Evaluating fecal inflammation biomarkers for non-invasive detection of weaning-related intestinal inflammation in piglets

Post-weaning diarrhea remains a major challenge without noninvasive diagnostic tools. Fecal biomarkers represent a promising alternative, as they reflect gut inflammation without invasive sampling. This study evaluated four biomarkers-calprotectin (fCal), lipocalin-2 (LCN-2), myeloperoxidase (MPO), and adenosine deaminase (ADA)-using a zinc oxide (ZnO) model to assess their value as gut-health indicators in piglets. 384 weaned piglets (23 days) were assigned to two diets: Control and ZnO-supplemented (1 g/kg feed). Piglets were weighed on days 0, 3, 8, and 14. On days 3 and 14, nine average-weight piglets per group were sampled for feces and intestinal tissue. fCal was measured by immunoturbidimetry, MPO by o-dianisidine oxidation, ADA by enzymatic colorimetry, and LCN-2 by ELISA. Microbiota was analyzed by 16S sequencing and tissues by qPCR and histology. On day 3, ZnO piglets showed fewer intraepithelial lymphocytes (P = 0.062), higher IL-10 expression (P = 0.029), and lower Escherichia-Shigella abundance (P = 0.045). By day 14, they were ∼300 g heavier (P = 0.071), had higher villus-to-crypt ratios (P = 0.026), maintained IL-10 upregulation (P = 0.083), and increased OCLN expression (P < 0.001). Their microbiota shifted toward Odoribacter and Akkermansia (P ≤ 0.002), with reductions in Clostridium sensu stricto 1 (P < 0.001). fCal and LCN-2 were reduced in the ZnO group on day 3 (P ≤ 0.002), but only fCal remained reduced by day 14 (P = 0.012). No diet effects were observed for MPO or ADA (P > 0.05). On day 3, fCal correlated with CRHR1 (R = 0.59, P = 0.025) and negatively with barrier-related genes MUC13 and OCLN (R ≤ -0.50, P ≤ 0.050). MPO tended to correlate with CRHR1, crypt depth (R ≥ 0.44, P ≤ 0.069), and Streptococcus abundance (R = 0.45, P = 0.073). LCN-2 was negatively associated with inflammatory genes such as TNF-α and NF-κB (R ≤ -0.55, P ≤ 0.041). ADA showed positive trends with potentially pathogenic phyla, such as Campilobacterota (R ≥ 0.43, P ≤ 0.084). By day 14, fCal, LCN-2, and MPO were correlated with immune-related genes (e.g., TNF-α, TGF-β; R ≥ 0.52, P ≤ 0.071), while ADA was associated with the stress-related gene HSD11B1 (R = 0.74, P = 0.002). Overall, fecal biomarkers were valuable indicators of post-weaning gut inflammation and barrier function. Specifically, fCal was the most consistent, LCN-2 and MPO may serve complementary roles, whereas ADA primarily reflected cellular stress rather than inflammation.