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 oren associated with the peak incidence of produc6on problems, metabolic disorders, infec6ous diseases, metri6s and mas66s. The aim of this study was to compare the milk's microbiota during the periparturient period in order to assess the possible implica6on on animal health and milk quality. Milk samples were collected from each quarters of six pluriparous Holstein cows at 340 ± 60 days of lacta6on (T1), at the day arer calving (T2) and between 7 to 10 days arer 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 soma6c 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 modifica6ons 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 addi6on, the presence of the inflamma6on markers cathelicidin and S100A9 was es6mated by western immunoblobng. Tests were done for all quarters and for the three lacta6on stages. Moreover, the expression of CD45 and KRT5 messengers in the isolated milk cells was analyzed in order to assess the modifica6on of leukocyte/exfoliated epithelial cell ra6o 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 modula6on and to check the ac6va6on of the mammary epithelial cells. Bacteriological analysis showed the absence of contagious bacteria such as Staph. aureus and S. agalac6ae. An interes6ng modifica6on of the leukocyte/exfoliated epithelial cell ra6o during peripartum was observed with an up\regula6on of PTX3 in the colostrum. Moreover, this study describes the rela6ve changes in milk protein abundance along lacta6on and according to composi6on of the microbial flora. These data have also been integrated with informa6on on protein markers of inflamma6on. To our knowledge, this is the first 6me that mammary gland metagenome was studied during the periparturient period.