Molecular Detection of Babesia caballi

Total DNA was extracted from B. caballi-infected blood using FTA^® elute cards and purification reagent (Whatman) following the manufacturer’s instructions. DNA was eluted and PCR was performed for amplification of the B. caballi sbp4 according to Mahmoud et al. [6 (link)] using primers Bcsbp4-For (5′-ATG GCT GCC TTC TCG ACC CGC TCC-′) and Bcsbp4-Rev (5′-CTC AGA CTT TTC GGC GGC TTC AGC-3′). The sequences of the primers were derived from the sbp4 gene identified in the B. caballi genome. Babesia caballi DNA positive control for PCR was kindly donated from the OIE equine piroplasmosis reference laboratory located in Pullman, WA, USA. The PCR amplicons were electrophoresed on 1.5% agarose gel and stained with SYBR Safe (Invitrogen, Waltham, USA). The length of the amplified products was estimated using a 1 Kbp DNA ladder (Invitrogen) and the amplified products were visualized with an UV trans-illuminator (Bachofer, Germany), and photographed using a gel documentation system (BioDocAnalyze-Biometra Analytik GmbH, Göttingen, Germany).