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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

2016 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
ARS scientists in Athens, Georgia, investigated environmental drivers of disease-causing bacteria isolated from all-natural poultry flocks raised on pasture. Research addressed the NP Action Plan Component to provide scientific knowledge to reduce the incidence of foodborne illnesses in the U.S. Twenty-seven poultry flocks were followed throughout their life cycle (in 2014-15) and 12 more are being followed in 2016, with samples taken at the beginning, middle, and end of their lives on pasture, during processing, and from the final retail product. Samples (feces, soil, ceca, carcass rinses) are being characterized physiochemically (e.g. pH, moisture, nutrients), culturally (targeting the zoonotic pathogens quantitatively and for subtype analysis), and molecularly (using qPCR and next-generation microbiomic sequencing analysis), as is pertinent environmental metadata (e.g. farm management, rainfall, temperature), using a polyphasic, systems-based approach. To date, over 1,500 environmental samples are being processed and molecularly analyzed for community analysis and pathogen detection, over 3,000 isolates of pathogenic (Salmonella, Campylobacter, Listeria) and indicator (E. coli) have been catalogued and are in the process of being characterized via subtyping/speciation/serotyping and antimicrobial sensitivity testing. The collected data is currently being analyzed to elucidate potential environmental drivers of pathogen survival, and to provide much needed environmental and food safety-related scientific data to stakeholders involved in this emerging and growing poultry farming system. ARS scientists in Athens, Georgia, designed a very-low-dose experimental model with egg laying hens to gain fresh insight into the transmission of organ-invasive Salmonella infections occurring in poultry on-farm. A stated objective of this project is to study the impact of Salmonella infections on organ invasion in hens exposed to very low dosages of the pathogen, which also includes incalculably low dose infections initiated by contact. Experimental research on transmission and invasion of Salmonella in the egg-laying hen in the past often used infectious dosages greater than 10exp5 per bird, in part because treatment groups of 10–30 hens gave positive results that could be analyzed with statistics. Factors complicating the use of the egg-laying hen in low-dose experimental models are the expense of raising them pathogen-free to maturity at 24 weeks of age, using enough hens to give results for statistical analyses based on positive results, use of a design that gives consistent results across different investigators and different genetic stock, and adhering to the principle of reducing the use of animals in research. As the infectious dose decreases, there is a need to include more hens because fewer birds are positive. In contrast, increasing the infectious dose results in less applicability to farm situations. The goal is to find the design that produces valid statistics and verifiable results with small groups of hens that are infected at very low dosages with less than 10exp4 cells per hen. The emphasis on initiating infection with low dosages came from previous research which suggested that J-curve statistics were present for Salmonella enterica serotype Enteritidis invading the organs of hens. In those experiments, decreasing the dosage below 10exp5 yielded more positive samples from some organs. The term “J-curve” statistic is used in biology when a small quantity of a substance causes more of an effect than a somewhat higher amount, but perhaps not as much as a very high dose. The resulting graph of such a phenomenon looks like the letter “J”, because the dose gets progressively greater on the X axis and the measurable value from the sample is recorded on the Y axis. A biological explanation for such an effect pertaining to serotype Enteritidis is that as the dose drops it escapes triggering the host’s innate immune response. In contrast, a somewhat higher triggers innate defense mechanisms and is potentially cleared. In comparison, a very high dose overwhelms the host and thus gives results not necessarily pertinent for understanding what happens on-farm. This research on J-curve statistics with Salmonella may have implications for understanding transmission and control of other infectious agents. The experimental design under investigation is to place 20 uninfected hens in cages underneath 20 hens deliberately infected with at least 10exp5 Salmonella. The samples of interest are organs collected from the uninfected hens. Thus each treatment group consists of 40 hens, but only 20 will yield data for analyzing the impact of low dosage on organ invasion. Another flexible aspect of the model is that serotypes can be used singularly, consecutively, or in mixtures. Results suggest that hens do produce granulomatous lesions in livers and ovarian pedicles following infection.

1. ARS scientists in Athens, Georgia, characterized antibiotic resistance profiles of bacteria from all-natural, antibiotic-free broilers. Isolates examined were Campylobacter, Listeria, and E. coli (no salmonella) and included 15 flocks raised on pasture from 6 different farms throughout the 2014 growing season. Isolates from a variety of environmental samples (feces, soil, carcass rinses, ceca) were collected. The antibiotic resistance patterns were found to be diverse among the different isolates, with resistance profiles being farm-specific under certain circumstances (e.g. Salmonella). These data highlight the importance of including background antibiotic resistance profiling in future studies because higher levels of resistance, and also multi-drug resistance, can be found on farms that have never used antibiotics during production. This research directly contributes to a better understanding of the larger problem of emerging antibiotic resistance.

2. ARS scientists in Athens, Geprgia, characterized acid resistance profiles of five serotypes of Salmonella associated with poultry. Fifty-one (51) datasets of metabolic microarrays were obtained from different strains of Salmonella serotypes Typhimurium, Enteritidis, Heidelberg, Infantis and Senftenberg. Statistical profiling across a microarray panel of 950 different metabolites and/or growth conditions was used to find strains with acid resistance at pH 4.5. Results suggest that acid resistance may be linked to emerging resistance to some common preservatives, such as Sodium Lactate and Sodium Chloride. Salmonella Enteritidis (SE), the world’s leading cause of poultry-associated food borne illness, had extreme variability in acid resistance profiles and acid resistance did not correlate to virulence in this study. This information will be used to direct future research on emerging resistance to common antimicrobial compounds used within the food supply to reduce bacterial growth.


Review Publications
Jones, D.R., Guard, J.Y., Gast, R.K., Buhr, R.J., Fedorka-Cray, P.J., Abdo, Z., Plumblee Lawrence, J.R., Bourassa, D.V., Cox Jr, N.A., Rigsby, L.L., Robison, C.I., Regmi, P., Karcher, D.M. 2016. Influence of commercial laying hen housing systems on the incidence and identification of Salmonella and Campylobacter. Poultry Science. 95:1116-1124.
Rothrock Jr, M.J., Hiett, K.L., Guard, J.Y., Jackson, C.R. 2016. Antibiotic resistance patterns of major zoonotic pathogens from all-natural, antibiotic-free, pasture-raised broiler flocks in the southeastern United States. Journal of Environmental Quality. 45(2):593-603.
Elder, J.R., Chiok, K.L., Paul, N., Haldorson, G.J., Guard, J.Y., Shah, D.H. 2016. The Salmonella Pathogenicity Island 13 contributes to pathogenesis in streptomycin pre-treated mice but not in day-old chickens. Veterinary Microbiology. Available:
Guard, J.Y., Abdo, Z., Byers, S.O., Kriebel, P., Rothrock Jr, M.J. 2016. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA). Foodborne Pathogens and Disease. 13(7)350-362.