Paratuberculosis is a chronic enteric disease that affects ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) an obligated intracellular acid-fast bacilli that usually infect macrophages in the intestine and lymph nodes and is able to survive and replicate inside phagosomes by inhibition of the phago-lysosome maturation. Paratuberculosis is characterized clinically by progressive emaciation and chronic diarrhea and causes major economic losses in many countries by reduced milk production, weight loss and death.The protective immunity against paratuberculosis is incompletely defined. However, it is well known that Th1 CD4+ T cells have a central role by activating the infected macrophages by producing IFN-γ during the antigen recognition process through the MHC class II- TCR interaction. On the other hand, there is a very little knowledge about other subgroups of CD4+ T cells like Th2, Treg and Th17, which produce cytokine like IL-4, IL-10 and IL-17A, respectively and their role in the protective immunity against paratuberculosis.The main aim of this thesis was to study the characteristics of reactive CD4+ T cells under MAP infection by optimising a method to isolate T cell lines and T cell clones from naturally infected goats. Different methods were tried to obtain a maximum growth of CD4+ T cells. Positive selection of CD4+ T cells isolated by MACS or Dynal magnetic beads increased the yield of cultivated CD4+ T cells and minimized the overgrowth of CD8+ and γδ T cells in the cultures. Media supplemented with 10% goat serum (GS) was better for the growth of T cells than the same concentration of foetal calf serum (FCS) and was further used for the culture of goat T cells. PHA at 1μg/ml was used as a mitogen, and the CellTiter-Glo assay was used instead of 3H-thymidin incorporation assay as a read out for cell proliferation in T cell recall response tests (T cell proliferation assay).Cytokine production was tested by different methods. A routine plasma ELISA method was used to measure IFN-γ production by PBMC after PPD-J stimulation of whole blood for 20 hours and confirmed the presence of MAP responsive T cells in the blood. Intracellular staining by flow cytometry demonstrated that both CD4+ and CD8+ T cells could secret IFN-γ, and that the highest production after PPD-J stimulation was in CD4+ T cells. RT-PCR for mRNA encoding IL-10, IFN-γ and IL-17 was established and an increase of IFN-γ and IL-17 mRNA expression after PPD-J stimulation was noted in one T -cell line. No increase of IL-10 mRNAXwas detected after PPD-J stimulation of T cell lines by either Q-RT-PCR or intracellular cytokine staining.The ability to culture antigen specific T cells was demonstrated after immunization with MAP specific peptides. Five CD4+ T cell lines had responses to three peptide pools that contained 20 peptides in each. Furthermore, responses to 11 individual peptides were demonstrated.In conclusion, we have established a method for cultivation of CD4+ T cells in vitro. This method can be used for detailed characterisation of both specificity and phenotype of this T cell in MAP infection.