Robust detection in faeces and in vivo quantification of bacteriophages infecting enterotoxigenic Escherichia coli by quantitative PCR

• developed a quantitative PCR (qPCR) method specific for the previously isolated ETEC phage ETEP21B and validated in vitro and in vivo • the qPCR assay exhibited excellent specificity even with non-target phages and in faecal matrices • in the in vivo PWD model, ETEP21B was detected in faecal samples from all pigs receiving the phage-supplemented diet, confirming successful passage through the gastrointestinal tract • phage administration did not significantly reduce diarrhoea or shedding compared with challenged controls, the qPCR assay highlighted delivery challenges that ensure further optimisation. With increasing restrictions on antimicrobials in pig production, bacteriophages (phages) offer an innovative and targeted approach for treating porcine post-weaning diarrhoea (PWD) caused by enterotoxigenic Escherichia coli (ETEC). An important step towards the use of phage-based interventions is developing reliable methods for quantifying phages throughout the animal digestive tract and in complex matrices, such as faeces. Here, we developed a quantitative PCR (qPCR) method with the primary objective of validating it for sensitive and specific quantification of the previously isolated ETEC phage ETEP21B, both in vitro and in vivo . We validated its performance using spiked faecal samples and an in vivo experimental model of PWD. The assay showed high sensitivity and specificity, detecting ETEP21B even in presence of abundant non-target phages and producing a near perfect calibration curve when phages were spiked into faeces. The in vivo experimental model consisted of three groups of animals (n=10 pigs/group) that were fed a standard diet from weaning, with one group receiving the diet supplemented with phage ETEP21B. Phage-fed pigs and one control group were challenged with ETEC F4, while the remaining control group received saline. Phage ETEP21B was detected in faeces from all pigs receiving phage-supplemented diet, confirming successful passage of the administered formulation through the gastrointestinal tract. However, no significant reduction in diarrhoea or F4 gene copy shedding was observed in phage-treated pigs compared with controls. Overall, the qPCR assay proved to be a robust and sensitive tool for tracking phage ETEP21B in vitro and in vivo . By precisely tracking phage fate in faeces, it helped discuss phage delivery issues that require optimisation to achieve therapeutic efficacy.