Evaluation of a Preharvest Bacteriophage Therapy for Control of Salmonella within Bovine Peripheral Lymph Nodes.

ABSTRACT: 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 (P < 0.01) 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 (P < 0.01) 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 (P > 0.10) 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.

Evaluation of the Efficacy of Three Direct Fed Microbial Cocktails To Reduce Fecal Shedding of Escherichia coli O157:H7 in Naturally Colonized Cattle and Fecal Shedding and Peripheral Lymph Node Carriage of Salmonella in Experimentally Infected Cattle.

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.

Methylsulfonylmethane exhibits bacteriostatic inhibition of Escherichia coli, and Salmonella enterica Kinshasa, in vitro.

AIMS: To evaluate antibacterial properties of methylsulfonylmethane (MSM) on Escherichia coli (MDRE21) and Salmonella enterica serovar Kinshasa (SK132). METHODS AND RESULTS: Bacterial proliferation analysis was measured spectrophotometrically during log phase growth with 0, 3, 5, 7, 10, 12 and 16% MSM. To assess the mechanism of inhibition, cultures were grown overnight with 0-16% MSM and enumerated on unmedicated brain-heart infusion agar (BHIA) or BHIA with 0-16% MSM. The long-term viability studies were done to evaluate the impact of 10% MSM. Absorbance data indicated a dose-dependent inhibition from 0 to 16% MSM. There was no growth of MDRE21 or SK132 on BHIA in 10-16% MSM. Both strains enumerated on unmedicated BHIA from overnight cultures with 10-16% MSM were able to partially recover. CONCLUSIONS: Recovery after MSM removal may be indicative of a bacteriostatic mechanism of inhibition. The long-term viability studies illustrated that neither MDRE21 nor SK132 could be rescued from 10% MSM after 5 or 6 days respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Methylsulfonylmethane antibacterial activity may prove useful during pre or postharvest food safety as a disinfectant. The primary benefit being, its clinical safety to humans. Comparisons to other disinfectants would also need to be done to determine if MSM was superior to those already on the market and would be cost effective.

Inhibition of multidrug-resistant Staphylococci by sodium chlorate and select nitro- and medium chain fatty acid compounds.

AIMS: Determine the antimicrobial effects of 5 µmol ml-1 sodium chlorate, 9 µmol ml-1 nitroethane or 2-nitropropanol as well as lauric acid, myristic acid and the glycerol ester of lauric acid Lauricidin® , each at 5 mg ml-1 , against representative methicillin-resistant staphylococci, important mastitis- and opportunistic dermal-pathogens of humans and livestock. METHODS AND RESULTS: Three methicillin-resistant Staphylococcus aureus and two methicillin-resistant coagulase-negative staphylococci were cultured at 39°C in 5 µmol ml-1 nitrate-supplemented half-strength Brain Heart Infusion broth treated without or with the potential inhibitors. Results revealed that 2-nitropropanol was the most potent and persistent of all compounds tested, achieving 58-99% decreases in mean specific growth rates and maximum optical densities when compared with untreated controls. Growth inhibition did not persist by cultures treated solely with chlorate or nitroethane, with adaptation occurring by different mechanisms after 7 h. Adaptation did not occur in cultures co-treated with nitroethane and chlorate. The medium chain fatty acid compounds had modest effects on all the staphylococci tested except the coagulase-negative Staphylococcus epidermidis strain NKR1. CONCLUSIONS: The antimicrobial activity of nitrocompounds, chlorate and medium chain fatty acid compounds against different methicillin-resistant staphylococci varied in potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that differential antimicrobial activities exhibited by mechanistically dissimilar inhibitors against methicillin-resistant staphylococci may yield potential opportunities to combine the treatments to overcome their individual limitations and broaden their activity against other mastitis and dermal pathogens.

Interactions of organic acids with vancomycin-resistant Enterococcus faecium isolated from community wastewater in Texas.

AIMS: Investigate the interactions of organic acids (OAs), acetic, butyric, citric, formic, lactic and propionic acid against 50 Gram-positive vancomycin-resistant Enterococcus faecium (VRE) strains to determine whether pH, undissociated or dissociated acid forms correlate with bacterial inhibition. METHODS AND RESULTS: Concentrations of undissociated and dissociated OAs at the molar minimum inhibitory concentrations (MICM s) of the VRE were calculated using the Henderson-Hasselbalch equation. The pH at the MICM s of all VRE strains against acetic, butyric, formic and propionic acids was similar, 4·66 ± 0·07, but there was a 1·1 pH unit difference for all six OAs. Inhibition of VRE by all six OAs did not appear to be solely dependent on pH or on the undissociated OA species. The inhibition of VRE by all six dissociated acids was within Δ = 3·1 mmol l-1 . CONCLUSIONS: Vancomycin-resistant Enterococcus faecium inhibition correlated with the dissociated OA species. A small decrease in the concentration of the dissociated OAs from optimum may result in allowing VRE strains to escape disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: When an OA is used to disinfect VRE strains, the concentration of the dissociated OA should be carefully controlled. A concentration of at least 20 mmol l-1 dissociated OA should be maintained when disinfecting VRE.

The horizontal transfer of Salmonella between the lesser mealworm (Alphitobius diaperinus) and poultry manure.

There is need to determine the nature of enduring reservoirs of Salmonella contributing to perpetual contamination within poultry flocks. The dispersal of Salmonella between birds, litter and the lesser mealworm has been established, but the extent that these act as critical components in the epidemiology of Salmonella infection during broiler grow-out and flock rotation has not been delineated; in particular, the level of participation by the lesser mealworm beetles (LMB) as agents of retention and dispersal. This study defines this route of transmission and provides empirical data on bacterial loads that facilitate Salmonella transfer. Results showed differential Salmonella transfer dependent on bacterial concentration. At 103  cfu/ml, only a small, but not significant, amount of Salmonella was transferred, from the LMB to the manure and back to uninfected LMB; while from 105 to 107  cfu/ml, a significant acquisition and transfer occurred both internally and externally to the LMB over 4 and 24 hr exposures. These data will be used in correlation with facility management practices to develop intervention strategies to mitigate the establishment and spreading of reservoir Salmonella populations contributing to pre-harvest contamination of poultry flocks.

Transferability of antimicrobial resistance from multidrug-resistant Escherichia coli isolated from cattle in the USA to E. coli and Salmonella Newport recipients.

OBJECTIVES: This study aimed to evaluate conjugative transfer of cephalosporin resistance among 100 strains of multidrug-resistant Escherichia coli (MDRE) to Salmonella enterica serotype Newport and E. coli DH5α recipients. METHODS: Phenotypic and genotypic profiles were determined for MDRE as well as for Salmonella Newport (trSN) and E. coli DH5α (trDH) transconjugants. RESULTS: Of 95 MDRE donor isolates, 26 (27%) and 27 (28%) transferred resistance to trSN and trDH recipients, respectively. A total of 27 MDRE (27%) were confirmed as extended-spectrum ß-lactamase (ESBL)-producers based on the double-disk synergy assay and whole-genome sequencing (WGS). WGS was performed on 25 of the ESBL-producing isolates, showing that 2 isolates carried blaCTX-M-6, 22 possessed blaCTX-M-32 and 1 was negative for blaCTX-M genes. Fourteen of the ESBLs sequenced were qnrB19. Differential transfer of IncA/C and IncN from MDRE32 was observed between trSN32 and trDH32. IncN-positive trDH32 displayed an ESBL phenotype, whereas IncA/C-positive trSN32 displayed an AmpC phenotype. The rate of ESBL transfer to trSN and trDH recipients was 11% and 96%, respectively. CONCLUSIONS: Twenty-seven MDRE were phenotypically identified as ESBL-producers. WGS of 25 MDRE revealed that 2 and 22 isolates carried blaCTX-M-6 and blaCTX-M-32, respectively. One multidrug-resistant isolate exhibited conversion from an AmpC phenotype to an ESBL phenotype with the transfer of only the IncN plasmid. The rate of resistance transfer to Salmonella or E. coli recipients was nearly identical. However, the ESBL phenotype was transferred with significantly greater prevalence to E. coli compared with Salmonella Newport (96% and 11%, respectively).

The identification of fungi collected from the ceca of commercial poultry.

Under normal conditions, fungi are ignored unless a disease/syndrome clinical signs are reported. The scientific communities are largely unaware of the roles fungi play in normal production parameters. Numerous preharvest interventions have demonstrated that beneficial bacteria can play a role in improving productions parameters; however, most researchers have ignored the impact that fungi may have on production. The goal of the present study was to record fungi recovered from commercial broiler and layer houses during production. Over 3,000 cecal samples were isolated using conventional culture methodology and over 890 samples were further characterized using an automated repetitive sequence-based PCR (rep-PCR) methodology. Eighty-eight different fungal and yeast species were identified, including Aspergillus spp., Penicillium spp., and Sporidiobolus spp, and 18 unknown genera were separated using rep-PCR. The results from the present study will provide a normal fungi background genera under commercial conditions and will be a stepping stone for investigating the impact of fungi on the gastrointestinal tract and on the health of poultry.

Salmonella Persistence within the Peripheral Lymph Nodes of Cattle following Experimental Inoculation.

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.

In Vitro Effects of Thymol-ß-D-Glucopyranoside on Salmonella enterica Serovar Typhimurium and Escherichia coli K88.

Although thymol is bactericidal against many pathogens in vitro, its in vivo effectiveness against pathogens in the lower gastrointestinal tract is limited because of its rapid absorption in the proximal gut. Thymol-ß-D-glucopyranoside (ß-thymol), a conjugated form of thymol, can deliver thymol to the lower gastrointestinal tract and has shown antibacterial effects. In the present study, we examined the in vitro effects of ß-thymol on Salmonella enterica serovar Typhimurium (ST) and Escherichia coli K88 (K88). We inoculated one-half strength Mueller-Hinton broth with 5.8 ± 0.09 log CFU/ml novobiocin- and naladixic acid-resistant (NN) ST (NVSL 95-1776) and 5.1 ± 0.09 log CFU ml(-1) NN-resistant K88, with or without porcine feces (0.1% [wt/vol]) (fecal incubations). The resultant bacterial suspensions were distributed under N2 to triplicate sets of tubes to achieve initial concentrations of 0, 3, 6, and 12 mM for ST treatments and 0, 3, 12, and 30 mM for K88 treatments. Samples were incubated at 39°C and then plated onto NN-containing brilliant green agar and NN-containing MacConkey agar; ST and K88 CFU concentrations were determined via 10-fold dilutions, and viable cell counts were performed at 0, 6, and 24 h. No differences in ST CFU counts were observed in ß-thymol-treated tubes without the added porcine feces (i.e., pure culture) at 6 or 24 h. However, in tubes that contained fecal incubations, ST CFU counts were reduced (P < 0.05) from controls at 6 h in tubes treated with 6 and 12 mM ß-thymol, whereas in tubes treated with 3, 6, and 12 mM ß-thymol the CFU counts were reduced (P < 0.05) at 24 h. No differences were observed in K88 CFU counts in pure culture or in fecal incubations at 6 h, but K88 CFU counts were reduced (P < 0.05) in both pure and fecal incubations at 24 h. The results from this study demonstrate that ß-thymol, in the presence of fecal suspensions, has anti-Salmonella and anti-E. coli effects, suggesting a role of ß-glycoside-hydrolyzing microbes for the release of bactericidal thymol from ß-thymol.

The Role of Direct-Fed Microbials in Conventional Livestock Production.

Supplementation of direct-fed microbials (DFM) as a means to improve the health and performance of livestock has generated significant interest over the past 15+ years. A driving force for this increased interest in DFM is to reduce or eliminate the use of low-dose antibiotics in livestock production. This increased attention toward DFM supplementation has generated an extensive body of research. This effort has resulted in conflicting reports. Although there has been considerable variation in the design of these studies, one of the main causes for this lack of consistency may be attributed to the variation in the experimental immune challenge incorporated to evaluate DFM supplementation. Taking into account the experimental immune challenge, there is strong evidence to suggest that DFM supplementation may have an impact on the immune response, overall health, and performance of livestock.

Evidence supporting vertical transmission of Salmonella in dairy cattle.

We set out to investigate whether Salmonella enterica could be recovered from various tissues of viable neonatal calves immediately following parturition. Eleven samples were aseptically collected from each of 20 calves and consisted of both left and right subiliac and prescapular lymph nodes (LN), mesenteric LN, spleen and liver, as well as intestinal tissue (including luminal contents) from the small intestine, caecum, spiral colon and rectum. In addition, a faecal sample was collected from 19 of the dams. Salmonella was recovered from at least one sample from 10 of the 20 neonates. Across all calves, Salmonella was recovered from 12·7% of all samples and from LN in particular, Salmonella was recovered from 10·0%, 5·0%, and 5·0% of subiliac, prescapular, and mesenteric LN, respectively. Within calves, Salmonella was recovered from 0% to 73% of samples and across tissues, estimates of Salmonella prevalence were greatest in the caecum (30%) but was never recovered from the right pre-scapular LN. These data provide evidence of vertical transmission from a dam to her fetus such that viable calves are born already infected and thereby not requiring faecal-oral exposure for transmission. This new knowledge ought to challenge - or at least add to - existing paradigms of Salmonella transmission dynamics within cattle herds.

Investigation into Possible Differences in Salmonella Prevalence in the Peripheral Lymph Nodes of Cattle Derived from Distinct Production Systems and of Different Breed Types.

Previous research demonstrated significant variation in the prevalence of Salmonella in peripheral lymph nodes (LNs) of feedlot cattle and cull cows, with greater prevalence in feedlot cattle. Therefore, we performed experiments to investigate whether these differences in Salmonella prevalence in subiliac LNs are due to, or influenced by, breed, which in many respects is a proxy for the production system in which the animal is derived. Holstein steers are a by-product of dairy systems, and beef steers are an intended product of commercial beef operations. For the first experiment, Holstein and beef steers originating from the same feedlot and harvested on the same day were sampled. Of the 467 Holstein and 462 beef cattle LNs collected, 62.1% of Holstein and 59.7% of beef cattle samples harbored Salmonella (P = 0.46; qualitative culture), with 51.2 and 48.9% of samples containing quantifiable concentrations (P = 0.49), respectively. The concentration of Salmonella within the LN followed a decreasing trend over the collection period (May to October), averaging 1.4 log CFU/g of LN for both Holstein and beef cattle samples (P = 0.78). In a second experiment, we compared 100% Brahman cattle to their beef cattle counterparts, as we hypothesized that the resistance of Brahman cattle to insects may reduce Salmonella transmission via biting insects. Of the 42 Brahman and 31 beef cattle LNs collected, the concentration of Salmonella within the LN averaged 3.0 log CFU/g for Brahman cattle and 2.9 log CFU/g for beef cattle samples (P = 0.30). Using qualitative culture, we recovered Salmonella from 100% of LNs from Brahman cattle and 97% of beef cattle samples (P = 0.25). Results of this research indicate that the differences observed are not due to breed and are likely a function of age, immune function, or other factors yet to be identified. Understanding which cattle are more likely to harbor Salmonella within LNs will aid in targeting both pre- and postharvest intervention strategies.

Oral salmonella challenge and subsequent uptake by the peripheral lymph nodes in calves.

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 (∼10(10); PCON), or 10 consecutive doses in water (∼10(3); 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 × 10(4) CFU/day) in experiment 1, Salmonella was recovered from one subiliac and one superficial cervical lymph node of calves that were deprived of feed and water (72 h). No treatment differences (P > 0.05) were observed between control and deprived calves. Based on the poor recovery of Salmonella from the PLNs in WAT-challenged calves in experiments 1 and 2, a higher challenge dose (average 1.2 × 10(7) CFU) was used in experiment 3. The increased dose resulted in the recovery of the challenge strain of Salmonella from the PLNs (70.8 and 75.0% of control and deprived calves, respectively). However, no treatment differences (P > 0.05) were observed between control and deprived calves. Results of this research demonstrated that a substantial oral challenge is required to produce Salmonella-positive PLNs. However, as the challenge periods examined herein were considerably shorter compared with the normal time spent by cattle in feedlots, increased exposure time to lower doses may produce the same effect observed in experiment 3.

Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994-2003.

AIMS: To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants. METHODS AND RESULTS: Pseudomonas aeruginosa isolates collected from dogs (n = 155) and other animals (n = 20) from sixteen states during 1994-2003 were tested for susceptibility. Most isolates were resistant to twenty-one antimicrobials tested, and the highest prevalence of resistance was to ß-lactams (93.8%) and sulphonamides (93.5%). Fluoroquinolone resistance did not increase from 1994 to 2003. Ciprofloxacin and enrofloxacin had a 5 and 16% prevalence of resistance, respectively, while sarafloxacin and nalidixic acid had a prevalence of resistance of 97 and 98%, respectively. Strains were pan-resistant to triclosan and chlorhexidine, were highly resistant to benzalkonium chloride and demonstrated high susceptibility to other disinfectants. Didecyldimethylammonium chloride was the most active ammonium chloride. Inducible resistance was observed to cetyl ammonium halides, chlorhexidine and benzyl ammonium chlorides, which formulate disinfectants used in veterinary clinics and dairies. Organic acid inhibition was associated with the dissociated acid species. CONCLUSIONS: Dissociated organic acids appear able to inhibit Ps. aeruginosa, and rates of fluoroquinolone resistance merit sustained companion animal isolate surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of Ps. aeruginosa susceptibility to 24 disinfectants and illustrates the high resistance of Ps. aeruginosa to both antibiotics and disinfectants.

Meat Science and Muscle Biology Symposium: Ecological and dietary impactors of foodborne pathogens and methods to reduce fecal shedding in cattle.

Pathogenic bacteria can live asymptomatically within and on cattle and can enter the food chain but also can be transmitted to humans by fecal or direct animal contact. Reducing pathogenic bacterial incidence and populations within live cattle represents an important step in improving food safety. A broad range of preslaughter intervention strategies are being developed, which can be loosely classified as 1) directly antipathogen strategies, 2) competitive enhancement strategies (that use the microbiome's competitive nature against pathogens), and 3) animal management strategies. Included within these broad categories are such diverse methods as vaccination against foodborne pathogens, probiotics and prebiotics, bacterial viruses (i.e., bacteriophages), sodium chlorate feeding, and dietary and management changes that specifically alter the microbiome. The simultaneous application of 1 or more preharvest strategies has the potential to reduce human foodborne illnesses by erecting multiple hurdles preventing entry into humans. However, economic factors that govern producer profitability must be kept in mind while improving food safety.

Evaluation of feeding distiller's grains, containing virginiamycin, on antimicrobial susceptibilities in fecal isolates of Enterococcus and Escherichia coli and prevalence of resistance genes in cattle.

Dried distiller's grains (DG) produced from ethanol fermentations dosed with 0 (control), 2, or 20 mg/kg virginiamycin-based product or spiked with virginiamycin (VM) postfermentation were fed to cattle and effects on antimicrobial susceptibility, and prevalence of antimicrobial resistance genes in commensal bacteria was examined. Biological activity assays of DG (from each fermentation) indicated a concentration of 0, 0.7, and 8.9 mg/kg VM, respectively. Twenty-four crossbred beef steers were fed 1 of 4 diets (containing 8% of each of the different batches of DG) and a fourth using 8% of the control DG (0 mg/kg VM) + 0.025 g/kg V-Max50 (positive control) for 7 wk. Fecal samples were collected weekly throughout the experimental period and cultured for Escherichia coli and Enterococcus, and isolates were examined for antimicrobial susceptibility, antimicrobial resistance genes (vatE, ermB, and msrC in Enterococcus), and integrons (E. coli). No treatment differences (P > 0.05) were observed in antimicrobial susceptibility of the E. coli isolates. Enterococcus isolates were resistant to more antimicrobials; however, this was influenced by the species of Enterococcus and not treatment (P > 0.10). The prevalence of ermB was greater (P < 0.05) in the control isolates after 4 and 6 wk while at wk 7, prevalence was greater (P < 0.01) in the 0.7 and 8.9 mg/kg VM treatments. Taken together, the minor treatment differences observed for the presence of ermB coupled with the lack of effect on antimicrobial susceptibility patterns suggest that feeding DG containing VM residues should have minimal if any impact on prevalence of antimicrobial resistance.

Development of a transdermal Salmonella challenge model in calves.

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.

Development of challenge models to evaluate the efficacy of a vaccine to reduce carriage of Salmonella in peripheral lymph nodes of cattle.

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 ∼10¹° 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 (P < 0.05). 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 ∼10¹° 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.

Interactions between oil substrates and glucose on pure cultures of ruminal lipase-producing bacteria.

The hydrolysis of free fatty acids from lipids is a prerequisite for biohydrogenation, a process that effectively saturates free fatty acids. Anaerovibrio lipolyticus 5s and Butyrivibrio fibrisolvens have long been thought to be the major contributors to ruminal lipolysis; however, Propionibacterium avidum and acnes recently have been identified as contributing lipase activity in the rumen. In order to further characterize the lipase activity of these bacterial populations, each was grown with three different lipid substrates, olive oil, corn oil, and flaxseed oil (3 %). Because different finishing rations contain varying levels of glycogen (a source of free glucose) this study also documented the effects of glucose on lipolysis. P. avidum and A. lipolyticus 5s demonstrated the most rapid rates (P < 0.05) of lipolysis for cultures grown with olive oil and flaxseed oil, respectively. A. lipolyticus, B. fibrisolvens, and P. avidum more effectively hydrolyzed flaxseed oil than olive oil or corn oil, especially in the presence of 0.02 % glucose. Conversely, P. acnes hydrolyzed corn oil more readily than olive oil or flaxseed oil and glucose had no effect on lipolytic rate. Thus, these bacterial species demonstrated different specificities for oil substrates and different sensitivities to glucose.