Survival of Campylobacter jejuni during in vitro culture with mixed bovine ruminal microorganisms in the presence of methanogen inhibitors.

Foodborne pathogen Campylobacter jejuni has been associated with ruminants. The objectives of this experiment were to determine C. jejuni survivability in mixed in vitro rumen microbial populations and the impact on methane production with or without methane inhibitors 2-bromosulfonate (BES) and/or sodium nitrate. When inoculated into rumen microbial populations without or with 0.5 mM BES, 5.0 mM nitrate or their combination, C. jejuni viability decreased from 4.7 ± 0.1 log10 colony forming units (CFU)/mL after 24 h. Loss of C. jejuni viability was greater (P < 0.05) when incubated under 100% CO2 compared to 50% H2:50% CO2, decreasing 1.46 versus 1.15 log units, respectively. C. jejuni viability was also decreased (P < 0.05) by more than 0.43 log units by the anti-methanogen treatments. Rumen microbial populations produced less methane (P = 0.05) when incubated with than without C. jejuni regardless of whether under 100% CO2 or 50% H2:50% CO2. For either gas phase, nitrate was decreased (13.2 versus 37.9%) by the anti-methanogen treatments versus controls although not always significant. C. jejuni-inoculated populations metabolized 16.4% more (P < 0.05) nitrate under H2:CO2 versus 100% CO2. Apparently, C. jejuni can compete for H2 with methanogens but has limited survivability under rumen conditions.

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.

Glycerol inhibition of ruminal lipolysis in vitro.

Supplemental glycerol inhibits rumen lipolysis, a prerequisite for rumen biohydrogenation, which is responsible for the saturation of dietary fatty acids consumed by ruminant animals. Feeding excess glycerol, however, adversely affects dry matter digestibility. To more clearly define the effect of supplemental glycerol on rumen lipolysis, mixed populations of ruminal bacteria were incubated with 6 or 20% glycerol (vol/vol). After 48-h anaerobic incubation of mixed culture rumen fluid, rates of free fatty acid production (nmol/mL per h) for the 6 and 20% glycerol-supplemented samples were decreased by 80 and 86%, respectively, compared with rates from nonsupplemented control cultures (12.4±1.0; mean ± SE). Conversely, assay of the prominent ruminal lipase-producing bacteria Anaerovibrio lipolyticus 5S, Butyrivibrio fibrisolvens 49, and Propionibacterium species avidum and acnes revealed no effect of 2 or 10% (vol/vol) added glycerol on lipolytic activity by these organisms. Supplementing glycerol at 6% on a vol/vol basis, equivalent to supplementing glycerol at approximately 8 to 15% of diet dry matter, effectively reduced lipolysis. However, the mechanism of glycerol inhibition of ruminal lipolysis remains to be demonstrated.

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.

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.

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.

Effect of method of applying fibrolytic enzymes or ammonia to Bermudagrass hay on feed intake, digestion, and growth of beef steers.

This study examined how different methods of applying a fibrolytic enzyme or ammonia affect the nutritive value of Bermudagrass hay and the performance of beef cattle. Fifty Angus x Brangus crossbred steers (mean initial BW 244 +/- 26 kg) were individually fed for ad libitum intake of a 5-wk regrowth of a mixture of Florakirk and Tifton 44 Bermudagrass [Cynodon dactylon (L.) Pers] hay for 84 d with a concentrate supplement (77% soybean hull pellets, 23% cottonseed meal (DM basis) fed at 1% of BW daily. The Bermudagrass was conserved as hay without treatment (control), with NH(3) (30 g/kg of DM), or with a fibrolytic enzyme (16.5 g/t, air-dry basis) that was applied immediately after cutting (Ec), at baling (Eb), or at feeding. Chromic oxide was dosed to steers for 10 consecutive days, and fecal Cr concentrations from the last 5 d were used to estimate apparent total tract digestibility. In situ ruminal DM degradability was measured by incubating ground (4-mm) hay samples in duplicate in each of 2 ruminally cannulated cows having ad libitum access to Bermudagrass hay and 500 g/d of soybean meal. Unlike the enzyme treatment, ammoniation increased (P < 0.001) the CP concentration and reduced (P < 0.001) NDF, hemicellulose, and lignin concentrations of hay. Total DMI was greater (P < 0.05) for steers fed hays treated with Ec or NH(3) than for those fed control hays. All additive treatments increased (P < 0.05) DM digestibility, and NH(3), Ec, and Eb treatments also increased (P < 0.01) NDF digestibility. The initial and final BW, ADG, BCS, G:F, and hip height of the steers were not affected (P > 0.05) by treatment. The wash loss fractions in hays treated with Ec and Eb were lower than that in the control hay, but the potentially degradable fraction, total degradable fraction, and the effective degradability were increased (P < 0.01) by NH(3) treatment. Application at cutting was the most promising method of enzyme treatment, and this treatment was almost as effective as ammonia for enhancing forage quality.