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Two experiments were conducted to evaluate the feeding of direct fed microbials (DFMs) on fecal shedding of Escherichia coli O157:H7 in naturally infected cattle (experiment I) and on Salmonella in the feces and peripheral lymph nodes (PLNs) of experimentally infected cattle (experiment II). Thirty cattle, 10 per treatment, were used in each experiment. Treatments in experiment I consisted of a control (lactose carrier only); DFM1, a 1:1 ratio of Enterococcus faecium and Lactobacillus animalis; and DFM2, a 1:1 ratio of Lactobacillus acidophilus and Pediococcus acidilactici. In Experiment II, DFM1 was replaced with DFM3, a 1:2 ratio of Lactobacillus reuteri and other Lactobacillus strains. Additives were mixed in water and applied as a top-dressing to each pen's daily ration for 50 days. Approximately half-way through each experiment, the DFM concentration was doubled for the remainder of the study. Fecal samples were collected throughout experiment I and cultured for E. coli O157:H7. Cattle in experiment II were inoculated intradermally with Salmonella Montevideo on days 32, 37, and 42 and then necropsied on days 49 and 50 (five cattle per treatment on each day). Innate immune function was assessed on days 29, 49, and 50. In experiment I, fecal concentration and prevalence of E. coli O157:H7 were not different (P > 0.10) nor was there an effect (P = 0.95) on the percentage of super shedders (cattle shedding ≥3.0 log CFU/g of feces). In experiment II, no treatment differences (P > 0.05) were observed for Salmonella in the PLNs except for the inguinal nodes, which had a significantly lower Salmonella prevalence in DFM-supplemented cattle than in the controls. Immune function, as measured by monocyte nitric oxide production and neutrophil oxidative burst, was decreased (P < 0.05) in the DFM treatment groups. Although results of this research indicate little to no effect of these DFMs on E. coli O157:H7 or Salmonella in cattle, an increase in the duration of administration to that similar to what is used for commercial cattle might elicit treatment differences.

A series of proof-of-concept studies were developed to determine whether a commercial bacteriophage cocktail could be utilized for the mitigation of Salmonella in bovine peripheral lymph nodes (LNs). The first objective sought to determine whether exogenous phage could be isolated from the LNs following administration. If isolation were successful, the second objective was to determine whether the phage in the LNs could effectively reduce Salmonella. Salmonella Montevideo was inoculated intradermally at multiple sites and multiple times, followed by delivery of the phage cocktail subcutaneously in two injections around each of the right and left prescapular and subiliac LNs. At the conclusion of each study, animals were euthanized, and the popliteal, prescapular, and subiliac LNs were examined. The inoculated phage was successfully isolated from the LNs; transmission electron microscopy revealed phages in the LNs of the treated cattle, and these phages were identical to those in the cocktail. Levels of phage were higher (P0.01) in the prescapular and subiliac LNs in the phage-treated than in the control cattle. In subsequent studies, the protocols were modified to increase Salmonella and phage levels within the LNs. Compared with the first study, overall Salmonella levels were increased in the LNs, and phage treatment decreased (P0.01) Salmonella in the some of the LNs. Phage levels were numerically but not significantly increased (P = 0.12) in the treated cattle. The final study was modified, hypothesizing that a 48-h postmortem period before LN removal would facilitate phage-Salmonella interaction; however, no differences (P0.10) in Salmonella levels were found among treatments. Salmonella-specific phages administered to live cattle can translocate to the LNs; however, these phages had limited to no effect on Salmonella in these LNs under these experimental conditions.

Previous attempts to infect peripheral lymph nodes (PLNs) with Salmonella via oral inoculation have been inconsistent. Therefore, we performed a series of experiments to determine whether multiple exposures to an oral challenge would result in Salmonella-positive PLN in cattle. In each of three experiments, calves were inoculated with Salmonella Montevideo. In the first experiment, calves were challenged with either no Salmonella (control), a single oral dose (~1010; PCON), or 10 consecutive doses in water (~103; WAT). The positive control treatment resulted in an increase (P < 0.05) in the percentage of Salmonella-positive PLNs, compared with the WAT-treated and control animals. Experiments 2 and 3 were designed to additionally determine if the stress associated with feed and water deprivation influences the systemic spread of Salmonella from the gastrointestinal tract to PLNs. Following 14 days of oral inoculation (average 7.1 × 104 CFU/day) in experiment 1, Salmonella was recovered from one subiliac an...

Because challenge models to infect peripheral lymph nodes (PLNs) with Salmonella have not been reported, we performed a series of experiments to develop and refine challenge models to evaluate an intervention applied at the animal level and to provide initial estimates of efficacy of an intervention (i.e., a vaccine) to aid in the design of future studies. In each of four experiments, steers (control or vaccinated) were inoculated with Salmonella strains Montevideo or Newport, and in experiment IV, Salmonella Senftenberg was also used. Calves were euthanized 14 to 42 days postinoculation, and PLNs were collected. In the first experiment, calves were challenged with ∼10¹⁰ Salmonella cells, and few treatment differences were observed 14 days postchallenge. However, by day 21, Salmonella Newport was recovered from fewer vaccinated calves than control calves (P0.05). In experiment II, calves were challenged with ∼10⁷ Salmonella cells and, after two necropsies (14 and 28 days postchallenge), only one lymph node was Salmonella positive; therefore, the study was terminated. In experiment III, calves were again challenged with ∼10¹⁰ Salmonella cells, and no significant effect of vaccine was observed in calves challenged with Montevideo or Newport strains. A transdermal route of challenge was explored in experiment IV, using a 10-lancet, allergy testing instrument. Sixteen steers were challenged with either Salmonella Newport or Salmonella Montevideo (Salmonella Newport right legs; Salmonella Montevideo left legs), and all steers were challenged on the lower abdomen with Salmonella Senftenberg. Transdermal inoculation resulted in predictably Salmonella-positive PLNs, and a modest vaccine effect was detected. Because it is well tolerated by the calves and results in predictable and regionally specific Salmonella recovery from PLNs, the transdermal route of challenge may be preferred by researchers wishing to evaluate the impact of interventions designed to reduce the carriage of Salmonella in PLNs.

Recent investigations have found that Salmonella can be routinely recovered from peripheral lymph nodes (PLNs) of cattle presented for harvest. When contained within the PLNs, this foodborne pathogen is protected from currently used postharvest, inplant intervention strategies and, therefore, PLNs harboring Salmonella may be a potential contaminant of ground beef. The objective of this work was to develop a challenge model that effectively and repeatedly results in Salmonella -positive PLNs. A 10-lancet skin-allergy instrument was inoculated with Salmonella, and calves were inoculated intra- and/or transdermally by applying the device over various ventral regions of the skin. Salmonella was successfully and predictably recovered from regionspecific PLNs up to 8 days postchallenge. Furthermore, serotypes inoculated within specific regions were only recovered from the PLNs draining those regions. This model provides a method to predictably infect PLNs with Salmonella. Further, this model makes it possible to determine the duration of infection and to evaluate candidate interventions that may shorten the duration of infection.

Utilizing a transdermal method of inoculation developed in our laboratory, the duration of infection of Salmonella in the peripheral lymph nodes of steers was examined. Thirty-six Holstein steers (mean body weight of 137 kg) were inoculated with Salmonella Montevideo (day 0) on each lower leg and both sides of the back and abdomen. Calves were euthanized beginning at 6 h and subsequently on each of days 1, 2, 4, 7, 9, 11, 14, and 21 postinoculation (four animals each time). The subiliac, popliteal, and superficial cervical (prescapular) lymph nodes were collected and cultured (quantitatively and qualitatively) for the challenge strain of Salmonella. The challenge strain was detected via direct culture within the lymph nodes at 6 h postinoculation and on each subsequent necropsy date. Salmonella levels in lymph node were 0.8 to 1.8 log CFU/g. Lymph nodes were generally positive after enrichment culture throughout the experiment. Salmonella elimination appeared to begin approximately 14 days postinoculation. However, elimination was not completed by day 21; therefore, a second experiment was conducted identical to the first except that the time from inoculation to necropsy was extended. Salmonella was recovered via direct culture on each of the necropsy days, and results in general were similar to those of experiment I, except that on days 20, 24, and 28 isolates from serogroups C2 and E1 were identified in addition to the inoculation strain C1 in multiple animals. The data from both experiments indicate that after a single inoculation event, Salmonella would be completely cleared by approximately 28 days. Further research with expanded times between inoculation and necropsy is required for verification.

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 (P0.0001) 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 (P0.01) 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.

Strategies aimed at reducing fecal shedding of Salmonella and other foodborne pathogens may be effective for limiting transmission of pathogens from food animals to humans. The objective of this study was to determine the effectiveness of gallium maltolate (GaM) against Salmonella in vitro and to determine whether oral administration of GaM would reduce fecal shedding of Salmonella in cattle. Gallium is a semimetal exhibiting antimicrobial properties against some pathogenic bacteria, including Salmonella, by exploiting their need for iron to survive and replicate. In vitro growth studies were performed in pure cultures of Salmonella and in mixed cultures from ruminal fluid. Inclusion of GaM in culture medium or in mixed cultures of ruminal fluid resulted in a significant reduction in growth of Salmonella, suggesting that GaM may be effective for limiting growth and survival in vivo. Therefore, we subsequently administered two doses of GaM to Holstein steers, experimentally infected them with Salmonella, and quantitatively and qualitatively monitored fecal shedding at 12-h intervals. Sixty hours after beginning treatment, cattle were euthanized, and luminal contents and tissue were aseptically harvested from the rumen, jejunum, spiral colon, cecum, and rectum. The luminal contents were processed for quantitative and qualitative analysis of the challenge strains of Salmonella, and tissue samples were enriched and plated for qualitative analysis. We found no significant differences between control and treated animals in quantitative levels of Salmonella in the feces or the luminal contents. Likewise, we observed no pattern between control and treated animals in the frequency of positive or negative results from enriched feces, luminal contents, or tissue samples. These results suggest that GaM was not effective for reducing Salmonella in cattle.

Escherichia coli O157:H7 is a virulent foodborne pathogen that causes severe human illness and inhabits the intestinal tract of food animals. Colicins are antimicrobial proteins produced by E. coli strains that inhibit or kill other E. coli. In the present study, the efficacy of three pore-forming colicins (E1, N, and A) were quantified in vitro against E. coli O157:H7 strains 86-24 and 933. Colicins E1 and N reduced the growth of E. coli O157:H7 strains, but the efficacy of each colicin varied among strains. Colicin E1 was more effective against both strains of E. coli O157:H7 than colicins A and N and reduced (P < 0.05) populations of E. coli O157:H7 at concentrations

Ruminant animals are a natural reservoir of the foodborne pathogen Escherichia coli O157:H7. Some foodborne pathogens (e.g., E. coli) are equipped with a nitrate reductase that cometabolically reduces chlorate. The intracellular reduction of chlorate to chlorite kills nitrate reductase-positive bacteria; however, species that do not reduce nitrate are not affected by chlorate. Therefore, it has been suggested that ruminants be supplemented with chlorate prior to shipment for slaughter in order to reduce foodborne illnesses in human consumers. Sheep (n = 14) were fed a high-grain ration and were experimentally infected with E. coli O157:H7. These sheep were given an experimental product (XCP) containing the equivalent of either 2.5 mM NaNO 3 and 100 mM NaCl (control sheep; n = 7) or 2.5 mM NaNO 3 and 100 mM NaClO 3 (chlorate [XCP]-treated sheep; n = 7). Control and XCP-treated sheep were treated for 24 h; XCP treatment reduced the population of inoculated E. coli O157:H7 (P < 0.05) from 10 2 , 105, and 10 5 CFU/g in the rumen, cecum, and rectum, respectively, to

The objective of the current research was to determine if feeding the citrus by-products(D) -limonene (DL) and citrus molasses would reduce the concentration and prevalence of Salmonella in weanling pigs experimentally infected with Salmonella Typhimurium. Twenty crossbred weanling pigs (average body weight [BW], 19.9 kg) were randomly assigned to one of four treatments: control, low-dose DL (1.5 ml/kg of BW per day), high-dose DL (3.0 ml/kg of BW per day), and citrus molasses (0.05 kg/kg of BW per day). Treatments were administered in the feed (twice daily) for 7 days, with one-half of the dose administered at each feeding. Fecal samples were collected twice daily (prior to administration of treatment) and cultured for quantitative and qualitative determination of the challenge strain of Salmonella. Upon termination of the study, pigs were euthanized and tissues from the stomach, ileum, cecum, spiral colon, and rectum, as well as luminal contents, were collected. In addition, the popliteal and ileocecal lymph nodes and liver, spleen, and tonsil tissue were collected for qualitative Salmonella culture. No significant treatment differences (P0.05) were observed among treatments for fecal concentration or prevalence of Salmonella throughout the 7-day collection period. Likewise, no treatment differences (P0.05) were observed for any of the tissue or luminal content samples collected. Salmonella was not cultured from the muscle-bound popliteal lymph node but was cultured from the mesenteric ileocecal lymph nodes. While there were no effects in the current experiment, future research may examine the effect of a lower challenge dose and/or different administration (dose or duration) of the citrus by-products.

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.

The present study examined the incidence of Escherichia coli 0157: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 0157: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 0157:H7 averaged 9 and 18%, respectively. One carcass swab was E. coli 0157: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 0157:H7, Salmonella, and fecal coliforms) were susceptible to ceftiofur, enrofloxacin, and trimethoprim-sulfamethoxazole. One E. coli 0157:H7 isolate cultured from a carcass swab was resistant to seven antibiotics, and seven wool E. coli 0157: H7 isolates were multidrug resistant. Results of this research demonstrate that feedlot sheep are naturally colonized with E. coli 0157: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.

The objectives of the current research were twofold: (i) to determine the prevalence of multidrug-resistant (MDR) Salmonella in the various classes of dairy cattle and (ii) to determine if comingling of calves from multiple farms at a heifer feedlot serves as a transmission vector for Salmonella back to the dairy farm. Four large commercial dairies in the southwestern United States were sampled in October 2005 and again in March 2006. Fecal samples were collected from hutch calves, 12- and 24-month-old heifers, lactating cows, dry cows, and cattle in the sick-fresh pen and cultured with brilliant green agar supplemented with novobiocin (BGA(nov)) to estimate the overall Salmonella prevalence, or with tetracycline (BGA(tet)) to estimate MDR Salmonella. Antimicrobial susceptibility testing was conducted with the National Antibiotic Resistance Monitoring System (NARMS) testing panel, and a portion of the isolates were serotyped. Salmonella prevalence among groups ranged from 0 to 96% positive, with the highest incidence observed in the hutch calves and cattle in the sick-fresh pen. Twenty-eight different serotypes were identified with serotype Reading accounting for the majority of isolates cultured on BGA(tet). Nearly all (100 of 103) isolates cultured on BGA(nov) and screened for antibiotic resistance were pan susceptible, whereas over one-half (64%) of the isolates cultured on BGA(tet) were MDR. Forty isolates displayed the ACSSuT resistance pattern, and 36 isolates displayed the MDR-AmpC pattern of the 72 isolates examined following culture on BGA(tet). The incidence of Salmonella cultured on BGA(tet) was low (9%) in all heifers and only one MDR isolate was cultured (from a 12-month-old heifer), suggesting the risk of transmission of Salmonella from the heifer feedlot back to the dairy is low. Results of this research suggest the incidence of MDR Salmonella, found primarily in hutch calves and cattle in the sick-fresh pen, is low in comparison to the overall Salmonella prevalence.

Our laboratory has developed a bacterial competitive-exclusion (CE) culture against enteropathogens (which are considered human foodborne pathogens) for use in swine. In this article, we document the effects of this CE culture, PCF1, on cecal colonization by and fecal shedding of Salmonella Choleraesuis in neonatal and weaned pigs and its effects on the horizontal transmission of this pathogen between weaned penmates. Piglets treated with the PCF1 culture twice within their first day of life and challenged with Salmonella 48 h after birth shed Salmonella at a significantly (P < 0.05) lower rate than did control pigs in experiment 1. Significant reductions of the pathogen were also observed in the cecum, the cecal contents, the ileocolic junction, and the colon contents (P < 0.05). In experiment 2, culture of the cecal contents and lymph nodes revealed a significant reduction in Salmonella isolated from PCF1-treated pigs (P < 0.05). Pigs in experiment 3 were treated as pigs in experiments 1 and 2 were: however, they were followed through day 10 postweaning. Significant reductions in shedding were noted for treated groups both pre- and postweaning (P < 0.05). Experiments 4 and 5 assessed the effects of PCF1 treatment on the horizontal transmission of Salmonella between littermates that were followed through day 14 postweaning. In these experiments, litters were divided into untreated contacts (UC), untreated seeders (US), treated contacts (TC), and treated seeders (TS). Overall, TC in experiment 4 shed Salmonella at a significantly lower rate than UC and US did (P < 0.05). In experiment 5, the transmission of Salmonella was significantly reduced for litters in which TS or TC were present, as evidenced by reduced shedding of Salmonella by both treated and untreated animals within these litters (P < 0.05). TS shed less often than US did, resulting in reduced levels of Salmonella shedding by both treated and untreated contacts (P < 0.05). Litters containing both TC and UC or both TC and US also shed Salmonella at lower rates than did litters in which only UC and US were present (P < 0.05).

Although there have been numerous studies investigating the prevalence of campylobacters in animals and raw meats, there are limited data on the persistence of these organisms in ready-to-eat (RTE) foodstuffs. Although poultry is now well established as a major reservoir of thermophilic campylobacters, it is widely assumed that hazard analysis critical control point (HACCP) controls in commercial and industrial settings are effective in eliminating this hazard through thorough cooking of RTE products. Therefore, it was the primary aim of this study to investigate the effectiveness of HACCP controls in eliminating campylobacters in such cooked RTE foods by attempting to isolate viable organisms from product. Concurrently, the results of this study demonstrate that local poultry is highly contaminated with campylobacters. Commercially available RTE foodstuffs (n = 2,030) consisting of 1,061 poultry-related cooked products and 969 other products were analyzed and were not found to contain thermophilic Campylobacter spp. In addition, 107 raw chickens (63 fresh birds and 44 frozen birds) were sampled, and 94% of the fresh birds and 77% of the frozen birds examined were demonstrated to be contaminated with campylobacters, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari accounting for 69, 30, and 1% of the contaminating organisms, respectively. In general, commercially available RTE foodstuffs, including cooked poultry, are not commonly contaminated with campylobacters and thus do not appear to represent a significant cause of clinical infection of Campylobacter spp. in Northern Ireland. However, raw poultry produce, including fresh and frozen chicken, frequently tested positive for campylobacters. Implementation of HACCP systems by food processors will help to minimize and/or eliminate the risk posed by this organism to the consumer.