Total genomic DNA was isolated from purified oocysts using a standard phenol/chloroform extraction

protocol following disruption using a Mini Beadbeater-8 as described previously (Blake et al., 2003). A summary of the PCR assays tested, and the primers used, is provided in Supplementary Table 1. The presence of Eimeria genus genomic DNA was tested by PCR amplification of the partial 18S rDNA sequence using the primers ERIB1 and ERIB10 as described elsewhere ( Schwarz et al., 2009). Briefly, each reaction contained 2 μl genomic DNA template, 25 pmol forward and reverse primer, 0.5 U Taq polymerase (Invitrogen, Paisley, UK), 10 mM Tris–HCl, 1.5 mM MgCl2, 50 mM KCl and 200 μM dNTPs. Standard cycle parameters were 1× (5 min at 94 °C), 30× see more (30 s at 94 °C, 30 s at 57 °C, 2 min at 72 °C) and 1× (10 min at 72 °C). Post-amplification PCR products were resolved by agarose gel electrophoresis.

The nested PCR protocol using ITS-1 primers was standardised for identification of Eimeria species of poultry. Primers amplifying the entire ITS-1 sequence with flanking partial 18S rDNA and 5.8S rDNA regions of Eimeria were used in the genus-specific PCR phase, while species-specific primers targeting the ITS-1 region were used to amplify the individual Eimeria species as described elsewhere ( Lew et al., 2003). Briefly, each 25.0 μl PCR reaction included 2 μl of genomic DNA, 25 pmol each of genus-specific primers, 1.25 U of Taq polymerase,

200 μM each of dNTPs, and 2.5 μl of PCR buffer containing 1.5 mM MgCl2. The thermal cycling was done with an initial denaturing step at 94 °C E7080 for 3 min followed by 30 cycles of 94 °C for 30 s, 55 °C for Calpain 30 s and 72 °C for 90 s and a final extension at 72 °C for 7 min. The product of the primary PCR (1.0 μl in 25.0 μl reaction mixture) was used as template for the nested PCR with species-specific primers in individual tubes using the same amplification conditions described above excepting different annealing temperatures for different Eimeria spp. (58 °C for E. mitis; 61 °C for E. necatrix and E. praecox; 65 °C for E. tenella; 71 °C for E. acervulina, E. maxima and E. brunetti). Negative, no-template controls were included with each assay using triple distilled water in place of template. The amplification of specific nested PCR product was checked by gel electrophoresis in 2% agarose gels stained with 0.5 μg/ml ethidium bromide. The multiplex PCR using SCAR primers for identification of the seven Eimeria species that infect chickens ( Fernandez et al., 2003) was standardised using pure DNA samples from the Houghton strains of each Eimeria spp. Initially, the PCR amplification was standardised separately for each species using specific primer pairs (0.55 μM for E. tenella, E. maxima and E. mitis; 0.7 μM for E. acervulina, E. necatrix and E. praecox; 0.85 μM for E. brunetti), 200 μM dNTP, 5.0 mM MgCl2, 3.