Location: Egg and Poultry Production Safety Research Unit
2024 Annual Report
Objectives
1. Identify factors within the hatchery and brooder phase that induce serotype diversity and homologous recombination within Salmonella enterica subspecies I for the purpose of facilitating reduction of colonization of chicks through environmental remediation.
1.A. Investigate Salmonella ecology within commercial hatchery environments through the use of bio-mapping.
1.B. Identify conditions that facilitate either Homologous Recombination (HR)
and/or Clonal Expansion (CX) by Salmonella enterica within the hatchery environment.
1.C. Develop intervention strategies for impeding emergence of new serotypes of
Salmonella enterica in the hatchery and brooder environments.
2. Identification, characterization, and application of probiotic commensal microbes as an alternative to antibiotics to reduce Salmonella prevalence within commercial poultry houses.
2.A. Identification of non-pathogenic bacterial species that are modulated upon
Salmonella Heidelberg infection.
2.B. Evaluate and characterize potential non-pathogenic strains to be included in the proLitterbiotic (pLb) culture collection.
2.C. Evaluate efficacy of proLitterbiotic (pLb) under live production scenarios.
3. Identify environmental and management drivers of foodborne pathogen ecology under pastured poultry rearing systems.
3.A. Environmental and microbiological characterization of pastured poultry farms to identify drivers of foodborne pathogen ecology.
3.B. Predict Salmonella prevalence during live production within pastured poultry flocks.
3.C. Evaluate the effectiveness of implementing probiotic/all-natural products
within the diet of very young chicks (<1 week of age) on poultry gut health and
product safety.
4. Determine pre-harvest environmental and management factors that drive the persistence of zoonotic bacterial pathogens within commercial-scale poultry production houses.
4.A. Assess the effect of pre-harvest environmental conditions and management practices on the identification, prevalence, and characterization of pathogens during live production.
4.B. Develop analytical models to predict the environmental drivers of pathogen prevalence and persistence within live poultry production systems to improve stakeholder pre-harvest data utilization and implementation.
Approach
Our goal is to reduce pathogenic and antibiotic resistant Salmonella in eggs and poultry products entering the processing/post-harvest environment by generating research that identifies the drivers of Salmonella ecology in pre-harvest environments. This investigation will begin at the nexus of commercial poultry management (hatchery) and extend onto the farms where the birds are reared to processing weight, and we will investigate the variables that genotypically and phenotypically affect the presence of Salmonella pre-harvest. A better understanding of Salmonella ecology and diversity through the pre-harvest phase of poultry production will reduce Salmonella loads entering the processing environment and result in a safer product for the consumer. We will test alternative hypotheses about which pre-harvest environmental factors and management practices influence genomic (clonality, diversity) and phenomic (growth potential, antibiotic resistance) attributes of Salmonella. We will develop and test a “proLitterbiotic” culture to evaluate its efficiency to reduce the development of multidrug resistant Salmonella in live broiler chickens. Live production studies will use pastured poultry farms as a model for poultry management as we have access to working and experimental pastured poultry farms for more controlled research experiments. Expected outcomes for regulatory agencies, the conventional and pastured poultry industries and the consumer include: i) data-supported approaches for identifying risks associated with contamination of poultry entering the processing/post-harvest environments; ii) tools that facilitate characterization of Salmonella serovars and how mixtures correlate to epidemiological trends; iii) new approaches to interventions intended to disrupt the ability of Salmonella to maintain an optimized genome; iv) correlation of genomic markers to antimicrobial resistances present between and within Salmonella serovars within pre-harvest environments; and v) identification of best pre-harvest practices and alternatives to antibiotics that will help producers reduce foodborne pathogens in consumer products. Levels of pathogens associated with poultry will be determined based on environmental conditions (e.g. dust, moisture, temperature, etc.) during commercial-scale poultry production. These isolated pathogens will then be further identified and characterized to evaluate microbial factors that influence their persistence with live production systems. Optimal environmental factors will be identified with the purpose of minimizing the presence and transmission of pathogens within commercial-scale poultry production houses. Microbiological, physiochemical, and management data from live poultry productions systems studies will be utilized as the variable data into appropriate multivariate predictive or machine learning/deep learning based algorithims. These models will be used to predict the variables that are the most influential drivers of zoonotic bacterial prevalence, persistence, and diversity within pre-harvest poultry live production and enable stakeholders to develop decision support tools to improve poultry food safety.
Progress Report
Determination of the efficacy of Bacillus velezensis for the reduction of antibiotic resistant foodborne pathogens. ARS scientists in Athens, Georgia, determined if a probiotic composed of four B. velezensis (BV) strains can reduce the population of Salmonella Heidelberg and multidrug resistant (MDR) E. coli strains. Ninety microcosms holding reused litter top-dressed with pine shavings were assigned to one of six treatments: low litter moisture (17.5%) + BV + S. Heidelberg + E. coli (T1), high litter moisture (40%) + BV + S. Heidelberg + E. coli (T2), low litter moisture (17.5%) + S. Heidelberg + E. coli (T3), high litter moisture (40%) + S. Heidelberg + E. coli (T4), low litter moisture (17.5%) + No bacteria added (T5), and high litter moisture (40%) + No bacteria added (T6). BV was applied at a rate of 2.7 x 109 cells/ft2 to T1 and T2 microcosms, while other treatments received equal amounts of sterile phosphate buffered saline.
Afterwards, microcosms were incubated in a growth chamber for three days (37°C and 65% relative humidity) before pathogen inoculation. A culture cocktail of three S. Heidelberg and three E. coli strains were inoculated into T1, T2, T3 and T4 microcosms before incubation continued for an added 21 days. Under low litter moisture, microcosms amended with BV (T1) harbored lower Salmonella (log10 1.17±0.01) and E. coli (log10 0.75±0.0006) abundance than unamended microcosms (T3) (Salmonella; log10 2.26±1.56, E. coli; log10 0.95 ± 0.34) after 21 days. Likewise, under high litter moisture, microcosms amended with BV (T2) carried lower levels of Salmonella (log10 3.72±0.67) and E. coli (log10 1.73±0.58) compared to unamended microcosms (Salmonella; log10 4.71±1.84, E. coli; log10 3.21 ± 2.53). The results indicate that amending litter with probiotics can reduce S. Heidelberg and MDR E. coli populations; however, litter moisture may influence probiotic efficacy.
Pastured poultry field trials to determine the effect of broiler breed on performance, gut microbiology, physiology, food safety, and welfare. ARS researchers in Athens, Georgia, working in collaboration with scientists at the University of Georgia and a local pastured poultry farmer are utilizing automated broiler coops in field trials to perform a systems-based comparison of the two major pastured poultry breeds (fast growing Cornish Cross and the slow growing Ranger) on the pre-harvest gut microbiology and the subsequent effects on performance, physiology, and welfare. For gut microbiology/physiology assessments, broilers are being sampled twice during brood (days 0 and 7), and three times during pasture grow-out (within days of being on pasture, halfway through, and day of processing) by sampling the feces on pasture, fresh feces, cecal, ileal, and jejunal contents, as well as from potential pathogen vectors on farm (water, feed, flying insects, wild bird feces). Feed and broiler weights are being measured throughout to determine performance estimates and camera systems are installed within the coops to perform broiler behavior/welfare assessments. Weather data (both for the farm site and within the coops themselves) is being collected throughout the study to assess potential meteorological effects on microbiological food safety, performance, and welfare variables. The results of this study will provide stakeholders with valuable datasets that directly link gut microbiology, performance, welfare, and climate data within poultry production systems that are becoming more prevalent throughout the country.
Isolation of Salmonella and Campylobacter from a vertically integrated poultry complex. Researchers at Auburn University longitudinally collected pullet, breeder, broiler houses, hatchery, trucks and a processing plant samples from a vertically integrated production complex. Altogether 17.32% (167/964) and 32.52% (239/735) of samples were positive for Salmonella and Campylobacter, respectively. Interestingly, the odds of occurrence of Salmonella in hatchery, transport, and processing plant samples was significantly higher (P<0.05) than breeder, pullet, and broiler farms, while the odds of occurrence of Campylobacter in broiler farms, transport, and processing plant samples was significantly (P<0.05) higher than breeders, pullets, and the hatchery. Results showed that of the 12 different sample types collected throughout the study, boot swab, sponge-stick swab, and carcass rinses (post-picking) were most useful for selecting both pathogens as compared to rest of the sample types. Among 167 Salmonella positive samples based upon PCR screening, 103 were also positive on culture while among the 239 PCR positive Campylobacter samples, 133 were also positive on culture. Both pathogens were recovered from all stages of production and sample types, which indicates that the surroundings of poultry houses and facilities are contaminated with these pathogens despite adopting various preventive measures. These results provide information about the contamination status of these pathogens in broiler production stages within a single vertically integrated complex.
Although Campylobacter is a microaerophilic pathogen, requiring a lower oxygen environment and has limited cellular mechanisms to survive in an oxygen-rich environment, recent reports suggest that Campylobacter is developing an ability to survive in aerobic conditions via expression of different oxidative stress genes. To further examine the ability of Campylobacter to grow in aerobic environments Auburn University scientists conducted a study to characterize the aerotolerance of six Campylobacter coli (C. coli) isolated from a poultry production system. First, the survival of the isolates in aerobic conditions were categorized into three categories: aero-sensitive (AS: does not survive for 12 h), intermediate aero-tolerance (IAT: survives for 12 h, but not 24 h), and hyper aerotolerant (HAT: survives for 24 h). Out of 6 tested strains, one isolate was AS, and 5 strains were IAT. Concentrations for all IAT strains decreased by 2-3 logs by 12 h; however, there was no growth after 24 h of incubation. Whole genome sequences from the 6 isolates were then screened for the presence of the 14 stress genes, and all isolates possessed 10 of them. These results indicate that intermediate aero-tolerant isolates could be prevalent in poultry production systems, potentially contributing to Campylobacter persistence in poultry environments.
Quantification method developed to target poultry-specific Salmonella serotypes of greatest human health concern. While Salmonella has >2300 serotypes, relatively few are considered serotypes of primary concern for outbreaks from poultry, and it is essential to be able to accurately determine the load of these serotypes within food-related samples. ARS researchers in Athens, Georgia, developed singleplex qPCR assays for five Salmonella enterica subsp. enterica serotypes of concern in the poultry industry (S. Enteritidis, S. Typhimurium, S. Kentucky, S. Infantis, and S. Heidelberg) based on whole genome assembly alignments. Unique genomic regions were identified and used for PCR assay testing to ensure 100% specificity among the five serovars. These specific regions were then utilized to develop TaqMan chemistry-based duplex qPCR assays. Each duplex assay incorporated an internal amplification control (IAC) to enhance the accuracy of serovar-specific detection. The assays were validated using a master standard curve comprising six independent instrumental standard curve runs. Preliminary results indicate that these standardized qPCR assays can accurately quantify different Salmonella serovars within complex poultry-related samples and these assays will be developed into a multiplex assay to simultaneously detect and quantify these 5 serotypes within a sample.
Accomplishments
1. Biomapping analyses reveal the potential role that the hatchery could have in developing the broiler-related microbiome during live production. Hatcheries represent a commercial broiler management nexus point between egg production and live production; therefore, it represents an ideal target for microbial ecology experiments to improve broiler food safety. ARS researchers in Athens, Georgia, biomapped general microbial communities (16S microbiome) and Salmonella populations (culturally) from a commercial broiler hatchery facility by targeting eggs from 2 flocks and collecting egg-, water-, air-, and facility-related samples from the pre-hatch, hatch, and post-hatch areas. Diversity estimates within the hatchery-associated microbiomes were significantly influenced by the sample type and the hatchery area (p = 0.001). Culturally, overall Salmonella prevalence was 26% (48/184), with prevalence being highest in floor drain and transport truck swabs (56%) and in the hatch and post-hatch hatchery areas (50%). Kentucky, Gaminara, and Alachua were the dominant Salmonella serotypes, with serotypes of greatest outbreak concern from chickens according to the CDC representing only 6.25% (3/48) of all recovered Salmonella isolates (Enteritidis). Taxa related to Salmonella in the microbiome dataset were significantly enriched in the post-hatch areas, and serotype diversity analyses showed that serotype Typhimurium, which was never isolated culturally, was present in the enrichments of the Salmonella positive post-hatch samples. These Typhimurium sequences matched the live vaccine strain applied directly onto the newly chicks. These findings highlight the complex diversity of commercial hatchery general microbial and Salmonella populations, including identifying facility floor drains and transport trucks as potentially important critical control points for hatchery managers to focus their Salmonella mitigation efforts to reduce loads and serotypes entering live production farms.
2. Fitness factors of Campylobacter isolates recovered from consecutively reused broiler litter. Campylobacter is a leading cause of foodborne illness in the United States due to consumption of contaminated or mishandled food products, often associated with chicken meat. Although there are studies investigating the genetic diversity of Campylobacter isolated from post-harvest chicken samples, there are limited data on the genome characteristics of isolates recovered from preharvest broiler production. ARS researchers in Athens, Georgia, in collaboration with computational biologists from Colorado State University, characterized the genetic diversity of Campylobacter isolates found in the litter of four broiler houses co-located on one farm. Campylobacter was detected in 9.38% (27/288) of litter samples and C. jejuni (n = 39) and C. coli (n = 5) were the main species found. Campylobacter fitness factors differed within and across houses but were explained by the flock raised on litter, Campylobacter species and Campylobacter sequence type. Fitness factors involved in the ability to invade and colonize host tissues and evade host defenses were present in C. jejuni isolates from flock 1 and 2 but absent in C. jejuni isolates from flock 3. Furthermore, C. jejuni isolates from house three harbored a significantly higher proportion of fitness genes with functions related to glycosylation and immune evasion than C. jejuni isolates from houses 1 and 2. All C. jejuni isolates were susceptible to all antibiotics tested while C. coli isolates (n = 4) were resistant to tetracycline. These results suggest that C. jejuni and C. coli differ in their carriage of fitness factors and antimicrobial resistance and the presence/absence of fitness factors needed for evasion of host defense mechanisms and gut colonization were important for characterizing Campylobacter strains.
3. Genetic characteristics of Salmonella isolates recovered from reused litter over three successive flocks. The severity of Salmonella infections is influenced by the presence of fitness factors such as antimicrobial resistance and virulence genes. While there are many studies that have investigated the genetic background of Salmonella strains isolated from chicken samples collected post-harvest, there is a gap in our understanding of the genetic properties that influence the persistence of Salmonella on chicken farms. ARS researchers in Athens, Georgia, in collaboration with computational biologists from Colorado State University used whole genome sequencing and hierarchical clustering to characterize and classify the genetic diversity of Salmonella isolates (n = 55) recovered from the litter of a commercial broiler farm over three consecutive flocks. Salmonella Enteritidis (n = 12), Kentucky (n = 40), and Senftenberg (n = 3) were the major serotypes found in litter. Metal resistance genes, including copper and silver, were harbored on the chromosome and plasmid of Senftenberg and Kentucky isolates, respectively, while serovar Enteritidis carried several fitness factors on plasmids. Kentucky isolates that carried metal resistance genes were the only Salmonella isolates recovered from the litter of each flock. These results suggest that there might be environmental selection for Salmonella strains carrying plasmid-linked fitness factors, which could play a role in their persistence in litter.
4. Evaluation of Campylobacter ecology within pastured poultry management systems. Prevalence of Campylobacter spp. in pastured poultry production systems can lead to the contamination of the final product, especially due to the exposure of these broilers to the environment. Therefore, understanding Campylobacter ecology along the pastured poultry farm-to-fork continuum is critical for implementing effective control measures to enhance the safety of these products. ARS researchers in Athens, Georgia, are determined the prevalence and diversity of Campylobacter populations inherent within 40 pastured poultry flocks from 9 pastured poultry farms over a four-year period. Campylobacter spp. was isolated and characterized from pre-harvest, post-harvest, and final product samples from flocks raised antibiotic-free. Campylobacter spp. were recovered from ~48% of the over 1800 samples, with the highest prevalence (95.5%) and load (4.64 log10 CFU/mL) and lowest prevalence (15.45%) and load (0.32 log10 CFU/mL) in the final product whole carcass rinses, suggesting that the Campylobacter load in the ceca may not be indicative of the Campylobacter load on the final product. In terms of diversity, ~75% were identified as C. jejuni while ~22% were C. coli, and even though these broilers were never exposed to antibiotics during production, both Campylobacter species demonstrated a high level of resistance to tetracycline (56% and 70%, respectively). Campylobacter load, prevalence, and diversity were more affected by farm location than by the type of sample from which the Campylobacter was isolated. Overall, these results indicated a need for farm-specific Campylobacter mitigation strategies to ensure the safety of these increasingly in-demand poultry products.
Review Publications
Woyda, R., Oladeinde, A.A., Endale, D.M., Strickland, T.C., Plumblee Lawrence, J.R., Abdo, Z. 2023. Virulence factors and antimicrobial resistance profiles of Campylobacter isolates recovered from consecutively reused broiler litter. Microbiology Spectrum. 11(6):1-18.
Xu, X., Rothrock Jr, M.J., Dev Kumar, G., Mishra, A. 2023. Relationship of the Poultry Microbiome to Pathogen Colonization, Farm Management, Poultry Production, and Foodborne Illness Risk Assessmen. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2023.100169.
Bodie, A.R., Wythe, L.A., Dittoe, D.K., Rothrock Jr, M.J., Obryan, C.A., Ricke, S.C. 2024. Alternative Additives for Organic and Natural Ready-to-Eat Meats to Control Spoilage and Maintain Shelf Life: Current Perspectives in the United States. Foods. https://doi.org/10.3390/foods13030464.
Stearns, R., Bowen, K., Taylor, R.L., Moritz, J., Matak, K., Tou, J., Freshour, A., Jaczynski, J., Boltz, T., Li, X., Shen, C. 2024. Microbial profile of broiler carcasses processed at a university scale mobile poultry processing unit. Poultry Science. https://doi.org/10.1016/j.psj.2024.103576.
Guo, Y., Aggrey, S.E., Oladeinde, A.A., Qiao, Y., Chai, L. 2023. Detecting broiler chickens on litter floor with the YOLOv5-CBAM deep learning model. Artificial Intelligence in Agriculture. 9:36-45.
Olson, E.G., Dittoe, D.K., Micciche, A.C., Stock, D.A., Rubinelli, P.M., Rothrock Jr, M.J., Ricke, S.C. 2024. Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods. Journal of Environmental Science and Health. https://doi.org/10.1080/03601234.2024.2353002.
Olson, E.G., Dittoe, D.K., Micciche, A.C., Stock, D.A., Rubinelli, P.M., Rothrock Jr, M.J., Ricke, S.C. 2024. Microbiome analyses of poultry feeds: Part II: Comparison of different poultry feeds. Journal of Environmental Science and Health. https://doi.org/10.1080/03601234.2024.2361596.
Ram Das, A., Pillai, N., Nanduri, B., Rothrock Jr, M.J., Ramkumar, M. 2024. Exploring Pathogen Presence Prediction in Pastured Poultry Farms through Transformer-Based Models and Attention Mechanism Explainability. Microorganisms. https://doi.org/10.3390/microorganisms12071274.
Woyda, R., Oladeinde, A.A., Endale, D., Strickland, T., Plumblee Lawrence, J.R., Abdo, Z. 2024. Genetic characteristics of Salmonella Isolates recovered from reused broiler. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2024.100236.
Ayoola, M., Pillai, N., Nanduri, B., Rothrock Jr, M.J., Ramkumar, M. 2023. Predicting Foodborne Pathogens and Probiotics Taxa within poultry-related microbiomes using a machine learning approach. Animal Microbiome. https://doi.org/10.1186/s42523-023-00260-w.