DNA-based quantification and counting of transmission stages provides different but complementary parasite load estimates: an example from rodent coccidia (Eimeria)

Alice Balard,Emanuel Heitlinger,Vivian Mittné,Víctor Hugo Jarquín-Díaz,Julia Mari Murata,Susana Carolina Martins Ferreira

doi:10.1186/s13071-021-05119-0

Abstract

BackgroundCounting parasite transmission stages in faeces is the classical measurement to quantify “parasite load”. DNA-based quantifications of parasite intensities from faecal samples are relatively novel and often validated against such counts. When microscopic and molecular quantifications do not correlate, it is unclear whether oocyst counts or DNA-based intensity better reflects biologically meaningful concepts. Here, we investigate this issue using the example of Eimeria ferrisi (Coccidia), an intracellular parasite of house mice (Mus musculus).MethodsWe performed an infection experiment of house mice with E. ferrisi, in which the intensity of infection correlates with increased health impact on the host, measured as temporary weight loss during infection. We recorded the number of parasite transmissive stages (oocysts) per gram of faeces (OPG) and, as a DNA-based measurement, the number of Eimeria genome copies per gram of faeces for 10 days post-infection (dpi). We assessed weight loss relative to the day of experimental infection as a proxy of host health and evaluated whether DNA or oocyst counts are better predictors of host health.ResultsAbsolute quantification of Eimeria DNA and oocyst counts showed similar but slightly diverging temporal patterns during 10 dpi. We detected Eimeria DNA earlier than the first appearance of oocysts in faeces. Additionally, Eimeria OPGs within each dpi did not explain parasite DNA intensity. Early dpi were characterized by high DNA intensity with low oocyst counts, while late infections showed the opposite pattern. The intensity of Eimeria DNA was consistently a stronger predictor of either maximal weight loss (1 value per animal during the infection course) or weight loss on each day during the experiment when controlling for between-dpi and between-individual variance.ConclusionsEimeria ferrisi oocyst counts correlate weakly with parasite intensity assessed through DNA quantification. DNA is likely partially derived from life-cycle stages other than transmissive oocysts. DNA-based intensities predict health outcomes of infection for the host more robustly than counts of transmissive stages. We conclude that DNA-based quantifications should not necessarily require validation against counts of transmissive stages. Instead, DNA-based load estimates should be evaluated as complementary sources of information with potential specific biological relevance for each host-parasite system.Graphical

Highlights

Counting parasite transmission stages in faeces is the classical measurement to quantify “parasite load”
Absolute quantification of Eimeria DNA is robust for different DNA extractions on multiple amplification platforms We first performed a series of experiments to assure the accurate quantification of Eimeria DNA from faecal samples
We constructed standard curves from defined numbers of sporulated oocysts of E. falciformis and E. ferrisi using Quantitative PCR (qPCR) amplification of a fragment of the c oxidase (COI) gene [16, 36]

Summary

Introduction

Counting parasite transmission stages in faeces is the classical measurement to quantify “parasite load”. When microscopic and molecular quantifications do not correlate, it is unclear whether oocyst counts or DNA-based intensity better reflects biologically meaningful concepts We investigate this issue using the exam‐ ple of Eimeria ferrisi (Coccidia), an intracellular parasite of house mice (Mus musculus). In the last decade, ‘molecular techniques’ based on the quantification of nucleic acids (DNA for eukaryotic parasites) are increasingly complementing or even replacing classical coprological estimates of parasite load. Such DNA-based techniques are often thought to still require validation against a classical standard procedure, such as the counting of parasite reproductive and transmissive stages [1,2,3]. Oocysts floated from faeces are the firmly established standard for quantification of Coccidia [8, 9]

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Discussion

Conclusion