Macrophage phenotypic adaptation critically regulates inflammatory balance during infection; however, progress in equine immunology has been limited by a lack of specific tools for standardized identification. To address this gap, we established a reliable, antibody-based detection method for characterizing equine macrophage M1-like and M2-like marker profiles. After initially validating canonical marker genes via qPCR, we developed specific monoclonal antibodies targeting the differentially expressed surface proteins CD80 (M1-like) and CD163 (M2-like). These novel antibodies enabled the creation of a multi-modal detection approach combining flow cytometry, western blotting, and qPCR. Applying this methodology in vitro revealed distinct pathogen-specific marker profiles: Salmonella abortus equi, equine herpesvirus (EHV-1), and equine arteritis virus (EAV) promoted an early M1-like profile, whereas an attenuated equine infectious anemia virus (EIAV) strain drove an M2-like phenotype. Ultimately, this work provides a validated detection system for equine macrophage phenotyping, establishing a critical foundation for future research into host-pathogen interactions and targeted therapeutics.IMPORTANCEMacrophage phenotypic adaptation plays a critical role in infectious diseases, as pathogens often manipulate these states to evade immune responses or drive damaging inflammation. Accurately monitoring these functional shifts is vital for guiding disease treatment and evaluating vaccines. However, standardized detection tools for equine macrophages have been lacking. In this study, we established a reliable, antibody-based method utilizing novel monoclonal antibodies against equine CD80 and CD163 to identify M1-like and M2-like marker profiles. This straightforward and highly specific approach overcomes the limitations of previous indirect methods, providing a critical, accessible tool for assessing macrophage responses to equine pathogens and advancing veterinary immunology.

Keywords: CD163; CD80; EAV; EHV; EIAV; equine macrophages; infection; macrophage polarization.