Contributed Papers: Oral Presentations Diagnosis and Epidemiology

Counting coccidia: a genomic approach

Damer P. Blake, Patricia Hesketh, Andrew Archer,
Martin W. Shirley and Adrian L. Smith.
Institute for Animal Health, Compton,
Berkshire, RG20 7NN, UK.
Email: damer.blake@bbsrc.ac.uk

Quantification of infection with the Eimeria spp. through monitoring pathological severity or magnitude of oocyst excretion has greatly informed our understanding of the Eimerian life cycle. However, attempts to quantify fluctuations in parasite reproduction within the host have largely relied upon labour-intensive microscopic analysis, creating a ‘grey box’. The development, validation and application of a quantitative real-time PCR assay has opened this biological ‘box’, permitting the sensitive and reproducible enumeration of intracellular parasite genomes present throughout the course of infection. Generic and species-specific quantitative PCR assays that target the conserved 5S ribosomal RNA coding sequence of nine avian and murine Eimeria species and the E. maxima MIC1 gene have been validated as capable of detecting >0.01 and >1 E. maxima genomes respectively. The generic 5S ribosomal RNA assay has been shown to enumerate the genomes of E. acervulina, E. mitis, E. tenella, E. pragensis and E. vermiformis with a comparable sensitivity and efficiency to that recorded for E. maxima over at least seven orders of magnitude. Preparation of samples collected in vivo with a commercial DNA extraction kit allowed the rapid enumeration of parasite genomes within an excised tissue section in just two working days. Parasite genomic DNA could first be detected within the host three days after infection with only 100 sporulated E. maxima oocysts.
Application of these complimentary generic and species-specific assays to study the dynamics of E. maxima replication within hosts representing ‘susceptible’ and ‘resistant’ genotypes indicate a brief period of differential killing four to five days after infection, suggesting that the third and fourth generation schizonts, but not the gametocytes, are targeted by the strain-specific host immune response. Other potential applications for the quantitative PCR assays described here include investigating the efficacy and point of action of existing and novel chemotherapeutic compounds and vaccine candidates, together with the continued characterisation of the Eimerian life cycle.

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