Defined Antigen Skin Test for Bovine Tuberculosis Retains Specificity on Revaccination With Bacillus Calmette-Guérin

Abstract

The Bacillus Calmette-Guérin (BCG) vaccination provides partial protection against, and reduces severity of pathological lesions associated with bovine tuberculosis (bTB) in cattle. Accumulating evidence also suggests that revaccination with BCG may be needed to enhance the duration of immune protection. Since BCG vaccine cross-reacts with traditional tuberculin-based diagnostic tests, a peptide-based defined antigen skin test (DST) comprising of ESAT-6, CFP-10, and Rv3615c to detect the infected among the BCG-vaccinated animals (DIVA) was recently described. The DST reliably identifies bTB-infected animals in experimental challenge models and in natural infection settings, and differentiated these from animals immunized with a single dose of BCG in both skin tests and interferon-gamma release assay (IGRA). The current investigation sought to assess the diagnostic specificity of DST in calves (Bos taurus ssp. taurus × B. t. ssp. indicus; n = 15) revaccinated with BCG 6 months after primary immunization. The results show that none of the 15 BCG-revaccinated calves exhibited a delayed hypersensitivity response when skin tested with DST 61 days post-revaccination, suggesting 100% diagnostic specificity (one-tailed lower 95% CI: 82). In contrast, 8 of 15 (diagnostic specificity = 47%; 95% CI: 21, 73) BCG-revaccinated calves were positive per the single cervical tuberculin (SCT) test using bovine tuberculin. Together, these results show that the DST retains its specificity even after revaccination with BCG and confirms the potential for implementation of BCG-based interventions in settings where test-and-slaughter are not economically or culturally feasible.

Keywords: Bacillus Calmette-Guérin (BCG) vaccine; DIVA; DST; bovine tuberculosis (bTB); specificity.

Figure 1

Timeline chart. Calves received the first Bacillus Calmette–Guérin (BCG) dose on day 0 of the previous trial (blue section) (20). The orange section represents the current trial. BCG revaccination was performed after 6 months of the first dose (day 0 of current trial). Blood was collected for interferon-gamma release assay (IGRA) just prior to revaccination with BCG and on days 15, 30, 45, and 60 post revaccination. Skin test was conducted on day 61 post-revaccination. The duration of the current trial from revaccination to skin test is 2 months.

Figure 2

PPDs- and DST-stimulated IFN-γ responses following Bacillus Calmette–Guérin (BCG) vaccination. In vitro IFN-γ responses of BCG vaccinates (closed circle, n = 12) and revaccinates (open circle, n = 14) to (A) PPD-A, (B) PPD-B, (C) PPD(B-A), and (D) DST. Results are expressed as background-corrected (delta) optical density (OD) values. Days post-BCG are shown in the x-axis. All calves were vaccinated with BCG on day 0. Blood was collected for interferon-gamma release assay (IGRA) just prior to injection of BCG on day 0, and on days 15, 30, 45, and 60 post-vaccination. The differences in responses induced between the various timepoints were determined using Tukey's multiple comparison test (*p < 0.05). The IGRA cutoff of 0.1 is shown as a dotted red line.

Figure 3

Skin test responses induced by PPD-B (SCT), PPD-A, CCT, and DST10 (10 μg per peptide constituent) were measured 72 h post injection in calves vaccinated with BCG for the first time (n = 14) and in revaccinates (n = 15). Results are expressed as the difference in skin thickness (in millimeters) between pre- and post-skin test readings, with the horizontal line providing the median [±95% confidence interval (CI)]. The statistical difference between the responses was determined using Tukey's multiple comparison test (****p < 0.0001, ***p < 0.001, **p < 0.01). The dotted horizontal lines at 2 and 4 mm are the cutoffs used for DST, and SCT and CCT, respectively.