Paratuberculosis or Johne's disease in ruminants is an infectious disease of the small intestine caused by Mycobacterium avium spp. paratuberculosis, and a global problem of the livestock industry. No therapy is available and the use of a whole bacterin vaccine is limited due to interference with tuberculosis diagnostics. In earlier studies we showed the protective effect of Hsp70/DDA subunit vaccination against paratuberculosis. In the current study, potential interference of this vaccination with immunodiagnostic procedures to detect bovine tuberculosis and paratuberculosis was studied. Vaccination of cattle with an Hsp70/DDA subunit vaccine has little side-effects, and does not interfere with current tuberculosis immunodiagnostic assays. Serological assays for paratuberculosis diagnostics can be adapted by inclusion of an Hsp70 pre-absorption step that enables differentiation between vaccinated and infected animals. In conclusion, the Hsp70/DDA subunit vaccine may contribute to paratuberculosis control strategies, without compromising diagnosis of bovine tuberculosis or paratuberculosis.