Comparative Effects of Raw Milk and Milk Replacer Feeding on Gut Microbiota Diversity and Function in Cryptosporidium parvum-Infected Neonatal Dairy Calves on a Japanese Farm

Neonatal diarrhea is a major health concern in the livestock industry, and <i>Cryptosporidium parvum</i> is a key pathogen responsible for this condition in calves. Milk management and gut microbiome regulation may play important roles in preventing cryptosporidiosis symptoms. This study analyzed the gut microbiota of neonatal calves fed raw milk (BM) or milk replacer (MR) using a total of 58 fecal samples collected on the same farm in 2022 and 2024. In milk replacer-fed calves, alpha diversity was significantly higher in <i>C. parvum</i>-positive (P) calves without diarrhea (N) (PN, n = 5) than in <i>C. parvum</i>-positive calves with diarrhea(D) (PD, n = 18) (Shannon <i>p</i> = 0.0358; Chao1 <i>p</i> = 0.0598). Beta diversity also differed between PN and PD (PERMANOVA, R² = 0.1763, <i>p</i> = 0.0092). Predicted microbial taxa such as <i>Faecalibacterium</i> (ALDEx2, effect size = 2.31, <i>p</i> = 0.00003) and <i>Butyricicoccus</i> (effect size = 1.31, <i>p</i> = 0.0041) were enriched in PN calves in MR. Comparison between milk types (BM vs. MR) further showed higher species richness in PN calves in MR than in those (n = 5) in BM(Chao1, <i>p</i> = 0.0088), along with significant differences in beta diversity (R² = 0.4112, <i>p</i> = 0.0069). These findings suggest that microbial diversity and the presence of specific taxa may be associated with reduced diarrheal symptoms. Predicted metabolic pathway profiling using a computational functional profiling approach showed the distinct metabolic pathways, including amino acid, carbohydrate, lipid, and vitamin biosynthesis, were enriched in healthier calves in both groups. These results suggest certain functional features of the microbiome could be associated with anti-inflammatory activity and short-chain fatty acid production, potentially mitigating diarrheal symptoms.