Source: UNIVERSITY OF ARKANSAS submitted to
CONTROLLING SALMONELLA THROUGH ENHANCED UNDERSTANDING OF HORIZONTAL TRANSMISSION AND A NOVEL AND SCALABLE VACCINATION STRATEGY IN BROILERS.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1028395
Grant No.
2022-67017-36936
Cumulative Award Amt.
$622,841.00
Proposal No.
2021-08164
Multistate No.
(N/A)
Project Start Date
Jan 1, 2022
Project End Date
Dec 31, 2026
Grant Year
2022
Program Code
[A1332]- Food Safety and Defense
Project Director
Kwon, Y.
Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703
Performing Department
Poultry Science
Non Technical Summary
The capacity of Salmonella to successfully colonize and transmit in chickens is the most critical feature contributing to public food safety concerns due to consumption of Salmonella-contaminated chicken meat. Although a great deal of effort has been made toward understanding gut colonization by Salmonella, the mechanisms underlying horizontal transmission remain largely unknown. In this project, we will develop a novel vaccination strategy to control Salmonella in chickens based on the two key concepts recently developed by our team. In Objective 1, we will optimize and evaluate the use of DNA-based aptamer adjuvant targeting CD40 as a universal bivalent system. In Objective 2, we will use a powerful functional genomics tool, transposon sequencing, to identify genetic factors in Salmonella Typhimurium required for horizontal transmission. In Objective 3, the novel vaccination strategy developed in Objective 1 will be adopted to target the transmission factors identified in Objective 2 to reduce horizontal transmission in a chicken flock. The results of this project will provide previously unknown mechanistic insights on Salmonella horizontal transmission and a versatile and universal vaccination platform that can be used for effective control of the horizontal transmission and thus the prevalence of Salmonella in a chicken flock.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113299109050%
3114010104050%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other; 4010 - Bacteria;

Field Of Science
1090 - Immunology; 1040 - Molecular biology;
Goals / Objectives
The long-term goal of this project is to reduce the prevalence of Salmonella serovars in broiler chickens at the early pre-harvest stage by developing a novel vaccination strategy that reduces horizontal transmission within a chicken flock. Salmonella continues to be a great public health concern and the consumption of chickens remains to be the major source of Salmonella infection for the human population. Despite the great efforts that have been made to reduce this burden, its overall negative impact remains unhampered and this situation demands the development of novel strategies that can cripple Salmonella colonization in chicken flocks. Our team has proved CD40 targeting is a universal strategy that can effectively enhance vaccination efficacy in chickens using agonistic CD40-specific monoclonal antibodies through experimental demonstration of its efficacy against avian influenza virus and Clostridium perfringens. More recently, our team expanded a way to apply this strategy at a commercial scale, overcoming the disadvantages associated with the use of monoclonal antibodies. In brief, we developed single-stranded DNA aptamers with specific binding affinity to the chicken CD40 extracellular domain, which were then utilized to produce rolling circle amplification (RCA) products that incorporate two of the most promising anti-CD40 aptamers (SEQ3 and SEQ4) in hundreds of copies. When this RCA product was conjugated to an antigen of interest, it significantly enhanced immune response to the antigen in broiler chickens. Most vaccines used to control Salmonella have been exclusively focused on reducing gut colonization, while horizontal transmission is another critical aspect contributing to Salmonella prevalence in final chicken meat products. This is largely due to the fact that our understanding of the horizontal transmission of Salmonella is limited, particularly in terms of the bacterial factors contributing to the process. In this project, our team will overcome this barrier by performing comprehensive screening to identify transmission factors of S. Typhimurium using a functional genomics tool, transposon sequence (Tn-seq). Once the transmission factors are identified and validated, we will develop the novel enhanced vaccination platform based on the aptamer RCA adjuvant that is designed to reduce horizontal transmission. We have the following 3 objectives to accomplish the long-term goal of this project.Objective 1. Evaluation of the universal CD40-targeting aptamer RCA adjuvant platform as a bivalent systemObjective 2. Comprehensive identification of the genetic factors in S. Typhimurium required for gut colonization and horizontal transmissionObjective 3. Vaccination targeting surface-exposed transmission factors in S. Typhimurium to reduce horizontal transmission in a chicken flockEach component/technique within this research proposal has been previously verified independently, but the union of these components to develop a more industry-functional and affordable system has yet to be accomplished. Successful completion of the proposed research will: 1) bring the universal aptamer RCA platform forward as a versatile and effective vaccination method that can be used for multiple pathogens in the poultry industry, 2) provide a safe option to vaccinate chickens against Salmonella, and 3) potentially serve as a multivalent/multi-serogroup Salmonella vaccination method, capable accommodating new outbreak strains.
Project Methods
Efficacy of an optimized DNA-based aptamer adjuvant targeting CD40 as a universal bivalent system will be investigated. Transposon sequencing will be used to identify genetic factors in Salmonella Typhimurium required for horizontal transmission. The the novel vaccination strategy developed will be adopted to target the transmission factors identified to reduce horizontal transmission in a chicken flock. This will be validated in laboratory-scale in vivo studies.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The results of the study were presented at the 2024 Annual PSA meeting to the audience group that consists of professors, staff, graduate students from academia, and scientists and personnel from poultry or related industries. Changes/Problems:One of the goals of the project was to identify the genetic factors in Salmonella Typhimurium that are required for horizontal transmission of this pathogen within the broiler flock and to use the proteins or their peptides as the antigens in combination with the DNA vaccine complexes. However, we encountered both intrinsic and technical challenges that are difficult to overcome. We decided to omit this component from the project but added the new component (HCR-based DNA complex) to improve the vaccine effcacy. What opportunities for training and professional development has the project provided?We trained 3 Ph.D. students through this project. They have acquired new knowledge to understand the project and skills to perform the experiments in this project. How have the results been disseminated to communities of interest?We presented the results from this project through presentations at the annual Poultry Science Association meeting. We presented two abstracts through oral presentations (shown below). Abigeal I Omolewu, Christine N Vuong, Santiago U Diaz, Adil S Al-Ogaili, Seong W Kang, Billy M Hargis, and Young Min Kwon. Development and Evaluation of DNA aptamer rolling circle amplification (RCA) platform as a vaccine complex to control Salmonella in broiler chickens. July 15-18, 2024, PSA Annual Meeting, Louisville, KY. (Oral presentation) Santiago Uribe-Diaz, Abigeal I. Omolewu, Jossie M. Santamaria, Chrysta N. Beck, Christine N. Vuong, Guillermo Tellez-Isaias, Gisela F. Erf, Billy M. Hargis, Young Min Kwon. July 15-18, 2024, PSA Annual Meeting, Louisville, KY. (Oral presentation) In addition, we are currently working on 2 manuscripts to submit to peer-reviewed journals soon. What do you plan to do during the next reporting period to accomplish the goals? We will complete the evaluation of the two DNA vaccine complexes (versions 3 and 5) using a chicken challenge study. The results will be collected, analyzed, and summarized to prepare the presentations and manuscripts. We will complete the sample analysis from the growing feather model study and the pilot chicken challenge study to determine the humoral immune responses triggered by the DNA complex. We will complete the evaluation for the new DNA complex design based on HCR. If the result is similar to or better than the DNA complex designs based on RCA, we will also perform further evaluations using the growing feather model and chicken challenge study.

Impacts
What was accomplished under these goals? Accomplishments The chicken challenge study was performed on a pilot scale to evaluate the two versions of the DNA complex (versions 3 and 5). The result showed that the group vaccinated with the version 3 DNA complex showed a significantly lower level of cecal colonization by the Major goals of the project Continued development of CD40-targeting DNA complexes for improved immune-enhancing capacity. Evaluation of the DNA vaccine complexes to control Salmonella through chicken challenge studies. What was accomplished under these goals? We established and have routinely used the macrophage activation assay using the HD-11 cell line. This assay gave reliable and consistent results on the level of macrophage activation through quantification of nitric oxide released from activated macrophage cells. We used this in vitro assay conveniently to screen and evaluate different design variations of CD40-targeting DNA complex. In addition to the previous versions of DNA complex, we evaluated new versions of DNA complex using HD-11 cell line assay and found that versions 3 and 5 produced the highest level of significant macrophage activation consistently. Therefore, these two versions of DNA complex (version 3 and 5) in combination with killed Salmonella cells were selected for further evaluation using 2 additional approaches: (1) evaluation of immune response using growing feather pulp cutaneous test model, and (2) chicken challenge model. The results from the growing feather model show that both the DNA complex (version 3) and commercial vaccine complex (mannosylated chitosan; positive control) induced heterophil and macrophage infiltration. However, only the DNA complex demonstrated a high and prolonged T- and B- lymphocyte infiltration during the recall immune response. challenge strain (S. Enteritidis) as compared to the group vaccinated with killed Salmonella cells. The main platform for producing DNA complex was the rolling circle amplification (RCA) product. In addition to RCA, we developed an alternative and potentially better platform using HCR (Hybridization Chain Reaction) to produce DNA complex, which may have certain advantages over the RCA-based platform. We will evaluate these two different platforms to compare the capacity to activate HD-11 cells.

Publications


    Progress 01/01/22 to 12/31/22

    Outputs
    Target Audience: Nothing Reported Changes/Problems:We had great difficulty recruiting and getting the second graduate student (PhD) in place. There were residual problems which were partially COVID-related and associatied with acquisition of his visa. Santiago Uribe is now in place (Since August 14th) and will begin the animal work very soon. He is currently assisting with aptamer testing in vitro. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?One graduate student presentation was made at the Annual Meeting of the Poultry Science Association in Philidelphia this year, publication is in progress. We anticipate building momentum in the next year. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Major goals of the project 1. Evaluation of the universal CD40-targeting aptamer RCA adjuvant platform as a bivalent system 2. Comprehensive identification of the genetic factors in Salmonella Typhimurium required for horizontal transmission What was accomplished under these goals? We established a chicken gut colonization model using 4 different serotypes of Salmonella enterica (S. Typhimurium, S. Enteritidis, S. Heidelberg, and S. Kentucky). These challenge models will be used for evaluation of the efficacy of the vaccination with the RCA complex we developed. We developed a rolling circle amplification (RCA) product that has a specific binding affinity to various Salmonella enterica serotypes based on published Salmonella-specific aptamers. This RCA product, called 3Sal-RCA, was used as one component in the vaccine complex. We developed 3 different versions of DNA-based adjuvant based on RCA that activates macrophage through CD40 targeting. We established a macrophage activation assay using the HD-11 cell line. This assay has been used to evaluate the capacity of CD40 activation by the RCA vaccine complexes. We found RCA vaccine complex version 2 and 3 activates HD-11 cells significantly as compared to negative controls. We also started Tn-seq experiment to identify Salmonella Typhimurium genes required for survival in chicken feces.

    Publications