SOLUTIONS FOR THE FOOD SAFETY THREAT POSED BY SALMONELLA IN THE LYMPH NODES OF CATTLE PRESENTED FOR HARVEST

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 0226989
• Grant No.: 2011-51110-31081
• Cumulative Award Amt.: $540,326.00
• Proposal No.: 2011-04799
• Project Start Date: Sep 1, 2011
• Project End Date: Aug 31, 2015
• Grant Year: 2011
• Program Code: [111]- National Integrated Food Safety Initiative

Recipient Organization
TEXAS TECH UNIVERSITY
(N/A)
LUBBOCK,TX 79409

Performing Department
International Center for Food Industry Excellence

Non Technical Summary
Informed regulatory oversight and improved process control through industry adoption of Pathogen Reduction-Hazard Analysis and Critical Control Points (HACCP) plans have dramatically reduced the incidence of disease caused by E. coli O157:H7. Despite this tremendous success as well as improved sanitary slaughter processes, control of Salmonella in beef remains frustratingly elusive. Our preliminary data indicate that Salmonella may be carried in the lymph nodes of cattle and this phenomenon appears to be the likely avenue by which Salmonella evades carcass decontamination. Our preliminary data also provide salient insights into opportunities to mitigate this food-safety threat. In our integrated, multi-disciplinary, and multi-state project, we will conduct a series of experimental and observations studies to better understand the biology, epidemiology, and ecology of Salmonella in lymph nodes. These studies will also be used to explore candidate control strategies and identify those that are most successful and adoptable. We will use our newly generated knowledge to frame best practices that effectively mitigate the risk posed by Salmonella in lymph nodes. We will engaged stakeholders in this process as interventions have to be viewed as implementable by those responsible for that implementation. To this end, stakeholders, through our proactive engagement, will have ownership of the process and more likely to see the practices as implementable. Once the best practices have been completed, we will disseminate them to relevant stakeholders and evaluate the effect of our dissemination. Outcomes/Impacts of in project include generation of new knowledge about Salmonella in the lymph nodes of cattle, identification of mitigation strategies, practical and implementable best-practices guidelines, and improved food safety for all Americans. Moreover, we have secured matching Beef Checkoff funds to support this NIFSI request; these funds highlight the stakeholder relevance of our team and approach, and will increase the scale, scope, and impact of our activities.

Animal Health Component

60%

Research Effort Categories

Basic

(N/A)

Applied

60%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	3310	1170	10%
307	3320	1100	10%
307	3320	3020	15%
307	3410	1100	10%
307	6010	3020	10%
307	6010	3080	10%
712	3320	1170	10%
712	3440	1170	10%
712	6010	3020	15%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 307 - Animal Management Systems;

Subject Of Investigation
3320 - Meat, beef cattle; 3310 - Beef cattle, live animal; 3440 - Meat, dairy cattle; 6010 - Individuals; 3410 - Dairy cattle, live animal;

Field Of Science
3020 - Education; 1100 - Bacteriology; 3080 - Sociology; 1170 - Epidemiology;

Keywords

systems approach

salmonella

lymph nodes

cattle

epidemiology

best practices

integrated project

behavior change

Goals / Objectives
Goal: To provide innovative and practical solutions to the beef industry to improve public health by mitigating bacterial food-borne threats Objectives: 1. Characterize variation in regional, seasonal, and animal-type burden of Salmonella harborage in lymph nodes of cattle presented for harvest; 2. Identify and describe important biological and epidemiological factors associated with Salmonella in lymph nodes of cattle; 3. Develop and test practical solutions for control of Salmonella in the lymph nodes of cattle; and 4. Develop and disseminate best practices to improve food safety through mitigation of Salmonella in lymph nodes of cattle Expected outputs: Activities - We will conduct a series of integrated activities to better understand the biological, epidemiological, and ecological factors that contribute to and may mitigate the risk associated with carriage of Salmonella in the lymph nodes of cattle. Within these activities, we will conduct observational and and experimental studies, and develop and disseminate best practices through producer engagement. Events - We will hold 2 producer engagement workshops. The first is to engage producers to develop best practices and provide them the opportunity to inform its content and the second workshop will be to disseminate the final best practices document. Products - A salient product of our activities will be a broad industry-university-academia network that works to provide solutions to public health needs. Other products include a model for stakeholder engagement to inform best practices and an adoptable and implementable best practice document that improves public health.

Project Methods
We will conduct a series of observational and experimental studies. Efforts: * The experimental studies will include challenge models to estimate duration of Salmonella survival in the lymph nodes of cattle as well as an evaluation of potential mitigation strategies. The knowledge gained will increase our understand of the biology of Salmonella carriage including likely routes of infection, duration of carriage, and efficacy of candidate mitigation strategies such as control through chemical, immunomodulation, and biological strategies both before and after harvest. * The observational studies will include an ongoing surveillance program to better describe risk in terms of animal-type, region, and season. Risk will be defined in a number of ways such as a) all Salmonella pose a risk; or b) only a phenotypic or genotypic subset of Salmonella pose a risk. Further, we further explore the surveillance data through targeted epidemiological evaluation to identify herd-level factors associated with carriage of Salmonella in the lymph nodes of animals presented for harvest. * We will also engage producers (pre- and post-harvest) to share findings and build a best-practices document. An initial stakeholder-engagement workshop will be conducted to frame the best practices so that recommendations are perceived to be implementable by those responsible for that implementation. A second workshop will be used to disseminate these best practices to the stakeholders. Evaluation: The primary product of these activities is a producer-developed best-practice document. An important aspect of our work will be to evaluate adoption of these practices and an understanding of the mitigation of risk it provides.

Progress 09/01/11 to 08/31/15

Outputs
Target Audience:This reporting period included presentations to industry to deliver applied information that they can implement in their daily activities. We were also able to effectively disseminate our work at a mix of scientific conferences both domestically and internationally. This was particularly benefitial for those international producers that export product to the US. In addition to group-level audience dissemination, information was prepared for individuals with specific needs that we could address because of the work performed in our integrated research endeavor. For example, we worked within individual beef slaughter establishments to establish procedures for removal of perippheral lymph nodes during the fabrication process. They have since put these learnings into action and this is helping to reduce Salmonella in beef products. Findings of this work are now included in graduate education efforts and changing knowledge of skills of our students. In addition, we have disseminated to audiences in the 'traditional' manner thought peer-reviewer publication. Where possible and applicable, we have included an open-access option in the publication so that we can 'democratize' the dissemination process rather than the usual subscription-based approach of many journals. This has greatly increased our ability to disseminate the findings to a wider group of users around the globe (for example, within a week of publication of an open access journal, discussions were had with a reader from Europe). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. This project supported the activities of various graduate students. Moreover, as we uncovered new aspects of the biology of Salmonella, it provided the opportunity for students to be engaged in novel aspects of scientific discovery and a broader array of disciplines from molecular biology, to microbiology, to epidemiology and entomology. 2. As a conseuqence of this project, students were able to study at the French Food Safety Agency (ANSES) in Maisons-Alfort (Paris), France. Here they worked on advanced aspects of surveillance, isolate characterization, and antimicrobial resistance. This opportunity - while a great experiential learning opportunity - was also a great opportunity to be challenged with different ideas and culture. The graduate students who participated in this are far more mature and will make a far greater impact because of this experience. 3. A post doc working on this project, Marie Bugarel, discovered that several isolates were incorrectly serotyped. Through molecular detective work, we identified a new serotype and this has been confirmed by the Pasteur Institute and has been called Salmonella Lubbock. 4. For the scientists involved, we have had the opportunity to challenge our very paradigms of how Salmonella interacts with livestock. It has forced us to closely collaborate and generate new hypothesis and models/approaches to test them. We have broadened our team to incldue bioinformaticists, entomologists, and molecular biologists. We have demonstrated that Salmonella has a much more complex ecology than previously believe and between-animal transmission dynamics includes vertical and transdermal routes of infection. 5. our research collaborations have greatly broadened to include close ties with colleages in the ANSES, the University of Cambridge in the UK, and the Roslin Institute in Scotland. All of these new colleagues are contributing to our broader understanding of the biology of Salmonella. How have the results been disseminated to communities of interest?We have disseminated results in a number of approaches: 1. Various peer-reviewed publications 2. Presentations at domestic and international scientific meetings 3. Presentations and workshops at industry meetings 4. Education of graduate students 5. Articles in lay publications 6. Individual meetings with domestic producers and those international producers who export to the US. 7. Media requests and interviews What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. We were able to document substantial variation in the prevalence of Salmonella harbored within peripheral lymph nodes (PLN) of cattle presented for slaughter. In particular, the prevalence was greatest in feedlot-fattened cattle, during the summer/fall months, and in the southern climes of the US. We successfully achieved objective 1. Objective 2. This is related to objective 1; however, we were able to develop this substantially with a challenge model that was developed with support of this award. In this challenge model, we were able to determine that the duration of infection is relatively short - somewhere in the order of 42 days. Consequently, the prevalence observed at slaughter is the result of repeated reinfections. Moreover, we observed substantial within-carcass diversity of Salmonella among PLNand we developed a hypothesis that Salmonella in infecting these animal primarily via a transdermal route. This lead to an investigation of biting flies and we successfully showed that infected flies can result in a regionally-draining PLN acquiring Salmonella. Lastly, we also provided novel evidence that suggests Salmonella can be passed from a dam to its fetus resulting in viable, Salmonella-positive calves at birth.We successfully achieved objective 2 (however, more biological questions have been raised because of our work). Objective 3. We explored a number of pre-slaughter approaches to control Salmonella in PLN. These have been marginally effective. We also developed - in cooperation with producers - procedures for removal of large PLN from carcasses during fabrication. We were partially successfuly in achieving this objective (the biology of Salmonella in cattle populations is far more complex that initial assumed). Objective 4. We successfully achieved this objective. best practices have been developed based on the procedures we developed.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Bugarel, M., den Bakker, H. C., Nightingale, K. K., Brichta-Harhay, D. M., Edrington, T. S., & Loneragan, G. H. (2015). Two draft genome sequences of a new serovar of Salmonella enterica, Serovar Lubbock. Genome Announcements, 3(2), 1-2.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Edrington, T. S., Loneragan, G. H., Genovese, K. J., & Nisbet, D. J. (submitted for publication). Salmonella persistence within the peripheral lymph nodes of cattle following experimental inoculations. Journal of Food Protection.
• Type: Journal Articles Status: Other Year Published: 2016 Citation: Webb, H. E., Loneragan, G. H., Gragg, S. E., Brashears, M. M., Nightingale, K. K., Arthur, T. M., & Brichta-Harhay, D. M. Salmonella in lymph nodes of cull and fed cattle at harvest. PLOS ONE.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Olafson, P. U., Lohmeyer, K. H., Edrington, T. S., & Loneragan, G. H. (2014). Survival and fate of Salmonella enterica ser. Montevideo in adult horn flies (Diptera: Muscidae). J Med Entomol, 51, 993-1001.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Gragg, S. E., Loneragan, G. H., Nightingale, K. K., Brictha-Harhay, D. M., Ruiz, H., Elder, J. R., & Brashears, M. M. (2013). Substantial within-animal diversity of Salmonella isolates from lymph nodes, feces, and hides of cattle at slaughter. Applied and Environmental Microbiology, 79(15), 4744-4750.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Edrington, T. S., Loneragan, G. H., Hill, J., Genovese, K. J., He, H., Callaway, T. R., & Nisbet, D. J. (2013). Development of a transdermal Salmonella challenge model in calves. Journal of Food Protection, 76(7), 1255-1258.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Edrington, T. S., Loneragan, G. H., Hill, J., Genovese, K. J., Brichta-Harhay, D. M., Farrow, R. L., & Nisbet, D. J. (2013). Development of challenge models to evaluate the efficacy of a vaccine to reduce carriage of Salmonella in peripheral lymph nodes of cattle. Journal of Food Protection, 76(7), 1259-1263.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Brown, T. R., Edrington, T.S., Loneragan, G. H., Hill, J., Genovese, K. J., Brichta-Harhay, D. M., &Nisbet, D. J. (accepted for publication 7-9-15). Investigation into possible differences in Salmonella prevalence in the peripheral lymph nodes of cattle derived from distinct production systems. Journal of Food Protection.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hanson DL, Loneragan GH, Brown TR, Nisbet DJ, Hume ME, Edrington TS (2015). Evidence supporting vertical transmission of Salmonella in dairy cattle. Epidemiol Infect, 30:1-6.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Transmission of Salmonella Senftenberg to bovine peripheral lymph nodes upon horn fly feeding: an on-animal study. Journal of Food Protection (submitted)

Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: Activities: To date, we have analyzed almost 4,000 lymph nodes as part of this project. These lymph nodes have been collected from 12 commercial packing plants from across the US. Through this process, we have developed a strong network for industry engagement and also included 3 graduate students directly in day to day activities providing opportunities for hands-on mentoring. In addition, we have developed 2 challenge models including a calf-oral challenge model and a more promising calf-transdermal challenge model. The latter model is a better representation of real-world events. With this model, we have developed early estimates of the duration of infection of Salmonella within lymph nodes. Further, our 3rd-party evaluators conducted a year-end formative evaluation and provided the PD with feedback. Events: Project directors have been included as invited presenters at a number of events in the US and international locations. At these events, we have shared initial results of our integrated project as well as plans for future research. Products: We have developed a large database of prevalence and concentration of Salmonella within lymph nodes. These data include pertinent covariates for further analysis. In addition, we have submitted 3 invention disclosures (related to Salmonella differentiation and a transdermal challenge model). Finally, we developed a method for challenging calves transdermally that provides a real-world representation for lymph node contamination. A PhD student who has worked on this project successfully defended her dissertation and will graduate December, 2012. Dissemination: Initial results have been presented at a variety of industry and scientific meetings in the US and at international locations. Participating industry personnel are each contacted weekly or biweekly to report results and prepare future samplings. In addition, manuscripts for peer-reviewed journals have been developed and are under review. PARTICIPANTS: Various individuals and trainees from Texas Tech have worked on this project and include: Guy H Loneragan, Mindy Brashears, Kendra Nightingale, Chance Brooks and Mike Ballou. Further, a post doctoral research associate, Marie Bugarel, has worked on this project. Finally, graduate students Hattie Webb, Sara Gragg, Tyson Brown, and Sarah Hazenfield have all contributed greatly to the success of this project. Our collaborators at USDA/ARS (Dayna Harhay at Clay Center and Tom Edrington at College Station) have also contributed meaningfully to this project. The 3rd party evaluates at the Office of Educational Innovation and Evaluation at Kansas State have performed a formative review of our first year's activities. TARGET AUDIENCES: We have focused on dissemination knowledge and opportunities for control to participants from the beef industry. In addition, we have engages other, non-collaborating scientists to expand opportunities for collaboration and allied industries such as biopharma companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: We have developed new fundamental knowledge that informs our understanding of the ecology and biology of Salmonella in groups of cattle. This knowledge is further informs our approaches to control this important food-borne pathogen. Change in actions: Industry participants are exploring opportunities and approaches to remove lymph nodes from carcasses based on our data. This adoption of improved skills will help reduce the burden of Salmonella in beef. Change in conditions: Plants that remove lymph nodes from beef carcasses appear to have reduced Salmonella in ground beef. Further, we have developed intellectual capital in the understanding of the ecology of Salmonella in animal populations which has expanded our collaborative network to include international participants and the biopharma industry.

Publications

• Webb1, H.E., G.H. Loneragan1, S.E. Gragg1, M.M. Brashears1, K.K. Nightingale1, T.M. Arthur2, J.M. Bosilevac2, N. Kalchayanand2, J.W. Schmidt2, R. Wang2, and D.M. Brichta-Harhay. Salmonella enterica in lymph nodes of cull and fed cattle at harvest (abstract). Conference of Research Workers in Animal Diseases, Chicago, Il December 1 to 3, 2012.
• Loneragan,G.H., Sara E. Gragg, Hattie E. Webb, Marie Bugarel, Kendra K. Nightingale, Mindy M. Brashears, Tom S. Edrington, Dayna M. Brichta-Harhay. Guy H. Loneragan, Sara E. Gragg, Hattie E. Webb, Marie Bugarel, Kendra K. Nightingale, Mindy M. Brashears, Tom S. Edrington, Dayna M. Brichta-Harhay (abstract). International Symposium on Salmonella and Salmonellosis, Saint Malo, France, May 27 to 29, 2012.
• Bugarel, M., Kendra K. Nightingale, Sara E. Gragg, Mindy M. Brashears, Guy H. Loneragan. Molecular categorization of Salmonella enterica isolated from bovine lymph nodes (abstract). International Symposium on Salmonella and Salmonellosis, Saint Malo, France, May 27 to 29, 2012.
• Edrington, T.S., G.H. Loneragan2, D.M. Brichta-Harhay3, J. Hill1, K.J. Genovese1, T.R. Callaway1, R.C. Anderson1, and D.J. Nisbet. Toward a Novel, Transdermal Challenge Model that Adequately Explains the Route of Infection of Peripheral Lymph Nodes by Salmonella (abstract). International Symposium on Salmonella and Salmonellosis, Saint Malo, France, May 27 to 29, 2012.