Development and validation of a multiplex, real-time PCR assay for Babesia rossi and Babesia vogeli.

Canine babesiosis is caused by tick-transmitted intraerythrocytic protozoan parasites occurring worldwide. In southern Africa, babesiosis is caused by Babesia rossi and B. vogeli and is one of the most common and important infectious diseases affecting dogs. There is no reliable, rapid and sensitive method for the detection of these parasites, especially when parasitaemia is low. The aim of this study was to develop a sensitive and specific multiplex TaqMan® MGB PCR assay for the diagnosis of canine babesiosis infections occurring in southern Africa, and to discriminate between Babesia rossi and B. vogeli. The fitness of purpose of the assay was to confirm diagnosis of suspect or clinical cases, and estimate prevalence of infection for research purposes. A total of 648 published sequences were used to design the assay. A set of group-specific canine Babesia spp. primers were designed to amplify a 117 nucleotide region of the 18S rRNA gene of all canine Babesia spp. Species-specific TaqMan® MGB probes were developed for B. rossi, B. vogeli, B. canis and B. gibsoni, but analytical validation was only performed for B. rossi and B. vogeli as a multiplex assay. The assay had a broad dynamic range and amplified B. rossi and B. vogeli efficiently (98.6% and 94.7% respectively). The assay was sensitive, with a 95% LOD of 10-2.67% parasitized erythrocytes (PE) for B. rossi and 10-2.03% PE for B. vogeli, and specific, with no cross reaction between B. rossi and B. vogeli and no detection of other haemoparasites that infect dogs, such as Ehrlichia canis and Anaplasma platys. Consistent repeatability within and between PCR runs was shown. This assay will be able to accurately and rapidly confirm babesiosis in canines and allow for treatment to be administered in the early stages of the disease, speeding up the recovery time in affected dogs.