Ppd a

Bovine tuberculin (PPD-B) and avian tuberculin (PPD-A) antigens were purchased from Thermo Fisher Scientific. The BOVIGAM^TM kit was used for the IFN-γ ELISA. The tuberculin antigens (purchased from Thermo Fisher), PPD-B and PPD-A, were used for in vitro stimulation of whole blood at a final concentration of 300 and 250 IU/ml, respectively, as per the kit instructions (antigens provided with the BOVIGAM^TM kit were not used in the assays). The DST was used at 10 μg/ml in in vitro assays. Blood was collected for IGRA just prior to BCG vaccination at week 0, and just prior to skin test at weeks 4, 6, and 16. Whole blood was stimulated overnight (37C, 5% CO₂) with antigens (PPDs and DST) in vitro. BOVIGAM^TM enzyme-linked immunosorbent assay-based kits (Thermo Fisher Scientific, USA) were used to determine IFN-γ concentrations in whole blood culture supernatants. Results for antigen-stimulated cultures were expressed as background-corrected optical density at 450 nm (i.e., ΔOD₄₅₀).

Bovine and Avian Purified Protein derivatives (PPD-B and PPD-A) were purchased from Thermo Fisher, UK. The DIVA skin test (DST) fusion protein consisting of the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c (DST-F) was obtained from Lionex (Braunschweig, Germany). A peptide cocktail of DST composed of 13 overlapping peptides covering the amino acid sequences of the same three antigens (DST-P) ^{24 (link)} was purchased as individual peptides from Genscript. Pokeweed mitogen (PWM) was obtained from Sigma, UK. All antigens were diluted in RPMI-1640 when used for in vitro whole blood stimulations. Due to reagent availability, DST-P was used in the experiment with 8 animals while DST-F in the pilot trial with 4 animals.

The SICCT test was conducted at four time points during the experiment, at the 6th week after vaccination or before exposure to reactor cows (month 0), and at the 4th, 8th, and 12th months post-exposure. This test was performed using bovine and avian purified protein derivatives (PPD-B and PPD-A) obtained from Thermo-Fisher (Prionics, Lelystad, the Netherlands). Briefly, 0.1 ml PPD-A (2,500 IU/ml) was administered intradermally on the left side of the neck at a site tip of ear reached and the same volume of PPD-B (3,000 IU/ml) was injected at a site 12 cm distant from the PPD-A site in the shoulder direction. The skin thicknesses were measured just before injection and at 72 h post-injection by the same operator using the same digital caliper, and the results were presented as change in skin thickness (mm) between the two readings. The differences in the increase of skin thickness at the bovine and avian PPD injection sites were determined. An animal was considered to be positive when the increase in skin thickness at the bovine PPD site was >4 mm more than the increase in skin thickness at the site of the avian injection. If the differential increase between the two sites were equal to or < 1mm, or between 1 and 4mm, the animal was considered negative and inconclusive, respectively (31 ).