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Targeting the gastrointestinal microbiome for improvement of feed efficiency and reduction of production costs is a potential promising strategy. However little progress has been made in manipulation of the gut microbiomes in dairy cattle to improve milk yield and milk quality. Even less understood is the milk microbiome. Understanding the milk microbiome may provide insight into how the microbiota correlate with milk yield and milk quality. The objective of this study was to characterize similarities between rumen, fecal, and milk microbiota simultaneously, and to investigate associations between microbiota, milk somatic cell count (SCC), and milk yield. A total of 51 mid-lactation, multiparous Holstein dairy cattle were chosen for sampling of ruminal, fecal, and milk contents that were processed for microbial DNA extraction and sequencing. Cows were categorized based on low, medium, and high SCC; as well as low, medium, and high milk yield. Beta diversity indicated that ruminal, fecal, and milk populations were distinct ( p  < 0.001). Additionally, the Shannon index demonstrated that ruminal microbial populations were more diverse ( p  < 0.05) than were fecal and milk populations, and milk microbiota was the least diverse of all sample types ( p  < 0.001). While diversity indices were not linked ( p  > 0.1) with milk yield, milk microbial populations from cows with low SCC demonstrated a more evenly distributed microbiome in comparison to cows with high SCC values ( p  = 0.053). These data demonstrate the complexity of host microbiomes both in the gut and mammary gland. Further, we conclude that there is a significant relationship between mammary health (i.e., SCC) and the milk microbiome. Whether this microbiome could be utilized in efforts to protect the mammary gland remains unclear, but should be explored in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Williamson, Callaway, Lourenco and Ryman.)

This study was conducted to investigate the effects of different dosages of tannic acid (TA) on growth performance, nutrient digestibility, gut health, immune system, oxidative status, microbial composition, volatile fatty acids (VFA), bone mineral density, and fat digestion and accumulation in broilers and to find optimal dosages of TA for efficient growth and gut health in broilers. A total of 320 male Cobb500 broilers were randomly distributed to 4 treatments with 8 replicates including 1) tannic acid 0 (TA0): basal diet without TA; 2) tannic acid 0.5 (TA0.5): basal diet with 0.5 g/kg TA; 3) tannic acid 1.5 (TA1.5); and 4) tannic acid 2.5 (TA2.5). Supplemental TA at levels greater than 972 mg/kg tended to reduce BW on D 21 ( p = 0.05). The TA2.5 had significantly lower apparent ileal digestibility (AID) of crude protein compared to the TA0 group. The AID of ether extract tended to be reduced by TA at levels greater than 525 mg/kg ( p = 0.08). The jejunal lipase activities tended to be reduced by TA at levels less than 595.3 mg/kg ( p = 0.09). TA linearly decreased goblet cell density in the crypts of the jejunum ( p < 0.05) and reduced mRNA expression of mucin two at levels less than 784.9 mg/kg and zonula occludens two at levels less than 892.6 mg/kg ( p < 0.05). The TA0.5 group had higher activities of liver superoxide dismutase compared to the TA0 group ( p < 0.05). Bone mineral density and contents tended to be linearly decreased by TA ( p = 0.05), and the ratio of lean to fat was linearly decreased ( p < 0.01). Total cecal VFA production tended to be linearly reduced by TA at levels greater than 850.9 mg/kg ( p = 0.07). Supplemental TA tended to increase the relative abundance of the phylum Bacteroidetes ( p = 0.1) and decrease the relative abundance of the phylum Proteobacteria ( p = 0.1). The relative abundance of the family Rikenellaceae was the lowest at 500 mg/kg TA, and the relative abundance of the family Bacillaceae was the highest at 1,045 mg/kg TA. Collectively, these results indicate that the optimum level of supplemental TA would range between 500 and 900 mg/kg; this range of TA supplementation would improve gut health without negatively affecting growth performance in broilers under antibiotic-free conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Choi, Yadav, Wang, Lorentz, Lourenco, Callaway and Kim.)

Johnsongrass [ Sorghum halepense (L.) Pers.] is a non-native, invasive species that causes substantial losses in row crops and hay fields, which could be minimized by using Johnsongrass as a conserved forage. Two experiments were conducted to evaluate the yield and quality of Johnsongrass ensiled at four maturities: harvested every 3 weeks (3WK), boot stage (BOOT), flower stage (FLOWER), and dough (DOUGH) stages. In experiment 1, yield, botanical composition, nutritive value, and fermentation characteristics of Johnsongrass were measured. In experiment 2, Johnsongrass silage was incubated for 48 h for assessment of gas production, pH, in vitro dry matter digestibility (IVDMD), and volatile fatty acids. The experimental area consisted of 16 plots (2.74 m × 4.57 m) divided into four blocks, and treatment was randomly assigned to plot within block. Each year, silage was prepared for each plot from the two cutting closest to July 1. After 10 weeks, the silos were opened, and silage samples were frozen for further analysis. Data from both experiments were tested for the effects of maturity stage and harvest timing (first and second harvest). The results from experiment 1 showed an increase ( P < 0.0001) in dry matter yield from 3WK stage to DOUGH. Johnsongrass, as a proportion of the total botanical composition, declined at the end of the growing season for 3WK but increased in FLOWER ( P = 0.0010). In the first harvest, 3WK and BOOT stage silages had the greatest concentrations of crude protein and total digestible nutrients and lowest of fiber (neutral detergent fiber and acid detergent fiber; P < 0.0001). In the second harvest, differences in nutrient content were significant only for 3WK silages, which showed the best nutritive value ( P < 0.0001). In experiment 2, IVDMD of silage followed the same trends described for nutritive value from experiment 1. Overall, these results demonstrate that Johnsongrass can be successfully ensiled, but to optimize forage nutritive value and quantity, Johnsongrass should be ensiled before it reaches the flower stage., (Published by Oxford University Press for the American Society of Animal Science 2022.)

The microbial population in the gastrointestinal tract of ruminant animals aids in the utilization of forages with high levels of secondary plant compounds. Two divergent bloodlines of meat goats have been selected by screening fecal samples with near-infrared reflectance spectroscopy to assess the goat's consumption of high or low levels of Juniperus sp. leaves containing several monoterpenes, including camphor. The mechanism by which these goats can consume greater concentrations of Juniperus spp. leaves than their counterparts is unclear, and therefore, this study was designed to determine if differences existed between the ruminal microbial populations of the low and high juniper-consuming bloodlines (LJC vs. HJC) by analyzing their ruminal microbiota and fermentation end products. In the present study, concentrations (0.00, 0.5, 0.99, 1.97, or 5.91 mM) of camphor were added to mixed ruminal microorganism fermentation. Five LJC and five HJC goats were fed a juniper-free diet ( n = 10), and five LJC and five HJC goats ( n = 10) were fed a diet that contained 30% fresh Juniperus ashei leaves for 21 d prior to ruminal fluid collection. In vitro fermentations used LJC and HJC, ruminal fluid inoculum added (33% v/v) to anoxic media in sealed Balch tubes. Camphor increased ( P < 0.05) total short-chain fatty acid (SCFA) concentrations for all but one experimental group. Between the main dietary and bloodline goat effects, the diet was significant for all SCFA results except butyrate. In contrast, bloodline was only significant for acetate and butyrate molar proportions. Rumen fluid from juniper-free-fed goats exhibited greater concentrations of Ruminococcaceae , whereas juniper-fed goats contained more Coriobacteriaceae . Results demonstrated that mixed ruminal microorganisms fermentations from HJC goats did not produce greater concentrations of SCFAs or have the ability to degrade camphor at a higher rate than did that from LJC goats. Results suggest that camphor tolerance from J. ashei , was related to hepatic catabolic mechanisms instead of ruminal microbial degradation; however, further in vivo work is warranted., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Encroaching plant species in West Texas continues to significantly reduce livestock production capacity and cause an imbalance between plant and livestock ecology. Juniperus ashei and Juniperus pinchotii are encroaching species rarely used by browsing animals, mainly due to phytochemical defenses. Juniperus spp. contain large concentrations of monoterpenes in their essential oil profiles to deter herbivory. Since 2003, two divergent bloodlines of meat goats have been selected to consume low or high (LJC or HJC) amounts of juniper foliage, through screening fecal samples with near-infrared reflectance spectroscopy. However, it remains unclear whether HJC goats expressed a physiological ability to consume a greater amount of juniper or if they were colonized by a ruminal microbial population that could detoxify juniper phytochemicals. Therefore, this study aimed to investigate the impact of 0.00 and 1.97 mM of camphor on the mixed ruminal microorganism fermentation after 0, 1, 2, 4, 12, and 24 h of incubation. Five LJC and five HJC goats were fed a juniper-free diet ( n = 10) and five LJC and five HJC goats ( n = 10), were fed a diet containing 30% fresh J. ashei leaves for 21 days prior to ruminal fluid collection. In vitro fermentations used LJC and HJC, ruminal fluid inoculum was added (33% v/v) to anoxic media in sealed Balch tubes. Total short-chain fatty acid (SCFA) production and acetate to propionate ratio were increased ( P < 0.05), but there was no effect on pH (6.56 ± 0.09). Goats that received the juniper-free diet had higher ( P < 0.05) SCFA production than juniper-containing diets. There was no consistent difference in LJC and HJC microbial fermentation end products caused by the addition of 1.97 mM of camphor, and goats receiving a juniper-free diet consistently generated more SCFAs in the presence of 1.97 mM of camphor. Furthermore, bloodline differences in juniper consumption were likely related to physiological adaption capacities within the animal and not a ruminal microbial detoxification advantage., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Dry seasons pose a major nutritional constraint on ruminant livestock production in tropical regions, which justifies forage conservation to meet the dry season feed requirement. Napier grass is a tropical forage that is used for silage in South Africa. The present objective was to determine the effects of Napier grass fertilization with bio-digester slurry (BDS) and the inclusion of fermentable carbohydrate additives at ensiling on the chemical composition and ruminal degradability of Napier grass silage. Napier grass was established in 5 × 4 m plots, replicated three times in a completely randomized design, and irrigated weekly with either BDS or water. After 12 weeks, the Napier was cut and ensiled for 90 days in 1-liter glass jars in a 2 (BDS, water) × 4 (no-additive, molasses, brown sugar, and maize meal) factorial arrangement replicated three times. The nutrient composition was determined using standard protocols. The ruminal degradability of dry matter (DM) and crude protein (CP) was determined using the nylon bag technique. Fertilization with BDS increased ( P < 0.05) pH and CP and reduced ( P < 0.05) fat content of fresh-cut Napier. Additives increased ( P < 0.01) silage DM content and reduced ( P < 0.01) acid detergent fiber, neutral detergent fiber content. The BDS fertilization with molasses inclusion increased ( P < 0.05) silage DM relative to the no-additive and maize meal inclusion, and decreased ( P < 0.05) fat content compared to the no-fertilizer, added maize meal silage. Molasses increased silage water-soluble carbohydrate and decreased the NH 3 -N content ( P < 0.05) compared to the no-additive and maize meal treatments. For DM, the BDS fertilized, no additive silage had the least "a" fraction ( P < 0.01), while the no BDS, no-additive silage had the least "b" fraction ( P < 0.01), with least ( P < 0.01) potential degradability (PD) observed for the no BDS, no-additive treatment. Fertilization increased ( P < 0.01) effective degradability of DM at outflow rates k = 0.02, 0.05, 0.08, with same effect for molasses and maize meal inclusion. Relative to the control, molasses inclusion increased ( P < 0.01) PD of silage CP. In conclusion, our results suggested BDS fertilization of Napier grass ensiling with added readily fermentable carbohydrate substrate, particularly from molasses, induced changes in silage chemical and fermentation characteristics likely to promote better forage preservation and ruminal microbial function., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The gastrointestinal microbiota of cattle is important for feedstuff degradation and feed efficiency determination. This study evaluated the fecal microbiome of Angus steers with distinct feed efficiencies during the feedlot-finishing phase. Angus steers ( n = 65), fed a feedlot-finishing diet for 82 days, had growth performance metrics evaluated. Steers were ranked based upon residual feed intake (RFI), and the 5 lowest RFI (most efficient) and 5 highest RFI (least efficient) steers were selected for evaluation. Fecal samples were collected on 0-d and 82-d of the finishing period and microbial DNA was extracted and evaluated by 16S rRNA gene sequencing. During the feedlot trial, inefficient steers had decreased ( p = 0.02) Ruminococcaceae populations and increased ( p = 0.01) Clostridiaceae populations. Conversely, efficient steers had increased Peptostreptococcaceae ( p = 0.03) and Turicibacteraceae ( p = 0.01), and a trend for decreased Proteobacteria abundance ( p = 0.096). Efficient steers had increased microbial richness and diversity during the feedlot period, which likely resulted in increased fiber-degrading enzymes in their hindgut, allowing them to extract more energy from the feed. Results suggest that cattle with better feed efficiency have greater diversity of hindgut microorganisms, resulting in more enzymes available for digestion, and improving energy harvest in the gut of efficient cattle.

Nematopsis messor infections severely impact on shrimp’s health with devastating economic consequences on shrimp farming. In a shrimp primary intestinal cells (SGP) model of infection, a sub-inhibitory concentration (0.5%) of natural antimicrobials (Aq) was able to reduce the ability of N. messor to infect (p < 0.0001). To prevent N. messor infection of SGP cells, Aq inhibits host actin polymerization and restores tight junction integrity (TEER) and the expression of Zo-1 and occluding. The oxidative burst, caused by N. messor infection, is attenuated by Aq through the inhibition of NADPH-produced H2O2. Simultaneous to the reduction in H2O2 released, the activity of catalase (CAT) and superoxide dismutase (SOD) were also significantly increase (p < 0.0001). The antimicrobial mixture inactivates the ERK signal transduction pathway by tyrosine dephosphorylation and reduces the expression of DCR2, ALF-A, and ALF-C antimicrobial peptides. The observed in vitro results were also translated in vivo, whereby the use of a shrimp challenge test, we show that in N. messor infected shrimp the mortality rate was 68% compared to the Aq-treated group where the mortality rate was maintained at 14%. The significant increase in CAT and SOD activity in treated and infected shrimp suggested an in vivo antioxidant role for Aq. In conclusion, our study shows that Aq can efficiently reduce N. messor colonization of shrimp’s intestinal cells in vitro and in vivo and the oxidative induced cellular damage, repairs epithelial integrity, and enhances gut immunity.

The gastrointestinal tract, or gut, microbiota is a microbial community containing a variety of microorganisms colonizing throughout the gut that plays a crucial role in animal health, growth performance, and welfare. The gut microbiota is closely associated with the quality and microbiological safety of foods and food products originating from animals. The gut microbiota of the host can be modulated and enhanced in ways that improve the quality and safety of foods of animal origin. Probiotics-also known as direct-fed microbials-competitive exclusion cultures, prebiotics, and synbiotics have been utilized to achieve this goal. Reducing foodborne pathogen colonization in the gut prior to slaughter and enhancing the chemical, nutritional, or sensory characteristics of foods (e.g., meat, milk, and eggs) are two of many positive outcomes derived from the use of these competitive enhancement-based treatments in food-producing animals.

The objective of this study was to investigate the effect of a diet supplemented with fresh amla fruit as a natural feed additive on blood metabolic parameters, milk antioxidant capacity, and milk fatty acid (FA) proportions in lactating dairy cows. Eight ruminally cannulated mid-lactation dairy cows were used in a repeated crossover design. The first group of four cows received total mixed ration (TMR) feed without fresh amla fruit (control group). The remaining four cows sequentially supplemented fresh amla fruit (FAF) at three levels (200, 400, then 600 g/d) (treatment group) at 14-day intervals. In second period, control and treatment groups were exchanged. The first ten days were adjusted to diet adaptation for each sub-period, and the last four days for sampling milk and blood. A total of 514 metabolites were detected from FAF using UPLC-ESI-MS/MS. The five main metabolites in FAF were phenolic acids (22%), flavonoids (20%), lipids (20%), amino acids and derivatives (9%), and tannins (7%). Amla fruit supplementation reduced total saturated fatty acid and the omega-6/omega-3 ratio at 200 or 400 g/d FAF dose compared to controls. In addition, amla fruit increased unsaturated FA, such as C 20:5 (Eicosapentaenoic acid, EPA) and C 22:6 (docosahexaenoic acid, DHA), and branched-chain FA in a dose-dependent manner at 200 or 400 g/d compared to controls. In addition, amla fruit increased the antioxidant capacity biomarkers in the blood, such as superoxide dismutase (SOD) and albumin; this confirms that amla fruit is an excellent antioxidant, inhibiting reactive oxygen species' (ROS) metabolism, and can thereby protect cells from oxidative stress. Moreover, the most remarkable improvement of ferric reducing-antioxidant power (FRAP) and total antioxidant capacity (TAC) in milk was recorded at 400 g/d FAF doses compared to controls. Therefore, fresh amla fruit doses for lactating cows at 400 g/d on an as-fed basis can be used as an alternative additive feed in dairy cow diets to improve antioxidant capacity, protein efficiency, butter quality, and to produce more desirable milk fatty acid profiles for human consumption.

The purpose of the study was to investigate effects of different inoculation dosages of E. tenella on growth performance, gastrointestinal permeability, oocyst shedding, intestinal morphology, fecal consistency, ileal apparent digestibility, antioxidant capacity, and cecal VFA profile in broiler chickens. Five different dosages (T0: 0, T1: 6250, T2: 12,500, T3: 25,000, and T4: 50,000) of E. tenella oocysts were inoculated via oral gavage to fourteen-day-old broilers. Inoculation of E. tenella linearly increased FCR ( p < 0.05), and feed intake was quadratically increased on 6 days post-infection (dpi; p = 0.08) and 7 dpi ( p = 0.09). Cecal lesion score of each treatment was T0: 0; T1: 0.39 ± 0.14; T2: 0.93 ± 0.21; T3: 1.25 ± 0.16; and T4: 1.58 ± 0.2. Cecal total VFA production was linearly reduced due to E. tenella infection on 6 dpi ( p < 0.01). E. tenella infection deepened cecal crypts depth on 6 dpi (CD; p < 0.05). Gastrointestinal permeability tended to be linearly increased ( p = 0.07). E. tenella infection tended to linearly reduce duodenal VH ( p = 0.1) and jejunal VH on 9 dpi ( p = 0.09). Different dosages of E. tenella modulated the tendency of fecal moisture content and oocyst shedding. Therefore, E. tenella infection impaired feed efficiency and small intestinal health mainly by reducing cecal VFA production and deepening cecal CD in broilers.

Numerous studies have examined the link between the presence of specific gastrointestinal bacteria and the feed efficiency of cattle. However, cattle undergo dietary changes during their productive life which can cause fluctuations in their microbial consortium. The objective of the present study was to assess changes in the fecal microbiome of beef steers genetically selected to be divergent in feedlot feed efficiency, to determine whether differences in their fecal microbiomes could be detected as early as weaning, and continued throughout the rearing process regardless of dietary changes. Fecal samples were collected at weaning, yearling age, and slaughter for a group of 63 steers. Based on their feedlot-finishing performance, the steers were selected and divided into two groups according to their residual feed intake ( RFI ): efficient steers (low-RFI; n = 7) and inefficient steers (high-RFI; n = 8). To ascertain the fecal microbial consortium and volatile fatty acid (VFA) content, 16S rRNA gene sequencing and VFA analysis were performed. Overall, bacterial evenness and diversity were greater at weaning compared to yearling and slaughter for both efficiency groups ( P < 0.001). Feedlot RFI linearly decreased as both Shannon diversity and Ruminococcaceae abundance increased ( R 2 = 65.6 and 60.7%, respectively). Abundances of Ruminococcaceae, Rikenellaceae , and Christensenellaceae were higher at weaning vs. yearling age and slaughter ( P < 0.001); moreover, these families were consistently more abundant in the feces of the low-RFI steers (for most of the timepoints evaluated; P ≤ 0.05), compared to the high-RFI steers. Conversely, abundances of Bifidobacteriaceae were numerically higher in the feces of the high-RFI steers throughout their lifespan. Total VFA concentrations increased at slaughter compared to weaning and yearling for both efficiency groups ( P < 0.001). The acetate:propionate ratio decreased linearly ( P < 0.001) throughout the life of the steers regardless of their efficiency, reflective of dietary changes. Our results indicate that despite fluctuations due to animal age and dietary changes, specific bacterial families may be correlated with feed efficiency of steers. Furthermore, such differences may be identifiable at earlier stages of the production cycle, potentially as early as weaning., Competing Interests: The authors declare that this study received funding from Brasstown Beef LLC. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. Brasstown Beef LLC graciously provided intake monitored pens and labor during the study., (Copyright © 2021 Welch, Lourenco, Krause, Seidel, Fluharty, Pringle and Callaway.)

This study investigated the use of carbadox in the diet of nursery pigs. Ten pens of weanling piglets were assigned to 2 treatments: one containing carbadox and another without it. From days 21 to 35 of age, the first group of piglets was fed carbadox at 55 mg/kg of diet; followed by 27.5 mg/kg from days 36 to 49; and 0 mg/kg from days 50 to 63. The second group of pigs was fed a control diet without carbadox from days 21 to 63 of age. On days 35, 49, and 63, fecal samples were collected directly from the rectum of 2 piglets in each pen, and the samples were subjected to microbial DNA sequencing and metagenomic functional analysis using the 16S rRNA gene. Feed conversion from days 21 to 63 was improved ( P = 0.04) in the group of piglets fed carbadox. Faith's phylogenetic diversity was similar ( P = 0.89) for both groups of piglets on day 35, but it was diminished ( P = 0.01) in the carbadox-fed group on day 49; however, following the complete removal of carbadox from their diets, this microbial diversity index was once again found to be similar ( P = 0.27) in both groups on day 63. Likewise, abundances of Slackia, Peptococcus, Catenibacterium, Coprococcus , and Blautia were all similar between the two groups ( P ≥ 0.40) on day 35, but were smaller in the carbadox group ( P ≤ 0.05) on day 49; however, on day 63, abundances of all these genera were once again similar ( P ≥ 0.29). Metabolic pathways involved in cellular growth, death, and genetic information processing (translation) were found to be similarly expressed in the microbiota of piglets from both groups on day 35 ( P ≥ 0.52), but decreased in the carbadox group on day 49 ( P ≤ 0.05), and were similar again in both groups on day 63 ( P ≥ 0.51). These results revealed that feeding carbadox to piglets during the first 4 weeks after weaning significantly affected their fecal microbiotas; however, 2 weeks after the removal of carbadox, those changes tended to disappear, indicating that the shifts were carbadox-dependent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lourenco, Hampton, Johnson, Callaway, Rothrock and Azain.)

Stroke is a major cause of death and long-term disability affecting seven million adults in the United States each year. Recently, it has been demonstrated that neurological diseases, associated pathology, and susceptibility changes correlated with changes in the gut microbiota. However, changes in the microbial community in stroke has not been well characterized. The acute stage of stroke is a critical period for assessing injury severity, therapeutic intervention, and clinical prognosis. We investigated the changes in the gut microbiota composition and diversity using a middle cerebral artery (MCA) occlusion ischemic stroke pig model. Ischemic stroke was induced by cauterization of the MCA in pigs. Blood samples were collected prestroke and 4 h, 12 h, 1 day, and 5 days poststroke to evaluate circulating proinflammatory cytokines. Fecal samples were collected prestroke and 1, 3, and 5 days poststroke to assess gut microbiome changes. Results showed elevated systemic inflammation with increased plasma levels of tumor necrosis factor alpha at 4 h and interleukin-6 at 12 h poststroke, relative to prestroke. Microbial diversity and evenness were reduced at 1 day poststroke compared to prestroke. Microbial diversity at 3 days poststroke was negatively correlated with lesion volume. Moreover, beta-diversity analysis revealed trending overall differences over time, with the most significant changes in microbial patterns observed between prestroke and 3 days poststroke. Abundance of the Proteobacteria was significantly increased, while Firmicutes decreased at 3 days poststroke, compared to prestroke populations. Abundance of the lactic acid bacteria Lactobacillus was reduced at 3 days poststroke. By day 5, the microbial pattern returned to similar values as prestroke, suggesting the plasticity of gut microbiome in an acute period of stroke in a pig model. These findings provide a basis for characterizing gut microbial changes during the acute stage of stroke, which can be used to assess stroke pathology and the potential development of therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Jeon, Lourenco, Kaiser, Waters, Scheulin, Fang, Kinder, Platt, Rothrock, Callaway, West and Park.)

The objective of this study was to determine if immunosuppression through daily dexamethasone (DEX) infusion altered Salmonella translocation from the gastrointestinal tract. Weaned Holstein steers ( n  = 20; body weight [BW] = 102 ± 2.7 kg) received DEX ( n  = 10; 0.5 mg/kg BW) or saline (control [CON]; n  = 10;) for 4 days (from day -1 to 2) before oral inoculation of naldixic acid-resistant Salmonella enterica Typhimurium (SAL; 3.4 × 10 6 colony-forming units [CFU]/animal) on day 0. Fecal swabs were obtained daily, and blood was collected daily for hematology. At harvest (day 5), ileum, cecal fluid, lymph nodes (ileocecal, mandibular, popliteal, and subscapular), and synovial (stifle, coxofemoral, and shoulder) samples were collected for isolation of the inoculated strain of SAL. White blood cell (WBC) and neutrophil concentrations were elevated ( p  < 0.01) in DEX calves following each administration event. Following inoculation, 100% of DEX calves shed the experimental strain of SAL for all 5 days, 90% of CON calves shed from day 1 to 3, and 100% of CON calves shed from day 4 to 5. Greater ( p  < 0.01) concentrations of SAL were quantified from the cecum of DEX calves (3.86 ± 0.37 log CFU/g) compared with CON calves (1.37 ± 0.37 log CFU/g). There was no difference in SAL concentrations between DEX and CON calves in ileal tissue ( p  = 0.07) or ileocecal ( p  = 0.57), mandibular ( p  = 0.12), popliteal ( p  = 0.99), or subscapular ( p  = 0.83) lymph nodes. Of the stifle samples collected, 3.3% were positive for SAL, highlighting a contamination opportunity during hindquarter breakdown. While more research is needed to elucidate the interactions of immunosuppression and pathogen migration patterns, these data confirm that orally inoculated SAL can translocate from the gastrointestinal tract and be harbored in atypical locations representing a food safety risk.

To evaluate the effects of a patented Bacillus subtilis probiotic, weaned Holstein steers, not shedding Salmonella ( n  = 40; ∼90 kg), were supplemented (CLO) or not (CON) with CLOSTAT ® (13 g/hd per day; Kemin Industries, Des Moines, IA) in a starter ration for 35 d. The calves were assigned to one of four treatments in a 2 × 2 factorial design with CLO and CON calves that were orally administered Salmonella (STM) or not (NoSTM). Calves were challenged with 1.6 × 10 6 colony-forming unit (CFU) Salmonella Typhimurium (resistant to 50 μg/mL nalidixic acid) in 1 L of milk replacer on day 0. Blood samples were collected through jugular catheters every 6 h for 96 h, and body temperature was measured every 5 min through indwelling rectal temperature recording devices. Five calves from each treatment were harvested 48 h postchallenge, and the remaining calves were harvested 96 h postchallenge. During necropsy, tissues were collected for the isolation and quantification of the inoculated STM from various tissues. The CLOSTM group had reduced STM concentrations in the jejunum, ileum, and transverse colon 48 h after the challenge ( p  ≤ 0.03), but were not different 96 h postchallenge ( p  > 0.05). Decreased ( p  < 0.01) pyrexia was observed after the challenge in CLOSTM calves when compared with CONSTM calves. White blood cells and lymphocyte counts were increased ( p  ≤ 0.05) in CLOSTM calves after the challenge in comparison with other treatments. In calves given STM, the CLO group had greater feed intake before and after the challenge ( p  < 0.01) compared with the CON group. Increased serum IL-6 and IFN-γ concentrations were observed in the CONSTM group compared with other treatments. Overall, CLO reduced Salmonella presence and concentrations in gastrointestinal tissues while simultaneously reducing the severity of the challenge as indicated by blood parameters and the reduced febrile response.

During the first week after hatch, young chicks are vulnerable to pathogens as the immune system is not fully developed. The objectives of this study were to determine if supplementing the starter diet with a microencapsulated feed additive containing citric and sorbic acids, thymol, and vanillin affects in vitro functional activity of peripheral blood leukocytes (PBLs). Day-old chicks (n = 800) were assigned to either a control diet (0 g/metric ton [MT]) or a diet supplemented with 500 g/MT of the microencapsulated additive. At 4 D of age, peripheral blood was collected (100 birds per treatment), and heterophils and monocytes isolated (n = 4). Heterophils were assayed for the ability to undergo degranulation and production of an oxidative burst response while nitric oxide production was measured in monocytes. Select cytokine and chemokine mRNA expression levels were also determined. Statistical analysis was performed using Student t test comparing the supplemented diet to the control (P ≤ 0.05). Heterophils isolated from chicks fed the microencapsulated citric and sorbic acids, thymol, and vanillin had higher (P ≤ 0.05) levels of degranulation and oxidative burst responses than those isolated from chicks on the control diet. Heterophils from the supplemented chicks also had greater (P ≤ 0.05) expression of IL10, IL1β, and CXCL8 mRNA than those from control-fed chicks. Similarly, nitric oxide production was significantly (P ≤ 0.05) higher in monocytes isolated from birds fed the supplement. The cytokine and chemokine profile in monocytes from the supplement-fed chicks showed a significant (P ≤ 0.05) drop in IL10 mRNA expression while IL1β, IL4, and CXCL8 were unchanged. In conclusion, 4 D of supplementation with a microencapsulated blend made up of citric and sorbic acids, thymol, and vanillin enhanced the in vitro PBL functions of degranulation, oxidative burst, and nitric oxide production compared with the control diet. Collectively, the data suggest feeding broiler chicks a diet supplemented with a microencapsulated blend of citric and sorbic acids, thymol, and vanillin may prime key immune cells making them more functionally efficient and acts as an immune-modulator to boost the inefficient and undeveloped immune system of young chicks., (Copyright © 2020. Published by Elsevier Inc.)

Stressors experienced by pigs at weaning may negatively impact health and productivity. Thus, supplements that enhance pig immunity during the early post-weaned period are of great interest to the swine industry. The objective of this experiment was to evaluate the performance and hematological responses of weaned pigs supplemented with yeast cell wall (YCW) when challenged orally with Salmonella Typhimurium . Weaned pigs were assigned to one of three treatments for 22d ( n = 13/treatment): Control diet, which was a non-medicated starter diet (Control); Control diet supplemented with YCW at 250 mg/kg BW (YCW250; Phileo Lesaffre Animal Care, Milwaukee, Wisconsin, USA); and Control diet supplemented with YCW at 500 mg/kg BW (YCW500). On d19 blood samples were collected from -6 to 72 h relative to oral Salmonella Typhimurium (1 × 10 6 cfu/pig) challenge. Gain:feed was greater ( P = 0.01) in YCW250 treatment compared to both Control and YCW500 pigs. Baseline intraperitoneal temperature was greater ( P < 0.001) in YCW250 pigs than Control or YCW500 pigs. There was a treatment x time interaction for the change in intraperitoneal temperature ( P < 0.01), post-challenge cortisol, white blood cell counts (WBC), neutrophils, and neutrophil:lymphocyte ratio ( P ≤ 0.03). Control pigs had greater ( P < 0.05) cortisol concentrations than both YCW-supplemented groups at 0 h, but Control pigs had reduced ( P < 0.05) cortisol compared to YCW500 pigs at 24 and 30 h post-challenge. Control pigs had greater ( P < 0.05) WBC counts than both YCW-supplemented groups 6 and 12 h post-challenge, and YCW250 pigs had reduced ( P < 0.01) WBC counts than Control and YCW500 pigs 18 h post-challenge. Neutrophil counts were greater ( P < 0.05) in Control pigs than both YCW-supplemented groups at 6 and 12 h post-challenge and were greater ( P = 0.02) than YCW250 pigs at 18 h post-challenge. Lymphocytes were greater ( P < 0.001) in Control and YCW500 pigs pre- and post-challenge compared to YCW250 pigs. Control pigs had the greatest ( P < 0.001) monocyte counts compared to YCW treatments. There was no effect of yeast supplementation on fecal shedding or Salmonella counts in the rectum, colon or cecum ( P ≥ 0.05). While some differences were observed in intraperitoneal temperature and some hematological variables, data suggests there were minimal effects of yeast supplementation on the acute immune response to Salmonella challenge.

A study was conducted to examine the effects of supplementing beef calves during their suckling phase (popularly known as creep feeding) with supplements that contained or did not contain the enzyme xylanase. Forty-two cow-calf pairs were divided into three groups and assigned to one of three treatments for a period of 105 days, as follows: (1) No supplemental feed for calves (control; CON); (2) Corn and soybean meal-based supplement feed for calves (positive control; PCON); and (3) Same feed regimen as PCON with xylanase added to the supplement (enzyme; ENZ). After 105 days, out of the 42 calves participating in the study, 25 male calves were randomly selected (8 from CON, 9 from PCON, and 8 from ENZ) and samples of their forestomach were collected by esophageal tubing. Immediately after this procedure, all calves were weaned, commingled, and placed in a common post-weaning diet for 4 weeks. At the end of this period, ruminal fluid was once again collected from the same 25 calves. All samples were subjected to DNA extraction and 16S rRNA gene sequencing. At weaning, most of the alpha diversity indexes were greater in CON; however, no differences ( P ≥ 0.23) in alpha diversity were observed in samples collected 4 weeks after weaning. Regardless of treatment, 2 phyla - Bacteroidetes and Firmicutes - comprised approximately 80% of the total bacterial abundance of samples collected on both days. At the genus level, an effect of diet ( P = 0.02) was observed for Prevotella in the samples collected at weaning; however, no differences were detected in the samples collected 4 weeks after weaning. Calf average daily gain (ADG) during the 105-day creep feeding trial tended ( P = 0.09) to be greater in the groups that received supplementation, with the greatest numerical value observed in ENZ. Moreover, there was a positive correlation (ρ = 0.43; P = 0.03) between ADG and abundance of Prevotella , indicating the importance of this bacterial group for ruminants. In summary, most of the significant differences found in this study were detected at weaning, and the majority of them disappeared 4 weeks after the calves were weaned and commingled.

Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term "super-shedder" has been applied to cattle that shed concentrations of E. coli O157:H7 ≥ 10⁴ colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates linked to human illness makes it imperative that the etiology of this phenomenon be characterized.

Foodborne pathogenic bacteria can live in the intestinal tract of food animals and can be transmitted to humans via food or indirectly through animal or fecal contact. Organic acid blend products have been used as nonantibiotic modifiers of the gastrointestinal fermentation of food animals to improve growth performance efficiency. However, the impact of these organic acid products on the microbial population, including foodborne pathogens, remains unknown. Therefore, this study was designed to examine the effects of a commercial organic acid and botanical blend product (OABP) on populations of the foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. Pure cultures (2×10(6) colony-forming units [CFU]/mL) of each pathogen were added to tubes that contained water-solubilized OABP added at concentrations of 0, 0.1, 0.5, 1, 2, 5, and 10% (vol/vol; n=3). Water-solubilized OABP reduced (p<0.05) the growth rate and final populations of E. coli O157:H7 and Salmonella Typhimurium in pure culture at concentrations >2%. E. coli O157:H7 and Salmonella Typhimurium were added (2×10(5) and 3×10(6) CFU/mL, respectively) to in vitro mixed ruminal microorganism fermentations that contained water-solubilized OABP at concentrations of 0, 1, 2, 5, and 10% (vol/vol; n=3) that were incubated for 24 h. OABP addition reduced (p<0.05) final populations of E. coli O157:H7 and Salmonella Typhimurium in the ruminal fluid at concentrations ≥5%. The acetate-to-propionate ratios from the in vitro fermentations were reduced (p<0.05) by OABP treatment ≥5%. Treatments to reduce foodborne pathogens must be economically feasible to implement, and results indicate that organic acid products, such as OABP, can enhance animal growth efficiency and can be used to reduce populations of pathogenic bacteria.

The use of luminescent plasmids in bacteria may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding patterns of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth characteristics of E. coli O157:H7 in mixed ruminal and fecal microbial fluid cultures would be altered when transformed with one of the two luminescent plasmids: pAK1-lux (PAK) or pXEN-13 (XEN). Transformants harboring the luminescent plasmids were compared to the non-transformed parental strain (wild type [WT]) after incubating in mixed ruminal or fecal microbial fluid media for 6 h in triplicate (n=3). The transformants and WT exhibited similar growth rates. Within mixed ruminal microbial fluid fermentations and mixed fecal microbial fluid, all transformants grew similarly (p=0.28) through the 6-h study. The reflective light unit (RLU; photons/pixel per second) photonic emissions of each plasmid within ruminal fluid differed at 0 h (p=0.002) and 2 h (p=0.02) and within fecal fluid at 0 h (p=0.009) and 2 h (p=0.04). The RLU remained the same within rumen fluid at 4 h (p=0.22) and 6 h (p=0.80) and within fecal fluid at 4 h (p=0.06) and 6 h (p=0.29). Growth of E. coli O157:H7 transformed with the bioluminescent plasmids was not altered in comparison to the WT, suggesting that both plasmids may serve as useful models for in vivo studies.

While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

Reducing Salmonella in poultry has been a paramount goal of the poultry industry in order to improve food safety. Inclusion of high-fiber fermentable feedstuffs in chicken diets has been shown to reduce the incidence of Salmonella colonization in laying hens, but no work has been performed in growing birds. Therefore, the present study was designed to quantify differences in artificially inoculated cecal Salmonella Typhimurium populations in growing layer chicks (n = 60 in each of two replications) fed 0%, 25%, and 50% of their diet (w/w) replaced with alfalfa meal from day (d) 7 to d14 after hatch. Alfalfa supplementation reduced cecal populations of Salmonella by 0.95 and 1.25 log10 colony-forming unit per gram in the 25% and 50% alfalfa groups compared to controls. Alfalfa feeding reduced (p < 0.05) the number of cecal- and crop-positive birds compared to controls. Increasing levels of alfalfa increased (p < 0.05) total volatile fatty acids (VFA) and the proportion of acetate in the cecum. Surprisingly, alfalfa inclusion did not negatively impact average daily gain (ADG) in birds over the 7-d feeding period. Alfalfa inclusion at 50% of the diet increased (p < 0.05) the number of bacterial genera detected in the cecum compared to controls, and also altered proportions of the microbial population by reducing Ruminococcus and increasing Clostridia populations. Results support the idea that providing a fermentable substrate can increase gastrointestinal VFA production and bacterial diversity which in turn can reduce colonization by Salmonella via natural competitive barriers. However, further studies are obviously needed to more fully understand the impact of changes made in diet or management procedures on poultry production.

Salmonella can live undetected in the gut of food animals and be transmitted to humans. Animal diets can impact intestinal populations of foodborne pathogens, including Salmonella spp. Orange juice production results in a waste product, orange peel and orange pulp, which has a high nutritive value and is often included in cattle diets as a least-cost ration ingredient. Here we show that the inclusion of orange peel products reduced Salmonella Typhimurium populations in the gut of experimentally inoculated sheep. Sheep (n=24) were fed a cracked corn grain-based high grain diet that was supplemented with a 50%/50% (dry matter [DM], w/w) mixture of dried orange pellet and fresh orange peel to achieve a final concentration (DM, basis) of 0%, 10%, or 20% orange product (OP) for 10 days before inoculation with Salmonella Typhimurium. Sheep were experimentally inoculated with 10(10) colony forming units Salmonella Typhimurium, and fecal samples were collected every 24 h after inoculation. Sheep were humanely euthanized at 96 h after oral Salmonella inoculation. Populations of inoculated Salmonella Typhimurium were numerically reduced by OP treatment throughout the gastrointestinal tract, and this reduction only reached significant levels in the cecum (p<0.05) of sheep fed 10% OP diets. Apparent palatability issues decreased the consumption of OP in sheep fed 20% OP to intake levels below that of 10% OP (approximately 7% dry matter intake [DMI]/d feed refusal), thereby reducing the potential effects of OP feeding at this higher level. Our results demonstrate that orange peel and pellets are environmentally friendly and low-cost products that can be used as a pre-harvest intervention as part of an integrated pathogen reduction scheme.

It is thought that antimicrobial resistance imposes a fitness cost on bacteria, so that a reduction in antimicrobial use may reduce the incidence of resistant bacteria. The objectives of the present study were to determine (1) whether multidrug resistant (MDR) Escherichia coli field strains with different plasmid profiles show disparate plasmid loss when grown over time without selection pressure; (2) whether the number of plasmids present in the cell affects growth. Nine β-hemolytic E. coli strains from swine (n=8) and cattle (n=1) were grown in separate continuous-flow vessels for 36 days without antimicrobial selection. Populations were enumerated on brain heart infusion agar and brain heart infusion agar with tetracycline on days 2, 5, 8, 15, 22, 29, and 36. Growth rates, plasmid profiles and susceptibility profiles of the strains were compared, and day 36 isolates (n=40, five for each MDR strain) were compared with their corresponding day 0 strains. Plasmid content of the nine field strains ranged from zero to eight with sizes from 3.2 to 165 kb. Changes in susceptibility profiles of day 36 isolates were observed among 20% (8 of 40) of the isolates. MDR E. coli largely maintained their original plasmid profiles, replicon types, and susceptibility profiles over 36 days of continuous culture. There was no significant difference in maximum specific growth rate among strains when compared with the plasmid-free strain or when day 36 isolates were compared with their own day 0 strain. This suggests that there is little fitness cost in the maintenance of multiple plasmids of various sizes under the conditions of this study. Other strategies rather than merely reducing antimicrobial usage are needed to combat the emergence of MDR bacteria., (© Mary Ann Liebert, Inc.)

Salmonella is a foodborne pathogenic bacterium that causes human illnesses and morbidity and mortality in swine. Bacteriophages are viruses that prey on bacteria and are naturally found in many microbial environments, including the gut of food animals, and have been suggested as a potential intervention strategy to reduce Salmonella levels in the live animal. The present study was designed to determine if anti-Salmonella phages isolated from the feces of commercial finishing swine could reduce gastrointestinal populations of the foodborne pathogen Salmonella Typhimurium in artificially inoculated swine. Weaned pigs (n = 48) were randomly assigned to two treatment groups (control or phage-treated). Each pig was inoculated with Salmonella Typhimurium (2 × 10(10) colony forming units/pig) via oral gavage at 0 h and fecal samples were collected every 24 h. Swine were inoculated with a phage cocktail via oral gavage (3 × 10(9) plaque forming units) at 24 and 48 h. Pigs were humanely killed at 96 h, and cecal and rectal intestinal contents were collected for quantitative and qualitative analysis. Fecal Salmonella populations in phage-treated pigs were lower (p < 0.09) than controls after 48 h. Phage treatment reduced intestinal populations of inoculated Salmonella Typhimurium in pigs compared to controls at necropsy. Cecal populations were reduced (p = 0.07) by phage treatment >1.4 log(10) colony forming units/g digesta, and rectal populations were numerically reduced. The number of pigs that contained inoculated Salmonella Typhimurium was reduced by phage treatment, but a significant (p < 0.05) reduction was only observed in the rectum. We conclude that phages can be a viable tool to reduce Salmonella in swine. Further research needs to be performed to determine the most efficacious dosing regimens and the most effective combinations of phages targeting the diverse Salmonella population found in swine before they can enter the food supply.

The existence of two separate genetic lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that lineage I could be more pathogenic toward human hosts than lineage II. We have previously shown that lineage I as a group expresses higher levels of Shiga toxin 2 (Stx2) than lineage II. To help evaluate why lineage II strains do not express appreciable levels of this toxin, whole-genome microarrays were performed using Agilent custom microarrays. Gene expression of the two representative bovine lineage II strains (FRIK966 and FRIK2000) were compared with gene expression of E. coli O157:H7 EDL933 (lineage I clinical type strain). Missing or differentially expressed genes and pathways were identified. Quantitative reverse transcription-polymerase chain reaction was performed to validate the microarray data. Draft genomes of FRIK966 and FRIK2000 were sequenced using Roche Applied Science/454 GS-FLX technology shotgun and paired-end approaches followed by de novo assembly. These assemblies were compared with the lineage I genome sequences from E. coli O157:H7 EDL933. The bacteriophage 933W, which encodes the Stx2 genes, showed a notable repression in gene expression. Polymerase chain reaction primers, based upon EDL933 genomic information, were also designed against all of the potentially missing genes of this bacteriophage. Most of the structural genes associated with the bacteriophage were found to be absent from the genome of the two bovine strains. These analyses, combined with evaluation of the genomic information, suggest that transposon (IS629) rearrangements may be associated with disruption of the bacteriophage genome in the FRIK strains. The results support the hypothesis that lineage II strains may be less of a risk as human foodborne pathogens. The microarray and genome data have been made available to the scientific community to allow continuing analysis of these cattle-isolated lineage II genomes and their gene expression.

The objectives of these studies were to determine cell yield and fermentation responses of a Salmonella enterica serovar Typhimurium poultry isolate using various dilution rates in steady state continuous culture incubations. S. enterica Typhimurium cells were propagated in continuous cultures with a total volume of 0.50 l of Luria Bertani medium containing 0.1% glucose. Dilution rates from 0.0125 to 1.44/h were used. Cell protein concentration generally increased linearly with increased dilution rate up to a rate of 0.54/h and declined at the higher dilution rates. Glucose consumption gave a similar pattern to cell protein concentration by declining at the three highest dilution rates. Short chain fatty acid production was inconsistently influenced by dilution rate. Acetate, the most predominant fatty acid produced, declined at the higher dilution rates, as did propionate. Ammonia production remained stable at the lowest dilution rates, but increased significantly at a dilution rates above 0.27/h.

An anaerobic continuous-flow culture of chicken gastrointestinal microflora (CCF) and pure cultures of Enterococcus faecalis I.2 were used for survivability studies of niche-adapted and non-niche-adapted Enterococcus faecium isolates. CCF eliminated non-CCF niche-adapted glycopeptide-resistant E. faecium 47 (GRE47) at a rate of 1.01 log(10) cfu/mL/day, whereas CCF niche-adapted E. faecium I.3(rif) survived in CCF at 4.5-6.5 log(10) cfu/mL. In continuous-flow monocultures of GRE47 (8.93 log(10) cfu/mL), the addition of 100 mL (9.5% total volume) of CCF resulted in the displacement of GRE47 in 14 days at a rate of 0.66 log(10) cfu/mL/day. Pure continuous-flow cocultures were used to assess a direct inhibitory effect of E. faecalis I.2 on E. faecium isolates. In cocultures of E. faecalis I.2 and GRE47, GRE47 was eliminated from the culture at a rate of 1.24 log(10) cfu/mL/day. In cocultures of E. faecalis I.2 and E. faecium I.3(rif), the E. faecium I.3(rif) population fluctuated, but was 6.86 log(10) CFU/mL on day 21. A fit subset of the E. faecium I.3(rif) population survived in CCF and with E. faecalis I.2 alone. No subset of the non-niche-adapted E. faecium GRE47 was able to survive under the same conditions. The mechanism by which E. faecium I.3(rif) is tolerant in CCF, and in E. faecalis coculture is unknown. E. faecium I.3(rif) and GRE47 possessed the cell wall adhesion factor efaAfm. E. faecalis I.2 was positive by polymerase chain reaction for gelE, efaAfs, cad, ccf, cdp, and cob, but not the cytolysin-associated gene cylMAB, suggesting that the mechanism of activity against E. faecium strains was due to factors other than the two-component cytolysin.

Escherichia coli O157 is an important foodborne pathogen affecting human health and the beef cattle industry. Contamination of carcasses at slaughter is correlated to the prevalence of E. coli O157 in cattle feces. Many associations have been made between dietary factors and E. coli O157 prevalence in cattle feces. Preharvest interventions, such as diet management, could reduce the fecal prevalence and diminish the impact of this adulterant. Dietary influences, including grain type and processing method, forage quality, and distillers grains have all been associated with E. coli O157 prevalence. In addition, several plant compounds, including phenolic acids and essential oils, have been proposed as in-feed intervention strategies. The specific mechanisms responsible for increased or decreased E. coli O157 shedding or survival are not known but are often attributed to changes in hindgut ecology induced by diet types. Some interventions may have a direct bacterial effect. Frequently, results of studies are conflicting or not repeatable, which speaks to the complexity of the hindgut ecosystem, variation in animal feed utilization, and variation within feed products. Still, understanding specific mechanisms, driven by diet influences, responsible for E. coli O157 shedding will aid in the development and implementation of better and practical preharvest intervention strategies.

The dicarboxylic acids malate and fumarate increase ruminal pH, reduce methane production, increase propionate and total volatile fatty acid (VFA) production, and reduce lactic acid accumulation in a manner similar to ionophores. These acids stimulate the ruminal bacterium Selenomonas ruminantium to ferment lactate to produce propionate. Thus, dicarboxylic acids have been suggested as nonantibiotic modifiers of the ruminal fermentation, but their impact on ruminal microbial ecology remains unknown. This study was designed to examine what effects these modifiers may have on intestinal pathogen populations such as Escherichia coli O157:H7 and S. enterica Typhimurium prior to their widespread incorporation into cattle rations. Pure cultures of E. coli O157:H7 strain 933 and S. enterica Typhimurium were grown with malate and fumarate added at 0, 1, 5, 10, and 20 mM (v/v; n = 3). Neither dicarboxylic acid inhibited (p > 0.1) the growth rate or final populations of E. coli O157:H7 or S. enterica Typhimurium. Ruminal fluid was collected from cattle (n = 2) and E. coli O157:H7 and S. enterica Typhimurium were added to separate ruminal fermentations incubated for 24 h at 39 degrees C. Fumarate and malate were added at concentrations of 0, 5, 10, and 20 mM (v/v; n = 2) and incubated for 24 h at 39 degrees C. Malate or fumarate addition did not affect (p > 0.1) populations of E. coli O157:H7 or S. enterica Typhimurium. However, the final pH was increased (p < 0.05), the acetate:propionate ratio was decreased (p < 0.05), and the total VFA production was increased (p < 0.05) by > or =10 mM dicarboxylic acid addition. These results confirm that dicarboxylic acids can modify ruminal fermentation, but they do not affect populations of critical foodborne pathogens.

Escherichia coli are commensal bacteria that can account for up to 1% of the bacterial population of the gut. Ruminant animals are reservoirs of the pathogenic bacteria E. colil strain O157:H7, and approximately 30% of feedlot cattle shed E. coli O157:H7. Feedlot and high-producing dairy cattle are fed high grain rations in order to increase feed efficiency. When cattle are fed high grain rations, some starch escapes ruminal microbial degradation and passes to the hindgut where it undergoes fermentation. Ten years ago researchers demonstrated that populations of total E. coli were higher in grain-fed than in forage-fed cattle, and when cattle were abruptly switched from a high grain diet to an all hay diet, total E. coli populations declined 1000-fold within 5 days and reduced the ability of the surviving E. coli to survive an acid shock mimicking passage through the human gastric stomach. This research provoked many questions about the effects of diet or E. coli O157:H7 populations that have not been conclusively answered to date. Subsequent research has shown that diet does affect E. coli O157:H7 populations, but the effects have varied in magnitude and impact. Further studies have demonstrated that the effects of forage feeding on E. coli O157:H7 populations may be due to concentrations of tannins and phenolic acids in forages. Other ration components such as rapidly ruminally fermented grains (e.g., barley) increase the shedding of E. coli O157:H7, and in some situations, feeding distillers grains can increase fecal shedding of E. coli O157:H7 due to VFA concentrations. Data from researchers across North America indicate that diet does impact STEC/EHEC populations in cattle prior to slaughter; however the economic, logistic and practical impacts of dietary changes must be examined and accounted for.

Due to increasing concerns about the development of antimicrobial resistance amongst pathogenic bacteria, alternative strategies have been sought that do not use antibiotics to reduce pathogenic bacteria from foods and patients. A natural compound that has potent antimicrobial properties is citrus peel, which contains a variety of essential oils that inhibit the growth of or kill pathogenic bacteria. In the present study, seven citrus-based natural antimicrobials were evaluated for their ability to inhibit the growth of the pathogen Escherichia coli O157:H7. Zones of inhibition of E. coli O157:H7 by the citrus-derived fraction (10 microL/6 mm disk) were determined by a disk-diffusion assay on Sorbitol-MacConkey agar. Inhibition zones were observed after 48 h lawn growth of E. coli O157:H7 cells at 37 degrees C. Two citrus-based fractions, orange CP VAL terpeneless FAB 968611 and Limonene 1x Dist FAB 955430, inhibited E. coli O157:H7 with inhibition zones of approx. 11-24 mm dia. The remaining other five citrus-derived extracts (orange oil FL VAL 1121 ARR 974760, Orange 5x Conc VAL 4121 ARR 968374, orange terpenes ESS 1120 ARR 986259, orange terpenes CP 1100 ARR 986255, and orange terpenes OEO HP 1100 ARR 986257) were noninhibitory to E. coli O157:H7, yielding no clear inhibition zones. These studies show that citrus-derived natural compounds differ in their inhibitory activity against E. coli O157:H7 and some have potential applications as inhibitory agents against E. coli O157:H7 in various pathogen reduction strategies.

Campylobacter are important human foodborne pathogens known to colonize the gastrointestinal tract of cattle. The incidence of Campylobacter in cattle may be seasonal and may vary among age groups and type (beef versus dairy). Less is known about other factors that could influence the prevalence, colonization site, and shedding of Campylobacter in cattle. The objectives of this study were to evaluate the prevalence and enumerate Campylobacter at two sites along the digestive tract of beef and dairy type cattle consuming either grass or feedlot diets. In an initial study, Campylobacter was not recovered from rumen samples of any of 10 ruminally cannulated (six dairy and four beef type) pasture-reared cattle and there was no difference (p > 0.05) between cattle types on fecal Campylobacter recovery, with 50% of each type yielding culture-positive feces (overall mean +/- SE, 0.75 +/- 0.001 SEM log(10) colony-forming units [CFU]/g feces). When calculated from Campylobacter culture-positive animals only, mean fecal concentrations were 1.50 +/- 0.001 SEM log(10) CFU/g. In a follow-up study with feedlot and pasture-reared cattle (n = 18 head each), 78% of rumen and 94% of fecal samples from pastured cattle were positive for Campylobacter while 50% of the rumen and 72% of the fecal samples were positive in concentrate-fed animals. Overall mean concentration of Campylobacter was greater in feces than ruminal fluid (p < 0.05). When only Campylobacter-positive animals were analyzed, concentrations recovered from feces were higher (p < 0.05) in concentrate-fed than in pasture-fed cattle (4.29 vs. 3.34 log(10) CFU/g, respectively; SEM = 0.29). Our results suggest that the rumen environment and its microbial population are less favorable for the growth of Campylobacter and that concentrate diets may provide a more hospitable lower gastrointestinal tract for Campylobacter.

Escherichia coli O157:H7 can live undetected in the gut of food animals and be spread to humans directly and indirectly. Bacteriophages are viruses that prey on bacteria, offering a natural, nonantibiotic method to reduce pathogens from the food supply. Here we show that a cocktail of phages isolated from commercial cattle feces reduced E. coli O157:H7 populations in the gut of experimentally inoculated sheep. A cocktail of phages was used in order to prevent the development of resistance to the phages. In our first in vivo study we found that our cocktail of phages reduced E. coli O157:H7 populations in the feces of sheep (p < 0.05) by 24 hours after phage treatment. Upon necropsy, populations of inoculated E. coli O157:H7 were reduced by phage treatment in both the cecum (p < 0.05) and rectum (p < 0.1). In our second in vivo study, several ratios of phage plaque-forming units (PFU) to E. coli O157:H7 colony-forming units (CFU) were used (0:1, 1:1, 10:1, and 100:1 PFU/CFU) to determine the most efficacious phage dose. A 1:1 ratio of phage to bacteria was found to be more effective (p < 0.05) than either of the higher ratios used (10:1 or 100:1). Ruminal levels of E. coli O157:H7 were not significantly reduced (p > 0.10) in any of the studies due to relatively low inoculated E. coli O157:H7 ruminal populations. Our results demonstrate that phage can be used as a preharvest intervention as part of an integrated pathogen reduction scheme.

Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer decreasing to very low levels in the winter. We hypothesize that this seasonal variation is a result of physiological responses within the host animal to changing day-length. To determine the effects of melatonin (MEL) on fecal shedding of E. coli O157:H7 in cattle, eight crossbred beef steers identified as shedding E. coli O157:H7, were allotted to treatment: control or MEL (0.5 mg/kg body weight (BW); 1x) administered orally daily for 7 days. After a 5-day period of no treatment, a second MEL dose (5.0 mg/kg BW; 10x) was administered daily for 4 days. Fecal samples were collected daily for qualification of E. coli O157:H7. No differences (P > 0.10) were observed in the percentage of E. coli O157:H7 positive fecal samples in steers receiving the 1x MEL dose, however the 10x dose decreased (P = 0.05) the percentage of fecal samples E. coli O157:H7 positive. Serum MEL concentrations were higher in the 1x, but not 10x, treated animals compared to control animals. Although it is difficult to explain, this may be a result of decreasing day-length increasing serum melatonin concentrations that may have masked any treatment effect on serum melatonin. In a second similar experiment, a second group of cattle (heifers and steers) were administered tryptophan (TRP) over a 17-day experimental period (5 g/head/day for 10 days followed by 10 g/head/day for 7 days). Tryptophan had no effect (P > 0.20) on the percentage of fecal samples positive for E. coli O157. Serum TRP (P < 0.05), but not MEL (P > 0.20), concentrations were elevated in TRP-treated animals. The decrease in the number of positive fecal samples observed in the first experiment, may be related to gastrointestinal MEL, affected by the 10x, but not 1x MEL dose.

Enterococcus faecalis and E. faecium are gram-positive human pathogens that can live in the gastrointestinal tract of food animals. Vancomycin-resistant enterococci are an increasing threat to humans as a nosocomial infection, as well as a reservoir of antibiotic resistance genes. Ionophores are feed-grade antimicrobials that are widely used to enhance the ruminal fermentation efficiency via inhibiting gram-positive bacteria by dissipating ion and proton gradients. Some bacteria can become resistant to ionophores, and this has prompted concerns about whether ionophore resistance can enhance antibiotic resistance in intestinal bacteria. Since enterococci are normal members of the ruminant intestinal tract and function as an antibiotic resistance reservoir, the present study investigated whether treatment with the most commonly used ionophores affected the growth of enterococci, and whether ionophore-resistant enterococci developed. Ionophores do inhibit the growth of enterococci in pure culture, but in our study did not alter populations in mixed ruminal bacterial culture. Ionophore-resistant isolates were not isolated during this study from pure or mixed cultures. Our results indicate that the role of ionophores in the dissemination of antibiotic resistance genes through the intestinal Enterococcus spp. appears to be limited.

Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer months and decreases to low or undetectable levels in the winter. We hypothesize that the seasonal variation of this pathogen is a result of physiological responses within the host animal to changing day length. The thyroid is an endocrine gland known to respond to changing day length. Two experiments were conducted to determine if a hyperthyroid status would initiate fecal shedding of E. coli O157 in cattle during the winter when shedding is virtually nonexistent (winter experiment) or influence cattle actively shedding E. coli O157 (summer experiment). Yearling cattle were group-penned under dry-lot conditions, adjusted to a high concentrate ration, and randomly assigned to treatment: control (1 mL corn oil injected s.c. daily) or triiodothyronine (T(3); 1.5 mg suspended in corn oil injected s.c daily). Cattle were individually processed daily for collection of fecal and blood samples. Treatment with exogenous T(3) produced a significant change in serum thyroid hormone concentrations indicative of a hyperthyroid status in both experiments. No differences (P>0.10) were observed in fecal shedding of E. coli O157 in the winter experiment. In the summer experiment, fecal shedding of E. coli O157 was decreased (P=0.05) by administration of T(3) during the treatment period (days 1-10), tended to be lower (P=0.08) during the following 7-day period of no treatment, and was lower (P=0.01) when examined across the entire experimental period. Results of this research indicate that the thyroid or its hormones may be involved in the seasonal shedding patterns of E. coli O157 in cattle.

The effects of the beta-agonist ractopamine, approved for use in finishing swine and cattle to improve carcass quality and performance, were examined on two important foodborne pathogens, Escherichia coli O157:H7 and Salmonella. Ractopamine, administered to sheep before and after oral inoculation with E. coli O157:H7, increased (P < 0.01) fecal shedding and tended to increase (P = 0.08) cecal populations of the challenge strain. Pigs receiving ractopamine in the diet and then experimentally infected with Salmonella Typhimurium, had decreased (P < 0.05) fecal shedding and fewer (P = 0.05) liver samples positive for the challenge strain of Salmonella. Pure cultures of E. coli O157:H7 (used in the present sheep study), E. coli O157:H19 (isolated from pigs with postweaning diarrhea), Salmonella Typhimurium (used in the present pig study), and Salmonella Choleraesuis were incubated with varying concentrations of ractopamine to determine if ractopamine has a direct effect on bacterial growth. No differences in growth rate were observed for either strain of E. coli or for Salmonella Typhimurium when incubated with increasing concentrations of ractopamine. The growth rate for Salmonella Choleraesuis was increased with the addition of 2.0 mug ractopamine/ml compared with the other concentrations examined. Collectively, these results indicate that ractopamine may influence gut populations and fecal shedding of E. coli O157:H7 and Salmonella. Because ractopamine is currently approved to be fed to finishing cattle and swine immediately before slaughter, any potential for decreasing foodborne pathogens has exciting food safety implications.

Escherichia coli O157 and Salmonella spp. are among the leading causes of food-borne illness in the United Sates and bacteria have been isolated from numerous ruminant animal sources. The objective of this study was to assess the incidence of E. coli O157 and Salmonella spp. in white-tailed deer (Odocoileus virginianus) and livestock simultaneously grazing the same rangeland. Escherichia coli O157 was found in 1.25% of cattle, 1.22% of sheep, and 5.00% of water all from samples taken in September; however, no E. coli O157 was found in other sampled months or any species. Salmonella spp. were found in the highest quantities in deer and sheep, 7.69% and 7.32%, respectively. Salmonella spp. were also found in sampled water troughs, goats, and cattle (5.00%, 3.70%, and 1.25%, respectively). Further research examining pathogen distribution is needed to determine if white-tailed deer are a natural reservoir for these bacteria.