Milk's microbiota during the periparturient period in holstein cows: possible implication on animal health and milk quality

Dairy cattle are exposed to a high risk for disease during periparturient period and this period is often associated with the peak incidence of production problems, metabolic disorders, infectious diseases, metritis and mastitis. The aim of this study was to compare the milk's microbiota during the periparturient period in order to assess the possible implication on animal health and milk quality. Milk samples were collected from each quarters of six pluriparous Holstein cows at 340 ? 60 days of lactation (T1), at the day after calving (T2) and between 7 to 10 days after calving (T3). To determine the udder health status, a standard bacteriological analysis on milk samples were performed; moreover, SCC was obtained by an automated fluorescent microscopic somatic cell counter. For the microbiome analysis, the bacterial DNA was extracted from the pool of the quarters for each animal using a protocol previously described (Cremonesi et al., 2005) with some modifications and the 16S rRNA gene (V3-V4 region) was analyzed by Miseq (Illumina). Milk proteins were evaluated by SDS-PAGE and by densitometric analysis for assessment of total protein profiles. In addition, the presence of the inflammation markers cathelicidin and S100A9 was estimated by western immunoblotting. Tests were done for all quarters and for the three lactation stages. Moreover, the expression of CD45 and KRT5 messengers in the isolated milk cells was analyzed in order to assess the modification of leukocyte/exfoliated epithelial cell ratio during peripartum, while the expression of IL-1b and TNFa mRNA in the isolated milk cells was studied for their capability to produce cytokines. Finally, the expression of PTX3 transcript in the milk fat globules was analyzed in order to evaluate its modulation and to check the activation of the mammary epithelial cells. Bacteriological analysis showed the absence of contagious bacteria such as Staph. aureus and S. agalactiae. An interesting modification of the leukocyte/exfoliated epithelial cell ratio during peripartum was observed with an up-regulation of PTX3 in the colostrum. Moreover, this study describes the relative changes in milk protein abundance along lactation and according to composition of the microbial flora. These data have also been integrated with information on protein markers of inflammation. To our knowledge, this is the first time that mammary gland metagenome was studied during the periparturient period.