Research ArticleHEALTH AND MEDICINE

A defined antigen skin test for the diagnosis of bovine tuberculosis

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaax4899
DOI: 10.1126/sciadv.aax4899
  • Fig. 1 In vitro and skin test responses of fusion protein and PCL.

    (A) Capacity of antigens (Fusion and PCL) to induce in vitro IFN-γ response in PBMCs collected from naturally M. bovis–infected cattle (n = 10), naïve cattle (n = 10), and BCG vaccinates (n = 10) was determined. The antigens were used at titrated dose concentrations, and AUC is plotted. The horizontal line provides the mean (±SD), and the statistical difference between the responses was determined by using two-tailed paired t test (**P < 0.01; ***P < 0.001). The background-corrected optical density (OD) values of the Fusion and PCL at each titrated concentration tested are shown in the inset. The PPD-B–induced IGRA responses of these animals tested in (A) are shown in fig. S1. (B) Skin test responses for Fusion, PCL, and PPD(B-A) were measured at 72 hours after injection in cattle experimentally infected with M. bovis (n = 24) and naïve controls (n = 20). Results are expressed as the difference in skin thickness (in millimeters) between the 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 analysis of variance (ANOVA) (****P < 0.0001). The solid horizontal lines at 2 and 4 mm are the cutoffs used for Fusion and PCL, and PPD(B-A) and PPD-B, respectively.

  • Fig. 2 Heat map representing the IFN-γ response elicited by the individual long (top) and short (bottom) peptides of ESAT-6, CFP-10, and Rv3615c.

    ELISpot/FluoroSpot assays were performed using PBMCs isolated from naturally M. bovis–infected cattle (n = 14). Z scores of individual peptide response are mapped. On the basis of overall peptide responder frequencies, a new peptide cocktail was formulated, PC-1. The peptides constituting PC-1 are marked with closed circles. Note: RL2 could not be synthesized due to technical difficulties, and RS5 was included as part of PCL to cover the gap in the overlap left by the absence of RL2.

  • Fig. 3 In vitro responses of PCL and PC-1.

    Capacity of PCL and PC-1 to induce in vitro IFN-γ responses in PBMCs collected from naturally M. bovis–infected cattle (n = 20), BCG vaccinates (n = 10), and controls (n = 10) was evaluated. The antigens were used at titrated dose concentrations, and AUC is plotted. The horizontal line provides the median (±95% CI), and the statistical difference between the responses for reactors and controls was determined using the Wilcoxon matched-pairs signed-rank test (***P < 0.001; **P < 0.01), while two-tailed t test was used for vaccinates (**P < 0.01). There is no significant difference between the responses induced by PCL and PC-1 in infected animals. The background-corrected OD values of PCL and PC-1 at each titrated concentration tested are shown in the inset, where PCL induced a significantly stronger response than PC-1 only at the highest titrated dose concentration of 10 μg/ml. PPD-B–induced IGRA responses of these animals are shown in fig. S2B.

  • Fig. 4 Skin test response to peptide cocktails in field reactors.

    (A) Peptide cocktails, PCL and PC-1, alongside PPD-A and PPD-B, were tested in naturally infected animals identified in Ethiopia (n = 25). Results are expressed as the difference in skin thickness (in millimeters) between the pre- and post-skin test readings, with the horizontal line providing the median (±95% CI). Positivity is defined as ≥2 mm for PCL and PC-1, and ≥4 mm for PPD(B-A) and PPD-B (denoted by the solid lines). The statistical difference between PCL and PC-1 responses was determined by using two-tailed t test (**P < 0.01). (B) The relative sensitivity of the peptide cocktails, SIT, and SICCT was calculated. (C) Animals represented by open red circles are positive by PCL only, closed blue circles are positive by PPD(B-A) only, open blue circles are negative on both, and closed red circles are positive on both. The peptide cocktail, PCL, identified nine (36%) presumed infected animals that were not detected by the standard SICCT.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaax4899/DC1

    Table S1. All short and long peptides used in the study are listed and sequences are provided.

    Fig. S1. PPD-B responses in the IGRA.

    Fig. S2. Comparison of performance of PCL with PC-1 in IGRA.

    Fig. S3. Skin test responses in field reactors.

    Fig. S4. Schematic representation of the approach to identify the dominant peptides based on overall responder frequency for the peptide cocktails.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. All short and long peptides used in the study are listed and sequences are provided.
    • Fig. S1. PPD-B responses in the IGRA.
    • Fig. S2. Comparison of performance of PCL with PC-1 in IGRA.
    • Fig. S3. Skin test responses in field reactors.
    • Fig. S4. Schematic representation of the approach to identify the dominant peptides based on overall responder frequency for the peptide cocktails.

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