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OBJECTIVES: High priority issues to be addressed include: -Determining qualitatively the presence or absence of SH and SHp in fecal contents of SPF birds given probiotics that are housed in the same area as birds that are positive for SH and SHp. The goal of this objective is to identify conditions (probiotic concentration and age of birds exposed to probiotics) conducive to reducing SH shedding and spread of SH within a poultry house in order to reduce exposure. -Assessing the virulence of SH and SHp based upon transcription of genes associated with virulence under different environmental conditions (i.e. pH, salt, temperature, cleaning solutions, bacteriophage). The goal of this objective is to identify conditions in the processing plant and in the packaged raw chicken that reduce the virulence of SH and SHp if present in the raw chicken. Both objectives are essential for disease control of pathogens and emerging diseases including antibiotic resistant food borne diseases.
<p>NON-TECHNICAL SUMMARY:<br/> Salmonella enterica is one of the most common causes of foodborne illnesses in humans (USDA, 2011 and 2012). In addition, surveillance conducted by the National Antimicrobial Resistance Monitoring System (NARMS) has shown a recent increase in antimicrobial resistance among Salmonella including Salmonella Heidelberg (SH) isolated from chickens and turkeys at the slaughter and retail level (FDA, 2012). While the majority of Salmonella infections are relatively mild and self-limiting, SH tends to cause a disproportionately high percentage of invasive infections in humans (CDC, 2008). This may be due to the observation that Salmonella are known to carry plasmids associated with antimicrobial resistance or virulence (Fernandez-Alarcon, 2011). Consequently, new and emerging strains of SH, obtained from the poultry environment, can be associated with
both high virulence and antimicrobial resistance. During the summer of 2012 an outbreak of (SH) linked to the consumption of chicken from Foster Farm Chickens was detected. A total of 134 cases from 13 states were identified including 33 patients who were hospitalized (CDC, 2013). Of the 14 clinical isolates that were tested by the National Antimicrobial Resistance Monitoring System (NARMS), two isolates were resistant to amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftofur, and ceftriaxone. Resistance was believed to be mediated by the presence of the IncI1 plasmid carrying the antiobiotic resistant blaCMY-2 gene (CDC, 2013). Resistance to third generation cephalosporins such as ceftriaxone is clinically important because these type of extended-spectrum cephalosporins are commonly used for treatment of severe Salmonellosis in children (Pickering, 2012). In addition, because
plasmids can be gained and lost relatively easily the potential for spread of virulent SH with antibiotic resistant plasmids exists (Fernandez-Alarcon, 2011) While the above trends and observations are relatively well characterized, mitigations of SH at the farm and processing level are poorly understood. <p>RESEARCH HYPOTHESIS: <br/>Our study is intended to identify mitigations of both SH shedding and virulence at the farm and processing plant level respectively. Reducing fecal shedding in live chickens can reduce the risk of Salmonellosis in humans (Alali, 2013). Previous work has shown a decrease in SH shedding in chicks fed probiotics (Menconi, 2011). We intend to investigate whether fecal shedding of SH and SHp (SH carrying a plasmid with the antibiotic resistant blaCMY-2 gene) can be mitigated with the addition of probiotics to the diet and thereby decrease the spread of SH within an adult
flock. In addition, the variability in virulence of SH under different environmental conditions is poorly understood (Singh, 2012 and Winter 2009). Using Next Generation Sequencing, the RNA expression profiles of genes associated with virulence (e.g. genes associated with pathogenicity island and type III secretion systems) can be analyzed (Solieri, 2012) under different processing level conditions (i.e. pH, salt, osmolarity, temperature) in order to identify conditions where SH virulence is mitigated.</p><p>
APPROACH: <br/>1. Reducing fecal shedding and transmission of SH and SHp: Control birds, birds exposed to SH or SHp (i.e. SH carrying a plasmid with a specific antimicrobial resistance and or virulence associated with it) and birds exposed to SH and SHp plus probiotics will be exposed to Specific Pathogen Free (SPF) birds. Fecal samples and or the cecal contents from the exposed birds will be tested for the presence of SH using standard microbiological isolation techniques. Experiments will be performed in self-contained HEPA filtered isolators in order to prevent cross-contamination. Results will help identify whether the transmission of SH in a flock can be mitigated by the addition of probiotics to the feed. SH and Shp samples will be obtained from our collaborators in the poultry industry and at the CAHFS lab. 2.Virulence of SH in the poultry farm and processing
environment: Next Generation Sequencing technology allows for the rapid comparison of transcribed sequences (e.g. the 'transcriptome') from one or multiple genomes. We propose to evaluate the transcriptomes of SH and SHp when exposed to a series of environmental conditions common to a poultry processing plant. In addition, we propose to do the same work using different conditions including changes in heat, pH, salt concentration, osmolarity, bacteriophage exposure, and different cleaning solutions and preservatives used to keep poultry fresh. RNA expression profiles will be compared between the normal conditions and the modified conditions. The goal is to find a series of conditions that mitigate the survivability and virulence of SH and SHp as detected by changes in expression of genes associated with virulence. Each experiment will be done in triplicate in order to understand
the variability of virulence gene expression.</p>