A Study on the Changing Law of Bacterial Communities in the Milk of Bactrian Camels with Subclinical Mastitis

Abstract

Subclinical mastitis is a critical disease affecting camel health and milk quality. However, research on shifts in milk bacterial communities following subclinical mastitis in camels is limited. We evaluated changes in bacterial communities following subclinical mastitis in Bactrian camels. Three portions of California Mastitis Test (CMT)-negative milk and five portions of CMT-positive milk were collected from each Jimunai County and Keping County using the CMT, and the bacterial community composition of the camel milk was analyzed using amplicon sequencing of the v34 region of the 16S rRNA gene. Subclinical mastitis induced genus-level differences in the core bacterial microbiota of Bactrian camel milk. To our knowledge, Delftia was identified in camel milk for the first time, predominantly in Jimunai County. Bacterial abundance in camel milk from Keping County was increased and altered. Alpha diversity analysis revealed that subclinical mastitis induced lower and higher bacterial abundance in milk from Jimunai County and Keping County, respectively, compared to that of healthy camels. Therefore, these findings provide direction for future research on pathogenic microorganisms for the prevention and control of subclinical mastitis in Bactrian camels.

Keywords: 16S rRNA amplicon sequencing; Bactrian camel; camel milk bacterial community; pathogenesis; subclinical mastitis.

Figure 1

Relative abundance of gate-level species in subclinical mastitis versus healthy camel milk: top 10 histograms. The horizontal axis (Sample Name) represents the sample name, the vertical axis represents the relative abundance, and “others” refers to sum of the relative abundances of all gates except for the 10 gates in the graph.

Figure 2

Relative abundance of the top 10 bacterial genera in milk from camels with subclinical mastitis versus healthy camels. The horizontal axis represents the sample names and the vertical axis indicates the relative abundance. “Others” signifies the combined relative abundance of all other genera not included among the ten genera depicted in the graph.

Figure 3

Relative abundance of the top 10 bacterial species in milk from camels with subclinical mastitis versus healthy camels. The horizontal axis represents the sample name, the vertical axis represents the relative abundance, and “others” refers to the sum of the relative abundances of all species, except for the 10 species in the graph.

Figure 4

Genus-level phylogenetic relationships between subclinical mastitis and healthy camel milk. The colors of the branches and sectors indicate their respective corresponding phyla, whereas the stacked bar chart positioned on the outer side of the sector ring displays the abundance of the bacterial genera across various samples. The legend on the left specifies the samples and the legend on the right specifies taxonomic classifications at the phylum level which correspond to those at the genus level.

Figure 5

Dilution curves of species in the milk from subclinical mastitis and healthy camelids.

Figure 6

Cumulative box plot of alpha diversity species.

Figure 7

Heat map of the Beta diversity index (UniFrac distance matrix) between subclinical mastitis and healthy camel milk. The numbers in the squares of the figure represent the dissimilarity coefficients between the pairs of samples. The smaller the dissimilarity coefficient, the lesser the difference in species diversity between the two samples. Within the same square, the upper and lower values represent the weighted and unweighted UniFrac distances, respectively.

Figure 8

UPGMA clustering tree at the phylum level based on weighted UniFrac distances between subclinical mastitis and healthy camel milk. The UPGMA clustering tree structure is shown on the left, whereas the graph on the right depicts the relative abundance distribution for each sample at the phylum level.

Figure 9

PCoA analyses comparing subclinical mastitis and healthy milk samples in Jimunai County (A) and Keping County (B). The horizontal axis represents one principal component, the vertical axis represents another principal component, and the percentage represents the contribution value of the principal component to the sample difference. Each point in the figure represents a sample, and samples in the same group are represented by the same color.

Figure 10

Analysis of species differences between subclinical mastitis and healthy milk using t-test groups. Panels (A,B) demonstrate the differential analysis of healthy and mastitis-infected Bactrian camels from Jimunai County and Keping County, respectively. Panels (C,D) show the differential analyses of milk from healthy and mastitis-infected camels, respectively. The left sides of the panels display the abundances of significantly different species between groups. Each bar in the graph represents the mean of significantly different abundant species between groups in each group. The right sides of the panels display inter-group confidence levels. The extreme left and right points of each circle in the panels represent the lower and upper limits of the 95% confidence interval for the mean difference, respectively. The center of the circles signifies the difference in means, while the circle’s color indicates the p-value from the significant difference test between groups for the corresponding species, * indicates p value < 0.05, ** indicates p value < 0.01.

Figure 11

PICRUSt2 functionally annotated relative abundance histogram of subclinical mastitis versus healthy milk samples. The horizontal axis represents the sample names; the vertical axis indicates the relative abundance; and “Others” signifies the sum of the relative abundances of all other functional information beyond the 10 shown in the figure.