Three versus seven days of apramycin treatment for post-weaning diarrhoea in piglets: clinical and resistance gene outcomes from a cluster-randomised field trial

Technical Summary: Three versus Seven Days of Apramycin Treatment for Post-Weaning Diarrhoea in Piglets

Problem Statement
Post-weaning diarrhoea (PWD), primarily associated with enterotoxigenic Escherichia coli (ETEC), is a leading driver of antimicrobial use in pig production. In Denmark, weaners are the most intensively treated age group, with aminoglycoside use (specifically apramycin and neomycin) rising significantly following the phase-out of medicinal zinc oxide and regulatory restrictions on other antimicrobials. This increase correlates with rising resistance, including the aac(3)-IV gene, which confers reduced susceptibility to apramycin and gentamicin.

While optimizing treatment duration is a recognized strategy to reduce antimicrobial selection pressure without compromising efficacy, EU Regulation (EU) 2019/6 mandates adherence to authorized treatment durations listed in the Summary of Product Characteristics (SPC). The current SPC for apramycin specifies a 7-day course. There is a regulatory and clinical need for controlled evidence to support potential label changes to shorter durations, as shorter courses could reduce total antimicrobial consumption while maintaining clinical cure.

Methodology
The study was a cluster-randomised, non-inferiority field trial conducted in a commercial Danish specific pathogen-free (SPF) herd between January and February 2026.

• Design: 28 nursery pens were randomized at the pen level to receive apramycin via drinking water for either 3 days (short-course) or 7 days (standard-course).
• Population: 518 diarrhoeic piglets (faecal score ≥3) were enrolled across two batches.
• Endpoints:
  • Primary: Clinical cure (faecal score ≤2) at End of Treatment (EOT). Note that EOT occurred at Day 4 for the 3-day group and Day 8 for the 7-day group.
  • Secondary: Clinical cure at a common Day 8 timepoint after treatment initiation for both groups.
  • Microbiological: Pen-level relative abundance of the apramycin resistance gene aac(3)-IV normalized to 16S rRNA, quantified via qPCR on pooled DNA samples from stratified subsets of piglets.
• Statistical Analysis: A non-inferiority margin of −10 percentage points was set for the risk difference (3-day minus 7-day). Analyses accounted for pen-level clustering using generalized estimating equations (GEE).

Key Results

• Clinical Efficacy:
  • At EOT: Non-inferiority was not demonstrated. The 3-day group had a cure rate of 71.6% (184/257) compared to 78.2% (204/261) in the 7-day group. The risk difference was −0.063 (95% CI −0.184 to 0.058), with the lower bound crossing the −0.10 non-inferiority margin.
  • At Day 8 (Common Timepoint): Non-inferiority was demonstrated. The 3-day group showed a cure rate of 83.7% (215/257) versus 78.2% (204/261) in the 7-day group. The risk difference was +0.055 (95% CI −0.076 to 0.184), with the entire confidence interval falling within the non-inferiority margin.
  • Supportive Data: Among cured piglets, the 3-day group had higher odds of achieving a faecal score of 1 (normal) versus 2 (soft) at both timepoints, though both scores represent non-diarrhoeic states. No significant differences were found in the severity of residual diarrhoea among non-cured piglets.
• Resistance Gene Dynamics (aac(3)-IV):
  • Both groups showed significant reductions in pen-level aac(3)-IV abundance from baseline to EOT. The median decline was greater in the 3-day group (−2.90) than the 7-day group (−1.70) at EOT (p = 0.024).
  • At the common Day 8 timepoint, the change from baseline did not differ significantly between groups (−1.23 vs −1.70; p = 0.701).
  • In the 3-day group, relative gene abundance increased between EOT and Day 8, suggesting a rebound effect after treatment cessation. However, at Day 8, the 3-day course did not result in higher resistance gene abundance compared to the 7-day course.
• Association with Cure: Pen-level aac(3)-IV abundance was not associated with clinical cure in adjusted models. Batch-level differences (likely reflecting pathogen composition, such as the presence of apramycin-resistant F18 ETEC in Batch 2) were more significant determinants of cure outcomes than resistance gene abundance.

Significance and Claims
The paper concludes that a 3-day apramycin course achieved clinical outcomes comparable to the standard 7-day course when evaluated at a common post-treatment timepoint (Day 8), without evidence of increased pen-level abundance of the aac(3)-IV resistance gene.

The authors argue that the failure to demonstrate non-inferiority at the primary EOT endpoint was likely due to the asymmetry in assessment timing (Day 4 vs. Day 8), which captured the groups at different stages of biological recovery, rather than a true lack of efficacy in the shorter course. The study supports the hypothesis that shortened treatment duration is a viable antimicrobial stewardship strategy for PWD, potentially reducing antimicrobial consumption by 57% without compromising clinical efficacy or increasing resistance gene abundance at a comparable recovery stage.

The findings suggest that current authorized treatment durations may be open to reassessment based on evidence from controlled field trials, aligning with the principle that treatment duration should be minimized where clinical efficacy is maintained. However, the authors note limitations, including the single-herd setting and the lack of an untreated control group, and call for further studies across different antimicrobial classes and herd contexts to validate these findings for regulatory purposes.