Some aspects of the acute phase immune response to a lipopolysaccharide (LPS) challenge are mitigated by supplementation with a Saccharomyces cerevisiae fermentation product in weaned beef calves.

This study was conducted to determine if feeding a Saccharomyces cerevisiae fermentation product (SCFP) to calves would alter the acute phase response to a lipopolysaccharide (LPS) challenge. Crossbred steer calves [n = 32; 274 ± 1.9 kg body weight (BW)] were randomly allotted to two treatment diets for 21 d: 1) control, fed RAMP (Cargill, Dalhart, TX) and 2) SCFP, fed the control ration supplemented with NaturSafe at 12 g/hd/d mixed into the TMR (NaturSafe, Diamond V, Cedar Rapids, IA). On day 22, steers were fitted with indwelling jugular catheters and rectal temperature monitoring devices and placed in individual bleeding stalls. On day 23, steers were challenged i.v. with 0.25 µg/kg BW LPS. Blood samples were collected at 0.5-h (serum) or 2-h (complete blood counts) intervals from -2 to 8 h and again at 24 h relative to the LPS challenge at 0 h. Sickness behavior scores (SBS) were recorded after the collection of each blood sample. Rectal temperatures were greater in SCFP steers from 6 to 11 h, at 13 h, from 15 to 20 h, and from 22 to 24 h following the LPS challenge compared to Control steers (treatment × time: P = 0.01). Additionally, SCFP-supplemented steers had reduced (P < 0.01) SBS compared to Control steers. Platelet concentrations remained greater in SCFP-supplemented steers compared to Control steers throughout the study (P = 0.05), while there was a tendency (P = 0.09) for SCFP steers to have greater white blood cells and eosinophils concentrations than Control steers. There was a treatment × time interaction for serum cortisol and glucose (P < 0.01). Specifically, cortisol was greater at 0.5 and 2 h postchallenge but was reduced at 3 h for SCFP steers compared to Control steers. Glucose was greater in SCFP steers at -0.5, 2, and 7.5 h compared to Control steers. Serum amyloid A was reduced in SCFP steers at 0.5 h, yet greater at 1 and 7.5 h postchallenge compared to Control steers (treatment × time: P < 0.01). Fibrinogen concentrations were greater (P < 0.01) in SCFP compared to Control steers. There was a treatment × time interaction (P < 0.01) for tumor necrosis factor-α (TNF-α) such that concentrations were reduced in SCFP steers from 1 to 2 h postchallenge compared to Control steers. Overall, these data suggest that supplementing calves with SCFP may have primed the innate immune response prior to the challenge, particularly platelets, which resulted in an attenuated sickness behavior and TNF-α response to LPS.

Effect of waste milk pasteurization on fecal shedding of Salmonella in preweaned calves.

The objective of the current research was to determine if pasteurization of nonsaleable waste milk influences fecal Salmonella concentrations and prevalence, or antimicrobial susceptibility and serotype of the cultured isolates. Holstein dairy calves (n = 211) were housed on a single commercial dairy in the southwestern United States and randomly allotted to be fed either pasteurized (PWM; n = 128 calves) or nonpasteurized waste milk (NPWM; n = 83 calves). Fecal samples were collected via rectal palpation or from freshly voided, undisturbed fecal pats, weekly during the first 4 wk of the animal's life and then again at weaning. Eight total collections were made and 1,117 fecal samples cultured for Salmonella. One isolate from each culture-positive fecal sample was preserved for antimicrobial susceptibility screening and serotyping. Sixty-nine percent of the fecal samples were culture positive for Salmonella with no difference due to treatment (67.7 and 69% Salmonella positive for PWM and NPWM treatments, respectively). Few fecal samples (178/1,117; 15.9%) contained Salmonella concentrations above the limit of detection (∼1 cfu/g of feces) with concentrations ranging from 1.0 to 6.46 cfu (log10)/g of feces. Concentration was not affected by treatment. Seventeen different serotypes were identified, the majority of which were Montevideo and Anatum. A greater percentage of Typhimurium (87 vs. 13%), Muenchen (88 vs. 12%), and Derby (91 vs. 9%) were recovered from calves fed PWM compared with NPWM-fed calves. Conversely, Newport (12.5 vs. 86%), Bredeney (22.2 vs. 77.8%), and Muenster (12.5 vs. 87.5%) were lower in PWM compared with NPWM treatments. The majority (66.7%) of isolates were susceptible to all of the antibiotics examined. Results from this one commercial dairy suggest that milkborne Salmonella is not an important vector of transmission in dairy neonates, nor does pasteurization of waste milk influence fecal shedding of this pathogen. Caution should be used, however, when extrapolating results to other farms as Salmonella contamination of milk on farm is well documented. The potential benefits of pasteurization in disease prevention outweigh the potential risks of feeding a nonpasteurized product and warrants incorporation into any calf-rearing program using nonsaleable waste milk for feeding young dairy neonates.

WGS accurately predicts antimicrobial resistance in Escherichia coli.

OBJECTIVES: The objective of this study was to determine the effectiveness of WGS in identifying resistance genotypes of MDR Escherichia coli and whether these correlate with observed phenotypes. METHODS: Seventy-six E. coli strains were isolated from farm cattle and measured for phenotypic resistance to 15 antimicrobials with the Sensititre(®) system. Isolates with resistance to at least four antimicrobials in three classes were selected for WGS using an Illumina MiSeq. Genotypic analysis was conducted with in-house Perl scripts using BLAST analysis to identify known genes and mutations associated with clinical resistance. RESULTS: Over 30 resistance genes and a number of resistance mutations were identified among the E. coli isolates. Resistance genotypes correlated with 97.8% specificity and 99.6% sensitivity to the identified phenotypes. The majority of discordant results were attributable to the aminoglycoside streptomycin, whereas there was a perfect genotype-phenotype correlation for most antibiotic classes such as tetracyclines, quinolones and phenicols. WGS also revealed information about rare resistance mechanisms, such as structural mutations in chromosomal copies of ampC conferring third-generation cephalosporin resistance. CONCLUSIONS: WGS can provide comprehensive resistance genotypes and is capable of accurately predicting resistance phenotypes, making it a valuable tool for surveillance. Moreover, the data presented here showing the ability to accurately predict resistance suggest that WGS may be used as a screening tool in selecting anti-infective therapy, especially as costs drop and methods improve.

Organic acid blend with pure botanical product treatment reduces Escherichia coli and Salmonella populations in pure culture and in in vitro mixed ruminal microorganism fermentations.

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.

Isolation of Escherichia coli O157:H7 and Salmonella from migratory brown-headed cowbirds (Molothrus ater), common Grackles (Quiscalus quiscula), and cattle egrets (Bubulcus ibis).

Zoonotic enteric pathogenic bacteria can live in the intestinal tract of birds and can be transmitted to food animals or humans via fecal contact. In the present study, cecal samples were collected from 376 migratory birds from species often associated with cattle during the fall migration in the Central Flyway of the United States. Brown-headed cowbirds (n=309, Molothrus ater), common grackles (n=51, Quiscalus quiscula), and cattle egrets (n=12, Bubulcus ibis) contained foodborne pathogenic bacteria in their ceca. Salmonella enterica was isolated from 14.9% of all samples, and Escherichia coli O157:H7 from 3.7%. Salmonella serotypes isolated included the following: Muenster, Montevideo, and Typhimurium. Our data suggest that migratory birds associated with cattle could be a vector for zoonotic enteric pathogenic bacteria to be disseminated across long distances.

Effects of dietary alfalfa inclusion on Salmonella typhimurium populations in growing layer chicks.

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.

Influence of vitamin D on fecal shedding of Escherichia coli O157:H7 in naturally colonized cattle.

Three experiments were conducted to evaluate the influence of vitamin D on fecal shedding of Escherichia coli O157:H7 in cattle. In the first experiment, two groups of cattle (beef and dairy) were assigned to a control treatment or to receive 0.5 × 10(6) IU vitamin D per day via oral bolus for 10 days. Fecal samples were collected before and throughout the dosing period for culture of E. coli O157:H7. No differences were observed for fecal shedding of E. coli O157:H7 among treatments for either beef or dairy animals. Serum concentrations of vitamin D were markedly higher (P < 0.0001) in treated beef cattle but only tended to be higher (P = 0.09) in the dairy cattle. In the second experiment, three successive vitamin D dosages (2,400, 4,800, and 9,600 IU/day; 14 days each) were administered to 14 dairy steers (7 steers served as controls), fecal samples were collected daily, and serum samples were collected weekly throughout the 42-day experimental period. No significant differences in fecal prevalence or serum vitamin D concentrations were observed for any of the vitamin D dosages. A third experiment sampled feedlot cattle (winter and summer) to determine whether serum vitamin D concentrations were correlated with fecal shedding of E. coli O157:H7. A fecal sample and a blood sample were obtained in each season from 60 randomly selected animals (total of 120 fecal samples and 120 corresponding blood samples). As expected, season was highly correlated (r = 0.66) with serum vitamin D concentration with higher concentrations (P < 0.01) observed in the summer. E. coli O157:H7 prevalence (percentage of positive samples) was not highly correlated (r = 0.16) with season, although the correlation tended to be significant (P = 0.08). The proportion of cattle shedding E. coli O157:H7 was 16.7 and 6.7% for the summer and winter collections, respectively. Results of this research do not support a correlation between vitamin D intake and E. coli O157:H7 shedding in cattle.

Escherichia coli O157:H7 populations in ruminants can be reduced by orange peel product feeding.

Foodborne pathogenic bacteria such as Escherichia coli O157:H7 are threats to the safety of beef. Citrus peel and dried orange pulp are by-products from citrus juice production that have natural antimicrobial effects and are often incorporated into least-cost ration formulations for beef and dairy cattle. This study was designed to determine if orange peel and pulp affected E. coli O157:H7 populations in vivo. Sheep (n = 24) were fed a cracked corn grain-based diet that was supplemented with a 50-50 mixture of dried orange pellet and fresh orange peel to achieve a final concentration (dry matter basis, wt/wt) of 0, 5, or 10% pelleted orange peel (OP) for 10 days. Sheep were artificially inoculated with 10(10) CFU of E. coli O157:H7 by oral dosing. Fecal shedding of E. coli O157:H7 was measured daily for 5 days after inoculation, after which all animals were humanely euthanized. At 96 h postinoculation, E. coli O157:H7 shedding was reduced (P < 0.05) in sheep fed 10% OP. Populations of inoculated E. coli O157:H7 were reduced by OP treatment throughout the gastrointestinal tract; however, this reduction reached significant levels in the rumen (P < 0.05) of sheep fed 10% OP diets. Cecal and rectal populations of E. coli O157:H7 were reduced (P < 0.05) by inclusion of both 5 and 10% OP diets. Our results demonstrate that orange peel products can be used as a preharvest intervention strategy as part of an integrated pathogen reduction scheme.

Orange peel products can reduce Salmonella populations in ruminants.

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.

Influence of weaning on fecal shedding of pathogenic bacteria in dairy calves.

The objectives of the current research were to determine the effect of weaning on fecal shedding of Salmonella and Escherichia coli O157:H7 in dairy calves and to examine cultured isolates (to include Enterococcus) for antimicrobial susceptibility. This research was conducted on one large commercial dairy (>3000 head) in the southwestern United States. Two collections were made, during the winter (January 2009) and summer (July 2009) seasons. For the winter collection, two groups of calves were sampled (group 1: n = 18 pens, 69 head, ∼12 weeks of age; group 2: n = 19 pens, 75 head, ∼10 weeks of age). Fecal samples were collected from all calves via rectal palpation 2 days pre- and again 2 days postweaning. For the summer collection, one group of calves housed in 40 pens were utilized and 79 and 76 calves sampled 7 days pre- and 5 days postweaning, respectively. Fecal samples were collected into sterile palpation sleeves, placed on ice, and shipped to our laboratory for bacterial culture of E. coli O157:H7, Salmonella, and Enterococcus. Antimicrobial susceptibility was determined on select isolates. No differences (p > 0.10) in prevalence of Salmonella or E. coli O157:H7 were observed due to weaning in the winter collection. In the summer collection, more (p < 0.01) fecal samples were Salmonella positive preweaning (15.2%) as compared to postweaning (2.6%). No differences were observed for antimicrobial susceptibility in isolates collected pre- as compared to postweaning in either winter or summer collections, with the exception that multidrug-resistant Enterococcus faecium isolates preweaning were resistant to six antibiotics compared to seven or eight antibiotics postweaning (summer collection). Results of the current research indicate that the weaning of dairy calves does not increase the prevalence of pathogenic bacteria or substantially modify antimicrobial susceptibility profiles of these bacteria.

Evaluation of phage treatment as a strategy to reduce Salmonella populations in growing swine.

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.

Occurrence of Salmonella-specific bacteriophages in swine feces collected from commercial farms.

Salmonella is one of the leading causes of human foodborne illness and is associated with swine production. Bacteriophages are naturally occurring viruses that prey on bacteria and have been suggested as a potential intervention strategy to reduce Salmonella levels in food animals on the farm and in the lairage period. If phages are to be used to improve food safety, then we must understand the incidence and natural ecology of both phages and their hosts in the intestinal environment. This study investigates the incidence of phages that are active against Salmonella spp. in the feces of commercial finishing swine. Fecal samples (n = 60) were collected from each of 10 commercial swine finishing operations. Samples were collected from 10 randomly selected pens throughout each operation; a total of 600 fecal samples were collected. Salmonella spp. were found in 7.3% (44/600) of the fecal samples. Bacteriophages were isolated from fecal samples through two parallel methods: (1) initial enrichment in Salmonella Typhimurium; (2) initial enrichment in Escherichia coli B (an indicator strain), followed by direct spot testing against Salmonella Typhimurium. Bacteriophages active against Salmonella Typhimurium were isolated from 1% (6/600) of the individual fecal samples when initially enriched in Salmonella Typhimurium, but E. coli B-killing phages were isolated from 48.3% (290/600) of the fecal samples and only two of these phages infected Salmonella Typhimurium on secondary plating. Collectively, our results indicate that bacteriophages are widespread in commercial swine, but those capable of killing Salmonella Typhimurium may be present at relatively low population levels. These results indicate that phages (predator) populations may vary along with Salmonella (prey) populations; and that phages could potentially be used as a food safety pathogen reduction strategy in swine.

Influence of wet distiller's grains on prevalence of Escherichia coli O157:H7 and Salmonella in feedlot cattle and antimicrobial susceptibility of generic Escherichia coli isolates.

The current research examined the inclusion of 20% wet distiller's grains (WDG) fed with steam-flaked corn (SFC) or dry-rolled corn (DRC) in diets fed to feedlot cattle on fecal prevalence of Escherichia coli O157:H7 and Salmonella. Crossbred beef heifers (n = 272; average initial body weight (BW) = 354 kg) were blocked by BW and pen size and randomly assigned to treatment. Fecal samples from freshly voided fecal pats were collected from each pen on the day cattle shipped for slaughter (237 fecal samples: 72, 125, and 40 from cattle 132, 160, and 181 days on feed, respectively). Fecal samples were cultured quantitatively and qualitatively for the above pathogens. Populations of E. coli O157:H7 and Salmonella were generally low with very few samples containing quantifiable populations. Similarly, after enrichment, few samples were E. coli O157:H7 positive in any collection with no treatment differences (p > 0.10). More samples were Salmonella positive during the first collection with an increased (p < 0.05) prevalence observed in the SFC and DRC treatments compared with DRC + WDG treatment. No other treatment differences were observed for Salmonella. Putative fecal coliform isolates (18 per treatment; first collection) were examined for antimicrobial susceptibility, and the majority were susceptible to all of the antibiotics examined. Most of the resistance was observed in the SFC (n = 3) and DRC (n = 4) treatments, and only one isolate in each of the two WDG treatments demonstrated resistance (one antibiotic each, streptomycin and tetracycline). All multidrug resistance (2-4 antibiotics) was observed in isolates cultured from the DRC and SFC treatments (n = 2 isolates in each treatment). Results of the current research found no significant effect of feeding WDG to feedlot cattle on fecal prevalence, at time of shipment for slaughter, of E. coli O157:H7, and only modest differences (decreases) in Salmonella prevalence with no apparent affect on antimicrobial susceptibility of fecal coliform isolates.

Influence of beta-agonists (ractopamine HCl and zilpaterol HCl) on fecal shedding of Escherichia coli O157:H7 in feedlot cattle.

Ractopamine HCl and zilpaterol HCl, beta-agonists recently approved for use in feedlot cattle to improve performance traits and carcass leanness, were examined for their effects on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. Fecal samples (n = 2,454) were obtained from four experiments (one ractopamine HCl, three zilpaterol HCl) over the course of a 3-year period, either by rectal palpation (ractopamine HCl experiment) or from pen-floor fecal pats. Samples were cultured quantitatively and qualitatively for E. coli O157:H7. No significant treatment differences were detected for fecal prevalence of E. coli O157:H7 in the ractopamine HCl experiment. Zilpaterol HCl feeding had no effect (P > 0.20) on fecal shedding in the first or second experiments, with overall E. coli O157:H7 prevalence relatively low (<7%). In the third zilpaterol HCl experiment, the percentage of fecal samples that were E. coli O157:H7 positive following qualitative culture was higher (P < 0.05) in the zilpaterol HCl treatment (10.3%) than for the control (6.1%). The current research showed minimal effects of beta-agonists on fecal shedding of E. coli O157:H7 and indicated that these compounds (fed immediately prior to slaughter) are not a cause for concern from a food safety standpoint.

Prevalence and antimicrobial resistance profiles of Escherichia coil O157:H7 and Salmonella isolated from feedlot lambs.

The present study examined the incidence of Escherichia coli O:H7 and Salmonella in feedlot lambs. Fifty-six feedlot lambs from eight sheep farming operations were grouped in a single drylot pen, fed, and managed as is typical in the southwestern United States. Fecal samples were collected on days 0, 46, 87, and 122 of the feeding period via rectal palpation. Wool samples (ventral midline) were collected one time only at the feedlot, immediately prior to shipping to the processing plant, and carcass swabs were collected following slaughter. All samples were cultured for E. coli O157:H7, Salmonella, and fecal coliforms, and select isolates were examined for antimicrobial susceptibility. Overall, the percentages of fecal and wool samples positive for E. coli O157:H7 averaged 9 and 18%, respectively. One carcass swab was E. coli O157:H7 positive. Of the 155 fecal samples collected, 11 (7%) were Salmonella positive. Salmonella was detected in nearly 50% of the wool samples collected prior to slaughter, while none of the carcasses were Salmonella positive 24 h postslaughter. All isolates (E. coli O157:H7, Salmonella, and fecal coliforms) were susceptible to ceftiofur, enrofloxacin, and trimethoprim-sulfamethoxazole. One E. coli O157:H7 isolate cultured from a carcass swab was resistant to seven antibiotics, and seven wool E. coli O157:H7 isolates were multidrug resistant. Results of this research demonstrate that feedlot sheep are naturally colonized with E. coli O157:H7 and Salmonella and wool can be a source of carcass contamination; however, in-plant processing procedures and intervention strategies were largely effective in preventing carcass contamination.

Conjugative transferability of the A/C plasmids from Salmonella enterica isolates that possess or lack bla(CMY) in the A/C plasmid backbone.

The objective of this study was to understand the conjugative transmissibility of resistance plasmids present in 205 Salmonella enterica isolates from bovine sources. Polymerase chain reaction (PCR)-based replicon typing was used to type plasmid replicons. Conjugation experiments were preformed in triplicate at 30 degrees C and 37 degrees C on solid medium. PCR mapping of the A/C transfer gene operon was done on 17 Salmonella Newport isolates that were only positive for A/C. Eighty-six percent (n = 177) of the Salmonella isolates were multidrug resistant (MDR) with resistance to 3-12 antimicrobial agents. Of these, 82% (n = 146) were resistant to extended-spectrum cephalosporins and possessed a bla(CMY) gene. A/C was the predominant replicon detected, present in 90% (n = 160) of the MDR isolates. Twenty-three percent (n = 37) of the A/C-positive strains were positive for a second replicon. Replicons coresident with A/C included I1, N, B/O, HI1, and HI2. Only 31% (n = 54) of the MDR isolates produced transconjugants, and most of these donors carried multiple replicons. A/C cotransferred with B/O, N, and I1 at both 30 degrees C and 37 degrees C and with HI2 at 30 degrees C. Seven Salmonella Newport isolates that produced transconjugants possessed only the single A/C replicon and lacked bla(CMY). PCR mapping of the A/C transfer gene operon in ten Salmonella Newport isolates that carried bla(CMY) revealed a bla(CMY) inverted repeat element integrated between the traA and traC genes. These results suggest that A/C may have been a conjugative plasmid before the integration of bla(CMY) into the transfer gene operon. Additionally, transfer deficient A/C replicons may be mobilized in the presence of certain compatible conjugative plasmids.

Influence of exogenous melatonin on horizontal transfer of Escherichia coli O157:H7 in experimentally infected sheep.

The objective of the current research was to determine if exogenous melatonin would exert a "protective" effect on the gastrointestinal tract of sheep and prevent or reduce the horizontal transfer of Escherichia coli O157:H7 from experimentally infected to noninfected or "naïve" sheep. Sixteen cross-bred ewes were housed indoors and adapted to a high concentrate ration. Ewes were randomly assigned to one of four rooms and treatment (three ewes/room, six ewes/treatment) and received either control (gelatin capsule only) or melatonin (5.0 mg/kg body weight [BW]/d). Four additional ewes served as "carrier" sheep (one/room) and were experimentally infected via oral gavage with E. coli O157:H7. Three days post-challenge, carrier ewes were housed with naïve sheep and remained with them for the remainder of the experimental period. Treatments were administered to the naïve sheep 1 day prior to introduction of the carrier sheep and on each of the remaining 7 days of the experimental period. Fecal samples were collected via rectal palpation from the carrier sheep daily throughout experiment and from the naïve sheep daily for 5 days, starting 2 days following introduction of the carriers. On day 8 of the experiment, all ewes were euthanized and tissues from the rumen, ileum, cecum, colon, and rectum as well as their respective lumen contents collected. The carrier sheep quickly infected the naïve ewes, which had similar fecal concentrations as the carrier animals throughout the 5-day sampling period. Melatonin treatment had no effect (p > 0.10) on daily fecal shedding, luminal content concentrations, or in the percentage of gastrointestinal tract tissue positive for the inoculated strain of E. coli O157:H7.

Pathogen prevalence and influence of composted dairy manure application on antimicrobial resistance profiles of commensal soil bacteria.

Composting manure, if done properly, should kill pathogenic bacteria such as Salmonella and Escherichia coli O157:H7, providing for an environmentally safe product. Over a 3-year period, samples of composted dairy manure, representing 11 composting operations (two to six samples per producer; 100 total samples), were screened for Salmonella and E. coli O157:H7 and were all culture negative. Nonpathogenic bacteria were cultured from these compost samples that could theoretically facilitate the spread of antimicrobial resistance from the dairy to compost application sites. Therefore, we collected soil samples (three samples per plot; 10 plots/treatment; 90 total samples) from rangeland that received either composted dairy manure (CP), commercial fertilizer (F), or no treatment (control, CON). Two collections were made appoximately 2 and 7 months following treatment application. Soil samples were cultured for Pseudomonas and Enterobacter and confirmed isolates subjected to antimicrobial susceptibility testing. Three species of Enterobacter (cloacae, 27 isolates; aeroginosa, two isolates; sakazakii, one isolate) and two species of Pseudomonas (aeruginosa, 11 isolates; putida, seven isolates) were identified. Five Enterobacter isolates were resistant to ampicillin and one isolate was resistant to spectinomycin. All Pseudomonas isolates were resistant to ampicillin, ceftiofur, florfenicol, sulphachloropyridazine, sulphadimethoxine, and trimethoprim/sulfamethoxazole and most isolates were resistant to chlortetracycline and spectinomycin. Pseudomonas isolates were resistant to an average of 8.6, 7.9, and 8 antibiotics for CON, CP, and F treatments, respectively. No treatment differences were observed in antimicrobial resistance patterns in any of the soil isolates examined. Results reported herein support the use of composted dairy manure as an environmentally friendly soil amendment.

Prevalence and concentration of Campylobacter in rumen contents and feces in pasture and feedlot-fed cattle.

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.

Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts.

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.