title: Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.
abstract: The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings.
authors.affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, University of London, Hatfield AL9 7TA, United Kingdom. Electronic address: mnolan@rvc.ac.uk.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Nolan
authors.affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, University of London, Hatfield AL9 7TA, United Kingdom. Electronic address: ftomley@rvc.ac.uk.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Tomley
authors.affiliation: The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, Scotland, United Kingdom. Electronic address: pete.kaiser@roslin.ed.ac.uk.
authors.forename: Pete
authors.initials: P
authors.lastname: Kaiser
authors.affiliation: Department of Pathology and Pathogen Biology, Royal Veterinary College, University of London, Hatfield AL9 7TA, United Kingdom. Electronic address: dblake@rvc.ac.uk.
authors.forename: Damer P
authors.initials: DP
authors.lastname: Blake
mesh_terms: D000818:Animals; D002645:Chickens; D003048:Coccidiosis / Q000469:parasitology / Q000662:veterinary*; D016054:DNA, Protozoan / Q000235:genetics; D016786:Eimeria tenella / Q000502:physiology*; D005243:Feces / Q000469:parasitology; D023281:Genomics; D033661:Oocysts; D011201:Poultry Diseases / Q000175:diagnosis / Q000469:parasitology*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms
chemical_list: D016054:DNA, Protozoan
keywords: Avian coccidiosis; Chicken; Cytoplasmic beta-actin gene (actb); RAPD-SCAR marker (Tn-E03-1161)