Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
(N/A)
Non Technical Summary
Summary:Salmonella infections are of significant concern for both animal and human health. In cattle, Salmonella infections can cause salmonellosis, which is characterized by fever, diarrhea, and rapid dehydration, leading to steep drops in milk production, reduced feed efficiency, abortions, and even death. With antimicrobial resistance on the rise and the emergence of multi-drug resistant Salmonella, the development of novel strategies to combat Salmonella infection is a high priority. In poultry, the novel strategy of pre-colonizing the gut with non-virulent Salmonella strains is already being commercially implemented to combat enteric disease. This is believed to occur through the process of competitive exclusion, an ecological principle that states if an organism is occupying a niche, it can prevent other closely related organisms from occupying that same niche. I hypothesize that the same strategy can be utilized in a bovine model by pre-colonizing calves with Salmonella enterica serovar Cerro (SC) to reduce the impact of salmonellosis. We have documented calves naturally being colonized with SC at Emmons Blaine Dairy at the UW-Madison Arlington Agricultural Research Station and have recorded cases of SC being isolated from the farm as far back as 2016. To assess the protective potential of SC, I will first document the persistence and genomic stability of SC at the farm by performing whole genome sequencing. Lastly, I propose the use of a simplified in vitro competition assay to assess the ability of SC to outcompete other serovars of Salmonella that are known to cause disease.Importance:The emergence of multi-drug resistant pathogens is of great concern in the United States. Decades of antimicrobial use in the animal food and health industry has resulted in increases in antimicrobial resistance. With Salmonella being a zoonotic pathogen, both the health of animals and humans are at risk when Salmonella develop multiple resistance to antibiotics. While vaccines against Salmonella infections exist, they are either narrowly specific to a single serotype or have proven to be equivocal in their effectiveness. Through utilization of the competitive exclusion principle and the use of pre-colonization of cattle with SC, it is our hope to use this novel approach to reduce the impact of salmonellosis such that vaccine use and antimicrobial use can be reduced, an important step toward mitigating the global epidemic of antimicrobial resistance.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
Salmonella infections are of significant concern for both animal and human health. In cattle, Salmonella infections can cause salmonellosis, which is characterized by fever, diarrhea, and rapid dehydration, leading to steep drops in milk production, reduced feed efficiency, abortions, and even death. With antimicrobial resistance on the rise and the emergence of multi-drug resistant Salmonella, the development of novel strategies to combat Salmonella infection is a high priority. In poultry, the novel strategy of pre-colonizing the gut with non-virulent Salmonella strains is already being commercially implemented to combat enteric disease. This is believed to occur through the process of competitive exclusion, an ecological principle that states if an organism is occupying a niche, it can prevent other closely related organisms from occupying that same niche. I hypothesize that the same strategy can be utilized in a bovine model by pre-colonizing cattlewith Salmonella enterica serovar Cerro (SC) to reduce the impact of salmonellosis. We have documented calves naturally being colonized with SC at Emmons Blaine Dairy at the UW-Madison Arlington Agricultural Research Station and have recorded cases of SC being isolated from the farm as far back as 2016. To assess the protective potential of SC, I will first document the persistence and genomic stability of SC at the farm by performing whole genome sequencing. Lastly, I propose the use of a simplified in vitro competition assay to assess the ability of SC to outcompete other serovars of Salmonella that are known to cause disease.Goal 1: Test the hypothesis that SC is persistent and genomically stable at Arlington over timeThe goal of this aim is to address gaps in knowledge about Salmonella enterica serovar Cerro (SC) persistence and genomic stability, which is necessary to understand the serovars competitive potential. I will collect samples from various environments (e.g. pens, manure pit, bedding, etc) biweekly from Emmons Blaine Dairy (Arlington) over the course of 12 months. Direct fecal grabs (n=12) of randomly sampled lactating members of the herd will also be collected biweekly. Salmonella will be isolated from samples via culturing and subjected to whole genome sequencing for serotyping. Sampling various environments will allow me to assess the impact of location on persistence and genomic stability. Historical SC isolates collected yearly from Arlington will also be subjected to whole genome sequencing. Sequencing from both timepoints will allow the assessment of long-term and short-term evolution of SC at a single location. This aim will provide valuable knowledge about the SC genome necessary to understand its persistence and competitive potential.Goal 2: Test the hypothesis that SC is capable of outcompeting other serovars of SalmonellaThe goal of this aim is to directly test the competitive potential of SC by competing it against other serovars that are known to cause disease. I will use factors such as growth rates and biofilm production as well as whole genome data to score prevalence and virulence before selecting and competing specific serovars. All serovars used in this aim will be those collected from Arlington to better assess how competition may have occurred in the natural farm environment. Serovars will be challenged in liquid culture in triplicate at various concentrations and plated on selective media to separate and quantify growth by counting colony forming units. This aim will address the competitive advantage of SC against more virulent serovars in a simplified model.
Project Methods
Aim 1: Test the hypothesis that SC is persistent and genomically stable at ArlingtonMethods: To evaluate the short-term evolution of the SC genome, samples will be collected from Arlington every other week beginning in February 2022 for the course of one year. Samples to be collected include one pooled environmental sample from each of the 5 lactating pens, calf area, maternity pen, and sick/cull pen respectively. Additionally, 12 direct fecal grabs will be collected from randomly selected lactating cows in the herd. Historical SC isolates collected by the Wisconsin Veterinary Diagnostics Lab (WVDL) yearly from Arlington as far back as 2016 will be subjected to whole genome sequencing as well and used to evaluate long-term evolution. All samples will be subjected to a Salmonella isolation protocol that includes enrichment in tetrathionate and Rappaport-Vassiliadis broth as well as streaking onto MacConkey and XLT4 agar to assess lactose and hydrogen sulfide production. All isolates will be tested via PCR for the invA gene to confirm Salmonella presence.Whole-genome sequencing using the Illumina MiSeq platform will be performed on all isolates identified as Salmonella positive. Trimmomatic will be used to remove sequencing adapters and low quality bases and then FastQC will be used to check the quality of reads before assembly using the SPAdes assembler. QUAST will be used to assess the quality of the generated draft genomes, and CRISPR-typing will be performed using Minced to determine serotype. Following serotyping, genomes identified as SC will be annotated using clusters of orthologous groups (COGs). The resulting annotations will be converted into a presence/absence matrix to determine the core and accessory genome. The accessory genome will be further analyzed via a principal components analysis (PCA) using the prcomp function in R-Studio to assess spatial and temporal differences between isolates. Anvio will then be used to characterize changes in core and accessory gene dynamics over time.Aim 2: Test the hypothesis that SC is capable of outcompeting other serovars of SalmonellaMethods: To evaluate the competitive potential of SC against other serovars of Salmonella in vitro, I will first perform a series of tests to evaluate basic measures of virulence before moving to direct challenges. I will evaluate growth rates by measuring the OD600 of all isolates at times 0hr, 1hr, 2hr, 3hr, 4hr, 5hr, 6hr, and 24hr post inoculation in Luria Broth (LB). All isolates will also be screened for biofilm production on solid media using congo red and calcofluor staining, and in liquid media using crystal violet staining to visualize the biofilm components of cellulose and curli. The results from these phenotypic tests will be used to select for the strain of SC with the largest competitive potential to be used in the challenges outlined below. A second strain of SC, the genomic strain found most abundantly on the farm (identified from the previous aim), will additionally be challenged. These two strains of SC will be challeneged against two known pathogenic strains ofSalmonellafound on the farm. Similar to the two strains of SC, one pathogenic Salmonella strain will be selected with the largest competitive potential (as previously described). The other pathogenic Salmonella strain will be the strain most abundantly found on the farm.SC is naturally susceptible to most antibiotics while pathogenic serovars can have multiple resistance. Genomic data as well as culturing methods will be used to assess antibiotic resistance, and these results will be taken into account for selecting isolates before challenge. In vitro competition assays will be performed in liquid culture as outlined in previous studies with poultry isolates. Briefly, selected challenge strains with varying antibiotic resistance will be grown individually in LB overnight at 37C. Cultures will be normalized to the same OD600 before being mixed in the proportions of 1:4, 2:4, and 3:4 of SC to challenge strain in triplicate. Mixed cultures will be allowed to incubate overnight at 37C before being plated on selective antibiotic media to separate the isolates. Plates will be incubated at 37C overnight and colony forming units (CFUs) will be counted to assess competition. The importance of prior colonization before challenge will also be investigated by performing the same series of tests but allowing SC and the challenge strain to grow overnight before having the established culture inoculated with the opposite strain.Evaluation and Efforts: An advisory board of stakeholders has already been established as part of the ongoing study of Salmonella enterica serovar Cerro at Arlington Research Station and will be leveraged to complete the current proposed study. This advisory board consists of veterinarians, farm managers, ruminant nutritionists, and salmonellosis experts. Regular face-to-face meetings occur with the advisory board for continued input in project decisions and guidance on data collection and reporting. The research proposed here willbe presented by the PD at the next 2 stakeholder meetings to obtain input on the project's significance to the dairy and food industry, and to hear any concerns they might have with study design, interpretation of results, or reporting. Continuation of the project beyond the funding period will be assured though the continuation of the PD's thesis work, which will continue to be funded past the funding period of the predoctoral fellowship.Throughout the duration of the project, quarterly meetings of study personnel will be scheduled to ensure that milestones are achieved in a timely manner. Decisions about the scientific direction, changes in actions or conditions, and re-allocation of financial resources of the project will be made by the PD and PM during these meetings. Daily decisions will be made by the PD with the undergraduate student, involving the PM only when necessary. A yearly PhD thesis committee meeting will be held between the PD, PM, and the PD's thesis committee to disseminate project progress and results. Data will be shared between study personnel through use of the secure university UW-Madison Box file server and local storage on the Suen lab server. In the event of apparent misunderstandings or conflicts among study personnel, the involved parties will be requested to contact the PD and PM to mediate the dispute. While unlikely, if unresolvable conflicts arise, facilitation will be requested from the human resource personnel at UW Madison.