Differential effects of rice bran cultivars to limit Salmonella Typhimurium in chicken cecal in vitro incubations and impact on the cecal microbiome and metabolome.

In this study, rice brans from different cultivars (Calrose, Jasmine, and Red Wells) were assessed for their ability to inhibit Salmonella enterica serovar Typhimurium using an in vitro mixed anaerobic culture system containing cecal microbiota obtained from broilers of different ages. Salmonella Typhimurium was added to controls (feed only, cecal only, and feed + cecal material) and treatments (feed + cecal + different rice brans) and S. Typhimurium populations were enumerated at 0, 24, and 48 h. Two experimental conditions were applied 1) unadapted condition in which S. Typhimurium was added at the beginning of the culture incubation and 2) adapted condition in which S. Typhimurium was added after a 24 hour pre-incubation of the cecal bacteria with the feed and/or rice bran. Among the three rice brans, only Calrose exhibited a rapid inhibition of S. Typhimurium, which decreased to undetectable levels after 24 h under the adapted incubation. Changes in microbiological composition and metabolites by addition of Calrose bran were also investigated with an Illumina MiSeq platform and gas chromatography-mass spectrometry, respectively. Addition of Calrose bran resulted in significant changes including decreased Firmicutes phylum abundance and an increased number of metabolites associated with fatty acid metabolism. In summary, it appears that rice bran from specific rice cultivars may be effective as a means to reduce Salmonella in the chicken ceca. In addition, Calrose rice bran inclusion leads to changes in cecal microbiological composition and metabolite profile.

Original XPCTM Effect on Salmonella Typhimurium and Cecal Microbiota from Three Different Ages of Broiler Chickens When Incubated in an Anaerobic In Vitro Culture System.

Feed supplements are utilized in the poultry industry as a means for improving growth performance and reducing pathogens. The aim of the present study was to evaluate the effects of Diamond V Original XPCTM (XPC, a fermented product generated from yeast cultures) on Salmonella Typhimurium ST 97 along with its potential for modulation of the cecal microbiota by using an anaerobic in vitro mixed culture assay. Cecal slurries obtained from three broiler chickens at each of three sampling ages (14, 28, and 42 days) were generated and exposed to a 24 h pre-incubation period with the various treatments: XPC (1% XPC, ceca, and feeds), CO (ceca only), and NC (negative control) group consisting of ceca and feeds. The XPC, CO, and NC were each challenged with S. Typhimurium and subsequently plated on selective media at 0, 24, and 48 h. Plating results indicated that the XPC treatment significantly reduced the survival of S. Typhimurium at the 24 h plating time point for both the 28 and 42 days bird sampling ages, while S. Typhimurium reduction in the NC appeared to eventually reach the same population survival level at the 48 h plating time point. For microbiome analysis, Trial 1 revealed that XPC, CO, and NC groups exhibited a similar pattern of taxa summary. However, more Bacteroidetes were observed in the CO group at 24 and 48 h. There were no significant differences (P > 0.05) in alpha diversity among samples based on day, hour and treatment. For beta diversity analysis, a pattern shift was observed when samples clustered according to sampling hour. In Trial 2, both XPC and NC groups exhibited the highest Firmicutes level at 0 h but the Bacteroidetes group became dominant at 6 h. Complexity of alpha diversity was increased in the initial contents from older birds and became less complex after 6 h of incubation. Beta diversity analysis was clustered as a function of treatment NC and XPC groups and by individual hours including 6, 12, 24, and 48 h. Overall, addition of XPC influenced microbiome diversity in a similar fashion to the profile of the NC group.

Microbial compositional changes in broiler chicken cecal contents from birds challenged with different Salmonella vaccine candidate strains.

Previously, we constructed and characterized the vaccine efficacy of Salmonella Typhimurium mutant strains in poultry with either inducible mviN expression (PBAD-mviN) or methionine auxotrophy (ΔΔmetRmetD). The aim of the present study was to assess potential impact of these Salmonella vaccine strains on the cecal microbiota using a next generation sequencing (NGS). The cecal microbial community obtained from unvaccinated (group 1) and vaccinated chickens (group 2, vaccinated with PBAD-mviN; group 3, vaccinated with wild type; group 4, vaccinated with ΔΔmetRmetD) were subjected to microbiome sequencing analysis with an Illumina MiSeq platform. The NGS microbiome analysis of chicken ceca revealed considerable changes in microbial composition in the presence of the different vaccine strains and exhibited detectable patterns of distinctive clustering among the respective groups (the R value of unweighted PCoA plot was 0.68). The present study indicates that different S. Typhimurium vaccine strains can differentially influence the microbiota of the ceca in terms of presence but not in the relative abundance of microbiota.

Sodium bisulfate and a sodium bisulfate/tannin mixture decreases pH when added to an in vitro incubated poultry cecal or fecal contents while reducing Salmonella Typhimurium marker strain survival and altering the microbiome.

The objective of the present study was to investigate the ability of animal feed-grade sodium bisulfate (SBS) and a mixture of sodium bisulfate/tannin to inhibit the growth of Salmonella using an anerobic in vitro mixed cecal culture to mimic the conditions within the chicken cecum. An initial inoculum of Salmonella Typhimurium was introduced to an anerobic dilution solution containing 1/3000 diluted cecal bacteria and solids consisting of ground chicken feed and different percentages of solid SBS or SBS/tannin, and surviving organisms were enumerated. Two different experimental designs were employed. In the "unadapted" treatment, the S. Typhimurium was added at the beginning of the culture incubation along with cecal bacteria and chicken feed/SBS or chicken feed/SBS/tannin. In the "adapted" treatment, S. Typhimurium was added after a 24 hour pre-incubation of the cecal bacteria with the chicken feed/SBS or chicken feed/SBS/tannin. Adding SBS resulted in reduction of pH in the cultures which paralleled with the reduction of S. Typhimurium. The SBS alone was found to be inhibitory to S. Typhimurium in the adapted treatment at all concentrations tested (0.25, 0.5, and 0.75%), and the degree of inhibition was concentration-dependent. Salmonella Typhimurium was completely killed in the adapted culture with 0.5% SBS after 24 and 48 h. The SBS/tannin mixture was less inhibitory than SBS alone at the same concentrations in side-by-side comparisons. Testing at a 0.5% SBS concentration, chicken age had little or no effect on log reduction of S. Typhimurium relative to age-matched control cultures without SBS, but age did affect the absolute number of S. Typhimurium surviving, with the greatest decreases occurring at 2 and 4 weeks of age (approx. 103 S. Typhimurium surviving) compared to 6 weeks of age (approx. 105 Salmonella surviving). Microbiome analysis with an Illumina MiSeq platform was conducted to investigate bacterial compositional changes related to the addition of SBS. The relative abundance of Firmicutes (at the phylum level) was decreased, and genera Lactobacillus and Faecalibacterium were increased when SBS was added to the anaerobic mixed culture containing either fecal or cecal material. The antimicrobial action of feed-grade SBS may represent a potential pre-harvest control measure for Salmonella in poultry production.

Reduction of Salmonella Typhimurium by Fermentation Metabolites of Diamond V Original XPC in an In Vitro Anaerobic Mixed Chicken Cecal Culture.

Fermentation metabolites of Diamond V Original XPC™ (XPC), a biological product derived from yeast fermentation, were evaluated for their ability to reduce the Salmonella Typhimurium population using an in vitro mixed anaerobic culture system containing cecal microbiota to simulate chicken hindgut conditions. Four different samples were prepared: anaerobic mixed culture containing (1) feed only, (2) cecal only (ceca were harvested from 42 days old broiler chickens), (3) feed and cecal contents, and (4) feed, cecal contents, and 1% XPC. Two experimental conditions were investigated: Group 1, in which the cecal content was added at the same time as a S. Typhimurium marker strain and Group 2, in which the cecal content was preincubated for 24 h prior to the inoculation with the S. Typhimurium marker strain. The mixed cultures were incubated anaerobically at 37°C, and the S. Typhimurium marker strain was enumerated at 0, 24, and 48 h. Analysis of short chain fatty acids was also conducted for 24 h. In the Group 1 experiment, adding XPC did not exhibit significant reduction of S. Typhimurium. However, the presence of XPC resulted in rapid reduction of S. Typhimurium in Group 2. S. Typhimurium was reduced from 6.81 log10 CFU/ml (0 h) to 3.73 log10 CFU/ml and 1.19 log10 CFU/ml after 24 and 48 h, respectively. These levels were also 2.47 log10 and 2.72 log10 lower than the S. Typhimurium level recovered from the control culture with feed and cecal contents, but without XPC. Based on these results, it appears that the ability of XPC to reduce S. Typhimurium requires the presence of the cecal microbiota. Short chain fatty acid analysis indicated that acetate and butyrate concentrations of cultures containing XPC were twofold greater than the control cultures by 24 h of anaerobic growth. Results from the present study suggest that dietary inclusion of XPC may influence cecal microbiota fermentation and has the potential to reduce Salmonella in the cecum. Implications of these findings suggest that XPC may decrease preharvest levels of Salmonella in broilers and layers.

Applications of In Ovo Technique for the Optimal Development of the Gastrointestinal Tract and the Potential Influence on the Establishment of Its Microbiome in Poultry.

As the current poultry production system stands, there is a period of time when newly hatched chicks are prevented from access to feed for approximately 48-72 h. Research has indicated that this delay in feeding may result in decreased growth performance when compared to chicks that are fed immediately post-hatch. To remedy this issue, in ovo methodology may be applied in order to supply the embryo with additional nutrients prior to hatching and those nutrients will continue to be utilized by the chick post-hatch during the fasting period. Furthermore, in ovo injection of various biologics have been researched based on the ability of not only supplying the chick embryo with additional nutrients that would promote improved growth but also compounds that may benefit the future health of the chicken host. Such compounds include various immunostimulants, live beneficial bacteria, prebiotics, and synbiotics. However, it is important to determine the site and age of the in ovo injection for the most productive effects. The primary focus of the current review is to address these two issues [the most effective site(s) and age(s) of in ovo injection] as well as provide the framework for the development of the gastrointestinal tract (GIT) of the chick embryo. Additionally, recent research suggests the colonization of the microbiota in the developing chick may occur during the late stages of embryogenesis. Therefore, we will also discuss the potentials of the in ovo injection method in establishing a healthy and diverse community of microorganisms to colonize the developing GIT that will provide both protection from pathogen invasion and improvement in growth performance to developing chicks.

An Introduction to the Avian Gut Microbiota and the Effects of Yeast-Based Prebiotic-Type Compounds as Potential Feed Additives.

The poultry industry has been searching for a replacement for antibiotic growth promoters in poultry feed as public concerns over the use of antibiotics and the appearance of antibiotic resistance has become more intense. An ideal replacement would be feed amendments that could eliminate pathogens and disease while retaining economic value via improvements on body weight and feed conversion ratios. Establishing a healthy gut microbiota can have a positive impact on growth and development of both body weight and the immune system of poultry while reducing pathogen invasion and disease. The addition of prebiotics to poultry feed represents one such recognized way to establish a healthy gut microbiota. Prebiotics are feed additives, mainly in the form of specific types of carbohydrates that are indigestible to the host while serving as substrates to select beneficial bacteria and altering the gut microbiota. Beneficial bacteria in the ceca easily ferment commonly studied prebiotics, producing short-chain fatty acids, while pathogenic bacteria and the host are unable to digest their molecular bonds. Prebiotic-like substances are less commonly studied, but show promise in their effects on the prevention of pathogen colonization, improvements on the immune system, and host growth. Inclusion of yeast and yeast derivatives as probiotic and prebiotic-like substances, respectively, in animal feed has demonstrated positive associations with growth performance and modification of gut morphology. This review will aim to link together how such prebiotics and prebiotic-like substances function to influence the native and beneficial microorganisms that result in a diverse and well-developed gut microbiota.

Regulated expression of virulence gene mviN provides protective immunity and colonization control of Salmonella in poultry.

Current live attenuated vaccines for control of Salmonella in poultry persist in the ceca and may persist in the environment. In this paper we report the construction and characterization of the vaccine efficacy of a Salmonella mutant strain with inducible mviN expression and rapid clearance from the host. The mutant was effective in oral immunization of the broiler chicken host against a virulent Salmonella oral challenge strain, having a mean 7×10(6)CFU/g in the ceca of unvaccinated controls compared to a mean 2×10(3)CFU/g in the ceca of vaccinated chickens at 4 weeks post-challenge (6 weeks of age). The mutant strain also demonstrated immunogenicity, reduced organ colonization, and rapid clearance in broiler chickens within 3 weeks of inoculation.