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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Egg Safety & Quality Research » 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 Safety & Quality Research

2017 Annual Report


1a. Objectives (from AD-416):
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.


1b. Approach (from AD-416):
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.


3. Progress Report:
Progress was made on the 3 remaining objectives, and objective 4 was finished. For objective 1 questionnaires/management surveys were conducted from 10 participating farms, and over 650 samples (feces, soil, ceca, whole carcass rinses) were collected and analyzed physiochemically and microbiologically (including isolate of Salmonella spp.). For Objective 2, we conducted 519 serotyping assays, and approximately 50% of these support meeting Objective 1 and the rest support Objective 2. For Objective 3, changes in approach were made to better approximate real world farm situations and to incorporate findings obtained from completing Objective 4 in the model of infection that uses the egg-laying hen. Objective 4 is finished, and it answered questions about the impact of lowering dose. Infections will now be by the oral route for the foreseeable future, and in the range of 10exp3 to 10exp5. ARS scientists in Athens, Georgia, investigated if Salmonella serotypes that do not often cause disease foodborne illness decrease the recovery of those that do from chickens. For Objectives 1 and 2, substantial progress was made in associating variability of Salmonella enterica in the environment of the chicken with illness in people. One of the mysteries of Salmonella enterica is why only a few serotypes out of over 2,500 frequently and persistently cause most of the foodborne salmonellosis in people. Serotype Enteritidis is the world’s leading cause of human salmonellosis, and it is highly organ invasive in chickens and can contaminate the internal contents of eggs. In contrast, serotype Kentucky is a common environmental serotype that rarely invades the organs of chickens or causes disease in people. Results from the egg-laying hen model support that prior colonization of hens with serotype Kentucky reduces serotype Enteritidis in the organs of hens, and also increases cecal carriage of serotype Kentucky while greatly reducing serotype Enteritidis. These results support and expand upon published research conducted at higher dosages and with other routes of exposure. Thus, the hypothesis that some serotypes of Salmonella enterica might protect against colonization of chickens by pathogenic serotypes that are potentially transmissible to people is at this time supported as true. These data suggest that there may be a way to pit one Salmonella serotype against the other on-farm to reduce the incidence of salmonellosis in people associated with consumption of contaminated eggs and poultry products. This research has in the past contributed to vaccine strategies by biologics companies for designing killed vaccines (bacterins) containing multiple serotypes. This research suggests choosing serotypes for inclusion in bacterins should take into consideration that some environmental Salmonella microbiota might be beneficial for reducing illness in people. ARS scientists in Athens, Georgia, investigated the prevalence of Salmonella serotypes along the farm-to-fork continuum on local, all-natural, pasture-raised poultry farms. Farm management data was collected from participating farms to better understand the management practices employed on pasture-raised farms that may have food safety implications. These data are important to collect since this type of management system is one of the alternative poultry management systems, when combined, constitute upwards of 20% of the poultry market in the U.S. Salmonella species (>200) were isolated from various farms, flocks, and sample types along the farm-to-fork continuum, including feces, soil, ceca, and whole carcass rinses (after processing and after storage/final product). In support of the importance of the basic research described above, Kentucky was the dominant serotype recovered (>90%), and antibiotic sensitivity testing using the Centers for Disease Control’s (CDC) National Antibiotic Resistance Monitoring System (NARMS) protocol revealed significant and unique resistance patterns correlated to originating farm. These initial data suggest that there are significant environmental drivers at work in these pasture-based management systems, and that these drivers can potentially be universal (e.g. serotype) or very farm-specific (e.g. NARMS data), but data from more flocks and also different types of analyses of these samples need to be performed to elucidate these drivers. ARS scientists in Athens, Georgia, collaborated with the Food and Drug Administration (FDA), to sequence Salmonella enterica serovar Enteritidis circulating in mice caught on poultry farms during the 1990s. Egg contamination by Salmonella enterica serotype Enteritidis was at its height between 1980-1995. To date 94 isolates of serotype Enteritidis cultured from the spleens and intestines of live-caught mice captured on-farm in the mid 1990s have been submitted for analysis by collaborating through the Genome Trakr pipeline. This set includes isolates used extensively for phenotypic analysis and in hens to determine virulence characteristics, and which were used to identify a set of 16 genes that correlated to naturally occurring phenotypic variation. A first genome announcement for 67 genomes has been submitted, and these sequences have been deposited at the National Center for Biotechnology Information (NCBI); 27 more are undergoing processing. Analysis will answer questions about genome content of serotype Enteritidis during peak years of egg contamination, help further correlate genomic content with phenotypic variability, and investigate if genomic variability of serotype Enteritidis cultured from mice is similar to that occurring within chicken and human populations.


4. Accomplishments
1. Infection of egg-laying hens with Salmonella enterica serovar Enteritidis has characteristics of J-curve statistics. Improving risk modeling is of interest to regulatory agencies, public health agencies, and producers in order to design effective sampling regimens for detection of pathogens. ARS researchers in Athens, Georgia, recovered more organs from hens that were positive for serotype Enteritidis when infected with 10exp3 cells than with 10exp5. J-curve statistics indicate that a non-linear relationship exists between dose and recovery of foodborne bacteria from organs, which challenges assumptions of linearity in the response of the infected host to the dose received. Risk models for transmission of serotype Enteritidis between hens should include iterations where as few as 10, 100, and 1,000 cells of the bacteria result in spreading the pathogen to 10% of hens within a flock as measured by culture positive spleens. It is possible that very low doses evade innate host immune responses and thus the pathogen maintains a presence within a flock without consequences for bird health and/or contamination of products.

2. Novel, 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. ARS researchers in Athens, Georgia, tested an in-package dielectric barrier discharge-cold plasma (DBD-CP) system to determine the most effective treatment times and in-package atmospheric conditions to reduce common poultry foodborne pathogens (Salmonella Typhimurium, Campylobacter jejuni) and spoilage (Pseudomonas fluorescens) in liquid culture. If the poultry products can be treated within the packaging, after processing, then the likelihood of recontamination within the processing environment is greatly reduced. The data suggest that the DBD-CP system completely inactivated all tested 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 major bacteria of economic interest to the poultry industry in terms of reduced spoilage (longer shelf life) and increased safety of food product.

3. High-throughput sequencing analyses help improve poultry litter and feces sampling methods and determine the microbiomes within a poultry house. The poultry industry wants to know more about the distinct microenvironments occurring in commercial facilities because they have been shown to vary substantially and in a manner that impacts the presence of pathogens on-farm. ARS researchers in Athens, Georgia, took litter and fecal samples from 4 distinct microenvironments within a poultry house and analyzed sequence data by two different sample pooling methods. Therefore, it is essential that poultry house microbiome sampling strategies be developed to account for this microbial diversity among these distinct microenvironments. These data showed that estimates (litter only) and relative abundances (litter and feces) were significantly affected by house microenvironment, and pooling method significantly effects the fecal microbiome. These results indicate that poultry house sampling strategies cannot be universally, applied, and consideration needs to be given to (1) sample type, (2) house microenvironments of interest, and (3) appropriate creation of “composite” pooled sample that is representative of the entire poultry house.

4. A retrospective study of Salmonella serotypes circulating in mice caught live on poultry farms during the 1990s shows diversity of serotype. More realistic models of how poultry respond to infection by Salmonella helps the poultry industry design better vaccines that protect the public health. ARS researchers in Athens, Georgia, performed a retrospective analysis of serotype on 152 isolates of Salmonella enterica obtained from mice caught on farm during the 1990s, which was a time when contaminated eggs were linked to increased salmonellosis in people worldwide. Only serotypes Enteritidis and Typhimurium, which have been the 2 most prevalent serotypes causing human disease for decades, were cultured from both spleens and intestines from within the same mouse. This finding suggests that flexibility of Salmonella enterica in adapting to the environment of the mouse gut and organs is a type of virulence attribute contributing to persistence of Salmonella in the food chain. Of the 9 genotypes isolated, 8 are within the CDC top 30 list of Salmonella serotypes persistently associated with human disease.


Review Publications
Guard, J.Y., Rothrock Jr, M.J., Shah, D., Jones, D.R., Gast, R.K., Sanchez-Ingunza, R., Madsen, M., El-Attrache, J., Lungu, B. 2016. Metabolic parameters linked by Phenotype MicroArray to acid resistance profiles of poultry-associated Salmonella enterica. Research in Microbiology. 167:745-756.

Locatelli, A., Hiett, K.L., Caudill, A., Rothrock Jr, M.J. 2017. Do fecal and litter microbiomes vary within the major areas of a commercial poultry house, and does this effect sampling strategies for whole house microbiomic studies? Applied Poultry Research. 26(3):325-336. doi:10.3382/japr/pfw076.

Burt, C.D., Cabrera, M.L., Rothrock Jr, M.J. 2017. Flue-gas desulfurization gypsum effects on urea-degrading bacteria and ammonia volatilization from broiler litter. Poultry Science. doi:10.3382/ps/pex044.