Project Summary

Collaborator II ‐ Investigation into the Origin of Salmonella in the Peripheral Lymph Nodes of Fed Beef Cattle at Slaughter in the Southwestern United States Using Whole Genome Sequencing

Principle Investigator(s):
Bugarel, M., den Bakker, H. C., Edrington, T. S., Loneragan, G. H.
Texas Tech University, Department of Animal and Food Sciences
Completion Date:
April 2017



Salmonella enterica is a foodborne pathogen, which inhabits and colonizes a large variety of hosts and environments. Recent studies (Gragg, Loneragan, Brashears, et al. 2013; Gragg, Loneragan, Nightingale, et al. 2013) have high‐ lighted the presence of Salmonella in bovine peripheral lymph nodes, which may be present in lymph nodes in   meat used to produce ground beef and therefore become a source of food contamination. In this study, whole genome sequencing (WGS) was used to characterize Salmonella isolates obtained from 5 animals at 5 different sites (hide, spleen, mesenteric lymph node, left sub‐iliac lymph node and right sub‐iliac lymph node) at slaughter to understand dispersal with the animals. Knowledge about the origin of Salmonella in peripheral lymph nodes will provide insight into the ecology of Salmonella within cohorts of cattle and offer direction for intervention opportunities.

This study was developed around 2 main objectives: (1) characterization of Salmonella by whole genome sequencing of fed beef cattle at slaughter, and (2) to use WGS data to infer the origin of Salmonella in peripheral lymph nodes of cattle. 


335 strains isolated from hide, feces and peripheral lymph nodes of cattle were sequenced using sequencing by synthesis technology. To do so, each strain was grown individually on brain‐heart infusion (BHI) agar. A single colony was used to inoculate 5mL of BHI and incubated overnight at 37°C with shaking. Bacteria from 1mL of overnight culture were harvested and genomic DNA was extracted using the GenElute Bacterial Genomic DNA kit (Sigma Aldrich) following manufacturer’s recommendations. All the gDNA extractions were quantified using fluorimetric quantification (Qubit, Thermofisher) and their concentration was adjusted to 0.20ng/mL. Then, libraries were prepared for each strain using 1ng of gDNA and were processed using the XT Nextera kits following manufacturer’s recommendations (Illumina).   

Libraries were then pooled, quantified, and sequenced on 2x75bp cartridges (Illumina) on a MiSeq sequencer. Raw sequence data were used directly to predict Salmonella serotype using SeqSero pipeline (Zhang et al. 2015) and SNP trees were inferred as outlined in Den Bakker et al. (Bakker et al. 2014).


The results of the study showed extreme genomic homogeneity of Salmonella between animals and within animals. This suggests rapid between and within animal dispersal of Salmonella strains but makes it difficult to infer within animal directionality of dispersal. 


Knowledge of the origin of Salmonella in peripheral lymph nodes could help offer direction for intervention opportunities. The research presented here shows WGS cannot be used to directly infer the origin of Salmonella in peripheral lymph nodes.

  • Den Bakker, Henk C, Marc W Allard, Dianna Bopp, Eric W Brown, John Fontana, Zamin Iqbal, Aristea Kinney, et al. 2014. “Rapid Whole‐Genome Sequencing for Surveillance of Salmonella Enterica Serovar Enteritidis..” Emerging Infec tious Diseases 20 (8): 1306–14. doi:10.3201/eid2008.131399.
  • Gragg, Sara E, Guy H Loneragan, Kendra K Nightingale, Dayna M Brichta‐Harhay, Henry Ruiz, Jacob R Elder, Lyda G Garcia, et al. 2013. “Substantial Within‐Animal Diversity of Salmonella Isolates From Lymph Nodes, Feces, and Hides of Cattle at Slaughter..” Applied and Environmental Microbiology 79 (15): 4744–50. doi:10.1128/ AEM.01020‐13.
  • Gragg, Sara E, Guy H Loneragan, Mindy M Brashears, Terrance M Arthur, Joseph M Bosilevac, Norasak Kalchayanand, Rong Wang, et al. 2013. “Cross‐Sectional Study Examining Salmonella Enterica Carriage in Subiliac Lymph Nodes of Cull and Feedlot Cattle at Harvest..” Foodborne Pathogens and Disease 10 (4): 368–74. doi:10.1089/ fpd.2012.1275. 
  • Zhang, Shaokang, Yanlong Yin, Marcus B Jones, Zhenzhen Zhang, Brooke L Deatherage Kaiser, Blake A Dinsmore, Collette Fitzgerald, Patricia I Fields, and Xiangyu Deng. 2015. “Salmonella Serotype Determination Utilizing High‐ Throughput Genome Sequencing Data..” Journal of Clinical Microbiology 53 (5): 1685–92. doi:10.1128/ JCM.00323‐15.