Eosin methylene blue agar

For enterobacteria and coliform counts, samples were serially diluted in Ringer’s lactate solution (Sigma-Aldrich, Madrid, Spain) and proper dilutions seeded in MacConkey agar (Oxoid; Madrid, Spain) and eosin methylene blue agar (Scharlab; Barcelona, Spain). Plaques were incubated for 24 h at 37°C and colonies were manually counted. From colon content and ileal mucosal scrapings, the total bacterial DNA was extracted using QIAmp DNA Stool Mini Kit (Qiagen; West Sussex, United Kingdom) following the manufacturer’s instructions. The ETEC F4 concentration was than assessed by qPCR targeting the gene coding the F4 fimbria of E. coli, according to the procedure described by Hermes et al. (2013) (link), using SYBR green dye with the ABI 7900 HT Sequence Detection System (PE Biosystems, Warrington, United Kingdom) with optical grade 96-well plates. The results were scored in five levels according to the number of gene copies per gram of fresh matter (FM). Scores were defined as following: negative = less than 4 logarithmic units of gene copies per gram FM; low = 4–5.5 logarithmic units of gene copies per gram FM; medium = 5.5–7 logarithmic units of gene copies per gram FM; high = 7–8.5 logarithmic units of gene copies per gram FM; and very high = more than 8.5 logarithmic units of gene copies per gram FM.

E. coli were isolated and quantified from the faecal samples by drop plate method^{56 (link)} on MacConkey agar (MCA) (Oxoid, United Kingdom). Lactose-positive, red, non-mucoid colonies were randomly selected from each sample and sub cultured onto Eosin Methylene Blue agar (EMB) (Scharlau, Spain) for biochemical confirmation. Species identity of E. coli was confirmed by standard biochemical properties followed by species-specific multiplex PCR^{57 (link)}. Primers for the uidA gene and flanking region of the uspA gene were used. The amplified PCR products were separated by electrophoresis at 70 V in a 2% agarose gel (Sigma-Aldrich, USA) containing ethidium bromide (AMRESCO, USA) followed by visualizing under UV light. Gene Ruler 100 bp Plus DNA Ladder (Thermo Fisher Scientific, USA) was used to standardize the PCR band images. Confirmed E. coli isolates were preserved at -80 ˚C after subculture onto the Blood agar plate (BA) (Oxoid, UK).