Groups (Figure 3A). The PLS-DA plot illustrated a clear separation with the two groups (Figure 3B). Regarding the prime ten dominant taxa at the genus and species levels,Animals 2021, 11,6 ofthe wholesome cows had a significantly greater abundance of Tetratrichomonas sp. 2003001 (FDR adjusted p 0.05) than the mastitic counterparts (Figure 3C,D).(A) (B)(C)(D)Figure 3. Ruminal protozoa and fungi composition had been identified by 18S rRNA sequencing. (A) The Chao1 richness estimator and Shannon’s diversity index. (B) Partial least squares discriminant evaluation (PLS-DA) plot according to the SB 218795 Protocol relative abundance of OTUs indicates a drastically different composition of healthier versus mastitis groups. Ellipses represent 95 confidence intervals for every single group. The best 10 (C) genera and (D) species identified in cow ruminal fluid, every bar refers to an individual cow.3.three. Identification of your Critical Ruminal Bacterial Biomarkers Related with Inflammation and Their Co-Occurring Patterns Since the NGS final results indicated that HC and MC may very well be distinguished by ruminal bacteria and archaea, as well as dominant taxa, the much less abundant but vital taxa associated with mastitis were identified as bacterial biomarkers by the LEfSe algorithm, revealing thirty influential taxonomic clades, which includes seven genera and 3 species (Figure 4A). Within the HC group, the critical biomarkers were the genera Ruminococcus 1, Ruminococcaceae UCG-014, Treponema 2, Fibrobacter, and Selenomonas 1, as well as the species Ruminococcus flavefaciens and Treponema saccharophilum. The genera Bacillus and PF-07321332 Protocol Sharpea and species Bacillus anthracis have been the important taxa in the MC group. The co-occurrence patterns in the biomarkers have been determined by constructing a bacterial network of your seven genera and three species, which was further correlated with the SCC and IL-6 levels. The SCC level positively correlated with Sharpea and negatively correlated with Ruminococcaceae UCG-014, Ruminococcus flavefaciens, and Treponema saccharophilum (p 0.05) (Figure 4B). Amongst the genus bacterial network, Sharpea negatively correlated with Selenomonas 1 (p 0.05), whereas, Ruminococcaceae UCG-014 positively correlated with Ruminococcus 1, Treponema 2, Fibrobacter, and Selenomonas 1 (p 0.05), indicating the important function of Ruminococcaceae UCG-014 within the network. In terms of the species bacterial network, Ruminococcus flavefaciens and Treponema saccharophilum have been positively correlated (p 0.05) (Figure 4B). IL-6 positively correlated with Sharpea and Bacillus and negatively correlated with Ruminococcaceae UCG-014, Treponema 2, Fibrobacter, Selenomonas 1, Ruminococcus flavefaciens, and Treponema saccharophilum (p 0.05) (Figure 4C).Animals 2021, 11,7 of(A)(B)RuminococcusGenusSpeciesRuminococcus flavefaciensSharpeaRuminococcaceae UCG-Abundance 5 1-5 Correlation 0.four | r | 0.6 | r | 0.6 Positive correlation (P 0.05) Negative correlation (P 0.05) Constructive correlated with somatic cell count (P 0.05) Adverse correlated with somatic cell count (P 0.05) 1BacillusTreponemaTreponema saccharophilum Selenomonas 1 FibrobacterBacillus anthracis(C)RuminococcusGenusSpeciesRuminococcus flavefaciensSharpeaRuminococcaceae UCG-Abundance 5 1-5 Correlation 0.four | r | 0.six | r | 0.6 Optimistic correlation (P 0.05) Unfavorable correlation (P 0.05) Constructive correlated with IL-6 (P 0.05) Negative correlated with IL-6 (P 0.05) 1BacillusTreponemaTreponema saccharophilumBacillus anthracis(D) GenusSelenomonasFibrobacterSpeciesFigure 4.