2005 Annual 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? 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. 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. 4c.List any significant activities that support special target populations. None. 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. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None. Review 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.