Development of a multiplex PCR assay targeting mitochondrial cytochrome oxidase subunit III (cox3) gene for simultaneous specific and sensitive detection of Babesia gibsoni and Babesia vogeli in dogs

Abstract

Canine babesiosis is a potential threat for the dog population worldwide. Rapid, sensitive, and specific identification of the etiological agent to the species is pivotal for initiating effective therapeutic and control measures. Co-infection with multiple species pathogens due to multiple vectors infesting dogs is not uncommon. A multiplex PCR (Bg-Bv mPCR) for simultaneous detection and differentiation of the two common Babesia species, B. gibsoni and B. vogeli has been developed targeting the mitochondrial cytochrome oxidase subunit III (cox3) gene. These two species are the species responsible for causing canine babesiosis in Indian subcontinent and Southern Asia. This cox3 gene is present in high copy number and the sequences are species specific and hence targeted to develop the diagnostic multiplex PCR. The multiplex PCR was able to detect up to 5 pg DNA of the Babesia species. No cross-amplifications were observed between the primers specific for either B. vogeli or B. gibsoni. The Bg-Bv mPCR resulted in significantly higher B. gibsoni positives (30/250) than existing 18S ribosomal RNA PCR (22/250). Similarly, the mPCR detected more B. vogeli (26/250) than the 18S rRNA PCR (18/250). The kappa statistics when applied to the results generated by each of the PCR tests also revealed a substantial to perfect agreement between the data. The multiplex PCR targeting cox3 gene is thus a rapid, sensitive, and specific method for simultaneous detection and differentiation of the B. gibsoni and B. vogeli. © 2025 Elsevier Inc.