Dietary Organic Acids Modulate Gut Microbiota and Improve Growth Performance of Nursery Pigs.

Feed additives have been suggested to improve animal growth performance through modulating the gut microbiota. The hypothesis of this study was that the combination of two organic acids would exert synergistic effects on the growth performance and gut microbiota of weaning pigs. To test this hypothesis, we followed 398 weaning pigs from two university experiment stations (University of Illinois at Urbana-Champaign (UIUC) and University of Arkansas (UA)) to determine the effects of increasing levels (0%, 0.035%, 0.070%, and 0.105%) of sodium butyrate combined with 0.5% benzoic acid on the growth performance of nursery pigs. At the UA, an additional negative control diet was included and the gut microbiota analysis was carried out. At both universities, increasing levels of sodium butyrate in a diet containing 0.5% benzoic acid improved growth performance, which reached a plateau in the pigs fed 0.035% (SBA0.035) or 0.070% (SBA0.070) butyrate. Gut microbiota analysis revealed that pigs fed the SBA0.035 diet had more diverse microbiota and contained more potentially beneficial bacteria such as Oscillospira, Blautia, and Turicibacter and reduced levels of Veillonella and Sarcina. Results of the present study indicated that the inclusion of sodium butyrate at moderate levels in a diet containing 0.5% benzoic acid improved growth performance of weaning pigs and established potential health benefits on gut microbiota.

ZnO Modulates Swine Gut Microbiota and Improves Growth Performance of Nursery Pigs When Combined with Peptide Cocktail.

Zinc has been very efficacious in reducing post-weaning diarrhea, whereas animal-derived peptides are suggested to improve the growth performance of weaned piglets. However, the combined effect of zinc and peptides on swine production and swine gut microbiota is still largely unknown. In this study, we followed 288 nursery pigs from the age of d30 to d60 to evaluate the growth performance and gut microbiota of weanling pigs subjected to different levels of a fish-porcine-microbial peptide cocktail (0.05%, 0.25%, and 0.5%) with or without the pharmaceutical level of zinc oxide (ZnO) (2500 ppm) supplementation in a nutrient-deficient diet. Rectal swab samples were collected from pigs with body weight (BW) approach average at each pen on d30, d42, and d60 to determine gut microbiota. Average daily gain (ADG) and BW in piglets fed high zinc (HZ) increased with increasing levels of peptide. The microbiota of the HZ group also diverged from those of the standard zinc (SZ) group from d30 to d60. Adding peptide did not alter community structure regardless of zinc supplementation. Collectively, these findings demonstrated that the pharmaceutical level of zinc as ZnO conditioned the gut community to the point where peptide could effectively restore growth performance in nursery pigs fed nutrient-deficient diets.

Acridine orange as an alternative to optical density to study growth kinetics of Lactobacillus bulgaricus ATCC 7517.

In this study we assessed the use of acridine orange as an alternative to optical density to quantify the growth of Lactobacillus bulgaricus ATCC 7517. The growth of bacteria in Lactobacillus de Man Rogosa Sharpe (MRS) medium was measured by both acridine orange (AO) and optical density (OD) measurements for 24 h. The relationship between both methods was compared via correlation analysis. The doubling time of bacteria based on the values of OD600 and AO obtained during 24 h growth were also calculated. The result shows strong correlation of cell growth between OD600 and AO during the first 10 hours of growth, but the correlation was less strong when analyzing the data from 0 to 24 hours. Growth rates, generation time and lag time were also similar. This study indicates that AO could be used in place of OD to prepare growth curves of Lactobacillus bulgaricus during the exponential phase of growth, and to compare growth rates, generation times or lag times.

Antimicrobial properties of three lactic acid bacterial cultures and their cell free supernatants against Listeria monocytogenes.

Control of Listeria monocytogenes in ready-to-eat (RTE) food products is a significant challenge and improved means for control are needed. In this study, the anti-listerial effects of three lactic acid bacteria (LAB) were investigated. Spot-on-lawn assays demonstrated the largest zones of inhibition against L. monocytogenes were produced by the Pediococcus acidilactici strain, with zone diameters ranging from 13 to 18 mm. Minimum inhibitory concentration (MIC) experiments using cell free supernatant (CFS) from the LAB revealed that while two of the strains were effective at inhibiting L. monocytogenes growth only up to a 1:4 dilution, P. acidilactici was able to inhibit growth up to a 1:256 dilution. Survival assays performed at 7°C determined that the P. acidilactici strain was capable of producing a 4.5 log reduction in L. monocytogenes counts and maintaining the reduction for 21 days. The effectiveness of P. acidilactici was reduced under log phase growth, autoclaving for longer than 15 min (121°C and 15 psi), and treatment with proteinase K (25 mg/mL).

Laser-induced inactivation of Plasmodium falciparum.

BACKGROUND: Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG). In the current study the feasibility of using haemozoin, constitutively stored in the parasite's food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated. METHODS: Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU). RESULTS: Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with -4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (-2-log reduction). A -1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 µM synthetic haemozoin and a -2-log reduction in the presence of 10 µM haemozoin. Minimal (≤ 15%) cell kill was observed in the presence of 10 µM haemin. CONCLUSIONS: Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient's blood through the skin or through dialysis tubing with a NIR laser.

Intermediate outcomes of fresh talar osteochondral allografts for treatment of large osteochondral lesions of the talus.

BACKGROUND: Large osteochondral defects of the talus present a treatment challenge. Fresh osteochondral allograft transplantation can be used for large lesions without the donor-site morbidity associated with other procedures such as autologous chondrocyte implantation or osteochondral autograft transfer. The goal of this study was to prospectively evaluate the intermediate outcomes of fresh osteochondral allografting for osteochondral lesions of the talus with use of validated outcome measures. METHODS: Sixteen patients (seventeen ankles) received a fresh osteochondral allograft, and all sixteen were available for follow-up. Data were prospectively collected with use of the Ankle Osteoarthritis Scale (AOS), Short Form-36 (SF-36), and American Academy of Orthopaedic Surgeons (AAOS) Foot and Ankle Module outcome measures. Postoperative American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot scale scores were also collected. All sixteen patients underwent radiographic and computed tomographic (CT) analyses preoperatively, and fifteen patients had these studies postoperatively. RESULTS: The average duration of follow-up was 4.1 years. The latest follow-up CT evaluation identified failure of graft incorporation in two of sixteen ankles. Osteolysis, subchondral cysts, and degenerative changes were found in five, eight, and seven ankles, respectively. Five ankles were considered failures, and two required a reoperation because of ongoing symptoms. The AOS Disability and the AAOS Foot and Ankle Core Scale scores significantly improved, but there was no significant change in the AOS Pain, AAOS Foot and Ankle Shoe Comfort Scale, or SF-36 scores. Overall, ten patients had a good or excellent result; however, persistent symptoms remained in six of these patients. Only four were symptom-free. CONCLUSION: The use of a fresh osteochondral allograft is a reasonable option for the treatment of large talar osteochondral lesions. The high reoperation rate (two of seventeen) and failure rate (five of seventeen) must be taken into consideration when one is choosing this procedure for the management of these lesions.

The combination of energy-dependent internal adaptation mechanisms and external factors enables Listeria monocytogenes to express a strong starvation survival response during multiple-nutrient starvation.

The goal of this study was to characterize the starvation survival response (SSR) of a wild-type Listeria monocytogenes 10403S and an isogenic DeltasigB mutant strain during multiple-nutrient starvation conditions over 28 days. This study examined the effects of inhibitors of protein synthesis, the proton motive force, substrate level phosphorylation, and oxidative phosphorylation on the SSR of L. monocytogenes 10403S and a DeltasigB mutant during multiple-nutrient starvation. The effects of starvation buffer changes on viability were also examined. During multiple-nutrient starvation, both strains expressed a strong SSR, suggesting that L. monocytogenes possesses SigB-independent mechanism(s) for survival during multiple-nutrient starvation. Neither strain was able to express an SSR following starvation buffer changes, indicating that the nutrients/factors present in the starvation buffer could be a source of energy for cell maintenance and survival. Neither the wild-type nor the DeltasigB mutant strain was able to elicit an SSR when exposed to the protein synthesis inhibitor chloramphenicol within the first 4 h of starvation. However, both strains expressed an SSR when exposed to chloramphenicol after 6 h or more of starvation, suggesting that the majority of proteins required to elicit an effective SSR in L. monocytogenes are likely produced somewhere between 4 and 6 h of starvation. The varying SSRs of both strains to the different metabolic inhibitors under aerobic or anaerobic conditions suggested that (1) energy derived from the proton motive force is important for an effective SSR, (2) L. monocytogenes utilizes an anaerobic electron transport during multiple-nutrient starvation conditions, and (3) the glycolytic pathway is an important energy source during multiple-nutrient starvation when oxygen is available, and less important under anaerobic conditions. Collectively, the data suggest that the combination of energy-dependent internal adaptation mechanisms of cells and external nutrients/factors enables L. monocytogenes to express a strong SSR.

Ciprofloxacin-resistant Campylobacter persists in raw retail chicken after the fluoroquinolone ban.

In 2005, the FDA withdrew approval for the use of fluoroquinolones in live poultry production. To assess any changes in countable numbers of ciprofloxacin-resistant Campylobacter before and after the fluoroquinolone withdrawal, retail whole raw chicken carcasses (RTCC) purchased in Northwest Arkansas from 2004 to 2006 were sampled for this purpose. Using a previously published direct-plating method developed in our laboratory, we quantified trends of Campylobacter and ciprofloxacin-resistant Campylobacter loads by direct plating whole chicken carcass rinses on Campylobacter agar (CA) or Campylobacter agar containing 8.6 mg/ml ciprofloxacin (CCA). Countable populations of Campylobacter were recovered from 74, 96, and 63% of carcasses sampled in 2004, 2005, and 2006 respectively. The percentages of carcasses with minimum detectable levels of ciprofloxacin-resistant Campylobacter were 20, 42 and 33%, sampled in 2004, 2005 and 2006, respectively. Our 3 year analysis in one geographical area indicated a persistence of Campylobacter and ciprofloxacin-resistant Campylobacter on retail raw chicken carcasses despite the cessation of fluoroquinolone use in poultry production.

Characterization of new hybridoma clones producing monoclonal antibodies reactive against both live and heat-killed Listeria monocytogenes.

The objective of this study is to develop high affinity monoclonal antibody (MAb) probes recognizing all major serotypes of Listeria monocytogenes cells. From 500 candidate hybridoma clones, 2 new monoclonal antibody-producing hybridomas were selected and evaluated. MAbs 22D10 and 24F6 reacted strongly with live cells of most serotypes of L. monocytogenes except 4c and 4e and with some L. innocua strains; MAb 22D10 reacted strongly with both live and heat-killed cells (100 degrees C for 20 min) of Listeria. Both MAbs 22D10 and 24F6 did not show any cross-reactions with the other non-Listeria G(+) bacteria tested in ELISA. The mixture of EM-7G1 and 22D10 or 24F6 reacted with all 13 major serotypes of live L. monocytogenes except serotype 4c, while none of these 3 MAbs when tested alone did so. MAb 22D10 mixed with 7G1 reacted with all heat-killed L. monocytogenes serotypes except 4c and 4e. In Western blots, MAbs 22D10 and 24F6 reacted with 1 major protein band of 66 kDa in extracts from L. monocytogenes, but with 2 major protein bands of 66 kDa and 76 kDa in extracts from L. innocua. These results suggest that MAbs 22D10 and 24F6 have high affinity for 11 of 13 serotypes of L. monocytogenes, both live and heat-killed cells. MAbs 22D10 and 24F6--in combination with species-specific MAb EM-7G1--should be useful candidates for use in an ELISA sandwich assays for detecting L. monocytogenes in RTE meat and poultry products.

Differential killing activity of cetylpyridinium chloride with or without bacto neutralizing buffer quench against firmly adhered Salmonella gaminara and Shigella sonnei on cut lettuce stored at 4 degrees C.

Cetylpyridinium chloride (CPC) activity was quenched with Bacto neutralizing buffer on inoculated cut iceberg lettuce. This protocol permitted comparison of the numbers of Salmonella Gaminara- or Shigella sonnei-inoculated cells on lettuce that survived 1 min of CPC treatment. Cut lettuce was inoculated with about 6 log of Salmonella or 9 log of Shigella and stored in Whirl-Pak bags at 4 degrees C for up to 4 days. Loosely adhered pathogen cells were washed off before CPC treatment. Firmly adhered cells of Salmonella Gaminara or S. sonnei on cut iceberg lettuce survived treatment with CPC even at the 0.4% CPC level if the CPC activity was quenched after 1 min by adding Bacto neutralizing buffer. The results confirm that there is extended killing activity of residual CPC against Salmonella Gaminara or S. sonnei if the residual CPC remaining in contact with the lettuce after the initial 1-min wash is not quenched. The CPC treatment was useful in reducing the numbers of these target pathogens on lettuce.

Concurrent quantitation of total campylobacter and total ciprofloxacin-resistant campylobacter loads in rinses from retail raw chicken carcasses from 2001 to 2003 by direct plating at 42 degrees C.

This is the first report on the use of a normally lethal dose of ciprofloxacin in a Campylobacter agar medium to kill all ciprofloxacin-sensitive Campylobacter spp. but allow the selective isolation and quantitation of naturally occurring presumptive ciprofloxacin-resistant Campylobacter CFU in rinses from retail raw chicken carcasses (RTCC). Thermophilic-group total Campylobacter CFU and total ciprofloxacin-resistant Campylobacter CFU (irrespective of species) were concurrently quantified in rinses from RTCC by direct plating of centrifuged pellets from 10 or 50 ml out of 400-ml rinse subsamples concurrently on Campylobacter agar and ciprofloxacin-containing Campylobacter agar at 42 degrees C (detection limit = 0.90 log(10) CFU/carcass). For 2001, 2002, and 2003, countable Campylobacter CFU were recovered from 85%, 96%, and 57% of RTCC, while countable ciprofloxacin-resistant Campylobacter CFU were recovered from 60%, 59%, and 17.5% of RTCC, respectively. Total Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.52, 0.90 to 4.58, and 0.90 to 4.48 log(10) CFU/carcass in 2001, 2002, and 2003, respectively. Total ciprofloxacin-resistant Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.06, 0.90 to 3.95, and 0.90 to 3.04 log(10) CFU/carcass in 2001, 2002, and 2003, respectively. Overall, total Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, 4 to 5 log(10) CFU/carcass, respectively, were recovered from 16%, 32%, 26%, and 5% of RTCC tested over the 2-year sampling period. For the same period, total ciprofloxacin-resistant Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log(10) CFU/carcass, respectively, were recovered from 24%, 11%, 7%, and 0.2% of RTCC tested. There was a steady decline in total Campylobacter and total ciprofloxacin-resistant Campylobacter loads in RTCC rinses from 2001/2002 to 2003.