Background This study investigated the validity of the DNA-marker based test to determine susceptibility to ETEC-F4 diarrhoea by comparing the results of two DNA sequencing techniques in weaner pigs following experimental infection with F4 enterotoxigenic Escherichia coli (ETEC-F4). The effects of diet and genetic susceptibility were assessed by measuring the incidence of piglet post-weaning diarrhoea (PWD), faecal E. coli shedding and the diarrhoea index. Results A DNA marker-based test targeting the mucin 4 gene ( MUC4 ) that encodes F4 fimbria receptor identified pigs as either fully susceptible (SS), partially or mildly susceptible (SR), and resistant (RR) to developing ETEC-F4 diarrhoea. To further analyse this, DNA sequencing was undertaken, and a significantly higher proportion of C nucleotides was observed for RR and SR at the Xba I cleavage site genotypes when compared to SS. However, no significant difference was found between SR and RR genotypes. Therefore, results obtained from Sanger sequencing retrospectively allocated pigs into a resistant genotype ( MUC4– ), in the case of a C nucleotide, and a susceptible genotype ( MUC4+ ), in the case of a G nucleotide, at the single nucleotide polymorphism site. A total of 72 weaner pigs (age ~ 21 days), weighing 6.1 ± 1.2 kg (mean ± SEM), were fed 3 different diets: (i) positive control (PC) group supplemented with 3 g/kg zinc oxide (ZnO), (ii) negative control (NC) group (no ZnO or HAMSA), and (iii) a diet containing a 50 g/kg high-amylose maize starch product (HAMSA) esterified with acetate. At days five and six after weaning, all pigs were orally infected with ETEC (serotype O149:F4; toxins LT1, ST1, ST2 and EAST). The percentage of pigs that developed diarrhoea following infection was higher ( P = 0.05) in MUC4+ pigs compared to MUC4– pigs (50% vs. 26.8%, respectively). Furthermore, pigs fed ZnO had less ETEC-F4 diarrhoea ( P = 0.009) than pigs fed other diets, however faecal shedding of ETEC was similar ( P > 0.05) between diets. Conclusion These results confirm that MUC4+ pigs have a higher prevalence of ETEC-F4 diarrhoea following exposure, and that pigs fed ZnO, irrespective of MUC4 status, have reduced ETEC-F4 diarrhoea. Additionally, sequencing or quantifying the single nucleotide polymorphism distribution at the Xba I cleavage site may be more reliable in identifying genotypic susceptibility when compared to traditional methods.

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