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

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

Location: Egg and Poultry Production Safety Research Unit

2018 Annual Report

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

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

Progress Report
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.

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Review 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. 6:e00171-18.
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. 4:125. doi:10.3389/fvets.2017.00125.
Afroj, S., Aldahami, K., Reddy, G., Guard, J.Y., Adesiyum, A., Samuel, T., Abdela, W. 2017. Simultaneous detection of multiple Salmonella serovars from milk and chicken meat by real-time PCR using unique genomic target regions. Journal of Food Protection. 800(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.
Guard, J.Y., Henzler, D.J., Ramadan, H., Jones, D.R., Gast, R.K., Davison, S., Allard, M.W. 2018. Serotyping of Salmonella enterica isolated from mice caught on US 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).