Lourens de Villiers1, Melvyn Quan2, Milana Troskie2, Joyce C Jordaan3, Andrew L Leisewitz4. 1. Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa. ldevilliers@unam.na. 2. Vector and Vector-Borne Diseases Research Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa. 3. Department of Statistics, Faculty of Natural and Agricultural Sciences, University of Pretoria, Private Bag X20, Hatfield, 0028, South Africa. 4. Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa.
Abstract
PURPOSE: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
PURPOSE: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
Entities:
Keywords:
Babesiosis; Canine; Light microscopy; Parasite density; SYBR Green I
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