A Comparison Between Manual Count, Flow Cytometry and Quantitative Real-Time Polymerase Chain Reaction as a Means of Determining Babesia rossi Parasitaemia in Naturally Infected Dogs



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 Babesia 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 polymerase chain reaction (qPCR).


Peripheral capillary and central venous blood were sampled from 40 naturally B. rossi-infected dogs and 10 healthy control dogs. Samples were analysed by reverse line blot hybridization assay to confirm a mono-B. rossi infection. Capillary blood parasite density was detected using light microscopic manual counting and venous blood parasitaemia detected by manual counts, flow cytometry and qPCR.


A significant correlation was found between the venous manual counts and flow cytometry (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), flow cytometry (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 detecting 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.

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Data Availability

The datasets generated during and/or analysed during the current study care are available in the University of Pretoria repository, http://hdl.handle.net/2263/66680.


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This work was supported by the National Research Foundation of South Africa under a grant held by ALL (CPRR13080726333), as well as by the Department of Companion Animal Studies, Faculty of Veterinary Science, Onderstepoort, South Africa. We thank Dr. Jeanne Rudman from Mamelodi Animal Health Clinic and Sr. Marizelle de Clercq from the Onderstepoort Animal Blood Bank for assistance in sample collections. We also thank Prof. Marinda Oosthuizen, Prof. Geoffrey Fosgate and Mr. Pieter de Villiers for their contributions. A special thanks to Doornpoort Animal Clinic for allowing us to sample at the clinic.


This work was supported by the National Research Foundation of South Africa under a grant held by ALL (CPRR13080726333), as well as by the Department of Companion Animal Studies, Faculty of Veterinary Science, Onderstepoort, South Africa.

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LDV wrote the manuscript, with input from ALL, as part of fulfilment of the requirements for obtaining the degree M.Sc. Veterinary Science. LDV assisted in sample collection and performed the light microscopic and flow cytometric laboratory work. ALL supervised the study and assisted with sample collections. MQ co-supervised the study and developed the qPCR protocol along with MT, who performed all the RLB and qPCR laboratory work. JCJ performed the statistical analysis for the study, with input from LDV.

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Correspondence to Lourens de Villiers.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with ethical required in terms of the University of Pretoria’s Code of ethics for researchers and the Policy guidelines for responsible research and ethical clearance was granted by the Animal Ethics Committee of the Faculty of Veterinary Science, Onderstepoort (Reference: V060-16).

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de Villiers, L., Quan, M., Troskie, M. et al. A Comparison Between Manual Count, Flow Cytometry and Quantitative Real-Time Polymerase Chain Reaction as a Means of Determining Babesia rossi Parasitaemia in Naturally Infected Dogs. Acta Parasit. 65, 128–135 (2020). https://doi.org/10.2478/s11686-019-00134-9

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  • Babesiosis
  • Canine
  • Light microscopy
  • SYBR Green I
  • Parasite density