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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Egg and Poultry Production Safety Research Unit » Research » Research Project #430357

Research Project: Reduction of Invasive Salmonella enterica in Poultry through Genomics, Phenomics and Field Investigations of Small Multi-Species Farm Environments

Location: Egg and Poultry Production Safety Research Unit

2021 Annual Report

Objective 1: Identify the environmental drivers impacting the presence and variability of Salmonella enterica serotypes and other common food borne pathogens within local, natural, multi-use poultry production systems. Objective 2: Determine the linkage between phenotypes and genotypes of Salmonella enterica to find markers associated with colonization or invasion in chickens, as well as patterns of antibiotic resistances present in the poultry production environment. Objective 3: Test mixtures of Salmonella enterica serotypes that vary in their ability to invade and colonize hens to determine the ability of commensal-like serotypes reduce the ability of pathogenic serotypes to colonize and persist. This information will be used to assess and improve vaccination strategies and reduce the use of antibiotics. Objective 4: Determine the impact of infectious dosage of the various Salmonella enterica isolates on their ability to colonize and persist in egg-laying hens to facilitate their detection and reduction in poultry.

Reducing pathogenic Salmonella enterica in eggs and poultry products is facilitated by generating research that bridges the gap between laboratory and field application. This project focuses on small farms and associated processing facilities, their management practices, and characteristics of Salmonella enterica in these environments. This research will investigate which contributes more to pathogenic Salmonella enterica on-farm, namely environmental factors and management practices versus the genetics of the pathogen. Focusing on local farms facilitates access, consistent sampling schedules and communication with participating farmers. Additional experimentation will focus on the interaction between types of Salmonella enterica that rarely cause disease with those that frequently cause disease. Specifically, we will address how the farm-prevalent serovar Kentucky impacts recovery of invasive serovar Enteritidis from internal organs of hens. Expected outcomes for regulatory agencies, the poultry industry and the consumer include: 1) data-supported approaches for identifying risks associated with contamination of end products; 2) tools that facilitate characterization of Salmonella serovars and how mixtures correlate to epidemiological trends; 3) correlation of genomic markers to antimicrobial resistances present between and within Salmonella serovars; and 4) identification of best practices that help the producer raising smaller flocks reduce pathogens in consumer products. A summary meeting will be held with participating farmers to inform them of results in a confidential setting, and how results might be used to advise management practices such as the decision to vaccinate and to raise mixed species of animals on-farm.

Progress Report
This is the final report, project replaced by 6040-32000-012-000D, "Reduction of Foodborne Pathogens and Antimicrobial Resistance in Poultry Production Environments." Refer to new project for additional information. Analyses of DNA sequences helped improve sampling methods of poultry. The poultry industry wants to know more about the microbiology occurring in commercial facilities because it impacts the presence of pathogens on-farm. ARS researchers took litter and fecal samples from microenvironments within a poultry house and analyzed sequence data by two different sample pooling methods. Estimates and relative abundances were significantly affected by house microenvironments, and the pooling method affected the fecal microbiological content (a.k.a. microbiome). Thus, poultry sampling strategies cannot be universally applied, and consideration needs to be given to sample type, house environments of interest, and “composite” pooled samples that are representative of the entire poultry house. A cold plasma-based system is efficacious for reducing common poultry foodborne pathogens and spoilage organisms. The poultry industry is interested in non-chemical treatments for reducing bacteria that cause disease and spoilage of packaged poultry products, because it can reduce the likelihood of recontamination within the processing environment. ARS researchers tested an in-package dielectric barrier discharge-cold plasma (DBD-CP) system to determine the most effective treatment times and in-package conditions to reduce common poultry foodborne pathogens and spoilage organisms in liquid culture. The data suggest that the DBD-CP system completely inactivated all bacteria, although the effect of treatment time and in-package atmospheric conditions on this inactivation was bacteria-specific. These results demonstrate the potential for DBD-CP treatment to inactivate bacteria of economic interest to the poultry industry in terms of reduced spoilage and increased safety of food product. Egg-contaminating Salmonella was biased against evolving antibiotic resistance. Evolving antibiotic resistance is a persistent threat to human and animal health. ARS scientists investigated the survival and growth of egg-contaminating Salmonella enterica serovar Enteritidis (SE), which varied in the presence of certain genes and antibiotic resistances, within egg yolks and egg whites. Genes were identified in strains that were associated with the ability of the pathogen to grow, invade internal organs of hens, and to form biofilm for bacterial adherence and survival. Antibiotic resistance genes decreased long term survival in egg white; in contrast, a cell surface molecule called sefD, which facilitates adherence, significantly increased growth in egg yolk. These findings help explain why SE has not developed the degree of antibiotic resistance observed for other serotypes that are often associated with foodborne illness. Non-pathogenic Salmonella enterica serovar Kentucky is an important reservoir for antibiotic resistance genes. In collaboration with researchers at Washington State University, Pullman, ARS researchers participated in the whole genome sequencing of Salmonella enterica serovar Kentucky ST198, which had rapidly and extensively disseminated globally and is an important source of hospital-acquired infections. ARS researchers participated by providing poultry-associated strains for analyzing single nucleotide polymorphisms associated with antibiotic resistance to ciprofloxacin. Two mutations in the gyrA gene together with one mutation in parC showed a genetic basis for emergence of fluoroquinolone resistance. Reference genomes are important for conducting epidemiological surveys of large numbers of isolates. Salmonella spp. isolated from pastured poultry farms in the United States are dominated by serotype Kentucky. ARS researchers followed 42 pastured poultry flocks over 4 years using a farm-to-fork approach to characterize Salmonella spp. from live production, processing, and final retail product. Salmonella spp. were recovered from ~17% of the >2100 samples collected. Serotype Kentucky was the dominant serotype recovered overall and on the final retail product (73.6% and 95.9 % of isolates, respectively). Serotypes belonging to the “top 30” human isolates only accounted for 15% of the Salmonella recovered. These results show the unique distribution of Salmonella within pastured poultry management systems, and the need for further research to determine the environmental drivers of the diversity of Salmonella spp. ARS researchers determined the diversity and distribution of Listeria spp. during a survey of pastured poultry farms. Although poultry are rarely a source of foodborne illness from Listeria, three prominent species of the bacterial pathogen was recovered from 15% of all fecal and soil samples tested over a 3-year period. Growth experiments of select L. innocua and L. monocytogenes isolates revealed that the enrichment media used in the USDA-FSIS MLG8.10 Listeria isolation method (UVM) allowed for the preferential growth of L. innocua over L.monocytogenes at low cell concentrations typically found in the environment (~100 Colony Forming Units per mL). These results indicate a potential underreporting of L. monocytogenes from poultry farms which needs to be considered in future epidemiological assessments of listeriosis outbreaks. ARS researchers found environmental drivers of pre- and post-harvest incidence of Listeria in poultry products. Predictive modeling showed that time of year the flock was on pasture and the age of the broiler flock were major on-farm drivers for Listeria prevalence in preharvest samples, while brood feed and chlorination status of processing rinse water were the most relevant drivers of Listeria prevalence in post-harvest samples. The variables that most closely correlated to Listeria prevalence in preharvest samples were average minimum temperature and average humidity over the 3-4 days prior to sampling. These findings help pastured poultry farmers understand the variables that impact the safety of their products. Hens receiving a low dose of egg-contaminating Salmonella were more at risk of producing contaminated eggs than those receiving a higher dose as measured by organ invasion. Salmonella enterica serovar Enteritidis (SE) is the world’s most frequent cause of foodborne illness in part because it has the unique ability out of 1500 subspecies I Samonella serotypes to efficiently contaminate and survive inside whole shell eggs. ARS scientists in Athens, GA found that lowering the infectious dosage given to hens to a thousand cells of SE resulted in a higher recovery from internal organs than a dose that was 100 times higher. The phenomenon of a lower dose having a larger impact than a higher dose on an organism is referred to in biology as a “J-curve statistic”. These results indicate that subclinical infection can thwart efforts to eradicate SE from flocks, and thus more sensitive methods for testing hens and their environment are needed. Genomes of egg-contaminationg Salmonella isolated from feral mice and other sources reveal evolutionary trends impacting the safety of food. Salmonella enterica in association with poultry products is a major source of foodborne illness in the United States. ARS researchers used whole genome sequencing, serotyping, and phenotype microarray to find evolutionary trends from 164 isolates collected from 821 mice captured live on poultry farms during the 1990s. Serotypes Enteritidis, Typhimurium, Heidelberg, Schwarzengrund, Braenderup, Agona and Manhattan were associated with feral mice; thus, rodent control on-farm is important. Acid tolerant isolates correlated to resistance to microbiocidal food additives sodium chloride and lactic acid; thus, common food preservatives could lose effectiveness. Thirteen genes were confirmed as contributing to phenotypic variation impacting organ invasiveness and environmental survival. These findings reveal that Salmonella enterica has the ability to constantly optimize a core genome for survival and growth in the environment of poultry. ARS researchers applied 16S rRNA microbiome sequencing to determine foodborne pathogen ecology within pastured poultry management systems. Pastured broiler flocks that were fed diets differing in soy content were followed from the fertilized egg within the hatchery to the final product purchased by the consumer. The intestinal flora, or microbiome, of the birds changed significantly throughout their life cycle; however, there was a core microbiome that was present in over 75% of all samples and it included Salmonella, Campylobacter, and Acinetobacter. The core microbiome correlated to the stage of growth of the live bird, which may aid targeting interventions to reduce bacteria. Diet had a significant impact on the final products post-harvest. Most notably, Campylobacter was significantly more prevalent on the product from the soy-free flocks; thus, specific proteins may reduce pathogens in pastured poultry. These data show a role for broiler feed in modulating poultry gut microbiomes to exclude major pathogens. Salmonella enterica serotype Kentucky colonization of hens interferes with detection of other Salmonella serotypes more likely to be associated with foodborne illness. Salmonella enterica serotype Kentucky (SK) is the most prevalent environmental Salmonellae found in the poultry environment. ARS researchers and collaborators at Washington State University found that previous infection of hens by SK can interfere with the detection of the egg-contaminating serotype Enteritidis (SE) in the intestine of birds. Genome analysis found that SK lost gene content to be more like the common inhabitant of the gut called non-toxigenic Escherichia coli. This finding is important because SE causes the most disease in people out of 1500 Salmonella serotypes.

1. Salmonella enterica serovar Kentucky strains that varied in antibiotic resistances also used carbohydrates differently. It is not evident why antibiotic resistances emerge within environments not necessarily associated with clinical or agricultural use of antibiotics. ARS researchers in Athens, Georgia, and collaborators found evidence that antibiotic resistances might be associated with the ability of pathogens to grow on alternative carbon sources. In the United States, Salmonella enterica serovar Kentucky causes human illness primarily in association with international travel; in contrast, other Salmonella pathogens cause foodborne illness. Phenotype microarrays found that domestic and international strains of serovar Kentucky that varied in antibiotic resistances also varied in metabolism. Continued monitoring is important because of the potential for international strains to displace domestic strains and result in more foodborne illness from Salmonella enterica.

2. Preharvest management practices predicted Campylobacter prevalence in pastured poultry flocks and associated poultry products. Building on previous predictive models for Salmonella and Listeria, ARS researchers in Athens, Georgia, used questionnaires and statistical analysis of farm management data to identify factors contributing to Campylobacter prevalence on pastured poultry farms. Preharvest samples (feces, pasture soil) showed that sources of feces present on pasture (broiler, layer, cattle, swine), the number of flocks grown during a single year, the presence of soy within the supplement feed on pasture and the type of broiler housing on pasture were all highly predictive of Campylobacter prevalence. For the postharvest samples (processing whole carcass rinses), older flocks (>11 weeks of age) and flocks reared in spring/early summer were predictive of higher Campylobacter prevalence. These data provide stakeholders with targets to monitor or change to reduce Campylobacter in a manner more specific to their management systems.

Review Publications
Shah, D., Board, M., Crespo, R., Guard, J.Y., Paul, N., Faux, C. 2020. The occurrence of Salmonella, ESBL producing E. coli and carbapenem resistant non-fermenting Gram-negative bacteria in a backyard poultry flock environment. Zoonoses and Public Health. 2020:00:1-2.
Rothrock Jr, M.J., Min, B., Castleberry, B., Waldrip, H., Parker, D.B., Brauer, D.K., Pitta, D., Indugu, N. 2021. Antibiotic resistance, antimicrobial residues and bacterial community diversity in pasture-raised poultry, swine and beef cattle manures. Journal of Animal Science.
Golden, C.E., Rothrock Jr, M.J., Abhinav, M. 2021. Mapping foodborne pathogen contamination throughout the conventional and alternative poultry supply chains. Poultry Science.
Xu, X., Rothrock Jr, M.J., Mohan, A., Kumar, G.D., Mishra, A. 2021. Using farm management practices to predict campylobacter prevalence in pastured poultry farm. Environmental Research.
Jeon, J., Lourenco, J., Kaiser, E.E., Waters, E.S., Scheulin, K.M., Fang, X., Kinder, H.A., Platt, S.R., Rothrock Jr, M.J., Callaway, T.R., West, F.D., Park, H. 2020. Dynamic changes in the gut microbiome at the acute stage of ischemic stroke in a pig model. PLoS ONE.
Krause, T.R., Lourenco, J.M., Welch, C.B., Rothrock Jr, M.J., Callaway, T.R., Pringle, D.T. 2020. The relationship between the rumen microbiome and carcass merit in Angus steers. Journal of Animal Science. 98(9):1-12.
Crippen, C., Paltry, R.T., Rothrock Jr, M.J., Sanchez, S., Szymanski, C.M. 2020. Multidrug resistant Acinetobacter isolates release resistance determinants through contact-dependent killing and bacteriophage lysis. Frontiers in Microbiology. 11:1918.
Welch, C.B., Lourenco, J.M., Davis, D.B., Krause, T.R., Carmichael, M.N., Rothrock Jr, M.J., Pringle, D.T., Callaway, T.R. 2020. The impact of feed efficiency selection on the ruminal, cecal, and fecal microbiomes of Angus steers from a commercial feedlot. Journal of Animal Science. 98(7):skaa230.