A Cohort Study of the Milk Microbiota of Healthy and Inflamed Bovine Mammary Glands From Dryoff Through 150 Days in Milk

Abstract

The objective of this longitudinal cohort study was to describe the milk microbiota of dairy cow mammary glands based on inflammation status before and after the dry period. Individual mammary quarters were assigned to cohorts based on culture results and somatic cell count (SCC) at dryoff and twice in the first 2 weeks post-calving. Mammary glands that were microbiologically negative and had low SCC (< 100,000 cells/mL) at all 3 sampling periods were classified as Healthy (n = 80). Microbiologically negative mammary glands that had SCC ≥150,000 cells/mL at dryoff and the first post-calving sample were classified as Chronic Culture-Negative Inflammation (CHRON; n = 17). Quarters that did not have both culture-negative milk and SCC ≥ 150,000 cells/mL at dryoff but were culture-negative with SCC ≥ 150,000 at both post-calving sampling periods were classified as Culture-Negative New Inflammation (NEWINF; n = 6). Mammary glands with bacterial growth and SCC ≥ 150,000 cells/mL at all 3 periods were classified as Positive (POS; n = 3). Milk samples were collected from all enrolled quarters until 150 days in milk and subjected to microbiota analysis. Milk samples underwent total DNA extraction, a 40-cycle PCR to amplify the V4 region of the bacterial 16S rRNA gene, and next-generation sequencing. Healthy quarters had the lowest rate of PCR and sequencing success (53, 67, 83, and 67% for Healthy, CHRON, NEWINF, and POS, respectively). Chao richness was greatest in milk collected from Healthy quarters and Shannon diversity was greater in milk from Healthy and CHRON quarters than in milk collected from glands in the NEWINF or POS cohorts. Regardless of cohort, season was associated with both richness and diversity, but stage of lactation was not. The most prevalent OTUs included typical gut- and skin-associated bacteria such as those in the phylum Bacteroidetes and the genera Enhydrobacter and Corynebacterium. The increased sequencing success in quarters with worse health outcomes, combined with the lack of bacterial growth in most samples and the high PCR cycle number required for amplification of bacterial DNA, suggests that the milk microbiota of culture-negative, healthy mammary glands is less abundant than that of culture-negative glands with a history of inflammation.

Negatively controlled, randomized clinical trial to evaluate use of intramammary ceftiofur for treatment of nonsevere culture-negative clinical mastitis

“…The proportion of culture-negative cases was similar to those reported by Pinzón-Sánchez and Olde Riekerink et al (2008). Failure to recover pathogens from milk samples collected from cases of CM include spontaneous bacteriological clearance be-fore detection of inflammation (Smith et al, 1985;de Haas et al, 2002), occurrence of false-negative microbiological results (Silva et al, 2005;Lago, 2009), or mastitis caused by a nonculturable bacteria (Metzger, 2018). Occurrence of spontaneous cure is often evident based on rapid resolution of QSCC after resolution of clinical signs; in one study, SCC of culture-negative cases returned to almost-normal values 21 to 28 d after case detection (de Haas et al, 2002).…”

The correlation and mechanism between rumen microbiota disturbance and mastitis in dairy cows

“…Evidence has shown that cow mastitis-associated pathogens such as Streptococcus uberis, although present in small quantities, are part of the normal milk microbiota; thus, the development of mastitis may be a dysbiosis of milk microbiota rather than a simple primary infection [37,38] . Others suggested that the microbiota community in milk is relevant to in ammation status and showed that the bacterial ora are more abundant but less diverse in milk of cows suffering from in ammation [39]. In the present study, we found that elevated the abundance of Stenotrophomonas in rumen was associated with mastitis during cows suffering SARA.…”

A core microbiota dominates a rich microbial diversity in the bovine udder and may indicate presence of dysbiosis

“…The most common taxa found in the quarter milk samples are frequently reported in previous milk microbiota studies 2 , 20 . The family Corynebacteriaceae , which includes only the genus Corynebacterium, has been previously described as part of the skin-associated microbiome in humans and it is also found in other bovine body sites, such as the teat canal and uterus 24 – 27 .…”

“…These included Aerococcaceae , families within the order Clostridiales, Streptococcaceae and Enterococcaceae. The sampling regime used in the study resulted in a similar or lower bacterial richness in our samples compared to previous studies 20 – 22 . However, comparisons with other studies cannot be conclusive due to different sampling procedures, filters and bioinformatics pipelines used between the studies.…”