Effects of Menthol Supplementation in Feedlot Cattle Diets on the Fecal Prevalence of Antimicrobial-Resistant Escherichia coli.

The pool of antimicrobial resistance determinants in the environment and in the gut flora of cattle is a serious public health concern. In addition to being a source of human exposure, these bacteria can transfer antibiotic resistance determinants to pathogenic bacteria and endanger the future of antimicrobial therapy. The occurrence of antimicrobial resistance genes on mobile genetic elements, such as plasmids, facilitates spread of resistance. Recent work has shown in vitro anti-plasmid activity of menthol, a plant-based compound with the potential to be used as a feed additive to beneficially alter ruminal fermentation. The present study aimed to determine if menthol supplementation in diets of feedlot cattle decreases the prevalence of multidrug-resistant bacteria in feces. Menthol was included in diets of steers at 0.3% of diet dry matter. Fecal samples were collected weekly for 4 weeks and analyzed for total coliforms counts, antimicrobial susceptibilities, and the prevalence of tet genes in E. coli isolates. Results revealed no effect of menthol supplementation on total coliforms counts or prevalence of E. coli resistant to amoxicillin, ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, and sulfamethoxazole; however, 30 days of menthol addition to steer diets increased the prevalence of tetracycline-resistant E. coli (P < 0.02). Although the mechanism by which menthol exerts its effects remains unclear, results of our study suggest that menthol may have an impact on antimicrobial resistance in gut bacteria.

Effects of crystalline menthol on blood metabolites in Holstein steers and in vitro volatile fatty acid and gas production.

Fifty-two Holstein steers (573 ± 9.92 kg BW) were used to determine if oral administration of crystalline menthol would induce changes in endogenous secretions of IGF-1 and circulating concentrations of glucose, lactate, and plasma urea nitrogen (PUN). Steers were blocked by BW and assigned within block to treatment. Treatments consisted of 0, 0.003, 0.03, or 0.3% crystalline menthol (DM basis) added to the diet. Animals were housed in individual, partially covered pens equipped with feed bunks and automatic water fountains. On d 1 of the experiment, blood samples were obtained via jugular venipuncture at 0, 6, 12, 18, and 24 h after feeding. Treatment administration commenced on d 2, and blood samples were again drawn at 0, 6, 12, 18, and 24 h after feeding. This blood-sampling schedule was repeated on d 9, 16, 23, and 30. Plasma was analyzed for PUN, glucose, and lactate concentrations. Serum was used to analyze IGF-1 concentration. Body weights were measured on d 1, 9, 16, 23, and 30. To accompany the live animal phase, in vitro fermentations were performed using ruminal fluid cultures. Measurements included VFA concentrations and fermentative gas production for cultures containing crystalline menthol at 0, 0.003, 0.03, or 0.3% of substrate DM. Addition of menthol to the diet of steers resulted in a treatment × day interaction ( < 0.01) for concentrations of IGF-1, PUN, and plasma glucose. Cattle fed 0 and 0.003% menthol had greater serum IGF-1 concentrations on d 2 compared with steers fed 0.03% menthol. Steers fed 0% menthol had greater serum IGF-1 concentrations on d 9 compared with steers fed 0.03 and 0.3% menthol, whereas no differences were observed on d 23 or 30. Plasma glucose was similar among treatments until d 23, when steers supplemented with 0.03% menthol had lower glucose concentrations. Plasma urea nitrogen concentrations were not different among treatments; however, PUN concentrations varied by day. A linear response was detected for BW ( = 0.03), with steers consuming 0% menthol having the greatest BW and steers that consumed 0.3% menthol having the lightest BW until d 30. A menthol × day interaction was observed for daily feed deliveries ( < 0.01): cattle fed 0.3% menthol consumed less feed from d 5 through 12. Furthermore, in vitro gas production and VFA concentrations were unaffected by addition of menthol ( > 0.21). In conclusion, menthol supplementation minimally affected blood parameters associated with growth or ruminal fermentative activity.

Effects of flaxseed encapsulation on biohydrogenation of polyunsaturated fatty acids by ruminal microorganisms: feedlot performance, carcass quality, and tissue fatty acid composition.

The objective of this study was to evaluate the efficacy of protecting PUFA within ground flaxseed against ruminal biohydrogenation by encapsulating them in a matrix consisting of a 1:1 blend of ground flaxseed and dolomitic lime hydrate (L-Flaxseed). Crossbreed heifers ( = 462, 346 ± 19 kg) were blocked by weight and randomly assigned to pens. Pens were assigned to 1 of 6 dietary treatments in a randomized complete block design. Treatment 1 consisted of a combination of 54.6% steam-flaked corn (SFC), 30.0% wet corn gluten feed, 8.0% roughage, and supplement (0% flaxseed). In treatments 2 and 3, a proportion of SFC was replaced with 3 and 6% flaxseed, respectively; in treatments 4, 5, and 6, SFC was replaced with 2, 4, or 6% L-Flaxseed, respectively. Cattle were fed for 140 or 168 d and then harvested in a commercial abattoir where carcass data were collected. Approximately 24 h after harvest, carcasses were evaluated for 12th-rib fat thickness, KPH, LM area, marbling score, and USDA yield and quality grades. Samples of LM were also obtained for determination of long-chain fatty acid profiles. Cattle that were fed diets with 4 and 6% L-Flaxseed consumed less feed than other treatments ( < 0.05), which adversely affected ADG. Compared with cattle fed 0% flaxseed, cattle in these treatments had lower final BW (18 and 45 kg less for the 4 and 6% L-Flaxseed treatments, respectively), less ADG (0.16 and 0.48 kg/day less for the 4 and 6% L-Flaxseed treatments, respectively), and lower carcass weights, dressing percentages, LM areas, backfat thicknesses, and marbling scores ( < 0.05). The addition of flaxseed or 2% L-Flaxseed did not affect performance or carcass traits ( > 0.05). Supplementation with flaxseed increased ( < 0.05) the concentration of α-linolenic acid (ALA) in meat (0.173, 0.482, 0.743 mg/g for 0, 3, and 6% flaxseed, respectively). Furthermore, proportionate increases in the ALA content of muscle tissue were 47% greater when flaxseed was encapsulated within the dolomitic lime hydrate matrix (0.288, 0.433, 0.592 mg/g for 2, 4, and 6% L-Flaxseed, respectively). Both products showed a linear response in ALA concentration ( > 99%; increases for Flaxseed and L-Flaxseed of 0.095 and 0.140 mg of ALA/g of tissue for each percentage of flaxseed added). This study indicates that a matrix consisting of dolomitic lime hydrate is an effective barrier to ruminal biohydrogenation of PUFA; however, adverse effects on DMI limit the amounts that can be fed.

Protection of polyunsaturated fatty acids against ruminal biohydrogenation: Pilot experiments for three approaches.

Three methods for protection of PUFA against biohydrogenation by ruminal microorganisms were evaluated. In method 1 a blend of ground flaxseed, calcium oxide, and molasses was processed through a dry extruder. In method 2, a blend of ground flaxseed, soybean meal, molasses, and baker's yeast was moistened and prewarmed, allowing enzymes from yeast to produce reducing sugars, and the mixture was subsequently processed through a dry extruder like in method 1. In method 3, ground flaxseed was embedded within a matrix of dolomitic lime hydrate (L-Flaxseed) as a protective barrier against biohydrogenation. Dolomitic lime was mixed with ground flaxseed, water was added, the mixture was blended in a high-speed turbulizer, and the resulting material was then dried to form a granular matrix. Methods 1 and 2 were tested in 1 study (study 1), and method 3 was tested in 2 studies (studies 2 and 3). In study 1, 60 crossbred yearling steers (BW = 475 ± 55 kg) were used in a randomized complete block design experiment. Steers were fed for 12 d with a diet consisting of 48.73% steam-flaked corn, 35% wet corn gluten feed, 12% corn silage, and 4.27% vitamins and minerals (Control). For the other 4 treatments, a portion of wet corn gluten feed was replaced with 5% of unprocessed or extruded mixtures as described for methods 1 and 2. Steers were weighed, and jugular blood samples were taken for analysis of long-chain fatty acids (LCFA) on d 0 and 12 of the study. Both methods failed to improve resistance of PUFA against biohydrogenation (P > 0.1). In study 2, in situ fatty acid disappearance was evaluated for ground flaxseed (Flaxseed) or L-Flaxseed using 6 ruminally fistulated Holstein steers. The proportion of α-linolenic acid (ALA) that was resistant to ruminal biohydrogenation was approximately 2-fold greater for L-Flaxseed than for Flaxseed (P < 0.05). In study 3, 45 steers (269 ± 19.5 kg initial BW) were used in a randomized complete block design. Steers were fed diets containing 0% Flaxseed (No Flaxseed), and in treatments 2 and 3, a portion of flaked corn was replaced with Flaxseed or L-Flaxseed. Animals were weighed and blood samples were taken on d 0, 7, and 14 of the study, and LCFA were analyzed. The use of L-Flaxseed in study 3 increased plasma concentrations of ALA to more than 4 times the level observed in cattle fed unprotected flaxseed, suggesting the dolomitic lime hydrate was effective as a protective barrier against biohydrogenation.

Effect of Megasphaera elsdenii NCIMB 41125 dosing on rumen development, volatile fatty acid production and blood ß-hydroxybutyrate in neonatal dairy calves.

Thirty calves were randomly assigned to two treatments and fed until weaning [42 days (d) of age]. Treatments were a control group (n = 15), which did not receive Megasphaera elsdenii (Me0) and a M. elsdenii group, which received a 50-ml oral dose of M. elsdenii NCIMB 41125 (10(8) CFU/ml) at day 14 day of age (Me14). Calves were given colostrum for the first 3 day followed by limited whole milk feeding. A commercial calf starter was offered ad libitum starting at day 4 until the end of the study. Fresh water was available throughout the study. Feed intake and growth were measured. Blood samples were collected via jugular venipuncture to determine ß-hydroxybutyrate (BHBA) concentrations. Fourteen male calves (seven per group) were euthanised on day 42 and digestive tracts harvested. Reticulo-rumen weight was determined and rumen tissue samples collected from the cranial and caudal sacs of the ventral and dorsal portions of the rumen for measurements of papillae length, papillae width and rumen wall thickness. Dosing with M. elsdenii NCIMB 41125 improved starter dry matter intake (DMI), weaning body weight (BW) and tended to improve average daily gain. Calves in Me14 group had greater plasma BHBA concentration than Me0-calves during the last 3 weeks of the trial and had at day 42 greater reticulo-rumen weight, papillae width and papillae density compared to Me0. No differences in rumen wall thickness or papillae length were observed between the two groups. Total volatile fatty acids, acetate and propionate production did not differ between treatments, but butyrate production was greater in Me14 than Me0. Dosing M. elsdenii NCIMB 41125 showed benefit for calves with improved feed intake and rumen development suggesting increased epithelium metabolism and improved absorption of digestive end products.

Effects of in-feed copper and tylosin supplementations on copper and antimicrobial resistance in faecal enterococci of feedlot cattle.

AIMS: The objective was to investigate whether in-feed supplementation of copper, at elevated level, co-selects for macrolide resistance in faecal enterococci. METHODS AND RESULTS: The study was conducted in cattle (n = 80) with a 2 × 2 factorial design of copper (10 or 100 mg kg(-1) of feed) and tylosin (0 or 10 mg kg(-1) of feed). Thirty-seven isolates (4·6%; 37/800) of faecal enterococci were positive for the tcrB and all were Enterococcus faecium. The prevalence was higher among cattle fed diets with copper and tylosin (8·5%) compared to control (2·0%), copper (4·5%) and tylosin (3·5%) alone. All tcrB-positive isolates were positive for erm(B) and tet(M) genes. Median copper minimum inhibitory concentrations (MICs) for tcrB-positive and tcrB-negative enterococci were 20 and 4 mmol l(-1) , respectively. CONCLUSIONS: Feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B)-mediated copper and tylosin-resistant faecal enterococci in feedlot cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: In-feed supplementation of elevated dietary copper has the potential to co-select for macrolide resistance. Further studies are warranted to investigate the factors involved in maintenance and dissemination of the resistance determinants and their co-selection mechanism in relation to feed-grade antimicrobials' usage in feedlot cattle.

Effects of feeding diets rich in α-linolenic acid and copper on performance, carcass characteristics, and fatty acid profiles of feedlot heifers.

Our objective was to evaluate whether feeding elevated Cu concentrations in conjunction with Linpro, a co-extruded blend of field peas and flaxseed, affected in vitro fermentation, performance, and plasma lipid profiles of fattening beef heifers. In study 1, 2 in vitro trials were conducted as randomized complete experiments with a 2×2 factorial treatment arrangement (10 or 100 mg/kg added Cu and 0 or 10% Linpro, DM basis) to determine VFA/gas production and IVDMD. Linpro contains 12% α-linolenic acid and added vitamins and minerals. In study 2, a randomized complete block experiment with a 2×2 factorial treatment arrangement was conducted with the same previously described treatment. Crossbred yearling heifers (n=261; 351±23 kg initial BW) were blocked by weight into heavy and light groups and randomly assigned to experimental pens containing 10 or 11 heifers each. In study 1, no interactions between levels of Cu and Linpro were observed. Copper concentration did not affect IVDMD (P>0.2) but increased (P<0.05) by 1.2% when Linpro was included. Final pH was not effected by added Cu (P>0.05), but pH increased when Linpro was added (P<0.05). Total VFA were greater in high-Cu treatments (P=0.038) and molar proportions were not affected (P>0.34). Linpro had no effect on total VFA (P=0.46) and molar proportions of propionate and isobutyrate increased whereas acetate and the acetate:propionate ratio decreased (P<0.01). Linpro increased the production of H2S (30% higher; P=0.05), and Cu inclusion slightly increased CO2 proportion (64.06 vs. 67.58% for Linpro vs. Cu treatments, respectively). In study 2, there were no interactions between levels of Linpro and supplemental Cu except for plasma n-6:n-3 ratio (P<0.01). Final BW were similar for cattle fed 0 and 10% Linpro (581 vs. 588 kg; P>0.20), but cattle fed diets with Linpro consumed less feed (14.08 vs. 13.59 kg/d; P<0.05) and were therefore more efficient (0.129 vs. 0.137 for 0 vs. 10% Linpro, respectively; P<0.01). Carcass traits were not affected by treatment. Feeding elevated levels of Cu did not appreciably alter PUFA proportions in plasma and LM. Plasma and LM concentrations of omega-3 fatty acids, including C18:3, C20:5, and C22:5, were greater for heifers fed Linpro (P<0.05). Increasing dietary Cu was not effective as a strategy for decreasing ruminal biohydrogenation and subsequent tissue deposition of PUFA.

Capacity of the bovine intestinal mucus and its components to support growth of Escherichia coli O157:H7.

Colonization of the gastrointestinal tract of cattle by Shiga toxin-producing Escherichia coli increases the risk of contamination of food products at slaughter. Our study aimed to shed more light on the mechanisms used by E. coli O157:H7 to thrive and compete with other bacteria in the gastrointestinal tract of cattle. We evaluated, in vitro, bovine intestinal mucus and its constituents in terms of their capacity to support growth of E. coli O157:H7 in presence or absence of fecal inoculum, with and without various enzymes. Growth of E. coli O157:H7 and total anaerobic bacteria were proportionate to the amount of mucus added as substrate. Growth of E. coli O157:H7 was similar for small and large intestinal mucus as substrate, and was partially inhibited with addition of fecal inoculum to cultures, presumably due to competition from other organisms. Whole mucus stimulated growth to the greatest degree compared with other compounds evaluated, but the pathogen was capable of utilizing all substrates to some extent. Addition of enzymes to cultures failed to impact growth of E. coli O157:H7 except for neuraminidase, which resulted in greater growth of E. coli O157 when combined with sialic acid as substrate. In conclusion, E. coli O157 has capacity to utilize small or large intestinal mucus, and growth is greatest with whole mucus compared with individual mucus components. There are two possible explanations for these findings (i) multiple substrates are needed to optimize growth, or alternatively, (ii) a component of mucus not evaluated in this experiment is a key ingredient for optimal growth of E. coli O157:H7.

Transit effects on fecal Escherichia coli O157 prevalence and coliform concentrations in feedlot cattle.

Our objectives were to evaluate the effects of transportation and lairage on fecal shedding of Escherichia coli O157 (E. coli O157), total Escherichia coli, and total coliforms in feedlot cattle, and the relationships between E. coli O157 prevalence and total E. coli population. The study was a randomized complete block design with a split-plot including 2 treatments: a nontransported group, which remained in its pen at all times, and a transported group, which was transported for 1 h in a trailer and subsequently unloaded in a different pen. The experiment was repeated on 3 different days (blocking factor) with 20 steers/d (10 steers/treatment, 60 total). Fecal samples were taken pretransport (h 0) and after 4 and 28 h, lairage from freshly voided fecal pats were taken from each animal. One gram of feces was transferred to a PBS tube, serially diluted, and plated onto Petrifilm for enumeration of total coliforms. Another sample (1 g) was added to gram-negative broth containing cefixime, cefsulodin, and vancomycin, and subjected to immunomagnetic separation. Resulting beads were plated onto MacConkey agar with sorbitol, cefixime, and tellurite. Nonsorbitol fermenting colonies were selected and tested for indole production and O157 antigen agglutination. Results were confirmed using an API 20E kit. Prevalence of E. coli O157 was transient across blocks. E. coli O157 prevalence revealed no treatment × sampling time interaction (P = 0.179) or sampling time effect (P = 0.937), but a tendency for a treatment effect (P = 0.092). Numbers of E. coli and other coliforms did not change across blocks. No effect of treatment (P > 0.7) was observed on total E. coli concentrations or total coliforms. However, tendencies for treatment × sampling time interactions were observed on both populations (P < 0.08), as well as a tendency for a sampling time effect on total E. coli (P = 0.087) and an effect on total coliforms (P = 0.004). Prevalence of E. coli O157 was not correlated with the concentration of total E. coli (P = 0.954). Results suggest that shedding of E. coli O157 and coliforms can vary within a period of 29 h. Greater statistical power and pathogen quantification, as well as hide sampling and stress-related measurements, are needed to be able to conclude on the effects of transport stress on E. coli O157 prevalence and the changes undergone in pathogen shedding patterns after transportation.

Interaction of Bacillus species and Salmonella enterica serovar Typhimurium in immune or inflammatory signaling from swine intestinal epithelial cells.

Previous research evaluated a laboratory strain of Bacillus licheniformis (BL) in a model swine epithelium and found it exerted antiinflammatory effects on Salmonella enterica serovar Typhimurium (Sal)-induced secretion of IL-8. The current investigation evaluated the antiinflammatory actions of Bacillus bacteria available commercially as feed additives for the swine industry. Three isolates were obtained from the product, 2 Bacillus subtilis (BS1 and BS3) and 1 BL (BL2). Swine jejunal epithelial IPEC-J2 cells were seeded into wells on permeable membrane supports and allowed to form confluent monolayers. Treatments included apical pretreatment with BL, BS1, BL2, or BS3 for 17 h without Sal, and the same Bacillus treatments but with 10(8) cfu of Sal added in the final hour of Bacillus incubation. Two additional treatments included negative control wells receiving no bacteria (control) and positive control wells receiving only Sal (10 total treatments). After bacterial incubation, wells were washed and fresh medium containing gentamicin was added. Cells were incubated for an additional 5 h, after which apical and basolateral media were recovered for determination of IL-8 and bacitracin. In addition, inserts with epithelial cells that had received Sal were lysed and lysates were cultured to determine treatment effects on Sal invasion. Exposure to Sal alone provoked an increase in IL-8 secretion from IPEC-J2 cells compared with control wells (P < 0.001 for both the apical and basolateral directions). Pretreatment with each Bacillus isolate followed by challenge with Sal reduced Sal-induced IL-8 secretion in both the apical and basolateral compartments compared with wells receiving only Sal (P < 0.001; except for BS3 apical, P < 0.01). The residual presence of bacitracin could be detected only in BL2 and BL2+Sal. Fewer Sal colonies could be cultured from lysates of BL2+Sal than from the Sal, BS1+Sal, and BS3+Sal treatments (P < 0.001). Results indicate that B. subtilis and BL have the ability to intervene in secretion of the neutrophil chemoattractant IL-8 from swine intestinal epithelial cells. This effect on chemokine secretion by gastrointestinal epithelial cells in vitro could not be explained solely by reduced invasion of epithelial cells by Sal.