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

Author:

Zhao Yuhao1234,Fei Wentao1234,Yang Li5,Xiang Zhijie1234,Chen Xi1234,Chen Yingyu1234ORCID,Hu Changmin1234,Chen Jianguo1234,Guo Aizhen1234

Affiliation:

1. National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

2. Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China

3. Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, China

4. Key Laboratory of Ruminant Bio-Products of Ministry of Agriculture and Rural Affairs, Huazhong Agriculture University, Wuhan 430070, China

5. Wuhan Keqian Biology Co., Ltd., Wuhan 430206, China

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.

Funder

Ministry of Science and Technology of the People's Republic of China

Publisher

MDPI AG

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