Non-Bacterial Microbiome Diversity and Signatures in Bovine Subclinical Mastitis

Introduction: Bovine subclinical mastitis (SCM) is traditionally studied through a bacterial lens, leaving the archaeome and virome of the mammary microbiome largely uncharacterized. These non-bacterial components may drive key ecological shifts between udder health, disease, and post-antibiotic recovery.

Methods: Shotgun metagenomic sequencing was performed on milk samples from three bovine mammary states: SCM (n=10), healthy mammary (HM, n=10), and antibiotic-treated (ANT, n=10). Reads were analyzed for species richness, inter-group overlap, and differential abundance to identify state-specific archaeal and viral signatures.

Results: The archaeome comprised 54 species and the virome 96 species, with 40.74% and 48.96% shared across groups, respectively. SCM was defined by Methanosarcina sp. Kolksee (21.31%) and Methanolacinia petrolearia (16.30%), the latter absent in HM. The HM state had a distinct biomarker, Halomicrobium mukohataei (15.96%), absent in ANT samples. ANT showed a significant bloom of Methanobrevibacter sp. YE315 (25.30%; p=0.014), indicating antibiotic-resilient archaeal enrichment. Virome analysis reflected bacterial dysbiosis in SCM through Streptococcus phage enrichment (5.58%). HM and ANT shared a restorative phage community; Lactococcus phage 98103 (12.22% and 12.13%) and Escherichia phage Penshu1 (6.59% and 8.86%) were both absent (<1.2%) in SCM.

Conclusions: Bovine mammary health, SCM, and antibiotic intervention carry distinct non-bacterial microbiome fingerprints. Antibiotic treatment appears to suppress SCM-associated archaeal taxa and facilitate a structural shift toward the HM virome profile, marked by resilient archaea and restorative phages. These non-bacterial signatures offer candidate biomarkers for SCM diagnosis and ecological recovery assessment.