The microbiome of common bedding materials before and after use on commercial dairy farms

Abstract

Background: Bovine mastitis is one of the most economically important diseases affecting dairy cows. The choice of bedding material has been identified as an important risk factor contributing to the development of mastitis. However, few reports examine both the culturable and nonculturable microbial composition of commonly used bedding materials, i.e., the microbiome. Given the prevalence of nonculturable microbes in most environments, this information could be an important step to understanding whether and how the bedding microbiome acts as a risk factor for mastitis. Therefore, our objective was to characterize the microbiome composition and diversity of bedding material microbiomes, before and after use.

Methods: We collected 88 bedding samples from 44 dairy farms in the U.S. Unused (from storage pile) and used (out of stalls) bedding materials were collected from four bedding types: new sand (NSA), recycled manure solids (RMS), organic non-manure (ON) and recycled sand (RSA). Samples were analyzed using 16S rRNA sequencing of the V3-V4 region.

Results: The overall composition as well as the counts of several microbial taxa differed between bedding types, with Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes dominating across all types. Used bedding contained a significantly different microbial composition than unused bedding, but the magnitude of this difference varied by bedding type, with RMS bedding exhibiting the smallest difference. In addition, positive correlations were observed between 16S rRNA sequence counts of potential mastitis pathogens (bacterial genera) and corresponding bedding bacterial culture data.

Conclusion: Our results strengthen the role of bedding as a potential source of mastitis pathogens. The consistent shift in the microbiome of all bedding types that occurred during use by dairy cows deserves further investigation to understand whether this shift promotes pathogen colonization and/or persistence, or whether it can differentially impact udder health outcomes. Future studies of bedding and udder health may be strengthened by including a microbiome component to the study design.

Keywords: Bedding; Differential abundance; Mastitis pathogens; Microbiome.

Fig. 1

100% stacked relative abundance bar plots for each bedding sample, at the phylum level, grouped by bedding status and type. Phyla comprising < 0.5% of the total sequence counts were grouped together. NSA new sand, ON organic non-manure, RMS recycled manure solids, RSA recycled sand

Fig. 2

Box plots of alpha diversity indices (richness, inverse Simpson and Pielou’s evenness), by bedding type and status, at the phylum, class, genus, and ASV levels. Boxes represent the 25th to 75th percentile; horizontal line represents the median; and whiskers indicate 1.5× the interquartile range (IQR). NSA new sand, ON organic non-manure, RMS recycled manure solids, RSA recycled sand bedding type

Fig. 3

Non-metric multidimensional scaling (NMDS) ordination plots based on Bray–Curtis distances for A used versus unused status for each bedding type; B bedding status across all bedding types; and C all bedding types. NSA new sand, ON organic non-manure, RMS recycled manure solids, RSA recycled sand bedding type

Fig. 4

Log₂-fold change (Log2FC) in abundance of phyla between used and unused bedding samples, separated by bedding type. Only phyla with an average abundance > 50th percentile within each bedding type are depicted. Red indicates phyla whose abundance was significantly different between used and unused bedding samples (i.e., adjusted P < 0.05). Circle diameter is proportional to the average abundance of each phylum across all samples within each bedding type. NSA new sand, ON organic non-manure, RMS recycled manure solids, RSA recycled sand bedding type

Fig. 5

Barplot of total number of sequence reads (“total count”, left-hand side) and proportion of potential mastitis pathogens out of all genus-level counts, grouped by bedding status and type. Only genera with > 0.1% of the total genus-level counts are depicted as individual colors within the bars; those representing < 0.1% are grouped together as “low count pathogens”. NSA new sand, ON organic non-manure, RMS recycled manure solids, RSA recycled sand bedding type