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. 2024 Apr 30;11(5):198.
doi: 10.3390/vetsci11050198.

The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle

Yuhao Zhao  1   2   3   4 , Wentao Fei  1   2   3   4 , Li Yang  5 , Zhijie Xiang  1   2   3   4 , Xi Chen  1   2   3   4 , Yingyu Chen  1   2   3   4 , Changmin Hu  1   2   3   4 , Jianguo Chen  1   2   3   4 , Aizhen Guo  1   2   3   4
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The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle

Yuhao Zhao et al. Vet Sci. .

Abstract

BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 μg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected calves but negative results in vaccinated calves. In conclusion, this DIVA method has broad prospects in differentiating BCG vaccination from natural infection to prevent bTB.

Keywords: BCG; bovine tuberculosis (bTB); differential antigens; differentiation of the infected from vaccinated animals (DIVA); interferon-gamma (IFN-γ) in vitro release assay (IGRA); regions of difference (RDs); vaccination.

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Conflict of interest statement

The authors declare no potential conflict of interest in the research, authorship, and/or publication of this article.

Figures

Figure A1
Figure A1
Purified RCE of 35 kDa was checked using 10% SDS-PAGE. Lane M represents the marker of the molecular mass; Lane 1 represents the purified RCE.
Figure 1
Figure 1
The ROC was used to determine the cut-off value of RCE-DIVA IGRA for bTB diagnosis. (A) Stimulation of blood with 2 μg/mL of RCE. (B) Stimulation of blood with 4 μg/mL of RCE. (C) Stimulation of blood with 6 μg/mL of RCE. (D) Stimulation of blood with 8 μg/mL of RCE. The commercial IGRA for bTB diagnosis was used to test 97 cattle blood samples with 74 bTB-positive and 23 bTB-negative cattle as the references.
Figure 2
Figure 2
Spearman’s correlation analysis of plasma samples from 86 cattle stimulated with PPD and RCE OD630 values. (A) The correlation between PPD-B–PBS and RCE-PBS OD630 values in the plasma samples. (B) The correlation between PPD-B–PPD-A and RCE-PBS OD630 values in plasma samples.
Figure 3
Figure 3
Differentiation of naturally infected and BCG-vaccinated calves via RCE and PPD-based whole blood IFN-γ release assay (IGRA). (A,B) IFN-γ levels of calves in infected, vaccinated, and PBS control groups detected via commercial PPD-IGRA (the red dashed line represents the cut-off value of 0.20). (C) IFN-γ levels of calves in infected, vaccinated, and PBS control groups detected via RCE-IGRA (the red dashed line represents the cut-off value of 0.15). ns, not significant. *, **, and **** represent p < 0.05, p < 0.01, and p < 0.0001, respectively.
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