Advances and Challenges in the Diagnosis of Vector-Borne Protozoal Infections in Veterinary Medicine

Vector-borne protozoal infections—including babesiosis, theileriosis, hepatozoonosis, trypanosomosis, and leishmaniosis—impose a substantial burden on livestock and companion animal health worldwide and carry important zoonotic and public health implications. Accurate diagnosis is essential yet challenging, given the diversity of parasite genera, their markedly different tissue tropisms, and the uneven distribution of diagnostic resources across veterinary settings. This review provides an integrated overview of the principal diagnostic approaches available, structured around the biological logic that guides test selection in practice. Microscopic examination remains the first-line method; its strengths and limitations are discussed for intraerythrocytic parasites (Plasmodium spp., Babesia spp., Theileria spp., Cytauxzoon spp.—the latter two with additional extra-erythrocytic schizont stages in leukocytes and tissue macrophages, respectively), leukocyte-associated forms (Hepatozoon spp.), extracellular trypanosomes, and tissue-stage parasites, including emerging applications of artificial intelligence. Serological methods—enzyme-linked immunosorbent assay (ELISA), indirect fluorescence antibody test (IFAT), and point-of-care lateral flow assays—are evaluated for their role in exposure detection, population screening, and international trade certification, with attention to cross-reactivity and the active-versus-past-infection distinction. Molecular diagnostics, encompassing conventional PCR, qPCR, droplet digital PCR, isothermal amplification, and next-generation sequencing, are reviewed with respect to target selection, sensitivity, and point-of-care applicability. Finally, diagnostic challenges are contextualised within a One Health framework, highlighting the fragmentation of veterinary surveillance and the need for integrated, cross-sector approaches to detect emerging threats.