To evaluate the inhibitory effects of drugs on the growth of Babesia gibsoni, relative quantification real-time PCR method was developed in this study. The 18S rRNA gene was used as a target gene for the 2-ΔΔCt method analysis. Additionally, chicken RNA was added to the parasitized blood before total RNA extraction. The chicken β-actin gene was selected as an internal control gene for the 2-ΔΔCt method analysis. The 100 µL parasitized blood samples with different percentages of parasitized erythrocytes (PPEs) (3%, 1.5%, 0.75%, 0.375% and 0.1875%) were prepared for relative quantification of B. gibsoni. Regression analysis results revealed significant linear relationships between the relative quantification value and parasitemia. 18S rRNA gene expression was significantly decreased after treatment with diminazene aceturate and artesunate in vitro drug sensitivity test. This result suggested that this relative quantification real-time PCR method can be used to evaluate the effects of drug inhibition.
The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.