The 16S rDNA high-throughput sequencing correlation analysis of milk and gut microbial communities in mastitis Holstein cows

Abstract

This study aimed to understand the changes in the milk and gut microbiota of dairy cows with mastitis, and to further explore the relationship between mastitis and the microbiota. In this study, we extracted microbial DNA from healthy and mastitis cows and performed high-throughput sequencing using the Illumina NovaSeq sequencing platform. OTU clustering was performed to analyze complexity, multi-sample comparisons, differences in community structure between groups, and differential analysis of species composition and abundance. The results showed that there were differences in microbial diversity and community composition in the milk and feces of normal and mastitis cows, where the diversity of microbiota decreased and species abundance increased in the mastitis group. There was a significant difference in the flora composition of the two groups of samples (P < 0.05), especially at the genus level, the difference in the milk samples was Sphingomonas (P < 0.05) and Stenotrophomonas (P < 0.05), the differences in stool samples were Alistipes (P < 0.05), Flavonifractor (P < 0.05), Agathobacter (P < 0.05) and Pygmaiobacter (P < 0.05). In conclusion, the microbiota of the udder and intestinal tissues of dairy cows suffering from mastitis will change significantly. This suggests that the development of mastitis is related to the endogenous pathway of microbial intestinal mammary glands, but the mechanisms involved need further study.

Keywords: 16S rDNA high-throughput sequencing; Gut microbial community; Holstein cow; Mastitis; Milk microorganism.

Fig. 1

Venn diagram representation of the OTUs shared among different groups

Fig. 2

Rarefaction Curve of each sample

Fig.3

Alpha diversity estimates of the bacterial communities A-D: Box plots of bacterial alpha diversity assessed by Shannon index, Simpson index, Chao index, and Ace index. Different numbers in the graph indicate different α-diversity indices and p-values between groups. The three lines from bottom to top are: first quartile, median and third quartile. Outliers are represented by Outliers are represented by “.”

Fig. 4

Analysis of differences between microbial groups in samples A Principal coordinate analysis (PCoA). B Anosim analysis of milk samples. C Anosim analysis of faecalis samples. The R-value and P-value in the graph represent the confidence level of the sample statistics, where R-value is between (-1, 1) and R-value is greater than 0, indicating a significant difference between groups; P < 0.05 indicates that the statistics are significant

Fig. 5

Relative abundances at the phylum level and genus level.  A&C Mastitis milk and normal milk. B&D Mastitis fecal and normal fecal

Fig. 6

T-test sample group interspecies variance analysis graph. A Mastitis milk and normal milk. B Mastitis fecal and normal fecal

Fig.7

Linear discriminant analysis Effect Size (LEfSe) Analysis of Samples. A Histogram of LDA value distribution of milk samples (LD < 4). B LEfSe cladogram of milk samples. C Histogram of LDA value distribution of rectal stool samples (LD < 4). D LEfSe cladogram of rectal stool samples