Establishment and evaluation of an interferon-gamma enzyme-linked immunospot method for the detection of Brucella-infected cattle and goats.

Brucellosis is classified as a class II animal disease in China, with recent years seeing an increasing prevalence of Brucella infections in livestock, posing a significant threat to public health. In this study, a novel IFN-γ enzyme-linked immunospot (ELISpot; Brucella purified protein derivative, Br-PPD) assay specifically tailored for detecting Brucella-infected cattle and goats was developed. This assay employed bovine and goat IFN-γ monoclonal antibodies, 3E3 and biotinylated 8D3, respectively, for capturing and detecting IFN-γ. This method demonstrated high sensitivity and specificity. When 10 spot-forming units was selected as the cut-off value, the sensitivity and specificity of the method were 96.9% and 90.6%, respectively. Compared with the ELISA method, the IFN-γ ELISpot assay showed ∼30 times greater sensitivity in detecting IFN-γ release from peripheral blood mononuclear cells. When applied to clinical samples from both cattle and goats, the ELISpot results strongly correlated with traditional antibody-based diagnostic methods, including the serum agglutination test (SAT), rose bengal plate test (RBPT), and competitive ELISA. The positive agreement rate exceeded 80%, and the negative agreement rate surpassed 90%. Notably, employing Brucella-vaccinated goat models, our study has confirmed that the ELISpot assay can detect Brucella infections earlier than the SAT and offers a more extended diagnostic window. As a cellular immunology-based diagnostic tool, the IFN-γ ELISpot (Br-PPD) assay holds considerable potential to improve early detection of Brucella-infected cattle and goats, addressing existing diagnostic shortcomings.