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.

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.

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.

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.

Evaluation of the potential antimicrobial resistance transfer from a multi-drug resistant Escherichia coli to Salmonella in dairy calves.

Previous research conducted in our laboratory found a significant prevalence of multi-drug resistant (MDR) Salmonella and MDR Escherichia coli (MDR EC) in dairy calves and suggests that the MDR EC population may be an important reservoir for resistance elements that could potentially transfer to Salmonella. Therefore, the objective of the current research was to determine if resistance transfers from MDR EC to susceptible strains of inoculated Salmonella. The experiment utilized Holstein calves (approximately 3 weeks old) naturally colonized with MDR EC and fecal culture negative for Salmonella. Fecal samples were collected for culture of Salmonella and MDR EC throughout the experiment following experimental inoculation with the susceptible Salmonella strains. Results initially suggested that resistance did transfer from the MDR E. coli to the inoculated strains of Salmonella, with these stains demonstrating resistance to multiple antibiotics following in vivo exposure to MDR EC. However, serogrouping and serotyping results from a portion of the Salmonella isolates recovered from the calves post-challenge, identified two new strains of Salmonella; therefore transfer of resistance was not demonstrated under these experimental conditions.

Development of colonic microflora as assessed by pyrosequencing in dairy calves fed waste milk.

The objective of the current study was to examine the effect of pasteurization of waste milk, used to feed dairy calves, on the bacterial diversity of their lower gut. Using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing, fecal samples from dairy calves, ages 1 wk to 6 mo old and fed either pasteurized or nonpasteurized waste milk, were analyzed for bacterial diversity. Calves were maintained on 2 separate farms and, aside from how the waste milk was treated, were housed and cared for similarly. Fifteen calves were sampled from each age group (1, 2, and 4 wk, and 2, 4, and 6 mo of age; n=90 samples per milk treatment, 180 total samples) on each farm via rectal palpation and the samples shipped and frozen before analysis. In general, bacterial diversity, as represented by the total number of different species, was greater for the calves fed pasteurized waste milk at all ages (except 1 wk of age) and increased with increasing age in both treatments. Proteobacteria, Bacteroidetes, and Firmicutes were the predominant phyla. Differences in phyla and class were observed among treatments and age of calf but with no consistent trends. Salmonella were detected in 9 out of 14 (64%) of the 1-wk-old calves fed nonpasteurized milk. Treponema, an important beneficial bacterium in cattle rumen, was more prevalent in the pasteurized waste milk-fed animals and became higher in the older animals from this group. Escherichia-Shigella were detected among treatments at all ages, and highest at 1 wk of age, averaging approximately 21 and 20% of all bacteria for calves fed pasteurized and nonpasteurized waste milk, respectively, and decreasing as calves aged (2.6 and 1.3%). The consistent detection of Salmonella in the younger animals fed nonpasteurized milk and its absence in all other groups is an important finding related to this feeding practice.

Lack of effect of feeding citrus by-products in reducing salmonella in experimentally infected weanling pigs.

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 (P > 0.05) were observed among treatments for fecal concentration or prevalence of Salmonella throughout the 7-day collection period. Likewise, no treatment differences (P > 0.05) 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.

Evaluation of the effect of gallium maltolate on fecal Salmonella 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.

Evaluation of bacterial diversity in the rumen and feces of cattle fed different levels of dried distillers grains plus solubles using bacterial tag-encoded FLX amplicon pyrosequencing.

Dietary components and changes cause shifts in the gastrointestinal microbial ecology that can play a role in animal health and productivity. However, most information about the microbial populations in the gut of livestock species has not been quantitative. In the present study, we utilized a new molecular method, bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) that can perform diversity analyses of gastrointestinal bacterial populations. In the present study, cattle (n = 6) were fed a basal feedlot diet and were subsequently randomly assigned to 1 of 3 diets (n = 2 cows per diet). In each diet, 0, 25, or 50% of the concentrate portion of the ration was replaced with dried distillers grain (DDGS). Ruminal and fecal bacterial populations were different when animals were fed DDGS compared with controls; ruminal and fecal Firmicute:Bacteroidetes ratios were smaller (P = 0.07) in the 25 and 50% DDG diets compared with controls. Ruminal pH was decreased (P < 0.05) in ruminal fluid from cattle fed diets containing 50% compared with 0% DDGS. Using bTEFAP, the normal microbiota of cattle were examined using modern molecular methods to understand how diets affect gastrointestinal ecology and the gastrointestinal contribution of the microbiome to animal health and production.

Decreased competiveness of the foodborne pathogen Campylobacter jejuni during Co-culture with the hyper-ammonia producing anaerobe Clostridium aminophilum.

Campylobacter spp. are a leading bacterial cause of human foodborne illness. When cocultured in anaerobic Bolton broth with the hyper-ammonia producing bacterium, Clostridium aminophilum, ammonia accumulation was greater and final growth of Campylobacter jejuni was reduced (CFU>or=1.4 log10/mL) compared to that obtained by pure culture controls. Co-culture with the less active ammonia-producing saccharolytic Prevotella albensis had no effect on final C. jejuni concentrations. When co-cultured similarly except with the addition of 10 micromol/L monensin, monensin-susceptible Cl. aminophilum was reduced by 2 to 4 log10 CFU/mL and concentrations of C. jejuni, which is insensitive to monensin, did not differ from its pure culture control. These results suggest that in the absence of added monensin, the hyper ammonia-producing Cl. aminophilum may be able to outcompete asaccharolytic C. jejuni for amino acid substrates and that this competitive ability was eliminated by addition on monensin.

Evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro.

Ruminant-derived foods contain high proportions of saturated fats as a result of ruminal biohydrogenation that rapidly saturates and thus limits the availability of free unsaturated fatty acids for assimilation. The objective of this study was to evaluate the effects of glycerol on ruminal free-fatty acid (FFA) production rates and in vitro fermentation kinetics of alfalfa hay. In vitro incubations demonstrated 48% and 77% reductions in rates of FFA accumulation in incubations supplemented with 2% and 20% glycerol as compared to controls. In vitro incubations with alfalfa hay demonstrated that increasing levels of glycerol did not affect NDF digestibility of the hay. Additionally, increasing amounts of glycerol decreased the acetate to propionate ratio in the rumen. These results suggest that inhibiting bacterial fat degradation may promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products.

Influence of body condition and forage type on prevalence of Escherichia coli O157:H7 and Salmonella in grazing beef cows.

AIM: To determine the influence of body condition (BC) and forage type on the prevalence of faecal shedding of Escherichia coli O157:H7 and Salmonella from beef cows. METHODS AND RESULTS: Thin or moderately conditioned cows (n = 115) were randomly assigned to graze either common bermudagrass (n = 3 pastures) or toxic endophyte-infected tall fescue (n = 3 pastures) for 62 days. Faecal samples were collected on day 0, 30 and 62. Overall percentage of faecal samples positive for E. coli O157:H7 was 2.6% and 2.0% for Salmonella. Percentage of cows positive for both E. coli O157:H7 and Salmonella on at least one occasion was 6.1%. BC, forage type or the interaction did not influence the prevalence of E. coli O157:H7 or Salmonella in the faeces of cows. CONCLUSIONS: BC at initiation of the grazing period or loss of BC in moderate conditioned cows during the grazing period did not influence faecal shedding of E. coli O157:H7 or Salmonella. Consumption of either forage type did not influence faecal shedding of either E. coli O157:H7 or Salmonella in beef cows of thin or moderate BC. SIGNIFICANCE AND IMPACT OF THE STUDY: Change in BC that typically occurs during the normal production cycle in grazing cows did not influence faecal shedding of pathogenic bacteria regardless of forage type.

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro.

AIMS: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. METHODS AND RESULTS: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar-Hinton broth supplemented with 0.25 micromol DIC or thymol ml(-1) but not with 0.01 micromol monensin ml(-1) or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log(10) CFU from controls after 24 h pure culture in Bolton broth supplemented with 0.25 or 1.0 micromol DIC ml(-1) or with 1.0 micromol thymol ml(-1). Similarly, each test Campylobacter strain was reduced >3 log(10) CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0.25 or 1.0 micromol DIC ml(-1) or with 1.0 micromol thymol ml(-1). Treatments with 0.25 micromol thymol ml(-1), 0.01 micromol monensin ml(-1) or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0.25 or 1.0 micromol DIC ml(-1) but only intermittently reduced, if at all, by the other treatments. CONCLUSIONS: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest a potential physiological characteristic that may be exploited to develop interventions.

Influence of sprinklers, used to alleviate heat stress, on faecal shedding of E. coli O157:H7 and Salmonella and antimicrobial susceptibility of Salmonella and Enterococcus in lactating dairy cattle.

AIMS: To determine the effect of sprinklers on faecal shedding of Escherichia coli (E. coli) O157:H7 and Salmonella in lactating dairy cattle. METHODS AND RESULTS: Sprinklers were applied to lactating dairy cattle on two farms at either the feedbunk or in the holding pen prior to milking. Faecal samples were collected approx. 1 and 4 weeks following initiation of sprinkler treatments for culture of E. coli O157:H7, Salmonella and Enterococcus. No treatment differences were observed for E. coli O157:H7. Salmonella was higher (P = 0.11) in the control treatment on day-7 whereas on day-28, the bunk sprinklers increased the number of Salmonella positive cows. Salmonella prevalence decreased (P = 0.0001) on day-5 and when examined across days in cows exposed to sprinklers prior to milking. Antimicrobial susceptibility screening found very few isolates that were multi-drug resistant. All Enterococcus isolates were susceptible to vancomycin. CONCLUSIONS: This study demonstrated a significant decrease in faecal prevalence of Salmonella in lactating cattle following exposure to sprinklers administered prior to milking. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of current dairy management techniques that are also effective in reducing on-farm prevalence of pathogenic bacteria could have significant food safety and environmental implications.