordinated activities of a diverse microbial community. Bacteroidota is a major phylum in this bovine rumen microbiota. They contain several clusters of genes called polysaccharide utilization loci (PULs) that encode proteins working in concert to capture, degrade, and transport polysaccharides. Despite the critical role of SusD-like proteins for efficient substrate transport, they remain largely unexplored. Here, we present the biochemical characterization of a SusD-like protein encoded by a β-glucan utilization locus from an Escherichia coli metagenomic clone previously isolated by functional screening of the bovine rumen microbiome. In this study, we show that clone 41O1 can grow on laminaritriose, cellotriose, and a mixture of cellobiosyl-cellobiose and glucosyl-cellotriose as sole carbon sources. Based on this, we used various in vitro analyses to investigate the binding ability of 41O1_SusD-like towards these oligosaccharides and the corresponding polysaccharides. We observed a clear binding affinity for β-1,6 branched β-1,3-glucans (laminarins, yeast β-glucan) and laminaritriose. Comparison of the AlphaFold2 model of 41O1_SusD-like with its closest structural homologs highlights a similar pattern of substrate recognition. In particular, three tryptophan residues are shown to be crucial for laminarin recognition. In the context of the cow rumen, we discuss the possible substrates targeted by the 41O1_PUL, such as the (1,3;1,4)-β-d-glucans present in cereal grains or the β-1,3- and (1,3;1,6)-β-d-glucans that are components of the cell wall of ruminal yeasts.IMPORTANCEThe rumen microbiota can majorly impact overall animal health, feed efficiency, and release of harmful substances into the environment. This microbiota is involved in the fermentation of organic matter to provide the host with valuable and assimilable nutrients. Bacteroidota efficiently captures, breaks down, and imports complex polysaccharides through the concerted action of proteins encoded by polysaccharide utilization loci (PULs). Within this system, SusD-like protein has proven necessary for the active internalization of the substrate. Nevertheless, the vast majority of SusD-like proteins characterized to date originate from cultured bacteria. With regard to the diversity and importance of uncultured bacteria in the rumen, further studies are required to better understand the role of polysaccharide utilization loci in ruminal polysaccharide degradation. Our detailed characterization of the 41O1_SusD-like therefore contributes to a better understanding of the carbohydrate metabolism of an uncultured Bacteroides from the cow rumen., Competing Interests: The authors declare no conflict of interest.

The development and growth of animals coincide with the establishment and maturation of their microbiotas. To evaluate the respiratory and fecal microbiotas of beef calves from birth to weaning, a total of 30 pregnant cows, and their calves at birth, were enrolled in this study. Deep nasal swabs and feces were collected from calves longitudinally, starting on the day of birth and ending on the day of weaning. Nasopharyngeal, vaginal, and fecal samples were also collected from cows, and the microbiotas of all samples were analyzed. The fecal microbiota of calves was enriched with Lactobacillus during the first 8 weeks of life, before being displaced by genera associated with fiber digestion, and then increasing in diversity across time. In contrast, the diversity of calf respiratory microbiota generally decreased with age. At birth, the calf and cow nasal microbiotas were highly similar, indicating colonization from dam contact. This was supported by microbial source-tracking analysis. The structure of the calf nasal microbiota remained similar to that of the cows, until weaning, when it diverged. The changes were driven by a decrease in Lactobacillus and an increase in genera typically associated with bovine respiratory disease, including Mannheimia , Pasteurella , and Mycoplasma . These three genera colonized calves early in life, though Mannheimia was initially transferred from the cow reproductive tract. Path analysis was used to model the interrelationships of calf respiratory and fecal microbiotas. It was observed that respiratory Lactobacillus and fecal Oscillospiraceae UCG-005 negatively affected the abundance of Mannheimia or Pasteurella .IMPORTANCEIn beef cattle production, bovine respiratory disease (BRD) accounts for most of the feedlot morbidities and mortalities. Metaphylaxis is a common management tool to mitigate BRD, however its use has led to increased antimicrobial resistance. Novel methods to mitigate BRD are needed, including microbiota-based strategies. However, information on the respiratory bacteria of beef calves prior to weaning was limited. In this study, it was shown that the microbiota of cows influenced the initial composition of both respiratory and fecal microbiotas in calves. While colonization of the respiratory tract of calves by BRD-associated genera occurred early in life, their relative abundances increased at weaning, and were negatively correlated with respiratory and gut bacteria. Thus, microbiotas of both the respiratory and gastrointestinal tracts have important roles in antagonism of respiratory pathogens and are potential targets for enhancing calf respiratory health. Modulation may be most beneficial, if done prior to weaning, before opportunistic pathogens establish colonization., Competing Interests: The authors declare no conflict of interest.

Shiga toxin-producing Escherichia coli (STEC) strains can colonize cattle for several months and may, thus, serve as gene reservoirs for the genesis of highly virulent zoonotic enterohemorrhagic E. coli (EHEC). Attempts to reduce the human risk for acquiring EHEC infections should include strategies to control such STEC strains persisting in cattle. We therefore aimed to identify genetic patterns associated with the STEC colonization type in the bovine host. We included 88 persistent colonizing STEC (STECper) (shedding for>4 months) and 74 sporadically colonizing STEC (STECspo) (shedding for≤2 months) isolates from cattle and 16 bovine STEC isolates with unknown colonization types. Genoserotypes and multilocus sequence types (MLSTs) were determined, and the isolates were probed with a DNA microarray for virulence-associated genes (VAGs). All STECper isolates belonged to only four genoserotypes (O26:H11, O156:H25, O165:H25, O182:H25), which formed three genetic clusters (ST21/396/1705, ST300/688, ST119). In contrast, STECspo isolates were scattered among 28 genoserotypes and 30 MLSTs, with O157:H7 (ST11) and O6:H49 (ST1079) being the most prevalent. The microarray analysis identified 139 unique gene patterns that clustered with the genoserotypes and MLSTs of the strains. While the STECper isolates possessed heterogeneous phylogenetic backgrounds, the accessory genome clustered these isolates together, separating them from the STECspo isolates. Given the vast genetic heterogeneity of bovine STEC strains, defining the genetic patterns distinguishing STECper from STECspo isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level. [ABSTRACT FROM AUTHOR]

This study investigated fecal specimens from 489 sheep and 537 cattle in multiple cities in northeast China for the prevalence and genetic characteristics of Enterocytozoon bieneusi by PCR and sequencing of the ribosomal internal transcribed spacer. Sixty-eight sheep specimens (13.9%) and 32 cattle specimens (6.0%) were positive for E. bieneusi. Sequence polymorphisms enabled the identification of 9 known genotypes (BEB4, BEB6, CM7, CS-4, EbpC, G, I, J, and OEB1) and 11 new genotypes (NESH1 to NESH6 and NECA1 to NECA5). The genotypes formed two genetic clusters in a phylogenetic analysis, with CS-4, EbpC, G, NESH1 to NESH3, and NECA1 to NECA5 distributed in zoonotic group 1 and BEB4, BEB6, CM7, EbpI, J, OEB1, and NESH4 to NESH6 distributed in potentially host-adapted group 2. Nearly 70% of cases of E. bieneusi infections in sheep were contributed by human-pathogenic genotypes BEB6, CS-4, and EbpC, and over 80% of those in cattle were by genotypes BEB4, CS-4, EbpC, I, and J. The cooccurrence of genotypes BEB4, CS-4, EbpC, I, and J in domestic ruminants and children in northeast China and the identification of BEB6 and EbpC in humans and water in central China imply the possibility of zoonotic transmission. This study also summarizes E. bieneusi genotypes obtained from ruminants worldwide and displays their host ranges, geographical distributions, and phylogenetic relationships. The data suggest a host range expansion in some group 2 genotypes (notably BEB4, BEB6, I, and J) that were previously considered to be adapted to ruminants. We should be concerned about the increasing zoonotic importance of group 2 genotypes with low host specificity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The qseBC-encoded quorum-sensing system regulates the motility of Escherichia coli O157:H7 in response to bacterial autoinducer 3 (AI-3) and the mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that autophosphorylates in response to AI-3, E, or NE and subsequently phosphorylates its cognate response regulator QseB. In the absence of QseC, QseB downregulates bacterial motility and virulence in animal models. In this study, we found that 8- to 10-month-old calves orally inoculated with a mixture of E. coli O157:H7 and its isogenic qseBC mutant showed significantly higher fecal shedding of the qseBC mutant. In vitro analysis revealed similar growth profiles and motilities of the qseBC mutant and the parental strain in the presence or absence of NE. The magnitudes of the response to NE and expression of flagellar genes flhD and fliC were also similar for the qseBC mutant and the parental strain. The expression of ler (a positive regulator of the locus of enterocyte effacement [LEE]), the ler-regulated espA gene, and the csgA gene (encoding curli fimbriae) was increased in the qseBC mutant compared to the parental strain. On the other hand, growth, motility, and transcription of flhD, fliC, ler, espA, and csgA were significantly reduced in the qseBC mutant complemented with a plasmid-cloned copy of the qseBC genes. Thus, in vitro motility and gene expression data indicate that the near-parental level of motility, ability to respond to NE, and enhanced expression of LEE and curli genes might in part be responsible for increased colonization and fecal shedding of the qseBC mutant in calves. [ABSTRACT FROM AUTHOR]

The diversity of protozoan-associated methanogens in cattle was investigated using five universal archaeal small-subunit (SSU) rRNA gene primer sets. Methanobrevibacter spp. and rumen cluster C (distantly related to Thermoplasma spp.) were predominant. Significant differences in species composition among libraries indicate that some primers used previously to characterize rumen methanogens exhibit biased amplification. [ABSTRACT FROM AUTHOR]

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT1O4) or U302. The cluster! isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VI! isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfon- amides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. !n the 19 isolates representative of cluster VH, the bIaTEM1 gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the blaTEM1-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan. [ABSTRACT FROM AUTHOR]

Shiga-toxigenic Escherichia coli (STEC) 0157 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 0157 was distributed along the entire GIT, though interanimal distribution was variable. Neither feces nor rectoanaljunction samples accurately predicted the STEC 0157-negative status of any particular animal. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cattle with high feed efficiencies (designated "efficient") produce less methane gas than those with low feed efficiencies (designated "inefficient"); however, the role of the methane producers in such difference is unknown. This study investigated whether the structures and populations of methanogens in the rumen were associated with differences in cattle feed efficiencies by using culture-independent methods. Two 16S rRNA libraries were constructed using ∼800-bp amplicons generated from pooled total DNA isolated from efficient (n = 29) and inefficient (n = 29) animals. Sequence analysis of up to 490 randomly selected clones from each library showed that the methanogenic composition was variable: less species variation (22 operational taxonomic units [OTUs]) was detected in the rumens of efficient animals, compared to 27 OTUs in inefficient animals. The methanogenic communities in inefficient animals were more diverse than those in efficient ones, as revealed by the diversity indices of 0.84 and 0.42, respectively. Differences at the strain and genotype levels were also observed and found to be associated with feed efficiency in the host. No difference was detected in the total population of methanogens, but the prevalences of Methanosphaera stadtmanae and Methanobrevibacter sp. strain AbM4 were 1.92 (P < 0.05) and 2.26 (P < 0.05) times higher in inefficient animals, while Methanobrevibacter sp. strain AbM4 was reported for the first time to occur in the bovine rumen. Our data indicate that the methanogenic ecology at the species, strain, and/or genotype level in the rumen may play important roles in contributing to the difference in methane gas production between cattle with different feed efficiencies. [ABSTRACT FROM AUTHOR]

Examines the role of cattle water troughs as environmental reservoirs of Escherichia coli O157:H7. Ability to infect cattle after phenotypic, metabolic and genetic changes; Effect of water chlorination on survival of coliform in previously contaminated water troughs; Temporal pattern of fecal excretion of coliform.

Investigates the glucose transport by mixed ruminal bacteria from a cow. Transport systems; Dominance of the high-affinity, low velocity transport system in the feeding condition; Non-involvement of the proton motive force in the transport system; Phosphoenolpyruvate-dependent phosphotransferase system as the main system; Inhibition of the high-affinity system by sucrose, mannose and fructose.

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.)

Genomic analyses were performed on florfenicol-resistant (FFN r ) Campylobacter coli isolates recovered from cattle, and the cfr (C) gene-associated multidrug resistance (MDR) plasmid was characterized. Sixteen FFN r C. coli isolates recovered between 2013 and 2018 from beef cattle were sequenced using MiSeq. Genomes and plasmids were found to be closed for three of the isolates using the PacBio system. Single nucleotide polymorphisms (SNPs) across the genome and the structures of MDR plasmids were investigated. Conjugation experiments were performed to determine the transferability of cfr (C)-associated MDR plasmids. The spectrum of resistance encoded by the cfr (C) gene was further investigated by agar dilution antimicrobial susceptibility testing. All 16 FFN r isolates were MDR and exhibited coresistance to ciprofloxacin, nalidixic acid, clindamycin, and tetracycline. All isolates shared the same resistance genotype, carrying aph (3')-III , hph , ΔaadE (truncated), bla OXA-61 , cfr (C), and tet (O) genes plus a mutation of GyrA (T86I). The cfr (C), aph (3')-III , hph , ΔaadE , and tet (O) genes were colocated on transferable MDR plasmids ranging in size from 48 to 50 kb. These plasmids showed high sequence homology with the pTet plasmid and carried several Campylobacter virulence genes, including virB2 , virB4 , virB5 , VirB6 , virB7 , virB8 , virb9 , virB10 , virB11 , and virD4 The cfr (C) gene conferred resistance to florfenicol (8 to 32 μg/ml), clindamycin (512 to 1,024 μg/ml), linezolid (128 to 512 μg/ml), and tiamulin (1,024 μg/ml). Phylogenetic analysis showed SNP differences ranging from 11 to 2,248 SNPs among the 16 isolates. The results showed that the cfr (C) gene located in the conjugative pTet MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to several antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. This report highlights the power of genomic antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies. IMPORTANCE Campylobacter is a leading cause of foodborne diarrheal illness worldwide, with more than one million cases each year in the United States alone. The global emergence of antimicrobial resistance in this pathogen has become a growing public health concern. Florfenicol-resistant (FFN r ) Campylobacter has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter coli isolates recovered from cattle in the United States. A gene [ cfr (C)] was found to be responsible for resistance not only to florfenicol but also to several other antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. The results showed that cfr (C) is located in a conjugative pTet MDR/virulence plasmid. This report highlights the power of antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.

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.)

Mycoplasma wenyonii is a hemotrophic mycoplasma that causes acute and chronic infections in cattle. Here, we announce the first complete genome sequence of this organism. The genome is a single circular chromosome with 650,228 bp and G+C% of 33.9. Analyses of M. wenyonii genome will provide insights into its biology. [ABSTRACT FROM AUTHOR]

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 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.)

Milk samples from dairy cows provide a ready source of material for measuring antibody responses to Mycobacterium bovis antigens. In this study, we evaluated the IDEXX enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody responses to M. bovis antigens MPB70 and MPB83 in milk samples from New Zealand cattle. Test sensitivities for individual milk and serum samples were assessed in samples collected from 44 M. bovis-infected cows, and test specificities were assessed in milk samples collected from 356 cows from tuberculosis (TB)-free herds. Milk vat samples were collected from 505 herds from regions with relatively high or low prevalences of infection. The ELISA had a sensitivity of 50% and a specificity of 97.5% for milk samples, and the test sensitivities for milk and serum samples were the same. Dilution of the positive test milk samples in milk from noninfected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Small differences were observed in the ELISA responses of milk samples from individual TB-free cows collected at different times during lactation. No significant differences were detected in the ELISA responses of milk vat samples collected from infected and noninfected herds. This study shows that milk samples can be substituted for serum samples for screening individual cows for M. bovis infection, and pooling of milk samples from 10 to 20 animals can result in a reduction in the sensitivity by approximately 50%. However, screening of milk vat samples is unlikely to be useful in countries with low prevalences of M. bovis in cattle and large herd sizes.

Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n = 5) and uninfected cows (n = 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease.

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.

Oscillibacter ruminantium strain GH1 was isolated from the rumen of Korean native cattle (HanWoo; Bos taurus coreanae). Here, we present the 3.07-Mb draft genome of this strain, which could reveal the presence of certain fiber-specific glycoside hydrolases and butyric acid-producing genes.

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.

An enteric bacterium, Escherichia coli W26 (KACC 16630), was isolated from feces from a healthy cow in South Korea. Here, we report the draft genome sequence of the isolate, which is closely affiliated with commensal strains belonging to E. coli phylogroup B1.

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1β was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.