Longitudinal evaluation of fecal microbiota transplantation for ameliorating calf diarrhea and improving growth performance

Calf diarrhea is a major cause of morbidity and mortality in early life, accounting for a large fraction of deaths within the first weeks. Etiologies include viral, bacterial, and protozoal pathogens as well as non-infectious stressors. Widespread antibiotic use to treat or prevent diarrhea has led to antibiotic resistance, residues in meat, disruption of beneficial gut microbes, and potential recurrence of diarrhea due to dysbiosis. Dysbiosis in preweaning calves may impair maturation of ruminal and intestinal microbiota with long-term negative effects on nutrient utilization and growth. Microbiota-based therapies, particularly fecal microbiota transplantation (FMT), have shown efficacy in human gastrointestinal disorders and promising results in swine, but have not been systematically tested in ruminants. This study tests the hypothesis that FMT from rigorously screened healthy donor calves can remediate diarrhea in preweaning calves by restoring eubiosis, and examines whether induced gut microbiota changes affect host metabolome and long-term growth performance.

Prior work highlights: (1) Antibiotics in livestock, while historically used for growth promotion and disease control, contribute to antimicrobial resistance, ecological impacts, and gut dysbiosis that can predispose to recurrent diarrhea. (2) Microbiota-targeted approaches, including FMT, are highly effective for Clostridioides difficile infection and have been explored in IBS/IBD in humans. In production animals, microbiota-derived factors (e.g., bacteriocins like gassericin A) and FMT have shown protective effects in piglet models (diarrhea resistance, NEC prevention). (3) Rumen transfaunation has long been used empirically in cattle to manage rumen dysfunctions and toxin exposures, suggesting feasibility of microbial transfers in ruminants. However, meaningful, longitudinal, community-level changes and clinical outcomes from FMT in ruminants had not been demonstrated prior to this study. These lines of evidence support investigating FMT as an antibiotic-sparing therapy for calf diarrhea and potentially as a modulator of growth-related microbiome features.

Study design and ethics: Controlled intervention in Korean brown cattle (Bos taurus coreanae) assessing FMT for diarrheic remission, gut microbiota/metabolome dynamics, and growth outcomes. Approved by Kyung Hee University IRBs [KHUASP(SE)-17-028, KHUASP(SE)-17-145] in accordance with WOAH/OIE guidelines. Animals and housing: Calves had free access to food and water; preweaning housed with dams, post-weaning mixed in 3×3 m stalls (five per stall) with standardized diets across groups to minimize environmental/diet confounders. Cohorts:

• Preliminary safety/feasibility trial: 7 calves total (1 donor, 6 recipients regardless of diarrhea). Recipients received oral FMT twice daily for safety assessment; fecal sampling at 0, 1, 2, 4, 8, 16 days. Environmental samples: feed pellets (n=2) and maternal milk (n=6).
• Validation trial: 6 rigorously screened healthy donors from 20 candidates; 57 diarrheic recipients randomized to FMT (n=20), saline control (CON, n=14), or antibiotics (ABX, n=23). Inclusion/exclusion detailed below. Fecal sampling at 0, 2, 4, 8, 16, 32, 48 days; additional 12-month sampling. Serum collected at 12 months; body weight measured at 6, 12, 24 months; dressed weight at 24 months. Donor selection (Table 1): Age 21–50 days; Bristol stool score (BSS) 3–4; well-nourished and in good physical condition; exclusion: any antimicrobial history, communicable livestock diseases, current diarrhea (BSS 6–7), rectal bleeding/mucus, and PCR-detected enteric pathogens (multiple bovine viral, bacterial, and protozoal agents including Eimeria zuernii). 6 donors (2F/4M) met criteria. Recipient selection (Table 1): Age 5–50 days; moderate-to-severe diarrhea (BSS 6–7) with rectal bleeding/mucosal appearance; exclusions: antimicrobial history, communicable disease, age outside range, BSS<6. 57 enrolled. Interventions:
• FMT: Per rectum feces collected from healthy suckling donors. Each 5 g sample homogenized in 45 mL buffer (4% saline with defined salts, glucose; 10% glycerol cryoprotectant). Final 0.1 g/mL concentration. Multidonor inocula prepared by mixing 3–4 donor samples per batch; thawed before use. Dosing: 50 mL bolus (5 g feces) at 0 h, 12 h, 24 h, day 8, and day 48 (total five administrations).
• CON: 50 mL isotonic saline at FMT time points.
• ABX: Oral neomycin at FMT time points; for recurrent diarrhea, intramuscular apramycin, sulfadiazine/trimethoprim, or enrofloxacin as needed. Outcomes and assessments:
• Clinical diarrhea: Daily BSS and documentation of remission and mortality through day 48.
• Microbiome profiling: 16S rRNA V3–V4 amplicon sequencing (Illumina MiSeq 2×300 bp). DNA extraction via repeated bead-beating plus column. Quality control with contamination checks and standard community. QIIME2 (2020.06) with DADA2 denoising to ASV features; taxonomy via SILVA v132 classifier. Diversity analyses (alpha/beta) using weighted/unweighted UniFrac; PCoA; PERMANOVA (999 permutations). LEfSe (LDA>3.5) for discriminant taxa; Mann–Whitney U for differential abundances. SourceTracker v1.0.1 to estimate donor contribution to recipients' microbiota.
• Metabolomics (untargeted GC-TOF-MS): Fecal metabolome at day 0 (n=54) and day 48 (n=54); serum metabolome at 12 months (n=50). Extraction protocols for feces and serum; derivatization (methoxyamine, MSTFA); analysis on Agilent 7890 GC and LECO Pegasus HT TOF MS. Data processing via LECO ChromaTOF, NetCDF export, MetAlign 3.0 alignment, SIMCA-P+ multivariate analyses. Clustering with Bray-Curtis PCoA and UPGMA; metabolites identified using standards and in-house library (levels 1–2). Discriminant variables by VIP>0.7.
• Targeted assays: Enzymatic quantification of total amino acids (L-Amino Acid kit) and branched-chain amino acids (BCAAs: leucine, isoleucine, valine; SIGMA MAK003) in feces (days 0 and 48; n=112) and serum at 6, 12, 24 months (n=150).
• Growth performance: Body weights at 6, 12, 24 months by sex and group; dressed weight at slaughter (~24 months).
• Functional inference: PICRUSt 1.1.4 on 12-month fecal ASVs, KEGG ortholog predictions. Statistics: Mann–Whitney U (two-tailed) for between-group comparisons; PERMANOVA for multivariate separations; Pearson correlations for multi-variable associations (BSS with Enterobacteriaceae and Porphyromonadaceae). Significance threshold P<0.05. Data visualization as box/dot plots and heatmaps. Data and code: 16S reads ENA PRJEB35993; metabolomics MetaboLights MTBLS2226; code at Zenodo doi:10.5281/zenodo.4176198.

Clinical efficacy: FMT markedly reduced diarrhea incidence and mortality versus controls by day 48. Complete remission rates: FMT 95.0% (19/20) vs CON 35.7% (5/14) and ABX 26.1% (6/23). Mortality: FMT 0% (0/20) vs CON 14.3% (2/14) and ABX 17.4% (4/23). BSS significantly lower in FMT vs controls at day 48 (Mann–Whitney U; P-values reported as significant in Fig. 1e). Microbiome reconstitution: Weighted UniFrac PCoA showed dysbiosis at baseline (day 0) across groups; FMT recipients’ communities progressively shifted toward donor profiles with reduced intra-group dissimilarity over time, unlike CON/ABX. Donor–recipient dissimilarity decreased significantly over time only in FMT; at day 48 FMT had lowest dissimilarity and highest donor-shared ASVs. SourceTracker: donor contribution increased over time in FMT (highest at day 48) but not in controls. Discriminant taxa (day 48): LEfSe indicated Bacteroidetes predominated in FMT, Proteobacteria in ABX, Verrucomicrobia in CON. Family-level: FMT enriched Ruminococcaceae, Bacteroidaceae, Paraprevotellaceae, and Porphyromonadaceae (e.g., Parabacteroides). Enterobacteriaceae and Lactobacillaceae enriched in ABX; Verrucomicrobiaceae enriched in CON. Temporal dynamics: Enterobacteriaceae decreased and Porphyromonadaceae increased over time, with significantly lower Enterobacteriaceae and higher Porphyromonadaceae in FMT vs CON/ABX at days 32 and 48. Correlations with diarrhea severity (BSS): Strong positive correlation between Enterobacteriaceae abundance and BSS across groups (CON r=0.673, P=0.049; ABX r=0.932, P=0.001; FMT r=0.834, P=0.010). Significant negative correlation between Porphyromonadaceae abundance and BSS only in FMT (r=−0.714, P=0.041). Negative correlation between Enterobacteriaceae and Porphyromonadaceae in FMT (r=−0.824, P=0.024), indicating maturation/eubiosis associated with remission. Fecal metabolome: Day 0 samples were dispersed irrespective of group; day 48 FMT metabolomes clustered distinctly from CON and ABX (PERMANOVA: CON vs FMT P=0.007; ABX vs FMT P=0.001). Multiple amino acids (alanine, leucine, valine, isoleucine, glycine, arginine, ornithine, glutamic acid) were reduced in FMT at day 48 versus both CON and ABX; decreases from baseline were significant only in FMT. Enzymatic assays confirmed lower total amino acids and BCAAs in FMT feces at day 48 (P<0.0001 vs controls). Growth performance: No group differences at 6 months; at 12 months, both sexes in FMT had higher body mass than CON and ABX (P<0.05); at 24 months, FMT had higher body and dressed masses than controls (P<0.05). Body mass gain from 6→12 and 12→24 months was significantly greater in FMT vs ABX (and male FMT > male CON). 12-month fecal microbiome PCoA separated FMT from CON/ABX (PERMANOVA: CON vs FMT P=0.001; ABX vs FMT P=0.001). LEfSe: FMT enriched Christensenellaceae, Clostridiaceae, Peptostreptococcaceae, Dehalobacteriaceae, Coriobacteriaceae; CON enriched Bacteroidaceae/Porphyromonadaceae; ABX enriched Lachnospiraceae. PICRUSt predicted enrichment in ribosome/DNA replication/repair and BCAA biosynthesis pathways in FMT. Serum metabolome and BCAAs: At 12 months, serum metabolomes differed by group (PERMANOVA: CON vs FMT P=0.001; CON vs ABX P=0.031; ABX vs FMT P=0.001); FMT showed distinct clustering and higher relative serum BCAAs. Enzymatic assays showed significantly higher serum BCAA concentrations in FMT cattle at 6, 12, and 24 months (all P<0.01), suggesting systemic metabolic impact associated with growth.

The study demonstrates that FMT can shift diarrheic calves from a dysbiotic to a eubiotic gut state, characterized by decreased Proteobacteria/Enterobacteriaceae and increased strict anaerobes including Porphyromonadaceae. These microbial changes correlated with reduced diarrhea severity and remission, indicating that restoration of community structure and function underlies clinical benefits. The negative association between Porphyromonadaceae and BSS, and its inverse relationship with Enterobacteriaceae in FMT recipients, suggests that elements of this family may curb expansion of facultative pathogens and promote maturation of the gut ecosystem. Concomitant metabolomic remodeling—especially reduced fecal free amino acids and BCAAs—aligns with improved fermentation efficiency and reduced malabsorptive diarrhea, consistent with literature linking dysbiosis to elevated fecal amino acids. Longitudinally, FMT-driven microbiota configurations persisted into the fattening period, associating with distinct functional potentials (e.g., biosynthesis and growth-related pathways) and higher systemic BCAAs, alongside superior weight gain and dressed weight. These findings position FMT as a promising antibiotic-sparing strategy that addresses both health (diarrhea control) and production (growth performance) goals in ruminants.

This first longitudinal FMT trial in ruminants shows that multi-donor FMT safely and effectively induces remission of calf diarrhea, reduces mortality, and reconstitutes the gut microbiota toward a donor-like, mature eubiotic state. FMT also remodels local (fecal) and systemic (serum) metabolomes and improves growth performance through 24 months, suggesting durable host benefits. The work provides a framework for microbiota-targeted therapy in livestock and indicates that microbial taxa such as Porphyromonadaceae may be central to diarrhea resolution and gut maturation. Future research should (1) elucidate the mechanisms by which specific Porphyromonadaceae members mediate protection; (2) resolve and culture key taxa to develop defined microbial therapeutics; (3) validate efficacy and safety in larger, diverse cohorts with deeper baseline phenotyping; and (4) explore personalized donor–recipient matching and defined consortia. Until broader cross-species data are available, clinical application should be focused on cattle.

• Mechanistic uncertainty: Specific species/strains and molecular mechanisms (e.g., within Porphyromonadaceae) driving remission are not fully defined.
• Taxonomic resolution and cultivation: Key protective members were not isolated/cultured; functional roles remain to be experimentally validated.
• Sample size and generalizability: Although larger than preliminary trials, cohort size (n=57 diarrheic calves) and single-breed/farm conditions may limit generalizability; larger, multi-site studies are needed.
• Recipient heterogeneity: Baseline host physiology and microbiota characteristics influencing response were not exhaustively profiled; predictors of response require further study.
• Comparator therapies: Antibiotic regimens varied upon recurrence (multiple agents), potentially introducing heterogeneity in ABX outcomes.
• Species translation: Findings pertain to cattle; extrapolation to other species (including humans) should be done cautiously.