A roadmap in detecting frequently reported bovine babesiosis: From blood smear to CRISPR.

Current diagnosis of Babesia bovis and B. bigemina relies on direct microscopy, nucleic acid detection, and serology. Light-microscopic analysis of Giemsa-stained smears still serves as the primary diagnostic modality at the point of care. However, carrier cattle, particularly those harbouring B. bovis, often carry parasites at levels far below the detection threshold, and such levels are sensitive only to DNA-based detection approaches. Early probe-hybridization techniques have been largely replaced by conventional Polymerase Chain Reaction (PCR), nested formats, and real-time quantitative PCR (qPCR)-which enable species-specific discrimination within closed-tube systems, thereby minimizing contamination risk. Species‑level identification is essential for clinical management, surveillance, and experimental studies. Duplex TaqMan qPCRs simultaneously distinguish B. bovis from B. bigemina, surpassing the analytical sensitivity offered by nested PCR (nPCR) methodologies. Reverse-line-blot (RLB) hybridisation broadens the diagnostic scope by concurrently detecting co-infections of B. bovis and B. bigemina and mixed haemoparasitic species in one workflow. However, the analytical sensitivity of RLB remains inferior to that of qPCR in detecting low-density and carrier-state infections of B. bigemina. RLB remains useful for retrospective genotyping when amplification is impractical or fails. Field-ready isothermal approaches have expanded the scope of molecular diagnostics beyond laboratory settings, facilitating field-level application and rapid on-site detection. Coupling Loop-mediated isothermal amplification (LAMP) with a lateral-flow dipstick (LFD) (LAMP-LFD) enables pen-side direct visual detection. Antibody tests are essential tools for herd-level surveillance. A recent chimeric Enzyme-Linked Immunosorbent Assay (ELISA) that combines three immunodominant B. bovis antigens broaden strain coverage and boosts diagnostic reliability. Recombinase polymerase amplification coupled to CRISPR-Cas12a cleavage has achieved single-target detection of B. bigemina from tick salivary-gland DNA, paving the way for innovative pen-side platforms, once cost and technical hurdles are overcome. Importantly, vector-based detection using appropriate tissues bearing an optimum level of ticks is species-dependent. B. bigemina sporozoites concentrate in nymph/adult salivary glands, whereas B. bovis sporozoites are produced mainly in larval salivary glands, so monitoring programs should stratify sampling accordingly.