This study investigates three Brucella virulence-related Tat substrate proteins—L,D-transpeptidase ErfK (A0577), linear amide C-N hydrolase YxeI (A1479), and thioesterase domain-containing protein EntF (B0249)—to systematically analyze their roles in the diagnosis of brucellosis in bovine and sheep sera. Here, TAT is linked to brucellosis.