Characterization of Microbiome on Feces, Blood and Milk in Dairy Cows with Different Milk Leucocyte Pattern

Mastitis is an inflammatory disease of the mammary gland, caused by the invasion of microorganism on this site, associated with an altered immune response. Recent studies in this field hypothesize that the origin of these pathogens can also be from the gastrointestinal tract, through the entero-mammary pathway in relation to an increase in gut permeability. In this study, we wanted to investigate if inflammatory status of the mammary gland is related to an alteration of gut permeability. The microbiome of feces, blood and milk of lactating cows, recruited on the basis of the total somatic cell count and of the percentage of polymorphonuclear neutrophils and lymphocytes, was studied. Cows were divided into healthy (G), at risk of mastitis (Y) and with mastitis (R) classifications. The bacterial DNA was extracted and the V3 and V4 regions of 16S rRNA sequenced. Moreover, the quantification of total bacteria was performed with quantitative real-time PCR. A non-parametric Kruskal–Wallis test was applied at the phylum, family and genera levels and beta biodiversity was evaluated with the unweighted UniFrac distance metric. Significant differences between groups were found for the microbial composition of feces (Clostridiaceae, Turicibacteriaceae for family level and Clostridium, Dorea, SMB53 and Turicibacter for genus level), blood (Tenericutes for phylum level and Mycoplasma for genus level) and milk (OD1 and Proteobacteria for phylum level, Enterobacteriaceae and Moraxallaceae for family level and Olsenella and Rhodococcus for genus level). The beta biodiversity of feces and blood did not change between groups. Significant differences (p < 0.05) were observed between the beta diversity in milk of G group and Y group and between Y group and R group. The number of taxa in common between feces, blood and milk were 8 at a phylum, 19 at a family and 15 at a genus level. From these results, the bacterial crossing from gut to milk in cows was not confirmed but remained hypothetical and deserves further investigation. Simple Summary Mastitis is an inflammation of the mammary gland caused by microorganisms and associated with an altered immune response. Recently, several studies hypothesized that a translocation of some bacteria from the gastrointestinal tract to the mammary gland can occur and that this bacterial crossing could be the cause of certain mastitis. The aim of this research is to investigate the bacteria translocation from the gut to the mammary gland, the so-called entero-mammary pathway, through the study of the fecal, blood and milk microbiome. Cows were recruited on the basis of their mammary gland health status and classified as healthy, at risk of mastitis and with mastitis. The microbial composition of feces, blood and milk were analyzed through high-throughput sequencing technique and the results were checked through a quantitative real-time PCR analysis. Although small differences were found in the microbiome of these three specimens between the groups of animals, beta biodiversity, that is, the ratio between whole and individual species diversity, highlighted a microbial community change in the milk of cows with different udder health conditions. The three matrices shared a high number of taxa; however, our results do not confirm a bacterial crossing from gut to milk, that still remains hypothetical.