A comparison between manual count, flow cytometry and qPCR as a means of determining Babesia rossi parasitaemia in naturally infected dogs

Abstract

Parasite quantification is crucial to understand disease pathogenesis. An automated method of determining parasite density would facilitate higher throughput and provide results that are more objective. The study objectives included: a) validating the use of flow cytometry to detect and quantify Babesia rossi nucleic acid; b) comparing B. rossi parasite density in venous blood quantified by manual count, flow cytometry and quantitative real-time PCR (qPCR) in the same dog; and c) comparing the parasite density of B. rossi in capillary blood (quantified by manual count), with the parasite density in venous blood, as determined by manual count, flow cytometry and qPCR in the same dog. Peripheral capillary and central venous blood was sampled from 40 naturally B. rossi-infected dogs and 10 healthy control dogs. Samples were analyzed by reverse line blot to confirm a mono-B. rossi infection. Capillary blood parasite density was quantified using light microscopy (manual counts) and venous blood parasitaemia quantified using manual counts, flow cytometry and qPCR. Flow cytometry, using SYBR Green I staining, showed promise in quantifying B. rossi nucleic acid in venous blood. Non-parametric methods were used for statistical analysis. Spearman’s rho revealed a significant correlation between the venous manual counts and both flow cytometry (rs = 0.467; P = 0.001) and qPCR (rs = -0.813; P < 0.001), as well as a significant correlation between the capillary manual counts compared to venous manual counts (rs = 0.793; P < 0.001), flow cytometry (rs = 0.400; P = 0.004) and qPCR (rs = -0.760; P < 0.001). Preliminary results suggest that both flow cytometry and qPCR may be of value as an alternative to the gold standard (manual count) for quantifying B. rossi parasitaemia in canine whole blood.