Project Summary

Impact of the Feedlot Environment Microbiome, Soil Properties and Weather on Salmonella Survival and Persistence and Evaluation of Mitigation Strategies to Remediate Salmonella in Soil

Principle Investigator(s):
Dale Woerner1, Diego Casas1, Tayler Hays1, Kendra Nightingale1, Alejandro Echeverry1, Lindsey Slaughter2, Noelle Noyes3, Angie Siemens4, and Tyson Brown4
Institution(s):
1Department of Animal and Food Sciences, Texas Tech University 
2Department of Plants and Soil Sciences, Texas Tech University 
3Department of Veterinary Population Medicine, University of Minnesota 
4Cargill Protein, North America
Completion Date:
June 2022
KEY TAKEAWAYS

  • The microbiome within each soil layer is distinct, and results indicated that the soil layer itself is the major driver of microbiome composition, with more minor contributions from season and feedlot.
  • Soil nutrient profile exploratory analysis showed a significant predictor effect in Salmonella presence, suggesting potassium, phosphorus, and calcium concentration in soil were positively correlated with Salmonella presence, whereas copper and magnesium were negatively correlated with Salmonella presence.
  • Total lethality was not reached with any intervention strategy, nor was any intervention different from the control at 28 d. Additionally, no intervention was different from itself from 14 to 21 d, suggesting the temperature change in either direction did not affect intervention efficacy.
  • Salmonella's seasonality is well evidenced in this study. Tailoring management practices in the feedlot and interventions within the processing environment during high prevalence months may lead to a safer food supply.

BACKGROUND

Previous demonstrations of survival and persistence of Salmonella in pre-harvest agricultural systems (i.e., swine, poultry, dairy, produce, tree nut and marine) suggests that persistent strains may have the ability to adapt and respond to diverse conditions in these niches to thrive in the environment. While previous studies have demonstrated the survival and persistence of Salmonella in various pre-harvest agriculture systems, no studies on the survival and persistence of Salmonella naturally present in the feedlot environment have been published to date to our knowledge. Additionally, no studies have mechanistically determined how feedlot environmental factors collectively impact Salmonella survival and transmission dynamics (i.e., environment, vectors, and animals) within a feedlot system. 

High levels of microbial diversity have been shown to decrease persistence of pathogens such as E. coli through competitive exclusion. Soil microbiome composition and soil properties, such as nutrient and water dynamics and weather, also likely play a role in Salmonella persistence. Routine Salmonella surveillance data trending in cattle slaughter facilities and a previous study we conducted indicate that the prevalence of Salmonella in beef lymph nodes and trim varies significantly by geographical location and season, where Salmonella prevalence peaks during the warmer months (summer and fall) and increases among a southern gradient. In addition, a few specific Salmonella serotypes predominate Salmonella isolated from lymph nodes and boneless beef (trim), suggesting that these strains have adapted to the environment and/or cattle in the feedlot system. Observations on the varied regional and seasonal prevalence of Salmonella in cattle lymph nodes and beef trim support the following hypotheses: (i) feedlot environmental factors (i.e., the microbiome, soil properties and weather) may impact the ability of Salmonella to survive and thrive in the feedlot environment, (ii) specific Salmonella serotypes (those commonly isolated from lymph nodes and trim) may have adapted to persist in the feedlot environment for extended periods of time and (iii) mitigation strategies to remediate Salmonella in soil may be effective in breaking the feedlot Salmonella environmental cycle and thus transmission to animals that enter the human food chain. 


Methodology   
Longitudinal Field Study:  Salmonella prevalence was monitored throughout one year in four feedlots located in the Texas panhandle. Weather data was monitored 7 days prior to every season sampling at each feedlot. Feedlot soil nutrient profiles were examined and correlated to Salmonella presence. Samples taken were diluted on a 1:10 ratio with modified tryptic soy broth. Secondary enrichment was done in Rappaport Vassiliadis and tetrathionate broths. Xylose lysine tergitol 4 was used as a selective agar for Salmonella isolation. Presumptive colonies were confirmed using BAX real-time Salmonella assay. 


Microbiome Study:  DNA was extracted from samples using the ZymoBIOMICS DNA/RNA Miniprep Kit to the manufacturer’s protocol and eluted using 50 uL of DNase/RNase free water. A total of 467 16S rRNA V4 libraries were generated, which included 447 actual samples and 20 negative controls. 

Benchtop Soil Intervention Challenge Study:  Autoclaved soil was weighed into conical tubes with 50 percent water holding capacity and inoculated with 100µL Salmonella cocktail at a high and low concentration. The application of Decon7, fertilizer, and L28 was added after a 1 h attachment time. To mimic seasonal differences, half of the samples were incubated at 35°C for the first 14 d and then incubated at 20°C for the remaining 14 d. The other half of the samples began at 20°C and were transferred to 35°C after 14 d. Samples were plated at 0d (1 h after Salmonella inoculation), 1 h (after intervention application), 7 d, 14 d, 21 d (first measurement after temperature change), and 28 d.


Findings   
Salmonella prevalence varied throughout the seasons, where the highest prevalence was observed in summer and fall seasons. A difference in prevalence between feedlots was observed. Surface pen samples had a higher presence of Salmonella than deeper soil stratum sampled. Soil nutrient profile exploratory analysis showed a significant predictor effect in Salmonella presence, suggesting potassium, phosphorus, and calcium concentration in soil were positively correlated with Salmonella presence, whereas copper and magnesium were negatively correlated with Salmonella presence. 

The largest difference in environmental species diversity was driven by sample type, with lagoon and perimeter samples clustering separately from other sample locations. Overall, sample type explained 32% of microbiome variation, compared to <4% for season, feedlot, and pen. Perimeter and lagoon samples contained a higher diversity of unique phyla compared to the soil samples taken from the pens. Within pen-environment samples, the specific layer of soil sampled had the biggest impact on the microbiome composition, explaining 19% of the variability compared to 5% explained by season. Season had a stronger impact on the bacterial composition of the more superficial soil-layer samples. The diversity of the microbiome varied significantly by both sample type and season, with diversity decreasing in the spring and winter samples as compared to the summer and fall samples, across all sample types. Confirmed Salmonella status explained a statistically significant, yet generally small amount of the variation observed in the microbiome composition of each sample. The number of samples with Enterobacteriaceae sequences did not differ greatly across feedlots. There was high discordance between the Enterobacteriaceae counts and the confirmed Salmonella status of each sample, suggesting that the Enterobacteriaceae counts were not a robust proxy for Salmonella status. 

All intervention × time interactions for Salmonella counts, soil pH and electrical conductivity (EC) were different in every inoculation × temperature combination. However, total lethality was not reached with any intervention, nor was any intervention different from the control at 28 d. Additionally, no intervention was different from itself from 14 to 21 d, suggesting the temperature change in either direction did not affect intervention efficacy. Following the same trend, soil pH was affected by the intervention × time interaction, and within intervention, soil pH gradually increased over time. EC was also affected by the intervention × time interaction, where L28 increased soil EC and fertilizer decreased soil EC, representing a positive and negative effect on soil quality, respectively.


Implications 
Further research will elucidate the effect specific nutrients in the soil have on Salmonella presence and persistence. Moreover, seasonal variation of Salmonella presence can lead to tailored interventions at different stages of the year based on the possible risk of Salmonella entrance into the beef processing environment and have a risk-based approach to Salmonella mitigation to further ensure food safety. This study further reinforces the adaptability of Salmonella spp. and warrants further investigation of individual and combinations of serovars found in the beef feedlot system and their reactions to intended environmental mitigation strategies.