To develop a therapeutic vaccine for use in animals and humans infected with MAP, we performed a targeted bioinformatic analysis of the MAP genome and selected two secreted and two membrane bound components each related to the pathogenic phenotype. A fusion construct including these four MAP antigens was assembled from overlapping 40mer oligonucleotides incorporating a sequence adjusted for optimal mammalian codon usage. Functional domains including potential cross-reacting human epitopes and hydrophobic transmembrane regions were excluded. A monoclonal antibody recognition peptide was added to the C-terminus and a short human ubiquitin leader sequence to the N-terminus. The construct was cloned into the pSG2 expression vector and inserted by homologous recombination into Modified Vaccinia Ankara (MVA). Expression of the 95 kDa polyprotein in cell culture was confirmed. Prime boost vaccination using single dose pSG2.rec (i.m.) followed by single dose MVA.rec (i.v.) resulted in significant antigen-specific IFN-γ T-cell responses in ELISPOT assays compared with control immunization using wild type vector or buffer only. No adverse effects occurred in the hyper-immune mice. In two subsequent studies, a modified protocol incorporating two priming doses of pSG2.rec (i.m.) followed by a single boost of MVA.rec (i.v.) was tested for its ability to attenuate pre-existing MAP infection and to protect against subsequent MAP infection compared with control groups. The burden of MAP infection was measured using IS900-specific quantitative real-time PCR on liver/spleen DNA extracts and by culture on solid medium for future determination of cfu. Prime boost vaccination four weeks after MAP infection resulted in a significant attenuation of infective load (qRT-PCR) in spleen and liver after 26 weeks compared with controls. Vaccination prior to MAP challenge showed a protective effect in a subgroup of animals. These early results provide a promising basis for further vaccination studies in larger animals.