Bacterial translocation as a source of imprinting in the bovine mammary gland: unraveling the enteromammary pathway

Abstract

Recent advances in molecular techniques have fueled a growing interest in exploring commensal communities of microorganisms in the bovine mammary gland, where these populations may influence the prevention or promotion of disease. The traditional belief in the sterility of the mammary gland is now challenged, with conflicting opinions in the literature. Some propose the presence of a mammary gland microbiota, while others argue for sterility. The possibility of enteromammary circulation in ruminants lacks robust evidence, but the pre-partum period, marked by hormonal changes, may facilitate the migration of immune system cells that transport viable bacteria to the mammary gland through the lymphatic system. The hypothesis arises that the bovine enteromammary pathway functions during the pre-partum period, facilitating bacterial translocation through immune system cells. To test this hypothesis, ten Holstein cows will be sampled at three times: M I) moment of drying; M II) 10 days before partum; M III) 3 days after partum. Samples will be taken from the teat apex, colostrum, rumen fluid, feces, blood, and lymph for bacteriological and shotgun analysis. The DNA extraction from all samples will be done using the MolYsis complete 5 kit. For shotgun analysis, Illumina Nextera XT will be used on Novaseq 6000 equipment. Sequences from the datasets will be processed through metaWRAP. High-quality reads will be assembled using the metaSPAdes paired libraries. The taxonomy of each contig will be estimated with blastn parameters with NCBI_nt as the database embedded in the MetaWRAP-Blobology module. Statistical analysis and graph construction will be performed with RStudio. Groups with parametric distribution will be analyzed by one-way ANOVA. The Wilcoxon test will be used to compare two unpaired groups. Differences will be considered significant when P < 0.05.