Heat shock protein 70 and bovine paratuberculosis

Abstract

Paratuberculosis, or Johne’s disease, is a chronic infection of the small intestine of cattle caused by Mycobacterium avium ssp. paratuberculosis (M.a.p.) , leading to an incurable form of protein loosing enteropathy. Young animals are orally infected with M.a.p., which resides and replicates in the macrophages of the gut. After a long period without symptoms, disease develops generally at the age of 4-6 years. Asymptomatic animals shedding bacteria with their feces contribute to infection of other young animals. Due to both difficulties of diagnosis of the disease in an early stage and high resistance of the bacteria against environmental influences, prevention of the disease is difficult. A good vaccine, together with management measures that are currently undertaken, would help in eradicating the disease.

Based on the knowledge on other intracellular bacteria, we hypothesized that a vaccine should induce a cytotoxic T cell (CTL) reaction in vaccinated animals. Secondly, we hypothesized that these CTL could be induced by using Heat Shock Protein 70 (Hsp70) of M.a.p. as a carrier protein to shuttle antigen into the MHC class I pathway.

In a longitudinal study on 20 infected calves we showed that antibody production was related to high frequent shedding of bacteria, while differences in cellular immune reactions between high and low frequent shedders were only detectable early in the study. For the use of Hsp70 as a CTL inducing protein we investigated if bovine antigen presenting cells (APC) interacted with the recombinant Hsp70 in vitro. Receptor mediated endocytosis of Hsp70 could be demonstrated, which was related to the type and the maturation stage of the APC (monocytes, macrophages, dendritic cells). Interaction of a fusion protein, consisting of the receptor binding domain of Hsp70 fused to enhanced Green Fluorescent Protein (GFP), with bovine APC could also be demonstrated, which made the fusion protein suitable for immunization of cattle. Recognition of the fusion protein could be demonstrated in vitro with lymphocyte stimulation tests, however, GFP specific CTL activity could not be detected with two different cytotoxicity assays. As dendritic cells (DC) are pivotal in initiating immune reactions, cytokine gene expression profiles of DC incubated with M.a.p., E.coli and Hsp70 were compared at 6 and 24 hours of incubation. Cytokine gene expression was largely similar for incubation with M.a.p. and E.coli, resulting mainly in the expression of pro-inflammatory cytokines, while especially IL-12 gene expression was detected later, after 24 hours of incubation only, when DC were incubated with Hsp70. In the last chapter of the thesis the results are discussed with emphasis on Hsp70 as a tool in disease prevention and as a dominant antigen during infection.