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Much work has been dedicated to identifying members of the microbial gut community that have potential to augment the growth rate of agricultural animals including chickens. Here, we assessed any correlations between the fecal microbiome, a proxy for the gut microbiome, and feed efficiency or weight gain at the pedigree chicken level, the highest tier of the production process. Because selective breeding is conducted at the pedigree level, our aim was to determine if microbiome profiles could be used to predict feed conversion or weight gain in order to improve selective breeding. Using 16s rRNA amplicon sequencing, we profiled the microbiomes of high and low weight gain (WG) birds and good and poor feed efficient (FE) birds in two pedigree lineages of broiler chickens. We also aimed to understand the dynamics of the microbiome with respect to maturation. A time series experiment was conducted, where fecal samples of chickens were collected at 6 points of the rearing process and the microbiome of these samples profiled. We identified OTUs differences at different taxonomic levels in the fecal community between high and low performing birds within each genetic line, indicating a specificity of the microbial community profiles correlated to performance factors. Using machine-learning methods, we built a classification model that could predict feed conversion performance from the fecal microbial community. With respect to maturation, we found that the fecal microbiome is dynamic in early life but stabilizes after 3 weeks of age independent of lineage. Our results indicate that the fecal microbiome profile can be used to predict feed conversion, but not weight gain in these pedigree lines. From the time series experiments, it appears that these predictions can be evaluated as early as 20 days of age. Our data also indicates that there is a genetic factor for the microbiome profile., Competing Interests: The authors have read the journal’s policies and have the following competing interests to declare: Funding for this study was provided by Cobb-Vantress, Inc. Authors RH and RO are commercially affiliated with the funder. The funder provided support in the form of salaries for SDS, and RH and RO, employed by the funder had active roles in the study as stated in the authors’ contributions. This does not alter our adherence to all PLOS ONE policies on sharing data and materials.

When prebiotics and other fermentation substrates are delivered to animals as feed supplements, the typical goal is to improve weight gain and feed conversion. In this work, we examined pasture flock chicken cecal contents using next generation sequencing (NGS) to identify and understand the composition of the microbiome when prebiotics and fermentation substrates were supplemented. We generated 16S rRNA sequencing data for 120 separate cecal samples from groups of chickens receiving one of 3 prebiotics or fiber feed additives. The data indicated that respective feed additives enrich for specific bacterial community members and modulate the diversity of the microbiome. We applied synthetic learning in microbial ecology (SLiME) analysis to interpret 16S rRNA microbial community data and identify specific bacterial operational taxonomic units (OTU) that are predictive of the particular feed additives used in these experiments. The results suggest that feed can influence microbiome composition in a predictable way, and thus diet may have indirect effects on weight gain and feed conversion through the microbiome., (© 2017 Poultry Science Association Inc.)

Previously, we reported the use of quinolones in broiler chickens resulted in residues in retail poultry meat obtained from nine districts in the Santiago Province of the Dominican Republic. Residues in poultry products are a concern due to consumer allergies and the potential to develop antibiotic-resistant bacteria. Given the use of quinolones in poultry production and our previous findings in poultry meat, the objective of this study was to evaluate the presence of quinolone residues in eggs. Samples were collected from 48 different farms located in three of the four municipalities (Moca, Cayetano Germosén, and Jamao) of the Espaíllat Province. Each farm was sampled three times between July and September for a total of 144 samples. Samples were evaluated qualitatively and quantitatively for quinolone residues using the Equinox test. Operation systems (cage or floor), seasonality, and location were considered along with egg-producer sizes that were defined as small scale, <30,000 eggs per day; medium scale, 30,000 to 60,000 eggs per day; or large scale, >60,000 eggs per day. From small-, medium-, and large-scale producers, 69, 50, and 40% of samples were positive for quinolone residues, respectively. A greater number of samples were positive (61%) in floor-laying hen producers compared with those using cages (40%). In the Jamao municipality, 67% of the samples were positive compared with Moca and Cayetano Germosén, where 56 and 25% of samples were positive, respectively. Sampling time had an effect on percent positives: samples collected in July, August, and September were 71, 19, and 63% positive, respectively. Overall, 51% of the samples obtained from eggs produced in the province of Espaíllat were positive for quinolone residues at levels higher than the maximum limits for edible tissue established by the regulatory agencies, including the European Union and U.S. Department of Agriculture. The results obtained from this research confirmed the presence of quinolone residue in eggs, which may present a health risk to some consumers.

Yerba mate (Ilex paraguariensis) is a tea known to have beneficial effects on human health and antimicrobial activity against some foodborne pathogens. Thus, the application of yerba mate as a feed additive for broiler chickens to reduce Salmonella colonization was evaluated. The first in vitro evaluation was conducted by suspending Salmonella Enteritidis and lactic acid bacteria (LAB) in yerba mate extract. The in vivo evaluations were conducted using preventative and horizontal transmission experiments. In all experiments, day-of-hatch chicks were treated with one of the following 1) no treatment (control); 2) ground yerba mate in feed; 3) probiotic treatment (Lactobacillus acidophilus and Pediococcus; 9:1 administered once on day of hatch by gavage); or 4) both yerba mate and probiotic treatments. At d 3, all chicks were challenged with Salmonella Enteritidis (preventative experiment) or 5 of 20 chicks (horizontal transmission experiment). At d 10, all birds were euthanized, weighed, and cecal contents enumerated for Salmonella. For the in vitro evaluation, antimicrobial activity was observed against Salmonella and the same treatment enhanced growth of LAB. For in vivo evaluations, none of the yerba mate treatments significantly reduced Salmonella Enteritidis colonization, whereas the probiotic treatment significantly reduced Salmonella colonization in the horizontal transmission experiment. Yerba mate decreased chicken BW and decreased the performance of the probiotic treatment when used in combination. In conclusion, yerba mate had antimicrobial activity against foodborne pathogens and enhanced the growth of LAB in vitro, but in vivo yerba mate did not decrease Salmonella Enteritidis colonization.

There is a gap in knowledge of microbiological quality in raw chicken products produced by nonconventional methods and no studies have reported the microbiological quality of turkeys produced under different rearing environments. Thus, the aim of this study was to compare the microbiological quality of conventionally and organically reared whole chicken and turkey carcasses purchased from 3 retail outlets in Knoxville, Tenn., U.S.A. A total of 100 raw broiler chickens organically (n = 50) and 50 raw turkey carcasses consisting of 3 brands reared either conventionally (n = 25) or organically (n = 25) were evaluated. The FDA BAM protocol for rinsing poultry carcasses was used to enumerate of aerobic bacteria, Campylobacter, and Staphylococcus spp., and for qualitative analysis of Salmonella. Organic chickens from one brand had the highest average counts of aerobic bacteria, Staphylococcus spp. and Campylobacter (4.8, 4.8, and 4.7 Log10 CFU/mL rinsate, respectively) while the other organic brand had the lowest average counts (3.4, 3.3, and 3.1, respectively) of all 4 brands evaluated. The organic turkeys had the highest average counts of these same bacteria (4, 3.9, and 3.8, respectively) compared to the 2 brands of conventional turkeys evaluated. Salmonella (5% prevalence) was isolated only from organic chickens and turkeys. From these data, it appears that the microbiological quality of the raw product was not dependent on rearing conditions and, thus, it cannot be assumed that organic raw poultry is safer than conventionally raised poultry in terms of microbiological quality., (© 2013 Institute of Food Technologists®)

Pasture-flock-raised poultry are becoming an increasingly popular product, but only limited options are currently available for maintaining gut health. For these producers, prebiotics are an attractive option because they are generally recognized as safe (GRAS) and can be mixed into the feed and thus do not require adjustments to production protocols. However, if prebiotic treatments reduce production performance, they would not be useful to producers. Thus, the objective of this study was to measure performance of pasture-raised broilers fed 1 of 3 prebiotic treatments. For these trials, 2 breeds of birds were used: Naked Neck slow-growing breeds and Cornish White Rock cross fast-growing breeds. The experimental design was replicated for each breed. A total of 340 birds were split into 4 groups, each group fed one feed additive: 1) galactoligosaccharides (2% wt/wt), 2) fructooligosaccharides (1% wt/wt), 3) plum fibers (1% wt/wt), or 4) no additives. During the 8-wk rearing period, 10 birds from each group were collected and euthanized to take small intestine samples. Histological preparations were made from the small intestine tissue, and 4 measurements of villi height and crypt depth from each cross section were taken. Throughout the study, mortality was monitored and BW measurements were taken at 2-wk intervals. For the Cornish White Rock cross, the group receiving the feed supplemented with fructooligosaccharides had higher (P < 0.05) 8-wk BW than those fed Plum; control and birds fed galactoligosaccharides were intermediate. For the Naked Neck breed, the group receiving the plum fibers had the highest final BW. It appears that all 3 feed supplements offered some protective effect for alterations in villi length and crypt depth due to feed withdrawal, but only for the Naked Neck breed. The data indicate the 3 prebiotics utilized in this study could be used without risk of decreasing production performance, but only for Naked Neck breeds.

The main objective of this trial was to set up a series of assays following quantified inoculation of Salmonella samples in 2 initial enrichment methods to ultimately determine the most effective and fastest detection method for recovery of Salmonella in a poultry environment matrix. Samples were randomly split into 2 different containers containing either buffered peptone water (BPW) + yeast extract, or tetrathionate broth (TT) with added iodine and Brilliant Green solution 0.1%. A frozen stock Salmonella culture was thawed and serially diluted 10-fold to inoculate 100 µL of the dilution into the enriched samples. The samples were incubated at 42 and 37°C, respectively, for 24 h and secondarily enriched in modified semi-solid Rappaport Vassiliadis (MSRV) incubated at 42°C. All samples then were reincubated under the same conditions. After secondary enrichment, the samples were streaked onto Chromogenic agar/ XLT4 bi-plates and incubated under the same conditions. After initial inoculation and each 24-h incubation, a portion of the enriched samples was analyzed using a real-time PCR assay. The results of this trial indicate that recovery of Salmonella in a culture-based assay may be enhanced by up to 3 logs by using the TT as the initial enrichment media compared with BPW. The incorporation of MSRV as a secondary cultural selective media after the TT gave the best recovery of Salmonella. These data indicate that considerable time can be saved by using TT as an initial media for Salmonella recovery.

Listeria monocytogenes is a ubiquitous, saprophytic, Gram-positive bacterium and occasional food-borne pathogen, often associated with ready-to-eat meat products. Because of the increased consumer interest in organic, all natural, and free range poultry products, it is important to understand L. monocytogenes in the context of such systems. Pasture-reared poultry were surveyed over the course of two 8-wk rearing periods. Cecal, soil, and grass samples were collected for Listeria isolation and characterization. Seven of 399 cecal samples (or 1.75%) were Listeria-positive. All positive cecal samples were obtained from broilers sampled at 2 wk of age. Grass and soil samples were collected from the pasture both before and after introduction of the poultry. Environmental samples collected after introduction of poultry were significantly more likely to contain Listeria (P < 0.001). The results of analytical profile index Listeria, sigB allelic typing, and hlyA PCR tests found that both L. monocytogenes and L. innocua, including hemolytic L. innocua, were recovered from the cecal and environmental (grass/soil) samples. The sigB allelic typing also revealed that (1) positive samples could be composed of 2 or more allelic types; (2) allelic types found in cecal samples could also be found in the environment; and (3) allelic types could persist through the 2 rearing periods. Our data indicate that both pasture-reared poultry and their environment can be contaminated with L. monocytogenes and hemolytic L. innocua.

Staphylococcus aureus can be carried on the skin and nasal passages of humans and animals as a commensal. A case of human methicillin-resistant S. aureus infection resulting from contact with pork has been reported. Poultry carcasses are sold at retail with the skin intact, but pork and beef typically are not. Thus, the risk of methicillin-resistant S. aureus human infection from whole raw poultry carcasses may be greater than that of exposure from pork or beef. The objective of this study was to isolate and characterize S. aureus from whole retail poultry carcasses and compare the isolates to S. aureus isolates from humans. A total of 25 S. aureus isolates were collected from 222 whole poultry carcasses. The isolates were characterized phenotypically with antibiotic resistance disc diffusion assays and genotypically using multilocus sequence typing. A total of 17 S. aureus isolates obtained from healthy humans were included and characterized in the same way as the poultry isolates. Staphylococcus spp. were recovered from all poultry carcasses. Only 25 poultry carcasses (11.2%) were contaminated with S. aureus. Of these 25 isolates, 36% were resistant to at least one of the antibiotics tested and 20% were resistant to two or more antibiotics tested. However, 100% of the human isolates were resistant to at least one of the antibiotics and 94% were resistant to two or more antibiotics. The results of the multilocus sequence typing indicate that most of the isolates grouped according to source. These results indicate a low prevalence of S. aureus present in poultry, and the isolates were not phenotypically similar to human isolates. The low number of S. aureus isolates from this study indicates that chicken carcasses would appear to not be a significant source of this bacterium.

Salmonella is the major foodborne bacterial pathogen worldwide. Among numerous serotypes, Salmonella Enteritidis (SE) is one of the most common Salmonella serotypes responsible for human infections in the United States. The main source of SE outbreaks is foods associated with raw or undercooked chicken eggs. Salmonella Enteritidis is the only serotype that routinely contaminates eggs. The transovarian transmission of SE and subsequent contamination of the eggs before egg shell formation is considered to be the main route of egg contamination by SE. To evaluate whether invasion of ovarian follicles is an important step during the production of eggs contaminated by SE, we used an in vitro invasion assay to determine ovarian follicle invasion by 5 SE strains. After inoculating the freshly collected ovarian follicles, all 5 SE strains were able to invade into the follicles after 2 h of incubation at 37°C. The mean percentage of SE invasion ranged from 0.016 to 0.034% and no significant difference was found among the SE strains. For Escherichia coli K-12 strain, which was used as a negative control, the mean percentage of invasion was 0.0003%. The in vitro follicle invasion by SE strains demonstrated in this study may reflect the ability of the strains to invade ovarian follicles in laying hens once SE cells reach ovaries through various routes.

Campylobacter is a leading cause of foodborne illness in the United States, and epidemiological evidence indicates poultry products to be a significant source of human Campylobacter infections. Caprylic acid, an eight-carbon medium-chain fatty acid, reduces Campylobacter colonization in chickens. How caprylic acid reduces Campylobacter carriage may be related to changes in intestinal microflora. To evaluate this possibility, cecal microbial populations were evaluated with denaturing gradient gel electrophoresis from market-age broiler chickens fed caprylic acid. In the first trial, chicks (n = 40 per trial) were assigned to four treatment groups (n = 10 birds per treatment group): positive controls (Campylobacter, no caprylic acid), with or without a 12-h feed withdrawal before slaughter; and 0.7% caprylic acid supplemented in feed for the last 3 days of the trial, with or without a 12-h feed withdrawal before slaughter. Treatments were similar for trial 2, except caprylic acid was supplemented for the last 7 days of the trial. At age 14 days, chicks were orally challenged with Campylobacter jejuni, and on day 42, ceca were collected for denaturing gradient gel electrophoresis and Campylobacter analysis. Caprylic acid supplemented for 3 or 7 days at 0.7% reduced Campylobacter compared with the positive controls, except for the 7-day treatment with a 12-h feed withdrawal period. Denaturing gradient gel electrophoresis profiles of the cecal content showed very limited differences in microbial populations. The results of this study indicate that caprylic acid's ability to reduce Campylobacter does not appear to be due to changes in cecal microflora.

In this study, we evaluated the ability of different Campylobacter phenotypes (biofilm versus planktonic) to colonize young poultry. It has been suggested that a persistent Campylobacter biofilm reservoir may be involved in the initial contamination of poultry flocks. Campylobacter jejuni cultured adherent to agar was utilized as the biofilm model and C. jejuni cultured in broth was evaluated as the planktonic model. In 2 independent trials, 1-d-old broiler chicks were given 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). Cecal contents of all birds were evaluated by culturing 12 d after the initial challenge with C. jejuni. In both trials, birds challenged with C. jejuni cultured in broth had approximately 3 to 4 log higher cecal Campylobacter concentration than birds challenged with C. jejuni cultured adherent to agar. Using 2 cell lines (INT 407 and DF1), virulence of C. jejuni cultured in broth versus adherent to agar also was evaluated by challenging monolayers of eukaryotic cells with 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). The virulence study also showed differences of C. jejuni cultured in broth or agar in attachment and invasion abilities to tissue culture cells, but differences were not as consistent as with the chick colonization study. This study indicates that phenotype may play a role in colonization of chickens and virulence by C. jejuni.

In this research we developed a real-time SYBR green assay to detect both Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in a single reaction. A total of 30,000 samples from broiler breeder flocks were screened using traditional serology (plate agglutination, enzyme-linked immunosorbent assay, hemagglutination inhibition) and polymerase chain reaction (PCR; traditional and real-time). It was determined that the real-time SYBR green PCR assay developed in this research was more rapid than all three methods tested and more sensitive and specific than culturing or serology. The SYBR green assay was optimized and could detect as few as 30 template copies of DNA per sample. In addition, the SYBR green assay was less expensive than traditional culturing and serology. MG and MS are infectious bacteria that can rapidly spread and infect commercial chicken flocks. These diseases can cause a significant loss to the poultry industry and especially to broiler breeders because infected flocks are destroyed under the National Poultry Improvement Plan MG and MS clean programs. The real-time SYBR green assay developed in this research has the potential to reduce the time it takes to reach a correct diagnosis and to arrest outbreaks of MG and MS.