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

Abstract

Light microscopic manual count is the current gold standard for parasite quantification. The ability to determine parasite density in whole blood is crucial to understanding disease pathogenesis and finding a suitable automated method of B. rossi parasite quantification would facilitate higher throughput and provide results that are more objective. This study investigated both peripheral capillary and central venous whole blood to estimate the correlations between light microscopy, flow cytometry and quantitative real-time PCR (qPCR). Furthermore, the use of SYBR Green I as a DNA marker for the detection and quantification of B. rossi by flow cytometric analysis was explored. The study objectives included: a) validating the use of SYBR Green I as DNA marker to detect and quantify Babesia rossi nucleic acid by flow cytometric analysis; b) correlate B. rossi parasite density in venous blood quantified by manual count, flow cytometry and qPCR in the same dog; and c) correlate the parasite density of B. rossi in capillary blood 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 were sampled from forty naturally B. rossi-infected dogs and ten healthy control dogs. Samples were analysed by reverse line blot hybridization to confirm a mono-B. rossi infection. Capillary blood parasite density was quantified using light microscopic manual counting and venous blood parasitaemia quantified by manual counts, flow cytometry and qPCR. Flow cytometry, using SYBR Green I staining, showed promise in quantifying B. rossi nucleic acid in venous blood. A significant correlation was found between the venous manual counts and adjusted flow cytometric results (rs = 0.465; P < 0.001), as well as qPCR (rs = -0.500; P < 0.001). A significant correlation was also observed between the capillary manual counts compared to venous manual counts (rs = 0.793; P < 0.001), adjusted flow cytometric results (rs = 0.399; P = 0.004), and qPCR (rs.= -0.526; P < 0.001). The study results suggest that qPCR is of value as an alternative to the gold standard manual count for quantifying B. rossi parasitaemia in canine whole blood and that flow cytometry may be useful with further refinement of issues such as background fluorescence and the influence of reticulocytes.