Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods.

Given extensive variability in feed composition, the absence of a dedicated DNA extraction kit for poultry feed underscores the need for an optimized extraction technique for reliable downstream sequencing analyses. This study investigates the impact of five DNA extraction techniques: Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA amplification and Illumina MiSeq sequencing were conducted. QIIME2-2021.4 facilitated data analysis, revealing significant diversity and compositional differences influenced by extraction methods. Qiagen exhibited lower evenness and richness compared to other methods. 1-Day Direct and PEG enhanced bacterial diversities by employing bead beating and lysozyme. Despite similar taxonomic resolution, the Qiagen kit provides a rapid, consistent method for assessing poultry feed microbiomes. Modified techniques (MQ and MQC) improve DNA purification, reducing bias in commercial poultry feed samples. PEG and 1-Day Direct methods were effective but may require standardization. Overall, this study underscores the importance of optimized extraction techniques in poultry feed analysis, with potential implications for future standardization of effective methods.

Poultry processing and the application of microbiome mapping.

Chicken is globally one of the most popular food animals. However, it is also one of the major reservoirs for foodborne pathogens, annually resulting in continued morbidity and mortality incidences worldwide. In an effort to reduce the threat of foodborne disease, the poultry industry has implemented a multifaceted antimicrobial program that incorporates not only chemical compounds, but also extensive amounts of water application and pathogen monitoring. Unfortunately, the pathogen detection methods currently used by the poultry industry lack speed, relying on microbiological plate methods and molecular detection systems that take time and lack precision. In many cases, the time to data acquisition can take 12 to 24 h. This is problematic if shorter-term answers are required which is becoming more likely as the public demand for chicken meat is only increasing, leading to new pressures to increase line speed. Therefore, new innovations in detection methods must occur to mitigate the risk of foodborne pathogens that could result from faster slaughter and processing speeds. Future technology will have 2 tracks: rapid methods that are meant to detect pathogens and indicator organisms within a few hours, and long-term methods that use microbiome mapping to evaluate sanitation and antimicrobial efficacy. Together, these methods will provide rapid, comprehensive data capable of being applied in both risk-assessment algorithms and used by management to safeguard the public.

Assessing the microbiomes of scalder and chiller tank waters throughout a typical commercial poultry processing day.

The commercial poultry processing environment plays a significant role in reducing foodborne pathogens and spoilage organisms from poultry products prior to being supplied to consumers. While understanding the microbiological quality of these products is essential, little is known about the microbiota of processing water tanks within the processing plant. Therefore, the goal of this study was to assess the microbiomes of the scalder and chiller tanks during a typical commercial processing d, and determine how bacterial populations, including foodborne pathogens and spoilage organisms, change during the processing day in relation to the bacterial communities as a whole. Additionally, considering this is the first microbiomic analysis of processing tank waters, 2 water sampling methods also were compared. Results of this study show that Proteobacteria and Firmicutes represented over half of the sequences recovered from both tanks at the phylum level, but the microbiomic profiles needed to be analyzed at the genus level to observe more dynamic population shifts. Bacteria known to predominate in the live production environment were found to increase in the scalder tank and gram negative spoilage-related bacteria were found to decrease in the chiller tank throughout the processing day. Directly sampling the scalder water, as compared to analyzing filtered samples, resulted in significantly different microbiomic profiles dominated by Anoxybacillus species. While no sequences related to major foodborne pathogens were found, further sampling collection and processing optimization should provide researchers and the poultry industry a new tool to understand the ecological role of spoilage and pathogenic bacteria within processing tank waters.

Recovery of ammonia from poultry litter using flat gas permeable membranes.

The use of flat gas-permeable membranes was investigated as components of a new process to capture and recover ammonia (NH3) in poultry houses. This process includes the passage of gaseous NH3 through a microporous hydrophobic membrane, capture with a circulating dilute acid on the other side of the membrane, and production of a concentrated ammonium (NH4) salt. Bench- and pilot-scale prototype systems using flat expanded polytetrafluoroethylene (ePTFE) membranes and a sulfuric acid solution consistently reduced headspace NH3 concentrations from 70% to 97% and recovered 88% to 100% of the NH3 volatilized from poultry litter. The potential benefits of this technology include cleaner air inside poultry houses, reduced ventilation costs, and a concentrated liquid ammonium salt that can be used as a plant nutrient solution.

Polymerase chain reaction detection of naturally occurring Campylobacter in commercial broiler chicken embryos.

Campylobacter, a foodborne pathogen closely associated with poultry, is recognized as a leading bacterial etiologic agent of human gastroenteritis in the United States. In this investigation, 2 trials were performed where tissues from 7-, 14/15-, and 19-d-old commercial broiler chicken embryos were tested for the presence of Campylobacter using both culturing methodology and PCR. Conventional culturing methods failed to detect Campylobacter from any samples tested during this investigation. Using a set of primers specific for the Campylobacter flagellinA short variable region (flaA SVR), Campylobacter DNA was amplified in 100, 80, and 100% of gastrointestinal tracts from 7-, 15-, and 19-d-old embryos, respectively, in the first trial. Similarly, Campylobacter DNA was detected in 100, 70, and 60% of gastrointestinal tracts of 7-, 14-, and 18-d-old embryos, respectively, in the second trial. In both trials, yolk sac, albumin, and liver/gallbladder samples from 19-d-old embryos all failed to produce amplicons indicative of Campylobacter DNA. Subsequent DNA sequence analyses of the flaA SVR PCR products were consistent with the amplicon arising from Campylobacter. Although a determination of whether the Campylobacter was living or dead within the embryos could not be made, these results demonstrate that Campylobacter-specific DNA is present within the gastrointestinal tract of broiler chicken embryos; however, the means by which it is present and the relative contribution to subsequent Campylobacter contamination of poultry flocks requires further investigation.

The effect of alum addition on microbial communities in poultry litter.

Alum [Al(2)(SO(4))(3).14H(2)O] is a common poultry litter amendment used to decrease water-soluble phosphorus or reduce ammonia volatilization, or both. Although the physiochemical effects of alum addition have been well researched, little attention has been given to the poultry litter microbial communities. The goal of this study was to use molecular biological methods [denaturing gradient gel electrophoresis (DGGE), community cloning, and quantitative real-time PCR] to characterize general, group-specific and pathogenic microbial communities in alum (10% wt/wt) and non-alum-treated litter. According to quantitative real-time PCR analyses, alum addition to the poultry litter resulted in significant reductions in both Campylobacter jejuni and Escherichia coli concentrations by the end of the first month of the experiment (3 log and 2 log, respectively). The concentrations of Salmonella spp. were below detection (<5 x 10(3) cell.g(-1) of litter) for the entire experiment. The DGGE analyses revealed significant reductions in the Clostridium/Eubacterium and low %GC gram-positive groups in the alum-treated litters by the end of the first month, with no bands detectable for either group after 8 wk of incubation. Conversely, minimal effects of alum addition were observed in the Actinomycetes community. The most significant shift in the microbial community (based on DGGE analyses) occurred in the fungal population, with a large increase in diversity and abundance within 1 mo of alum addition (1 dominant band on d 0 to 9 dominant bands at 4 wk). Specifically, the incidence of Aspergillus spp. increased from 0 to 50% of the sequences in fungal clone libraries (n = 80) over the course of the experiment. This suggests that the addition of alum to poultry litter potentially shifts the microbial populations from bacterially dominated to dominated by fungi. The ramifications of this shift in dominance are still unknown, and future work will be aimed at characterizing these fungi and elucidating their role in the acidified litter environment.

Development of a quantitative real-time polymerase chain reaction assay to target a novel group of ammonia-producing bacteria found in poultry litter.

Ammonia production in poultry houses has serious implications for flock health and performance, nutrient value of poultry litter, and energy costs for running poultry operations. In poultry litter, the conversion of organic N (uric acid and urea) to NH(4)-N is a microbially mediated process. The urease enzyme is responsible for the final step in the conversion of urea to NH(4)-N. Cloning and analysis of 168 urease sequences from extracted genomic DNA from poultry litter samples revealed the presence of a novel, dominant group of ureolytic microbes (representing 90% of the urease clone library). Specific primers and a probe were designed to target this novel poultry litter urease producer (PLUP) group, and a new quantitative real-time PCR assay was developed. The assay allowed for the detection of 10(2) copies of target urease sequences per PCR reaction (approximately 1 x 10(4) cells per gram of poultry litter), and the reaction was linear over 8 orders of magnitude. Our PLUP group was present only in poultry litter and was not present in environmental samples from diverse agricultural settings. This novel PLUP group represented between 0.1 to 3.1% of the total microbial populations (6.0 x 10(6) to 2.4 x 10(8) PLUP cells per gram of litter) from diverse poultry litter types. The PLUP cell concentrations were directly correlated to the total cell concentrations in the poultry litter and were found to be influenced by the physical parameters of the litters (bedding material, moisture content, pH), as well as the NH(4)-N content of the litters, based on principal component analysis. Chemical parameters (organic N, total N, total C) were not found to be influential in the concentrations of our PLUP group in the diverse poultry litters Future applications of this assay could include determining the efficacy of current NH(4)-N-reducing litter amendments or in designing more efficient treatment protocols.

Spatial shifts in microbial population structure within poultry litter associated with physicochemical properties.

Microbial populations within poultry litter have been largely ignored with the exception of potential human or livestock pathogens. A better understanding of the community structure and identity of the microbial populations within poultry litter could aid in the development of management practices that would reduce populations responsible for toxic air emissions and pathogen incidence. In this study, poultry litter air and physical properties were correlated to shifts in microbial community structure as analyzed by principal component analysis (PCA) and measured by denaturing gradient gel electrophoresis (DGGE). Litter samples were taken in a 36-point grid pattern at 5 m across and 12 m down a 146 m x 12.8 m chicken house. At each sample point, physical parameters such as litter moisture, pH, air and litter temperature, and relative humidity were recorded, and samples were taken for molecular analysis. The DGGE analysis showed that the banding pattern of samples from the back and water/feeder areas of poultry house were distinct from those of samples from other areas. There were distinct clusters of banding patterns corresponding to the front, middle front, middle back, back, and waterer/feeder areas. The PCA analysis showed similar cluster patterns, but with more distinct separation of the front and midhouse samples. The PCA analysis also showed that moisture content and litter temperature (accounting for 51.5 and 31.5% of the separation of samples, respectively) play a major role in spatial diversity of microbial community in the poultry house. Based on analysis of DGGE fingerprints and cloned DGGE band sequences, there appear to be differences in the types of microorganisms over the length of the house, which correspond to differences in the physical properties of the litter.

Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells in Dictyostelium discoideum.

A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH3) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.