Effect of crowding stress and Escherichia coli K88+ challenge in nursery pigs supplemented with anti-Escherichia coli K88+ probiotics.

Under commercial conditions and during production periods, pigs are generally exposed to a number of stressors that may have direct or indirect influence on their performance and general health. As a result, environmental stressors can influence the productivity as well as the onset and severity of infectious diseases. The aim of this study was to investigate the effects of crowding stress on performance and some immunological responses in piglets challenged with enterotoxigenic Escherichia coli (ETEC) K88(+) and fed diets containing anti-ETEC probiotic strains derived from E. coli in a 3-wk trial. Ninety-six piglets at 20 ± 1 d of age were randomly assigned to 6 experimental treatments with 3 pigs/pen and 5 replicates/treatment and fed a basal mash diet. To model stress, piglets were housed in 2 kinds of pens: standard floor allowance (SFA; no stress) and half standard floor allowance (HSFA; stressed). The treatments were 1) control pigs with SFA, 2) control pigs with HSFA, 3) pigs with SFA and challenged with ETEC (ESFA), 4) pigs with HSFA and challenged with ETEC (EHSFA), 5) pigs with ESFA and supplemented with a cocktail of E. coli probiotics (PSFA), and 6) pigs with EHSFA and supplemented with a cocktail of E. coli probiotics (PHSFA). After 4 d acclimatization, crowding stress was started on d 5, E. coli probiotics were given daily from d 5, and ETEC challenge was administered on d 8 and 10. Body weight and feed disappearance were determined weekly and severity of diarrhea was characterized daily. Blood samples were collected for biochemistry and inflammatory analysis and pigs were euthanized to obtain digesta for bacterial enumeration. The ADG and ADFI decreased (P < 0.05) in stressed and ETEC-challenged pigs 5 d after ETEC challenge. The ETEC population was higher in both ileal and colon digesta of stressed pigs whereas ETEC-challenged and probiotic-fed pigs had higher fecal consistency scores 96 h and 7 d after ETEC challenge. Stressed pigs had higher (P < 0.001) levels of neutrophils, neutrophil:lymphocyte ratio, albumin:globulin ratio, fibrinogen, total protein:fibrinogen ratio, and cortisol. Generally, the levels of cortisol, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) differed over time among some treatment groups. In conclusion, reduced space allowance stress and ETEC challenge depressed certain aspects of performance and immunological responses and increased E. coli counts; however, probiotics feeding had little positive effects on these measures suggesting further research.

Targeted 16S rRNA high-throughput sequencing to characterize microbial communities during composting of livestock mortalities.

AIM: A comprehensive understanding of the microbial community is necessary to ensure a significant reduction in pathogens during the composting process. METHODS AND RESULTS: Two biosecure, static composting systems containing cattle mortalities were constructed at subzero temperatures. Temperature at each sampling site was measured continuously and samples were grouped as either ≤50 or ≥55°C, based on temperature exposure required for effective pathogen inactivation during composting. High-throughput 454 sequencing was used to characterize the bacterial communities within each sample. Clustering of bacterial communities was observed according to temperature. However, neither richness nor diversity differed between temperature groups. Firmicutes was the most abundant phylum within both temperature groups but was more pronounced (63·6%) in samples ≥55°C (P < 0·05). Similarly, members of Clostridia, Clostridium sensu stricto (3·64%), Clostridium XI (0·59%), UF (Clostridiaceae 1) (5·29%) and UF (Clostridiales Incertae Sedis XI) (6·20%), were prominent at ≥55°C (P < 0·05), likely a reflection of spore survival and/or anaerobic microenvironments within passively aerated compost piles. Members of Thermobifida (3·54%), UO (Actinomycetales) (12·29%) and UO (Bacillales) (19·49%) were also prominent at ≥55°C (P < 0·05). CONCLUSION: Substantial spatial diversity exists within bacterial communities in field-scale compost piles. Localized temperature at the site of sampling may be one of the factors contributing to this phenomenon. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to describe the microbial community profile with the use of targeted 16S rRNA high-throughput sequencing in passively aerated composted livestock mortalities.

Screening of condensed tannins from Canadian prairie forages for anti-Escherichia coli O157:H7 with an emphasis on purple prairie clover (Dalea purpurea Vent).

Tannins from forages grown (n = 10) on the Canadian prairie, as well as from Quebracho, Rhus semialata, and brown seaweed (Ascophyllum nodosum), were screened for anti-Escherichia coli O157:H7 activity against E. coli O157:H7 strain 3081 at a concentration of 400 µg/ml for each tannin type, except for brown seaweed, which was at 50 µg/ml. Growth of the bacteria was assessed by measuring the optical density at 600 nm over 24 h. Tannin from seaweed at a concentration of 50 µg/ml inhibited growth of strain 3081. Among the terrestrial forages, only condensed tannins (CT) from purple prairie clover (Dalea purpurea Vent; PPC) increased (P < 0.05) the lag time and reduced (P < 0.05) the growth rate of E. coli O157:H7. The anti-E. coli O157:H7 activity of PPC CT was further assessed by culturing E. coli strain ATCC 25922 and eight strains of E. coli O157:H7 with PPC CT at 0, 25, 50, 100, or 200 µg/ml. Selected strains were enumerated after 0, 6, and 24 h of incubation, and fatty acid composition was determined after 24 h of incubation. E. coli strain 25922 was cultured with 0, 50, or 200 µg of CT per ml and harvested during the exponential growth phase for examination by transmission electron microscopy. Increasing CT concentration linearly increased (P < 0.001) the lag times of seven strains and linearly reduced (P < 0.001) the growth rates of eight E. coli O157:H7 strains. Proportions of unsaturated fatty acids in the total fatty acids were decreased (P < 0.01) by CT at 50 µg/ml. Transmission electron microscopy showed that CT disrupted the outer membrane structure. Anti-E. coli O157:H7 activity of PPC CT at levels of up to 200 µg/ml was bacteriostatic rather than bactericidal, and the mechanism of anti-E. coli activity may involve alteration in the fatty acid composition and disruption of the outer membrane of the cell.

Board-invited review: Rumen microbiology: leading the way in microbial ecology.

Robert Hungate, considered the father of rumen microbiology, was the first to initiate a systematic exploration of the microbial ecosystem of the rumen, but he was not alone. The techniques he developed to isolate and identify cellulose-digesting bacteria from the rumen have had a major impact not only in delineating the complex ecosystem of the rumen but also in clinical microbiology and in the exploration of a number of other anaerobic ecosystems, including the human hindgut. Rumen microbiology has pioneered our understanding of much of microbial ecology and has broadened our knowledge of ecology in general, as well as improved the ability to feed ruminants more efficiently. The discovery of anaerobic fungi as a component of the ruminal flora disproved the central dogma in microbiology that all fungi are aerobic organisms. Further novel interactions between bacterial species such as nutrient cross feeding and interspecies H2 transfer were first described in ruminal microorganisms. The complexity and diversity present in the rumen make it an ideal testing ground for microbial theories (e.g., the effects of nutrient limitation and excess) and techniques (such as 16S rRNA), which have rewarded the investigators that have used this easily accessed ecosystem to understand larger truths. Our understanding of characteristics of the ruminal microbial population has opened new avenues of microbial ecology, such as the existence of hyperammonia-producing bacteria and how they can be used to improve N efficiency in ruminants. In this review, we examine some of the contributions to science that were first made in the rumen, which have not been recognized in a broader sense.

Interactions of Saccharomyces cerevisiae fermentation product and in-feed antibiotic on gastrointestinal and immunological responses in piglets challenged with Escherichia coli K88+.

Interactive effects between in-feed antibiotics (PC; 0.04% chlortetracycline + 0.004% Denagard) and a Saccharomyces cerevisiae fermentation product (XPC) were investigated. Five pens of 3 pigs/pen were randomly allotted to one of six diets consisting of control (NC; no additives), XPC alone (0.2%), PC alone, or PC plus one of three levels of XPC (0.1, 0.2, or 0.4%). On day 7, all pigs were bled for baseline serum and then orally inoculated with a 6-mL dose of 2 x 10(9) cfu/mL of ciprofloxacin-resistant Escherichia coli K88(+) (ETEC). Severity of diarrhea, using fecal score (FS), was assessed on days 8, 9, and 10. On day 10, pigs were bled and 1 pig/pen was killed to obtain gastrointestinal (GIT) samples. Remaining pigs were monitored for another 4 d and then killed to obtain GIT samples. Pigs fed PC and XPC in combination of PC had a lower (P = 0.02) FS than NC. The PC pigs had lower (P = 0.02) serum interleukin-6 and XPC pigs had higher (P < 0.01) serum tumor necrosis factor-α than NC pigs. Ileal mucosal ETEC count was lower on day 10 (P = 0.04) and day 14 (P < 0.01) when additives were fed in combination. Microbiome analysis of day 10 digesta using pyrosequencing showed that pigs fed the PC with XPC had enriched (P = 0.01) Streptococcaceae family than PC fed alone. On day 10, pigs fed PC alone and in combination with XPC at a lower dose had shallower (P = 0.01) crypt depth than those fed higher doses of XPC, XPC alone, or NC. Pigs fed XPC had a thicker colon wall on day 10 (P < 0.01) compared to those fed non-XPC diets. Ileal and colonic digesta NH(3) concentration was lower (P < 0.05) for pigs fed additives-containing diets on days 10 and 14. Feeding PC or XPC reduced diarrhea and the number of ETEC attached to the ileal mucosa; however, responses suggest independent and interactive effects that might be associated with the nature of these additives.

Effects of subacute ruminal acidosis challenges on fermentation and endotoxins in the rumen and hindgut of dairy cows.

The effects of a grain-based subacute ruminal acidosis (SARA) challenge (GBSC) and an alfalfa-pellet SARA challenge (APSC) on fermentation and endotoxins in the rumen and in the cecum, as well as on endotoxins in peripheral blood, were determined. Six nonlactating Holstein cows with cannulas in the rumen and cecum were used in the study. A 3×3 Latin square arrangement of treatments with 4-wk experimental periods was adopted. During the first 3 wk of each experimental period, all cows received a diet containing 70% forages [dry matter (DM) basis]. In wk 4 of each period, cows received 1 of the following 3 diets: the 70% forage diet fed during wk 1 to 3 (control), a diet in which 34% of the dietary DM was replaced with grain pellets made of 50% ground wheat and 50% ground barely (GBSC), or a diet in which 37% of dietary DM was replaced with pellets of ground alfalfa (APSC). Rumen pH was monitored continuously using indwelling pH probes, and rumen fluid, blood, cecal digesta, and fecal grab samples were collected immediately before feed delivery at 0900 h and at 6 h after feed delivery on d 3 and 5 of wk 4. The time for which rumen pH was below 5.6 was 56.4, 225.2, and 298.8 min/d for the control, APSC, and GBSC treatments, respectively. Compared with the control, SARA challenges resulted in similar reductions in cecal digesta pH, which were 7.07, 6.86, and 6.79 for the control, APSC, and GBSC treatments, respectively. Compared with the control, only GBSC increased starch content in cecal digesta, which averaged 2.8, 2.6, and 7.4% of DM for the control, APSC, and GBSC, respectively. Free lipopolysaccharide endotoxin (LPS) concentration in rumen fluid increased from 10,405 endotoxin units (EU)/mL in the control treatment to 30,715 and 168,391 EU/mL in APSC and GBSC, respectively. Additionally, GBSC increased the LPS concentration from 16,508 to 118,522 EU/g in wet cecal digesta, and from 12,832 to 93,154 EU/g in wet feces. The APSC treatment did not affect LPS concentrations in cecal digesta and feces. All concentrations of LPS in blood plasma were below the detection limit of >0.05 EU/mL of the technique used. Despite the absence of LPS in blood, only GBSC increased the concentration of LPS-binding protein in blood plasma, which averaged, 8.9, 9.5, and 12.1mg/L for the control, APSC, and GBSC treatments, respectively. This suggests that GBSC caused translocation of LPS from the digestive tract but that LPS was detoxified before entering the peripheral blood circulation. The higher LPS concentration in cecal digesta in the GBSC compared with the APSC suggests a higher risk of LPS translocation in the large intestine in GBSC than in APSC.

Weaned pig responses to Escherichia coli K88 oral challenge when receiving a lysozyme supplement.

Lysozyme is a low-molecular-weight protein with antimicrobial properties. An experiment was conducted to investigate the response of piglets receiving a water-soluble lysozyme supplement [Entegard (EG), Neova Technologies Inc., Abbotsford, British Columbia, Canada; 4,000 lysozyme units/mg] after oral challenge with enterotoxigenic Escherichia coli (ETEC). A total of 36 individually housed weanling pigs were randomly allotted to 1 of the 4 treatments, with 9 replicates per treatment. Treatments were a control (CONT, no additive), antibiotic (AB; 2.5 g/kg of feed of antibiotic with chlortetracycline, sulfamethazine, and penicillin), and EG delivered in the drinking water at concentrations of 0.1% (EG1) and 0.2% (EG2). All pigs received a basal diet similar in composition and nutrients, except for pigs receiving the AB diet, which had an added antibiotic. Pigs were acclimated to treatments for a 7-d period to monitor growth performance. On d 8, blood samples were collected from each pig to obtain serum, and each pig was gavaged with 6 mL (2 × 10(9) cfu/mL) of ETEC solution. Pigs were monitored for another 7 d to assess incidences of diarrhea and growth performance, and then all pigs were killed to obtain intestinal tissue and digesta samples. Treatments did not influence growth performance throughout the study. Greater ETEC counts were observed in the ileal mucosal scrapings (P = 0.001) and colonic digesta (P = 0.025) of pigs in the CONT group compared with pigs in the AB and EG1 groups. Pigs receiving AB and EG1 had greater (P < 0.05) small intestinal weights and ileal villus heights than pigs receiving CONT; however, the ileal villus height-to-crypt depth ratio was greater in pigs fed the AB diet (1.69) compared with those fed the CONT diet (1.34), whereas pigs receiving EG1 were intermediate. Pigs in the EG1 group showed greater (P < 0.001) serum tumor necrosis factor α and IL-6 concentrations before ETEC challenge; however, at 7 d postchallenge, pigs receiving EG2 showed the least (P < 0.05) circulating tumor necrosis factor α and IL-6 concentrations. Overall, better intestinal growth and development, as well as decreased ETEC counts on the intestinal mucosa and serum proinflammatory cytokines, suggest that EG can maintain gut health and function in piglets commensurate with antibiotics. However, it is noteworthy that at the largest dose tested, EG seemed to have a dramatic effect on proinflammatory cytokines but had a minimal or no effect on the other response criteria.

Growth performance and gastrointestinal microbial ecology responses of piglets receiving Saccharomyces cerevisiae fermentation products after an oral challenge with Escherichia coli (K88).

The effects of Saccharomyces cerevisiae fermentation products (YFP) on growth performance and gastrointestinal (GIT) microbial ecology in 90 weanling pigs orally challenged with Escherichia coli K88(+) (ETEC) were investigated. The YFP were an original YFP product (XPC) and a water-suspendable yeast fermentation prototype (WSYFP) from a commercial company. Treatments consisted of a negative control (NC, no in-feed or in-water additive), carbadox (AB, 55 mg of carbadox/kg of feed), XPC (in feed, 0.2%), and WSYFP (in water, 0.5, 1, or 2 g/pig per day), and each was allotted to 5 pens (3 pigs/pen). The diets met the 1998 NRC specifications. Pigs were acclimated to treatments for a 7-d period before an ETEC challenge. On d 8, blood was collected from pigs to determine the baseline packed cell volume (PCV) measurement, and pigs were orally challenged with ETEC. At various time points postchallenge, blood samples were taken, performance measures and fecal consistency scores were recorded, and gut digesta and tissue samples were taken to evaluate GIT morphology, microbial ecology, and metabolites. Preplanned contrasts were used for comparison. Pigs receiving YFP had greater ADFI than NC pigs on d 3 (424 vs. 378 g/d; P = 0.01) and d 7 (506 vs. 458 g/d; P = 0.03) postchallenge. This effect of YFP on ADFI was similar to that of AB on d 3, but pigs receiving AB ate more (576 vs. 506 g/d; P = 0.03) at d 7 than pigs receiving YFP. Pigs exhibited reduced (P < 0.001) PCV upon ETEC challenge; however, pigs receiving additives sustained a greater (P < 0.05) PCV at 72 h compared with the NC group. Compared with the NC pigs, pigs receiving YFP showed a smaller (P < 0.05) number of ileal mucosa adherent ETEC and prevalence of the order Enterobacteriales in the ileal digesta, which corresponded to less (5.09 vs. 6.97 mg/dL; P = 0.03) colonic ammonia on d 7 postchallenge. Most of the indices for ileal digesta bacterial richness and diversity were greater (P < 0.01) for YFP pigs compared with NC pigs. However, results also indicated that the influence of YFP on the piglet intestinal microenvironment might differ when given in feed or water during ETEC challenge. In conclusion, pigs receiving YFP showed a better appetite in the presence of ETEC, which, together with the greater ileal digesta bacteria richness and diversity and decreased ETEC adhering to the mucosa and reduced colonic ammonia, indicates a healthier GIT environment.

Population structure of rumen Escherichia coli associated with subacute ruminal acidosis (SARA) in dairy cattle.

Previous studies indicated that only subacute ruminal acidosis (SARA), induced by feeding a high-grain diet, is associated with an inflammatory response and increased abundance of Escherichia coli in the rumen. We hypothesized that ruminal E. coli in grain pellet-induced SARA carried virulence factors that potentially contribute to the immune activation during SARA. One hundred twenty-nine E. coli isolates were cultured from the rumens of 8 cows (4 animals per treatment) in which SARA had been nutritionally induced by feeding a high-grain diet (GPI-SARA) or a diet containing alfalfa pellets (API-SARA). The population structure of the E. coli was evaluated with the ABD genotyping system and repetitive sequence-based (rep)-PCR fingerprinting. Twenty-five virulence factors were evaluated with PCR. Escherichia coli numbers were higher in the GPI-SARA treatment than in the API-SARA treatment. The genetic structure of the E. coli was significantly different between SARA challenge models. Isolates from GPI-control (46%), API-control (70%), and API-SARA (53%) were closely related and fell into one cluster, whereas isolates from GPI-SARA (54%) grouped separately. The ABD typing indicated a shift from an A-type E. coli population to a B1-type population only due to GPI-SARA. Of the 25 virulence factors tested, curli fiber genes were highly associated with GPI. Curli fibers were first identified in E. coli mastitis isolates and are potent virulence factors that induce a range of immune responses. Results suggest that under low rumen pH conditions induced by a grain diet, there is a burst in the number of E. coli with virulence genes that can take advantage of these rumen conditions to trigger an inflammatory response.

Response of nursery pigs to a synbiotic preparation of starch and an anti-Escherichia coli K88 probiotic.

Postweaning diarrhea in pigs is frequently caused by enterotoxigenic Escherichia coli K88 (ETEC). The aim of this study was to test the efficacy of E. coli probiotics (PRO) in young pigs challenged with E. coli K88. We also tested the synbiotic interaction with raw potato starch (RPS), which can be used as a prebiotic. Forty 17-day-old weaned piglets were randomly assigned to four treatments: treatment 1, positive-control diet (C), no probiotics or RPS but containing in-feed antibiotics; treatment 2, probiotic (PRO), no feed antibiotics plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7; treatment 3, 14% RPS, no antibiotics (RPS); treatment 4, 14% RPS plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7, no antibiotics (PRO-RPS). The pigs were challenged with pathogenic E. coli K88 strains on day 7 of the experiment (24-day-old pigs) and euthanized on day 10 of the experiment (35-day-old pigs). Probiotic and pathogenic E. coli strains were enumerated by selective enrichment on antibiotics, and microbial community analysis was conducted using terminal restriction length polymorphism analysis (T-RFLP) of 16S rRNA genes. The combination of raw potato starch and the probiotic had a beneficial effect on piglet growth performance and resulted in a reduction of diarrhea and increased microbial diversity in the gut. We conclude that the use of E. coli probiotic strains against E. coli K88 in the presence of raw potato starch is effective in reducing the negative effects of ETEC in a piglet challenge model.

Susceptibility of meat starter cultures to antimicrobials used in food animals in Canada.

Lactic acid bacteria (LAB) are extensively used in the food industry for fermentation processes. However, it is possible that these bacteria may serve as a reservoir for antibiotic resistance genes that can be transferred to pathogens, giving rise to public health concerns. Animal operations that use antimicrobials as growth promotants have been linked to the origin of resistance due to the selective effect of low levels of antimicrobial used in this management strategy. The objective of this study was to determine the antimicrobial susceptibilities and mechanisms of resistance for 30 isolates of meat starter cultures commonly used in dry sausage fermentations to 20 antimicrobial agents. Susceptibility tests were performed by broth microdilution using Iso-Sensitest broth (90%, vol/vol) and de Man Rogosa Sharpe (MRS) broth (10%, vol/vol). The results showed that all 30 isolates exhibited resistance to at least three antimicrobials regardless of antimicrobial class while 17 or 30% of strains were resistant to antibiotics in three or six different classes, respectively. The incidence of antimicrobial resistance was higher among Pediococcus pentosaceus and lower for Staphylococcus carnosus strains. Genetic determinants for the lincosamide, macrolide, and tetracycline antimicrobials were not found using PCR. Phenotypic resistance in the absence of known resistance genes found here suggests that other mechanisms or genes might have contributed to the negative results. Further studies are needed to explore the genetic mechanisms underlying the prevalence of antibiotic resistance in Pediococcus species.

Effect of dietary protein level on growth performance, indicators of enteric health, and gastrointestinal microbial ecology of weaned pigs induced with postweaning colibacillosis.

The effect of dietary CP level on performance, enteric health, and gastrointestinal microbial ecology of weaned pigs challenged with enterotoxigenic Escherichia coli (ETEC) K88 was investigated in a 14-d study. Forty weaned pigs (BW = 5.32 +/- 0.24 kg, mean +/- SD), housed 4 per pen, were randomly assigned to 2 diets (5 pens/diet): 1) 22.5% CP or 2) 17.6% CP supplemented with AA. Diets contained the same amount of ME and standardized ileal digestible Lys, Met + Cys, Thr, and Trp based on the ideal protein ratio. Isoleucine and Val were added to the 17.6% CP diet up to the level in the 22.5% CP diet. On d 8 postweaning, pigs were challenged with 6 mL of ETEC suspension (10(10) cfu/mL) by gavage. Feed disappearance and BW were measured on d 7, 9, 10, 12, and 14 for determination of ADG, ADFI, and G:F. One pig from each pen was serially slaughtered on -1, 3, and 7 d postchallenge (10 pigs/d of slaughter) to evaluate gut morphology, and gut microbial ecology and metabolites. Pigs fed the 22.5% CP diet had greater (P < 0.05) ADG and G:F than those fed the 17.6% CP diet before infection, but performance was similar between the 2 diets after ETEC challenge and overall. On d 3 after challenge, ETEC was not detected in the ileal digesta of pigs fed the 17.6% CP diet but was detected in the ileal digesta of 80% of pigs fed the 22.5% CP diet (5.22 +/- 1.07 cfu/g, mean +/- SD). Pigs fed the 17.6% CP diet had a greater (P < 0.01) prevalence of order Clostridiales (73 vs. 50%), family Lachnospiraceae (43 vs. 18%), and genus Roseburia (13 vs. 3%) in the colon digesta 7 d after challenge compared with those fed the 22.5% CP diet. The richness and diversity of bacteria in the colon digesta were less (P < 0.05) in pigs fed the 17.6% CP diet than in those fed the 22.5% CP diet at -1, 3, and 7 d postchallenge. Pigs fed the 22.5% CP diet had greater (P < 0.05) ammonia N concentration in the colon digesta on -1 and 7 d after challenge compared with those fed the 17.6% CP diet. Pigs fed the 22.5% CP diet had deeper (P < 0.05) crypts 1 d before challenge, shorter villi 3 d after challenge, and reduced villus height:crypt depth 1 d before and 3 d after challenge compared with those fed the 17.6% CP diet. In conclusion, a reduction in the dietary CP level of weaned pigs from 22.5 to 17.6% with AA supplementation impaired growth performance before, but not after, the ETEC challenge and increased the relative composition of butyrate producing bacteria in the colon digesta after ETEC challenge.

Development and in vitro evaluation of an Escherichia coli probiotic able to inhibit the growth of pathogenic Escherichia coli K88.

Weaned piglets commonly suffer from gastroenteritis caused by enterotoxigenic Escherichia coli K88. Our aim was to produce E. coli strains that inhibited the growth of E. coli K88 and could be used as a probiotic against postweaning diarrhea. The inclusion criteria for the probiotics were that in addition to being able to inhibit E. coli K88, they also needed to be negative for virulence genes commonly associated with E. coli. A total of 463 E. coli isolates from the cattle rumen, cattle feces, swine feces, and soil were screened against 18 E. coli K88 clinical isolates using an agar diffusion technique. Growth inhibition of the most sensitive K88 indicator strain 2-12 occurred for 121/463 isolates: 96/358 from cattle feces, 0/33 from rumen fluid, 9/35 from swine feces, and 16/37 from soil. Of the 121 positive strains, 71/121 were negative for toxin genes (LT, STa, STb, VT1, and VT2). The 14 most inhibitory strains were screened against a range of substrates to assess the ability to utilize carbohydrates that could be included in the diet to enhance their ability to compete in the gut. Two strains, UM-2 and UM-7, were weak utilizers of starch and inulin. In vitro competition assays between the probiotic strains and E. coli K88 strain 2-12 were conducted with glucose as the only carbon source (minimal medium; MM), MM + 2% starch, or MM + 2% inulin. The UM-2 and UM-7 strains were able to outcompete strain 2-12 when glucose was the only carbon source, indicating that inhibitory activity was produced against 2-12 independent of carbon source. The UM-2 strain outcompeted strain 2-12 in assays in which potato starch or inulin was the only carbon source; the ability of 2-12 to maintain its concentrations in the culture were probably the result of cross feeding of breakdown sugars of starch and inulin that could be utilized by 2-12. In contrast, UM-7 did not grow as well as UM-2 on starch and inulin and 2-12 declined rapidly in successive cultures likely because of the lack of breakdown products of starch and inulin produced by UM-7. We conclude that probiotic E. coli without known toxins and that produce inhibitory activity against E. coli that cause postweaning diarrhea can be produced. In addition, the ability to utilize starch or inulin is an important phenotype because it likely gives the probiotic a competitive advantage in the gut.

Alfalfa pellet-induced subacute ruminal acidosis in dairy cows increases bacterial endotoxin in the rumen without causing inflammation.

A study was conducted to determine if subacute ruminal acidosis (SARA) induced by feeding alfalfa pellets results in increases in free bacterial lipopolysaccharide (LPS) in rumen fluid and peripheral blood, and acute phase proteins in plasma, and to determine the effect of alfalfa pellet-induced SARA on feed intake, rumen fermentation characteristics, milk production and composition, and blood metabolites. Eight lactating Holstein cows, 4 of which were ruminally cannulated, were used in a 6-wk experiment and were fed once daily at 0900 h. During wk 1, cows received a diet containing 50% of DM as concentrate and 50% of DM chopped alfalfa hay. Between wk 2 and wk 6, alfalfa hay was gradually replaced with alfalfa pellets at the rate of 8% per week to reduce rumen pH. Rumen pH was monitored continuously in the ruminally cannulated cows using indwelling pH probes. Rumen fluid and peripheral blood were sampled 15 min before feed delivery and at 6 h after feed delivery. Based on adopted threshold of SARA of at least 180 min/d below pH 5.6, SARA was induced from wk 3 onwards. Replacing 40% of alfalfa hay with alfalfa pellets quadratically increased the DMI from 18.1 kg/d in wk 1 to 23.4 kg/d in wk 6. This replacement linearly decreased milk yield (32.7 vs. 35.9 kg/d) and milk fat percentage and yield (2.32 vs. 3.22%, and 0.77 vs. 1.14 kg/d, respectively), but increased milk protein percentage and yield (3.80 vs. 3.04%, and 1.23 vs. 1.07 kg/d, respectively). This gradual replacement also linearly increased the daily averages of total volatile fatty acids (90 to 121.9 mM), acetate (53.9 to 66.8 mM), propionate (21.5 to 39.6 mM), and osmolality (277.7 to 293.8 mmol/kg) in the rumen and decreased the acetate to propionate ratio from 2.62 to 1.73. Replacing alfalfa hay with alfalfa pellets linearly increased blood lactate from 1.00 mM in wk 1 to a peak of 3.46 mM in wk 5. Induction of SARA in this study increased free rumen LPS concentration from 42,122 endotoxin unit (EU)/mL in wk 1 to 145,593 EU/mL in wk 6. However, this increase was not accompanied by an increase in LPS (<0.05 EU/mL) and in acute phase proteins serum amyloid-A, haptoglobin, and LPS-binding protein in peripheral circulation. Results suggest that SARA induced by alfalfa pellets increased LPS in the rumen without causing translocation of LPS and an immune response.

A grain-based subacute ruminal acidosis challenge causes translocation of lipopolysaccharide and triggers inflammation.

The effects of a grain-based subacute ruminal acidosis (SARA) challenge on translocation of lipopolysaccharide (LPS) into the peripheral circulation, acute phase proteins in blood and milk, feed intake, milk production and composition, and blood metabolites were determined in 8 lactating Holstein cows. Between wk 1 and 5 of 2 successive 6-wk periods, cows received a total mixed ration ad libitum with a forage to concentrate (F:C) ratio of 50:50. In wk 6 of both periods, the SARA challenge was conducted by replacing 21% of the dry matter of the total mixed ration with pellets containing 50% wheat and 50% barley. Rumen pH was monitored continuously using indwelling pH probes in 4 rumen cannulated cows. Rumen fluid samples were collected 15 min before feed delivery and at 2, 4, 6, 12, 14, 16, 18, and 24 h after feed delivery for 2 d during wk 5 (control) and wk 6 (SARA). Peripheral blood samples were collected using jugular catheters 15 min before feeding and at 6 and 12 h after feeding at the same days of the rumen fluid collections. The SARA challenge significantly reduced average daily pH from 6.17 to 5.97 and increased the duration of rumen pH below pH 5.6 from 118 to 279 min/d. The challenge reduced dry matter intake (16.5 vs. 19 kg/d), milk yield (28.3 vs. 31.6 kg/d), and milk fat (2.93 vs. 3.30%, 0.85 vs. 0.97 kg/d), and tended to increase milk protein percentage (3.42 vs. 3.29%), without affecting milk protein yield (1.00 vs. 0.98 kg/d). The challenge also increased the concentration of free LPS in rumen fluid from 28,184 to 107,152 endotoxin units (EU)/mL. This was accompanied by an increase in LPS in peripheral blood plasma (0.52 vs. <0.05 EU/mL) with a peak at 12 h after feeding (0.81 EU/mL). Concentrations of the acute phase proteins serum amyloid A, haptoglobin, and LPS-binding protein (LBP) in peripheral blood as well as LBP concentration in milk increased (438.5 vs. 167.4, 475.6 vs. 0, 53.1 vs. 18.2, and 6.94 vs. 3.02 microg/mL, respectively) during SARA. The increase in LBP in combination with the increase in LPS in peripheral blood provides additional evidence of translocation of LPS. Results suggest that the grain-based SARA challenge resulted in translocation of LPS into the peripheral circulation, and that this translocation triggered a systemic inflammatory response.

Raw potato starch in weaned pig diets and its influence on postweaning scours and the molecular microbial ecology of the digestive tract.

We evaluated the effect of raw potato starch (RPS) on growth performance, postweaning diarrhea, and gastrointestinal microbial populations in weaned piglets. Eighty-four piglets were weaned at 17 +/- 2 d of age with an average BW of 6.0 +/- 0.9 kg. Pigs were blocked by BW and assigned to 1 of 4 diets in a randomized complete block design with 7 replicate pens per diet and 3 pigs per pen. Treatments were 1) a positive control (PC) containing an antibiotic, 2) a negative control (NC) with no RPS and no antibiotic, 3) NC + 7% RPS (7% RPS), and 4) NC + 14% RPS (14% RPS). Diets were corn-wheat-soybean meal-based and formulated to meet NRC (1998) recommendations. The ADG, ADFI, and G:F ratio were determined weekly. Fecal consistency (FC) scoring was determined daily. After wk 3, 1 pig with a BW closest to the pen mean was killed to evaluate ileal and colonic mucosal-attached Escherichia coli and lactic acid bacteria, as well as digesta pH, VFA, and ammonia N concentrations. The DNA was extracted from ileum and colon digesta and used for molecular microbial evaluations using terminal-RFLP analysis of 16S rDNA genes. The ADG for wk 1 was greater (P < 0.01) for the PC diet, but diet had no effect on ADG during wk 3. The ADFI did not differ among treatments during the first 2 wk, and ADFI was least for 7% RPS diet during wk 3. The NC diet had a greater (P < 0.05) FC score during wk 1 than other treatments, but diet had no effect on FC score during wk 2 and 3. Diets had no effect on the colon lactic acid bacterial counts; however, the PC diet had decreased (P < 0.05) colon E. coli counts than other treatments. Ileum and colon digesta pH and total VFA concentrations did not differ among treatments. Pigs fed with 7 and 14% RPS diets had greater (P < 0.05) ileum ammonia N concentration compared with pigs fed with other diets. There was more diarrhea (P < 0.05) in the 14% than the 7% RPS and control treatments at d 21. This difference correlated with a decline (P < 0.05) in microbial diversity in the colon. We concluded that 7% RPS can be used to prevent postweaning diarrhea in weaned piglets, but there are no effects on growth performance.

Subacute ruminal acidosis in dairy cows: the physiological causes, incidence and consequences.

During subacute ruminal acidosis (SARA) rumen pH is depressed for several hours per day due to accumulation of volatile fatty acids and insufficient rumen buffering. Surveys suggested an incidence of SARA of between 19% and 26% in early and mid-lactation dairy cows. Causes of SARA include feeding excessive amounts of non-structural carbohydrates and highly fermentable forages, and insufficient dietary coarse fiber. Consequences of SARA include feed intake depression, reduced fiber digestion, milk fat depression, diarrhea, laminitis, liver abscesses, increased production of bacterial endotoxin and inflammation characterized by increases in acute phase proteins. The increase in endotoxin is similar among methods for SARA induction, but depends on the diet fed before induction. Increases in acute phase proteins vary among methods of SARA induction, even when the methods result in similar rumen pH depressions. This suggests that the inflammatory response might not be solely due to bacterial endotoxin in the rumen.

Evaluation of alternatives to antibiotics using an Escherichia coli K88+ model of piglet diarrhea: effects on gut microbial ecology.

Enterotoxigenic Escherichia coli is a major problem in the swine industry and results in scouring, increased mortality, and poor performance in the period immediately postweaning. The traditional way to control this problem is to include subtherapeutic antibiotics in the feed, but this is no longer acceptable to consumers; thus, alternatives to antibiotics are needed. One of the supplements that has been effective in reducing scouring in the absence of antibiotics is animal blood products produced from the rendering process. This is also becoming a problem because of concerns regarding the transfer of transmissible spongiform encephalopathies to humans from animals. In this research, we investigated the effects of spray-dried porcine plasma (SDPP), a Bacillus subtilis direct-fed microbial (DFM), a blend of organic acids, and sweeteners on E. coli-induced scouring. A total of 108 pigs of approximately 17 d of age were assigned to 6 treatments, with 3 pigs per pen, in 2 blocks, with each block having 3 replicates. The 2 blocks were initiated approximately 2 mo apart, because a sufficient number of pigs were not available that met our inclusion criteria in the first block. Diet 1 was a negative control containing no antibiotics (NC). Diet 2 was the positive control and included the same ingredient composition as NC except that antibiotics (110 mg/kg of chlortetracycline, 110 mg/kg of sulfamethazine, and 55 mg/kg of penicillin) were added (PC). Diet 3 was equal to the NC, but with a B. subtilis probiotic (DFM). Diet 4 was the NC to which SDPP was added. Diet 5 was the NC plus a combination of SDPP and DFM (SDPP + DFM). Diet 6 was the NC plus a combination of supplements, including SDPP and a blend of organic acids, DFM, and a sweetener (Blend). At 24 d of age, the pigs were experimentally infected with 6.3 x 10(9) cfu/mL of E. coli K88. All pigs were euthanized 7 d after infection and tissues were obtained for analysis. There were no significant differences among treatments for ADG, ADFI, G:F, plasma urea nitrogen, alpha-acid glycoprotein, tumor necrosis factor-alpha, intestinal ammonia, pH, or VFA. However, the PC and DFM treatments showed decreased (P < 0.05) scours at 24 h postinfection compared with the NC, SDPP, and SDPP + DFM diets. Mortality in the NC treatment, which did not contain antibiotics, was greater (P < 0.05) than in the other treatments. Terminal restriction fragment length polymorphism analysis of the 16S rDNA genes of digesta showed a greater incidence (P < 0.05) of Bacteroidetes in the PC and DFM diets than in the NC diet. When SDPP and DFM were included in the diet, the incidence of Bacteroidetes was also greater than in the NC diet (P < 0.05).

Response of piglets weaned from sows fed diets supplemented with conjugated linoleic acid (CLA) to an Escherichia coli K88+ oral challenge.

Seventy-eight Cotswold piglets weaned from sows receiving 0% or 2% conjugated linoleic acid (CLA)-supplemented rations from day 85 of gestation through lactation were allocated to nursery diets (ND) according to their dam's lactation ration (LR) as follows (1) 0%-0% (0% CLA LR: 0% CLA ND, n = 17); (2) 0%-2% (0% CLA LR: 2% CLA ND, n = 17); (3) 2%-0% (2% CLA LR: 0% CLA ND, n = 23); and (4) 2%-2% (2% CLA LR: 2% CLA ND, n = 21). At 28 ± 2 days of age all piglets received an oral Escherichia coli K88+ (enterotoxigenic Escherichia coli, ETEC) challenge and were subsequently monitored for scour development and overall health until 36 ± 2 days of age, after which blood and tissue samples were collected. Piglet BW was not affected by dietary CLA supplementation to LR (P > 0.05). However, by day 36 piglets receiving 2% CLA-supplemented ND were significantly lighter (P < 0.05) than piglets receiving control diets. Average daily gain and feed efficiency were not affected by CLA supplementation. Average daily feed intake (ADFI) was greater for piglets weaned from 2% CLA-supplemented sows from day 17 to 28 (P < 0.05), otherwise ADFI was unaffected by dietary CLA supplementation (P > 0.05). The development of scours was less severe in piglets weaned from 2% CLA-supplemented sows at 8, 24, 48 and 56 h after ETEC challenge (P < 0.05). Intestinal coliform and lactic acid bacteria populations post challenge were not affected by CLA supplementation. However, cecal ammonia-N was numerically greatest in 0%-0% piglets compared to the other treatment groups, and the total volatile fatty acid production was numerically lower in 0%-0% and 0%-2% piglets compared to 2%-0% and 2%-2% piglets. In addition, piglets weaned from 2% CLA-supplemented sows had increased serum immunoglobulin A (P < 0.001) and G (P < 0.05) levels and reduced (P < 0.05) intestinal mucosal inflammation compared to piglets weaned from control sows. Although there were no obvious additional health effects observed when CLA was provided in ND, supplementing sow rations with 2% CLA from mid-gestation through weaning appears to have immune-stimulating carry-over effects post weaning. Thus, supplementing sow rations with CLA may be a practical strategy for enhancing passive immune transfer and improving the immune status and overall gut health of nursery piglets.

Ruminal lipopolysaccharide concentration and inflammatory response during grain-induced subacute ruminal acidosis in dairy cows.

The effects of grain-induced subacute ruminal acidosis (SARA) in lactating dairy cows on free ruminal lipopolysaccharide (LPS) and indicators of inflammation were determined. Four mid lactation dairy cows were divided into 2 groups of 2 cows and used in a repeated switchover design. During each period, SARA was induced in 2 animals for 5 subsequent days by replacing 25% of their total mixed ration (dry matter basis) with a concentrate made of 50% wheat and 50% barley. The other 2 cows acted as controls and were fed a total mixed ration diet in which 44% of dry matter was concentrate. On average, inducing SARA did not affect milk composition, increased the duration of rumen pH below 5.6 from 187 to 309 min/d, and increased free ruminal LPS concentration from 24,547 endotoxin units (EU)/mL to 128,825 EU/mL. Averaged across treatments, milk fat yield and milk protein yield were 0.66 and 1.00 kg/d, respectively. Rumen pH and milk fat data suggest that control cows also experienced ruminal acidosis, albeit a milder form of this disease than SARA cows. Serum LPS concentration in both control and SARA cows was less than the detection limit of <0.01 EU/mL for the assay. Induction of SARA elevated serum amyloid A concentration from 286.8 to 498.8 mug/mL, but did not affect other markers of inflammation including haptoglobin, fibrinogen, serum copper, or white blood cells. These results suggest that grain-induced SARA in mid lactation dairy cows increases the lysis of gram-negative bacteria and activates an inflammatory response.