Isolate fecal dna

Eimeria species were identified by qPCR using specific primers targeting SCAR markers derived from RAPD fragments for E. acervulina, E. tenella, and E. necatrix, and microneme protein gene 1 for E. maxima [19 (link)]. Oocysts from positive fecal samples were isolated, purified, and concentrated with a saturated salt solution [20 ]. Then, oocysts were washed three times with distilled water by repeated centrifugation [21 (link)]. Genomic DNA was extracted from 0.25 mg fecal sample and 100 μL oocysts pellets using the Isolate Fecal DNA (Bioline) kit according to the manufacturer’s instructions. The qPCR reactions were performed in the CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, London, UK) using IQ Multiplex Powermix (Bio-Rad, London, UK) in a final volume of 20 µL. The amplification conditions were similar to the conditions described by Blake et al. [19 (link)] as follows: 1 × initial denaturation at 95 °C for 20 s; 40 × 95 °C denaturation, 15 s, and primer hybridization combined with 60 °C extension, 30 s. Data were collected at the end of each cycle.

DNA extractions from stool samples were performed using the Isolate fecal DNA (Bioline) or QIAamp DNA Stool (Qiagen) kits following the manufacturer’s instructions and carried out at the Hopkirk Research Institute. DNA extraction required disruption of the oocyst/cysts using a beadbeater (Tissue Lyser II, Qiagen) at 30 Hz for 5 min. DNA quantification was measured using the Qubit fluorometer. A fragment of the gp60 and gdh genes were PCR amplified using a combination of external and internal primers (S1 Table) for Cryptosporidium [43 (link), 44 (link)] and Giardia [31 (link)], respectively. Both strands of the amplification products were sequenced using Big Dye Terminator version 3.1 reagents and an ABI 3730XL automated DNA sequencer (Applied Biosystems, Foster City, California, USA).