| Summary: | The equine respiratory and reproductive tract microbiomes are complex and subject to constant fluctuations. Among the microbial inhabitants, <i>Streptococcus equi</i> subsp. <i>zooepidemicus</i> (SEZ) is recognized as the dominant bacterium. It is an opportunistic pathogen that may occasionally lead to various types of infections. A key virulence factor of SEZ is the streptolysin S (SLS) toxin, which is responsible for the characteristic β-hemolysis on blood agar and tissue damage. Viruses and bacteria may interact and aggravate lesions and disease. This study aimed to evaluate the impact of an SLS-containing supernatant from SEZ on the nasal and vaginal mucosa and the subsequent replication of equine herpesviruses. The SLS-containing supernatant was prepared, and three 10-fold dilutions (optical density “OD” 10<sup>−2</sup>, 10<sup>−3</sup>, 10<sup>−4</sup>) were applied to equine nasal and vaginal explants. Untreated and EGTA-treated explants served as controls. Epithelial integrity was assessed by measuring the thickness and intercellular spaces. Nasal explants were inoculated with EHV-1 and EHV-4, while vaginal explants received EHV-1 and EHV-3. Viral replication was estimated via immunofluorescence staining and confocal microscopy. SLS-containing supernatants 10<sup>−2</sup> and 10<sup>−3</sup> compromised epithelial integrity. Viral replication increased in explants treated with SLS 10<sup>−3</sup>, demonstrating SLS’s damaging effects on the epithelium, facilitating equine herpesvirus replication.
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