Non Technical Summary
Salmonella is a significant public health threat and economic concern as foodborne illness continues to be an overwhelming challenge in the U.S. While numerous intervention strategies are used in poultry processing to reduceSalmonellapopulations with some success, p live bird production remains a critical stepforSalmonellacontrol. Using8 to12 hour feed withdrawal (FW) before thebroilers enterthe processing plantto reduce fecal contamination appears to be a major factor to increases in Salmonella. Our research proposal represents an newway for Salmonella control with atargeted bioengineering designof a multiple phage population with an optimizedSalmonella-killingimpact that can be applied during FW to reduce most if not allSalmonella serovars and strains.Ourgeneral hypothesisis that bioengineering phages with different Salmonellaattachment sites allow for their maximum and continuous exposure to targetSalmonella preventinghorizontalSalmonellatransmissionamong birds during FW reducing Salmonella prevalence at the processing plant.Our specific objectives:Objective 1- Bioengineer bacteriophages for optimal efficacy againstSalmonellaserovars during FW.Objective 2 -Screen the resulting bioengineered phage candidates fromObjective 1against multipleSalmonellaserovars using in vitro crop and cecal incubations.Objective 3- Determine the efficacy ofSalmonellaphage cocktail identified in Objective 2 supplemented in the drinking water of commercial broilers on reducingSalmonellatransmission during FW and Salmonella load at the processing plant.The results from this research occur at a most opportune time for the meat and poultry industry as the industry is now considering bacteriophages as a potential approach for limiting pathogens. Moreover, the industry is desperate for viable options. Our collaborative approach conceptually represents a highly targeted strategy by bioengineering more efficacious phage for the poultry GIT and tailored for application during FW before poultry processing. In addition, our bioengineered phage approach represents a broad-spectrum intervention to most serovars both currently prevalent ones as well as emerging serovars. Poultry is only the beginning. Salmonella is also prevalent in swine, cattle, and nonmeat foods such as vegetables. With traditional interventions, Salmonella can be especially difficult to tackle in swine and cattle once they invade deep tissues where phage application could be practical. Finally, from more of a long-term standpoint, this approach opens the floodgates to tackle other pathogens which are also highly problematic for the poultry industry, such as Campylobacter, which resides in the chicken gut as part of the microbiome and is nearly impossible to target with antimicrobials.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3260	1100	100%

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

Subject Of Investigation
3260 - Poultry meat;

Field Of Science
1100 - Bacteriology;

Goals / Objectives
Salmonella is a significant public health threat and economic concern as foodborne illness continues to be an overwhelming challenge in the U.S. Every year, human salmonellosis accounts for the highest number of outbreaks, illnesses, hospitalizations, and the second-highest deaths. Poultry remains the primary source of salmonellosis, accounting for 25% of the outbreaks. While numerous intervention strategies are used in poultry processing to reduceSalmonellapopulations with some success, bacterial burden and transmission duringpre-harvest live bird production remain the primary challenges forSalmonellacontrol. The management practice of 8-12 hour feed withdrawal (FW) prior to transporting broilers to the processing plant in order to reduce fecal contamination appears to be a major contributing factor to increases in Salmonella in flocks entering the processing plant. Our research proposal represents an entirelyinnovativeand radical way of tackling pathogen control with atargeted bioengineering designof a multiple phage population with an optimizedSalmonella-killingimpact that can be administered during FW to reduce most if not allSalmonella serovars and strains.Ourgeneral hypothesisis that bioengineering phages with different Salmonellaattachment sites allow for their maximum and continuous exposure to targetSalmonella and would preventhorizontalSalmonellatransmissionamong birds during FW and transportation to the processing plant, thereby reducing Salmonella prevalence at the processing plant.We plan to execute this proposal with specific objectives:Objective 1- Bioengineer bacteriophages for optimal efficacy againstSalmonellaserovars during FW and establish dosage levels.Objective 2 -Screen the resulting bioengineered phage candidates fromObjective 1against multipleSalmonellaserovars using in vitro crop and cecal incubations.Objective 3- Determine the efficacy ofSalmonellaphage cocktail identified in Objective 2 supplemented in the drinking water of commercial broilers on reducingSalmonellatransmission during FW and Salmonella load at the processing plant.

Project Methods
1) Our first goal will be to engineer phages that target S. Typhimurium LT2 with refined thermostability, pH stability, and host-eliminating capabilities. We will screen large non-biased variant libraries of Salmonella phages Felix01 and P22c2 against panels of representative target strains of Salmonella (including the serovars to be tested in Objectives 2 and 3). Using a combination of genome-wide and locus-specific mutagenesis joined with machine learning, we will engineer phages that (a) have optimal activity and host range, (b) can overcome resistance, and (c) are highly stable in the live bird internal gut environment. We anticipate the final phage pools should consist of >105 total unique combinations of engineered phages, each with between 5 to 100 tailored mutations across the phage genome.2) At this point it will be critical to test the phage candidates against multiple Salmonella serovars routinely associated with poultry that are also considered of the greatest public health concern. This is why the bioengineered phage approach proposed in this grant that can target more than one serovar will be invaluable versus more conventional approaches which have a limited Salmonella serovar range. Therefore, we will use a cocktail of 5 Salmonella serovars using DNA barcoding to quantitatively distinguish each within the cocktail. We expect thein vitroscreening to identify the suitable bioengineered phage candidates fromObjective 1that most effectively decrease allSalmonellaserovars in the gut contents with minimal disruption of gut microbes.3) We will apply bioengineered phage selected from Objective 2 and examine the following using standard Salmonella methodology and broiler grower and processing protocols:We will determine if Salmonella is reduced in infected birds.We will determine if Salmonellatransmission is preventedtinfected birds ("seeder") tonon-infected birds in the same group.Wewill examine if Salmonellacontamination is decreased on carcasses in the processing plant.We will examine bioengineered phage haveminimal impact on the non-Salmonellamicrobiota in live birds and on carcasses.The overall outcome of this is whether bioengineered Salmonella bacteriophage will reduce Salmonella contamination on broilers entring the processing plant. Each goal/step will be evaluated for sufficient success to move to the next goal.