Misconfigured Polymerase Chain Reaction Assay Leads to Erroneous Detection of <i>Babesia</i> spp. in <i>Theileria</i> -Positive Samples

Infections with tick-borne protozoa such as Babesia and Theileria spp.are major causes of bovine anemia and mortality in endemic regions.Molecular diagnostics, particularly polymerase chain reaction (PCR) assays, are widely used owing to their sensitivity and specificity; however, their accuracy heavily depends on primer design and assay specificity.Here, we describe cases in Korea in which a PCR-based assay targeting the 18S ribosomal RNA (rRNA) gene produced a false-positive result for Babesia spp. in four anemic Holstein cows that were actually infected with T. orientalis.Sequencing confirmed Theileria infection, and genotype-specific PCR identified the Ikeda and Chitose genotypes.We infer that overreliance on 18S rRNA-based PCR without confirmatory sequencing may result in misdiagnosis and misdirect subsequent case management, in addition to distorting the overall epidemiological data of bovine tick-borne diseases.Therefore, this case highlights the critical diagnostic pitfall of a misconfigured PCR assay, emphasizing the need to validate PCR protocols using species-specific markers such as the major piroplasm surface protein gene to improve field diagnostics.