Progress 04/02/04 to 03/15/09
Outputs
Progress Report Objectives (from AD-416) Define the effects of Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis (Bb12) colonization on the immune system. 2. Determine the effects of LGG or Bb12 on the development of a local allergic response in pigs as a model for human disease. 3. Determine the impact of probiotic bacteria in modulating a worm-induced colitis and enteritis caused by Salmonella typhimurium. 4. Determine the production of volatile organic compounds in the GI tract of animals consuming probiotics and prebiotics and the effect of these products on GI function. Approach (from AD-416) It is hypothesized that probiotic microbes induced a more balanced maturation of mucosal immune system. These studies are difficult to conduct in humans and an animal model to demonstrate a 'proof of principle' for the effectiveness of dietary probiotics on neonatal maturation and modulation of mucosal immunity is needed. The overall approach of this project is to identify and measure the effect of probiotics as a dietary component for early induction of mucosal immune system development in newborn swine as a model for nutrient conditioning of a balanced immune system in humans. On the basis of the epidemiological studies done with humans we predict that it will be possible to enhance development and modulate the immune system by administration of probiotics. Significant Activities that Support Special Target Populations During the final 6 months of this project, a study was conducted to evaluate the effect of three pre-biotic preparations of milk-based formula on survival of the probiotic Bifidobacterium lactis and impact on host immune markers in neonatal pigs, as well as developing plans for further experiments. The focus of this project has been to detect the presence of probiotic bacteria in the intestine of pigs fed a daily ration of probiotics commercially available to humans, and determine the effect of these probiotics immune markers (e.g., cytokines, cytokine receptors, mediators of immune function) in the host and on immune pathways. Recent data demonstrated species-dependent effects on the host that differentiate probiotic efficacy. Studies conducted as part of the project have determined that probiotic strains need to be tested individually to evaluate the influence of host age, site of development, type of diet, probiotic dose, length of exposure, and environmental factors that influence the establishment, maintenance, and effect of the probiotic strain in the host. Quantitative measurement of bacterial load was determined based on the identification of single bacterial genes that track the presence of probiotic strains within the endogenous gastrointestinal flora and their effect on expression of host markers of immune and physiological function. Studies were initiated to colonize baby pigs with specific human-derived probiotic strains and measure local changes in host gene expression as an animal model for humans. A procedure was established to mimic the colonization of newborn infants from mothers treated with probiotics during gestation. Optimal host responses were observed in pigs born to mothers treated during the last trimester of pregnancy and then orally treated with probiotics from birth. This model was used to test the effect of probiotics on a prototype allergic response induced by food proteins and of parasitic infection to test the modulating effect of distinct probiotic strains on gut function after challenge exposure to allergens. In addition, quantitative detection methods were used to compare beneficial species within the genus Lactobacillus and Bifidobacterium in the intestinal microflora of healthy children versus children with immune-related conditions such as asthma and chronic diarrhea to identify major population differences indicative of health status.
Impacts
(N/A)
Publications
- Norris, H., Peterson, M., Stebbins, C., Wetzel, B., Bundoc, V., Anthony, R. , Urban Jr, J., Long, E., Keane-Myers, A. 2007. Infiltration of Neutrophils and Eosinophils during Allergic Inflammation is Regulated by the Inhibitory Receptor gp-49B. Journal of Leukocyte Biology. 82(6):1531- 41.
- Solano Aguilar, G., Dawson, H.D., Restrepo, M., Andrews, K., Vinyard, B.T., Urban Jr, J.F. 2008. Detection of Bifidobacterium lactis (Bb12) in the intestine after feeding sows and their piglets. Applied and Environmental Microbiology.[epub ahead of print] http://aem.asm.org/cgi/reprint/AEM. 00309-08vl?view=long&pmid=18689506190901
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Define the effects of Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis (Bb12) colonization on the immune system. 2. Determine the effects of LGG or Bb12 on the development of a local allergic response in pigs as a model for human disease. 3. Determine the impact of probiotic bacteria in modulating a worm-induced colitis and enteritis caused by Salmonella typhimurium. 4. Determine the production of volatile organic compounds in the GI tract of animals consuming probiotics and prebiotics and the effect of these products on GI function. Approach (from AD-416) It is hypothesized that probiotic microbes induced a more balanced maturation of mucosal immune system. These studies are difficult to conduct in humans and an animal model to demonstrate a 'proof of principle' for the effectiveness of dietary probiotics on neonatal maturation and modulation of mucosal immunity is needed. The overall approach of this project is to identify and measure the effect of probiotics as a dietary component for early induction of mucosal immune system development in newborn swine as a model for nutrient conditioning of a balanced immune system in humans. On the basis of the epidemiological studies done with humans we predict that it will be possible to enhance development and modulate the immune system by administration of probiotics. Significant Activities that Support Special Target Populations Work completed in the project demonstrates that certain probiotic bacterial strains induce activation of the immune system by up-regulation of components of innate immunity, while other probiotic strains induce a generalized down-regulation of pro-inflammatory mediators. In addition, there is a demonstrated beneficial effect on intestinal permeability during disease conditions that include infection and food allergen. A Trust Fund Cooperative Agreement (58-1235-7-096FU) was initiated this year with the University of Antioquia in Columbia to study the microflora composition of children with intestinal disorders in Columbia. The lead scientist spent 10 weeks working in Columbia to identify the level of Lactobacillus and Bifidobacterium species in children in three locations. This project provides data to support a request for extramural funding and represents a paradigm for similar studies with children that can be conducted in the United States. An allergic sensitization of neonatal pigs with bovine milk induced a change in intestinal permeability following feeding that was reversed when the pigs were fed probiotic bacteria. The results demonstrate that the pig is a useful model for human milk allergy and the effects of probiotics to reverse the harmful effects of reactions in the intestine. The lead scientist will also present a summary of work on a cooperative agreement with Yakult Corporation at the �Fifth Yakult Shirota Conference on Probiotics� in Tokyo, Japan this fall. Accomplishments Probiotic bacteria have beneficial effects on intestinal immune and mucosal barrier function: Probiotic (Bifidobacterium lactis)-treated pigs showed changes in gene expression of immune mediators indicating local activation of the innate immune system at the intestinal mucosa and induction of changes that improve intestinal permeability; the response is enhanced in probiotic- treated pigs from probiotic-treated mothers. This probiotic in the diet ameliorated inflammatory responses against intestinal parasites and bovine milk used as a food allergen. Other probiotic strains tested showed down-regulation of the host innate immune system indicating that there are important differences among probiotic strains. Some strains down-regulate genes in distal sites in the small intestine mucosa, while others have a more general pattern of down-regulation through out the intestine. This research promotes the concept that probiotics in the diet differentially affect immune components associated with resolution of infectious and chronic disease. These findings address the National Program 107 Action Plan Component 6, Relationship between Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease. Technology Transfer Number of New CRADAS and MTAS: 1 Number of Active CRADAS and MTAS: 2 Number of Non-Peer Reviewed Presentations and Proceedings: 5
Impacts
(N/A)
Publications
- Harris, N.L., Spoerri, I., Schopfer, J.F., Nembrini, C., Merky, P., Massacand, J., Urban Jr, J.F., Lamarre, A., Burki, K., Odermatt, B., Zinkernagel, R.M., Macpherson, A.J. 2006. Mechanisms of neonatal mucosal antibody protection. Journal of Immunology. 177(9):6256-6262.
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Progress 10/01/05 to 09/30/06
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? This project addresses objectives of the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations." Identification of the beneficial and/or adverse biological effects of probiotics and their derivatives is the first step in understanding the potential impact they will have on health. It also addresses the Action Plan for Food Safety (animal and plant products - 108) related to objective to "Develop immunological based interventions, including vaccines and non-specific immune stimulants." The prevalence of allergic and inflammatory bowel diseases has increased in Western societies over the last 50 years. It has been proposed that the widespread use of antibiotics and processed foods have limited human exposure
to bacterial antigens; however, appropriate animal models to test these claims have not been developed. The current proposal tests the hypothesis that probiotic microbes derived from humans can establish in pigs and induce beneficial effects on immunity to infection and control of inflammation and allergy. Increasing consumer awareness of the link between diet and health has promoted the introduction of probiotic bacteria into the diet of Americans; they have been commonly used in the diet of many European and Asian societies for decades. Probiotics are a class of microorganisms mainly from the Lactobacillus and Bifidobacterium species that have been originally isolated from the normal flora of Humans and animals. These microorganisms can establish and grow in a compartment of the host after consumption and may provide some positive health benefits including protection against pathogenic microorganisms and viruses, stimulation of the gut immune system and enhanced disease resistance,
correction of some bowel diseases, reduced allergic disease, and protection against carcinogens. The basis of such claims, however, is often confounded by a lack of demonstrable growth and function of the probiotic strain in the gut. Scientific validation of many of the claims of probiotic activity is missing. Consumer concern about probiotic reliability, efficacy, and safety can be addressed by hypothesis-based testing of probiotic strains under controlled experimental conditions with adequate statistical testing of the data. Therefore, the overall objective of this research focuses on testing if dietary probiotics can safely enhance immune function and gut health by preventing the onset of allergenic responses and improving the immune response against infectious agents of the GI tract. Sound scientific evidence for probiotic efficacy will provide the food industry and regulatory agencies with relevant information for concerned consumers. Those at risk of chronic nutrition- related
diseases will directly benefit. Development of an animal model to test the unique nature of various probiotics species and strains and their efficacy against particular disease conditions will benefit the functional food industry and health care providers that recommend dietary interventions to improve healthy outcomes. 2. List by year the currently approved milestones (indicators of research progress) Milestone 1 (1-3 years) - The research will define 1) the colonization potential of probiotic bacteria in the porcine gut, and 2) the activation of immune system in the pig after probiotic treatment to validate the pig as a large animal model for human-derived probiotics on immune development. Milestone 2 (1-4 years) - The research will characterize the role of dietary probiotics on the development and severity of allergic disease and test the hypothesis that the moderate activation of pro-inflammatory cytokines can regulate immediate-type hypersensitivity reactions. Milestone 3 (2-4
years) - This research will determine the impact of dietary probiotics in prevention of bacterial-induced pathologies of the GI tract. Milestone 4 (3-5 years) - This research will provide evidence to support the effect of dietary fiber (barley) on probiotic colonization of the pig intestine, and biological markers of host mucosal health and barrier function. 4a List the single most significant research accomplishment during FY 2006. Probiotics activate local innate-immune-system: This accomplishment addresses the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations". The immune response of probiotic treated pigs measured by changes in gene expression of immune mediators indicated that there is a local activation of the innate-immune-system at the intestinal mucosa. This response is enhanced in Bifidobacterium lactis-treated pigs from treated sows. The results suggested that administration of
probiotics in the diet may be an alternative stimulant of the neonatal immune system improving responses to subsequent infectious agents. Other strains from the Lactobacillus species have been tested. The level of host innate-immune-system activation was down- regulated compared to vehicle-treated control pigs over several sites in the large and small intestinal mucosa. These results indicate that there are important differences among probiotic strains. Certain probiotic strains induce innate immune activation in intestinal sites where numbers of bacteria are high and can down-regulate genes in more distal sites in the small intestine mucosa. Other strains may have a more general pattern of down regulation of innate-immune gene expression throughout the intestine. This research promotes the concept that probiotics in the diet can differentially affect immune components that can enhance resolution of infectious and chronic disease. 4b List other significant research accomplishment(s),
if any. Pig model for the study of milk allergy: This accomplishment addresses the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations." A neonatal pig model has been developed to test the effect of dietary probiotics on the immune response to cow's milk allergy. Food allergy is a growing problem in children and can be expressed throughout life. Cow's milk was fed to sows and their piglets at one to three, and 5 to 7 days after birth, and expression of allergic disease was determined by clinical and molecular scoring at subsequent times after a challenge exposure to cow's milk. A partial evaluation of the data acquired has shown that glucose absorption in the intestine can be used as a marker of an allergic response and that reduced glucose absorption is corrected by use of a probiotic in the diet. This is part of a Trust Fund Agreement with Nestle USA, Inc., as part of 1235-52000-054-01T that
addresses the issue of control over milk allergy in pediatric human populations through the uses of early probiotic feeding in the diet. 4d Progress report. The focus of the project has been on measuring the presence of probiotic bacteria in intestine of treated animals and the effect that this colonization has on the immune markers (cytokines, receptors, or mediators of the immune response) of the host. Flow cytometric assays have been developed to determine changes in cellular phenotype of probiotic-treated animals versus controls. Certain probiotic bacterial strains induce activation of the immune system by up regulation of components of the innate immune response. Other probiotic strains induce a generalized down regulation of pro-inflammatory mediators. This effect would be particularly useful when inflammatory responses need to be reduced to minimize overt tissue damage that often accompanies infectious and chronic diseases. In addition, probiotics may have some beneficial
effect in improving gut function. This experimental system provides opportunities for examining the role of diet and nutritional status from the fetus to mature adult in an animal species that has immune and physiological systems that function closely to that of humans. 5. Describe the major accomplishments to date and their predicted or actual impact. The feasibility of using the pig as a large animal model for human nutritional interventions (i.e., use of probiotics) has been tested in the current research project. Emerging new technologies such as quantitative gene expression using real-time PCR measurements and cDNA and protein microarray surveys have been used to assess the effect of nutritional status on pigs infected with parasitic worms as a surrogate for allergic responses at mucosal sites in the animal. These rapid and sensitive assays of immune status of healthy animals will be applied to studies of effective dietary interventions in humans and will provide robust
identification of biomarkers of immune function that are responsive to changes in nutrition including probiotics in diet. These studies will provide quantitative data on the effect of probiotics and related functional foods on immune system development, and appropriate responses to allergic diseases and infectious agents that affect mucosal surfaces. The impact will be in the form of a large animal model to evaluate hypotheses on the effect of functional foods on healthy outcomes and the definition of definitive biomarkers. This research will provide identifiable dietary procedures that use probiotics to benefit the growing number of children that develop food allergy; it addresses the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations." 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? The interest in this area of research is supported by a Trust Fund Cooperative Agreement with Nestle U.S.A., as part of project 1235-52000- 054-01T, to study the effect of probiotics on immune function in neonatal pigs. This agreement has been expanded with additional funding to study the effect of a probiotic strain on the immune response against cow's milk allergy. A Trust Fund Cooperative Agreement with Yakult Central Institute for Microbiological Research, Japan, as part of project 1235- 52000-054-02T is also in place to study the effect of Lactobacillus casei strain shirota on the development of immune function in neonatal pigs.
Impacts
(N/A)
Publications
- Solano Aguilar, G., Dawson, H.D., Shea-Donohue, T., Madden, K., Jones, Y.L. , Beshah, E., Restrepo, M., Schoene, N.W., Hare Jr, W.R., Urban Jr, J.F. 2006. Biofiobacterium lactis enhances toll-like receptor (tlr) pathway gene expression locally in the colon and enhances glucose uptake in the small intestine of pigs infected with parasitic nematode ascaris sum. American Society for Microbiology Proceedings. 106:45A.
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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? The prevalence of allergic and inflammatory bowel diseases has increased in Western societies over the last 50 years. It has been proposed that the widespread use of antibiotics and processed foods have limited human exposure to bacterial antigens; however, appropriate animal models to test these claims have not been developed. The current proposal will test the hypothesis that probiotic microbes derived from humans can establish in pigs and induce beneficial effects on immunity to infection, and control of inflammation and allergy. Increasing consumer awareness of the link between diet and health has promoted the introduction of probiotic bacteria into the diet of Americans; they have been commonly used in the diet of many European and Asian societies for decades. Probiotics are a class of
microorganisms mainly from the Lactobacillus and Bifidobacterium species that have been originally isolated from the normal flora of humans and animals. These microorganisms can establish and grow in a compartment of the host after consumption and provide some positive health benefits including protection against pathogenic microorganisms and viruses, stimulation of the gut immune system and enhanced disease resistance, correction of some bowel diseases, reduced allergic disease and protection against carcinogens. The basis of such claims, however, is often confounded by a lack of demonstrable growth and function of the probiotic strain in the gut. Scientific validation of many of the claims of probiotic activity is missing. Consumer concern about probiotic reliability, efficacy and safety can be addressed by hypothesis-based testing of probiotic strains under controlled experimental conditions with adequate statistical testing of the data. Therefore, the overall objective of this
research will focus on testing if dietary probiotics can safely enhance immune function and gut health by preventing the onset of allergenic responses and improving the immune response against infectious agents of the gastrointestinal (GI) tract. Sound scientific evidence for probiotic efficacy will provide the food industry and regulatory agencies with relevant information for concerned consumers. Those at risk of chronic nutrition-related diseases will directly benefit. Development of an animal model to test the unique nature of various probiotics species and strains and their efficacy against particular disease conditions will benefit the functional food industry and health care providers that recommend dietary interventions to improve healthy outcomes. This project addresses objectives of the National Human Nutrition Action Plan (107) related to Performance Goal 3. 1.3.2; "Nutritious plant and animal products: Develop more nutritious plant and animal products for human
consumption" and "Identification of the beneficial and/or adverse biological effects of these food components is the first step in understanding the potential impact they will have on health." It also addresses the Action Plan for Food Safety (animal and plant products 108) related to objective 1.4.1.1, "Develop immunological based interventions, including vaccines and non-specific immune stimulants." 2. List the milestones (indicators of progress) from your Project Plan. Milestone 1 (12 - 36 months) - Probiotic strains of human origin [i.e., Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis (Bb12)] will be evaluated for their ability to colonize and activate the immune system of pigs. Milestone 2 (12 - 48 months) - Changes in the GI function (absorption, motility) animals consuming both probiotics and prebiotics will be assessed. Milestone 3 (24 - 48 months) - The effect of probiotic bacteria on local allergenic response models will be assessed. Milestone 4 (24 - 48
months) - The prophylactic effect of dietary probiotics in a worm-induced colitis and secondary bacterial invasion in the colon will be evaluated. Milestone 5 (48 - 60 months) - Completion of technology transfer with recommendations for specific probiotic strains that promote appropriate immune function development. 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. Milestone 1 (12 - 36 months) - Probiotic strains of human origin [ie., Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis (Bb12) will be evaluated for their ability to colonize and activate the immune system of pigs. Milestone Substantially Met 2. Milestone 2 (12 - 48 months) - Changes in the GI function (absorption, motility) animals consuming both probiotics and prebiotics will be assessed. Milestone Substantially 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? Milestone 1 (FY-06) - The colonization patterns of probiotic bacteria in neonatal pigs fed probiotic strains in the diet or from indirect colonization through fed sows will be determined. Data analysis is currently ongoing. It is anticipated that this milestone will be completed ahead of schedule and with no activity needed in FY07. Milestone 2 (FY-06) - Changes in gastrointestinal physiology (absorption and motility) after probiotic treatment will be measured; immune and bacteriological data from the same experiment will be collated. A replicate experiment has recently been completed and data is under analysis. (FY-07) A manuscript summarizing the data will be submitted. Milestone 3 (FY-06) - Analysis of two replicate experiments measuring the effect of probiotic on an immune response against a worm-induced reaction in pigs will be completed and a manuscript will
be finalized. (FY-07) The characteristic immune response in the lungs of worm-infected pigs will be measured following exposure to probiotic bacteria in the diet. (FY-08) Replicate studies will be completed, if necessary, and the data summarized for publication. Milestone 4 (FY-06) - The effects of probiotics as a prophylactic regimen that inhibits worm-induced colitis and secondary bacterial invasion in the colon will be assessed using a defined model of Trichuris suis infection and natural exposure to Campylobacter jejuni. A contingency model will utilize a chemically induced acute colitis. (FY-07) Molecular and culture technique will test for secondary C. jejuni infection and scoring system for pathology and localized immunity will be evaluated. (FY-09) Replicate studies will be completed as needed and a manuscript summarizing the data will be finalized. 4a What was the single most significant accomplishment this past year? Probiotic preparations (in the form of lyophilized
bacteria) containing the most commonly used microorganisms of the Lactic Acid Bacteria and Bifidobacterium species in humans were fed to neonatal pigs. Colonization of probiotic organisms and quantitative cytokine mRNA expression levels for 46 genes were assessed using real time polymerase chain reaction (PCR) . The immune response of probiotic treated animals measured by changes in gene expression of immune mediators indicated that there is a local activation of the innate immune system at the intestinal mucosa. This response is enhanced in B. lactis-treated pigs from treated mothers. These results indicate that administration of probiotics in the diet may be an alternative stimulant of the neonatal immune system improving responses to subsequent infectious agents. In addition, probiotic Bifidobacterium can improve the absorption of glucose in the intestine during an immune response to parasitic nematode infection in pigs which provides an opportunity to explore a mechanism of
action of probiotics on physiological responses in the intestine. These results demonstrate that pigs can be colonized by probiotic strains isolated from humans and that there are positive consequences in the form of enhanced innate immunity and physiological improvements in absorption. The impact of the model on human health will be developed as the conditions for optimal colonization are determined and the positive health benefits cataloged. Recommendations for clinical trials in humans would be a logical outcome of these studies. 4b List other significant accomplishments, if any. Species-specific primers and fluorogenic probes for real time PCR detection have been designed for the quantification of probiotic- bacterial deoxyribonucleic acid (DNA)in mucosal and fecal samples. Animals that have been fed either of the two probiotics strains have shown an activation of genes associated with innate immune response. Tissue samples collected from 15 different anatomical sites have been
processed for gene expression of 48 immune markers using real time PCR. Whole blood differentials, serum biochemistry and immune cell phenotype analysis by flow cytometry have been done during the first 5 weeks of age in pigs treated with B.lactis. 4d Progress report. The focus of the project has been on measuring the presence of probiotic bacteria in intestine of treated animals and the effect that this colonization has on the immune markers (cytokines, receptors or mediators of the immune response) of the host. Flow cytometric assays have been developed to determine changes in cellular phenotype of probiotic-treated animals vs. controls. Certain probiotic bacterial strains induce activation of the immune system by up regulation of components of the innate immune response. In addition, probiotics may have some beneficial effect in improving gut function. After an oral challenge with Ascaris suum, gut function was not as severely affected in probiotic treated pigs as compared to
pigs that did not receive the dietary probiotic. This experimental system provides opportunities for examining the role of diet and nutritional status from the fetus to mature adult in an animal species that has immune and physiological systems that function closely to that of humans. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The feasibility of using the pig as a large animal model for human nutritional interventions (i.e., use of probiotics) has been tested in the current project. It has already implemented the use of emerging new technologies such as quantitative gene expression using real-time PCR measurements and cDNA and protein microarray surveys to assess the effect of nutritional status on pigs infected with parasitic worms as a surrogate for allergic responses at mucosal sites in the animal. These rapid and sensitive assays of immune status of healthy animals will be applied to studies of effective dietary
interventions in humans and will provide robust identification of bio-markers of immune function that is responsive to changes in nutrition including probiotics in diet. These studies will provide quantitative data on the effect of probiotics and related functional foods on immune system development, and appropriate responses to allergic diseases and infectious agents that affect mucosal surfaces. The impact will be in the form of a large animal model to evaluate hypotheses on the effect of functional foods on healthy outcomes and the definition of definitive biomarkers. These studies will target nutritionists and health care providers. 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? The interest in this area of study is indicated by a Trust Fund
Cooperative Agreement 58-1235-3-135 to study the effect of probiotics on the immune function of neonatal pigs, and a Trust Fund Cooperative Agreement 1235-52000-054-02T to study the effect of Lactobacillus casei strain shirota on the immune response of neonatal pigs.
Impacts
(N/A)
Publications
- Solano Aguilar, G., Ledbetter, T., Dawson, H.D., Andrews, K., Harvey, R.B., Schoene, N., Urban Jr, J.F. 2004. The effect of probiotic bacteria on the immune system of weaned pigs monitored by Real time PCR. Proceedings 18th International Pig Veterinary Society Meeting, Hamburg, Germany, October 2004. p. 380.
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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? The prevalence of allergic and inflammatory bowel diseases has increased in Western Societies over the last 50 years. It has been proposed that the wide-spread use of antibiotics and processed foods have limited human exposure to bacterial antigens; however, appropriate animal models to test these claims have not been developed. The current proposal will test the hypothesis that probiotic microbes derived from humans can establish in pigs and induce beneficial effects on immunity to infection, and control of inflammation and allergy. Increasing consumer awareness of the link between diet and health has promoted the introduction of probiotic bacteria into the diet of Americans; they have been commonly used in the diet of many European and Asian societies for decades. Probiotics are a class of
microorganisms mainly from the Lactobacillus and Bifidobacterium species that have been originally isolated from the normal flora of Humans and animals. These microorganisms can establish and grow in a compartment of the host after consumption and provide some positive health benefits including protection against pathogenic microorganisms and viruses, stimulation of the gut immune system and enhanced disease resistance, correction of some bowel diseases, reduced allergic disease and protection against carcinogens. However, the basis of such claims is often confounded by a lack of demonstrable growth and function of the probiotic strain in the gut. Scientific validation of many of the claims of probiotic activity is missing. Consumer concern about probiotic reliability, efficacy and safety can be addressed by hypothesis-based testing of probiotic strains under controlled experimental conditions with adequate statistical testing of the data. Therefore, the overall objective of this
research will focus on testing if dietary probiotics can safely enhance immune function and gut health by preventing the onset of allergenic responses and improving the immune response against infectious agents of the GI tract. Sound scientific evidence for probiotic efficacy will provide the food industry and regulatory agencies with relevant information for concerned consumers. Those at risk of chronic nutrition-related diseases will directly benefit. Development of an animal model to test the unique nature of various probiotics species and strains and their efficacy against particular disease conditions will benefit the functional food industry and health care providers that recommend dietary interventions to improve healthy outcomes. This project addresses objectives of the National Human Nutrition Action Plan (107) related to Performance Goal 3.1.3.2; "Nutritious plant and animal products: Develop more nutritious plant and animal products for human consumption" and
"Identification of the beneficial and/or adverse biological effects of these food components is the first step in understanding the potential impact they will have on health." It also addresses the Action Plan for Food Safety (animal and plant products-108) related to objective 1.4.1.1 "Develop immunological based interventions, including vaccines and non-specific immune stimulants." 2. List the milestones (indicators of progress) from your Project Plan. Milestone 1 (12-36 months)- Probiotic strains of human origin [i.e., Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis (Bb12)] will be evaluated for their ability to colonize and activate the immune system of pigs. Milestone 2 (12-48 months)- Changes in the GI function (absorption, motility) animals consuming both probiotics and prebiotics will be assessed. Milestone 3 (24-48 months)- The effect of probiotic bacteria on local allergenic response models will be assessed. Milestone 4 (24-48 months)- The prophylactic effect of
dietary probiotics in a worm-induced colitis and secondary bacterial invasion in the colon will be evaluated. Milestone 5 (48-60 months)- Completion of technology transfer with recommendations for specific probiotic strains that promote appropriate immune function development. This research will contribute to our efforts to reduce the incidence of chronic diseases that may be prevented by nutritional interventions such as the addition of probiotics to the diet. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plan to do so. Milestone 1 (12-36 months)- Probiotic strains of human origin [i.e., Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis (Bb12)] have been evaluated for their ability to colonize and activate the immune system of pigs. Animals that have been fed either of the
two strains to demonstrate an activation of genes associated with innate immune response; tissues samples prepared for gene expression of relevant immune mediators are being evaluated using real time PCR measurements. Immune cell and serum patterns have been collected and the data is under analysis. Milestone 2 (12-48 months)- The effect of probiotic bacteria (Bifidobacterium lactis) on physiological changes in the intestine to an allergenic response has been evaluated using a parasite model where a strong allergenic response is induced in pigs after infection with a parasitic worm Ascaris suum that mimics local mucosal responses to allergens. The effect of probiotic bacteria in ameliorating the parasite induced alteration in intestinal function (i.e., absorption) has been demonstrated and the data correlated with immune patterns. Pre-biotic formulations will not be tested until the results of the probiotic exposure are clarified. B. List the milestones that you expect to address
over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish year by year, over the next 3 years under each milestone. Milestone 1 (12-36 months)- Analysis of pig tissue samples collected after oral administration of probiotics and recovery of probiotic strains from animal tissues and fecal content will be standardize for DNA quantification and genotyping. The colonization of probiotic bacteria in neonatal pigs fed probiotic strains in the diet or from indirect colonization through fed sows will be determined. Samples from different sections of the small and large intestine will be analyzed to correlate bacterial colonization with changes in the phenotype and function of cells associated with the intestinal and respiratory mucosa (immune phenotype, mRNA expression, immune function assays). Milestone 2 (12-48 months)- Changes in gastrointestinal physiology (absorption, motility) after probiotic treatment have been completed and the data will be correlated with the
functional immune and bacteriological data. Physiological measures such as intestinal motility and glucose absorption are indicators of nutrient assimilation. Milestone 3 (24-48 months)- The effect of probiotic on a polarized Th2 type immune response typically seen in allergenic reactions as Ascaris suum-induced hypersensitivity reaction in pigs will be used as a model for human allergic reactions. The characteristic eosinophilia and Th2- cytokine driven immune response will be measured when the infection is reproduced in animals previously exposed to probiotic bacteria in the diet. Milestone 4 (24-48 months)- The effects of probiotics as a prophylactic regimen that inhibits worm-induced colitis and secondary bacterial invasion in the colon will be assessed using a defined model of Trichuris suis infection and natural exposure to Campylobacter jejuni. A contingency model will utilize a chemically induced acute colitis. 4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment During FY 2004. Anecdotal accounts of the benefit of Lactic Acid Producing Probiotics on enhanced mucosal immune system development and improve nutritional absorption in the intestine are the rule. There is a need to verify the benefit of dietary probiotics through a cause and effect relationship between colonization by the probiotic in the host and a measurable immunological effect such as enhanced development or greater response to infection. Probiotic preparations (in the form of lyophilized bacteria) containing the most commonly used microorganisms of the Lactic Acid Bacteria and Bifidobacterium species in humans were fed to neonatal pigs. Colonization of probiotic organisms was tracked by real-time PCR using highly sensitive and specific probes. The immune response of probiotic treated animals measured by changes in gene expression of immune mediators indicated that there is an activation of the innate immune system. These results
indicate that administration of probiotics in the diet may be an alternative stimulant of the neonatal immune system improving responses to subsequent infectious agents. In addition, probiotic Bifidobacterium can improve the absorption of glucose in the intestine during an immune response to parasitic nematode infection in pigs which provides an opportunity to explore a mechanism of action of probiotics on physiological responses in the intestine. These results demonstrate that pigs can be colonized by probiotic strains isolated from humans and that there are positive consequences in the form of enhanced innate immunity and physiological improvements in absorption. The impact of the model on human health will be developed as the conditions for optimal colonization are determined and the positive health benefits cataloged. Recommendations for clinical trials in humans would be a logical outcome of these studies. B. Other Significant Accomplishments. The characterization of health
benefits of feeding dietary probiotics to humans and livestock require quantitative molecular and cellular measures of health status and immune function. The use of swine as a model for probiotic research requires development of novel tools or modification of procedures used to evaluate health in other species including man. A variety of new molecular probes for quantitative gene expression of pig cytokines and markers of innate immunity (i.e., Toll-like receptor pathway) were developed and tested in multiple tissues dissected from probiotic-treated animals. This technology can now be applied to animals and extrapolated to humans as indicators of specific markers of immune activation after nutritional interventions. In addition, a new immune histological staining technique for detection of specific carbohydrate side chains on mucins produced in the intestine of pigs was developed to examine changes in mucosal immunity. These assays can be used to define the health consequences of
probiotic application in pigs and suggest similar measurements when probiotics are tested in humans. C. Significant Activities that Support Special Target Populations. None. D. Progress Report. The focus of the project has been on measuring the presence of probiotic bacteria in intestine of treated animals and the effect that this colonization has on the immune markers (cytokines, receptors or mediators of the immune response) of the host. Flow cytometric assays have been developed to determine changes in cellular phenotype of probiotic-treated animals vs. controls. Certain probiotic bacterial strains induce activation of the immune system by up regulation of components of the innate immune response. In addition, probiotics may have some beneficial effect in improving gut function. After an oral challenge with Ascaris suum, gut function was not as severely affected in probiotic treated pigs as compared to pigs that did not receive the dietary probiotic. This experimental system
provides opportunities for examining the role of diet and nutritional status from the fetus to mature adult in an animal species that has immune and physiological systems that function closely to that of humans. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Cris project 1235-52000-041-00D examined the role that oxidative stress plays in elevating risk-factor metabolites associated with heart disease. It was redirected after the retirement of the CRIS project leader, Meira Fields, in 2000. The new project leader is Gloria Solano-Aguilar since the fall of 2001; with OSQR approval of a new CRIS in September 2004 that will study the interaction between probiotics and health. A bridge between information acquired from earlier work on oxidative stress and expression of metabolic disease to the regulation of these responses against mucosal infectious agents is the objective of the current research project. The feasibility of
using the pig as a large animal model for human nutritional interventions (i.e., use of probiotics) has been tested in the current Cris project. It has already implemented the use of emerging new technologies such as quantitative gene expression using real-time PCR measurements and cDNA and protein microarray surveys to assess the effect of nutritional status on pigs infected with parasitic worms as a surrogate for allergic responses at mucosal sites in the animal. These rapid and sensitive assays of immune status of healthy animals will be applied to studies of effective dietary interventions in humans and will provide robust identification of bio-markers of immune function that is responsive to changes in nutrition including probiotics in diet. These studies will provide quantitative data on the effect of probiotics and related functional foods on immune system development, and appropriate responses to allergic diseases and infectious agents that affect mucosal surfaces. The impact
will be in the form of a large animal model to evaluate hypotheses on the effect of functional foods on healthy outcomes and the definition of definitive biomarkers. 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? Probiotics can act as a prophylactic against infection by enhancing the mucosal immune response against certain pathogens. In addition, the "hygiene hypothesis" proposes that an increase in allergy is reduced by exposure to beneficial microbes early in life by altering the immune balance. The regulatory role of normal intestinal microbes may be crucial for the development of protective mechanisms against certain allergens. The specific objectives of the project will generate data to support the use of probiotics in neonates to limit food
related diseases, such as allergies and microbial pathogens, and a description of basic mechanisms for enhanced mucosal immune and barrier function. Probiotic products (in the form of yogurt, lyophilized bacteria or fermented products) containing the most commonly used microorganisms of the lactic acid bacteria (LAB) in humans will be orally administered to neonatal pigs. Comparative studies of the digestive processes in milk fed-piglets and human infants have shown that digestive processes are similar thus data generated in this large animal model can easily be extrapolated to humans. The administration of a highly allergenic dietary product (i.e., cow's milk), to suckling pigs in conjunction with Lactobacilli have been shown to degrade proteins into tolerogenic peptides. The effect of probiotic bacteria in the reduction of the food allergy by promoting endogenous barrier mechanisms and shifting immune activation will be evaluated. Probiotic application to human diet is a major food
supplement in Europe, but is only gaining favor in a small niche in the United States. However, interest is growing and several emerging and established food companies have begun to develop probiotics for use by the U.S. population. These companies are very interested in the outcome of our projected studies because they will describe molecular and immunological events that are not within the scope of industrial food research. Probiotics also have a following in the animal and human health industry because of the concept of "competitive exclusion" of pathogenic bacteria and "enhanced mucosal barrier function" to prevent invasion of the tissue by intestinal pathogens. These are major concerns of both of these sectors especially as an alternative to the use of antibiotics to control infection. The interest in this area of study is indicated by a Trust Fund Cooperative Agreement 58-1235-3-135 that was signed with Nestle U.S. (June 2003 through May 2004) to study the effect of probiotics
on the immune function of neonatal pigs.
Impacts
(N/A)
Publications
- Solano Aguilar, G., Dawson, H.D., Ledbetter, T., Shea Donohue, P.T., Schoene, N.W., Call, J., Beshah, E., Hare Jr, W.R., Urban Jr, J.F. 2003. The effects of dietary probiotics on immunity to ascaris suum in pigs [abstract]. Proceedings of American Association of Veterinary Parasitologists. Paper No. 56-54.
- Solano Aguilar, G., Dawson, H.D., Ledbetter, T., Shea Donohue, P.T., Schoene, N.W., Call, J.E., Beshah, E., Hare Jr, W.R., Urban Jr, J.F. 2003. The effect of human derived probiotic bacteria on the immune and intestinal function of pigs [abstract]. Allen D. Leman Swine Conference. 30:26.
- Solano Aguilar, G., Dawson, H.D., Ledbetter, T., Shea Donohue, P.T., Schoene, N.W., Call, J., Beshah, E., Hare Jr, W.R., Urban Jr, J.F. 2003. Human derived probiotic bacteria species can regulate intestinal responses to parasitic nematodes in pigs [abstract]. 19th International Conference of the World Association for the Advancement of Veterinary Parasitology. p. 199.
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