Multi-omics integration identifies regulatory factors underlying bovine subclinical mastitis.

BACKGROUND: Mastitis caused by multiple factors remains one of the most common and costly disease of the dairy industry. Multi-omics approaches enable the comprehensive investigation of the complex interactions between multiple layers of information to provide a more holistic view of disease pathogenesis. Therefore, this study investigated the genomic and epigenomic signatures and the possible regulatory mechanisms underlying subclinical mastitis by integrating RNA sequencing data (mRNA and lncRNA), small RNA sequencing data (miRNA) and DNA methylation sequencing data of milk somatic cells from 10 healthy cows and 20 cows with naturally occurring subclinical mastitis caused by Staphylococcus aureus or Staphylococcus chromogenes. RESULTS: Functional investigation of the data sets through gene set analysis uncovered 3458 biological process GO terms and 170 KEGG pathways with altered activities during subclinical mastitis, provided further insights into subclinical mastitis and revealed the involvement of multi-omics signatures in the altered immune responses and impaired mammary gland productivity during subclinical mastitis. The abundant genomic and epigenomic signatures with significant alterations related to subclinical mastitis were observed, including 30,846, 2552, 1276 and 57 differential methylation haplotype blocks (dMHBs), differentially expressed genes (DEGs), lncRNAs (DELs) and miRNAs (DEMs), respectively. Next, 5 factors presenting the principal variation of differential multi-omics signatures were identified. The important roles of Factor 1 (DEG, DEM and DEL) and Factor 2 (dMHB and DEM), in the regulation of immune defense and impaired mammary gland functions during subclinical mastitis were revealed. Each of the omics within Factors 1 and 2 explained about 20% of the source of variation in subclinical mastitis. Also, networks of important functional gene sets with the involvement of multi-omics signatures were demonstrated, which contributed to a comprehensive view of the possible regulatory mechanisms underlying subclinical mastitis. Furthermore, multi-omics integration enabled the association of the epigenomic regulatory factors (dMHBs, DELs and DEMs) of altered genes in important pathways, such as 'Staphylococcus aureus infection pathway' and 'natural killer cell mediated cytotoxicity pathway', etc., which provides further insights into mastitis regulatory mechanisms. Moreover, few multi-omics signatures (14 dMHBs, 25 DEGs, 18 DELs and 5 DEMs) were identified as candidate discriminant signatures with capacity of distinguishing subclinical mastitis cows from healthy cows. CONCLUSION: The integration of genomic and epigenomic data by multi-omics approaches in this study provided a better understanding of the molecular mechanisms underlying subclinical mastitis and identified multi-omics candidate discriminant signatures for subclinical mastitis, which may ultimately lead to the development of more effective mastitis control and management strategies.

DNA methylation haplotype block signatures responding to Staphylococcus aureus subclinical mastitis and association with production and health traits.

BACKGROUND: DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis. RESULTS: Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield). CONCLUSIONS: In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures.

Gene co-expression in response to Staphylococcus aureus infection reveals networks of genes with specific functions during bovine subclinical mastitis.

Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection.

Genome-Wide DNA Methylation and Transcriptome Integration Associates DNA Methylation Changes with Bovine Subclinical Mastitis Caused by Staphylococcus chromogenes.

Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis.

BACKGROUND: Mastitis caused by different pathogens including Streptococcus uberis (S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due to S. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurring S. uberis subclinical mastitis and healthy control cows (n = 3/group) were studied. RESULTS: Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower in S. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGET P-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S. uberis infection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG. CONCLUSION: The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Long-Term Persistence of Arbuscular Mycorrhizal Fungi in the Rhizosphere and Bulk Soils of Non-host Brassica napus and Their Networks of Co-occurring Microbes.

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that improve the nutrition and health of their host. Most, but not all the crops form a symbiosis with AMF. It is the case for canola (Brassica napus), an important crop in the Canadian Prairies that is known to not form this association. From 2008 to 2018, an experiment was replicated at three locations of the Canadian Prairies and it was used to assess the impact of canola on the community of AMF naturally occurring in three cropping systems, canola monoculture, or canola in two different rotation systems (2-years, canola-wheat and 3-years, barley-pea-canola). We sampled canola rhizosphere and bulk soils to: (i) determine diversity and community structure of AMF, we expected that canola will negatively impact AMF communities in function of its frequency in crop rotations and (ii) wanted to assess how these AMF communities interact with other fungi and bacteria. We detected 49 AMF amplicon sequence variants (ASVs) in canola rhizosphere and bulk soils, confirming the persistence of a diversified AMF community in canola-planted soil, even after 10 years of canola monoculture, which was unexpected considering that canola is among non-mycorrhizal plants. Network analysis revealed a broad range of potential interactions between canola-associated AMF and some fungal and bacterial taxa. We report for the first time that two AMF, Funneliformis mosseae and Rhizophagus iranicus, shared their bacterial cohort almost entirely in bulk soil. Our results suggest the existence of non-species-specific AMF-bacteria or AMF-fungi relationships that could benefit AMF in absence of host plants. The persistence of an AMF community in canola rhizosphere and bulk soils brings a new light on AMF ecology and leads to new perspectives for further studies about AMF and soil microbes interactions and AMF subsistence without mycotrophic host plants.

Inter-Kingdom Networks of Canola Microbiome Reveal Bradyrhizobium as Keystone Species and Underline the Importance of Bulk Soil in Microbial Studies to Enhance Canola Production.

The subterranean microbiota of plants is of great importance for plant growth and health, as root-associated microbes can perform crucial ecological functions. As the microbial environment of roots is extremely diverse, identifying keystone microorganisms in plant roots, rhizosphere, and bulk soil is a necessary step towards understanding the network of influence within the microbial community associated with roots and enhancing its beneficial elements. To target these hot spots of microbial interaction, we used inter-kingdom network analysis on the canola growth phase of a long-term cropping system diversification experiment conducted at four locations in the Canadian Prairies. Our aims were to verify whether bacterial and fungal communities of canola roots, rhizosphere, and bulk soil are related and influenced by diversification of the crop rotation system; to determine whether there are common or specific core fungi and bacteria in the roots, rhizosphere, and bulk soil under canola grown in different environments and with different levels of cropping system diversification; and to identify hub taxa at the inter-kingdom level that could play an important ecological role in the microbiota of canola. Our results showed that fungi were influenced by crop diversification, which was not the case on bacteria. We found no core microbiota in canola roots but identified three core fungi in the rhizosphere, one core mycobiota in the bulk soil, and one core bacterium shared by the rhizosphere and bulk soil. We identified two bacterial and one fungal hub taxa in the inter-kingdom networks of the canola rhizosphere, and one bacterial and two fungal hub taxa in the bulk soil. Among these inter-kingdom hub taxa, Bradyrhizobium sp. and Mortierella sp. are particularly influential on the microbial community and the plant. To our knowledge, this is the first inter-kingdom network analysis utilized to identify hot spots of interaction in canola microbial communities.

Similar Arbuscular Mycorrhizal Fungal Communities in 31 Durum Wheat Cultivars (Triticum turgidum L. var. durum) Under Field Conditions in Eastern Canada.

Wheat is among the important crops harnessed by humans whose breeding efforts resulted in a diversity of genotypes with contrasting traits. The goal of this study was to determine whether different old and new cultivars of durum wheat (Triticum turgidum L. var. durum) recruit specific arbuscular mycorrhizal (AM) fungal communities from indigenous AM fungal populations of soil under field conditions. A historical set of five landraces and 26 durum wheat cultivars were field cultivated in a humid climate in Eastern Canada, under phosphorus-limiting conditions. To characterize the community of AMF inhabiting bulk soil, rhizosphere, and roots, MiSeq amplicon sequencing targeting the 18S rRNA gene (SSU) was performed on total DNAs using a nested PCR approach. Mycorrhizal colonization was estimated using root staining and microscope observations. A total of 317 amplicon sequence variants (ASVs) were identified as belonging to Glomeromycota. The core AM fungal community (i.e., ASVs present in > 50% of the samples) in the soil, rhizosphere, and root included 29, 30, and 29 ASVs, respectively. ASVs from the genera Funneliformis, Claroideoglomus, and Rhizophagus represented 37%, 18.6%, and 14.7% of the sequences recovered in the rarefied dataset, respectively. The two most abundant ASVs had sequence homology with the 18S sequences from well-identified herbarium cultures of Funneliformis mosseae BEG12 and Rhizophagus irregularis DAOM 197198, while the third most abundant ASV was assigned to the genus Paraglomus. Cultivars showed no significant difference of the percentage of root colonization ranging from 57.8% in Arnautka to 84.0% in AC Navigator. Cultivars were generally associated with similar soil, rhizosphere, and root communities, but the abundance of F. mosseae, R. irregularis, and Claroideoglomus sp. sequences varied in Eurostar, Golden Ball, and Wakooma. Although these results were obtained in one field trial using a non-restricted pool of durum wheat and at the time of sampling, that may have filtered the community in biotopes. The low genetic variation between durum wheat cultivars for the diversity of AM symbiosis at the species level suggests breeding resources need not be committed to leveraging plant selective influence through the use of traditional methods for genotype development.