Targeted quantitative polymerase chain reaction-based bacterial and fecal metabolite markers show early stability in healthy puppies after 8 weeks of age.

OBJECTIVE

To assess longitudinal stability of targeted quantitative PCR (qPCR)-based fecal microbial and metabolomic markers in healthy puppies from 8 weeks to 1 year of age.

METHODS

This prospective longitudinal cohort study was conducted at the Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, University of Zurich, Switzerland, from August 2023 to January 2025. Clinically healthy client-owned puppies were sampled at 8 to 12 weeks, 16 weeks, 6 months, and 1 year. Eligible dogs had no gastrointestinal signs, no previous systemic antibiotics, and negative parasitology. Primary outcomes were the qPCR-based dysbiosis index, selected qPCR-targeted bacterial taxa, fecal bile acids, long-chain fatty acids, and sterols, quantified by mass spectrometry. Mixed-effects models assessed time point associations.

RESULTS

23 dogs contributed 62 fecal samples. Dysbiosis index values and most targeted bacterial taxa remained within reference intervals, with no significant longitudinal changes. Bacteroides spp abundance was lower at 8 to12 weeks than at 6 months (difference, -0.73 logDNA copies/g feces; 95% CI, -1.40 to -0.06; Tukey-adjusted P = .028). Secondary unconjugated bile acids dominated at all ages. The concentration of 12-oxo-lithocholic acid was lower at 16 weeks than at 1 year (ratio, 0.33; 95% CI, 0.14 to 0.79; Tukey-adjusted P = .007) but not after multiple-testing adjustment. Long-chain fatty acid and sterol profiles showed no age-related changes.

CONCLUSIONS

The qPCR-based dysbiosis index and targeted microbial and metabolomic markers were largely stable from 8 weeks to 1 year, supporting early functional maturation of measured markers.

CLINICAL RELEVANCE: These findings establish age-specific baseline information for fecal bacterial and metabolite markers, supporting interpretation in young dogs with gastrointestinal disease.