[Show abstract][Hide abstract] ABSTRACT: Previously, our laboratory has screened and identified Bacillus spp. isolates as direct-fed microbials (DFM). The purpose of the present study was to evaluate the cellulase and xylanase production of these isolates and select the most appropriate Bacillus spp. candidates for DFM. Furthermore, an in vitro digestive model, simulating different compartments of the gastrointestinal tract, was used to determine the effect of these selected candidates on digesta viscosity and Clostridium perfringens proliferation in different poultry diets. Production of cellulase and xylanase were based on their relative enzyme activity. Analysis of 16S rRNA sequence classified two strains as Bacillus amyloliquefaciens and one of the strains as Bacillus subtilis. The DFM was included at a concentration of 108 spores/g of feed in five different sterile soybean-based diets containing corn, wheat, rye, barley, or oat. After digestion time, supernatants from different diets were collected to measure viscosity, and C. perfringens proliferation. Additionally, from each in vitro simulated compartment, samples were taken to enumerate viable Bacillus spores using a plate count method after heat-treatment. Significant (P < 0.05) DFM-associated reductions in supernatant viscosity and C. perfringens proliferation were observed for all non-corn diets. These results suggest that antinutritional factors, such as non-starch polysaccharides from different cereals, can enhance viscosity and C. perfringens growth. Remarkably, dietary inclusion of the DFM that produce cellulase and xylanase reduced both viscosity and C. perfringens proliferation compared with control diets. Regardless of diet composition, 90% of the DFM spores germinated during the first 30 min in the crop compartment of the digestion model, followed by a noteworthy increased in the intestine compartment by ~2log10, suggesting a full-life cycle development. Further studies to evaluate in vivo necrotic enteritis effects are in progress.
[Show abstract][Hide abstract] ABSTRACT: Antibiotics as growth promoters (AGP) in the feed of different animal species have been used for many years to improve feed efficiency and growth. The mechanisms of how antibiotics can promote growth are not well known; nevertheless several studies have focused on interactions between antibiotics and intestinal microflora. However, many scientists have suggested that there may be other modes of action of some antibiotics, including the capacity of these antibiotics to produce immunomodulatory and anti-inflammatory effects. It has been demonstrated that antibiotics are able to inhibit one or more functions of inflammatory cells and pro-inflammatory cytokine production. Consequently, AGP may prevent immunologic stress that is associated with metabolic changes, allowing more energy to be available for muscle development thus, improving growth. Although the use of AGP in animal production has advantages, its extensive use has been suggested to have contributed to the emergence of antimicrobial resistance in zoonotic pathogens, resulting in a ban by the European Union with suggestive restrictions by other countries. The withdrawal of AGP from livestock production has led to an increase in cost, a decrease in production, and an increase in disease incidence, forcing food animal producers to find alternatives including probiotics, which have been used to enhance intestinal health. The major functional effect of probiotics may be the balance of both gastrointestinal microflora and immune-stimulation, particularly by maintaining the equilibrium of pro-inflammatory and anti-inflammatory cytokines. This review describes the immuno-modulation and anti-inflammatory effects of antibiotic growth promoters versus probiotics in the intestinal tract.
[Show abstract][Hide abstract] ABSTRACT: Two independent trials were conducted to evaluate the utilization of rye as energy source on bacterial translocation (BT), intestinal viscosity, gut integrity, gut microbiota composition, and bone mineralization, when compared with a traditional cereal (corn) in broiler chickens. In each experiment, day-of-hatch, broiler chickens were randomly assigned to either a corn or a rye diet (n = 20 chickens/group). At 10 d of age, in both experiments, 12 chickens/group were randomly selected, and given an oral gavage dose of fluorescein isothiocyanate dextran (FITC-d). After 2.5 h of oral gavage, blood samples were collected to determine the passage of FITC-d. The liver was collected from each bird to evaluate BT. Duodenum, ileum, and cecum gut sections were collected to evaluate intestinal viscosity and to enumerate gut microbiota. Tibias were collected for observation of bone parameters. Broilers fed with rye showed increased (p < 0.05) intestinal viscosity, BT, and serum FITC-d. Bacterial enumeration revealed that chickens fed with rye had increased the number of total lactic acid bacteria in all three sections of the gastrointestinal tract evaluated when compared to chickens fed with corn. Chickens fed with rye also had significantly higher coliforms in duodenum and ileum, whereas the total number of anaerobes increased only in duodenum. A significant reduction in bone strength and bone mineralization was observed in chickens fed with rye when compared with corn fed chickens. In conclusion, rye evoked mucosal damage in chickens that alter the intestinal viscosity, increased leakage through the intestinal tract, and altered the microbiota composition as well as bone mineralization. Studies to evaluate dietary inclusion of selected DFM candidates that produce exogenous enzymes in rye fed chickens are currently being evaluated.
Frontiers in Genetics 09/2014; 5:339. DOI:10.3389/fgene.2014.00339
[Show abstract][Hide abstract] ABSTRACT: Experimental and epidemiological evidence have indicated the respiratory route to be a potential portal of entry for salmonellae in poultry. The purpose of this study was to evaluate and compare the infectivity of Salmonella enterica serovar Senftenberg following oral gavage (OR), intratracheal (IT) or intravenous (IV) challenge in chickens. Seven-day-old chicks were challenged with either 104 or 106 CFU of S. Senftenberg /chick, OR, IT or IV, respectively in two independent trials. Chickens were humanely killed 24 h post challenge and S. Senftenberg was cultured and enumerated from cecal contents, ceca-cecal tonsils and liver and spleen (LS). In both trials, IT delivery of S. Senftenberg was the only route that allowed colonization of the ceca of the chickens when compared with OR or IV challenge in a dose response fashion (P < 0.05). Low levels of S. Senftenberg recovery from selectively enriched LS samples were found only after high dose administration of S. Senftenberg by either route in both trials. The results of the present study suggest that S. Senftenberg entering the blood is likely to be cleared and will not be able to colonize ceca to the same extent as compared to IT challenge. Clarification of the potential importance of the respiratory tract for transmission of salmonellae under field conditions may be of critical importance to develop intervention strategies to reduce its transmission in poultry.
[Show abstract][Hide abstract] ABSTRACT: Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative cells or spores is advisable.
[Show abstract][Hide abstract] ABSTRACT: Abstract 1. An experiment was designed to evaluate the effect of different doses of oocysts of E. acervulina on intestinal absorption and skin deposition of xanthophylls (XA) in broilers. 2. A total of 192 broiler chickens were randomly assigned to 4 groups: an uninfected control group and three groups inoculated with either 1x102, 1x104, or 1x105 sporulated oocysts of E. acervulina by gavaging at 21 d. There were 4 replicate pens (2 male and 2 female) per group. 3. Plasma xanthophyll (PX) and skin yellowness (SY) were measured in live birds weekly. At 42 d of age, SY was measured in the breast and abdomen after chilling and in the breast 24 h post processing on refrigerated carcasses. 4. In general, in all challenged treatments, and for the duration of the study, the average PX decreased by 0.02 μg/ml (R2 = 61.6%) for every 1000 inoculated oocysts, whereas PX increased by 1.26 μg/ml/d in uninfected birds. 5. The average SY in live birds from 21 to 42 d of age decreased by 0.019 b*/every 1000 oocysts administered, while SY of uninfected controls increased by 0.57 b*/d. It was also noted that in all treatments females had a greater SY (6.17 b*) than males for the duration of the study. The SY of the breast and abdomen was correlated (r = 0.76) in chilled carcasses. Breast SY in 24 h refrigerated carcasses was greater in the control group and for female birds. 6. Oocyst excretion was different between inoculated treatments only on 7 d post inoculation (PI). Coccidia lesion scores in the duodenum averaged 1+ in infected birds and 2+ in birds given the highest oocyst dose.
British Poultry Science 04/2014; 55(2). DOI:10.1080/00071668.2013.879095 · 0.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Experimental and epidemiological evidence suggests that primary infection of Salmonella is by the oral-fecal route for poultry. However, the airborne transmission of Salmonella and similar enteric zoonotic pathogens has been historically neglected. Increasing evidence of Salmonella bioaerosol generation in production facilities and studies suggesting the vulnerabilities of the avian respiratory architecture together have indicated the possibility of the respiratory system being a potential portal of entry for Salmonella in poultry. Presently, we evaluated this hypothesis through intratracheal (IT) administration of Salmonella Enteritidis and Salmonella Typhimurium, as separate challenges, in a total of 4 independent trials, followed by enumeration of cfu recovery in ceca-cecal tonsils and recovery incidence in liver and spleen. In all trials, both Salmonella Enteritidis and Salmonella Typhimurium, challenged IT colonized cecae to a similar or greater extent than oral administration at identical challenge levels. In most trials, chickens cultured for cfu enumeration from IT-challenged chicks at same dose as orally challenged, resulted in an increase of 1.5 log higher Salmonella Enteritidis from ceca-cecal tonsils and a much lower dose IT of Salmonella Enteritidis could colonize ceca to the same extent than a higher oral challenge. This trend of increased cecal colonization due to IT challenge was observed with all trails involving week-old birds (experiment 2 and 3), which are widely considered to be more difficult to infect via the oral route. Liver-spleen incidence data showed 33% of liver and spleen samples to be positive for Salmonella Enteritidis administered IT (10(6) cfu/chick), compared with 0% when administered orally (experiment 2, trial 1). Collectively, these data suggest that the respiratory tract may be a largely overlooked portal of entry for Salmonella infections in chickens.
[Show abstract][Hide abstract] ABSTRACT: Bacterial contamination of raw, processed poultry may include spoilage bacteria and foodborne pathogens. We evaluated different combinations of organic acid (OA) wash solutions for their ability to reduce bacterial contamination of raw chicken skin and to inhibit growth of spoilage bacteria and pathogens on skin during refrigerated storage. In experiment 1, raw chicken skin samples were dipped into a suspension of either 10(8) cfu/mL of Salmonella Typhimurium, Escherichia coli O157:H7, or Listeria monocytogenes for 30 s and then immersed in PBS or an OA wash solution mixture of 0.8% citric, 0.8% acetic, and 0.8% propionic acid (at equal wt/vol concentrations) for an additional 30 s. In experiment 2, three different concentrations of the OA wash solution (0.2, 0.4, and 0.6% at equal wt/vol concentrations) were tested against chicken skin samples contaminated with Salmonella Typhimurium. Viable pathogenic bacteria on each skin sample were enumerated after 1 and 24 h of storage at 4°C in both experiments. In experiment 3, skin samples were initially treated on d 1 with PBS or 2 concentrations of the OA mixture (0.4 and 0.8%), and total aerobic bacteria were enumerated during a 2-wk storage period. In all experiments, significant (P < 0.05) differences were observed when skin samples were treated with the OA wash solution and no spoilage organisms were recovered at any given time point, whereas increasing log10 numbers of spoilage organisms were recovered over time in PBS-treated skin samples. These results suggest that 0.2 to 0.8% concentrations of an equal-percentage mixture of this OA combination may reduce pathogens and spoilage organisms and improve food safety properties of raw poultry.
[Show abstract][Hide abstract] ABSTRACT: Bacteriophages used to treat infections are typically amplified in a pathogenic host. However, this practice introduces the risk of administering any remaining bacteriophage-resistant pathogen during bacteriophage application if separation techniques are less than perfect. In this study, bacteriophage isolates capable of replicating in both Salmonella and Klebsiella oxytoca were identified and applied to poultry carcasses. These Wide-Host-Range bacteriophages (WHR) were amplified using the non-pathogenic bacteria, Klebsiella oxytoca in tryptic soy broth until a titer of approximately 10⁹ PFU/mL was obtained. WHR and Klebsiella oxytoca were not separated prior to treatment of carcasses. Fresh processed chicken carcasses were inoculated with either Salmonella enteritidis (SE) or S. typhimurium (ST), sprayed with 5 mL of WHR and rinsed with sterile water. Samples were enriched, plated on XLD agar and evaluated for Salmonella-typical colonies. In four separate trials, WHR significantly reduced the recovery of SE. No SE was detected in two trials and a greater than 70% reduction was seen in the other two trials. ST was also significantly reduced in the two trials in which it was included (p<0.05). These experiments suggest that WHR could be an inexpensive and safe method for the reduction of Salmonella on broiler carcasses.
International Journal of Poultry Science 10/2007; 6(10). DOI:10.3923/ijps.2007.754.757
[Show abstract][Hide abstract] ABSTRACT: Survival of bacteriophages through the upper gastrointestinal tract (UGIT) and persistence in the lower gastrointestinal tract (LGIT) is essential for treatment of enteric bacterial infections. We have hypothesized that non-pathogenic Alternative Host Bacteria (AHB), originally isolated from poultry cecal samples, could be used to protect bacteriophages during UGIT passage and to provide host cells for continued amplification in the LGIT. We selected two previously-identified Wide Host Range (WHR) bacteriophages (WHR-8 and WHR-10) and their respective AHB for use in the present studies. For each of the bacteriophage-host combinations, combination of the bacteriophage with the AHB prior to oral gavage had little effect on the concentration of recovered bacteriophages from the cecal contents during the three days post-administration. Furthermore, continuous administration of the AHB in the drinking water had little effect on intestinal bacteriophage recovery during the three days of evaluation. Bacteriophages were also tested for differences in anaerobic and aerobic lysis of Salmonella enteritidis as a possible reason for decreased persistence in the LGIT. Differences in lysis between anaerobic and aerobic environments were significant, however levels were not likely different enough to have significant in vitro effects. These results suggest that selection of AHB to protect or amplify enteric bacteriophage populations is not necessarily a simple process. Survival of the AHB and ability of the AHB to replicate in the LGIT of the target animals are among considerations that should be made in future investigations.
International Journal of Poultry Science 10/2007; 6(10). DOI:10.3923/ijps.2007.758.761
[Show abstract][Hide abstract] ABSTRACT: Effective Competitive Exclusion (CE) cultures have been shown to accelerate development of normal microflora in chicks and poults, providing increased resistance to infection by some enteric bacterial pathogens. Our objective was to develop a CE culture for prophylaxis and reduced horizontal transmission of Salmonella enteritidis (SE) in broiler chickens. In the present study, seven members of the family Enterobacteriaceae and 2 lactic acid bacteria isolates, each capable of in vitro and in vivo inhibition of SE, were selected and combined to form the putative CE culture. In the first experiment, day-of-hatch chicks were randomly divided into four pens. All treated chicks were orally gavaged with the CE culture and 3 pens were treated with the CE culture in the drinking water for four consecutive days. Treated and control-non treated chicks were challenged with SE on day 4. All 3 groups of birds that were treated with the CE culture had a significant decrease (p<0.05) in cecal colonization compared with non-CE-treated SE-challenged chicks. Two additional experiments were designed to measure the efficacy of the CE culture in reducing SE horizontal transmission from infected to uninfected chicks when commingled. SE was recovered in the cecal tonsils with a significantly lower incidence at days 7 and 14 in Experiment 2 and day 7 in Experiment 3 from the groups that received the CE in the drinking water as compared to controls respectively. These results suggest that a relatively simple and defined CE culture can reduce SE colonization in neonatal chicks.
International Journal of Poultry Science 07/2007; 6(7). DOI:10.3923/ijps.2007.489.492
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to select appropriate bacteriophages that survive in the gastrointestinal tract of neonatal poultry and utilize those bacteriophages to reduce intestinal colonization of Salmonella enteritidis phage type 13A (SE) in challenged birds. Broiler chicks served as an in vivo biological filter to preferentially select bacteriophages from our bacteriophage library capable of surviving the gastrointestinal environment. A mixture of bacteriophage isolates designated PHL 1-71 was administered orally to three SE challenged chicks on three consecutive days. Each day, bacteriophages were recovered from the ileum, ileocecal junction and ceca for sequential administration the following day. The recovered bacteriophages were then administered to SE-infected turkey poults. In the first experiment, two-day old poults were challenged with 10 cfu SE and treated 48 h later with 5mM Mg (OH) followed by 2.5×10 plaque 4 9 2 forming units (pfu) of bacteriophages in 1mM Mg (OH) solution. This treatment numerically reduced SE 2 recovered from cecal contents at 12 and 24 h after treatment as compared to untreated controls. In a second experiment, two-day old poults were challenged with 1.6×10 cfu SE and treated with 5mM Mg (OH) followed 4 2 by 7.5×10 pfu phage in 1mM Mg (OH) solution 48 h post-challenge. We recovered 79,728 cfu of SE per g 9 2 of cecal contents in the control group and 11,224 cfu/g in the phage treated group 24 h post treatment. These data were not significantly different, but they suggest that bacteriophages can be preferentially selected in vivo to increase survival in the avian gastrointestinal tract. However, improved efficacy is required prior to useful application of the approach for reducing Salmonella infection.
International Journal of Poultry Science 03/2007; 6(3):163-168. DOI:10.3923/ijps.2007.163.168
[Show abstract][Hide abstract] ABSTRACT: SUMMARY We evaluated the effect of ionized reactive oxygen species created using Binary Ionization Technology (BIT) for disinfection of broiler carcasses, table eggs, and treatment of fertile eggs. Previous research has indicated that BIT creates a high concentration of reactive oxygen species (ROS) that lyse bacterial cells on contact. Application of BIT to broiler carcasses that had been intentionally inoculated with 1.58 × 106 Salmonella enterica Enteritidis (SE) caused a 1 to 3 log reduction in recoverable SE, depending on the duration of the treatment. Additionally, after inoculation of table eggs with 6.8 × 108 cfu of SE, we recovered SE from 95% fewer eggs following enrichment and found significantly fewer (7.77 and 7.41 log reduction) colony-forming units recovered from eggs treated with BIT compared with nontreated control eggs. We also evaluated whether application of the BIT treatment had any effect on hatchability of broiler breeder eggs to determine whether use of this technology could be feasible in a hatchery environment for disinfection of eggs. There were no significant effects of BIT on the hatchability (of total set) of treated eggs as compared with nontreated control eggs; however, there was a slight numerical increase in hatchability, between 5 and 10% in 2 trials. These data suggest that application of BIT technology to carcasses and table eggs could reduce contamination with pathogens and that the application to fertile eggs may not have effects on hatchability of eggs set.
The Journal of Applied Poultry Research 12/2005; 14(4). DOI:10.1093/japr/14.4.716 · 0.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aspergillus meal is a feed additive used to improve broiler gut health and performance. Trials reported here indicate that Aspergillus meal may offer a protein sparing effect when used with low protein diets. Aspergillus meal might offer better results when the level of protein and amino acids is lower than those recommended by NRC or applied in commercial flocks. Lower levels of protein and amino acids as compared with those recommended by NRC or applied in commercial flocks are also a potential environmental benefit. Thus, low protein diets are of interest and important for feed additive evaluation and animal performance.
The Journal of Applied Poultry Research 12/2005; 14(4):665-669. DOI:10.1093/japr/14.4.665 · 0.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intentional early colonization of the intestinal tract with beneficial microflora, known as competitive exclusion, has been shown to successfully protect poultry from selected enteric pathogens. Although effective cultures have been produced and are available, an inexpensive, air-tolerant, and completely defined culture is needed. Presently, we developed an in vitro competition assay to select for individual facultative anaerobes of poultry enteric origin that could exclude Salmonella. Using this assay, 24 isolates were selected and stored individually. These 24 isolates were amplified in batch culture (tryptic soy broth, 4 h at 40 degrees C) and administered at final dilutions of 10, 100, or 1,000 cfu to day-of-hatch poults. Forty-eight hours later, poults were challenged with 100 to 1,000 cfu antibiotic-resistance-marked Salmonella enteritidis PT 13A by oral gavage. Five days later, all poults were killed, and cecal tonsils were aseptically removed for tetrathionate enrichment (24 h at 37 degrees C) followed by selective plating with marker antibiotics. Selected lactose-negative, antibiotic-resistant colonies typical of Salmonella were further confirmed by serogrouping. Treatment-related protection ranged from 0 to 100% in three experiments. Greatest protection was related to the lowest concentrations of the protective microflora in each experiment. These data suggest that effective combinations of competitive enteric microflora can be identified by appropriate in vitro selection methods.