Paratuberculosis, or Johne's disease, is a disease of domestic and wild ruminants that culminate with a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis. The aim of this work was to evaluate the type of immune response, Th1 or Th2, induced by DNA vaccinations in lambs of Sarda breed. Twenty-five lambs, seronegative for paratuberculosis, were selected at birth from equally seronegative mothers. The lambs were inoculated with three different mycobacterial antigens cloned into a mammalian expression vector as fusion protein with the enhanced green fluorescent protein (pEGFP-N1). The animals at 5 months were divided in 5 groups containing each 5 lambs. Each group was vaccinated as following (A: physiological solution, B: Gudair™, C: p-85A-Mav, D: p-85A-BCG, E: p-Hsp65). Immune response was evaluated by measuring the expression of INF-γ (Th1 type response) and IL-10 (Th2 type response) by Real Time PCR. Gene expression was estimated by comparing the results with that of β-actin. Each animal was vaccinated by intramuscular inoculation with three doses. RNA extracted from peripheral lymphocytes stimulated with Johnin PPD (purified protein derivate) and from lymphocytes not stimulated, was extracted and used as templates in reverse transcription reaction. The cDNA obtained was investigated in order to evaluate gene expression by Real Time PCR. INF-γ expression level was increased in lambs vaccinated with plasmids codifying mycobacterial antigens (in particular with Hsp65) in comparison with the controls suggesting a Th1 immune response as that supported by natural infection by M. paratuberculosis. Moreover, vaccinated animals were challenged after 3 months with M. paratuberculosis. Animals were infected orally with a single dose of 20 ml of 10⁸ bact/ml. Sheep were sacrificed after one year from the challenge, histological analysis was performed and correlated with expression of INF-γ and IL-10 in order to verify in vivo the protection level of the different vaccines.

Sponsorship

Attendance to this Congress was sponsored by the EU-funded project SSPE-CT-2004-501903