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BackgroundAntimicrobial resistance (AMR) is a significant global public health concern associated with millions of deaths annually. Agriculture has been attributed as a leading factor in AMR and multidrug resistance (MDR) associated with swine production estimated as one of the largest agricultural consumers of antibiotics. Therefore, studying and understanding AMR in swine has global relevance. AMR research has received increased attention in recent years. However, we are still building our understanding of genetic variation within a complex gut microbiome system that impacts AMR and MDR. In order to evaluate the gut resistome, we evaluated genetic variation before, during, and after antibiotic treatments. We studied three treatment groups: non-antibiotic controls (C), chlortetracycline (CTC) treated, and tiamulin (TMU) treated. We collected fecal samples from each group and performed metagenomic sequencing for a longitudinal analysis of genetic variation and functions.ResultsWe generated 772,688,506 reads and 81 metagenome assembled genomes (MAGs). Interestingly, we identified a subset of 11 MAGs with sustained detection and high sustained entropy (SDHSE). Entropy described genetic variation throughout the MAG. Our SDHSE MAGs were considered MDR as they were identified prior to, throughout, and after CTC and TMU treatments as well as in the C piglets. SDHSE MAGs were especially concerning as they harbored relatively high variation. Consistently high variation indicated that these microbial populations may contain hypermutable elements which has been associated with increased chance of AMR and MDR acquisition. Our SDHSE MAGs demonstrated that MDR organisms (MDRO) are present in swine, and likely additional hosts contributing to global AMR. Altogether, our study provides comprehensive genetic support of MDR populations within the gut microbiome of swine.

Liver abscesses in feedlot cattle are a polymicrobial infection with Fusobacterium necrophorum and Trueperella pyogenes as the primary and secondary etiologic agents, respectively. Cattle with liver abscesses do not exhibit clinical signs and the abscesses are detected only at slaughter. The objective was to conduct metabolomics analysis of purulent materials of liver abscesses to identify biochemicals. Liver abscesses from crossbred cattle (n = 24) and Holstein steers (n = 24), each fed high-grain finishing diet with tylosin (n = 12) or no tylosin (n = 12), were included in the study. Abscess purulent materials were analyzed by ultrahigh-performance liquid chromatography-tandem mass spectroscopy. A total of 759 biochemicals were identified and were broadly categorized into carbohydrates, energy metabolism pathways intermediates, peptides, amino acids and their metabolites, lipids and their metabolites, nucleotides, vitamins and cofactors, xenobiotics, and partially characterized molecules. The top 50 biochemicals identified included amino acids, lipids, nucleotides, xenobiotics, peptides, and carbohydrates and their metabolites. Among the 15 amino acid metabolites in the top 50 biochemicals, four were tryptophan metabolites, indoleacrylate, indolepropionate, tryptamine, and anthranilate. The 3-phenylpropionate, a product of phenylalanine metabolism, was the predominant metabolite in purulent materials. Between the four treatment groups, a two-way ANOVA analysis identified biochemicals that exhibited significant main effects for cattle type and in-feed tylosin use and their interactions. A total of 59 and 85 biochemicals were different (P < 0.05) between the cattle type (crossbred vs. Holstein steers) and in-feed tylosin use (tylosin vs. no tylosin), respectively. Succinate, an intermediate of lactate fermentation by some bacterial species, was one of the top 30 biochemicals that differentiated the four treatment groups. A number of lysophospholipids, indicative of bacterial and host cell membrane lyses, were identified in the purulent materials. In conclusion, to our knowledge this is the first report on the metabolome of liver abscess purulent materials and several biochemicals identified were related to metabolic activities of the bacterial community, particularly F. necrophorum and T. pyogenes. Biochemicals unique to liver abscesses that appear in the blood may serve as biomarkers and be of diagnostic value to detect liver abscesses of cattle before slaughter.

Monensin and virginiamycin are included in beef cattle finishing diets as prophylaxis to minimize the incidence of ruminal acidosis and liver abscesses. Due to different and probably complementary modes of action, this study aimed to determine the effects of a combination of monensin and virginiamycin, both included in the diet at recommended doses, on ruminal health, the occurrence of liver abscesses, and growth performance of feedlot-finished cattle. One hundred and forty-four steers (6 animals/pen) were fed 1 of 3 corn-based finishing diets containing 30 mg of monensin (MN), 25 mg of virginiamycin (VM), or 30 and 25 mg of monensin and virginiamycin (MN + VM), respectively, per kilogram of dry matter. Ruminal pH probes were inserted into two animals per pen and set to record pH every 10 min. On d 100, animals were slaughtered, and rumens and livers were recovered, on which occurrence and degree of ruminal damage, prevalence and number of liver abscesses, and liver scores (A−: livers with no more than two small abscesses; A+: livers with at least one large abscess or more than four medium abscesses; A: any other abscessed liver) were determined. Simultaneous inclusion of monensin and virginiamycin resulted in a 4.3% decrease (P < 0.04) in dry matter intake (DMI; 8.8, 9.2, and 9.2 ± 0.19 kg/d for MN + VM, MN, and VM-fed animals, respectively) and similar (P > 0.13) average daily body weight gain (ADG; 1.49 ± 0.021 kg/d) and hot carcass weight (HCW; 269 ± 1.7 kg), compared with feeding diets containing one additive or the other. Therefore, in terms of ADG, a 9.4% improvement (P < 0.01) in feed efficiency was observed in MN + VM-fed animals. Backfat thickness (5.6 ± 0.08 mm) and ribeye area (69.9 ± 0.53 cm2) remained unaffected (P ≥ 0.74), as well as the minimum (4.98 ± 0.047), mean (6.11 ± 0.037), and maximum ruminal pH (7.23 ± 0.033) values and the time (125 ± 22.3 min/d), area (57.67 ± 12.383 pH × h), and episodes (22 ± 3.8 bouts) of pH below 5.6 (P ≥ 0.12). Overall, prevalence (24 ± 3.4%) and the number of liver abscesses (1.6 ± 0.14 abscesses/abscessed liver), liver scores (20 ± 3.1% of A− and 4 ± 1.8% of A livers), and prevalence (67 ± 3.5%) and degree of damage to the ruminal epithelium (2.5 ± 0.22% affected surface) were similar (P ≥ 0.18) across treatments; however, the occurrence of ruminal lesions tended (P ≤ 0.07) to be associated with that of liver abscesses and reduced ADG when feeding monensin alone.

Age and diet are among the factors that influence the community composition of the fecal microbiome. Additionally, antimicrobial use can alter the composition of bacterial communities. An 86-d study with finisher pigs aimed to evaluate age-related dynamics (day 98 to 177 of age), effects of types and levels of dietary fiber, and injectable antimicrobials on the fecal microbiome and antimicrobial resistance (AMR) was conducted. A total of 287 pigs, housed in 36 pens, with 7 to 8 pigs per pen, fed a corn grain and soybean meal-based basal diet, formulated to contain 8.7% neutral detergent fiber (NDF), were randomly assigned to one of three treatments: 1) basal diet with no supplement, 2) basal diet supplemented with 20% distillers dried grains with solubles (DDGS) formulated to contain 13.6% NDF, or 3) basal diet supplemented with 14.5% sugar beet pulp (SBP) formulated to contain 13.6% NDF. Five finisher pigs from each treatment group were selected randomly, and fecal samples were collected on days 98, 110, 144, and 177 of age. In addition, fecal samples were collected from pigs that were injected intramuscularly ceftiofur hydrochloride or penicillin G on days 1 and 3 along with pen-mate-untreated controls on day 1. Fecal samples were subjected to 16S rRNA amplicon-based microbiome analysis and culture methods to quantify the abundance of total AMR coliforms and enterococci populations. The alpha-diversity, such as species richness, increased with age, and the overall bacterial composition changed with age (P =0.001) and diet (P = 0.001). Diet-associated shifts in the specific bacterial taxa were observed. The richness, diversity, and evenness of bacterial taxa did not differ between pigs that were injected with ceftiofur vs. their untreated pen mates or by dietary treatments but differed in pigs that received penicillin G injection. Both antimicrobial treatments contributed to changes in the overall fecal bacterial composition at the genus level. Collectively, the data demonstrate that both age and the diet (control vs. DDGS-, control vs. SBP-, or DDGS- vs. SBP-based diets) were associated with the overall bacterial community composition, and the impact of age on variations in fecal microbiome composition was greater than the diet. Antibiotic treatment had minimal effect on bacterial diversity and relative abundance of taxa. Furthermore, diets and antimicrobial treatment had minimal impact on the overall counts of AMR coliforms and enterococci populations in feces.Bacterial communities in the gut and the feces are strongly influenced by a number of factors, particularly the age of the animal and the diet. In addition, antibiotic administration routinely used to treat bacterial diseases can also affect the community composition. A study with finisher pigs was conducted to evaluate age-related changes, effects of types—distiller’s dried grains with solubles (DGGS) or sugar beet pulp (SBP)—and levels of dietary fiber, and injectable antibiotics on the fecal bacterial composition and antibiotic resistance in fecal bacteria. Fecal samples were collected from five pigs in each of the three dietary treatment groups, control diet with no supplement or supplemented with DDGS or SBP, on days 98, 110, 144, and 177 of age and on days 1 and 3 after the first injection of antibiotics, ceftiofur or penicillin G. Samples were analyzed to identify the bacterial community composition and prevalence of antibiotic resistance in fecal bacteria. Data generated suggested that the overall bacterial composition changed with age and diet, and age appeared to have a greater impact than diet. Antibiotics had only a modest impact on the bacterial community and had minimum impact on antibiotic resistance of fecal bacteria.

Enterococcus faecium is one of the more commonly used bacterial species as a probiotic in animals. The organism, a common inhabitant of the gut of animals and humans, is a major nosocomial pathogen responsible for a variety infections in humans and sporadic infections in animals. In swine and cattle, E. faecium-based probiotic products are used for growth promotion and gut functional and health benefits. The objective of this study was to utilize whole genome sequence-based analysis to assess virulence potential, detect antimicrobial resistance genes, and analyze phylogenetic relationships of E. faecium strains from commercial swine and cattle probiotics. Genomic DNA extracted from E. faecium strains, isolated from commercial probiotic products of swine (n = 9) and cattle (n = 13), were sequenced in an Illumina MiSeq platform and analyzed. Seven of the nine swine strains and seven of the 13 cattle strains were identified as Enterococcus lactis, and not as E. faecium. None of the 22 probiotic strains carried major virulence genes required to initiate infections, but many carried genes involved in adhesion to host cells, which may benefit the probiotic strains to colonize and persist in the gut. Strains also carried genes encoding resistance to a few medically important antibiotics, which included aminoglycosides [aac(6')-Ii, aph(3')-III, ant(6)-Ia], macrolide, lincosamide and streptogramin B (msrC), tetracyclines [tet(L) and tet(M)], and phenicols [cat-(pc194)]. The comparison of the genotypic to phentypic AMR data showed presence of both related and unrelated genes in the probiotic strains. Swine and cattle probiotic E. faecium strains belonged to diverse sequence types. Phylogenetic analysis of the probiotic strains, and strains of human (n = 29), swine (n = 4), and cattle (n = 4) origin, downloaded from GenBank, indicated close clustering of strains belonging to the same species and source, but a few swine and cattle probiotic strains clustered closely with other cattle and human fecal strains. In conclusion, the absence of major virulence genes characteristic of the clinical E. faecium strains suggests that these probiotic strains are unlikely to initiate opportunistic infection. However, the carriage of AMR genes to medically important antibiotics and close clustering of the probiotic strains with other human and cattle fecal strains suggests that probiotic strains may pose risk to serve as a source of transmitting AMR genes to other gut bacteria.Probiotics, also called direct-fed microbials, are widely used in swine and cattle production systems, as an alternative for antibiotics. The benefits of feeding probiotic products include growth promotion and gut functional benefits. One of the more common bacterial species used in swine and cattle commercial probiotic products is Enterococcus faecium. The species is also a member of the normal flora of hindgut of humans and animals. In recent years, the species has emerged as a major hospital-acquired infection in humans, mainly because of the propensity to become resistant to antibiotics. In the United States, the species is considered as generally recognized as safe. In this study, the virulence and antimicrobial resistance genes profiles of 9 and 13 E. faecium strains isolated from commercial swine and cattle probiotics, respectively, were assessed by sequencing the whole genome DNA. The analysis indicated that 14 of 22 strains were Enterococcus lactis, and not E. faecium. The absence of major virulence genes characteristic of the clinical E. faecium strains suggests that the strains are unlikely to initiate opportunistic infection. However, the carriage of genes that confer resistance to medically important antibiotics suggests that probiotic strains may pose risk as a source of antimicrobial resistance genes to other bacteria.

Shiga toxin-producing Escherichia coli (STEC), particularly seven serogroups O26, O45, O103, O111, O121, O145, and O157, are major food borne pathogens. Cattle are a major reservoir in which STEC colonize the hindgut and are shed in the feces. Most cattle shed the organisms at low concentrations and a small proportion, called super shedders, shed at high concentrations (≥ 104 per g). Our objective was to evaluate the effect of a commercial DFM product containing lactic acid and propionic acid bacteria (Microbios Direct LA = 1 x 107 CFU and Direct PRO = 5 x 107 CFU/animal/d). The study was conducted in a commercial feedlot and a total of 16 pens were randomly assigned to control or DFM-treated group. Twenty pen-floor fecal samples were collected from each pen on 3 occasions, at 2-week intervals, approximately 100 d after DFM supplementation. Fecal samples were subjected to culture and PCR methods to quantify E. coli O157 and determine prevalence of the 6 non-O157 serogroups and 4 major virulence genes, Shiga toxins 1 and 2, intimin (an adhesin) and enterohemolysin. Data were analyzed using PROC MIXED procedure with pen as the experimental unit. The treatment means comparison within sampling day were carried out for variables that showed significant treatment and sampling day interaction. The overall fecal shedding of E. coli O157 was higher in the control group than in the DFM group, and the difference was significant in the third sampling day. Among the top-6 STEC, O26 and O103 were the dominant serogroups. The prevalence of the 4 major STEC virulence genes and the non-O157 STEC serogroups were not affected by inclusion of the DFM in the diet. In conclusion, DFM supplementation appeared to reduce fecal shedding E. coli O157, but prevalence of the 6 non-O157 serogroups and virulence genes were not affected.

The Fusobacterium genus contains Gram-negative, rod-shaped anaerobic bacteria. Fusobacterium necrophorum and its two subspecies, necrophorum and funduliforme, are normal ruminal inhabitants of cattle. Subsp. necrophorum is the primary causative agent of liver abscesses in feedlot cattle. Fusobacterium varium is also a normal rumen inhabitant, but its role in ruminal fermentation and involvement in liver abscesses is unknown. Currently, no PCR assays exist to quantify F. varium and both subspecies of F. necrophorum. Therefore, our objective was to develop, validate, and determine the applicability of a quantitative PCR assay for quantification of F. varium and the two subspecies of F. necrophorum. Assay targets were the species-specific hgdA gene, encoding for (R)-2-hydroxyglutarylCoA dehydratase subunit alpha, and the subspecies-specific promoter sequence of the leukotoxin gene, lktA. Assay specificity was validated with different species and strains of Fusobacterium, and other bacterial species. Assay sensitivity was determined using pure cultures. Applicability of the assay for quantification of F. necrophorum and F. varium was determined using ruminal fluid from slaughtered cattle. Species and subspecies-level specificity was confirmed. Pure culture quantification limits (CFU/ml) were 2.1x103 for the F. necrophorum species, 5.6x102 for subsp. necrophorum, 1.5x103 for subsp. funduliforme, and 1.8x103 for F. varium. Analysis of ruminal fluid collected from cattle with healthy livers (n=83) and abscessed livers (n=68) indicated that 97% of samples contained F. funduliforme, with mean concentrations of 7.8 x103, and 8.1x103, respectively. Additionally, subsp. necrophorum and F. varium were more prevalent in ruminal fluid from cattle with abscessed livers (41% and 59%) compared with samples from cattle with healthy livers (31% and 41%). In conclusion, subsp. funduliforme appears to be the normal inhabitant of the rumen, while subsp. necrophorum and F. varium are more prevalent in the rumen of cattle with abscessed livers than those with healthy livers.

The objective of this study was to evaluate the effectiveness of a direct-fed microbial (DFM) product in reducing fecal shedding of

All livestock animal species harbour complex microbial communities throughout their digestive tract that support vital biochemical processes, thus sustaining health and productivity. In part as a consequence of the strong and ancient alliance between the host and its associated microbes, the gut microbiota is also closely related to productivity traits such as feed efficiency. This phenomenon can help researchers and producers develop new and more effective microbiome-based interventions using probiotics, also known as direct-fed microbials (DFMs), in Animal Science. Here, we focus on one type of such beneficial microorganisms, the yeast Saccharomyces. Saccharomyces is one of the most widely used microorganisms as a DFM in livestock operations. Numerous studies have investigated the effects of dietary supplementation with different species, strains and doses of Saccharomyces (mostly Saccharomyces cerevisiae) on gut microbial ecology, health, nutrition and productivity traits of several livestock species. However, the possible existence of Saccharomyces which are indigenous to the animals' digestive tract has received little attention and has never been the subject of a review. We for the first time provide a comprehensive review, with the objective of shedding light into the possible existence of indigenous Saccharomyces of the digestive tract of livestock. Saccharomyces cerevisiae is a nomadic yeast able to survive in a broad range of environments including soil, grass and silages. Therefore, it is very likely that cattle and other animals have been in direct contact with this and other types of Saccharomyces throughout their entire existence. However, to date, the majority of animal scientists seem to agree that the presence of Saccharomyces in any section of the gut only reflects dietary contamination; in other words, these are foreign organisms that are only transiently present in the gut. Importantly, this belief (i.e. that Saccharomyces come solely from the diet) is often not well grounded and does not necessarily hold for all the many other groups of microbes in the gut. In addition to summarizing the current body of literature involving Saccharomyces in the digestive tract, we discuss whether the beneficial effects associated with the consumption of Saccharomyces may be related to its foreign origin, though this concept may not necessarily satisfy the theories that have been proposed to explain probiotic efficacy in vivo. This novel review may prove useful for biomedical scientists and others wishing to improve health and productivity using Saccharomyces and other beneficial microorganisms.

Holstein steers raised for beef production consistently have a higher prevalence and more severe form of liver abscesses than cattle of beef breeds. A study was conducted to compare bacterial flora of liver abscesses collected from multiple abattoirs from 4 groups of cattle, arranged in a 2 × 2 factorial design, consisting of crossbred cattle and Holstein steers, and each group fed a finishing diet supplemented with or without tylosin. A total of 383 liver abscess samples, consisting of 94 and 81 from crossbred cattle and 89 and 119 from Holstein steers fed finishing diets with or without tylosin, respectively, were subjected for anaerobic and aerobic bacterial isolations. The minimum inhibitory concentrations (MIC) of tylosin to the predominant bacterial species were determined. The likelihood chi-square test was performed to assess unadjusted differences in bacterial prevalence proportions between the 2 types of cattle (crossbred and Holstein steers) and feed type (tylosin or no tylosin). There was no interaction between cattle type and tylosin inclusion on the prevalence of any of the bacterial species isolated. Liver abscesses from Holstein steers yielded a higher total number of isolates compared to liver abscesses from crossbred cattle (1060 vs. 788). subsp. was isolated from all abscesses. The prevalence of subsp. was 19.1% and was not affected by the cattle type or tylosin. The prevalence of was higher (0.01) in crossbred cattle (73.7%) compared to Holstein steers (29.8%). Also, the prevalence of was higher in abscesses from tylosin-fed (66.1%) cattle than no tylosin-fed cattle (35%). The overall prevalence of was 25.3% and was similar ( = 0.58) between cattle type, but the prevalence was lower (0.01) in tylosin-fed (16.9%) compared to no tylosin-fed group (33%). Mean MIC of tylosin for and were similar across both cattle types and tylosin inclusion. Although bacterial flora of liver abscesses from Holstein steers appeared to be more diverse than that of crossbred cattle, there was no difference in the prevalence of the and and in fact, prevalence of was higher in crossbred than Holstein steers. Therefore, the difference in bacterial flora is not the likely reason for higher prevalence and severity of liver abscesses in Holstein steers than crossbred beef cattle.

Condensed tannins (CT), prior dietary CT exposure, animal species, and antimicrobial inclusion effects on 48 h extent of in vitro fermentation were measured in an experiment with a 3 × 2 × 2 × 3 factorial arrangement of treatments. Treatments included species of inoculum donor (Bos taurus, Ovis aries, or Capra hircus; n = 3/species), prior adaptation to dietary CT (not adapted or adapted), culture substrate (low-CT or high-CT), and antimicrobial additive (none, bacterial suppression with penicillin + streptomycin, or fungal suppression with cycloheximide). Low-CT or high-CT substrates were incubated in vitro using inoculum from animals either not exposed (period 1) or previously exposed to dietary CT (period 2). The extent of IVDMD after 48 h of incubation was greater (P < 0.001) for cultures with low-CT substrate (21.5%) than for cultures with high-CT substrate (16.5%). Cultures with high-CT substrate or with suppressed bacterial activity had less (P < 0.001) gas pressure than cultures with low-CT substrate or cultures with suppressed fungal activity. Total VFA concentrations were greater (P < 0.001) in low-CT cultures when inoculum donors were without prior CT exposure (83.7 mM) than when inoculum was from CT-exposed animals (79.6 mM). Conversely, total VFA concentrations were greater (P < 0.001) in high-CT cultures with tannin-exposed inoculum (59.4 mM) than with nonexposed inoculum (52.6 mM). As expected, CT and suppression of bacterial fermentative activities had strong negative effects on fermentation; however, prior exposure to dietary CT attenuated some negative effects of dietary CT on fermentation. In our experiment, the magnitude of inoculum-donor species effects on fermentation was minor.

The enterohemorrhagic pathotype represents a minor proportion of the Escherichia coli O103 strains shed in the feces of cattle. We report here the genome sequences of 43 strains of enterohemorrhagic E. coli (EHEC) O103:H2 isolated from feedlot cattle feces. The genomic analysis will provide information on the genetic diversity and virulence potential of bovine EHEC O103.

Previous in vitro data showed that was inhibited by limonene. We further evaluated effects of limonene on growth of in vitro as well as on ruminal concentrations of in vivo. With in vitro cultivation in anaerobic brain-heart infusion broth, limonene decreased growth of . Thymol also reduced growth of , but it was less effective than limonene. Tylosin effectively reduced growth of in vitro. Although the response over fermentation times and concentrations of antimicrobials differed somewhat between tylosin and limonene, the 2 antimicrobial agents yielded similar inhibitory effects on growth of at concentrations ranging from 6 to 24 mg/L. The effects of limonene on ruminal concentration in vivo were tested in 7 ruminally cannulated heifers (225 kg initial BW) used in a 7 × 4 Youden square design. Treatments included: 1) control, 2) limonene at 10 mg/kg diet DM, 3) limonene at 20 mg/kg diet DM, 4) limonene at 40 mg/kg diet DM, 5) limonene at 80 mg/kg diet DM, 6) CRINA-L (a blend of essential oil components) at 180 mg/kg diet DM, and 7) tylosin at 12 mg/kg diet DM. Each period included 11 d with 10 d washouts between periods. Samples of ruminal contents were collected before treatment initiation and after 4, 7, and 10 d of treatment for measuring by the most probable number method using selective culture medium. Limonene linearly decreased ( = 0.03) ruminal concentration, with the lowest concentration achieved with 40 mg of limonene/kg dietary DM. Limonene tended ( ≤ 0.07) to linearly reduce ruminal molar proportions of propionate and valerate while tending to linearly increase ( ≤ 0.10) those of butyrate and 2-methyl butyrate. Limonene did not affect ruminal NH concentrations or degradation rates of lysine. Neither CRINA-L ( = 0.52) nor tylosin ( = 0.19) affected ruminal concentrations. CRINA-L significantly decreased ruminal concentrations of NH and molar proportions of 3-methyl butyrate, whereas tylosin significantly decreased molar proportions of propionate while increasing those of butyrate and tending to increase those of acetate. Limonene supplementation reduced ruminal concentrations of suggesting that it may have the potential to reduce the prevalence of liver abscesses, although further research is needed to assess the effect of limonene in feedlot cattle.

A total of 480 pigs (PIC 327 × 1050; initially 48.7 ± 2.3 kg) were used to determine the interactive effects of supplemental Cu, Zn, and ractopamine HCl (RAC) on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria. Treatments were arranged in a 2 × 2 × 2 factorial with the main effects of added Cu (CuSO; 0 vs. 125 mg/kg Cu), Zn (ZnO; 0 vs. 150 mg/kg Zn), and RAC (0 vs. 10 mg/kg during the last 28 d prior to marketing). All diets contained 11 mg/kg Cu and 73 mg/kg Zn from the trace mineral premix. Pens of pigs were balanced and blocked on initial BW and then randomly allotted to 1 of the 4 mineral treatment diets. At 28 d prior to marketing, pens within each block and mineral treatment were randomly assigned to receive either 0 or 10 mg/kg RAC in addition to the mineral treatment. Adding either Cu or Zn alone did not improve ADG or ADFI yet resulted in numerical improvements in overall G:F and caloric efficiencies, but improvements were not additive (Cu × Zn, = 0.057, = 0.068, and = 0.064 for G:F and caloric efficiency on a ME and NE basis, respectively). Ractopamine improved (0.001) overall ADG, G:F, and caloric efficiency, thereby increasing final BW by 3% with no change in ADFI. Ractopamine also increased (0.001) HCW, percentage carcass yield, G:F, loin depth, and percent fat-free lean and decreased ( = 0.014) backfat. Adding Zn or Cu alone to diets containing RAC numerically improved percent yield and HCW G:F, but this effect was absent when the Cu or Zn was added to the control diet or when Cu and Zn were fed in combination in RAC diets (Cu × Zn × RAC, = 0.011 and = 0.018 for yield and HCW G:F, respectively). Fecal samples were collected on d 0 and at the conclusion of the finishing period (d 90) for bacterial isolation and antimicrobial susceptibility determinations according to Clinical and Laboratory Standards Institute minimal inhibitory concentrations breakpoints. spp. and isolates displayed varying levels of resistance to certain antibiotics prior to initiation of treatments on d 0. Resistance to most antibiotics decreased (0.05) over time or was stable for those that had a low baseline percentage of resistance. Neither Zn nor RAC adversely affected antimicrobial resistance. However, extended feeding of 125 mg/kg Cu throughout the finishing period seems to decrease enterococcal susceptability to tetracycline, tylosin, and quinupristin/dalfopristin.

Liver abscesses are the primary liver abnormality of feedlot cattle at slaughter. The incidence of liver abscesses is highly variable, but generally ranges from 10% to 20%. The incidence of total and the proportion of severely abscessed livers (A+) are greater in Holsteins fed for beef production and culled dairy cows than in beef breeds. The reason for the greater incidence of liver abscesses in Holstein steers is not known, but one of the reasons is likely because of increased days on feed. The high prevalence in cull cows is likely because no specific intervention, such as use of tylosin in the feed, is approved for use in dairy cows. Liver abscesses are generally a sequela to ruminal acidosis and rumenitis in cattle fed diets high in readily-fermentable carbohydrates and low in roughages; thus, the term "acidosis-rumenitis-liver abscess complex." Liver abscesses are almost always polymicrobial infections with Gram negative anaerobes constituting the predominant flora. Almost all studies have concluded that , a ruminal bacterium, is the primary causative agent and (formerly ) is the secondary pathogen. A limited number of studies have been done on the bacterial flora of liver abscesses of culled dairy cows and Holstein feedlot steers. A recent study has reported on isolation of from liver abscesses of Holstein cattle. The control of liver abscesses in feedlot cattle has depended on the use of antibiotics, particularly tylosin, in the feed combined with sound nutritional management to minimize occurrence of acidosis and subsequent rumenitis. Although there is no evidence of resistance development in , the future of tylosin use as a feed additive in feedlot cattle is uncertain. Regardless, beginning January 2017, the use of tylosin in feedlot cattle for the prevention of liver abscesses will be under veterinary oversight. Although tylosin is widely used in the feedlot industry, there is considerable interest in evaluating antibiotic alternatives, such as essential oils and vaccines, to control liver abscesses. Because liver abscess is a bacterial infection and the pathogenicity and virulence factors of have been studied widely, there have been considerable interest and efforts to develop an efficacious vaccine. The 2 antigens that have been targeted for vaccine production are leukotoxin and outer membrane proteins of .

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10(9) colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10(-1) to 10(-4)) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37 degrees C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37 degrees C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 microg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 x 10(4) cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.

Robert Hungate, considered the father of rumen microbiology, was the first to initiate a systematic exploration of the microbial ecosystem of the rumen, but he was not alone. The techniques he developed to isolate and identify cellulose-digesting bacteria from the rumen have had a major impact not only in delineating the complex ecosystem of the rumen but also in clinical microbiology and in the exploration of a number of other anaerobic ecosystems, including the human hindgut. Rumen microbiology has pioneered our understanding of much of microbial ecology and has broadened our knowledge of ecology in general, as well as improved the ability to feed ruminants more efficiently. The discovery of anaerobic fungi as a component of the ruminal flora disproved the central dogma in microbiology that all fungi are aerobic organisms. Further novel interactions between bacterial species such as nutrient cross feeding and interspecies H2 transfer were first described in ruminal microorganisms. The complexity and diversity present in the rumen make it an ideal testing ground for microbial theories (e.g., the effects of nutrient limitation and excess) and techniques (such as 16S rRNA), which have rewarded the investigators that have used this easily accessed ecosystem to understand larger truths. Our understanding of characteristics of the ruminal microbial population has opened new avenues of microbial ecology, such as the existence of hyperammonia-producing bacteria and how they can be used to improve N efficiency in ruminants. In this review, we examine some of the contributions to science that were first made in the rumen, which have not been recognized in a broader sense.

Distillers grains (DG), a co-product of ethanol production used as protein and energy supplements in cattle diets, have been shown to increase fecal shedding of Escherichia coli O157:H7, a major foodborne pathogen. The reason for the positive association is not known. Because DG often replaces grain in the diet, decreased starch content and flow to the hindgut may create a favorable environment for E. coli O157:H7. Our objective was to determine whether the addition of starch to a corn DG-supplemented diet negates the effects of DG on fecal shedding of E. coli O157:H7. We conducted a study with 21 steers fed 1 of 3 diets: a corn grain-based basal diet (CON), basal diet supplemented with 25% corn dried DG (DDG), and basal diet supplemented with 25% DDG with corn starch (DDG+S) added at a level intended to increase starch concentration to that of the CON diet. Steers, housed individually in a biosafety level 2 animal facility, were randomly allocated to treatment diets and orally inoculated with a 5-strain mixture (10(9) cfu per steer) of nalidixic acid-resistant (Nal(R)) E. coli O157:H7. Fecal samples were collected for 5 wk, and on d 35, steers were euthanized and necropsied to collect gut content samples. Fecal or gut samples were cultured to determine prevalence and concentrations of Nal(R) E. coli O157:H7. Dietary starch concentrations, based on feed analysis, were 46.3% in the CON compared with 43.3 and 41.3% in the DDG and DDG+S diets, respectively. Steers fed DDG or DDG+S diets shed Nal(R) E. coli O157:H7 more often (P = 0.0027 and 0.0003, respectively) and at greater concentrations (1.9 or 2.0 cfu/g; P = 0.0025 and 0.0006) than those fed CON diet (1.4 cfu/g), but no difference was observed between DDG and DDG+S diets. Cumulative prevalence and concentrations of Nal(R) E. coli O157:H7 were greater in gut samples collected at necropsy in steers fed DDG and DDG+S diets compared with those fed CON diet, but no difference was observed between DDG and DDG+S diets. The lack of effect of starch addition to the DDG diet on fecal shedding of E. coli O157:H7 may be because either the decreased starch content in the DG-supplemented diet is not a factor in the increased shedding of E. coli O157:H7 or inclusion of pure starch in the diet may not have achieved our intended goal to have starch flow into the hindgut similar to that of corn grain. The study confirms our previous finding of the positive association between feeding DG and fecal shedding of E. coli O157:H7.

Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Non-O157 STEC, particularly O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. Our objective was to develop a multiplex PCR to distinguish O157 and the 'top six' non-O157 serogroups (O26, O45, O103, O111, O121, and O145) and evaluate the applicability of the multiplex PCR to detect the seven serogroups of E. coli in cattle feces. Published sequences of O-specific antigen coding genes, rfbE (O157) and wzx and wbqE-F (non-O157), were analyzed to design serogroup-specific primers. The specificity of amplifications was confirmed with 138 known STEC strains and the reaction yielded the expected amplicons for each serogroup. In feces spiked with pooled 7 STEC strains, the sensitivity of the detection was 4.1 × 10(5)CFU/g before enrichment and 2.3 × 10(2) after 6h enrichment in E. coli broth. Additionally, 216 fecal samples from cattle were collected and tested by multiplex PCR and cultural methods. The multiplex PCR revealed a high prevalence of all seven serogroups (178 [O26], 108 [O45], 149 [O103], 30 [O111], 103 [O121], 5 [O145], and 160 [O157]) of 216 samples in fecal samples. Cultural procedures identified 33.1% (53/160) and 35.5% (11/31) of PCR-positive samples for E. coli O157 and non-O157 serogroups, respectively. Samples that were culture-positive were all positive by the multiplex PCR. The multiplex PCR can be used to identify serogroups of putative STEC isolates.

Inclusion of distillers grains (DG) in cattle diets has been shown to increase fecal shedding of Escherichia coli O157:H7. It is hypothesized that altered gut fermentation by DG may be responsible for the positive association. Therefore, feed additives affecting ruminal or hindgut fermentation of DG also may affect fecal shedding of E. coli O157:H7. The objectives of the study were to evaluate effects of monensin (33 or 44 mg/kg of DM), supplemental urea (0, 0.35, or 0.70% of DM), and ractopamine (0 or 200 mg/steer daily administered during the last 42 d of finishing) in a steam-flaked corn grain-based diet containing 30% wet sorghum DG on fecal shedding of E. coli O157:H7. Seven hundred twenty crossbred beef steers, housed in 48 pens (15 steers/pen), were assigned to dietary treatments in a randomized complete block design with a 2 × 3 × 2 factorial treatment arrangement. Fresh pen floor fecal samples (10 per/pen) were collected every 2 wk for 14 wk (July through November) and cultured for E. coli O157:H7. Isolation of E. coli O157:H7 was by selective enrichment of fecal samples in an enrichment broth, immunomagnetic separation, followed by plating onto a selective medium. Samples that yielded sorbitol-negative colonies, which were positive for indole production, O157 antigen agglutination, and contained rfbE, fliC, and stx2 were considered positive for E. coli O157:H7. Fecal prevalence data were analyzed as repeated measures using negative binomial regression to examine effects and interactions of sampling day, urea, monensin, and ractopamine. Mean fecal prevalence of E. coli O157:H7 was 7.6% and ranged from 1.6 to 23.6%. Cattle fed monensin at 44 mg/kg of feed had less (P = 0.05) fecal E. coli O157:H7 prevalence than cattle fed 33 mg/kg (4.3 vs. 6.8%). Although the reason for the reduction is not known, it is likely because of changes in the microbial ecosystem induced by the greater amount of monensin in the hindgut. Supplemental urea at 0.35 or 0.70% had no effect (P = 0.87) on fecal shedding of E. coli O157:H7. Fecal prevalence of E. coli O157:H7 were 5.3, 5.7, and 5.9% for groups fed 0, 0.35, and 0.7% urea, respectively. The inclusion of ractopamine at 0 or 200 mg/(animal•d) had no effect (P = 0.89) on fecal prevalence of E. coli O157:H7 (4.4 vs. 4.0%). Additional research is needed to confirm the reduction in fecal shedding of E. coli O157:H7 in cattle fed monensin at 44 mg/kg of feed compared with cattle fed 33 mg/kg of feed.

We used four pregnant Holstein cows to delineate ruminal adaptations as cows transitioned from one lactation to the next. Cows were fed typical diets through far-off and close-up dry periods and lactation. We measured ruminal characteristics on day 72 (late lactation), 51 (far-off dry), 23 and 9 (close-up dry) prepartum and on days 6, 20, 34, 48, 62, 76 and 90 postpartum (early lactation). Measurements included: ruminal fill (weight of actual contents), ruminal capacity (volume of rumen when fully filled), digestibilities and ruminal passage rates. Ruminal capacity tended to increase linearly during early lactation but was stable during dry and transition periods. Both total and liquid fill decreased linearly during the dry period, increased across parturition, and increased linearly through early lactation. Dry matter fill decreased as cows were fed the close-up diet at day 23 prepartum then increased near parturition and continued to increase across early lactation. Solid passage rate was greatest when cows were fed the close-up diet, and decreased throughout the transition period. In lactation, solid passage rate responded quadratically with peak at day 48 followed by decreases through day 90 postpartum. Liquid passage increased linearly across the transition period. Total tract organic matter digestibilities increased linearly over the dry period with significant increases prior to or immediately after parturition, then they remained relatively stable over early lactation until they increased at day 90. Fibre digestibilities demonstrated quadratic responses over early lactation, being higher on day 6 and day 90 than at other times. Starch digestibilities decreased linearly across both the dry and transition periods with decreases in lactation until day 62 followed by increases until day 90. High producing lactating dairy cows go through a multitude of ruminal adaptations, in terms of digestion, passage, capacity and fill, as they transition from one lactation to the next.

Cattle are a primary reservoir of Escherichia coli O157:H7, a major foodborne pathogen. The organism causes haemorrhagic colitis which can lead to serious complications, including haemolytic-uraemic syndrome. Although E. coli O157:H7 is widely prevalent in cattle and cattle environments, the number of human cases remain relatively low, suggesting possible strain diversity and differences in virulence between human and bovine strains. Shiga toxins, Stx1 and Stx2, are the major virulence factors. Differences in Stx2 production between human and bovine strains have been demonstrated previously, and isolates possessing the stx2 gene, but not producing Stx2 [toxin non-producing (TNP) strains] have been identified. In this study, 150 isolates (56 human, 94 bovine) were tested by PCR for stx2 upstream regions associated with TNP and the Q933 gene, which has been previously associated with toxin production. A reverse passive latex agglutination test was used to evaluate 107 isolates (50 human, 57 bovine) for Stx1 and Stx2 production. The percentages of human and bovine isolates positive for presence of the TNP regions were similar (57.1% and 53.1% respectively), while a higher percentage of human isolates was positive for Q933 gene (89.3% versus 54.3%). Stx2 production of ≥ 1:8 was found in 86.0% of human isolates compared with 26.3% of bovine isolates. Bovine isolates with the presence of the TNP regions were associated with significantly lower Stx2 production (P 0.05), while the Q933 gene was associated with higher Stx2 production (P 0.05). However, the presence of the TNP region was not associated (P 0.05) with low Stx2 production in human isolates. Therefore, Q933 was a better indicator of high Stx2 production by human and bovine isolates and may be a useful screening method to assess their potential to cause human disease.

In cattle, Escherichia coli O157 generally persists in the large intestine more often than in the rumen. In contrast to the rumen, the large intestine is lined by an epithelial membrane that secretes mucus. We hypothesize that substrates contained in intestinal mucus may constitute a source of energy that is preferentially used by E. coli O157. Therefore, our objective was to test the effects of mucin and its carbohydrate constituents on in vitro growth of E. coli O157 in ruminal or fecal microbial fermentations. Ruminal contents and feces were collected from a ruminally cannulated donor steer fed a corn grain-based finishing diet. Ruminal contents were strained through 2 layers of cheesecloth and incubated at 39 degrees C for 1 h; the floating hay mat was removed with a vacuum suction; and the remaining material was utilized as rumen microbial inoculum. Feces were suspended in physiologic saline to increase fluidity, blended, and strained through 2 layers of cheesecloth. The resulting fluid was utilized as fecal microbial inoculum. Fermentations (50 mL) were performed in serum bottles with a 2:1 mineral buffer to microbial inoculum ratio. Substrates (mucin, fucose, galactose, mannose, gluconic acid, galacturonic acid, glucuronic acid, galactosamine, and glucosamine) were added at 10 mg/mL. A mixture of 5 strains of nalidixic acid-resistant (Nal(R)) E. coli O157 strains was added to each fermentation and concentrations were determined after 0, 6, 12, and 24 h of incubation. In ruminal fermentations, fucose, mannose, glucuronic acid, galacturonic acid, glucosamine, galactosamine, and mucin had no effect on Nal(R) E. coli O157 concentration compared with the control (no substrate added) fermentation. At 24 h of fermentation, the mean concentration of Nal(R) E. coli O157 in fermentations with galactose was less than the control. However, including gluconic acid as substrate increased Nal(R) E. coli O157 concentration at 24 h. In fecal fermentations, mannose, galactose, gluconic acid, glucuronic acid, galacturonic acid, glucosamine, and mucin increased Nal(R) E. coli O157 growth compared with control at 24 h, whereas galactosamine and fucose did not. Gluconic acid was the most stimulatory substrate, increasing Nal(R) E. coli O157 by more than 1.0 log in ruminal fermentations and 2.0 log in fecal fermentations. In summary, availability of mucous constituents, particularly gluconic acid, may explain the greater prevalence of E. coli O157 in the large intestine compared with the rumen of the digestive tract.

Distillers grains, a coproduct of ethanol production from cereal grains, are composed principally of the bran, protein, and germ fractions and are commonly supplemented in ruminant diets. The objective of this study was to assess the effect of feeding wet distillers grains with solubles (WDGS) and monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal foodborne and commensal bacteria in feedlot cattle. Cattle were fed 0 or 25% WDGS in steam-flaked corn-based diets with the addition of no antimicrobials, monensin, or monensin and tylosin. Fecal samples were collected from each animal (n = 370) on d 122 and 136 of the 150-d finishing period and cultured for Escherichia coli O157. Fecal samples were also pooled by pen (n = 54) and cultured for E. coli O157, Salmonella, commensal E. coli, and Enterococcus species. Antimicrobial resistance was assessed by determining antimicrobial susceptibilities of pen bacterial isolates and quantifying antimicrobial resistance genes in fecal samples by real-time PCR. Individual animal prevalence of E. coli O157 in feces collected from cattle fed WDGS was greater (P0.001) compared with cattle not fed WDGS on d 122 but not on d 136. There were no treatment effects on the prevalence of E. coli O157 or Salmonella spp. in pooled fecal samples. Antimicrobial susceptibility results showed Enterococcus isolates from cattle fed monensin or monensin and tylosin had greater levels of resistance toward macrolides (P = 0.01). There was no effect of diet or antimicrobials on concentrations of 2 antimicrobial resistance genes, ermB or tetM, in fecal samples. Results from this study indicate that WDGS may have an effect on the prevalence of E. coli O157 and the concentration of selected antimicrobial resistance genes, but does not appear to affect antimicrobial susceptibility patterns in Enterococcus and generic E. coli isolates.

Five Holstein steers (235 kg of BW) fitted with ruminal, duodenal, and ileal cannulas were used in a 5 x 5 Latin square design experiment to determine the effects of supplemental fat source on site and extent of nutrient digestion and ruminal fermentation. Treatments were diets based on steam-flaked corn containing no supplemental fat (control) or 4% (DM basis) supplemental fat as tallow, dried full-fat corn germ (corn germ), corn oil, or flax oil. Fat supplementation decreased (P0.08) ruminal starch digestion but increased (P0.03) small intestinal starch digestion as a percentage of intake. Feeding corn germ decreased (P0.09) ruminal starch digestion and increased (P0.03) large intestinal starch digestion compared with steers fed corn oil. Large intestinal starch digestion was less (P0.04), and ruminal NDF digestion was greater (P0.09) for steers fed tallow compared with steers fed other fat sources. Small intestinal (P0.08) and total tract NDF digestibilities were greater (P0.02) for steers fed corn germ than for those fed corn oil. Feeding tallow increased total ruminal VFA (P0.03) and NH(3) (P0.07) concentrations compared with steers fed the other fat sources. Feeding corn germ led to a greater (P0.02) rate of ruminal liquid outflow compared with corn oil. A diet x hour interaction (P0.04) occurred for ruminal pH, with steers fed corn oil having the greatest ruminal pH 18 h after feeding, without differences at other time points. Fat supplementation increased (P0.09) ruminal concentrations of Fusobacterium necrophorum. Duodenal flow of C18:3n-3 was greater (P0.01) for steers fed flax oil compared with those fed corn oil. Feeding corn germ led to less (P0.01) ruminal biohydrogenation of fatty acids compared with corn oil. Steers fed tallow had greater small intestinal digestibility of C14:0 (P0.02) and C16:1 (P0.04) than steers fed the other fat sources. Fat supplementation decreased (P0.06) small intestinal digestibility of C18:0. Feeding corn germ decreased (P0.10) small intestinal digestibility of C18:1 compared with corn oil. It appears that source of supplemental fat can affect the site and extent of fatty acid and nutrient digestion in steers fed diets based on steam-flaked corn.

Manipulation of cattle diets has been proposed as a possible preharvest control measure for Escherichia coli O157. Altering hindgut fermentation through diet changes may be a means to reduce fecal shedding of E. coli O157. In Exp. 1, the objective was to determine whether fecal shedding of E. coli O157 was related to fecal starch concentration. Beginning on d 20, and every week thereafter until d 61, steers in 54 pens (6 to 7 steers per pen) were sampled (n = 122) by fecal collection and rectoanal mucosal swabs (RAMS) for E. coli O157 and fecal starch concentration determinations. Escherichia coli O157 prevalence was 3.3% in fecal samples, 4.1% as measured by RAMS, and 4.9% by fecal or RAMS samples. Steers positive for E. coli O157 contained 21% more (P0.05) fecal starch than steers that were negative for E. coli O157. In Exp. 2, we attempted to alter the concentration of starch escaping rumen fermentation by feeding finishing diets based on steam-flaked corn (SFC) and dry-rolled corn (DRC) to 30 heifers prescreened for being culture positive for fecal E. coli O157. Beginning on d 13, heifers were sampled (feces and RAMS) weekly to monitor fecal pH and starch concentration, and prevalence of E. coli O157. Prevalence of E. coli O157 remained above 30% for the first 13 d, but declined (P0.05) over the entire 7-wk period. Based on RAMS, the prevalence of E. coli O157 tended to be greater (P = 0.08) for heifers fed SFC than for those fed the DRC diet. After d 20, heifers fed DRC had greater (P0.05) fecal starch and lower (P0.05) fecal pH than heifers fed SFC. Fecal pH was negatively correlated (r = - 0.34; P0.05; n = 143) with fecal starch concentration. Fecal starch concentration and pH were not different (P0.05) for heifers that were positive or negative for E. coli O157. Our data suggest that fecal shedding of E. coli O157 was not related to fecal pH or starch concentration in cattle fed grain-based diets.

Hindgut is a major colonization site for Escherichia coli O157 in cattle. In this study, diets were formulated to effect changes in hindgut fermentation to test our hypothesis that changes in the hindgut ecosystem could have an impact on fecal shedding of E. coli O157. Feedlot heifers (n = 347) were prescreened for the prevalence of E. coli O157 by fecal and rectoanal mucosal swab cultures. A subset of 40 heifers identified as being positive for fecal shedding of E. coli O157 was selected, housed in individual pens, and randomly allocated to 4 dietary treatments. Treatments were arranged as a 2 x 2 factorial, with factor 1 consisting of grain type (sorghum or wheat) and factor 2 being method of grain processing (steam-flaking or dry-rolling). Four transition diets, each fed for 4 d, were used to adapt the animals to final diets that contained 93% concentrate and 7% roughage. The grain fraction consisted of dry-rolled sorghum, steam-flaked sorghum, a mixture of dry-rolled wheat and steam-flaked corn, or a mixture of steam-flaked wheat and steam-flaked corn. Wheat diets contained 52% wheat and 31% steam-flaked corn (DM basis). Fecal and rectoanal mucosal swab samples were obtained 3 times a week to isolate (enrichment, immunomagenetic separation, and plating on selective medium) and identify (sorbitol negative, indole production, and agglutination test) E. coli O157. The data were analyzed as repeated measures of binomial response (positive or negative) on each sampling day. Method of processing (dry-rolled vs. steam-flaked), sampling day, and the grain type x day interaction were significant (P0.05), but not the method of processing x grain type interaction. The average prevalence of E. coli O157 from d 9 was greater (P0.001) in cattle fed steam-flaked grains (65%) compared with those fed dry-rolled grains (30%). Average prevalence in cattle fed sorghum (51%) or wheat (43%) were similar (P0.10) on most sampling days. Results from this study indicate that feeding dry-rolled grains compared with steam-flaked grains reduced fecal shedding of E. coli O157. Possibly, dry-rolling allowed more substrate to reach the hindgut where it was fermented, thus making the hindgut inhospitable to the survival of E. coli O157. Dietary intervention to influence hindgut fermentation offers a simple and practical mitigation strategy to reduce the prevalence of E. coli O157 in feedlot cattle.

The objective of this study was to compare the concentration and duration of fecal shedding of Escherichia coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation. Eighteen 1-wk-old Holstein calves were orally inoculated with a strain of E. coli O157:H7 (3.6 x 10(8) cfu/calf) made resistant to nalidixic acid (NA). Rectal samples were obtained three times weekly for 8 wk following oral inoculation. Fecal shedding of NA-resistant E. coli O157:H7 was quantified by direct plating or detected by selective enrichment procedure. Eight weeks after inoculation, calves were killed, necropsied, and tissues (tonsils, retropharyngeal and mesenteric lymph nodes, and Peyer's patches) and gut contents (rumen, omasum, abomasum, ileum, cecum, colon, and rectum) were sampled to quantify or detect NA-resistant E. coli O157:H7. The percentage of calves shedding NA-resistant E. coli O157:H7 in the feces in the antibiotic-fed group was higher (P0.001) early in the study period (d 6 and 10) compared with the control group fed no antibiotics. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of fecal shedding between treated and untreated calves showed no significant difference between groups. At necropsy, E. coli O157:H7 was recovered from the rumen and omasum of one calf in the control group and from retropharyngeal lymph node and Peyer's patch of two calves in the antibiotic group. Supplementation of milk replacer with antibiotics may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect seems to be of low magnitude and short duration.

The effect of altering supplementation frequency on host N balance and key N transactions in the ruminal ecosystem were monitored. Four ruminally fistulated beef steers (BW = 513 kg; SEM = 6.5) were used in a 2 x 2 crossover design with two periods and two supplementation frequency treatments. Supplementation frequencies were 2 and 7 d/wk. Steers were fed tallgrass prairie hay (73.1% NDF, 5.3% CP) ad libitum. Supplement (42% CP; DM basis) was fed at 0.36% BW/d to steers supplemented 7 d/wk. Steers supplemented 2 d/wk received the same amount of supplement per week, but it was equally split among the two supplementation events. Steers supplemented 7 d/wk had higher forage (P0.02) and total digestible OM intake (P0.06), total N intake, fecal N excretion, and N retention. Although both supplementation frequencies were characterized by positive N balance, the decrease in N retention in the steers supplemented 2 d/wk was due to higher (P0.01) urinary N loss. Ruminal fluid was sampled at 0, 2, 4, 6, 12, 24, 48, and 72 h after supplementation beginning on a day when both treatments were supplemented. Frequency x hour interactions (P0.02) were observed for ruminal N metabolism criteria. Counts of peptide- and AA-fermenting bacteria peaked at 2 h and returned to nadir by 12 h for steers supplemented 7 d/wk. Steers supplemented 2 d/wk peaked at 6 h with a greater population and returned to nadir at 48 h. Ruminal ammonia concentrations followed a similar trend. Specific activity of ammonia production was lower (Por = 0.05) immediately after supplementation for steers supplemented 2 d/wk, but by 12 h was the same as for 7 d/wk steers. Ruminal peptides and free AA peaked at 2 h for steers supplemented 2 d/wk and were generally higher (Por = 0.05) during the first 6 h compared with steers supplemented 7 d/wk. Total VFA concentration was not different (P = 0.35) due to supplementation frequency. Frequency x hour interactions (P0.01) were observed for all molar proportions of VFA. The molar proportion of acetate and acetate:propionate ratio were lower (P0.01) and the molar proportions of propionate and butyrate were higher for steers supplemented 2 d/wk from 4 h to 24 h. In conclusion, forage use and N balance improved with supplementation 7 d/wk, but supplementation 2 d/wk was associated with some desirable shifts in select ruminal events that may contribute to moderating potential negative impacts of supplementing infrequently.

Six ruminally cannulated steers (345 +/- 20 kg initial BW) were used in a 6 x 6 Latin square to evaluate effects of diet and antibiotics on ruminal protein metabolism. Two diets and three antibiotic treatments were arranged factorially. One diet contained (DM basis) 72% dry-rolled corn, 12% soybean meal, 10% alfalfa hay, and 4% molasses (SBM), and the other contained 63% dry-rolled corn, 30% wet corn gluten feed, and 5% alfalfa hay (WCGF). Antibiotic treatments included control, virginiamycin (175 mg/d; VM), and monensin/tylosin (250 and 100 mg/d, respectively; MT). Steers were fed at 12-h intervals at a rate of 2.4% of empty BW daily. Each period included 18 d of adaptation and 3 d of ruminal fluid collections. Samples were collected at 0, 2, 4, 6, 8, and 10 h after the morning feeding on d 19 and 20. On d 21, rumens were dosed 2 h after the morning feeding with 350 g of solubilized casein to evaluate in vivo ruminal protease and deaminase activities. Ruminal fluid samples were collected 1, 2, 3, 4, and 6 h after the casein dose. On d 19 and 20, antibiotics had no effect on ruminal pH or concentrations of VFA, lactate, ammonia, ciliated protozoa, alpha-amino nitrogen (AAN), or peptide N, but VM reduced (P0.01) the concentration of isovalerate compared to MT and control. After casein dosing (d 21), peptide N concentration was unaffected by antibiotics, but AAN were higher (P0.01) for VM than MT and control. Relative to MT and control, VM reduced ruminal isovalerate (P = 0.05) and increased ruminal propionate (P0.01) on d 21. Ruminal pH was lower (P0.01) in steers fed SBM than in steers fed WCGF, but lactate concentrations were unaffected by diet. Steers fed SBM had higher (P0.05) ruminal concentrations of total VFA and propionate. Ammonia concentrations were lower before feeding and higher after feeding for steers fed WCGF (P0.01). Steers fed WCGF had higher counts of total ciliated protozoa than steers fed SBM (P0.05) due to greater Entodinium sp. (P0.05). Steers fed WCGF had higher (P0.01) ruminal AAN and peptide N concentrations than those fed SBM on d 19 and 20. After casein dosing, ruminal peptide N concentrations were similar, but AAN were lower (P0.01) for WCGF than SBM. Overall, VM appeared to depress ruminal deaminase activity, and MT had minimal effects on ruminal fermentation products. The protein in WCGF appeared to be more readily degradable than that in SBM.

To determine effects of tylosin on ruminal concentrations of Fusobacterium necrophorum and fermentation products in cattle during rapid adaptation to a high-concentrate diet.6 steers fitted with ruminal cannulas.Steers were assigned randomly to 2 treatment groups and switched from a 0 to an 85% concentrate diet during a 4-day period. Cattle received this diet, with or without tylosin (90 mg/steer/d), for 4 weeks. Samples of ruminal contents were collected daily beginning 2 days before the treatment protocol and in the first week of concentrate feeding. Four subsequent samples were collected at weekly intervals. Concentration of F. necrophorum in samples was determined, using the most-probable-number technique. Ruminal pH and concentrations of volatile fatty acids (VFA), lactate, and ammonia also were determined. All steers received both treatments separated by 4 weeks (cross-over design), during which time they were fed alfalfa hay only.In control steers, concentration of F. necrophorum increased in response to the high-concentrate diet. Tylosin-fed steers had lower concentrations of F. necrophorum than control steers at all times during concentrate feeding. However, ruminal pH and concentrations of lactate, VFA, and ammonia did not differ between treatment groups.Tylosin caused a significant reduction in ruminal concentrations of F. necrophorum during rapid adaptation to a high-concentrate diet but had no effect on fermentation products. The reduction in ruminal concentration of F. necrophorum helps explain the reduction in prevalence of hepatic abscesses reported in tylosin-fed feedlot cattle.

Actinomyces pyogenes is the second most frequently encountered pathogen, next only to Fusobacterium necrophorum, in liver abscesses of feedlot cattle. Ninety-one isolates, presumptively identified as A. pyogenes, isolated from liver abscesses of cattle were studied. Biochemical characteristics determined by the API 20 Strep kit were similar to those reported previously for A. pyogenes isolated from other infections, except that 18% of isolates hydrolyzed esculin. Nine isolates that resembled A. pyogenes in morphology and in certain biochemical characteristics, but fermented mannitol and/or raffinose, were called A. pyogenes-like (APL) organisms. The five antimicrobial agents, bacitracin, chlortetracycline, oxytetracycline, tylosin, and virginiamycin were inhibitory to all strains of A. pyogenes and APLs. Generally, APL organisms had higher mean hemolytic and leukotoxic activities than A. pyogenes. All isolates of A. pyogenes and APLs produced proteases and neuraminidases. Ribotyping with endonucleases, including BstEII, ClaI, EcoRI, EcoRV, HaeIII, MboI, PvuII, SalI, and SmaI alone or in combinations, showed considerable genetic heterogeneity in both A. pyogenes and APLs. No specific ribopattern characteristic of each group was observed with any of the endonuclease used. The origin of A. pyogenes and APLs and the relative importance of APLs in causing liver abscesses in feedlot cattle are not known.