Development and evaluation of a bead-based xMAP immunoassay for differential serodiagnosis of Babesia bovis and B. bigemina in cattle.

Bovine babesiosis is caused by Babesia bovis and Babesia bigemina and requires species-resolved serosurveillance to guide control programs. With this goal in mind, we developed an indirect bead-based liquid microarray (xMAP/Luminex) immunoassay using recombinant SBP4 (B. bovis) and RAP-1 (B. bigemina). Antigens immunoreactivity was confirmed by dot-blot and western blot. and antigens were covalently coupled to magnetic microspheres. Assay conditions were optimized with anti-bovine IgG-phycoerythrin detection, and diagnostic performance was assessed in 89 bovine sera previously classified by antigen-matched ELISAs; cut-offs were defined as the mean signal of negatives plus two standard deviations. The assay discriminated against species-specific antibodies and achieved sensitivity/specificity of 79.17%/94.12% for B. bovis (SBP4) and 96.92%/95.65% for B. bigemina (RAP-1). These results indicate high specificity and promising sensitivity, with inherent scalability to multiplex panels and high-throughput testing. Larger, geographically diverse validations-ideally including PCR as an independent reference-are warranted to refine cut-offs, quantify agreement, and assess intra-/inter-assay reproducibility for routine surveillance.