GOALS:The major goals of this project are to determine whereMycoplasma ovipneumoniae(M. ovi) is localized in the sheep respiratory tract and how biofilm formation affects both antibiotic resistance and immune system evasion.OBJECTIVES:This project is the first to demonstrate the capacity of the ovine respiratory pathogenM. ovito form biofilms and to explore the contributions of M. ovibiofilms to persistent colonization of the ovine respiratory mucosa. Biofilms are multicellular aggregates of bacteria embedded in an extracellular polymeric matrix (EPM). Many bacterial pathogens including Mycoplasma spp.form biofilms on mucosal surfaces. Biofilms are clinically important, since they have an inherent resistance to antimicrobial agents and hostimmune mechanisms. However, whether M. ovi can form biofilms and whether M. ovibiofilms contribute to chronic infection and pathogen transmission has not yet been explored systematically. We have generated preliminary data that demonstrate robust formation of M. ovibiofilms in vitro. Here, we seek to test the hypothesis that biofilm formation by M. ovi is acentral mechanism for immune evasion and antibiotic resistance by addressing three aims:Aim 1: Map the distribution of M. ovi and M. ovi biofilms in the ovine respiratory tract. Asymptomatic adult M. ovi carriers are the main source for M. ovi infections in lambs. However, the distribution of M. ovi across the different regions of the respiratory tract is poorly defined, and biofilm formation in situ has not been demonstrated. We will identify the specific sites in the respiratory tract where M. ovi establishes colonization and biofilm formation using quantitative PCR, immunohistochemistry, and fluorescent dyes that detect biofilm EPM. To perform these analyses, we have collected an extensive set of samples from the upper and lower respiratory tract of domestic specific-pathogen-free (SPF) lambs experimentally infected with M. ovi.Aim 2: Determine to what extent biofilm formation by M. ovi alters its susceptibility to antibiotic treatment. Recent data from our lab and others indicate that M. ovi infection is resistant to antibiotic treatment in vivo in spite of strong antibiotic susceptibility in vitro, aphenomenon that could be linked to biofilm formation. Therefore, we seek to determine the effects that M. ovi biofilm formation has on resistance to antibiotic treatment.Aim 3: Identify the mechanisms by which M. ovi biofilms impact phagocyte function. Alveolar macrophages (AMs) are the major immune cells of the respiratory mucosa and regulate homeostasis of the airways by killing pathogens and regulating tissue repair. Biofilms mayimpair alveolar macrophage function through release of immunomodulatory products and because the size of the biofilms can prevent phagocytic clearance. We have developed a phagocytosis assay for sheep AMs that we will use to determine if M. ovi biofilms interfere with phagocytosis of M. ovi as well as a second respiratory pathogen, Mannheimia haemolytica. Should M. ovi biofilms result in reduced phagocytosis, this might be a novel method of immune evasion and a factor in increased susceptibility to other respiratory infections.These aims align with the AFRI priority area Animal Health and Production by furthering the understanding of the factors that cause respiratory disease in domestic sheep. As a long-term goal, we hope that understanding the contributions of biofilms to M. ovi persistence in the respiratory tract will inform the development of preventive or therapeutic strategies that specifically target biofilms.