Schizochytrium sp. and lactoferrin supplementation alleviates Escherichia coli K99-induced diarrhea in preweaning dairy calves.

Calf diarrhea, a common disease mainly induced by Escherichia coli infection, is one of the main reasons for nonpredator losses. Hence, an effective nonantibacterial approach to prevent calf diarrhea has become an emerging requirement. This study evaluated the microalgae Schizochytrium sp. (SZ) and lactoferrin (LF) as a nutrient intervention approach against E. coli O101:K99-induced preweaning calve diarrhea. Fifty 1-d-old male Holstein calves were randomly divided into 5 groups (n = 10): (1) control, (2) blank (no supplement or challenge), (3) 1 g/d LF, (4) 20 g/d SZ, or (5) 1 g/d LF plus 20 g/d SZ (LFSZ). The experimental period lasted 14 d. On the morning of d 7, calves were challenged with 1 × 1011 cfu of E. coli O101:K99, and rectum feces were collected on 3, 12, 24, and 168 h postchallenge for the control, LF, SZ, and LFSZ groups. The rectal feces of the blank group were collected on d 14. Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). The E. coli K99 challenge decreased the average daily gain (ADG) and increased feed-to-gain ratio (F:G) and diarrhea frequency (control vs. blank). Compared with the control group, the LFSZ group had a higher ADG and lower F:G, and the LFSZ and SZ groups had lower diarrhea frequency compared with the control group. In addition, the LFSZ and SZ groups have no differences in diarrhea frequency compared with the blank group. Compared with the control group, the blank group had lower serum nitric oxide (NO), endothelin-1, d-lactic acid (D-LA), and lipopolysaccharide (LPS) concentrations, as well as serum IgG, IL-1ß, IL-6, IL-10, and TNF-α levels on d 7 and 14. On d 7, compared with the control group, all treatment groups had lower serum NO level, the SZ group had a lower serum D-LA concentration, and the LF and LFSZ groups had lower serum LPS concentration. On d 14, compared with the control group, the fecal microbiota of the blank group had lower Shannon, Simpson, Chao1, and ACE indexes, the LFSZ group had lower Shannon and Simpson indexes, the SZ and LFSZ groups had a higher Chao1 index, and all treatment groups had a higher ACE index. In fecal microbiota, Bifidobacterium and Actinobacteria were negatively associated with IL-10 and d-lactate, while Akkermansia was negatively associated with endothelin-1 and positively correlated with LPS, fecal scores, and d-lactate levels. Our results indicated that LF and SZ supplements could alleviate E. coli O101:K99-induced calf diarrhea individually or in combination. Supplementing 1 g/d LF and 20 g/d SZ could be a potential nutrient intervention approach to prevent bacterial diarrhea in calves.

Invited review: "Probiotic" approaches to improving dairy production: Reassessing "magic foo-foo dust".

The gastrointestinal microbial consortium in dairy cattle is critical to determining the energetic status of the dairy cow from birth through her final lactation. The ruminant's microbial community can degrade a wide variety of feedstuffs, which can affect growth, as well as production rate and efficiency on the farm, but can also affect food safety, animal health, and environmental impacts of dairy production. Gut microbial diversity and density are powerful tools that can be harnessed to benefit both producers and consumers. The incentives in the United States to develop Alternatives to Antibiotics for use in food-animal production have been largely driven by the Veterinary Feed Directive and have led to an increased use of probiotic approaches to alter the gastrointestinal microbial community composition, resulting in improved heifer growth, milk production and efficiency, and animal health. However, the efficacy of direct-fed microbials or probiotics in dairy cattle has been highly variable due to specific microbial ecological factors within the host gut and its native microflora. Interactions (both synergistic and antagonistic) between the microbial ecosystem and the host animal physiology (including epithelial cells, immune system, hormones, enzyme activities, and epigenetics) are critical to understanding why some probiotics work but others do not. Increasing availability of next-generation sequencing approaches provides novel insights into how probiotic approaches change the microbial community composition in the gut that can potentially affect animal health (e.g., diarrhea or scours, gut integrity, foodborne pathogens), as well as animal performance (e.g., growth, reproduction, productivity) and fermentation parameters (e.g., pH, short-chain fatty acids, methane production, and microbial profiles) of cattle. However, it remains clear that all direct-fed microbials are not created equal and their efficacy remains highly variable and dependent on stage of production and farm environment. Collectively, data have demonstrated that probiotic effects are not limited to the simple mechanisms that have been traditionally hypothesized, but instead are part of a complex cascade of microbial ecological and host animal physiological effects that ultimately impact dairy production and profitability.

Effects of cashew nut shell extract supplementation on production, rumen fermentation, metabolism, and inflammatory biomarkers in transition dairy cows.

Cashew nut shell extract (CNSE) is a byproduct of the cashew nut industry, containing bioactive compounds that alter rumen fermentation patterns. Therefore, study objectives were to evaluate the effects of CNSE (59% anacardic acid and 18% cardol) on production, rumen fermentation variables, metabolism, and inflammation in transition dairy cows. A total of 51 multiparous Holstein cows were used in a randomized design and assigned to treatment based on their previous 305-d mature equivalent milk and parity. Cows were assigned to 1 of 2 treatments 21 d before expected calving: (1) CON (control diet; n = 17) or (2) CNSE-5.0 (control diet and 5.0 g/d CNSE granule [containing 50% CNSE]; n = 34). Following parturition, 17 cows (preselected at initial treatment assignment) from the CNSE-5.0 treatment were reallocated into a third treatment group: CNSE-2.5 (control diet and 2.5 g/d CNSE granule; n = 17), resulting in 3 total treatments postpartum: (1) CON, (2) CNSE-2.5, and (3) CNSE-5.0. Prepartum rumen pH was unaltered by treatment; however, postpartum rumen pH was increased (0.31 units) in CNSE cows relative to CON. Prepartum rumen ammonia N concentration tended to be decreased (34%) in CNSE-5.0 cows compared with CON, and there tended to be a quadratic effect on postpartum ammonia N, as it was decreased in CNSE-2.5 compared with CON and CNSE-5.0. Prepartum dry matter intake (DMI) was unaffected by treatment; however, postpartum DMI was increased (8%) in CNSE cows relative to CON. No treatment differences were observed in pre- or postpartum digestibility measurements. Milk and protein yields from cows fed CNSE tended to be increased (6% and 7%, respectively) relative to CON. No treatment differences were detected for energy-corrected milk, feed efficiency, body weight, body condition score, energy balance, milk composition, milk urea nitrogen, or somatic cell count. Prepartum fecal pH decreased (0.12 units) in CNSE-5.0 cows relative to CON cows but was similar between treatments postpartum. Supplementing CNSE did not affect prepartum glucose, nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), or insulin. However, prepartum circulating blood urea nitrogen tended to be decreased and glucagon was decreased in CNSE-5.0 cows compared with CON (9 and 20%, respectively). Additionally, CNSE supplementation decreased glucose and insulin concentrations postpartum relative to CON cows (6% and 20%, respectively). Quadratic effects were detected for postpartum circulating NEFA and BHB such that their levels were increased in CNSE-2.5 cows relative to CON and CNSE-5.0. Pre- and postpartum circulating serum amyloid A, lipopolysaccharide-binding protein, and haptoglobin were unaffected by treatment. Overall, CNSE influenced some key rumen fermentation variables, altered postabsorptive metabolism, and increased production parameters in transition dairy cows.

Effects of abomasally infused rumen fluid from corn-challenged donor cows on production, metabolism, and inflammatory biomarkers in healthy recipient cows.

Subacute rumen acidosis may cause postruminal intestinal barrier dysfunction, but this does not appear to be due to increased hindgut fermentation. Alternatively, intestinal hyperpermeability may be explained by the plethora of potentially harmful substances (e.g., ethanol, endotoxin, and amines) produced in the rumen during subacute rumen acidosis, which are difficult to isolate in traditional in vivo experiments. Therefore, objectives were to evaluate whether abomasal infusion of acidotic rumen fluid collected from donor (Donor) cows elicits systemic inflammation or alters metabolism or production in healthy recipients. Ten rumen-cannulated lactating dairy cows [249 ± 63 d in milk; 753 ± 32 kg of body weight (BW)] were randomly assigned to 1 of 2 abomasal infusion treatments: (1) healthy rumen fluid (HF; 5 L/h; n = 5) or (2) acidotic rumen fluid (AF; 5 L/h; n = 5) infused. Eight rumen-cannulated cows [4 dry, 4 lactating (lactating = 391 ± 220 d in milk); 760 ± 70 kg of BW] were used as Donor cows. All 18 cows were acclimated to a high-fiber diet (46% neutral detergent fiber; 14% starch) during an 11-d prefeeding period during which rumen fluid was collected for the eventual infusion into HF cows. During period (P) 1 (5 d), baseline data were obtained and on d 5 Donor were corn-challenged (2.75% BW ground corn after 16 h of 75% feed restriction). Cows were fasted until 36 h relative to rumen acidosis induction (RAI), and data were collected through 96 h RAI. At 12 h RAI, an additional 0.50% BW of ground corn was added, and acidotic fluid collections began (7 L/Donor every 2 h; 6 M HCl was added to collected fluid until pH was between 5.0 and 5.2). On d 1 of P2 (4 d), HF/AF cows were abomasally infused with their respective treatments for 16 h, and data were collected for 96 h relative to the first infusion. Data were analyzed in SAS (SAS Institute Inc.) using PROC MIXED. Following the corn challenge in the Donor cows, rumen pH only mildly decreased at nadir (pH = 5.64 at 8 h RAI) and remained above the desired threshold for both acute (5.2) and subacute (5.6) acidosis. In contrast, fecal and blood pH markedly decreased to acidotic levels (nadir = 4.65 and 7.28 at 36 and 30 h RAI, respectively), and fecal pH remained below 5 from 22 to 36 h RAI. In Donor cows, dry matter intake remained decreased through d 4 (36% relative to baseline) and serum amyloid A and lipopolysaccharide-binding protein markedly increased by 48 h RAI in Donor cows (30- and 3-fold, respectively). In cows that received the abomasal infusions, fecal pH decreased in AF from 6 to 12 h relative to the first infusion (7.07 vs. 6.33) compared with HF; however, milk yield, dry matter intake, energy-corrected milk, rectal temperature, serum amyloid A, and lipopolysaccharide-binding protein were unaffected. Overall, the corn challenge did not cause subacute rumen acidosis but markedly decreased fecal and blood pH and stimulated a delayed inflammatory response in the Donor cows. Abomasal infusion of rumen fluid from corn-challenged Donor cows decreased fecal pH but did not cause inflammation, nor did it create an immune-activated phenotype in recipient cows.

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.

Effects of close-up dietary energy level and supplementing rumen-protected lysine on energy metabolites and milk production in transition cows.

The objective of this study was to investigate the effects of dietary energy levels and rumen-protected lysine supplementation on serum free fatty acid levels, ß-hydroxybutyrate levels, dry matter (DM) intake, and milk production and composition. Treatments were arranged in a 2 × 2 factorial design with 2 dietary energy levels [high net energy for lactation (NEL) = 1.53 Mcal/kg of DM vs. low NEL = 1.37 Mcal/kg of DM; HE vs. LE) fed either with rumen-protected lysine (bypass lysine; 40 g/cow per day) or without rumen-protected lysine (control). Sixty-eight third-lactation Holstein dairy cows entering their fourth lactation were randomly allocated to 4 treatments groups: HE with bypass lysine, HE without bypass lysine, LE with bypass lysine, and LE without bypass lysine. Groups were balanced based upon their expected calving date, previous milk yields, and body condition score. All cows were fed the same diet (NEL = 1.34 Mcal/kg of DM) during the dry period prior to the trial. Rumen-protected lysine was top-dressed on a total mixed ration to deliver 9.68 g/d of metabolizable lysine to pre- and postpartum cows. After calving, all cows received the same TMR (1.69 Mcal/kg of DM). Blood samples were collected at -21, -14, -7, 0, 3, 7, 14, and 21 d relative to calving, and free fatty acids and ß-hydroxybutyrate concentrations were measured. Amount of feed offered and orts were collected and measured for individual cows 4 d/wk. Milk samples were collected once per week following calving, and milk composition was analyzed. Feeding high NEL to close-up cows decreased the concentrations of free fatty acid and ß-hydroxybutyrate in prepartum cows but not in postpartum cows. Addition of rumen-protected lysine increased postpartum DM intake, and decreased serum free fatty acid and ß-hydroxybutyrate concentrations. Neither energy nor rumen-protected lysine supplementation nor their interaction affected milk yield or fat or lactose yields. However, cows in the group receiving HE with bypass lysine tended to produce more milk compared with other groups and had a lower blood ß-hydroxybutyrate concentration postpartum. These results indicate that feeding a high-energy diet together with rumen-protected lysine improved DM intake and lowered serum free fatty acid and ß-hydroxybutyrate concentrations in transition cows.

Isolation and screening of aflatoxin-detoxifying yeast and bacteria from ruminal fluids to reduce aflatoxin B1 contamination in dairy cattle feed.

AIMS: To obtain yeast and bacteria from ruminal fluids that possess aflatoxin B1 (AFB1 ) detoxifying ability for use in animal feed. METHODS AND RESULTS: Sources of isolation were ruminal fluids of three nonfistulated dairy cows, fed diets containing cassava pulp, rice straw or distillery yeast sludge. The isolation was carried out to screen for the isolates that were active in both anaerobic and aerobic conditions. Three yeast and three bacteria isolates were selected. Up to 85% of AFB1 was detoxified by yeast isolates and up to 60% AFB1 reduction was evident by bacteria isolates. Two yeast isolates were identified as Kluyveromyces marxianus and one isolate as Pichia kudriavzevii. The three bacteria isolates were identified as Enterococcus faecium, Corynebacterium phoceense and C. vitaeruminis. All strains showed high biomass production when cultivated in medium with 80 g l-1 glucose. CONCLUSIONS: The isolated yeast and bacteria with AFB1 detoxifying ability showed a good potential to be applied as an aflatoxin-detoxifying agent to ingredients used to feed dairy cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The abilities of isolates to survive and be active in anaerobic and aerobic conditions rendered them to be active in cattle's rumen. Their biomass could be produced in bulk and used as feed supplement for aflatoxin detoxification in dairy cattle.

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.

Forages and pastures symposium: forage biodegradation: advances in ruminal microbial ecology.

The rumen microbial ecosystem provides ruminants a selective advantage, the ability to utilize forages, allowing them to flourish worldwide in various environments. For many years, our understanding of the ruminal microbial ecosystem was limited to understanding the microbes (usually only laboratory-amenable bacteria) grown in pure culture, meaning that much of our understanding of ruminal function remained a "black box." However, the ruminal degradation of plant cell walls is performed by a consortium of bacteria, archaea, protozoa, and fungi that produces a wide variety of carbohydrate-active enzymes (CAZymes) that are responsible for the catabolism of cellulose, hemicellulose, and pectin. The past 15 years have seen the development and implementation of numerous next-generation sequencing (NGS) approaches (e.g., pyrosequencing, Illumina, and shotgun sequencing), which have contributed significantly to a greater level of insight regarding the microbial ecology of ruminants fed a variety of forages. There has also been an increase in the utilization of liquid chromatography and mass spectrometry that revolutionized transcriptomic approaches, and further improvements in the measurement of fermentation intermediates and end products have advanced with metabolomics. These advanced NGS techniques along with other analytic approaches, such as metaproteomics, have been utilized to elucidate the specific role of microbial CAZymes in forage degradation. Other methods have provided new insights into dynamic changes in the ruminal microbial population fed different diets and how these changes impact the assortment of products presented to the host animal. As more omics-based data has accumulated on forage-fed ruminants, the sequence of events that occur during fiber colonization by the microbial consortium has become more apparent, with fungal populations and fibrolytic bacterial populations working in conjunction, as well as expanding understanding of the individual microbial contributions to degradation of plant cell walls and polysaccharide components. In the future, the ability to predict microbial population and enzymatic activity and end products will be able to support the development of dynamic predictive models of rumen forage degradation and fermentation. Consequently, it is imperative to understand the rumen's microbial population better to improve fiber degradation in ruminants and, thus, stimulate more sustainable production systems.

Forage degradation in the rumen is critical to producing ruminant animals. For many years, scientists were limited to biochemical techniques to understand how ruminal microbes degraded forage, impairing our understanding of which microbes were involved with degrading which forage components. However, we have understood that as the ruminant opened up plant cells to microbial activity, a succession of microbes was involved in colonizing and breaking fiber into increasingly smaller pieces. The recent development of sequencing techniques has allowed a more detailed understanding of changes in the microbial population of the rumen during forage degradation and the types of degradative enzymes produced by this complex microbial ecosystem. We described the enzymes involved in the degradation of specific forage components, how their end products impact the microbial population through cross-feeding interactions, and how fermentation products can impact food animal production.

Competitive effect of commensal faecal bacteria from growing swine fed chlortetracycline-supplemented feed on ß-haemolytic Escherichia coli strains with multiple antimicrobial resistance plasmids.

AIMS: To determine the differences in competitive fitness among Escherichia coli strains with different plasmid profiles when grown in suspension with commensal faecal bacteria from growing swine fed chlortetracycline-supplemented or unsupplemented diets. METHODS AND RESULTS: Five multiple drug-resistant (MDR) E. coli strains that possessed 0, 2, 6 or 8 plasmids were inoculated into anoxic faecal cultures from swine fed an unsupplemented (control) or chlortetracycline (50 g ton(-1))-supplemented (experimental) diet. On days 21 of chlortetracycline supplementation, faecal growth competition studies were performed. MDR E. coli were enumerated at 0, 6 and 24 h. The plasmid-free strain was below culturable limits in both the control and experimental cultures by 24 h. For each plasmid-bearing strain, there was no statistically significant difference in population CFU ml(-1) (P < 0.05) between the control and experimental cultures. CONCLUSIONS: There was no significant effect on the faecal microflora, owing to the inclusion of chlortetracycline, in the swine diets, that affected the growth of E. coli in the competition studies employed. Furthermore, these results suggest that the cost of maintaining plasmids in these E. coli strains had little influence on survivability. SIGNIFICANCE AND IMPACT OF STUDY: Mutations that led to antimicrobial resistance may have a greater impact on survivability than multiple plasmid carriage.

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.

Association of pre-treatment somatic cell counts with bacteriological cure following diagnosis of intramammary infection.

Antibiotic administration is crucial to ensure the health and productivity of dairy cattle. Mastitis is a disease that is typically a result of an intramammary infection (IMI), and antibiotic regimens are implemented to aid in curing IMI. Diagnosis is usually by detection of elevated milk somatic cell counts (SCC) and/or presence of culturable pathogens in the milk. Antibiotic treatment success is associated with the SCC at the time of treatment, though this correlation is still poorly understood. The objective of this project was to evaluate pre-treatment SCC and its association with IMI cure incidence following a standard antibiotic treatment. We hypothesized that pre-treatment SCC would be significantly lower in cases where the IMI ultimately cured compared to cases where the IMI failed to cure. Milk samples were collected aseptically from lactating cow quarters experiencing clinical or subclinical mastitis (n = 52). Clinical mastitis was diagnosed by a trained milking technician and subclinical mastitis was diagnosed at the quarter level as a SCC > 200,000 cells/mL and presence of bacterical growth in milk at time of treatment. After collection of the day 0 (D0) milk samples, the SCC was enumerated, and the milk sample cultured. Intramammary antibiotic therapy Cetftiofur hydrochloride (Spectramast® LC) was administered once/day for 5 days. Post-treatment samples were collected 14 d (D14) and 28 d (D28) later. A bacteriological cure was confirmed when both the D14 and D28 samples were free of culturable pathogens. The overall cure rate was 46.2%. Interestingly, the cure rates of antibiotic therapy decreased as pre-treatment SCC increased. Quarters that experienced bacteriological cure demonstrated a lower pre-treatment SCC (507,041 cells/mL ± 127.86 SEM, P = 0.01) compared to cows that did not cure, which had high pre-treatment SCC (1,640,392 cells/mL ± 333.28 SEM). Quarters that failed to cure had higher SCC values 28 days post-treatment in comparison to quarters that cured (P < 0.001). Future studies should investigate whether we can develop unique SCC-dependent mastitis treatment protocols which increase mastitis cure rates and enhance overall mammary health.

Evaluation of in vitro gas production and rumen bacterial populations fermenting corn milling (co)products.

The objective of this study was to evaluate the fermentation dynamics of 2 commonly fed corn (co)products in their intact and defatted forms, using the in vitro gas production (IVGP) technique, and to investigate the shifts of the predominant rumen bacterial populations using the 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) technique. The bTEFAP technique was used to determine the bacterial profile of each fermentation time at 24 and 48 h. Bacterial populations were identified at the species level. Species were grouped by substrate affinities (guilds) for cellulose, hemicellulose, pectin, starch, sugars, protein, lipids, and lactate. The 2 (co)products were a dried distillers grain (DDG) plus solubles produced from a low-heat drying process (BPX) and a high-protein DDG without solubles (HP). Chemical analysis revealed that BPX contained about 11.4% ether extract, whereas HP contained only 3.88%. Previous studies have indicated that processing methods, as well as fat content, of corn (co)products directly affect fermentation rate and substrate availability, but little information is available regarding changes in rumen bacterial populations. Fermentation profiles of intact and defatted BPX and HP were compared with alfalfa hay as a standard profile. Defatting before incubation had no effect on total gas production in BPX or HP, but reduced lag time and the fractional rate of fermentation of BPX by at least half, whereas there was no effect for HP. The HP feed supported a greater percentage of fibrolytic and proteolytic bacteria than did BPX. Defatting both DDG increased the fibrolytic (26.8 to 38.7%) and proteolytic (26.1 to 37.2%) bacterial guild populations and decreased the lactate-utilizing bacterial guild (3.06 to 1.44%). Information regarding the fermentation kinetics and bacterial population shifts when feeding corn (co)products may lead to more innovative processing methods that improve feed quality (e.g., deoiling) and consequently allow greater inclusion rates in dairy cow rations.

Grain feeding and the dissemination of acid-resistant Escherichia coli from cattle.

The gastric stomach of humans is a barrier to food-borne pathogens, but Escherichia coli can survive at pH 2.0 if it is grown under mildly acidic conditions. Cattle are a natural reservoir for pathogenic E. coli, and cattle fed mostly grain had lower colonic pH and more acid-resistant E. coli than cattle fed only hay. On the basis of numbers and survival after acid shock, cattle that were fed grain had 10(6)-fold more acid-resistant E. coli than cattle fed hay, but a brief period of hay feeding decreased the acid-resistant count substantially.

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.

Acyl-homoserine-lactone autoinducer in the gastrointestinal [corrected] tract of feedlot cattle and correlation to season, E. coli O157:H7 prevalence, and diet.

Acyl-homoserine-lactone autoinducer (AHL) produced by nonenterohemorrhagic Escherichia coli species in cattle appears to be required for enterohemorrhagic E. coli (EHEC) colonization of the gastrointestinal tract (GIT). The current research aimed to examine the effect of season, diet, EHEC shedding, and location within the GIT on AHL prevalence in the ruminant. Luminal content samples were collected from the rumen and rectum of feedlot cattle at slaughter in the spring, summer, fall, and winter for culture of E. coli O157:H7 and AHL determination. During the spring collection, samples were additionally collected from the cecum and small intestine, but these samples all were AHL negative and therefore not examined again. To assess the influence of diet on AHL prevalence, 14 lambs were fed either 100% forage or 80% concentrate diets and experimentally inoculated with EHEC. At 8 days after infection, all the lambs were killed, and necropsies were taken, with luminal contents collected from the GIT. The collections from the feedlot cattle had AHL in 100% of the rumen content samples from the spring, summer, and fall, but not in any of the winter samples. No other GIT samples from feedlot cattle were AHL positive, and all the samples from the sheep study were AHL negative. The cattle seemed to show a weak correlation between ruminal AHL and EHEC prevalence. This research found AHL only in the rumen and not in the lower GIT of feedlot cattle. However, it is unclear whether this is because the pH of the lower gut destroys the AHL or because a lack of certain bacteria in the lower gut producing AHL.

Fate of Escherichia coli O157:H7 and Salmonella from contaminated manure slurry applied to soil surrounding tall fescue.

AIM: To investigate the potential transfer of Escherichia coli O157:H7 and Salmonella from contaminated manure slurry into the tissue of tall fescue plants. METHODS AND RESULTS: Tall fescue plants (n = 50) were fertilized with a manure slurry inoculated with E. coli O157:H7 and Salmonella. Soil was collected and tall fescue plants (n = 10 per day) harvested on day 1, 2, 4, 8, and 14 after manure slurry fertilization. Soil samples were positive for E. coli O157:H7 on all days and on day 1, 2, 8, and 14 for Salmonella. None of the plant tissue samples were positive for E. coli O157:H7 on day 1 or 2; however, 20%, 30% and 40% of plant tissue samples were positive for E. coli O157:H7 on day 4, 8, and 14, respectively. CONCLUSIONS: It may be possible that E. coli O157:H7 can become transmitted and internalized into tall fescue plant tissue within 4 days after exposure to an E. coli O157:H7-contaminated manure slurry. Salmonella did not appear to be transferred to tall fescue plant tissue. SIGNIFICANCE AND IMPACT OF THE STUDY: Faeces contaminated with E. coli O157:11H7 may be one means by which grazing ruminants spread bacterial pathogens to additional animals.

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.

Foodborne Salmonella ecology in the avian gastrointestinal tract.

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.

Evaluation of the bacterial diversity in cecal contents of laying hens fed various molting diets by using bacterial tag-encoded FLX amplicon pyrosequencing.

Laying hens are typically induced to molt to begin a new egg-laying cycle by withdrawing feed for up to 12 to 14 d. Fasted hens are more susceptible to colonization and tissue invasion by Salmonella enterica serovar Enteritidis. Much of this increased incidence in fasted hens is thought to be due to changes in the native intestinal microflora. An alternative to feed withdrawal involves feeding alfalfa meal crumble to hens, which is indigestible by poultry but provides fermentable substrate to the intestinal microbial population and reduces Salmonella colonization of hens compared with feed withdrawal. The present study was designed to quantify differences in the cecal microbial population of hens (n=12) fed a typical layer ration, undergoing feed withdrawal, or being fed alfalfa crumble by using a novel tag bacterial diversity amplification method. Bacteroides, Prevotella, and Clostridium were the most common genera isolated from all treatment groups. Only the ceca of hens undergoing feed withdrawal (n=4) contained Salmonella. The number of genera present was greatest in the alfalfa crumble-fed group and least in the feed withdrawal group (78 vs. 54 genera, respectively). Overall, the microbial diversity was least and Lactobacillius populations were not found in the hens undergoing feed withdrawal, which could explain much of these hens' sensitivity to colonization by Salmonella.