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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., 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.

Use of mixed crop-livestock farms (MCLFs) is one of the oldest and most traditional farming methods practiced all over the world, and MCLFs are still one of the major systems of food production, particularly for organic foods. On these typically small farms, livestock are reared primarily on grass and naturally grown crops, while composted animal wastes are used to fertilize the soil for growing crops. Specific to organic MCLFs, biosecurity challenges arise from the fact that animals are reared outdoors, which increases potential for contact with disease vectors including wild birds, rodents, and insects. Organic regulations do not allow the use of chemicals and antibiotics; therefore, alternative methods for control of disease and zoonotic pathogens must be used. Due to the biosecurity challenges and the complexity of the MCLF environment, methods for control of zoonotic pathogens need to be carefully considered in order to be effective and to abide by organic regulations if required. The objectives of this study are to define the complex routes of transmission, as well as the prevalence of potential zoonotic and possible interruption strategies of these pathogens among the food animals and crops produced on MCLFs.

The development of antibiotic resistant pathogens has resulted from the use of sub-therapeutic concentrations of antibiotics delivered in poultry feed. Furthermore, there are a number of consumer concerns regarding the use of antibiotics in food animals including residue contamination of poultry products and antibiotic resistant bacterial pathogens. These issues have resulted in recommendations to reduce the use of antibiotics as growth promoters in livestock in the United States. Unlike conventional production, organic systems are not permitted to use antibiotics. Thus, both conventional and organic poultry production need alternative methods to improve growth and performance of poultry. Herbs, spices, and various other plant extracts are being evaluated as alternatives to antibiotics and some do have growth promoting effects, antimicrobial properties, and other health-related benefits. This review aims to provide an overview of herbs, spices, and plant extracts, currently defined as phytobiotics as potential feed additives.

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.

Data indicate that prevalence of specific serovars ofSalmonella entericain human foodborne illness is not correlated with their prevalence in feed. Given that feed is a suboptimal environment forS. enterica, it appears that survival in poultry feed may be an independent factor unrelated to virulence of specific serovars ofSalmonella. Additionally,S. entericaserovars appear to have different host specificity and the ability to cause disease in those hosts is also serovar dependent. These differences among the serovars may be related to gene presence or absence and expression levels of those genes. With a better understanding of serovar specificity, mitigation methods can be implemented to controlSalmonellaat preharvest and postharvest levels.

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., Research funding was awarded to author SCR by the USDA/CSREES (Cooperative State Research, Education, and Extension Service, Washington DC) National Integrated Food Safety Initiative (NIFSI, Washington DC) grant # 406 2008 51110, USDA CREES Food Safety Consortium grant, the Plum Board (Sacramento, CA), and a donation of FOS and GOS prebiotics from GTC Nutrition Co. (Golden, CO).

Background : Probiotic strains of bacteria can prevent Salmonella from causing disease by preventing the pathogen from colonizing the intestines. Two strains of probiotics, Lactobacillus acidophilius and Pediococcus spp, that were obtained from poultry fecal samples have been shown to be efficacious in poultry. The objective of this study was to determine if these strains of probiotics could prevent salmonellosis in a mouse model. Methods : First, both strains of probiotics were evaluated for in vitro efficacy to inhibit the growth of and interfere with virulence gene regulation in Salmonella enterica. For in vivo efficacy, mice was used which models Typhoid illness. Mice were divided into 2 groups: Control and treatment, Lactobacillus and Pediococcus (LP; 108 Log CFU). Two experiments were conducted. In the first experiment, the mice were treated with LP in water for the first two days of the experiment and challenged with Salmonella at day three. In the second experiment, the LP treatment was given in the water for 10 days and challenge was performed on day 11. In both experiments, at day 20 post-challenge, all mice were sacrificed, intestinal tracts and organs removed and cultured for Salmonella. Results : The probiotic strains inhibited the growth of Salmonella and down-regulation of virulence genes was noted, but dependent on the strain of Salmonella being evaluated. For the in vivo experiment, the probiotics did not afford the mice protection from infection and increasing the length of time the probiotics were administered did not improve the efficacy of the probiotics. Conclusions : It appears that these strains of probiotic bacteria are effective against Salmonella in vitro. However, these isolates did not afford protection from Salmonella infection to mice which may be due to host specifity as these isolates were obtained from poultry. Keywords : Salmonella, Probiotic, Lactobacillus, Pediococcus, Mice

To evaluate potential bacterial runoff from poultry litter, litter was applied to test plots and exposed to simulated rainfall 1, 8 or 15 d after litter application. Runoff samples were tested for Salmonella and Campylobacter, two bacterial pathogens commonly associated with poultry, as well as common fecal indicators such as coliforms, enterococci and Escherichia coli. The runoff samples were evaluated from treatments of no litter (control), or the equivalent of 1, 2 or 4 ton/acre of untreated poultry litter. Additionally, runoff samples from treatments of 2 tons/acre of alum-treated litter, 2 tons/acre composted litter and 2 ton/acre deep-stacked litter were compared for bacterial content. Three replicates of the treatments were performed, for a total of 21 test plots. No Campylobacter was isolated from any of the samples and the majority of samples tested negative for Salmonella. Although Salmonella was detected in runoff from many of the plots, it may have originated from sources other than the applied litter (rodents, birds, etc.) since it was detected in two of the unfertilized control plots.

Feed components have low water activity, making bacterial survival difficult. The mechanisms of Salmonella survival in feed and subsequent colonization of poultry are unknown. The purpose of this research was to compare the ability of Salmonella serovars and strains to survive in broiler feed and to evaluate molecular mechanisms associated with survival and colonization by measuring the expression of genes associated with colonization (hilA, invA) and survival via fatty acid synthesis (cfa, fabA, fabB, fabD). Feed was inoculated with 1 of 15 strains of Salmonella enterica consisting of 11 serovars (Typhimurium, Enteriditis, Kentucky, Seftenburg, Heidelberg, Mbandanka, Newport, Bairely, Javiana, Montevideo, and Infantis). To inoculate feed, cultures were suspended in PBS and survival was evaluated by plating samples onto XLT4 agar plates at specific time points (0 h, 4 h, 8 h, 24 h, 4 d, and 7 d). To evaluate gene expression, RNA was extracted from the samples at the specific time points (0, 4, 8, and 24 h) and gene expression measured with real-time PCR. The largest reduction in Salmonella occurred at the first and third sampling time points (4 h and 4 d) with the average reductions being 1.9 and 1.6 log cfu per g, respectively. For the remaining time points (8 h, 24 h, and 7 d), the average reduction was less than 1 log cfu per g (0.6, 0.4, and 0.6, respectively). Most strains upregulated cfa (cyclopropane fatty acid synthesis) within 8 h, which would modify the fluidity of the cell wall to aid in survival. There was a weak negative correlation between survival and virulence gene expression indicating downregulation to focus energy on other gene expression efforts such as survival-related genes. These data indicate the ability of strains to survive over time in poultry feed was strain dependent and that upregulation of cyclopropane fatty acid synthesis and downregulation of virulence genes were associated with a response to desiccation stress.

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. Practical Application Our data support other published studies demonstrating that organic and free-range pasture chickens are not superior in terms of microbiological quality. In addition, our study examined organic turkeys, which has not been previously studied, and also found raw organic turkey meat does not have improved microbiological quality compared to conventionally raised turkeys. Thus, the data collected in this study implies that microbiological quality of raw chicken and turkeys is not a function of production and rearing practices and that raw organic chicken and turkey products should not be considered safer in terms of microbiological quality.

Blackberries contain anthocyanins and phenolic acids derived from anthocyanin fragments that have an important role in the small intestine for retaining gut health. It has been hypothesized that supplementing diets with blackberries may improve gut health through a prebiotic mechanism. Unfortunately identification of gut bacteria is difficult since only 20% of bacterial species can be cultured using current techniques. Therefore, a non-culture based technique was utilized to examine shifts in gut bacteria of mice fed one of five diet treatments: (1) control, (2) aged blackberries supplemented at 5% (w/w), (3) aged blackberries supplemented at 10% (w/w), (4) fresh blackberries supplemented at 5% (w/w), and (5) fresh blackberries supplemented at 10% (w/w). Mice feces were initially collected before supplementing blackberries as a control (0 week) and were collected every 2 weeks for up to 10 weeks. Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-based DGGE) was performed and further identification of bacteria was conducted by excising and sequencing target bands. Banding patterns were more consistent over time in those groups that had a diet supplemented with 10% aged or fresh blackberries compared to the 2 groups fed a diet supplemented with 5% of fresh or aged blackberries. From the sequencing data, Lactobacillus johnsonii was present in all groups after 2 weeks of sampling. The Aerococcus spp. and Actinomycetes were increased in blackberries treatments as well as Enterobacter spp., Klebsiella pneumonia and Erwinia spp. were present in aged blackberry treatment. These data indicate that supplementing a diet with aged or fresh blackberries causes shifts in the gastrointestinal microflora.

Campylobacter jejuni is one of the leading causes of human gastroenteritis. Campylobacter jejuni requires special conditions and media in the laboratory for its growth. In nature, however, this organism is able to survive in very diverse and hostile environments and produce disease in humans and animals. The different mechanisms by which C. jejuni survives stressful conditions in the environment still remain unclear. Stress-adaptation may be one of the factors helping this organism to survive stresses. Some C. jejuni strains have been found to have increased antibiotic resistance in last several years. To determine the effect of acid adaptation on the antibiotic sensitivity profile of C. jejuni, 4 different isolates of C. jejuni (a human isolate and 3 poultry isolates) were exposed to an acid pH of 5.5 and then rechallenged with different stresses. The antibiotic sensitivity profiles of C. jejuni after stress-adaptation were compared with antibiotic sensitivity profiles of non-stressed C. jejuni using the Kirby Bauer agar disc diffusion assay. The antibiotic sensitivity profiles of the C. jejuni isolates used in this study were found to change when the acidadapted bacteria were subjected to further stresses such as an acidic pH of 4.5, aerobic atmosphere and starvation. In the majority of the cases, antibiotic-resistant C. jejuni isolates were found to be more sensitive to antibiotics after stress-adaptation, but in a few cases C. jejuni showed increased resistance. These results indicate that increasing various stresses in a sequential pattern may, in some cases, reduce antibiotic resistance of C. jejuni isolates.

The zoonotic pathogen, Campylobacter, is the leading cause of foodborne illness worldwide. It colonizes the intestinal tract of chickens, its primary host. In chickens, this pathogen is considered commensal and does not cause illness or have adverse impacts on production performance. A number of methods aim at controlling Campylobacter in the farm. This chapter discusses the general features and epidemiology of Campylobacter, improving biosecurity in the farm, antimicrobial therapy and increased host resistance, boosting the immune response, and DNA vaccines for preventing campylobacteriosis. It is concluded that, along with biosecurity, preventative treatments for the birds can act synergistically to significantly reduce the risk of contamination.

Although consumer research indicates that heart health is the main health concern, sales of digestive health products are by far stronger and outsell heart health products. Due to its essential function, the heart is well protected, and the impact that ingested materials can have on the heart is typically limited. However, many indirect factors exist which negatively affect the heart, and we are now learning that the digestive microbiota profile can define health in nearly all the organs of the body, including the heart. This chapter will discuss these direct and indirect factors and how probiotics can possibly improve cardiac health.

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.

Aims: The objectives of this work were to evaluate immunomagnetic beads and a reverse transcriptase (RT)-PCR method for the detection of Salmonella inoculated into feed. In addition, a reverse transcriptase (RT)-PCR method was evaluated for quantifying virulence gene hilA expression of Salmonella ssp. in poultry feed matrices and utilized to determine the influence of poultry feed environmental factors on Salmonella hilA expression. Methods and Results: An immunomagnetic separation technique was evaluated for increased recovery of Salmonella from feed. Salmonella cultures were inoculated into feed samples and exposed to heat treatments of 70°C and sampled periodically. From these samples, RNA was collected and hilA gene expression was measured relative to the housekeeping 16S rRNA gene. The immunomagnetic bead protocol increased recovery by 1 log. The up-regulation of hilA was demonstrated after 5 and 10 min of inoculated feed samples being exposed to heat treatment. Conclusions: From this work, the data indicate that the ability to detect live Salmonella cells in feed samples may be increased by targeting the hilA gene. Significance and Impact of the Study: Foodborne salmonellosis originating from poultry is a major problem, and feed is a leading source of contamination in poultry, but detection in feed is complicated by low concentrations. The assays and experiments in this study examine possible improvements to recovery and detection of Salmonella in feed.

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.

A collection of 125 Salmonella enterica poultry isolates (71 serovar Kentucky isolates, and the remainder belonging to serovars Alachua, Enteritidis, Hadar, Heidelberg, Montevideo, Mbandaka, Senftenberg, Typhimurium, and Worthington) were tested for the ability to grow on tryptic soy agar containing sodium arsenite [As(III)] or arsenate [As(V)]. All serovar Kentucky isolates and 18 of the non-Kentucky isolates were able to grow in the presence of 0.1 mM As(III), and 69 grew in the presence of 1 mM As(V). Thirty of the non-Kentucky isolates did not grow at these As(III) and As(V) concentrations, but seven grew at 1 mM As(III) and 10 mM As(V). PCR-based analysis demonstrated the presence of arsB and arsD sequences in all Kentucky isolates, whereas one or both of these sequences were present in only 30 of the other isolates. It remains to be determined if these arsenic-resistance determinants benefit Salmonella exposed to man-made arsenic-containing compounds in poultry environments.

Aims: While considerable foodborne pathogen research has been conducted on conventionally produced broilers and turkeys, few studies have focused on free-range (organic) or pastured poultry. The current surveillance study was designed to isolate, identify and genetically characterize Salmonella from pastured poultry farm environment and from retail samples. Methods and Results: In this study, 59 isolates were collected from two pastured poultry farms (n = 164; pens, feed, water and insect traps) and retail carcasses (n = 36) from a local natural foods store and a local processing plant. All isolates were serotyped and analysed phenotypically (antimicrobial resistance profiles) and genotypically (DNA fingerprints, plasmid profiles and integron analysis). Salmonella enterica was detected using standard microbiological methods. Salmonella Kentucky was the most prevalent serotype detected from the sampled sources (53%), followed by Salmonella Enteritidis (24%), Bareilly (10%), Mbandaka (7%), Montevideo (5%) or Newport (2%). All isolates were resistant to sulfisoxazole and novobiocin, and the majority (40/59) possessed class I integrons shown by PCR detection. Each Salmonella serotype elicited a distinct pulsed-field gel electrophoresis fingerprint profile, and unique differences were observed among the serotypes. Conclusions: The findings of this study show that Salmonella serotypes isolated from pasture-raised poultry exhibit antimicrobial resistance and class I integrons. Significance and Impact of the Study: This study demonstrates that despite the cessation of antibiotic usage in poultry production, antibiotic resistant Salmonella may still be recovered from the environment and poultry products.

Post chill whole poultry carcasses from a commercial processing plant were stored in a processing combo at room temperature (70 °F/21 °C) for 54 h to mimic the scenario of temperature abuse before further processing. Temperature data were collected in 1-min intervals and averaged each hour by 9 temperature data loggers. Two linear regressions were developed for the combo and internal breast temperature and slopes were nearly identical. Microbial data was collected by performing whole bird carcass rinses that were enumerated for aerobic plate count (APC), Enterobacteriaceae, Escherichia coli, and total coliform. Samples were collected from the chiller chute at time zero for initial bacterial counts. Carcass sampling continued once the internal breast temperature achieved 45 °F (7 °C 10 h) and continued every 2 h until the final internal breast temperature was 63 °F (17 °C 54 h). Linear regressions were developed for the first 26 h, which exhibited no statistically significant growth except for Enterobacteriaceae. A 2nd linear regression (28 to 54 h) exhibited significant growth for all analyses. Overall, APC increased from a log10 colony forming unit (CFU)/mL count of 2.86 to 7.02, Enterobacteriaceae increased from 0.66 to 6.64, coliform increased from 0.72 to 4.81, and E. coli increased from 0.53 to 4.45. Denaturing gradient gel electrophoresis was performed to detect changes in the bacterial populations, which indicated 95% similarity within sampled groups, but the overall percent similarity among samples collected over 54 h was 8%. From the data, microbial growth demonstrates a period of 26 h for minimal growth; therefore, the product could be further processed rather than designated as waste.

UNLABELLED The growing interest in organic and natural foods warrants a greater need for information on the food safety of these products. In this study, samples were taken from 2 pasture flock farms (N = 178; feed, water, drag swabs, and insect traps), pasture flock retail carcasses (N = 48) and 1 pasture flock processing facility (N = 16) over a period of 8 mo. A total of 105 Campylobacter isolates were obtained from 53 (30%), 36 (75%), and 16 (100%) samples from the farms, retail carcasses, and processing facility, respectively. Of the 105 isolates collected, 65 were C. jejuni, 31 were C. coli, and 9 were other Campylobacter spp. Using PCR, the C. jejuni isolates were further analyzed for virulence genes involved in colonization and survival (flaA, flaC, cadF, dnaJ, racR, cbrR), invasion (virB11, ciaB, pldA), protection against harsh conditions (sodB, htrA, clpA), toxin production (cdtA, cdtB, cdtC), siderophore transport (ceuE), and ganglioside mimicry (wlaN). In addition, the short variable region of the flaA locus (flaA SVR) was sequenced to determine the genetic diversity of the C. jejuni isolates. The flaA SVR diversity indices increased along the farm to carcass continuum. PCR-based analysis indicated a low prevalence of 5 genes involved in colonization (dnaJ, ciaB, pldA, racR, virB11). The results of this survey indicate that the prevalence of Campylobacter on organic retail carcasses is similar to prevalence reports of Campylobacter on conventional retail carcasses. However, the genetic diversity of the flaA SVR genotypes increased along the farm to carcass continuum that contrasted with conventional poultry studies. PRACTICAL APPLICATION Campylobacter jejuni is a leading cause of foodborne illness with poultry and poultry products being leading sources of infection. Free-range and pasture flock chickens are becoming more popular; however, there is an inherent biosecurity risk that can increase the prevalence of foodborne pathogens in these flocks. This study aimed to determine sources and characterize C. jejuni isolated from pasture flocks.

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.

Pseudomonas genus-specific primers targeting the 16s rRNA gene were used in a real-time polymerase chain reaction (PCR) assay for rapid analysis of Pseudomonas isolated from retail chicken carcasses. A multiplex PCR assay was also designed using specific primers targeting the gyrase B sub-unit gene to rapidly distinguish between several species of poultry significant Pseudomonads. The assays were used to evaluate the species and level of spoilage Pseudomonads on raw chicken carcasses over an 8-day storage period. No Pseudomonas were detected on the chicken carcasses until 4 days after storage using culturing and plating techniques, but the PCR-based assays developed in this research were more sensitive and detected Pseudomonas in carcass rinses performed immediately after processing. With the multiplex PCR assay, it was determined that most of the Pseudomonas spoilage was because of Pseudomonas fluorescens and Pseudomonas fragi. PRACTICAL APPLICATIONS Economic losses as a result of spoilage are estimated between 5 and 17 billion dollars annually. In the poultry industry, the Pseudomonads not only cause the majority of this spoilage, but are also primary biofilm formers which may harbor and spread pathogens. Currently, only biochemical assays are available in the food industry for detection and differentiation of Pseudomonads. However, biochemical assays require 5 days for results, are limited to detection of a few species, and often produce erroneous results. The PCR assays developed in this work can reduce detection time to a few hours. Furthermore, these nucleic acid-based assays have the potential to simplify the identification process, reduce food spoilage and foodborne illness, and speed the diagnosis of ill birds.

Salmonella is a leading cause of foodborne illness in the United States, with poultry and poultry products being a primary source of infection to humans. Poultry may carry some Salmonella serovars without any signs or symptoms of disease and without causing any adverse effects to the health of the bird. Salmonella may be introduced to a flock by multiple environmental sources, but poultry feed is suspected to be a leading source. Detecting Salmonella in feed can be challenging because low levels of the bacteria may not be recovered using traditional culturing techniques. Numerous detection methodologies have been examined over the years for quantifying Salmonella in feeds and many have proven to be effective for Salmonella isolation and detection in a variety of feeds. However, given the potential need for increased detection sensitivity, molecular detection technologies may the best candidate for developing rapid sensitive methods for identifying small numbers of Salmonella in the background of large volumes of feed. Several studies have been done using polymerase chain reaction (PCR) assays and commercial kits to detect Salmonella spp. in a wide variety of feed sources. In addition, DNA array technology has recently been utilized to track the dissemination of a specific Salmonella serotype in feed mills. This review will discuss the processing of feeds and potential points in the process that may introduce Salmonella contamination to the feed. Detection methods currently used and the need for advances in these methods also will be discussed. Finally, implementation of rapid detection for optimizing control methods to prevent and remove any Salmonella contamination of feeds will be considered.

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.

Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis, but little is known concerning how this fastidious bacterium survives stressful conditions. C. jejuni strains were exposed to acid conditions (pH = 4.5), acid conditions and aerobic atmosphere, or starvation stress, allowed to recover, and again challenged with acid stress. The counts of acid-adapted, acid- and aerobic-adapted, and starvation-adapted cells that survived further acid stress were higher than those of nonstressed cells in some strains, but adaptation and survival was time dependent. Expression of the virulence gene cadF was up-regulated by starvation stress while cdtB and ciaB were down-regulated. The proteomic study revealed that six proteins were synthesized due to starvation stress and eight proteins appeared to be absent. These results indicate C. jejuni surviving stress can increase chances of surviving further stress such as passage through the human gastrointestinal tract and stress may be a significant factor in inducing some virulence genes. PRACTICAL APPLICATIONS The mechanisms of survival of Campylobacter jejuni outside the host are not fully understood. This research identifies adaptive tolerance responses (ATRs) as a mechanism of survival. Understanding how sub-lethal treatments and stress may further promote the survival of C. jejuni is necessary for evaluating food preservation methods because some methods may have unwanted effects on bacterial virulence, such as inducing virulence gene expression. This research determined a correlation between virulence and stress in C. jejuni that could have an impact on food preservation methods chosen, disease outcome and subsequent treatment options. The molecular mechanisms of disease by C. jejuni are still not fully understood, but by studying the survival of C. jejuni in response to specific stresses, insight may be gained into mechanisms of colonization and virulence in the human host. Understanding mechanisms of survival by studying global response to stress is an effective way to develop methods of control and targets for therapeutic medicines.

The cytolethal distending toxin (CDT) and ability to invade cells are known virulence factors of Campylobacter jejuni. The cdtB subunit was sequenced in isolates obtained from humans (n = 19), chickens (n = 50), and turkeys (n = 22) to determine if pathogenic C. jejuni could be detected using the cdtB gene. Most of the C. jejuni isolates (87%) were found to carry the cdtB gene with a highly conserved sequence. However, 4 human, 5 chicken, and 2 turkey isolates had mutations resulting in sequences being 30% or less homologous to the consensus sequence. Some of these mutations had minor effects on cytotoxicity and isolates still were able to kill 18–67% of HeLa cells. In this study, there was no correlation in invasion abilities and cytotoxicity. The results indicated C. jejuni may utilize more than one mechanism in causing disease and pathogenicity but is strain dependent. Also, the utility of the cdtB as an indicator of pathogenicity is doubtful. PRACTICAL APPLICATIONS Campylobacter jejuni is a leading cause of foodborne illness in the U.S.A. and creates a 1.2 billion dollar annual burden. Being able to detect and distinguish pathogenic from nonpathogenic strains is important for assessing the risk to consumers of contracting campylobacteriosis. Understanding which pathogenicity factors are important in causing disease can give information to develop targets for detecting pathogenic bacteria, intervention strategies and therapeutic medicines. This work points out that the ability of C. jejuni to cause disease varies by strain and one or more mechanisms may be used in causing disease. The results of this study indicate multiple targets may be necessary for designing a molecular assay used to detect pathogenic strains of C. jejuni. In order to reduce C. jejuni on poultry products, the incidences of campylobacteriosis and consequent economic burden, detection and intervention methods need to be developed.

The most recent outbreaks of listeriosis have been traced back to contaminated ready-to-eat (RTE) poultry and meat products. However, Listeria monocytogenes can be isolated from every food group, including fresh vegetables. This review is focused on one of the most popular RTE vegetable products, precut prepackaged lettuce. The available literature concerning Listeria contamination of vegetables is reviewed, and possible reasons why no recent outbreaks or sporadic cases of listeriosis due to contaminated precut prepackaged lettuce are explored.

Due to the significance of the microbiome on human health, much of the current data available regarding microbiome functionality is centered on human medicine. For agriculturally important taxa, the functionality of gastrointestinal bacteria has been studied with the primary goals of improving animal health and production performance. With respect to cattle, the digestive functions of bacteria in cattle are unarguably critical to digestion and positively impact production performance. Conversely, some research suggests that the gastrointestinal microbiome in chickens competes with the host for nutrients and produces toxins that can harm the host resulting in decreased growth efficiency. Concerning many other species including reptiles and cetaceans, some cataloging of fecal bacteria has been conducted, but the functionality within the host remains ambiguous. These taxa could provide interesting gastrointestinal insight into functionality and symbiosis considering the extreme feeding regimes (snakes), highly specialized diets (vampire bats), and living environments (polar bears), which warrants further exploration.

Campylobacter is one of the leading causes of bacterial foodborne illness in the United States and control of this pathogen has proven difficult due to its ability to colonize and spread quickly through a poultry flock. This chapter will discuss Campylobacter from its discovery to the current understanding of virulence, resistance, and epidemiological tracking methods. It also examines the role poultry production plays in human infection and how it influences the genome of Campylobacter . Resistance phenotypic and genotypic traits of Campylobacter gained by the usage of antibiotics and coccidiostats in poultry production are also discussed.

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.

The aim of this study was to determine the influence of acid-adaptation on the survival as well as adhesion and invasion of human intestinal cells by nine Campylobacter jejuni strains after exposure to different stress conditions. Acid-adapted and nonadapted C. jejuni were exposed to different secondary stress conditions such as acid (pH 4.5), starvation (phosphate-buffered saline, pH 7.2), or salt (3% wt/vol NaCl). After exposure to the secondary stress, the adhesion and invasion abilities of the strains were evaluated in vitro in tissue culture using the human intestinal cell line INT 407. The survival rates of acid-adapted cells of some strains of C. jejuni exposed to different secondary stresses were found to be significantly higher than the non-acid-adapted cells. Similarly, some strains also showed an increase in adhesion and invasion (p

The era of molecular biology and automation of the Sanger chain-terminator sequencing method has led to discovery and advances in diagnostics and biotechnology. The Sanger methodology dominated research for over 2 decades, leading to significant accomplishments and technological improvements in DNA sequencing. Next-generation high-throughput sequencing (HT-NGS) technologies were developed subsequently to overcome the limitations of this first generation technology that include higher speed, less labor, and lowered cost. Various platforms developed include sequencing-by-synthesis 454 Life Sciences, Illumina (Solexa) sequencing, SOLiD sequencing (among others), and the Ion Torrent semiconductor sequencing technologies that use different detection principles. As technology advances, progress made toward third generation sequencing technologies are being reported, which include Nanopore Sequencing and real-time monitoring of PCR activity through fluorescent resonant energy transfer. The advantages of these technologies include scalability, simplicity, with increasing DNA polymerase performance and yields, being less error prone, and even more economically feasible with the eventual goal of obtaining real-time results. These technologies can be directly applied to improve poultry production and enhance food safety. For example, sequence-based (determination of the gut microbial community, genes for metabolic pathways, or presence of plasmids) and function-based (screening for function such as antibiotic resistance, or vitamin production) metagenomic analysis can be carried out. Gut microbialflora/communities of poultry can be sequenced to determine the changes that affect health and disease along with efficacy of methods to control pathogenic growth. Thus, the purpose of this review is to provide an overview of the principles of these current technologies and their potential application to improve poultry production and food safety as well as public health.

Campylobacter jejuni is a leading cause of foodborne illness, with poultry and poultry products being leading sources of infection. Epidemiological efforts to trace Campylobacter can be challenging because of the extreme genetic diversity of this bacterium relative to other foodborne pathogens. To enhance tracking and epidemiological efforts, whole-genome sequencing has been used for other foodborne pathogens but not yet been evaluated for practicality with Campylobacter. Thus, the purpose of this study was to evaluate whole-genome sequencing as a genotyping method for C. jejuni by comparing it with 2 commonly used genotyping methods, namely pulsed-field gel electrophoresis (PFGE) and flaA typing. Whole-genome sequence data were generated using the Roche-454 sequencing platform to map Campylobacter strains (VOL_3, VOL_5, VOL_8, VOL_11, and VOL_20) isolated from conventional and organic poultry. Five additional isolates with published genomes were also compared. The PFGE profiles were created using Sma I digestion. For the flaA short variable region sequencing, standard PCR methods were used and high-quality Sanger reads were generated. The PFGE profiles of strains VOL_3 and VOL_11 were found to be indistinguishable, and strain VOL_20 was found indistinguishable from NCTC 11168. Whole-genome comparisons between strains VOL_20 and 11168 were in agreement with the obtained PFGE profiles, as these 2 isolates had very similar genome sizes, a number of shared genes (1,580), and very similar % G-C content (30.6). Of the 8 strains, 2 strains (VOL_3 and VOL_11) had identical flaA types. Whole-genome sequencing was the most discriminatory of the typing methods. However, the cost and time effort needed to sequence and assemble the genomes may hinder efforts, and therefore, we conclude that more bioinformatics tools need to be developed for whole-genome sequencing to be used as an epidemiological tool.

There is a need to develop and implement more economical delivery approaches for multiple-hurdle antimicrobial interventions that can be applied to food matrices such as retail meats. Recently, potential opportunities have emerged to use nanoscience and nanoengineering principles to develop antimicrobial carriers for controlling the major foodborne pathogens such as Salmonella in meat and food preservation systems. The overall goal of this review is to explore the potential of nanoparticle-based composite systems for practical and economical antimicrobial interventions to inhibit and decontaminate such pathogens on cooked ready-to-eat (RTE) poultry and red-meat products. The opportunities for specific systems such as chitosan-nanoparticle-based nanocomposite systems containing ɛ-polylysine peptide dispersed in organic acids are discussed. A second focus of this chapter is the potential health hazards that arise from the use of nanoparticles.

Salmonella enterica is the leading cause of foodborne illness with poultry and poultry products being primary sources of infection. The 2 most common S. enterica serovars associated with human infection are Typhimurium and Enteritidis. However, Kentucky and Heidelburg and the 2 most prevalent serovars isolated from poultry environments. Given the prevalence of other serovars in poultry products and environments, research is needed to understand virulence modulation in response to stress in serovars other than Typhimurium and Enteritidis. Thus, the objective of this research was to compare hilA gene expression (a master regulator of the virulence pathogenicity island) in response to acid stress among different strains and serovars of Salmonella. A total of 11 serovars consisting of 15 strains of S. enterica were utilized for these experiments. Cultures were suspended in tryptic soy broth (TSB) adjusted to pH 7.2, 6.2, or 5.5 with HCl or acetic acid. Total RNA was extracted from cultures at specific time points (0, 2, 4, and 24 h). Gene expression of hilA was measured with quantitative reverse transcriptase real time PCR (qRT-PCR). Growth and pH were measured throughout the 24 h time frame. Regulation of hilA in response to acid stress varied by serovar and strain and type of acid. The results of these experiments indicate that hilA regulation may have some impact on virulence and colonization of S. enterica. However, these results warrant further research to more fully understand the significance of hilA regulation in response to mild acid stress in S. enterica.

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 (P0.05) 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.

Although wild ruminants have been identified as reservoirs of Shiga-toxin producing Escherichia coli (STEC), little information is available concerning the role of Salmonella spp. and Campylobacter spp. in large game species. We evaluated the presence of these pathogens in faeces (N=574) and carcasses (N=585) sampled from red deer (N=295), wild boar (N=333) and other ungulates (fallow deer, mouflon) (N=9). Animal sampling was done in situ from 33 hunting estates during two hunting seasons. Salmonella spp. and Campylobacter spp. strains associated with human campylobacteriosis were infrequently detected indicating that both pathogens had a limited zoonotic risk in our study area. The overall STEC prevalence in animals was 21% (134/637), being significantly higher in faeces from red deer (90 out of 264). A total of 58 isolates were serotyped. Serotypes O146:H- and O27:H30 were the most frequent in red deer and the majority of isolates from red deer and wild boar were from serotypes previously found in STEC strains associated with human infection, including the serotype O157:H7. The STEC prevalence in red deer faeces was significantly higher with the presence of livestock (p, This study was partially supported by a grant from the Instituto Nacional de Investigación y Tecnología Agraria and Ministerio de Ciencia e Innovación (FAU2008-00021-C03), by Red Española de Investigación en Patología Infecciosa (REIPI RD06/0008/1016-1018) and grants PS09/01273 (Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, Gobierno de España), AGL-2008-02129 (Ministerio de Ciencia e Innovación, Gobierno de España) and 09TAL007261PR, 10MRU261023PR and 2007/000044-0 (Xunta de Galicia and The European Regional Development Fund, ERDF). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for his fellowship (09/02-C). S. Díaz-Sánchez acknowledges the Junta de Comunidades de Castilla-La Mancha for her PhD research grant (AG/07). D. Vidal acknowledges the Consejo Superior de Investigaciones Científicas for her JAE-Doc contract. A. Mora acknowledges the Ramón y Cajal programme from the Spanish Ministerio de Ciencia e Innovación.

Mycoplasmas sp. are prokaryote pseudo bacteria that lack a cell wall but have a cell membrane. The name Mycoplasma is derived from this characteristic, molli meaning “soft” and cute meaning “skin”. Mycoplasmas are taxonomically placed in the Class Mollicutes, Order Mycoplasmatales, and Family Mycoplasmataceae. This genus is distinguished from the other genera in the family by a growth requirement for cholesterol and an inability to hydrolyze urea. Members of the genus have a small genome (580 to 1350 Kb) and relatively low G+C % content (Papaszi et al. 2003). The small genome size is clearly reflected by the reduced metabolic capabilities of Mycoplasmas. Mycoplasmas lack pathways for cell wall production and biosynthesis of purines and also lack a functional tricarboxylic acid (TCA) cycle and a cytochromemediated electron transport–chain system. These organisms must obtain many of the necessary nutrients needed to sustain the organism from the environment. For this reason, Mycoplasmas are obligate parasites. This characteristic is also reflected in the ideal culturing temperature (370C) the same body temperature as that of humans and many animals.

A total of 142 samples of game meat and ready-to-eat meat products from red deer and wild boar were analysed in order to assess the presence of Shiga toxin-producing Escherichia coli (STEC). Shiga-toxin encoding genes (stx genes) were detected by PCR in 36 (25.4%) of the samples and STEC was isolated from 8 (5.6%) of the same samples. None of the samples tested positive for E. coli O157:H7. Four different serotypes were found among the 8 STEC isolates, with serotype O27:H30 being predominant (62.5%, 5/8). The PCR assay indicated the presence of the stx2 gene in all of the STEC isolates and further subtyping resulted in detection of three different subtypes: stx2a, stx2b and stx2g. The only stx1-positive isolate was further subtyped as stx1c. The ehxA gene was detected in 3 (37.5%) of the isolates and none of them contained the eae gene. All STEC isolates were sensitive to the 13 antibiotics tested. Some isolates possessed serotypes and virulence gene profiles previously associated with STEC infections in humans. The isolation of a STEC strain carrying the stx2. a subtype from a ready-to-eat meat product from deer suggests the role of these products as a potential source of STEC infections in humans., This study was supported by a grant from the Instituto Nacional de Investigación y Tecnología Agraria and Ministerio de Ciencia e Innovación (FAU2008-00021-C03). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for this fellowship (09/02-C). S. Díaz-Sánchez acknowledges the Junta de Comunidades de Castilla-La Mancha for her PhD research grant (AG/07). D. Vidal acknowledges the Consejo Superior de Investigaciones Científicas for her JAE-Doc contract.

Considering that most commercial poultry operations house up to 30,000 birds in one house, additives in feed are the most efficient means for delivering treatments, improving health and treating diseases. Typically, antibiotics used in feed are not specifically for therapeutic purposes but rather for growth promotion and improvement of poultry gut health. There is concern that this practice will ultimately result in an increase in antibiotic-resistant bacteria that may be difficult to treat. This chapter discusses the use of antimicrobials in feed, primarily poultry feed, and the benefits of antibiotic usage. The disadvantages, including the development of antibiotic-resistant bacteria, will also be addressed.