Figure 1.
Western blot demonstrating Ag85B expression in BCG Pasteur ΔleuD.
Lane 1, whole-cell lysates of rBCG transformed with pUP410::fbpB; lane 2, culture supernatant of rBCG transformed with pUP410::fbpB; lane 3, whole-cell lysates of wild-type BCG; lane 4, culture supernatant wild-type BCG.
Figure 2.
Protective efficacy as measured by gross pathology.
The calves were euthanized sixteen weeks after infection and thin slices of lungs and lymph nodes were analysed looking for granuloma formations. Pathology scores were established using the scoring system described in Material and Methods (A) Mean pathology scores of lungs in vaccinated and nonvaccinated groups. (B) Mean pathology scores of total lesions (head lymph nodes, respiratory tract-associated lymph nodes and lungs) in vaccinated and nonvaccinated groups. Pathology scores for individual animals are plotted. Horizontal lines indicate median values. Significance was determined by Mann Whitney test: * Statistically significantly different, P<0.05.
Figure 3.
Protective efficacy as measured by histopathology in lungs.
Histopathology scores in lungs of individual animals in vaccinated and nonvaccinated groups are plotted. Tissue samples from lungs and lymph nodes were obtained sixteen weeks after infection. Histopathology scores were established using the scoring system described in Material and Methods. Horizontal lines indicate mean values. Significance was determined by Mann Whitney test, * Statistically significantly different, P<0.05.
Figure 4.
IFN-γ production in PPDB-stimulated PBMCs.
(A) IFN-γ responses in vaccinated and nonvaccinated animals following vaccination and after challenge. IFN-γ production was measured in PPDB-stimulated blood from animals vaccinated with BCG (squares), ΔleuD BCG-85B (triangles) or nonvaccinated (circles) at different time points (0, 30 and 60, 120 and 150 days post-vaccination). Animals were infected eight weeks after vaccination and sacrificed sixteen weeks post-challenge. Arrow indicates the challenge time point. Significance was determined by ANOVA test. Statistically significantly different to that for the nonvaccinated group, *P<0.05. (B) Correlation between IFN-γ production in PPDB-stimulated PBMCs and disease severity. Results are expressed as IFN-γ 40 days post-challenge of individual animals in relation to the corresponding total gross pathology scores. Solid line indicates linear regression; dashed lines indicate 95% confidence intervals. Values for r2 and p of linear regression analysis are indicated.
Figure 5.
Mean tuberculin skin test responses to PPDB.
Animals were vaccinated with BCG (n = 5, squares), ΔleuD BCG-85B (n = 5, triangles) or inoculated with PBS (n = 6, circles) and challenged after eight weeks with M. bovis. Values indicate skin thickness at one month post-vaccination and prior to challenge (A), and three months post-challenge (B). Horizontal lines indicate mean values. Data were analysed using a Mann Whitney test,*P<0.05, **P<0.005.
Figure 6.
Determination of lymphocyte subsets in PPDB-stimulated PBMCs.
Percentages of lymphocyte cell subsets CD4+ (A) and CD8+ (B) expressing CD25 for PPDB stimulated-PBMCs from animals vaccinated with BCG (n = 5, triangles), ΔleuD BCG-85B (n = 6, circles) or nonvaccinated (n = 6, squares) at 15, 30 and 60 days after vaccination and 20, 40, 70 and 100 days after challenge. The arrow indicates the challenge time point. Data were analysed using Mann-Whitney test for comparison between groups. (Statistically significantly different to that for the nonvaccinated group *P<0.05 and ** P<0.01).
Figure 7.
Relative cytokine gene expression.
Gene expression of IFN-γ (A), IL-2 (B), IL-4 (C) and IL-17 (D) was measured in PPDB-stimulated PBMCs from animals vaccinated with BCG (gray), ΔleuD BCG-85B (white) or nonvaccinated (black) at different time points (15 and 30 days post-vaccination and 20, 40, 70 and 100 days post-challenge). Transversal line indicates the challenge time point. Relative gene expression was calculated using the 2-ΔΔCt method with E correction, using gadph mRNA expression as reference gene and the pre-immune condition as the calibrator. Data were analysed using Mann-Whitney test, statistically significantly different, * P<0.05 and ** P<0.01. The bars indicate the median fold change.
Figure 8.
Correlation between IL-17 and IFN-γ mRNA expression in PPDB-stimulated PBMCs and disease severity.
Results are expressed as mean increases in IL-17 (A) and IFN-γ (B) transcription at 15 days post-vaccination and IFN-γ transcription at 40 days post-challenge (C) of individual animals in relation to the corresponding total gross pathology scores (head lymph nodes, respiratory tract-associated lymph nodes and lungs). Animals vaccinated with either BCG (squares) or ΔleuD BCG-85B (triangles) and nonvaccinated (circles) were infected eight weeks after vaccination and sacrificed sixteen weeks post-challenge. Solid lines in panels A and B indicate linear regression; dashed lines indicate 95% confidence intervals. Values for r2 and p of linear regression analysis are indicated.