Mycobacterium avium subsp . paratuberculosis (Map) causes paratuberculosis (PTB), a granulomatous enteritis in ruminants that exerts high economic impact on the dairy industry worldwide. Current vaccines have shown to be cost-effective against Map and in some cases confer beneficial non-specific effects against other pathogens suggesting the existence of trained immunity. Although Map infection is mainly transmitted by the fecal-oral route, oral vaccination has not been deeply studied. Therefore, the aim of this study was to compare the oral route with a set of mycobacterial and non-mycobacterial vaccines with a subcutaneously administered commercially available vaccine. Training effects on polymorphonuclear neutrophils (PMNs) and homologous and heterologous in vivo protection against Map were investigated in the rabbit infection model. Oral vaccination with inactivated or live vaccines was able to activate mucosal immunity as seen by elevation of serum IgA and the expression of IL4 in peripheral blood mononuclear cells (PBMCs). In addition, peripheral PMN phagocytosis against Map was enhanced by vaccination and extracellular trap release against Map and non-related pathogens was modified by both, vaccination and Map-challenge, indicating trained immunity. Finally, PBMCs from vaccinated animals stimulated in vitro with Map antigens showed a rapid innate activation cytokine profile. In conclusion, our data show that oral vaccination against PTB can stimulate neutrophil activity and both innate and adaptive immune responses that correlate with protection.

Paratuberculosis (PTB) is an enteric granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that mainly affects ruminants. Current vaccines have shown to be cost–effective control reagents, although they are restricted due to cross-interference with bovine tuberculosis (bTB). Therefore, novel vaccination strategies are needed and this study is focused on evaluating alternative vaccination routes and their effect on the local immune response. The MAP oral challenge rabbit model was used to evaluate and compare an experimental inactivated MAP vaccine through oral (VOR) and intradermal (VID) routes. The VID group presented the highest proportion of animals with no visible lesions and the lowest proportion of animals with MAP positive tissues. Immunohistochemistry analysis revealed that the VID group presented a dominantly M1 polarized response indicating an ability to control MAP infection. In general, all vaccinated groups showed lower calprotectin levels compared to the non-vaccinated challenged group suggesting less active granulomatous lesions. The VID group showed some degree of skin test reactivity, whereas the same vaccine through oral administration was completely negative. These data show that PTB vaccination has an effect on macrophage polarization and that the route influences infection outcome and can also have an impact on bTB diagnosis. Future evaluation of new immunological products against mycobacterial diseases should consider assaying different vaccination routes.