Source: AGRICULTURAL RESEARCH SERVICE submitted to
INVESTIGATION OF IMMUNOREGULATION IN REDUCING FOODBORNE PATHOGEN COLONIZATION IN POULTRY
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0430194
Grant No.
(N/A)
Project No.
3091-32000-034-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 3, 2016
Project End Date
Aug 8, 2017
Grant Year
(N/A)
Project Director
KOGUT M H
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
COLLEGE STATION,TX 77845
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3083210109035%
7123220109043%
3083230109010%
7123520109012%
Goals / Objectives
Objective 1: Define the differential host-pathogen interactions between Salmonella and chicken and poultry mucosal immune systems using genomic technologies. Sub-objective 1.A. Screen two lines of chickens and turkeys to identify individual sires and dams that have inherently higher and lower levels of key pro-inflammatory cytokines/chemokines (IL-6, CXCLi2, and CCLi1) and perform specific matings to produce a high and low line of chickens/turkeys and evaluate this novel selection method for increased resistance against Salmonella enterica serovar Enteritidis. Sub-objective 1.B. Evaluate the mucosal immune response and gut microbiome in differentially selected immune lines of chickens and turkeys. Objective 2: Determine the relationship between foodborne pathogens and the mucosal innate immune response focusing on epigenetic reprogramming of host immune genes in persistent infections. Objective 3: Develop new vaccination strategies based on innate immunity to reduce Salmonella contamination in broiler chickens and turkeys. Objective 4: Develop strategies to reduce foodborne pathogens by targeting host immune-metabolic signaling pathways affected by Salmonella and Campylobacter virulence factors. Sub-objective 4.A. Characterize the immune-metabolic pathways through which Salmonella and Campylobacter infection induce a local "tolerogenic" environment in the intestine that controls T regulatory cell development and mediates long-term persistent infection. Sub-objective 4.B. Characterize the immune-metabolic signaling pathways in the ceca of chickens and turkeys treated with various immune modulators that protect birds against Salmonella and Campylobacter infections. Objective 5: Analyze and characterize both host and Salmonella proteins that are modulated in expression during infection using quantitative proteomics. Sub-objective 5.A. Construct a Salmonella proteomic array to identify common Salmonella-specific antigen targets using immune sera from chickens and turkeys infected with different serovars of Salmonella. Sub-objective 5.B. Develop a high-throughput assay to screen small molecules for their ability to inhibit virulence factors produced by various serovars of Salmonella enterica.
Project Methods
Poultry meat products are a major source of human foodborne illness caused by Salmonella and Campylobacter. With poultry producers under increasing pressure to reduce their use of antibiotics to control disease and enhance production, the development of cost-effective, pre-harvest immunological interventions to reduce these microbial pathogens in poultry products would be of great value to the food industry and to the consumer. Immune modulation is one approach for new anti-infective therapies, whereby natural mechanisms in the host can be exploited to strengthen therapeutic benefit. The stimulation of innate immunity has considerable potential to induce a profound and rapid cross-protection against multiple serovars of bacteria. Using "omic" techniques, including functional genomics, epigenetics, proteomics, and metabolomics, we will identify effective modulators of innate immunity to control infections, especially in situations where vaccination is not appropriate. Further, metabolism and host immunity are essential requirements for survival. Mounting an immune response requires major changes to metabolic processes. Thus, the integration of central metabolic pathways and nutrient sensing with antibacterial immunity alters cellular energy homeostasis and contributes to the prevention or resolution of infectious diseases. Hence, immune and metabolic response processes govern infectious diseases. A greater understanding of the critical nodes of immunometabolism during Salmonella and Campylobacter infections will provide opportunities to break the tight connection of defects in metabolism and immunity that propagate persistent infections resulting in improved safety of food products without the use of antibiotics.

Progress 01/03/16 to 08/08/17

Outputs
Progress Report Objectives (from AD-416): Objective 1: Define the differential host-pathogen interactions between Salmonella and chicken and poultry mucosal immune systems using genomic technologies. Sub-objective 1.A. Screen two lines of chickens and turkeys to identify individual sires and dams that have inherently higher and lower levels of key pro-inflammatory cytokines/chemokines (IL-6, CXCLi2, and CCLi1) and perform specific matings to produce a high and low line of chickens/ turkeys and evaluate this novel selection method for increased resistance against Salmonella enterica serovar Enteritidis. Sub-objective 1.B. Evaluate the mucosal immune response and gut microbiome in differentially selected immune lines of chickens and turkeys. Objective 2: Determine the relationship between foodborne pathogens and the mucosal innate immune response focusing on epigenetic reprogramming of host immune genes in persistent infections. Objective 3: Develop new vaccination strategies based on innate immunity to reduce Salmonella contamination in broiler chickens and turkeys. Objective 4: Develop strategies to reduce foodborne pathogens by targeting host immune-metabolic signaling pathways affected by Salmonella and Campylobacter virulence factors. Sub-objective 4.A. Characterize the immune-metabolic pathways through which Salmonella and Campylobacter infection induce a local "tolerogenic" environment in the intestine that controls T regulatory cell development and mediates long-term persistent infection. Sub-objective 4.B. Characterize the immune-metabolic signaling pathways in the ceca of chickens and turkeys treated with various immune modulators that protect birds against Salmonella and Campylobacter infections. Objective 5: Analyze and characterize both host and Salmonella proteins that are modulated in expression during infection using quantitative proteomics. Sub-objective 5.A. Construct a Salmonella proteomic array to identify common Salmonella-specific antigen targets using immune sera from chickens and turkeys infected with different serovars of Salmonella. Sub-objective 5.B. Develop a high-throughput assay to screen small molecules for their ability to inhibit virulence factors produced by various serovars of Salmonella enterica. Approach (from AD-416): Poultry meat products are a major source of human foodborne illness caused by Salmonella and Campylobacter. With poultry producers under increasing pressure to reduce their use of antibiotics to control disease and enhance production, the development of cost-effective, pre-harvest immunological interventions to reduce these microbial pathogens in poultry products would be of great value to the food industry and to the consumer. Immune modulation is one approach for new anti-infective therapies, whereby natural mechanisms in the host can be exploited to strengthen therapeutic benefit. The stimulation of innate immunity has considerable potential to induce a profound and rapid cross-protection against multiple serovars of bacteria. Using "omic" techniques, including functional genomics, epigenetics, proteomics, and metabolomics, we will identify effective modulators of innate immunity to control infections, especially in situations where vaccination is not appropriate. Further, metabolism and host immunity are essential requirements for survival. Mounting an immune response requires major changes to metabolic processes. Thus, the integration of central metabolic pathways and nutrient sensing with antibacterial immunity alters cellular energy homeostasis and contributes to the prevention or resolution of infectious diseases. Hence, immune and metabolic response processes govern infectious diseases. A greater understanding of the critical nodes of immunometabolism during Salmonella and Campylobacter infections will provide opportunities to break the tight connection of defects in metabolism and immunity that propagate persistent infections resulting in improved safety of food products without the use of antibiotics. Hosts have evolved countermeasures to pathogen invasion, establishment, and replication through two types of defenses: resistance and tolerance. Resistance functions to control pathogen invasion and reduce or eliminate the invading pathogen. Work during FY 2017 primarily concentrated on resistance mechanisms that are mediated by the immune system. Tolerance is mediated by different mechanisms that limit the damage caused by a pathogen's growth without affecting or reducing pathogen numbers or loads. The mechanisms of tolerance appear to be separated into those that protect host tissues from the virulence factors of a pathogen and those that limit or reduce the damage caused by the host immune and inflammatory responses to the pathogen (Objective 2). Some pathogens, such as Salmonella, have evolved the capacity to survive the initial robust immune response and persist within the animal. The persistent phase of a Salmonella infection in the avian host usually involves a complex balance of protective immunity and immunopathology. Salmonella is able to stay in the avian ceca for months without triggering clinical signs. Chronic colonization of the intestinal tract is an important aspect of persistent Salmonella infection because it results in a silent propagation of bacteria in poultry stocks due to the impossibility of isolating contaminated animals. Overall, work by project scientists in FY 2017 supports the hypothesis that Salmonella has evolved a unique survival strategy in poultry that minimizes host defenses (disease resistance) during the initial infection, and then exploits and/or induces a dramatic immunometabolic reprogramming in the cecum that alters the host defense to disease tolerance (Objective 5). This disease tolerance is a primary cause of the ongoing human food safety dilemma as related to Salmonella in meat and poultry products. Accomplishments 01 Breeding chickens for resistance to Salmonella and Campylobacter. The breeding for bacterial resistance, along with vaccination, is a potential long-term intervention for controlling these pathogens in broiler chicken production. New approaches are needed to produce poultry that are not colonized by these harmful bacteria, given that the absence of the pathogens in living birds will largely translate into pathogen-free meat products for human consumption. ARS researchers at College Station, Texas, working with collaborators at North Carolina State University and West Virginia University, have identified a population of roosters from the Athens Canadian Random Bred lineage, a 1950s meat-type chicken with differential expression of key immune markers. These roosters are serving as sires for the breeding of chickens that will have a more efficient innate immune responsiveness. Pathogen resistant chickens will be a major step forward in enhancing the microbial safety of poultry products reaching the consumer. 02 Mechanism of Salmonella-exposed chicken resistance to clinical disease. Chickens infected with Salmonella do not develop clinical disease which may be the result of important host interactions with key virulence proteins. ARS researchers at College Station, Texas, working with collaborators from the University of Delaware and the Emory University School of Medicine, inoculated chickens with mutant Salmonella typhimurium that lacked the virulence protein named AvrA and which moderates the host immune response. The leukocyte (white blood cell) migration pathway was altered by AvrA�ST mutants that allowed greater gut barrier permeability and invasion by the mutant. This accomplishment provides important insight into why chickens tolerate the Salmonella bacterium while such tolerance is not observed in many other species. The immunological insights gained by this work are important in ongoing efforts to minimize Salmonella colonization of poultry and thus enhance food safety.

Impacts
(N/A)

Publications

  • Kogut, M.H., Swaggerty, C.L., Byrd, J.A., Selvaraj, R., Arsenault, R.J. 2016. Chicken-specific kinome array reveals that Salmonella enterica serovar Enteritidis modulates host immune signaling pathways in the cecum to establish a persistent infection. International Journal of Molecular Sciences. 17(8):1-20.
  • Kogut, M.H., Arsenault, R.J. 2016. Gut health: The new paradigm in food animal production. Frontiers in Veterinary Science. 3:71. doi: 10.3389/ fvets.2016.00071.
  • Kogut, M.H., Byrd, J.A. 2016. The relationship between the immune response and susceptibility to Salmonella enterica serovar Enteritidis infection in the laying hen. In: Ricke, S.C., Gast, R.K., editors. Producing Safe Eggs. London, UK: Elsevier Inc. p. 209-234.
  • Swaggerty, C.L., Pevzner, I.Y., He, L.H., Genovese, K.J., Kogut, M.H. 2017. Selection for pro-inflammatory mediators produces chickens more resistant to Campylobacter jejuni. Poultry Science. 96(6):1623-1627.
  • Kogut, M.H., Arsenault, R.J. 2017. Immunometabolic phenotype alterations associated with the induction of disease tolerance and persistent asymptomatic infection of Salmonella in the chicken intestine. Frontiers in Immunology. 8:372. doi: 10.3389/fimmu.2017.00372.


Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): Objective 1: Define the differential host-pathogen interactions between Salmonella and chicken and poultry mucosal immune systems using genomic technologies. Sub-objective 1.A. Screen two lines of chickens and turkeys to identify individual sires and dams that have inherently higher and lower levels of key pro-inflammatory cytokines/chemokines (IL-6, CXCLi2, and CCLi1) and perform specific matings to produce a high and low line of chickens/ turkeys and evaluate this novel selection method for increased resistance against Salmonella enterica serovar Enteritidis. Sub-objective 1.B. Evaluate the mucosal immune response and gut microbiome in differentially selected immune lines of chickens and turkeys. Objective 2: Determine the relationship between foodborne pathogens and the mucosal innate immune response focusing on epigenetic reprogramming of host immune genes in persistent infections. Objective 3: Develop new vaccination strategies based on innate immunity to reduce Salmonella contamination in broiler chickens and turkeys. Objective 4: Develop strategies to reduce foodborne pathogens by targeting host immune-metabolic signaling pathways affected by Salmonella and Campylobacter virulence factors. Sub-objective 4.A. Characterize the immune-metabolic pathways through which Salmonella and Campylobacter infection induce a local "tolerogenic" environment in the intestine that controls T regulatory cell development and mediates long-term persistent infection. Sub-objective 4.B. Characterize the immune-metabolic signaling pathways in the ceca of chickens and turkeys treated with various immune modulators that protect birds against Salmonella and Campylobacter infections. Objective 5: Analyze and characterize both host and Salmonella proteins that are modulated in expression during infection using quantitative proteomics. Sub-objective 5.A. Construct a Salmonella proteomic array to identify common Salmonella-specific antigen targets using immune sera from chickens and turkeys infected with different serovars of Salmonella. Sub-objective 5.B. Develop a high-throughput assay to screen small molecules for their ability to inhibit virulence factors produced by various serovars of Salmonella enterica. Approach (from AD-416): Poultry meat products are a major source of human foodborne illness caused by Salmonella and Campylobacter. With poultry producers under increasing pressure to reduce their use of antibiotics to control disease and enhance production, the development of cost-effective, pre-harvest immunological interventions to reduce these microbial pathogens in poultry products would be of great value to the food industry and to the consumer. Immune modulation is one approach for new anti-infective therapies, whereby natural mechanisms in the host can be exploited to strengthen therapeutic benefit. The stimulation of innate immunity has considerable potential to induce a profound and rapid cross-protection against multiple serovars of bacteria. Using "omic" techniques, including functional genomics, epigenetics, proteomics, and metabolomics, we will identify effective modulators of innate immunity to control infections, especially in situations where vaccination is not appropriate. Further, metabolism and host immunity are essential requirements for survival. Mounting an immune response requires major changes to metabolic processes. Thus, the integration of central metabolic pathways and nutrient sensing with antibacterial immunity alters cellular energy homeostasis and contributes to the prevention or resolution of infectious diseases. Hence, immune and metabolic response processes govern infectious diseases. A greater understanding of the critical nodes of immunometabolism during Salmonella and Campylobacter infections will provide opportunities to break the tight connection of defects in metabolism and immunity that propagate persistent infections resulting in improved safety of food products without the use of antibiotics. This is a new project that replaced 3091-32000-031-00D, and which is expanding upon the work of the precursor project. Work during FY 2016 showed that upon infection with Salmonella, the regulation of immune and metabolic pathways in the chicken cecum are altered. During an early (4- 48 h) and late (4-14 d) infection with S. enteritidis, three separate immune-metabolic phases associated with different times post-infections were observed. The first observation was an inflammatory phase 1-3 days post-infection, characterized by the up-regulation of pro-inflammatory cytokine mRNA transcription and the induction of mTOR-mediated anabolic metabolism (protein synthesis, glycogen synthesis, and fatty acid synthesis). The second phase appears around 4 days post-infection and is characterized by profound changes in cecal immunity and metabolism. In general, the local immune microenvironment changed from pro-inflammatory to anti-inflammatory, exemplified by a decrease in pro-inflammatory cytokine mRNA transcription to control levels and a dramatic increase in anti-inflammatory cytokine mRNA expression accompanied by a significant increase in the number and function of Tregs in the cecum. In addition, there was a remarkable metabolic reprogramming in the cecal tissue at this time with a shift from anabolic to catabolic reactions mediated by the significant phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). It is during this phase that Salmonella takes advantage of the lack of host response to infection to begin to establish a persistent cecal colonization. The third phase appears to begin shortly after day 4 post-infection with a gradual return to homeostasis. The number of Tregs in the cecum remains elevated over non-infected tissue and IL-10 and TGF-� appear to regulate a more tolerant microenvironment. Lastly, there is a final metabolic reprogramming from catabolic to homeostasis with no difference in metabolism between infected and non-infected birds. It appears that, at this stage, the Salmonella has established itself as a part of the normal microbiome of the cecum and is no longer recognized by the host immune system. Accomplishments 01 Broiler chickens resistant to Salmonella and Campylobacter. Breeding chickens resistant to Salmonella and Campylobacter infection is considered, along with vaccination, to be a potential long-term intervention in controlling these bacteria in broiler chicken production. New approaches are needed to produce poultry that are not colonized by these harmful bacteria given that absence of the pathogens in living birds will largely translate into pathogen-free meat products for human consumption. ARS researchers at College Station, Texas, have identified a population of roosters from the Athens Canadian Random Bred (ACRB) lineage, a 1950s meat-type chicken, with differential expression of key immune markers to serve as sires for the generation of a F1 population of chickens selected for a more efficient innate immune responsiveness. Development of microbial pathogen-resistant birds would be a dramatic success in enhancing the microbial safety of poultry meat products reaching the consumer.

Impacts
(N/A)

Publications