Assessment of live candidate vaccines for paratuberculosis in animal models and macrophages

Abstract

Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the causative agent of paratuberculosis, a chronic enteritis of ruminants. To control the considerable economic effect that paratuberculosis has on the livestock industry, a vaccine that induces protection with minimal side effects is required. We employed transposon mutagenesis and allelic exchange to develop three potential vaccine candidates, which were then tested for virulence with macrophages, mice, and goats. All three models identified the WAg906 mutant as being the most attenuated, but some differences in the levels of attenuation were evident among the models when testing the other strains. In a preliminary mouse vaccine experiment, limited protection was induced by WAg915, as evidenced by a reduced bacterial load in spleens and livers 12 weeks following intraperitoneal challenge with M. paratuberculosis K10. While we found macrophages and murine models to be rapid and cost-effective alternatives for the initial screening of M. paratuberculosis mutants for attenuation, it appears necessary to do the definitive assessment of attenuation with a ruminant model.

FIG. 1.

Survival of mutant M. paratuberculosis strains within bovine macrophages. Macrophages were harvested for bacterial culture at day 0 (black bar), day 7 (white bar), and day 14 (gray bar). The data are shown as the mean number of CFU (±standard error [SE]), and the significance of the difference between the mutant strains and their parent wild type was determined by least-significant-difference ANOVA (*, P < 0.05).

FIG. 2.

Apoptosis of bovine macrophages infected with M. paratuberculosis strains. Macrophages from the two animals used are represented by black and gray bars; stau, staurosporine-positive control; uninf, uninfected macrophages. The data are representative of 1 of 2 experiments. Data are shown as mean percent apoptosis (±SE); *, significantly different from wild type (P < 0.05).

FIG. 3.

Production of IL-10 by bovine macrophages infected with M. paratuberculosis strains. Data are shown as the mean of IL-10 production (±SE) from macrophages of two animals indicated by black and gray bars; pos, positive control; uninf, uninfected; *, significantly different from wild type (P < 0.05).

FIG. 4.

Persistence of mutant M. paratuberculosis strains in mice. The data represent the mean CFU values (±standard deviation) from the spleens (A) and livers (B) of 5 mice. The growth of mutant strains WAg906 (▴), WAg913 (×) and WAg915 (○) was compared to that of the wild-type strains 989 (▪) and K10 (□). *, significantly different from wild type (P < 0.05).