**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** PRELIMINARY DATA DEVELOPED IN OUR LAB HAS INDICATED SELECT MICROBIAL SPECIES AFFECT THE MATURATION OF THE BOVINE IMMUNE SYSTEM. SPECIFICALLY, MICROBES DETECTED IN THE DISTAL GIT WHERE JEJUNAL AND ILEO-CECAL PEYER'S PATCHES (THE PRIMARY LYMPHATIC/IMMUNOLOGICAL TISSUES OF CATTLE) ARE KNOWN TO EXIST, WERE FOUND TO COLONIZE AND BECOME MORE ABUNDANT IN CONCERT WITH MEASURES OF IMMUNOLOGICAL MATURATION. WE HYPOTHESIZE THEY ARE IMPORTANT TO BOVINE NEONATAL HEALTH AND BY DIRECTLY INTERACTING WITH THESE GUT-ASSOCIATED LYMPHATIC TISSUES (GALT) PROMOTE IMMUNE MATURATION AND SUPPORT IMMUNE FUNCTION; A PROCESS THAT IS WELL ESTABLISHED IN HUMAN'S AND MICE. THE PROPOSED WORK SEEKS TO EXAMINE THIS PHENOMENON. SPECIFICALLY, THE PROPOSED WORK INCLUDES:SPECIFIC AIM 1. DETERMINE THE ABILITY OF IDENTIFIED MICROBES TO MODULATE BOVINE SMALL INTESTINAL CELL IMMUNE RESPONSIVENESS.THIS AIM WILL ASSESS THE INFLUENCE OF EACH MICROBE IDENTIFIED TO BE ASSOCIATED WITH MATURATION OF THE IMMUNE FUNCTION ON THE ABILITY OF A BOVINE SMALL INTESTINAL CELL LINE'S ABILITY TO RESPOND TO AN E. COLI CHALLENGE AND TO RESTORE HOMEOSTASIS FOLLOWING AN ENDOTOXIN CHALLENGE IN COCULTURE EXPERIMENTS THAT EXAMINE THE TEMPORAL EXPRESSION OF CYTOKINES AND TIGHT JUNCTION (TJ; THESE GENES AFFECT BARRIER FUNCTION WHICH IS A MEASURE OF IMPERMEABILITY OF THE GIT EPITHELIUM TO POTENTIAL TOXINS) GENES. SPECIFIC AIM 2. DETERMINE THE IN VIVO IMPACT OF 'NATURAL IMMUNOBIOTICS' TO INFLUENCE GIT MUCOSAL BIOLOGY. UP TO THREE MICROBES IDENTIFIED IN AIM 1 TO POSITIVELY AFFECT THE IMMUNORESPONSIVENESS AND BARRIER FUNCTION-RELATED GENE EXPRESSION OF A BOVINE SMALL INTESTINAL CELL LINE (DESCRIBED IN THE GRANT AS NATURAL IMMUNOBIOTICS), WILL BE INOCULATED TO COLOSTRUM-DEPRIVED CALVES (TO MINIMIZE EXOGENOUS MICROBIAL AND IMMUNE FACTORS) BEGINNING IMMEDIATELY FOLLOWING BIRTH AND CALVES WILL BE ASSESSED FOR IMMUNOBIOTIC COLONIZATION, THE DEVELOPMENT OF PEYER'S PATCHES, BARRIER FUNCTION, AND HISTOCHEMISTRY AT 0, 90, AND 180 DAYS OF AGE. AIM 3. EVALUATE THE IMMUNE-MEDIATED PROTECTION AGAINST ENTEROTOXIGENIC E. COLI IMPARTED TO DOMESTIC BEEF CALVES BY TARGET MICROBES.A THIRD AIM THAT WILL EVALUATE THE IMMUNE-MEDIATED PROTECTION AGAINST AN ENTEROTOXIGENIC E. COLI CHALLENGE.THE PROPOSED WORK WOULD BE THE FIRST DIRECT EXAMINATION OF THE CAPABILITY OF A SELECTION OF NATURALLY OCCURRING RUMINANT GIT MICROBES TO COLONIZE THE SMALL INTESTINAL MUCOSAL EPITHELIUM, ALTER THE DEVELOPMENT OF LYMPHATIC TISSUE, AND IMPROVE BARRIER FUNCTION. COMPLETION OF THE PROPOSED OBJECTIVES WILL PROVIDE A WEALTH OF NEW AND IMPORTANT KNOWLEDGE THAT COULD BOTH PROPEL THE RUMEN MICROBIOLOGICAL FIELD TO A NEW AND MORE SOPHISTICATED LEVEL OF UNDERSTANDING AND POTENTIATES TECHNOLOGICAL INNOVATIONS THAT SIGNIFICANTLY IMPROVE THE HEALTH AND MANAGEMENT OF CATTLE.