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United States Department of Agriculture

Agricultural Research Service

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Research Project: Rapid Molecular Pathotyping of Major Salmonella Enterica Serotypes Based on Single-Nucleotide Polymorphisms (Snps) in the Adenylate Cyclas

Location: Egg Safety and Quality

2013 Annual Report


1a.Objectives (from AD-416):
1. Identify single nucleotide polymorphisms (SNPs) within the Salmonella enterica cyaA gene unique to the major poultry food safety-related serotypes or S. Enteritidis pathotypes, design primers/probes targeting those SNPs, and develop a hybridization protocol based on the Luminex MagPlex® system.

2. Determine the efficacy and efficiency of this hybridization protocol in detecting the presence, quantity, and relative distribution of the targeted S. enterica serotypes under different cultural and environmental conditions.

3. Expand the utility of the hybridization protocol to include one, or a combination of the following: (1) Additional SNPs within the cyaA gene; (2) SNPs within other target genes such as SEN4316, SEN3898, SEN4256, and SEN1164; (3) Detection of serotypes on environmental samples across the “food to fork” continuum within the poultry industry.


1b.Approach (from AD-416):
Previous work at the USDA-ARS in Athens, GA has shown that small scale genetic changes within the cyaA gene, known as single nucleotide polymorphisms (SNPs), can be used to effectively pathotype different strains of S. Enteritidis (Morales et al., 2007). When combined with just 4 other SNPs, cyaA is central for identifying 7 different types of S. Enteritidis. As a first step to the development of the hybridization protocol, we identified conserved sequence regions upstream (positions 2087-2106) and downstream (positions 2365 – 2387) of this SNP-containing region to create a 300-bp amplicon. Using the cyaA gene sequence alignment single SNPs unique to the 5 strains we are targeting will be identified, and we will design 20-bp probes to target each of these SNPs. Once each individual SNP-probe set is validated, we will sequentially combine multiple SNP probe sets and validate their efficacy for qualitatively identifying different mixtures of S. enterica within a sample until a single, optimized multiplex assay is created to include all 5 SNP probe sets.


3.Progress Report:

This research relates to inhouse Objective 2: Characterize and evaluate poultry processing conditions associated with processed broiler carcasses that are heavily contaminated by Salmonella. This objective will include determining microbial recovery/extraction rates for poultry rinse sampling and examining survival characteristics for Salmonella serotypes that survive processing operations. Sampling should include whole carcasses that are high shedders, and mechanically-separated poultry parts; and Objective 4: Collaborate with the QSARU at Athens to develop emerging technologies for process control and correlate molecular and cultural analysis of microbial communities in poultry with process control and pathogen contamination.

Development of S. enterica serotyping/pathotyping SNP detection assay. ARS scientists in Athens, GA developed a serotyping/Pathotyping assay based on single base pair genetic differences (known as SNPs) within the adeylate cyclase (cyaA) gene and optimized for use with the Luminex MagPlex® system. The assay successfully differentiated between three S. Enteritidis serotypes, as well as S. Typhimurium and S. Kentucky, and was able to successfully type strains within pure cultures and in mixed cultures containing up to the 5 reference strains in varying concentrations). Environmental Salmonella isolates (recovered from various sections of the production/processing chain) were also used to successfully validate the efficacy of this novel assay. Results of this study will provide a new serotyping/pathotying tool to researchers and commercial producers/processors to rapidly identify Salmonella strains isolated from throughout the poultry production spectrum.

Development of a second-generation SNP assay to further delineate between Salmonella enterica serotypes relevant to poultry food safety. ARS scientists in Athens GA are developing a secondary SNP-based assay to further differentiate important food-safety related Salmonella found within the poultry production/processing chain. The SNP assay previously developed by these scientists (based on the cyaA gene) was unable to distinguish between the two of the major Salmonella zoonoses in poultry processing (Typhimurium and Heidelberg), therefore a new SNP assay was developed based on the UDP sugar hydrolase (ushA) gene that distinguishes between Heidelberg and Typhimurium (as well as the Enteritidis, Kentucky and Infantis serotypes). Results of this study, when combined with the previous SNP assay developed for this project, will provide researchers and the poultry industry with a powerful new tool to rapidly identify major zoonotic Salmonella strains isolated from within the poultry production/processing environment.


Last Modified: 11/27/2014
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