Evaluation of serological assays for intra vitam diagnosis of Bovine Tuberculosis in Water Buffalo (Bubalus bubalis)

Sara Giovannozzi¹Alessandra Martucciello²Maria Mercedes Dominguez³Inmaculada Moreno³Mery Boifava⁴Lorena Schiavo²Anna Viscito²Giovanni Parisio⁴Nicoletta Vitale⁵Javier Bezos⁶Esterina De Carlo⁷Maria Beatrice Boniotti^4*

• ¹1National Reference Centre for Bovine Tuberculosis (CNR-bTB), Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy
• ²National Reference Centre for Hygiene and Technologies of Water Buffalo Farming and Productions, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Salerno, Italy
• ³Unidad de Inmunología Microbiana, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain
• ⁴National Reference Centre for Bovine Tuberculosis (CNR-bTB), Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy
• ⁵Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta, Turin, Italy
• ⁶Animal Health Department of Veterinary Faculty, Complutense University of Madrid, Madrid, Spain, VISAVET Health Surveillance Centre,, Madrid, Spain
• ⁷Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Italy

Bovine tuberculosis (TB) in water buffalo (Bubalus bubalis) is primarily diagnosed using intra vitam tests, as the intradermal tuberculin test (IDT) and the interferon-gamma release assay (IGRA), both of which detect cell-mediated immunity (CMI). However, a subset of infected animals fails to mount a detectable CMI response, posing a significant risk of undetected transmission. Serological tests assessing the humoral immune response could provide a valuable complementary tool for identifying infected animals that escape detection through traditional CMI-based assays. This study analyzed 895 serum samples from water buffaloes, including 393 from TB-free herds and 502 from TB-infected herds. Animals from TB-free herds were tested using IDT and an ELISA assay, whereas those from infected herds were also tested using IGRA. We developed an ELISA assay targeting MPB70, MPB83, ESAT6, CFP10, PPDB, and P22 antigens to investigate the role of the humoral response in TB diagnosis. The ELISA showed a specificity of 98,2%. However, sensitivity differed based on the antigen used: among the most reactive proteins, sensitivity was 67.5% for MPB70, 69.8% for P22, and 74.4% for PPDB. Moreover, approximately 70% of samples with discordant IDT and IGRA results, as well as those with positive IDT but inconclusive IGRA results, tested positive by serology, highlighting the potential of antibody-based detection to improve TB diagnosis in buffaloes. Our findings suggest that integrating serological testing with standard diagnostic methods could enhance the detection of infected animals, ultimately contributing to better TB control in buffalo populations.