Progress 01/03/01 to 01/02/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Salmonella and Campylobacter are the leading causes of human food-borne illness caused by bacteria. There are an estimated 2-4 million cases of bacterially derived human gastroenteritis treated each year, costing the United States over $4 billion annually in lost wages and medical costs. Poultry meat products are a major source of human food-borne illness caused by both Salmonella and Campylobacter. More than eight billion broiler chickens are produced and commercially processed in the United States each year. Salmonella and/or Campylobacter contaminate as much as 35% of all processed poultry meat products purchased by consumers. This project aims to develop non-traditional, non-antibiotic, immunologically based strategies to prevent bacterial diseases in poultry. This research
contributes directly to the Microbial Pathogens component of National Program 108 (Food Safety), by developing practical technology for the reduction of microbial pathogens in live poultry that can ultimately contaminate processed poultry food products and cause human illness or death. The work focuses on enhancing the birds' own host defenses during the first week after hatch which is the period of highest susceptibility. This project will provide new technology that will allow production of microbiologically safer meat products leading to a decrease in human food- borne illness. The work is of importance to all involved in the production, processing, distribution, and preparation of products from poultry agriculture, and will directly benefit the American consumer through the availability of microbiologically safer poultry food products. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2002): 1. Complete evaluation of the cytokines
identified and cloned (interferon- gamma, interleukin 8, and p33) from Salmonella enteritidis-immune lymphokine (SILK) in poultry models. 2. Begin design and production of Toll-like receptor probes and optimize cell isolation techniques. 3. Optimize cell isolation techniques to facilitate production of molecular probes. Year 2 (FY 2003): 1. Complete evaluation of the components of innate immune signaling pathways biomarkers of immune competency and/or immune responsiveness of poultry at time of hatch. Year 3 (FY 2004): 1. Identify and produce molecular probes for pattern recognition receptors (PRRs) on cells of the innate immune system. 2. Define pathogen associated molecular patterns (PAMPs) on food-borne bacteria that are recognized by these pattern recognition receptors. Year 4 (FY 2005): 1. Define innate immune response gene expression through the use of functional genomic tools such as microarrays and quantitative RT-PCR. 2. Finalize definition of NF-kB signaling pathway
as biomarkers of immune competency. Year 5 (FY 2006): 1. Establish potentiation of innate immunity by in ovo cytokines or PAMP injection. 2. Adapt knowledge/technology developed to practical enteropathogen reduction strategies. 3. Facilitate efforts of industry partner in finalizing development of commercial enteropathogen-resistant poultry lines. 4a List the single most significant research accomplishment during FY 2006. Innate Immune Response in Poultry Defined by Molecular Techniques: State-of-the-art molecular biology techniques were used to assess gene activation in poultry as related to immune response to infection of birds with Salmonella, an important food poisoning microorganism. In order to develop more effective procedures and protocols for increasing the resistance of poultry to infection by food poisoning microbes such as Salmonella and Campylobacter, a better understanding is required of how the birds' immune system functions. Scientists in the Food and Feed Safety
Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, in collaboration with scientists at the Institute for Animal Health, Compton, United Kingdom, and at the University of Delaware, utilized the molecular techniques of microarray technology and qRT-PCR to identify candidate genes that are activated during the avian innate immune response to Salmonella infections. This accomplishment is important because it provides a clearer understanding of how poultry can potentially defend themselves against invading bacteria; the accomplishment also provides essential knowledge for use in selection of new microbe-resistant lines of poultry and for development of new therapies that could protect the birds against these pathogens. (NP 108; Component 1, Microbial Pathogens; ARS Strategic Plan Performance Measure 3.1.1) 4b List other significant research accomplishment(s), if any. Food Poisoning Bacteria Recognition Receptors Identified in Chickens: Receptors on
an important type of immune cell in chickens were defined, and methods to activate these cells against invading bacteria were identified. To better protect poultry species against invading bacteria that can subsequently cause human food poisoning, methods to stimulate the birds' own innate immune defenses need to be developed. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, were the first to demonstrate a class of receptors (Toll-like) on an important type of chicken cell (heterophil) associated with innate resistance against infection by bacterial pathogens; these heterophil receptors could be stimulated to destroy living bacteria by certain bacterial membrane components (called PAMPS), and also by bacterial DNA. This accomplishment is important because it clearly demonstrated that bacterial infection of baby chickens can be reduced and likely eliminated by stimulating their immune system to act
aggressively against invading microbes; the accomplishment may lead to practical immune-based treatments to prevent bacterial infections of poultry, perhaps as vaccine adjuvants to increase producer acceptance and to greatly reduce costs associated with treatment. (NP 108; Component 1, Microbial Pathogens; ARS Strategic Plan Performance Measure 3.1.1) 5. Describe the major accomplishments to date and their predicted or actual impact. Significant accomplishments were made in understanding the immune system of poultry and how it can be stimulated so that the birds will be more resistant to infection by disease-causing and food poisoning bacteria. Foundational studies were completed to facilitate SILK (Salmonella Immune LymphoKine) technology being used by the poultry industry to treat at- hatch birds so as to render them resistant or even immune to infection by the Salmonella organism. The biochemical mechanisms by which the immune system of newly hatched chicks attacks and kills
enteropathogenic bacteria were largely defined. Cross-breeding studies in chickens have enhanced Salmonella-resistant lines and dramatically facilitated joint ARS/industry efforts to develop commercial poultry lines that are largely immune to disease-causing and food poisoning microorganisms, while retaining performance and quality traits required by producers and demanded by consumers. The genetic basis by which chickens respond, at the cellular level, to Salmonella has been defined, which is foundational for practical exploitation of the immune system in raising poultry free of harmful food-borne bacteria. Project research gave definitive proof that Salmonella DNA fragments, flagellin, and other components of the cell walls of Gram-positive and Gram-negative bacteria can be used to stimulate birds to resist colonization by living Salmonella, an accomplishment that provides the basis for practical development of new bacterial intervention technology which will greatly enhance
human food safety. Genomic analysis of innate cells have used microarray and qRT- PCR technology to identify candidate genes that are activated during the avian innate immune response to Salmonella infections. Results of this research yield a clearer understanding of the innate host defenses to pathogen colonization in the intestine and extra-intestinal organs, provide essential information for the selection of new microbe-resistant lines of poultry, and provide the platform for new therapies that will protect birds against these pathogens. All accomplishments made under this project are fully consistent with relevant milestones listed in the Project Plan, and with the relevant research components as defined in the National Program 108 Action Plan. Accomplishments under this project contribute to the achievement of ARS Strategic Plan Goal 3, Objective 1, Performance Measure 1, in that project accomplishments contribute substantially to attainment of the Agency FY 2007 target of
developing practices and/or products that reduce pathogen contamination of major animal-derived food products. This project expired in FY 2006 but was replaced by 6202-32000-021-00D which will continue and expand upon the work. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? ,One of the products (referred to as SILK) identified by this project and which protects poultry against pathogenic microbes has been provided to other scientists both in the U.S. and abroad for use in research on the prevention and control of a large number of viral, bacterial, and protozoan diseases of poultry. Now that some of the major active components of SILK have been identified, preliminary discussions with pharmaceutical companies interested in developing a purified
immune modulator can begin. A Cooperative Research and Development Agreement (CRADA) with a large poultry breeding firm has been implemented to exploit the innate immune system of poultry in development of new poultry strains that have natural resistance to important pathogenic microorganisms such as Salmonella, Campylobacter, and Escherichia coli. Technology on development of pathogen-resistant poultry lines has been successfully transferred to the supporting CRADA partner. We foresee no significant constraints to the development, within 2-3 years, of practical technology, procedures, and pathogen-resistant poultry lines that will reach the marketplace and that will have a major impact in assuring that poultry are reared as pathogen free as is practically attainable, and that will result in significant enhancement in the microbiological safety of poultry products reaching the American consumer. A second CRADA with a veterinary vaccine firm has been implemented to evaluate new
bacterial vaccines and vaccine adjuvants on the avian host innate immune system to confer protection against important pathogenic microorganisms such as Salmonella, Campylobacter, Clostridium and Escherichia coli. We anticipate that, within 2 years, new practical vaccine adjuvants will reach the marketplace and provide added protective value to multiple vaccines against poultry disease agents. This project expired in FY 2006 but was replaced by 6202-32000-021-00D which will continue and expand upon the work; we expect that significant technological advances to enhance the microbiological safety of poultry food products will be achieved under the new project.
Impacts
(N/A)
Publications
- Kogut, M.H., Iqbal, M., He, H., Philbin, V., Kaiser, P., Smith, A. 2005. Expression and function of toll-like receptors in chicken heterophils. Developmental and Comparative Immunology. 29:791-807.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Salmonella and Campylobacter are the leading causes of human food-borne illness caused by bacteria. There are an estimated 2-4 million cases of bacterially derived human gastroenteritis treated each year, costing the United States over $4 billion annually in lost wages and medical costs. Poultry meat products are a major source of human food-borne illness caused by both Salmonella and Campylobacter. More than eight billion broiler chickens are produced and commercially processed in the United States each year, and Salmonella and/or Campylobacter contaminate as much as 35% of all processed poultry meat products purchased by consumers. This project aims to develop non-traditional, non-antibiotic, immunologically based strategies to prevent bacterial diseases in poultry. This research contributes
directly to the Microbial Pathogens component of National Program 108 (Food Safety), by developing practical technology for the reduction of microbial pathogens in live poultry that can ultimately contaminate processed poultry food products and cause human illness or death. The work focuses on enhancing the birds own host defenses during the first week after hatch which is the period of highest susceptibility. This project will provide new technology that will allow production of microbiologically safer meat products, leading to a decrease in human food-borne illness. This work is supported, in part, by outside funds via a Trust Agreement with Cobb-Vantress, Inc.; these funds are facilitating progress of the work at an accelerated pace. The work is of importance to all involved in the production, processing, distribution, and preparation of products from poultry agriculture, and will directly benefit the American consumer through the availability of microbiologically safer poultry
food products. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2002): 1. Complete evaluation of the cytokines identified and cloned (interferon-gamma, interleukin-8, and p33) from Salmonella enteritidis- immune lymphokine (SILK) in poultry models. 2. Begin design and production of Toll-like receptor probes and optimize cell isolation techniques. 3. Optimize cell isolation techniques to facilitate production of molecular probes. Year 2 (FY 2003): 1. Complete evaluation of the components of innate immune signaling pathways as biomarkers of immune competency and/or immune responsiveness of poultry at time of hatch. Year 3 (FY 2004): 1. Identify and produce molecular probes for pattern recognition receptors (PRRs) on cells of the innate immune system. 2. Define pathogen associated molecular patterns (PAMPs) on food-borne bacteria that are recognized by these pattern recognition receptors. Year 4 (2005): 1. Define innate immune response gene
expression through the use of functional genomic tools such as microarrays and quantitative RT-PCR. 2. Finalize definition of NF-kB signaling pathway as biomarkers of immune competency. Year 5 (2006): 1. Establish potentiation of innate immunity by in ovo cytokines or PAMP injection. 2. Adapt knowledge/technology developed to practical enteropathogen reduction strategies. 3. Facilitate efforts of industry partner in finalizing development of commercial enteropathogen-resistant poultry lines. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Define innate immune response gene expression through the use of functional genomic tools such as microarrays and quantitative RT-PCR. Milestone Fully Met 2. Finalize definition of NF-kB signaling pathway as biomarkers of immune competency. Milestone Fully Met 3b List the milestones that you expect to address over the
next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 (Year 5): 1. Establish potentiation of innate immunity by in ovo cytokines or PAMP injection: After identification of genes expressed during infections and activation of innate immunity, selected genes will be cloned and DNA vaccines will be prepared for injection in ovo. Immune responses will be evaluated over the first 2-3 weeks post-hatch using project-developed assays. 2. Adapt knowledge/technology developed to practical enteropathogen reduction strategies: The signaling events and gene activation patterns will be defined so we can identify appropriate targets for the selective activation or inhibition of specific pathways. 3. Facilitate efforts of industry partner in finalizing development of commercial enteropathogen-resistant poultry lines: The heritability of innate immune function in different genetic lines of chickens will be
assessed using custom microarrays; appropriate technology will be transferred to industry partner. This project will expire in FY 2006. The project plan for the replacement project has been developed, and OSQR review of the new project is scheduled for late FY 2005 or early FY 2006. Milestones for FY 2007 and FY 2008 are recorded in the new project plan. Work under the replacement project will focus on exploiting the genetic make-up of poultry to enhance innate immunity to food poisoning microorganisms, to develop new procedures and technology to minimize gut colonization by the organisms, and to develop new, commercially viable genetic lines of birds that are resistant to colonization by these microbes. 4a What was the single most significant accomplishment this past year? Development of Pathogen-Resistant Lines of Poultry: Candidate genes in chickens that are related to host resistance to pathogenic microorganisms were identified. At the present state of knowledge, gene
discovery is one of the most important research areas that can be employed to hasten knowledge acquisition of avian host immunity to pathogenic microbes for application in pathogen reduction strategies. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, in collaboration with scientists in the United Kingdom and at the University of Delaware, used the sophisticated molecular techniques of microarray technology and qRT-PCR to identify candidate genes that are activated during the avian innate immune response to Salmonella infections. This accomplishment has provided, and will continue to provide as the work progresses, essential information both for the selection of new pathogen- resistant lines of poultry and for new poultry treatment therapies for management of important food poisoning microbes. 4b List other significant accomplishments, if any. Modulation of the Immune System to Stimulate Pathogen
Resistance in Poultry: Methods to stimulate the avian immune system were developed that result in the birds having resistance to colonization/infection by food poisoning microorganisms such as Salmonella. Because of the seriousness of food poisoning microorganisms that can colonize the gut of living poultry and ultimately contaminate poultry food products reaching the consumer, new strategies are needed to permit rearing of pathogen-free birds. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, demonstrated the presence of receptors on avian cells that are associated with the ability of the birds to express immunity to pathogenic microbes; these receptors could be stimulated by bacterially derived components (PAMPS) or by other stimulants. The practical benefits of this accomplishment include the likelihood that PAMP-based products can be developed and safely used to stimulate living birds to resist
colonization by a variety of microbes such as Salmonella and Campylobacter; such products could also be used as novel additives for many poultry vaccines to increase vaccine effectiveness. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Significant accomplishments have been made in understanding the immune system of poultry and how it can be stimulated so that the birds will be more resistant to infection by disease-causing and food poisoning bacteria. Foundational studies were completed to facilitate SILK technology being used by the poultry industry to treat at-hatch birds so as to render them resistant or even immune to infection by the Salmonella organism. The biochemical mechanisms by which the immune system of newly hatched chicks attacks and kills enteropathogenic bacteria were largely defined. Cross-breeding studies in chickens has enhanced Salmonella- resistant lines and dramatically facilitated joint
ARS/industry efforts to develop commercial poultry lines that are largely immune to disease- causing and food-poisoning microorganisms, while retaining performance and quality traits required by producers and demanded by consumers. The genetic basis by which chickens respond, at the cellular level, to Salmonella has been defined, which is foundational for practical exploitation of the immune system in raising poultry free of harmful food- borne bacteria. Project research gave definitive proof that Salmonella DNA fragments, flagellin, and other components of the cell walls of Gram- positive and Gram-negative bacteria can be used to stimulate birds to resist colonization by living Salmonella, an accomplishment that provides the basis for practical development of new bacterial intervention technology which will greatly enhance human food safety. Genomic analysis of innate cells has identified candidate genes that are activated during the avian innate immune response to Salmonella
infections. Project research has provided a clearer understanding of the innate host defenses to pathogen colonization in the intestine and extra- intestinal organs, has provided essential information for the selection of new microbe-resistant lines of poultry, and has developed the platform for new therapies that could provide specific protection against these pathogens. All accomplishments made under this project are fully consistent with relevant milestones listed in the Project Plan, and with the relevant research components as defined in the National Program 108 Action Plan. Accomplishments under this project contribute to the achievement of ARS Strategic Plan Goal 3, Objective 1, Performance Measure 1, in that project accomplishments contribute substantially to attainment of the Agency FY 2007 target of developing practices and/or products that reduce pathogen contamination of major animal-derived food products. 6. What science and/or technologies have been transferred and to
whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? One of the products (referred to as SILK) identified by this project and which protects poultry against pathogenic microbes has been provided to other scientists both in the U.S. and abroad for use in research on the prevention and control of a large number of viral, bacterial, and protozoan diseases of poultry. Now that some of the major active components of SILK have been identified, preliminary discussions with pharmaceutical companies interested in developing a purified immune modulator can begin. A Cooperative Research and Development Agreement (CRADA) with a large poultry breeding firm is ongoing and focuses on exploiting the innate immune system of poultry in development of new poultry strains that have natural resistance to important pathogenic
microorganisms such as Salmonella, Campylobacter, and Escherichia coli. Technology on development of pathogen-resistant poultry lines has been successfully transferred to the supporting CRADA partner. A second CRADA with a veterinary vaccine firm has been implemented to evaluate both new bacterial vaccines and vaccine adjuvants on the avian host innate immune system and on protection against important pathogenic microorganisms such as Salmonella, Campylobacter, Clostridium, and Escherichia coli. We foresee no significant constraints to the development, within 1-3 years, of practical technology, procedures, pathogen-resistant poultry lines, and vaccine adjuvants that will reach the marketplace and that will have a major impact in assuring that poultry are reared as pathogen free as is practically attainable, and that will result in significant enhancement in the microbiological safety of poultry products reaching the American consumer.
Impacts
(N/A)
Publications
- Kogut, M.H., Rothwell, L., Kaiser, P. 2005. IFN-y priming of chicken heterophils upregulates the expression of proinflammatory and TH1 cytokine mRNA following receptor-mediated phagocytosis of Salmonella enterica serovar enteritidis. Journal of Interferon and Cytokine Research. 25:73-81.
- He, H., Lowry, V.K., Ferro, P.J., Kogut, M.H. 2005. CpG- oligodeoxynucleotide-stimulated chicken heterophil degranulation is serum cofactor and cell surface receptor dependent. Developmental and Comparative Immunology. 29:255-264.
- Ferro, P.J., Swaggerty, C.L., Kaiser, P., Pevzner, I.Y., Kogut, M.H. 2004. Heterophils isolated from chickens resistant to extra-intestinal Salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mRNA following infection than heterophils from susceptible chickens. Epidemiology and Infection. 132:1029-1037.
- Ferro, P.J., Swaggerty, C.L., He, H., Rothwell, L., Kaiser, P., Kogut, M.H. 2005. Recombinant chicken IL-6 does not activate heterophils isloated from day-old chickens in vitro. Developmental and Comparative Immunology. 29:375-383.
- Swaggerty, C.L., Lowry, V.K., Ferro, P.J., Pevzner, I.Y., Kogut, M.H. 2005. Disparity in susceptibility to vancomycin-resistant Enterococcus organ invasion in commercial broiler chickens that differ in innate immune responsiveness. Food and Agricultural Immunology. 16:1-15.
- Swaggerty, C.L., Kogut, M.H., Ferro, P.J., Rothwell, L., Pevzner, I.Y., Kaiser, P. 2004. Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens. Immunology. 113:139-148.
- Swaggerty, C.L., Ferro, P.J., Pevzner, I.Y., Kogut, M.H. 2005. Heterophils are associated with resistance to systemic Salmonella enteritidis infections in genetically distinct chicken lines. FEMS Immunology and Medical Microbiology. 43:149-154.
- Lowry, V.K., Farnell, M.B., Ferro, P.J., Swaggerty, C.L., Bahl, A., Kogut, M.H. 2005. Purified beta-glucan as an abiotic feed additive up-regulates the innate immune response in immature chickens against Salmonella enterica serovar enteritidis. International Journal of Food Microbiology. 98:309-318.
- He, H., Lowry, V.K., Swaggerty, C.L., Ferro, P.J., Kogut, M.H. 2004. In vitro activation of chicken leukocytes and in vivo protection against Salmonella enteritidis organ invasion and peritoneal S. enteritidis infection-induced mortality in neonatal chickens by immunostimulatory CpG oligodeoxynucleotide. FEMS Immunology and Medical Microbiology. 43:81-89.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Salmonella and Campylobacter are the leading causes of human foodborne illness caused by bacteria. There are an estimated 2-4 million cases of bacterially derived human gastroenteritis treated each year, costing the United States over $4 billion annually in lost wages and medical costs. Poultry meat products are a major source of human foodborne illness caused by both Salmonella and Campylobacter. More than eight billion broiler chickens are produced and commercially processed in the United States each year. Salmonella and/or Campylobacter contaminate as much as 35% of all processed poultry meat products purchased by consumers. The goal of this project is to develop non-traditional, non-antibiotic, immunologically based strategies to prevent bacterial diseases in poultry. The work focuses on
enhancing the birds' own host defenses during the first week after hatch which is the period of highest susceptibility. This research contributes directly to the Microbial Pathogens component of National Program 108 (Food Safety), by developing practical technology for the reduction of microbial pathogens in live poultry that can ultimately contaminate processed poultry food products and cause human illness or death. This work is supported, in part, by outside funds via a Trust Agreement with Cobb-Vantress, Inc.; these funds are facilitating progress of the work at an accelerated pace. This project will provide new technology to assure production of microbiologically safer meat products leading to a decrease in human foodborne illness. The work is of importance to all involved in the production, processing, distribution, and preparation of products from poultry agriculture, and will directly benefit the American consumer through the availability of microbiologically safer poultry
food products. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2002): 1. Evaluate cytokines identified and cloned (interferon-gamma, interleukin-8, and p33) from Salmonella enteritidis-immune lymphokine (SILK) in poultry/Salmonella models. 2. Begin design and production of Toll-like receptor (TLR) probes. 3. Optimize cell isolation techniques to facilitate production of molecular probes. Year 2 (FY 2003): 1. Evaluate components of innate immune signaling pathways as biomarkers of immune competency and/or immune responsiveness of poultry at time of hatch. Year 3 (FY 2004): 1. Produce molecular probes for TLRs. 2. Identify pattern recognition receptors (PRRs) on cells of the innate immune system. 3. Define pathogen associated molecular patterns (PAMPs) on food-borne bacteria that are recognized by the identified pattern recognition receptors. Year 4 (FY 2005): 1. Finalize definition of NF-kB signaling pathway as biomarkers of immune competency. 2.
Define innate immune response gene expression through the use of functional genomic tools including microarrays and quantitative RT-PCR. Year 5 (FY 2006): 1. Establish potentiation of innate immunity by in ovo cytokine or PAMP injection. 2. Adapt knowledge/technology developed to practical enteropathogen reduction strategies. 3. Facilitate efforts of industry partner in finalizing development of commercial enteropathogen-resistant poultry lines. 3. Milestones: A. Milestones scheduled to be addressed in FY 2004: 1. Produce molecular probes for TLRs. 2. Identify pattern recognition receptors (PPRs) on cells of the innate immune system. 3. Define pathogen associated molecular patterns (PAMPs) on food-borne bacteria that are recognized by the identified pattern recognition receptors. All of the FY 2004 milestones were fully met. B. Milestones to be addressed in FY 2005, FY 2006, and FY 2007: FY 2005 (Year 4): 1. Finalize definition of NF-kB signaling pathway as biomarkers of immune
competency: Use specific pharmacological inhibitors to prevent the phosphorylation of the NF-kB pathway and assess immune function of cells of the innate immune system (heterophils). 2. Define innate immune response gene expression through the use of functional genomic tools including microarrays and quantitative RT-PCR: Profile TLR, cytokine, and chemokine genes in heterophils following activation with bacteria and TLR agonists. FY 2006 (Year 5): 1. Establish potentiation of innate immunity by in ovo cytokine or PAMP injection: After identification of genes expressed during infections and activation of innate immunity, selected genes will be cloned and DNA vaccines will be prepared for injection in ovo. Immune response will be evaluated over the first 2-3 weeks post-hatch using project-developed assays. 2. Adapt knowledge/technology developed to practical enteropathogen reduction strategies: Define the signaling events and gene activation patterns so that we can identify
appropriate targets for the selective activation or inhibition of specific pathways. 3. Facilitate efforts of industry partner in finalizing development of commercial enteropathogen-resistant poultry lines: Assess heritability of innate immune function in different genetic lines of chickens using custom microarrays; transfer technology to industry partner. FY 2007: This project will expire in FY 2006. The Project Plan for the replacement project is under preparation, and the OSQR review of the new project is scheduled for mid-FY 2006. We anticipate FY 2007 milestones under the new Project Plan to include: 1. Evaluate avian heterophil-secreted antimicrobial peptides following activation of Toll-like receptors: Identify both anti-bacterial (defensins and cathelocidins) and anti-viral proteins secreted by heterophiils in response to TLR agonists. Establish anti-microbial activity using in vitro cell culture models. 2. Assess heritability of innate immune function in different genetic
lines of chickens: Use custom microarrays containing polymorphic immune response genes to evaluate crosses of chickens that have been bred for innate immune responsiveness. 3. Profile differential expressed genes in Salmonella-exposed chicken immune cells using Suppression Subtractive Hybridization: Construct expressed sequence tags (ESTs) representing differentially expressed genes from cDNA libraries of heterophils isolated from differentially innate immune responsive lines of chickens by subtracting those sequences common to both normal and stimulated heterophils. Add these genes to micoarrays used in evaluating genetic lines of chickens for innate immune responses. 4. Identify genes required by Salmonella and Campylobacter to induce heterophil infiltration in chickens and turkeys: Define the rapid resistance to bacterial invasion mediated by heterophils and identify practical mechanisms for use in preventing colonization. Use chicken epithelial cell transmigration models to
screen bacterial strains and serotypes for their capacity to induce heterophil migration. Use RNA from infected and uninfected epithelial cells to probe microarrays of immune and epithelial cell genes. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: Certain cells in the chicken were stimulated in a manner such that the cells then exhibited the ability to kill enteropathogenic bacteria. This represents a potentially new method for increasing resistance of birds to colonization by important food-poisoning microorganisms such as Salmonella and Campylobacter. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, in collaboration with scientists at the Institute for Animal Health, Compton, United Kingdom, showed that chicken heterophils can be induced by treatment with Toll-like receptor agonists so as to cause the heterophils to exhibit
strong enteropathogenic microbial killing activity. This accomplishment provides more understanding of the innate host defenses of poultry to Salmonella and other pathogen colonization, and provides essential information for the development of new vaccines and also the platform for new therapies that could provide effective protection against these pathogens. B. Other significant accomplishment(s): New lines of poultry that differ significantly in their innate ability to resist colonization by enteropathogenic bacteria were identified. Exploiting bacterial resistance phenomena in poultry could provide very efficient and cost-effective means of minimizing bird colonization by food-poisoning bacteria which would greatly enhance poultry food safety. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, working with CRADA partner Cobb-Vantress, Inc., evaluated inherent resistance factors in chickens for
subsequent selection of immunologically efficient lines of chickens that would be resistant to bacterial colonization. The ultimate impact of this work, done in close collaboration with one of the Nation's major pedigree broiler breeder companies, will be commercial poultry lines that are largely if not totally resistant to colonization by disease-causing and food-poisoning microorganisms such as Salmonella. C. Significant activities that support special target populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Significant accomplishments have been made under this project in understanding the immune system of poultry and how it can be stimulated so that the birds will be more resistant to infection by disease-causing and food-poisoning bacteria. Foundational studies were completed to facilitate SILK technology being utilized by the poultry industry to treat at-hatch birds so as to render them resistant
or even immune to infection by the Salmonella organism. The biochemical mechanisms by which the immune system of newly hatched chicks attacks and kills enteropathogenic bacteria were largely defined. Cross-breeding studies in chickens has enhanced Salmonella-resistant lines and dramatically facilitated joint ARS/industry efforts to develop commercial poultry lines that are largely immune to disease-causing and food-poisoning microorganisms, while retaining performance and quality traits required by producers and demanded by consumers. The genetic basis by which chickens respond, at the cellular level, to Salmonella has been defined, which is foundational for practical exploitation of the immune system in raising poultry free of harmful food-borne bacteria. Project research gave definitive proof that Salmonella DNA fragments can be used to stimulate birds to resist colonization by living Salmonella, an accomplishment that provides the basis for practical development of new
bacterial intervention technology which will greatly enhance human food safety. All accomplishments made under this project are fully consistent with relevant milestones listed in the Project Plan, and with the relevant research components as defined in the National Program 108 Action Plan. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? One of the products (referred to as SILK) identified by this project and which protects poultry against pathogenic microbes has been provided to other scientists both in the U.S. and abroad for use in research on the prevention and control of a large number of viral, bacterial, and protozoan diseases of poultry. Now that some of the major active components of SILK have been identified, discussions with
pharmaceutical companies interested in developing a purified immune modulator will begin. A Cooperative Research and Development Agreement with a large poultry breeding firm has been implemented to exploit the innate immune system of poultry to develop new poultry strains that have natural resistance to important pathogenic microorganisms such as Salmonella, Campylobacter, and Escherichia coli. Technology on development of pathogen resistant poultry lines has been successfully transferred to the supporting CRADA partner. We foresee no significant constraints to the development, within 2-3 years, of practical technology, procedures, and pathogen- resistant poultry lines that will reach the marketplace and that will have a major impact in assuring that poultry are reared as pathogen free as is practically attainable, and that will result in significant enhancement of the microbiological safety of poultry products reaching the American consumer.
Impacts
(N/A)
Publications
- Crippen, T.L., Pevzner, I.Y., Lowry, V.K., Farnell, M.B., Kogut, M.H. 2003. Innate immunoprofiling of commercial broiler chicken lines. Online Journal of Veterinary Research. 7:78-90. Available: http://www.cpb.uokhsc. edu/ojvr/crippen2003/immunoprofilingbroilers/immunobroilers200.html.
- Swaggerty, C.L., Pevzner, I.Y., Ferro, P.J., Crippen, T.L., Kogut, M.H. 2003. Association between in vitro heterophil function and the feathering gene in commercial broiler chickens. Avian Pathology. 32:483-488.
- Farnell, M.B., He, H., Genovese, K.J., Kogut, M.H. 2003. Pharmacological analysis of signal transduction pathways required for oxidative burst in chicken heterophils stimulated by a toll-like receptor 2 agonist. International Immunopharmacology. 3:1677-1684.
- Kogut, M.H., Rothwell, L., Kaiser, P. 2003. Priming by recombinant chicken interleukin-2 induces selective expression of IL-8 and IL-18 MRNA in chicken heterophils during receptor-mediated phagocytosis of opsonized and nonopsonized Salmonella enterica serovar enteritidis. Molecular Immunology. 40:603-610.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Salmonella and Campylobacter are the leading causes of human foodborne illness caused by bacteria. There are an estimated 2-4 million cases of bacterially derived human gastroenteritis treated each year, costing the United States over four billion dollars annually in lost wages and medical costs. Poultry meat products are a major source of human foodborne illness caused by both Salmonella and Campylobacter. This project aims to develop non-traditional, non-antibiotic, immunologically based strategies to prevent bacterial diseases in poultry. The work focuses on enhancing the birds' own host defenses during the first week after hatch which is the period of highest susceptibility. 2. How serious is the problem? Why does it matter? More than eight billion broiler chickens are produced and commercially processed in the United States each year. Salmonella and/or Campylobacter contaminate
as much as 35% of all processed poultry meat products purchased by consumers. This project will provide new technology that will allow production of microbiologically safer meat products leading to a decrease in human foodborne illness. The work is of importance to all involved in the production, processing, distribution, and preparation of products from poultry agriculture, and will directly benefit the American consumer through the availability of microbiologically safer poultry food products. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research contributes directly to the Microbial Pathogens component of National Program 108 (Food Safety), by developing practical technology for the reduction of microbial pathogens in live poultry that can ultimately contaminate processed poultry food products and cause human illness or death. Portions of the research contribute to the Strategies to Control Infectious and
Non-Infectious Disease Component of National Program 103 (Animal Health) by developing research information and therapeutic approaches that will better assure animal health. This project is supported, in part, by outside funds through a Trust Agreement with a cooperating industry partner, which is facilitating research progress at an accelerated pace. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003: Poultry meat products are a major source of human foodborne illness; there is need for new and more effective strategies to prevent poultry colonization by food poisoning bacteria such as Salmonella. Scientists in the Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, College Station, Texas, in collaboration with the Institute for Animal Health, Compton, United Kingdom, used functional genomics techniques to determine that the heterophil, the first cell type encountering Salmonella
infection in the intestine, is capable of rapid changes in cytokine gene expression following receptor-mediated phagocytosis. The research provided good insights into how heterophils can respond to different phagocytic stimuli and how, by controlling cytokine gene expression, they can function in regulating inflammatory and subsequent acquired immune responses. This accomplishment is important because it suggests that the production of cytokines by heterophils may play a significant role in orchestrating not only pro- or anti-inflammatory activities, but also immunoregulatory actions in chickens which can be exploited to make the birds resistant to colonization by food poisoning bacteria. B. Other Significant Accomplishment(s), if any: Because certain Salmonella strains that grow in the gut of poultry can escape the gut and infect the birds' organs and tissues, new methods are needed to prevent this occurrence and thus better assure human protection from these food poisoning
microbes. Scientists in the Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, College Station, Texas, studied the effects of administration of bacterial DNA fragments from the Salmonella organism on organ invasion by living Salmonella. The results showed that the DNA fragments (known as unmethylated CpG) were highly effective in protecting newly hatched chicks from organ invasion by Salmonella. This accomplishment provides clear evidence that the birds' immune system can recognize certain isolated genetic components from Salmonella, that these components stimulate the birds to develop "resistance" to the spread of Salmonella within the body, and that this phenomenon can likely be exploited to develop effective technology to protect birds from Salmonella and thus enhance human food safety. Salmonella is a dangerous food poisoning bacterium that can infect humans via contaminated poultry food products; new technologies are needed to assure that poultry
are protected from Salmonella during the critical first few days of life. Scientists in the Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, College Station, Texas, in collaboration with Texas AM University, discovered that white blood cells of day-of-hatch poultry (specifically heterophils and monocytes) possess the genes for pattern recognition receptors (PRRs) that alert the host innate immune system to the presence of bacterial infections, which in turn activates a protective immune response. These genes are capable of being up-regulated to stimulate the production of more receptors on the cells, thus the induction of a better immune response. This accomplishment has significantly advanced knowledge toward identifying target sites that regulate the ability of natural mechanisms within poultry to fight off bacterial infection; ultimately, better methods to protect birds from infection with disease-causing and human food poisoning microorganisms
will result. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. During the life of this project, significant accomplishments have been made in understanding the immune system of poultry and how it can be stimulated so that the birds will be more resistant to infection by disease-causing and food poisoning bacteria. It was definitively established that natural body chemicals (called cytokines) can protect birds against Salmonella infection. Other natural body chemicals (called T lymphocytes) can be stimulated in a manner so as to protect both newly hatched chickens and turkeys from a particularly dangerous species of Salmonella (S. enteritidis). One of the most significant accomplishments arising out of this project was the demonstration that a cytokine "soup" administered to young poultry by any of a variety of methods protects the
birds from many different Salmonella types, and also from infection by other harmful microorganisms including E. coli and Pasturella. Development of novel cytokine-based intervention strategies will benefit the poultry industry by providing another tool to reduce and/or prevent microbial numbers in food products. Further, these cytokines could also be used as adjuvants in current or new vaccines. Project accomplishments have thus far met the 1- and 2-year milestones as defined in the Project Plan by identification of the components of our Salmonella-immune lymphokine (SILK) that appear to be important in inducing the protective innate immunity against multiple bacterial infections in day-of-hatch chickens and turkeys, identifying Toll-like receptors on cells of the innate immune system, developing assays to measure signal transduction pathways in cells of the innate system, identifying news PAMPs that can be used to induce protection against Salmonella infections in day-of- hatch
chickens, and identifying innate immune response genes that are activated during the induction of an avian innate immune response. These accomplishments contribute significantly to the goals of enhancing microbial food safety of poultry food products as defined in the Food Safety National Program Action Plan (NP 108) and position the project well to reach the milestones defined for years 3 to 5 of the project. 6. What do you expect to accomplish, year by year, over the next 3 years? In FY 2004, genetic studies will accomplish the isolation, cloning, and purification of cytokine receptors as a prerequisite to developing effective protocols to prevent Salmonella infection in poultry. Salmonella vaccination/immunization studies with appropriate immune system components of poultry will be completed. In FY 2005, we will identify molecular events and biochemical pathways altered in chicken innate immune cells during Salmonella and Campylobacter infections in day- of-hatch chicks. In FY
2006, work on developing appropriate "markers" for evaluating the immune response of newly hatched poultry against Salmonella will be accomplished. Molecular studies will be completed on the definition of heterophil receptor phenomena that act to protect birds from Salmonella, E. coli, and Campylobacter. Practical and commercially viable gene transfer technology will be developed that will induce (via egg inoculation) Salmonella resistance in the resulting chicks. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? One of the products (referred to as SILK) identified by this project and which protects poultry against pathogenic microbes has been provided to other scientists both in the U.S. and abroad for use in research on the prevention and control
of a large number of viral, bacterial, and protozoan diseases of poultry. Now that some of the major active components of SILK have been identified, preliminary discussions with pharmaceutical companies interested in developing a purified immune modulator will begin. A Cooperative Research and Development Agreement with a large poultry breeding firm has been implemented to exploit the innate immune system of poultry in development of new poultry strains that have natural resistance to important pathogenic microorganisms such as Salmonella, Campylobacter, and E. coli. We foresee no significant constraints to the application of the accomplishments of this project in development of practical technology for reducing food poisoning microorganisms in poultry. We anticipate that commercially viable, effective products will reach the marketplace within 2-4 years. 8. List your most important publications in the popular press and presentations to organizations and articles written about
your work. (NOTE: This does not replace your peer-reviewed publications listed below). Kogut, M.H. Cytokine responses: What do we know and their affect on future vaccine development (oral presentation). American Association of Avian Pathologists Symposium on Poultry Vaccines and Vaccination Practices. Nashville, TN. 2002. Kogut, M.H. Activation of innate immunity and reduction of intestinal food-borne pathogens on poultry (oral presentation). National Turkey Federation Roundtable on On-Farm Pathogen Reduction Strategies-How Can the Industry Reduce Pathogens With On-Farm Interventions? San Antonio, TX. 2003. Kogut, M.H. Immunomodulation of the avian innate immune response to prevent bacterial infections in poultry (oral presentation). 26th Annual European Technical Turkey Conference. Manchester, UK. 2003. Crippen, T.L. The modulation of innate immunity to enhance disease resistance in poultry (oral presentation). FSIS Meat and Poultry Inspection Seminar for International Government
Officials. College Station, TX. 2003.
Impacts
(N/A)
Publications
- He, H., Kogut, M.H. CpG-ODN-induced nitric oxide production is mediated through clathrin-dependent endocytosis, endosomal maturation, and activation of PKC, MEK1/2 and p38 MAPK, and NF-kB pathways in avian macrophage cells (HD11). Cellular Signaling. 2003. v. 15. p. 911-917.
- Farnell, M.B., He, H., Kogut, M.H. Differential activation of signal transduction pathways mediating oxidative burst by chicken heterophils in response to stimulation with lipopolysaccharides and lipoteichoic acid. Inflammation. 2003. v. 27. p. 233-239.
- Kogut, M.H., Rothwell, L., Kaiser, P. Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2. Developmental and Comparative Immunology. 2002. v. 26. p. 817-83.
- He, H., Farnell, M.B., Kogut, M.H. Inflammatory agonist stimulation of oxidative burst by neonatal chicken heterophils. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology. 2003. v. 135. p. 177-184.
- Crippen, T.L., Bischoff, K.M., Lowry, V.K., Kogut, M.H. rP33 bacterial killing by chicken peripheral blood heterophils. Journal of Food Protection. 2003. v. 66. p. 787-792.
- Farnell, M.B., Crippen, T.L., Haiqi, H., Swaggerty, C.L., Kogut, M.H. Oxidative burst mediated by toll-like receptors (TLR) and CD14 on avian heterophils stimulated with bacterial toll agonists. Developmental and Comparative Immunology. 2003. v. 27. p. 423-429.
- Swaggerty, C.L., Pevzer, I.Y., Lowry, V.K., Farnell, M.B., Kogut, M.H. Functional comparison of heterophils isolated from commercial broiler chickens. Avian Pathology. 2002. v. 32. p. 95-102.
- Crippen, T.L., He, H., Sheffield, C.L., Lowry, V.K., Kogut, M.H. Differential nitric oxide production by chicken immune cells. Developmental and Comparative Immunology. 2003. v. 27. p. 603-610.
- He, H., Crippen, T.L., Farnell, M.B., Kogut, M.H. Identification of CpG oligonucleotide motifs that stimulate nitric oxide and cytokine production in avian macrophage and peripheral blood mononuclear cells. Developmental and Comparative Immunology. 2003. v. 27. p. 621-627.
- Kogut, M.H., Lowry, V.K., Farnell, M.B. The use of selective pharmacological inhibitors to delineate signal transduction pathways activated during complement receptor-mediated degranulation in chicken heterophils. International Immunopharmacology. 2003. v. 3. p. 693-706.
- Kogut, M.H., Rothwell, L., Kaiser, P. Differential regulation of cytokine gene expression by avian heterophils during receptor-mediated phagocytosis of opsonized and nonopsonized Salmonella enteritidis. Journal of Interferon and Cytokine Research. 2003. v. 23. p. 319-327.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Salmonella and Campylobacter are the leading causes of human foodborne illness caused by bacteria. There are an estimated 2-4 million cases of bacterially derived human gastroenteritis treated each year, costing the United States over $4 billion annually in lost wages and medical costs. Poultry meat products are a major source of human foodborne illness caused by both Salmonella and Campylobacter. This project aims to develop non-traditional, non-antibiotic, immunologically based strategies to prevent bacterial diseases in poultry. The work focuses on enhancing the birds' own host defenses during the first week after hatch, which is the period of highest susceptibility. 2. How serious is the problem? Why does it matter? More than eight billion broiler chickens are produced and commercially processed in the United States each year. Salmonella and/or Campylobacter contaminate as much as
35% of all processed poultry meat products purchased by consumers. This project will provide new technology that will allow production of microbiologically safer meat products, leading to a decrease in human foodborne illness. The work is of importance to all involved in the production, processing, distribution, and preparation of products from poultry agriculture, and will directly benefit the American consumer through the availability of microbiologically safer poultry food products. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? This research contributes directly to the Microbial Pathogens component of National Program 108 (Food Safety), by developing practical technology for the reduction of microbial pathogens in live poultry that can ultimately contaminate processed poultry food products and cause human illness or death. Portions of the research contribute to the Strategies to Control Infectious and
Noninfectious Disease component of National Program 103 (Animal Health) by developing research information and therapeutic approaches that will better assure animal health. This project is supported by outside funds through a Trust Agreement with a cooperating industry partner, which is facilitating research progress at an accelerated pace. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: Poultry meat products are a major source of human foodborne illness; therefore, there is need for new and more effective strategies to prevent poultry colonization by food-poisoning bacteria such as Salmonella. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, TX, in collaboration with the Institute for Animal Health, Compton, United Kingdom, determined the components of the naturally occurring Salmonella-immune lymphokines (SILK) in poultry that
provide significant protection against Salmonella infections in day-old chickens and turkeys. The research resulted, for the first time, in the active components of SILK being characterized and their occurrence correlated in Salmonella-immune versus nonimmune chickens. This accomplishment provides the necessary foundational information for subsequent work that will result in SILK technology being developed and used by the poultry industry to produce birds that are highly resistant or even immune to infection by the Salmonella organism. B. Other Significant Accomplishment(s), if any: Newly hatched poultry have poorly functioning immune systems and the birds are very susceptible to bacterial infections; new technologies are needed to assure bird health during the critical first few days of life. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, in collaboration with Texas A&M University, determined the
interactions between Salmonella and the receptors on certain bacteria-killing cells (called heterophils) that occur naturally in the birds. Depending upon the way the Salmonella bacterium interacted with receptors of the heterophil, the heterophil itself reacted in different ways to achieve potential killing of the bacterium. This accomplishment has significantly advanced knowledge toward identifying target sites that regulate the ability of natural mechanisms within poultry to fight off bacterial infection; ultimately, better methods to protect birds from infection with disease-causing and human food poisioning microorganisms will result. Because certain Salmonella strains that grow in the gut of poultry can escape the gut and infect the birds' organs and tissues, new methods are needed to prevent this occurrence and thus better assure human protection from these food-poisoning microbes. Scientists in the Food and Feed Safety Research Unit at the Southern Plains Agricultural
Research Center, College Station, Texas, studied the effects of administration of certain DNA fragments from the Salmonella organism on organ invasion by living Salmonella. The results showed that the DNA fragments (known as unmethylated CpG dinucleotides) were highly effective in protecting newly hatched chicks from organ invasion by Salmonella. This accomplishment provides clear evidence that the birds' immune system can recognize certain isolated genetic components from Salmonella, that these components stimulate the birds to develop "resistance" to the spread of Salmonella within the body and, furthermore, that this phenomenon can likely be exploited to develop effective technology to protect birds from Salmonella and thus enhance human food safety. C. Significant Accomplishments/Activities that Support Special Target Populations: None 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? During the life of this project,
significant accomplishments have been made in understanding the immune system of poultry and how it can be stimulated so that the birds will be more resistant to infection by disease-causing and food-poisoning bacteria. It was definitively established that natural body chemicals (called cytokines) can protect birds against Salmonella infection. Other natural body chemicals (called T lymphocytes) can be stimulated in a manner so as to protect both newly hatched chickens and turkeys from a particularly dangerous species of Salmonella (S. enteritidis). One of the most significant accomplishments arising out of this project is the demonstration that a cytokine "soup" administered to young poultry by any of a variety of methods protects the birds from many different Salmonella types, and also from infection by other harmful microorganisms including Escherichia coli and Pasturella. Accomplishments arising from this project will ultimately result in development of vaccine-like products
that will both protect poultry from costly diseases and also assure microbiologically safer poultry products for the American consumer. 6. What do you expect to accomplish, year by year, over the next 3 years? Normal progression of the work during the next three years is anticipated. In FY-03, further definition of the receptors on birds' immune cells, and how they act to destroy Salmonella, will be accomplished. The physiological and chemical basis of heterophil response to both Salmonella and Campylobacter (a serious food poisioning microorganism) will be defined. Appropriate systems to assure effective expression of critical SILK components in protecting birds from Salmonella infection will be developed. In FY-04, genetic studies will accomplish the isolation, cloning, and purification of cytokine receptors as a prerequisite to developing effective protocols to prevent Salmonella infection in poultry. Salmonella vaccination/immunization studies with appropriate immune system
components of poultry will be completed. In FY- 05, work on developing appropriate "markers" for evaluating the immune response of newly hatched poultry against Salmonella will be accomplished. Molecular studies will be completed on the definition of heterophil receptor phenomena that act to protect birds from Salmonella, Escherichia coli, and Campylobacter. Practical and commercially viable gene transfer technology will be developed that will induce (via egg inoculation) Salmonella resistance in the resulting chicks. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? One of the products (referred to as SILK) identified by this project and which protects poultry against pathogenic microbes has been provided to other scientists both in the U.S. and abroad for use in research on the prevention and
control of a large number of viral, bacterial, and protozoan diseases of poultry. A Cooperative Research and Development Agreement with a large poultry breeding firm has been implemented to exploit the innate immune system of poultry in development of new poultry strains that have natural resistance to important pathogenic microorganisms such as Salmonella, Campylobacter, and Escherichia coli.
Impacts
(N/A)
Publications
- Crippen, T.L., Ripley, L.H., Farnell, M.B., Lowry, V.K., Sheffield, C.L., Kogut, M.H. Differential nitric oxide production by immune cells of chickens. Journal of Leukocyte Biology. 2001. v. 70(Suppl.): Abstract p. 45.
- Kogut, M.H. Signal transduction pathways activated by engaging immunoglobulin Fc receptors on chicken heterophils. Developmental and Comparative Immunology. 2001. v. 25. p. 639-646.
- Bischoff, K.M., Pishko, E.J., Genovese, K., Crippen, T.L., Holtzapple, C., Stanker, L.H., Nisbet, D.J., Kogut, M.H. The chicken mim-1, P33, is a heterophil chemotactic factor present in Salmonella enteritidis-immune lymphokine. Journal of Food Protection. 2001. v. 64. p. 1503-1509.
- Kogut, M.H. Dynamics of a protective avian inflammatory response: The role of an IL-8-like cytokine in the recruitment of heterophils to the site of organ invasion by Salmonella enteritidis. Comparative Immunology, Microbiology, and Infectious Diseases. 2001. v. 25. p. 159-172.
- Kogut, M.H., Crippen, T.L., Lowry V.K., Farnell, M. Fc receptor signaling of chicken heterophil degranulation involves p38 mitogen-activated protein kinase, phospholipase C, phosphatidylinositol 3'-kinase, and calcium mobilization. International Immunopharmacology. 2002. v. 2. p. 963-973.
- Kogut, M.H. Immunomodulation of the avian innate immune response to prevent bacterial infections in poultry. Available from: http://www. feedinfo.com/asp/scientific/science_res.asp (2002).
- Kogut, M.H., Crippen, T.L., Lowry, V.K., Farnell, M. Fc receptor signaling of chicken heterophil degranulation involves p38 mitogen-activated protein kinase, phospholipase C, phosphatidylinositol 3'-kinase, and calcium mobilization. Veterinary Immunology International Symposium. 2001. Abstract p. 102.
- Farnell, M.B., Crippen, T.L., He, H., Kogut, M.H. Oxidative burst mediated by Toll-like receptors and CD14 in avian heterophils stimulated with bacterial components. Journal of Leukocyte Biology. 2001. v. 70(Suppl.): Abstract p. 38.
- Swaggerty, C.L., Farnell, M.B., Kogut, M.H. Differential functions stimulated by the activation of Toll-like receptors in chicken heterophils. Journal of Leukocyte Biology. 2001. v. 70(Suppl.): Abstract p. 66.
- He, H., Farnell, M.B., Crippen, T.L., Kogut, M.H. Activation of chicken immune cells by unmethylated CpG dinucleotide motif of bacterial DNA. Journal of Leukocyte Biology. 2001. v. 70(Suppl.): Abstract p. 38.
- Lowry, V.K., Farnell, M.B., Swaggerty, C.L., Kogut, M.H. Detection of protein tyrosine kinase in immature chicken heterophils. Journal of Leukocyte Biology. 2001. v. 70(Suppl.): Abstract p. 56.
- Kogut, M.H., Kaiser, P. Cytokine responses: What do we know and their affect on future vaccine development. Symposium on Poultry Vaccines and Vaccination Practices. 2002. Abstract p. 4-8.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
Salmonella and Campylobacter are the leading causes of human food-borne illness caused by bacteria. There are an estimated 2-4 million cases of bacterially derived human gastroenteritis treated each year, costing the United States over four billion dollars annually in lost wages and medical costs. Poultry meat products are a major source of human food- borne illness caused by both Salmonella and Campylobacter. Our research attempts to develop non-traditional, non-antibiotic, immunologically based strategies to prevent bacterial diseases in poultry. The work focuses on up regulating the birds' own host defenses during the first week after hatch which is the period of highest susceptibility.
2. How serious is the problem? Why does it matter?
More than eight billion broiler chickens are produced and commercially processed in the United States each year. Salmonella and/or Campylobacter contaminate as much as 35% of all processed poultry meat products purchased by consumers. This research will provide new technology that will allow production of microbiologically safer meat products leading to a decrease in human food-borne illness. The work is of importance to all involved in the production, processing, distribution, and preparation of products from poultry agriculture, and will directly benefit the American consumer through the availability of microbiologically safer poultry food products.
3. How does it relate to the National Program(s) and National Component(s)?
This research is covered under the Food Safety (Animal and Plant Products) National Program Action Plan 108, with research directed towards the preharvest microbial pathogen reduction objective. Our research will focus on two primary preharvest activities: 1.3. ecology and the host-pathogen relationship - emphasis will be placed on defining the mechanisms of local mucosal immunity and the role of the pathogen in stimulating this response; 1.4. intervention strategies - emphasis on objectives 1.4.1.1 developing immunologically-based interventions, and 1.4.1.4 developing strategies to minimize pathogens, changes in drugs and vaccines. Portions of the research contribute to National Program 103 (Animal Health).
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2001 year: As part of our attempts to improve the ability of newly-hatched poultry to fight off bacterial infections, we determined: (a) the interactions between Salmonella and the specific receptors on chicken heterophils and (b) the cascade of biochemical reactions in the host cell that results in the ingestion and killing of the pathogen. This work was done in collaboration with researchers from Texas A&M University. We found that specific, but different signal events are activated inside the heterophils depending upon the route of ingestion of the Salmonella through either their Fc-, complement-, or Toll-receptors that result in the death of the bacteria. These findings are significant, not only in providing us with a basic understanding of the signals that regulate heterophil functional activity, but also in suggesting ways to maximize the microbicidal activities of heterophils and, thus, the innate response. B. Other
Significant Accomplishment(s), if any: none C. Significant Accomplishments/Activities that Support Special Target Populations: none
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
We demonstrated the successful cytokine-mediated protection of neonatal poultry against salmonellosis. We found that the intraperitonal (ip) injection of conditioned medium prepared from Con A-stimulated T lymphocytes isolated from the spleen of chickens immunized against Salmonella enteritidis (SE), and protected newly-hatched chicks from oral or ip challenge with S. enteritidis. This protection was also induced in day-old turkey poults. The cytokine-mediated protection was generated when administered prophylactically either in ovo at 18 days of embryogenesis, orally, subcutaneously, or nasally at hatch. Poultry administered this cytokine soup were protected against vertical and horizontal transmission of paratyphoid and typhoid salmonellae infections. This cross-protection between serotypes, regardless of antigenic differences, is in contrast to most Salmonella vaccines.
6. What do you expect to accomplish, year by year, over the next 3 years?
This project replaced 6202-42000-014-00D. We anticipate normal progression of the work during the next three years. In FY-02, we will begin to design and produce probes for Toll-like receptors on the surface of avian heterophils and NK cells. These probes will be used as DNA markers to identify immunologically competent birds in response to Salmonella. In FY-03, we will evaluate potentiation of innate immunity by in ovo cytokine injection and the effect of these cytokines on protecting neonatal poultry against challenges with Salmonella and Campylobacter. In FY-04, we will develop gene transfer technology for use in stimulating the innate response of birds in order to protect them from colonization by microbes such as Salmonella and Camplylobacter.
7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product?
One of the products we have identified that protects poultry against pathogenic microbes (referred to as SILK) has been provided to other scientists both in the U.S. and abroad for use in research on the prevention and control of various viral, bacterial, and protozoal diseases of poultry. We have developed a new Cooperative Research and Development Agreement (CRADA) with a poultry breeder to investigate the potential use of innate immunity as a selection tool for improved host resistance to pathogens that live in the gut of poultry. We are also developing a new CRADA partner with a major pharmaceutical company to evaluate the effect of micronutrients on innate immunity in newly- hatched poultry.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Impacts
(N/A)
Publications
- Kogut, M.H., Pishko, E.J., Kaspers, B., Weining, K.C. Modulation of functional activities of chicken heterophils by recombinant chicken IFN- gamma. Journal of Interferon and Cytokine Research. 2001. v. 21. p. 85- 92.
- Kogut, M.H., Genovese, K.J., Lowry, V.K. Differential activation of signal transduction pathways mediating phagocytosis, oxidative burst, and degranulation by chicken heterophils in response to stimulation with opsonized Salmonella enteriditis. Inflammation. 2001. v. 25. p. 7-15.
- Kogut, M.H., Genovese, K.J., Stanker, L.S. Immune lymphokine-mediated control of salmonellosis in Swine. 2001. U.S. Patent 6,221,348.
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