Effect of Paratuberculosis Vaccination on the in Vitro Release of Neutrophil Extracellular Traps in a Sheep Model

Background: Vaccination of domestic ruminants against paratuberculosis has shown beneficial results in the control of the disease, even though protection is incomplete. This is partly due to the unawareness about the mechanisms taking part during protective immune response. In this sense, despite of the increased number of evidences that point out the participation of neutrophils in the homologous and heterologous protection, its key role is still unclear. Therefore, we evaluate the effect of paratuberculosis vaccination on the release of neutrophil extracellular traps (NETs), through its quantification and visualization after their in vitro infection with Mycobacterium avium subsp. paratuberculosis ( Map ) and non-related pathogens in a sheep model. Methods Ovine neutrophils were obtained from non-vaccinated ( n = 5) and vaccinated sheep ( n = 5) and infected in vitro with Map , Staphylococcus aureus ( SA ) and Escherichia coli ( EC ). After 4 hours, NETs release was quantified and visualized by fluorimetry and immunofluorescence microscopy, respectively. Results NETs components were confirmed by co-localization of extracellular DNA with neutrophil elastase and myeloperoxidase visualized by immunofluorescence. Infection of ovine neutrophils with Map ( P > 0.05), SA ( P < 0.001) and EC ( P < 0.01) induced the formation of NETs before vaccination as well as after 15- and 30-days post-vaccination. However, neither vaccination nor time post-vaccination did modify the generation of NETs. Conclusions These results show that the release of NETs did not play a fundamental role in the early homologous and heterologous protective immune response after paratuberculosis vaccination, although, other antimicrobial mechanisms not evaluated in this study may participate in the protective immune response.