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ARS Home Southeast Area Athens, Georgia U.S. National Poultry Research Center Egg and Poultry Production Safety Research Unit Research





- Rothrock, Michael
- Oladeinde, Adelumola - Ade
- 2023
- 2022
- 2021
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Reduction of Foodborne Pathogens and Antimicrobial Resistance in Poultry Production Environments **Location:
                            Egg and Poultry Production Safety Research Unit** 2023 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**
                                

Biomapping of a commercial broiler hatchery showed that microbial community composition and diversity were both dependent on the facility are and the type of samples tested within the facility. Hatcheries represent a nexus point in commercial broiler management between broiler egg production and live broiler production, therefore it represents an ideal area to target for in-depth microbial ecology experiments to improve commercial broiler food safety. ARS researchers in Athens, Georgia, biomapped a commercial broiler hatchery facility by targeting eggs from 2 flocks in peak production and sampling the major hatchery areas (egg inventory, pre in-ovo incubation, post in-ovo incubation, chick processing, and chick transport). For each area, 4 major sample types were recovered: eggs/chick; air filters/fans; floor drains; and rodent bait boxes. Sample diversity, richness, and evenness withing the hatchery-associated microbiomes were significantly influence by the sample type and by the area of the hatchery from which the sample originated (p = 0.001), and significant differences were found between all pairwise comparisons between the different sample types and facility areas (q = 0.01). Previous work from this study showed that Salmonella was recovered from 27.1% (39/144) of the samples, so we also investigated the differential abundance of taxa within the microbiome data based on the cultural Salmonella status of the samples. We found that there were 6 taxa significantly enriched in the Salmonella positive samples and 3 taxa significantly enriched in the Salmonella negative samples, potentially identifying taxa that are protagonistic or antagonistic to Salmonella, respectively. Additionally, taxa related to Salmonella in the microbiome dataset were also found to be significantly enriched in the feces samples from the chick pads of the crates used to transport the newly-hatched chicks to the live production farms. These results define the microbial communities inherent to the different major areas and sample types of a commercial broiler hatchery and indicate potential critical control points within the hatchery facility (chick pads from the transport areas) to reduce Salmonella levels entering the live production farms and improve overall commercial broiler food safety.

Complete genome assembly and characterization of four Bacillus velezensis strains for probiotic development. ARS researchers in Athens, Georgia, completed the characterization of four Bacillus velezensis strains isolated from reused broiler litter. These B. velezensis strains were selected for probiotic development because of their ability to limit the transfer of antimicrobial resistance between Salmonella and E. coli. Also, B. velezensis is classified as a safe microorganism for animal and human consumption by the European Food Safety Authority. A combination of long and short read sequencing was used to perform a complete whole genome assembly of the four strains. The strains had an Average Nucleotide Identity (ANI) of 96 – 100 % with each other which confirms that they belong to the same species, but strain-level genomic differences were still present. None of the strains harbored a plasmid, antimicrobial resistance or virulence gene. All four strains harbored gene clusters to produce antimicrobials including non-ribosomal peptides, polyketides, bacteriocins, and terpenes. These results suggests that the four B. velezensis can safely be used as probiotics for food-borne pathogen reduction in chickens.

Determination of B. velezensis survival and application rate in broiler litter. ARS researchers in Athens, Georgia, used quantitative PCR to determine the abundance of B. velezensis population after inoculation into twenty-four microcosms containing reused broiler litter top-dressed with fresh pine shavings (11 g of reused litter and 4 g of fresh pine shavings). Live culture of four B. velezensis strains were applied at a rate of 2.7 x 109 cells/ft2. Sterile phosphate buffered saline was applied to twenty-four uninoculated control microcosms. Afterwards, inoculated and uninoculated microcosms were incubated in growth chambers for 7 days at 37oC and 60% relative humidity. The abundance of B. velezensis was determined daily using PCR primers specific for B. velezensis. The results showed that B. velezensis population was 2 -log10 higher in inoculated microcosms compared to uninoculated microcosms throughout the course of the experiment. Furthermore, B. velezensis population abundance increased for the first 3 days after application. There was no significant difference in litter moisture, water activity and pH between inoculated and uninoculated microcosms. This result shows that B. velezensis can survive in litter and could be applied to litter 3 – 7 days before new flock placement.

Use of automated coops to rear pasture-raised broilers under controlled field trials to assess pre-harvest microbial safety and welfare of pasture poultry management systems. ARS researchers in Athens, Georgia, working in collaboration with animal welfare scientists at the University of Georgia and a local poultry coop engineering company, are utilizing automated broiler coops in field trials to assess the environmental and management variables to influence pre-harvest microbial communities using pastured poultry rearing practices. Previous work from following 42 broiler flocks on 11 active pasture poultry farms identified potential meteorological (e.g. max temperature, max humidity) and management (e.g. age of flock, starter/grower feed components) parameters that predict the probability of foodborne pathogen prevalence (Salmonella, Campylobacter, Listeria) in natural farm settings. To perform more controlled field studies, broiler coops were designed where the movement on pasture, feeding rate, and watering rate can be controlled remotely, with mounted weather stations to collect localized meteorological data throughout grow-out. Additionally, bird performance (based on measure weight gain and feed conversion ratio estimates) and welfare (using camera systems to monitor the welfare of the broilers within the coops) data will be collected alongside the meteorological, management, and microbiological data. The results of these studies will allow for a systems-based assessment of pre-harvest environmental drivers of foodborne pathogens within pastured poultry management systems in a scientifically controlled farm setting, while also assessing pastured broiler performance and welfare. **Accomplishments**
                                

1. Using machine learning algorithms to identify pastured poultry management variables that are predictive of pre-harvest Salmonella prevalence. Prevalence of Salmonella in pastured poultry production systems can lead to contamination of the final product. Therefore, the identification of farm practices that affect Salmonella prevalence is critical for implementing control measures to ensure the safety of these products. ARS researchers in Athens, Georgia, in collaboration with computer scientists from Mississippi State University, developed predictive models based predominantly on deep learning approaches to identify key pre-harvest management variables in pastured poultry farms that contribute to Salmonella prevalence. This ensemble approach utilizing five different machine learning techniques predicted that physiochemical parameters of the soil and feces (metals such as sodium (Na), zinc (Zn), potassium (K), copper (Cu), and electrical conductivity), the number of years that the farms have been in use, and flock size significantly influenced pre-harvest Salmonella prevalence. Egg source, feed type, breed, and manganese (Mn) levels in the soil/feces are other important variables identified to contribute to Salmonella prevalence on larger (at least 3 flocks reared per year) farms, while pasture feed and soil carbon-to-nitrogen ratio are predicted to be important for smaller/hobby (<3 flocks reared per year) farms. Predictive models such as the ones described here are important for developing science-based control measures for Salmonella to reduce the environmental, animal, and public health impacts from these types of poultry production systems. 2. A DNA-based method for rapid initial screening for Salmonella serotypes impacting the health of humans and/or animals. ARS researchers in Athens, Georgia, updated and improved DNA-based method for rapid initial screening for Salmonella serotypes impacting the health of humans and/or animals. Intergenic Sequence Ribotyping (ISR) assigns serotype to Salmonella enterica subspecies I in coordination with information freely available at the National Center for Biotechnology Information. The 2022 database has 268 sequences and 40 of these were assigned new accession numbers not previously available. However, 99 unique sequences with no alignment to the database indicate substantial genomic heterogeneity remains to be characterized; in addition, combining ISR with adenylate cyclase sequencing appears useful for detecting evolution within Salmonella. Analysis also suggested that homologous recombination is a major evolutionary mechanism involved in generating unique serotypes that pose a persistent threat to the safety of food and the health of people and animals. 3. Environmental conditions in a broiler house influence the microbiome of broiler chickens. Broiler house environment is one of the most important management factors that has been shown to significantly affect broiler performance, welfare, and health. However, there is limited data on how changes in environmental factors affect the microbiome of broiler chickens. ARS researchers in Athens, Georgia, investigated the dynamics of the ceca and litter microbiome of chickens raised in two separate houses from post-hatch through pre-harvest. The overall microbial community structure of the ceca and litter consistently changed throughout the course of the grow-out and was correlated with some of the environmental parameters measured. The ceca and litter microbiome of chickens were similar in the two houses at the beginning of the experiment, but over time, the microbial community changed and differed between the houses. Differences in microbial community between houses was correlated with significant differences in house morning temperature, morning humidity, and ammonia. These analyses showed that the environment inside a broiler house not only affects broiler performance, welfare and health, but also influences the microbiome of chickens, and that these processes are interconnected. **Review Publications**
                            *Pillai, N., Ayoola, M.B., Nanduri, B., Rothrock Jr, M.J., Ramkumar, M. 2022. An ensemble learning approach to identify pastured poultry farm practice variables and soil constituents that promote Salmonella prevalence. Heliyon. 8(11):e11331. https://doi.org/10.1016/j.heliyon.2022.e11331.*
                                *Woyda, R., Oladeinde, A., Abdo, Z. 2023. Chicken production and human clinical Escherichia coli isolates differ in their carriage of antimicrobial resistance and virulence factors. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.01167-22.*
                                *Oladeinde, A., Babafela, A., Woyda, R., Abdo, Z., Endale, D., Strickland, T., Plumblee Lawrence, J.R., Cudnik, D., House, S.L., Cook, K.L. 2022. Management and environmental factors influence the prevalence and abundance of food-borne pathogens and commensal bacteria in peanut hull-based broiler litter. Poultry Science. https://doi.org/10.1016/j.psj.2022.102313.*
                                *Guo, Y., Aggrey, S.E., Wang, P., Oladeinde, A.A., Chai, L. 2022. Monitoring behaviors of broiler chickens at different ages with deep learning. Animals. https://doi.org/10.3390/ani12233390.*
                                *Guard, J.Y., Jones, D.R., Gast, R.K., Garcia, J.S., Rothrock Jr, M.J. 2022. Serotype screening of salmonella enterica subspecies I by intergenic sequence ribotyping (ISR): Critical updates. Microorganisms. 11(1):97. https://doi.org/10.3390/microorganisms11010097.*
                                *Zwirzitz, B., Oladeinde, A.A., Johnson, J., Zock, G., Milfort, M.C., Fuller, L.A., Ghareeb, A., Foutz, J., Teran, J., Woyda, R., Abdoa, Z., Looft, T., Plumblee Lawrence, J.R., Aggrey, S.E. 2023. Temporal dynamics of the cecal and litter microbiome of chickens raised in two separate broiler houses. Frontiers in Physiology. https://doi.org/10.3389/fphys.2023.1083192.*
                                *Jeon, J.H., Kaiser, E.E., Water, E.S., Yang, X., Lourenco, J.M., Fagan, M.M., Schuelin, K.M., Sneed, S.E., Shin, S.K., Kinder, H.A., Kumar, A., Platt, S., Anh, J., Duberstein, K.J., Rothrock Jr, M.J., Callaway, T.R., Xie, J., West, F.D., Park, H. 2023. Tanshinone IIA-loaded nanoparticles and neural stem cell combination therapy improves gut homeostasis and recovery in a pig ishemic stroke model. Nature Scientific Reports. https://doi.org/10.1038/s41598-023-29282-9.*


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