Industrial animal farming, crucial for meeting global food demands, faces challenges from diseases like piglet diarrhea. This disease leads to high mortality and substantial economic losses. While antibiotics offer temporary relief, they often disrupt the gut microbiota, potentially leading to further health issues and antibiotic resistance. This study explores a natural alternative, utilizing specific *Bacillus* strains as a probiotic treatment to address piglet diarrhea and promote gut microbiome restoration. The historical context of animal breeding, from its Neolithic origins to modern industrial practices, highlights the growing need for sustainable and effective health management in livestock. The over-reliance on antibiotics necessitates the exploration of novel, sustainable approaches to livestock health management, minimizing the negative impacts on animal welfare, the environment, and the development of antibiotic resistance.

Piglet diarrhea, or scour, is a common and debilitating disease affecting piglets, typically caused by various pathogens such as *Coccidia*, *Clostridia*, *E. coli*, *Salmonella*, and *Brachyspira*. Conventional treatments using antibiotics have limitations, including adverse effects on animals and humans, the development of antibiotic resistance in bacterial strains, and detrimental impacts on the gut microbiome. Alternative strategies, such as feed additives (acidifiers, prebiotics, yeast products, plant oils), have been proposed, but some exhibit side effects. Probiotics, particularly *Bacillus* and *Lactobacillus* strains, show promise in improving gut health, stimulating digestive enzyme activity, enhancing gut integrity, strengthening the immune system, and improving growth performance in swine. This study builds on this existing research by focusing on a specific *Bacillus* cocktail and comprehensively analyzing its impact on piglet diarrhea and the gut microbiome.

Eighty piglets were divided into four groups (n=20 each): a healthy control group, a diarrhea group, an antibiotic-treated group (Colistin and Kitasamycin), and a *Bacillus*-treated group (microecosystem). The microecosystem group received a probiotic formula containing *Bacillus subtilis* Y-15, *B. amyloliquefaciens* DN6502, and *B. licheniformis* SDZD02. The treatment lasted for seven days. Fecal samples were collected after seven days and subjected to 16S rRNA gene sequencing using the Illumina HiSeq2500 platform. Data analysis included OTU clustering, taxonomic classification (phylum, class, order, family, genus, species), Venn diagrams, principal component analysis (PCA), OTU rank curves, heatmaps, and alpha diversity calculations (Sobs, Chao1, ACE, Shannon, Simpson). Phylogenetic analysis was also performed. Statistical comparisons were conducted using Wilcoxon Rank-Sum and Kruskal-Wallis tests. MicrobiomeAnalyst toolkits were used for alpha and beta diversity analysis. The entire process, from piglet selection and treatment to microbiome analysis and data interpretation, was performed rigorously according to established protocols and ethical guidelines. Specific details regarding DNA extraction, PCR amplification, sequencing procedures, and data processing are also provided.

The study revealed significant differences in the gut microbiome composition across the four groups. The *Bacillus*-treated group showed a marked improvement in gut health, similar to the healthy control group. The number of Operational Taxonomic Units (OTUs) was significantly higher in the healthy control and *Bacillus*-treated groups compared to the diarrhea and antibiotic-treated groups. Principal component analysis (PCA) and OTU rank curves demonstrated the similarity of the *Bacillus*-treated and healthy control groups. Taxonomic analysis revealed that the diarrhea group had high levels of *Bacteroidetes* and *Fusobacteria*, and low levels of *Firmicutes* and *Actinobacteria*. The antibiotic treatment failed to significantly alter this profile. In contrast, the *Bacillus* treatment significantly increased *Firmicutes* and restored the levels of other bacterial phyla to those observed in the healthy control group. Heatmaps and phylogenetic analysis further illustrated the distinct clustering of the *Bacillus*-treated group with the healthy control group, unlike the antibiotic-treated group, which clustered with the diarrhea group. Alpha diversity indices (Sobs, Chao1, ACE, Shannon, Simpson) indicated greater bacterial diversity in the healthy control and *Bacillus*-treated groups compared to the other two groups. The *Bacillus* treatment specifically increased beneficial bacteria like *Bilophila*, and decreased harmful bacteria like *Ruminococcus gnavus*, *Prevotella stercorea*, and *E. coli*. This result was further supported by comparing groups A (healthy and microecosystem) and B (ill and antibiotics) using microbiome analyst toolkits. The analysis showed significantly higher alpha diversity values (Chao1, ACE, Shannon, and Simpson indices) in group A compared to group B, indicating greater species richness and diversity in the healthy and *Bacillus*-treated groups. Beta diversity analysis (NMDS ordination) clearly separated the gut microbiota of group A from that of group B, further highlighting the similarity between the healthy and *Bacillus*-treated piglets.

The findings demonstrate that the *Bacillus*-based probiotic formula effectively cured piglet diarrhea and restored a healthy gut microbiome. Unlike antibiotic treatments, the probiotic approach not only resolved the symptoms but also promoted beneficial bacterial colonization, resembling the microbiota profile of healthy piglets. The study highlights the importance of gut microbiome composition in piglet health, emphasizing the Firmicutes/Bacteroidetes ratio and specific bacterial species (*P. copri*, *P. stercorea*, *E. coli*) as indicators of health status. The success of the *Bacillus* treatment suggests a promising, natural alternative to antibiotics, addressing the growing concerns of antibiotic resistance and the negative impact of antibiotics on gut health. This approach contributes to the sustainable development of the swine industry and potentially influences broader applications in animal and human health.

This study provides compelling evidence for the efficacy of a *Bacillus*-based probiotic formula in curing piglet diarrhea and restoring a healthy gut microbiome. The probiotic treatment represents a safe and effective alternative to antibiotics, mitigating issues associated with antibiotic resistance and gut dysbiosis. Further research should focus on the specific mechanisms of action of the probiotic formula, exploring its potential applications in other animal species and its translation into human health interventions. Future studies could also investigate the long-term effects of the probiotic treatment, optimize the probiotic formulation, and conduct large-scale field trials to validate the findings in diverse farming settings.

The study was conducted on a specific breed of piglets in controlled experimental farm settings. The results may not be directly generalizable to other pig breeds or farming environments. A longer-term follow-up could provide further insights into the long-term impact of the probiotic treatment on piglet health and gut microbiome stability. Additionally, the study focused primarily on bacterial composition, and future research could expand to include other aspects of the gut microbiome, such as fungal and viral components and metabolomic analysis.