Source: AGRICULTURAL RESEARCH SERVICE submitted to
REDUCTION OF INVASIVE SALMONELLA ENTERICA IN POULTRY THROUGH GENOMICS, PHENOMICS AND FIELD INVESTIGATIONS OF SMALL MULTI-SPECIES FARM ENVIRONMENTS
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
0430357
Grant No.
(N/A)
Project No.
6040-32000-011-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Feb 1, 2016
Project End Date
Jan 18, 2021
Grant Year
(N/A)
Project Director
GUARD J Y
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
ATHENS,GA 30613
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113210104028%
7123220110044%
7223270117028%
Goals / Objectives
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.
Project Methods
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 10/01/19 to 09/30/20

Outputs
Progress Report 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. 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. ARS researchers in Athens, Georgia, and Gainesville, Florida, find a genomic nucleotide motif that appears to distinguish Salmonella enterica from other eubacteria. Previous research had identified that single nucleotide (homopolymer) strings of adenines and thymines (specifically, AAAAAAAA or TTTTTTTT, and longer strings) might be prone to spontaneous mutation in Salmonella enterica. To characterize these motifs further, homopolymers were catalogued within the genomes of serotypes Typhimurium, Gallinarum, and Enteritidis. The prototypical reference strain for all foodborne Salmonella is Typhimurium LT2, and it had a total of 298 AT homopolymers that were present at close to the same percentage within genes and outside of genes. After analyzing other gram-positive and gram- negative pathogens, AT homopolymers appear to be a motif that varies greatly between different bacterial genera that are separated by evolutionary lineage and disease-causing potential. Results suggest that analysis of AT homopolymers is an efficient way to identify genes undergoing natural mutation across serotypes of Salmonella enterica; in addition, patterns of homopolymers may be prognostic for disease-causing potential of eubacteria. ARS researchers in Athens, Georgia, develop foodborne-pathogen predictive models for pastured, poultry-based management systems. Pastured poultry flocks are exposed to numerous environmental variables, and thus there is great interest in understanding which variables affect the safety of the poultry products throughout the poultry production chain. Previous work in our lab developed Listeria spp. and Salmonella enterica predictive models, and these models are being expanded to include (1) Campylobacter data and (2) using pathogen-specific microbiome 16S sequence data. The use of sequence data, as opposed to the cultural recovery data, allowed us to include microbial taxa relative abundance data in the models, expanding on the potential meteorological and farm management predicting variables currently employed. Preliminary results suggest that several environmental factors may be predictive of Campylobacter prevalence in both pre-harvest (fecal) and post-harvest (final product, whole-carcass rinses) samples, and that microbiome 16S sequence data shows the potential to greatly expand the utility of these models. Record of Any Impact of Maximized Teleworking Requirement: Maximized Teleworking has prevented accomplishment of FY2020 Milestone 1, which was to hold a meeting with farmers and producers to discuss results obtained by accessing their facilities. The meeting will be delayed until such time as it is safe to hold it. On-line meeting platforms are not appropriate due to a lack of experience of some participants with suce events. While maximum teleworking schedules have facilitated requirements for producing publications and reports, physical access to laboratories and field situations to begin additional experimentation has not been possible. Thus, we anticipate that maximum teleworking has the potential to impede completion of some research and anticipated publications next year. Accomplishments 01 Low-dose infection of the egg-laying hen with Salmonella enterica serotype Enteritidis has risk for egg contamination as much or more than somewhat higher doses. Salmonella enterica serovar Enteritidis is the leading cause of salmonellosis in people, and modeling of infections in chickens is used to identify intervention strategies. To address a lack of information on the impact of low dose infections impacting organ invasion in the hen at lay, ARS researchers in Athens, Georiga, conducted two experiments in triplicate. Experiment A hens were infected intramuscularly with 1,000, 100,000, and 10,000,000 cells, and hens in Experiment B were infected orally with 5000 cells with 4 strains from different genomic clades. Results from sampling the liver, spleen, ovarian pedicle, and paired ceca indicated that dosages of 1000 cells in both experiments produced positive samples. The kinetics of infection appeared to differ between low and high dosages suggestive of a J-curve response, which meant a very small dose of 1000 cells has potential to result in more positive organs than a somewhat higher dose of 100,000 cells. Thus, the risk for hens becoming orally infected and producing eggs contaminated by Salmonella enterica serotype Enteritidis is still present even as the number of bacterial cells in the poultry environment is decreased. 02 Application of predictive algorithms throughout the pastured poultry farm-to-fork continuum was effective for predicting Salmonella spp. prevalence. In order to predict the prevalence of Salmonella during pastured poultry production, ARS researchers in Athens, Georgia, used random forest modeling in combination with questionnaire-based farm- management data and meteorological data for the origin farms. The predictive modeling showed that years farming, broiler flock age, and dominant feed components were major farm management drivers for Salmonella prevalence in preharvest samples, while dominant feed components was the most relevant drivers of Salmonella prevalence in post-harvest samples. Average temperature, humidity and high wind gust speeds prior to sampling were the meteorological variables that most closely correlated to Salmonella prevalence in preharvest samples. These data provide stakeholders with target variables to monitor to determine potential Salmonella food safety risks within their management systems.

Impacts
(N/A)

Publications

  • Rothrock Jr, M.J., Locatelli, A., Feye, K.M., Caudill, A.J., Guard, J.Y., Hiett, K., Ricke, S.C. 2019. A microbiomic analysis of a pasture-raised broiler flock elucidates foodborne pathogen ecology along the farm-to-fork continuum. Frontiers in Veterinary Science. 6:260.
  • Hwang, D., Rothrock Jr, M.J., Pang, H., Kumar, G., Mishra, A. 2020. Farm management practices that affect the prevalence of Salmonella in pastured poultry farms. LWT - Food Science and Technology. v. 127.
  • Guard, J.Y., Rothrock Jr, M.J., Jones, D.R., Gast, R.K. 2020. Low dose infection of hens in lay with Salmonella enterica serovar Enteritidis from different genomic clades. Avian Diseases. 64(1):7-15.
  • Hwang, D., Rothrock Jr, M.J., Pang, H., Guo, M., Mishra, A. 2020. Predicting Salmonella prevalence associated with meteorological factors in pastured poultry farms in southeastern United States. Science of the Total Environment. 713.


Progress 10/01/18 to 09/30/19

Outputs
Progress Report 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. 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. Predictive modeling algorithms were applied to weather and farm management parameters; in addition the microbiological effects of removing soy from pasture-raised broiler flocks was investigated. Genes were linked to a bacterial behavior (phenotype) associated with efficient cecal colonization of the egg-laying hen by applying extensive genome analysis to results obtained from infection of hens. An acid resistant subpopulation was identified in Salmonella enterica serovar Braenderup. Assessed mixtures of Salmonella serotypes Enteritidis and Kentucky for competitive colonization of chickens and investigated the impact of low dosages on the egg-laying hen, were completed by end of calendar year 2018. ARS researchers in Athens, Georgia, found colonization of the chicken by egg-associated Salmonella Enteritidis may be linked to genes in the sulfur assimilation pathway. Egg-associated Salmonella Enteritidis became the world⿿s most common cause of foodborne salmonellosis by the 1990s and it remains so today. Genome sequencing of 91 strains from mice caught on- farm during the 1990s identified a set of 18 naturally-occurring high- consequence mutations that disrupted genes. Infection experiments conducted in egg-laying hens and in combination with genome sequencing identified that some gene mutations were more likely to be present in strains that efficiently colonize the cecum of chickens. Mutations were in 3 sulfur assimilation and degradation genes, namely tcyP, dsdA and cysN. Due to the unexpected prevalence of sulfur-associated genes with cecal colonization, a possible intervention strategy for producers is to manipulate the sulfur content of feed and litter to reduce Salmonella Enteritidis in chickens. ARS researchers in Athens, Georgia applied 16S rRNA microbiome sequencing to determine bacterial foodborne pathogen ecology throughout the pastured poultry farm-to-fork continuum. The safety of poultry products is influenced by not only the processing environment, but also by the pre-harvest farm environment. To elucidate the shifts in bacterial populations throughout the life cycle of a broiler flock, a single pastured poultry broiler flock was followed from embryonic development within the hatchery environment to the final product purchased by the consumer. Using microbiome analyses, it was found that poultry-related microbiomes changed significantly throughout the life cycle of the broilers, but there was a core microbiome that was present in over 75% of all samples, and known (Salmonella, Campylobacter) and emerging (Acinetobacter) pathogenic genera were part of this core microbiome. The data collected illustrate the need for more comprehensive farm-to-fork foodborne pathogen ecology studies, as well as a more detailed assessment of dynamics of the species/serotypes/strains for these genera, to develop intervention strategies to improve poultry food safety. ARS researchers in Athens, Georgia, investigated the prevalence of major bacterial foodborne pathogens 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 constituting upwards of 20% of the poultry market in the U.S. Salmonella, Campylobacter, and Listeria were isolated from various farms, flocks, and sample types along the farm-to-fork continuum. Kentucky was the dominant Salmonella serotype recovered, and antibiotic resistance profiling revealed significant and unique resistance patterns correlated to originating farm. While Campylobacter was the most prevalent foodborne pathogen (recovered from >65% of all samples), they exhibited the lowest rate of antibiotic resistance, while Listeria, at only 15% recovery, exhibited the highest rates of antibiotic resistance and multidrug resistance. These data suggest that there are significant environmental drivers at work in these pasture-based management systems, and that these drivers can potentially be universal or very farm-specific, and on-going statistical and modeling analyses are focused on elucidating these drivers. ARS researchers in Athens, Georgia, found acid resistance in egg- associated Salmonella enterica serovar Braenderup. A collaboration was begun other federal agencies to analyze egg-associated outbreak strains of Salmonella Braenderup for distinctive phenotypes. Analysis of 1200 different conditions revealed that some isolates appeared to be resistant to biocides such as acidic pH, lactic acid and salts. Biocide resistance is a safety concern because these compounds are commonly used in processing to reduce the presence of bacteria that cause food borne illness. Phenotype analysis showed that an acid resistant subpopulation could be recovered from every isolate under examination. This information was given to the FDA to guide their research on how to approach whole genome sequencing of Salmonella Braenderup to capture the acid resistant subpopulation. ARS researchers in Athens, Georgia, found chromosomal exchange contributes to frequent serotype diversification of Salmonella enterica. One of the mysteries of Salmonella enterica is how it continuously generates new serotypes numbering in the thousands while only about 3 dozen are persistently associated with foodborne illness and/or certain animal health problems. To address the issue of serotype generation, a rapid sequencing method for serotyping Salmonella enterica was used to conduct a review of the NCBI database with 1,264 complete genome sequences. Within the ISR database of 242 serotypes, homologous recombination and/or mobile elements transferred serotype in 30 instances (12.4%).This finding indicates the importance of finding on-farm and processing plant environments that facilitate exchange of DNA between different serotypes of Salmonella enterica. Accomplishments 01 Application of predictive algorithms throughout the pastured poultry farm-to-fork continuum was effective for predicting Listeria spp. prevalence. In order to predict the prevalence of Listeria spp. during pastured poultry production, ARS researchers in Athens, Georgia, used random forest modeling of Listeria culture data in combination with questionnaire-based farm management data and meteorological data for the origin farms. The predictive modeling showed that time of year the flock was on pasture and the age of the broiler flock were major farm management drivers for Listeria prevalence in preharvest (feces, soil) samples, while brood feed and chlorination of the processing rinse water were the most relevant drivers of Listeria prevalence in post- harvest (final product whole carcass rinse) samples. The meteorological 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. The further development and validation of these models will help pastured poultry farmers understand the variables that include the safety of their products, with the ultimate goal of providing them with management targets that can most easily be changed to reduce food safety issues within their flocks. 02 Microbiological effects (gut microbiome, product food safety) of removing soybean as the main protein source in pasture-raised broiler flocks. Soy-free diets for broiler chickens had significant effects on post-harvest microbial populations associated with carcasses. With the current trend in organic farming and animal welfare, pasture-raised broiler flocks are increasing in the U.S. ARS researchers in Athens, Georgia, investigated the effects of removing soybean as the main protein source on pasture-raised broiler⿿s gut microbiome and product food safety. The elimination of soy-based protein from alternatively grown broiler diets is increasing, but the animal and public health effects of this diet shift is relatively unknown. While changes were observed in the general gut microbiomes between pastured broilers fed soy and soy-free diets during live production, most of these changes were attributable to maturation of the broilers during their time on pasture, and not the protein source in the feed. But, when observing the post-harvest processing (ceca, whole carcass rinses) and final product (whole carcass rinses) samples, significant microbiome changes were observed between diets, with Campylobacter prevalence being significantly higher on the final product samples from the soy-free flocks. These studies show the need for greater research into the effects of removing soy-based proteins from pasture-raised broiler diets and the downstream food safety implications this may have.

Impacts
(N/A)

Publications

  • Shi, Z., Rothrock Jr, M.J., Ricke, S.C. 2019. Applications of microbiome analyses in alternative poultry broiler production systems. Frontiers in Veterinary Science. 6:157.
  • Lourenco, J.M., Rothrock Jr, M.J., Fluharty, F.L., Callaway, T.R. 2019. The successional changes in the gut microbiome of pasture-raised chickens fed soy-containing and soy-free diets. Frontiers in Sustainable Food Systems. 3:35.
  • Micciche, A., Rothrock Jr, M.J., Yang, Y., Ricke, S.C. 2019. Essential oils as an intervention strategy to reduce Campylobacter in poultry production: A review. Frontiers in Microbiology. 10:1058.
  • Rothrock Jr, M.J., Locatelli, A. 2019. Importance of farm environment to shape poultry-related microbiomes throughout the farm-to-fork continuum of pasture-raised broiler flocks. Frontiers in Sustainable Food Systems. 3:48.
  • Lourenco, J.M., Rothrock Jr, M.J., Sanad, Y.M., Callaway, T.R. 2019. The effects of feeding a soybean-based or a soy-free diet on the gut microbiome of pasture-raised chickens throughout their lifecycle. Frontiers in Sustainable Food Systems. 3:36.
  • Rothrock Jr, M.J., Micciche, A.C., Bodie, A.R., Ricke, S.C. 2019. Listeria occurrence and potential control strategies in alternative and conventional poultry processing and retail. Frontiers in Sustainable Food Systems. 3:(33).
  • Golden, C.E., Rothrock Jr, M.J., Misra, A. 2019. Comparison between random forest and gradient boosting machine methods for predicting Listeria spp. prevalence in the environment of pastured poultry farms. Food Research International. 122:47-55.
  • Bodie, A.R., Micciche, A.C., Atungulu, G.G., Rothrock Jr, M.J., Ricke, S.C. 2019. Current trends of rice milling byproducts for agricultural applications and alternative food production systems. Frontiers in Sustainable Food Systems. 3:47.
  • Golden, C.E., Rothrock Jr, M.J., Misra, A. 2019. Using farm practice variables as predictors of Listeria spp. prevalence in pastured poultry farms. Frontiers in Sustainable Food Systems. 3(15).
  • Elder, J.R., Narayan, P., Burin, R., Guard, J.Y., Shah, D.H. 2018. Genomic organization and role of SPI-13 in nutritional fitness of Salmonella. International Journal of Medical Microbiology. 308(8):1043-1052
  • Guard, J.Y., Cao, G., Luo, Y., Baugher, J.D., Davison, S., Yao, K., Hoffman, M., Zhang, G., Likens, N., Bell, R., Zheng, J., Brown, E., Allard, M. 2019. Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s. Genomics. GENO_2018_447_R1.


Progress 10/01/17 to 09/30/18

Outputs
Progress Report 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. 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. Earlier research finished in 2016 answered questions about the impact of lowering the dose of Salmonella enterica serovar Enteritidis (SE) on organ invasion in hens. Results suggest it is possible to see strain differences at very low infectious doses of 10exp3 to 10exp5 when whole spleens and one liver section from groups of 20 hens are cultured at 5 to 8 days post-infection. Questionnaires and management surveys were conducted from 11 participating farms, and over 1200 samples (feces, soil, ceca, whole carcass rinses) were collected and analyzed physiochemically and microbiologically (including isolates of Salmonella spp.). We conducted 545 serotyping assays. Two of three experiments were completed at low oral doses of hens with SE to study the difference between 4 strains from different genetic lineages, including one that harbored antibiotic resistance genes. ARS researchers at Athens, Georgia, investigated if Salmonella serotypes that do not often cause foodborne illness decrease the recovery of those that do from chickens. 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 (SE) 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 (SK) is a common environmental serotype that rarely invades the organs of chickens or causes disease in people. Results suggest that SE remains easily recoverable from organs collected from hens previously exposed to SK. These results support and expand upon published research conducted at higher dosages and with other routes of exposure. This research has in the past contributed to vaccine strategies by biologics companies for designing killed vaccines (bacterins) containing multiple serotypes. ARS researchers at Athens, GA investigated the impact of nalidixic acid resistance on growth characteristics of Salmonella. In a collaborative effort between two OSQR projects, 3 biofilm-forming strains of Salmonella enterica serotype Enteritidis (SE) that lacked antibiotic resistance to nalidixic acid were gradually made resistant through successive culturing in the presence of increasing quantities. After resistance was acquired, biofilm formation was greatly reduced as was the ability to grow in 6% Sodium chloride (NaCl). These results suggest that part of the reason biofilm formation by bacteria contributes to food safety issues is that it facilitates resistance to salt. ARS researchers at Athens, Georgia, investigated the prevalence of Salmonella serotypes along the farm-to-fork continuum on local, all- natural, pasture-raised poultry farms. Salmonella species (>300) 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). Serotype Kentucky (SK) was the dominant serotype recovered (>73%), 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 differences between pasture-based management systems, and that environmental influences on Salmonella can be shared between farms or be farm specific. Environmental modeling analyses are currently underway to investigate the farm management, physiochemical, and meteorological variables that most significantly influence the prevalence of Salmonella within alternative poultry production systems. ARS researchers at Athens, Georgia, collaborated with the University of Georgia, Athens (UGA), to investigate microbiomic differences in gastrointestinal tracts between broilers with high and low feed conversion rates. As poultry production management systems move away from antibiotic-based growth promotion, it is vital to better understand the gastrointestinal tract (GIT) microbiome and investigate how to better leverage this information to improve feed efficiency during production. Broilers that differed in feed conversion rates (FCR) were identified, and 4 main sections of the gastrointestinal tract (duodenum, jejunum, ileum, cecum) were removed. The sections were then used to inoculate different metabolites in an array format (EcoPlates) to look for significant differences in metabolism of sugars and amino acids by broilers that had different FCR. Preliminary data showed highly significant metabolic differences between the high and low FCR birds, and a shift in microbial communities from anaerobic dominant to aerobic dominant along the length of the tract was observed. Birds that were better at FCR had a more anaerobic tract than birds with lower FCR, especially in the first two upper GIT sections (duodenum and jejunum) as compared to the lower GIT sections (ileum and cecum). Further work will be performed to determine which carbon sources might impact FCR, and 16S microbiome analysis will be applied to identify which types of bacteria are present. The development of this model microbiome/metabolome system will be further utilized to look at the effects of feed supplementation and pathogen challenge on the broiler GIT. Accomplishments 01 Genes causing variation in the survival of Salmonella enterica serovar Enteritidis in egg albumen and growth in egg yolk were identified. Contamination of the internal contents of eggs is a substantial food safety problem impacting public health. ARS researchers at Athens, Georgia, found genes in Salmonella enterica serovar Enteritidis (SE) that impacted contamination of eggs. 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 agree with current findings about the epidemiology of egg contamination because, respectively, emergence of antibiotic resistance has not been as prevalent for SE as it has for other Salmonella, and only Salmonella in the lineage of SE have sefD. Strains that had no antibiotic resistances and a complete sefD gene had, as a group, uniform survival and growth in albumen and yolk respectively. Genome analysis showed that the antibiotic resistance genes under evaluation and adherence-associated sefD both undergo natural variation in SE. These results strongly suggest that some strains of SE are more adept at causing egg contamination than others. 02 Mice were identified as historical carriers of Salmonella enterica serovar Braenderup in egg-laying operations. ARS researchers at Athens, Georgia, used retrospective serotyping of a collection of 164 isolates obtained from 821 mice captured live on poultry farms during the 1990s revealed that Salmonella enterica serovar Braenderup, the causative agent of a recent outbreak of Salmonella in people associated with eggs, was recovered in 4 samples (2.4% of isolates processed). Later analysis by the FDA of risk factors impacting the contamination of eggs by serovar Braenderup concluded that poor rodent control was a major factor. These results suggest that the group of serovars found circulating in mice in the 1990s, namely Enteritidis, Typhimurium, Heidelberg, Schwarzengrund, Braenderup, Agona and Manhattan could have an association with mice that is especially challenging for maintaining the safety of the food supply. Historical isolates of serovar Braenderup will be made available to FDA for analysis. 03 The relatively 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 at Athens, Georgia, participated in the whole genome sequencing of Salmonella enterica serovar Kentucky ST198 that has rapidly and extensively disseminated globally and is of public health concern in hospitals. ARS researchers participated by providing poultry- associated strains that were useful for analyzing single nucleotide polymorphisms associated with antibiotic resistance to ciprofloxacin resistant SK. Two mutations in the gyrA gene together with one mutation in parC showed a genetic basis for emergence of high-level fluoroquinolone resistance. Completed reference genomes are important for conducting genome-based epidemiological surveys of large numbers of isolates. 04 Salmonella spp. isolated from pastured poultry farms are dominated by serotype Kentucky. As alternative poultry management systems increase in prevalence (accounting for ~20% of U.S. poultry market), more research needs to be conducted to understand the ecology of foodborne pathogens within these unique production/processing environments. ARS researchers at Athens, Georgia, followed 42 pastured poultry flocks over 4 years using a farm-to-fork approach in an attempt to isolate and characterize Salmonella spp. from live production (feces, soil), processing (cecal contents, whole carcass rinses) and final retail product (whole carcass rinses) samples. Overall, Salmonella spp. were recovered from ~17% of the >2100 samples collected, and serotype Kentucky (SK) was the most dominant serotype recovered overall and on the final retail product (73.6% and 95.9 % of recovered Salmonella, respectively). This is of concern since ciprofloxacin resistant SK is a problem within hospital settings. Serotypes belonging to the CDC �top 30� human isolates only accounted for 15% of the overall Salmonella spp. recovered, and only 0.4% of the Salmonella spp. isolated from final retail product (2 Infantis isolates from a single farm during a single year). These results show the unique distribution of Salmonella spp. within pastured poultry management systems, and the need for further research to determine the environmental drivers of the diversity of Salmonella spp. within these systems. 05 Potential culturing bias for Listeria innocua over Listeria monocytogenes isolated from poultry farms using the USDA-FSIS 8.10 method. While Listeria monocytogenes represents one of the major bacterial foodborne pathogens in the U.S., listeriosis outbreaks are rarely attributed to the poultry industry (especially outside of the processing environment) even though Listeria spp. can be found on poultry farms. ARS researchers at Athens, Georgia determined the diversity and distribution of Listeria spp. during a survey of pastured poultry farms, showing that Listeria were recovered from 15% of all fecal and soil samples tested over a 3-year period, and included L. innocua, L. monocytogenes, and L. welshimeri (65.7%, 17.4%, and 15.1% of all Listeria spp. recovered, respectively). Monoculture and co- culture 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 (~10^2 CFU/mL). These results indicate a potential underreporting of L. monocytogenes from poultry farms being tested using the UVM-based isolation method, which needs to be considered in future epidemiological assessments of listeriosis outbreaks.

Impacts
(N/A)

Publications

  • Gast, R.K., Guard, J.Y., Guraya, R., Locatelli, A. 2018. Multiplication in egg yolk and survival in egg albumen of genetically and phenotypically characterized Salmonella Enteritidis strains. Journal of Food Protection. 81:876-880.
  • Gast, R.K., Guraya, R., Jones, D.R., Guard, J.Y., Anderson, K.E., Karcher, D.M. 2017. Frequency and duration of fecal shedding of Salmonella serovars Heidelberg and Typhimurium by experimentally infected laying hens housed in enriched colony cages at different stocking densities. Avian Diseases. 61:366-371.
  • Rothrock Jr, M.J., Fan, P., Jeong, K.C., Kim, S.A., Ricke, S.C., Park, S. 2018. Complete genome sequence of Listeria monocytogenes strain MR310, isolated from a pastured-flock poultry farm system. Genome Announcements. doi:10.1128/genomeA.00171-18.
  • Guard, J.Y., Henzler, D.J., Ramadan, H., Jones, D.R., Gast, R.K., Davison, S., Allard, M.W. 2018. Serotypes of Salmonella enterica subspecies I isolated from mice caught on poultry farms 1995 through 1998. Journal of Food Safety. 6:44-50.
  • Guard, J.Y., Cao, G., Kastanis, G., Davison, S., Mcclelland, M., Leon, M.S. , Zheng, J., Brown, E., Allard, M. 2017. Draft genome sequences of 64 Salmonella enterica subtype Enteritidis isolates obtained from wild mice. Genome Announcements. 5(36):e00953-17.
  • Rothrock Jr, M.J., Davis, M.L., Locatelli, A., Bodie, A., Mcintosh, T., Donaldson, J.R., Ricke, S.C. 2017. Listeria Occurrence in Poultry Flocks: Detection and Potential Implications. Frontiers in Veterinary Science. doi:10.3389/fvets.2017.00125.
  • Afroj, S., Aldahami, K., Reddy, G., Guard, J.Y., Adesiyum, A., Samuel, T., Abdela, W. 2017. Development of Real Time PCR Using Novel Genomic Target for Detection of Multiple Salmonella Serovars from Milk and Chickens. Journal of Food Protection. 80(11):1944-1957.
  • Locatelli, A., Lewis, M.A., Rothrock Jr, M.J. 2017. The distribution of Listeria in pasture-raised broiler farm soils is potentially related to University of Vermont medium enrichment bias toward Listeria innocua over Listeria monocytogenes. Frontiers in Veterinary Science. 4:227. 10.3389/ fvets.2017.00227.
  • Burt, C., Cabrera, M., Rothrock Jr, M.J., Kissel, D.E. 2018. Urea hydrolysis and calcium carbonate precipitation in gypsum-amended broiler litter. Journal of Environmental Quality. 47(1):162.169. 10.2134/jeq2017. 08.0337.
  • Shah, D.H., Paul, N.C., Guard, J.Y. 2018. Complete genome sequence of a ciprofloxacin resistant Salmonella enterica subsp. enterica serovar Kentucky sequence of a ciprofloxacin strain, PU131, isolated from a human patient in Washington State. Genome Announcements. 6(9):e00125-18.


Progress 10/01/16 to 09/30/17

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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.

Impacts
(N/A)

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.


Progress 10/01/15 to 09/30/16

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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.

Impacts
(N/A)

Publications

  • Jones, D.R., Guard, J.Y., Gast, R.K., Buhr, R.J., Fedorka-Cray, P.J., Abdo, Z., Plumblee, J., Bourassa, D.V., Cox Jr, N.A., Rigsby, L.L., Robinson, 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(5):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.