Peptidyl moiety of cell wall lipopentapeptide (L5P) specific of Mycobacterium avium subsp paratuberculosis (Map) is immunogenic and a target for specific humoral response in Map infected animals. A chemically synthesized L5P is able to induce specific cell mediated immune response (CMIR) in IFN-γ release assay (IGRA) in selected cows from Map infected herds comparatively to non-infected or M. bovis infected. Following these observations, the aim of this study was to evaluate if L5P was an antigen of early specific immune response and potentially a predictive tool of Map infection. 113 heifers of 6 herds were included in a two years’ longitudinal study: 71 animals from three Map culture-confirmed herds, 11 animals from a Map infected herd Silirum® vaccinated during the study and 31 animals from two certified Map free herds. The analysis of the CMIR was investigated by IGRA following whole blood stimulation with synthetic L5P or mycobacterium purified protein derivative (PPD) from M. avium (PPDa), M. bovis (PPDb), Map (PPDj) and M. phlei (PPDp). Humoral immune response was quantified by L5P-based ELISA using an internal procedure and two commercial Map kits. Moreover, bacilli excretion was estimated by isolation and culture from faecal sample. PPDs’ CMIR was more or less high depending of infected herds context, became high over 2 S/P ratio for 10/11 animals just after Silirum® vaccination and was low, less than 0.1 S/P ratio, in certified Map free herds. L5P CMIR was observed in 9 of 71 animals from Map culture-confirmed herds. These 9 animals with a L5P CMIR positive between 0.05 and 0.6 S/P ratio were from the same herd, knowing that L5P CMIR was previously detected in all included Map culture-confirmed herds. L5P CMIR was fluctuant as already described for PPD but was significantly correlated with PPDj CMIR. For 2 of the 9 animals, the L5P CMIR was predictive of the Map positive serology, whereas it was concomitant with seropositivity for 2 others and that for 5 animals was several times observed without seroconversion. And no seroconversion was observed in other herds. The continuation of this study would assess the predictive potential of L5P CMIR for paratuberculosis diagnosis.