Introduction

Bovine paratuberculosis is caused by infection of young calves with Mycobacterium avium ssp. paratuberculosis (MAP). MAP causes intracellular infection in macrophages and results in chronic granulomatous enteritis. The aim of the present study was to investigate whether a heat shock protein (Hsp) based delivery system could be used to generate cytotoxic T cells in cattle as a tool to aid the elimination of MAP infected macrophages, as described in murine model systems.

Materials and methods

A recombinant fusion protein, rHsp70-GFP, was produced according to previously published methods (Huang et al, 2000, J. Exp. Med. 191, 403) with some modifications. In the current study, the N-terminal receptor binding part of recombinant MAP Hsp70 was fused to enhanced green fluorescent protein (GFP), the latter serving as a model antigen. Five cows were immunized 4 times with the rHsp70-GFP fusion protein, an additional cow was immunized with GFP only. Peripheral blood mononuclear cells were isolated using density gradient centrifugation and used in lymphocyte stimulation assays. Target cells were pulsed with the rHsp70-GFP, GFP protein or GFP peptides. Cytotoxicity was measured using chromium release assays and a flowcytometric method using different effector target ratios. Serum was used to determine antibody responses in ELISA.

Results and Conclusion

Immunisation with rHsp70-GFP fusion protein elicits cellular and humoral immune responses to GFP, GFP peptides and Hsp70-GFP but as assessed by chromium release assay and a flowcytometric assay, no cytotoxic T cell reactivity in cattle.