OUR HYPOTHESIS IS THAT CHRONIC BOVINE RESPIRATORY DISEASE (BRD) IS A BACTERIAL BIOFILM DISEASE, WHICH MAKES THE BACTERIAL PATHOGENS ASSOCIATED WITH BRD MORE RESISTANT TO ANTIBIOTICS AND HOST IMMUNE DEFENSES. A BIOFILM IS AN ORGANIZED STRUCTURE OF BACTERIAL CELLS THAT ARE ENCASED IN A NETWORK OF POLYSACCHARIDE, NUCLEIC ACIDS, AND PROTEINS. THIS MATRIX MATERIAL CONTAINS WATER CHANNELS TO PROVIDE NUTRIENTS TO CELLS DEEP WITHIN THE BIOFILM, BUT THE CELLS THEMSELVES, THOUGH VIABLE, GROW VERY SLOWLY. AS A RESULT, BACTERIA WITHIN A BIOFILM ARE MORE RESISTANT TO ANTIBIOTICS AND HOST IMMUNE FACTORS DUE TO INACCESSIBILITY AND THE SLOW GROWTH OF THE BACTERIA (ANTIBIOTICS USUALLY ONLY KILL ACTIVLY GROWING CELLS). THE PRIMARY BACTERIAL PATHOGENS ASSOCIATED WITH BRD ARE HISTOPHILUS SOMNI, PASTEURELLA MULTOCIDA, MANNHEIMIA HAEMOLYTICA, AND MYCOPLASMA SPP., AND THEY MAY OCCUR ALONE, IN COMBINATION WITH EACH OTHER, OR WITH VIRAL AGENTS. THIS PROJECT FOCUSES ON THE RELATED BACTERIA H. SOMNI, P. MULTOCIDA, AND M. HAEMOLYTICA. THESE ORGANISMS RESIDE ONLY IN HOST MUCOSAL SITES, AND H. SOMNI AND M. HAEMOLYTICA RESIDE ONLY IN RUMINANTS (PRIMARILY BOVINES AND SHEEP). WE AND OTHERS HAVE ESTABLISHED THAT EACH OF THESE OPPORTUNISTIC PATHOGENS ARE CAPABLE OF FORMING BIOFILMS ON COVER SLIPS, TUBES, CULTURE PLATE WELLS, AND IN THEIR NATURAL HOST. WE HAVE ALSO SHOWN THAT H. SOMNI AND P. MULTOCIDA FORM A POLY-MICROBIAL BIOFILM TOGETHER IN LABORATORY VESSELS AND IN HOST TISSUES. HOWEVER, IT IS ESSENTIAL TO UNDERSTAND IF ALL 3 BACTERIA INTERACT TO FORM A BIOFILM TOGETHER, AND HOW SINGLE- AND POLY-MICROBIAL BIOFILMS DEVELOP AND BECOME ESTABLISHED IN THE HOST. ALTHOUGH CURRENT VACCINES DO INDUCE ANTIBODIES TO THESE BACTERIA, THAT ARE NOT ADEQUATELY EFFECTIVE AGAINST BRD. ANTIMICROBIALS ARE EXPENSIVE, AND EVEN IF EFFECTIVE, REQUIRE LONG PERIODS OF TREATMENT. WE PROPOSE THAT THE INEFFECTIVENESS OF CURRENT VACCINES AND ANTIMICROBIALS IS BECAUSE THE BACTERIA HAVE BECOME ESTABLISHED IN A BIOFILM, BUT THERE IS A LACK OF UNDERSTANDING OF HOW THESE BIOFILMS DEVELOP IN HOST TISSUES, AND HOW TO ELIMINATE OR REDUCE THE BIOFILM. THIS PROPOSAL AIMS TO FILL SOME OF THOSE GAPS IN KNOWLEDGE, AND IDENTIFY COMPOUNDS THAT MAY ENHANCE THE EFFICACY OF VACCINES AND ANTIMICROBIAL AGENTS.WE WILL FIRST DETERMINE IF ALL 3 OF THE BACTERIAL PATHOGENS MENTIONED ABOVE FORM A POLY-MICROBIAL BIOFILM TOGETHER (OUR FIRST AIM), AND THEN HOW THIS BIOFILM BECOMES ESTABLISHED AND DEVELOPS IN THE HOST THROUGH THE USE OF 3-D TISSUE CULTURE USING BOVINE RESPIRATORY EPITHELIAL CELLS (OUR SECOND AIM). THIS TYPE OF CELL CULTURE CAN MOST CLOSELY APPROXIMATE WHAT OCCURS IN THE BOVINE USING A MODEL THAT CAN BE OBSERVED ON AN HOURLY OR DAILY BASIS. WE WILL ALSO SCREEN AN EXTENSIVE LIBRARY OF THOUSANDS OF COMPOUNDS FOR THEIR CAPABILITY TO BREAK UP A SINGLE OR POLY-MICROBIAL BIOFILM, AND THOSE COMPOUNDSTHAT ARE DETERMINED TO BE SAFE FOR USE IN ANIMALS WILL BE FURTHER TESTED TO BREAK UP THE BIOFILM IN THE 3-D TISSUE CULTURE MODEL.,THEREAFTER, USING AN ANTIMICROBIAL BIOFILM DEVICE ASSAY, WE WILL DETERMINE IF THE BACTERIA WITHIN A BIOFILM ARE SIGNIFICANTLY MORE SUSCEPTIBLE TO A COMBINATION OF THE ANTIMICROBIAL AND THE ANTI-BIOFILM COMPOUND, THAN THE ANTIMICROBIAL ALONE (OUR THIRD AIM). IF SO, FURTHER DEVELOPMENT OF THESE COMPOUNDS WILL BE EXPLORED WITH PHARMACEUTICAL COMPANIES.IF SUCCESSFUL, THIS WORK WILL HAVE BENEFITS TO SOCIETY THATEXTEND MUCH FARTHER THAN ONLY TO IMPROVED TREATMENT OF BRD. BIOFILMS ARE COMMON IN MANY BACTERIAL DISEASES OF ANIMALS AND HUMANS, INCLUDING SWINE ATROPHIC RHINITIS, HUMAN CYSTIC FIBROSIS, ENDOCARDITIS, OSTEOMYELITIS, AND OTHERS. WE HOPE THAT THIS WORK WILL LEAD TO TREATMENT AND REMOVAL OF THE BIOFILM TO OBTAIN IMPROVED RESOLUTION OF CHRONIC BACTERIAL INFECTIONS.