Project : USDA ARS

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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

2019 Annual Report

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.

Approach
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
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
1. 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.

2. 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.

U.S. DEPARTMENT OF AGRICULTURE

Egg and Poultry Production Safety Research Unit: Athens, GA