A Defined Antigen Skin Test That Enables Implementation of BCG Vaccination for Control of Bovine Tuberculosis: Proof of Concept

Abstract

In most low- and middle-income countries (LMICs), bovine tuberculosis (bTB) remains endemic due to the absence of control programs. This is because successful bTB control and eradication programs have relied on test-and-slaughter strategies that are socioeconomically unfeasible in LMICs. While Bacillus Calmette-Guérin (BCG) vaccine-induced protection for cattle has long been documented in experimental and field trials, its use in control programs has been precluded by the inability to differentiate BCG-vaccinated from naturally infected animals using the OIE-prescribed purified protein derivative (PPD)-based tuberculin skin tests. In the current study, the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated (Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0 week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test (100% specificity; one-tailed lower 95% CI: 82). In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) (specificity in vaccinated animals = 33%; 95% CI: 12, 62). Taken together, the results provide strong evidence that the DST is highly specific and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible.

Keywords: BCG; DIVA; DST; bovine tuberculosis; specificity.

Figure 1

Timeline chart. Following arrival of calves on site, they were acclimatized for a period of 2 weeks. Vaccination of calves at week 0 denotes start of trial. Blood was collected for Interferon-gamma release assay (IGRA) just prior to injection of BCG, and at weeks 4, 6, and 16 post-vaccination. Skin tests were conducted at weeks 6 and 16.

Figure 2

IFN-γ responses following BCG vaccination. Responses of BCG vaccinates (closed circle, n = 15) and controls (open circle, n = 15) to (A) PPD-B, (B) PPD-A, and (C) DST. The background-corrected (delta) optical density (OD) values are plotted. Time is shown as weeks post-vaccination in the x-axis. Calves were vaccinated with BCG at week 0. Blood was collected for IGRA just prior to injection of BCG, and at weeks 4, 6, and 16 post-vaccination. Skin test using PPDs and DST was conducted at weeks 6 and 16. The pairwise difference between pre- and post-vaccination time points was determined using the Wilcoxon matched pairs signed rank test (**P < 0.01; *P < 0.05). The dotted red line represents the IGRA cutoff of 0.1. (D) The bottom right panel provides the area under the curves (21), where the horizontal line provides the median, and the statistical difference between the responses was determined using Friedman (non-parametric) test (****P < 0.0001).

Figure 3

Skin test responses induced by DST, PPD-B, and PPD-A were measured 72 h post-injection in calves vaccinated with BCG (n = 15) and naïve controls (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 Friedman test (***P < 0.001). The dotted horizontal lines at 2 and 4 mm are the cutoffs used for DST, and PPD-B and PPD (B-A), respectively. The two control animals that are SIT positive are the same two that show > 4 mm PPD-A responses.