# 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

## TL;DR

This study shows that feeding neonatal calves milk replacer instead of raw milk can influence gut microbiota diversity and function, potentially reducing symptoms of cryptosporidiosis.

## Contribution

The study reveals that milk type and microbial diversity are linked to reduced diarrheal symptoms in calves infected with Cryptosporidium parvum.

## Key findings

- Calves without diarrhea had higher gut microbiota diversity regardless of milk type.
- Milk replacer-fed calves showed enriched beneficial taxa like Faecalibacterium and Butyricicoccus.
- Microbial diversity and specific taxa may help mitigate cryptosporidiosis symptoms through anti-inflammatory and short-chain fatty acid pathways.

## Abstract

Neonatal diarrhea is a major health concern in the livestock industry, and Cryptosporidium parvum is a key pathogen in affected calves. This study characterized the gut microbiota of neonatal calves with cryptosporidiosis fed either raw milk or milk replacer. A total of 58 fecal samples were analyzed from calves reared on the same farm in 2022 and 2024. Milk management influenced both microbial composition and predicted metabolic pathways. Infected calves without diarrhea showed higher alpha diversity, regardless of milk type, suggesting infected calves without diarrhea symptoms have protective microbial profiles. Beta diversity differed between milk types, indicating milk type influenced overall community structure. Functional predictions in gut microbiome further indicated that enriched taxa in healthier calves may contribute to anti-inflammatory activity and short-chain fatty acid production. These findings suggest that no single “diarrhea-resistant” microbiome exists; instead, maintaining microbial diversity and enriching beneficial taxa may help mitigate cryptosporidiosis symptoms.

Neonatal diarrhea is a major health concern in the livestock industry, and Cryptosporidium parvum 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 C. parvum-positive (P) calves without diarrhea (N) (PN, n = 5) than in C. parvum-positive calves with diarrhea(D) (PD, n = 18) (Shannon p = 0.0358; Chao1 p = 0.0598). Beta diversity also differed between PN and PD (PERMANOVA, R2 = 0.1763, p = 0.0092). Predicted microbial taxa such as Faecalibacterium (ALDEx2, effect size = 2.31, p = 0.00003) and Butyricicoccus (effect size = 1.31, p = 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, p = 0.0088), along with significant differences in beta diversity (R2 = 0.4112, p = 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.