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Staphylococcus aureus (S. aureus) is an opportunistic pathogen frequently associated with subclinical mastitis and accounts for a large proportion of the economic losses due to mastitis on Canadian dairy farms. Despite a plethora of investigations on the molecular mechanisms of mastitis, little information is available on the roles of regulatory noncoding RNAs (ncRNA). This study aimed to uncover the regulatory roles of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in the response to subclinical mastitis due to S. aureus of cows. Transcriptome sequencing (RNASeq and miRNA-Seq) was conducted on milk somatic cells from 13 cows with S. aureus subclinical mastitis [high somatic cell counts (SCC) of ≥350,000 cells/mL for ≥3 consecutive months and positive for S. aureus only], and 5 healthy cows (low SCC< 100,000 cells/mL for ≥3 consecutive months and negative for mastitis pathogens). RNASeq and smRNASeq nf-core bioinformatics pipelines were used to process the data. Results showed that 97 miRNAs (36 up-regulated and 61 down-regulated) and 1,565 lncRNAs (617 up-regulated and 948 down-regulated) were differentially expressed (DE; FDR< 0.05) between S. aureus subclinical mastitic cows and healthy controls. Competing endogenous networks comprising co-expressed differentially expressed mRNAs (DEGs), miRNAs (DEMs), and lncRNAs (DELs) were constructed. CytoHubba, a Cytoscape plugin, was utilized to identify the top hub regulatory ncRNAs including bta-miR-2387, btamiR-331, bta-miR-95, bta-miR-744, bta-miR-455, bta-novel-miR-60, bta-miR-3533, bta-miR-500, btamiR-1249 and bta-novel-miR-66). The results revealed that the main hubs (downregulated bta-miR-2387 and upregulated bta-miR-331) potentially regulate numerous DEGs during S. aureus subclinical mastitis such as NFKB1, IL10RA, IL17RA, TLR10, LOC112442665, LOC112448481 and LOC112442015, etc., and TUNAR, LOC112445429, LOC112444484 and LOC101907369, respectively. Functional analysis of bta-miR-331 target genes revealed roles in several KEGG pathways (e.g., tight junction, bacterial invasion of epithelial cells) and biological processes gene ontology (GO) terms (e.g., cell adhesion, cell locomotion and cell motility). Similarly, roles for bta-miR-2387 in KEGG pathways like JAK-STAT signaling pathway, NOD-like receptor signaling pathway and inflammatory bowel disease, and biological process GO terms such as inflammatory response, neutrophil migration, leucocyte migration and response to cytokine were revealed. MiR-331 has been found to promote cell proliferation, migration, and invasion of breast cancer. Furthermore, bta-miR-2387 has been shown to regulate several immune pathways which aid in improving the movement of spermatids across the epithelium and preleptotene spermatocytes across the blood-testis barrier during spermatogenesis. This supports our findings and suggests that bta-miR-2387 and bta-miR-331 could be pivotal regulators of immune-related genes and pathways during S. aureus subclinical mastitis. Thus, they hold potential as biomarkers for the development of therapeutic and diagnostic tools for managing subclinical mastitis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Staphylococcus chromogenes (SC), a frequent non-aureus staphylococcus on Canadian herds causes both clinical and subclinical forms of mastitis and contributes to large economic losses on Canadian farms. However, little research attention has been directed to uncovering the molecular mechanisms underlying SC mastitis pathogenesis, including the roles of small nucleolar RNAs (snoRNAs). SnoRNAs function to regulate host ribosomal RNAs (rRNAs) biogenesis and processing, and are known to have regulatory functions in several human diseases, but no study has examined their regulatory functions in bovine mastitis. To understand the regulatory roles of snoRNAs in cows with SC subclinical mastitis, the snoRNA transcriptome of milk somatic cells from three SC infected cows [positive for SC only, have high somatic cell counts (SCC =350,000 cells/mL) for = 3 months consecutively, only one positive udder sampled], and four healthy cows [low SCC (< 100,000 cells/mL) for =3 months consecutively, compositive sample of all quarters] was characterized by sequencing and bioinformatics analysis. Among 232 known snoRNAs found in the samples, 33 were differentially expressed (DE; 14 downregulated and 19 upregulated; FDR < 0.05). Out of these, SnoRA3/snoRA45 (upregulated) belongs to H/ACA snoRNA box type while SNORD14 (downregulated) belongs to C/D box snoRNA box type. According to RNAsnoop analysis, SnoRA3/snoRA45 interacted with 18S-1667 rRNA [minimum free energy (MFE) = -36Kcal/Mol] while SNORD14 interacted with 28S-3926 rRNA (MFE = -15.40Kcal/Mol). The observed strong MFE suggests that SnoRA3/snoRA45 and SNORD14 might potentially participate in rRNA modifications which could affect important functional sites of the ribosome with implications on the pathogenesis of bovine SC mastitis. SnoRA3/snoRA45 have been reported to guide site specific pseudouridylation of rRNA by upregulating the ribosomal pathway as well as have important roles in cell cycle progression in human. SNORD14 is expressed from the intron of its host gene RPS13, which is a ribosomal protein involved in protein biosynthesis. Also, SNORD14 has been shown to be potentially involved in intracellular protein transport and positive regulation of MAPK cascade. Our results suggest that the DE snoRNAs could potentially regulate the host rRNA biogenesis and processing in response to mastitis due to SC. To the best of our knowledge, this is the first study to report dysregulated snoRNA expression and potential involvement in bovine SC subclinical mastitis. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is one of the most prevalent pathogens of bovine subclinical mastitis and a challenging pathogen in dairy production. DNA methylation involvement in regulating mammary gland inflammatory defense has been documented. This study adopted whole genome bisulfite sequencing and RNA-sequencing technologies to profile the genome-wide DNA methylation and transcriptome landscapes, respectively, of milk somatic cells (MSCs) from 15 cows with S. aureus-induced subclinical mastitis (SACs) and 13 healthy cows (HCs). Bioinformatics processing of data with standard tools indicated that the global DNA methylation patterns of MSCs demonstrated inverse correlations between gene expression and DNA methylation levels at promoter, first exon and first intron regions, suggesting potential regulatory roles of DNA methylation in transcriptional activities. Totally, 26,289 differential methylation regions (DMRs) were identified between SACs and HCs (1,000 bp window, 1,000 bp step, q-value < 0.05, >20% methylation difference and =3 differentially methylated cytosines). A total of 887, 334 and 3,414 DMRs were overlapped with promoter, first exon, and first intron regions, respectively, including 363 DMRs harboring transcription start sites. Among them, the methylation levels of 956 DMRs were strongly correlated with the expression levels of their overlapped genes (|Spearman's correlation coefficient| >0.5, adjusted-pvalue< 0.05). Functional annotation of the overlapped genes indicated significant (adjusted-p-value < 0.05) enrichment in 18 GO terms and 1 KEGG pathway related to cellular activities, notably in cell migration, epithelial cell differentiation and tight junction, among others (Figure 1). These results suggest the involvement of DNA methylation in mammary gland defense against S. aureus-invasion. Applying DIABLO method to integrate correlated DMRs and genes, a total of 20 DMR and 10 gene candidate markers were found to discriminate SACs from HCs. The identified candidate markers could serve as reference for enhancing breeding for mastitis resistance and for developing new mastitis management strategies. [ABSTRACT FROM AUTHOR]

DNA methylation involvement in the regulation of mammary gland inflammatory defense against pathogens, including Staphylococcus aureus(SA) has been documented. However, the mechanisms are not clear. The possible influence of DNA methylation of first introns (DNA-MeFI) on transcriptional activities during subclinical mastitis was studied by profiling the methylome (whole genome methylation sequencing) and transcriptome (RNA-sequencing) of milk somatic cells from cows(n=15) with SA subclinical mastitis (SACs) and healthy cows (HCs)(n=13), followed by bioinformatics processing with standard tools. The DNA-MeFI was calculated, for each of 19411 genes found as expressed in the samples, by averaging the methylation levels of all CpG sites in the first intron. At genome-wide, the DNA-MeFIs was inversely correlated with gene expression (GE) (Pearson's r=-0.173, p=1.60×10-122)(Figure 1-A). Similarly, the difference in the DNA-MeFI and GE between SACs and HCs was also inversely correlated (Pearson's r=-0.149, p=4.25×10-97)(Figure 1-B). Application of machine learning by Gaussian Mixed model (GMM) using scikit-learn in python to the changes of DNA-MeFI and GE between SACs and HCs revealed 2866 outliers (GMM's p< 0.005) that showed significant changes in the DNA-MeFI and/or GE. 644 genes with >10% difference in DNA-MeFI and |log2FC| >1 in GE were selected and referred to as differentially methylated and expressed genes (DME-genes). 644 DME-genes were significantly enriched in 5 GO terms and 11 KEGG pathways related to diseases and immune functions, including Staphylococcus aureus infection and Natural killer cell mediated cytotoxicity (Table 1). Furthermore, 74.84% DME-genes showed inverse changes in DNA-MeFI and GE. Besides, the DNA-MeFI of 264 DME-genes were found to correlate significantly with their GE (|rho| >0.3, FDR<0.05), including 225 DME-genes(85.23%) with inverse correlations(rho< -0.3, FDR< 0.05). 39 DME-genes with positive correlations were significantly enriched in 21 GO terms mainly related to metabolic processes. In conclusion, the DNA-MeFI possibly participate in the regulation of gene expression during bovine subclinical mastitis caused by SA. [ABSTRACT FROM AUTHOR]

Near infrared reflectance spectroscopy (NIRS) holds promise for rapid assessment of soil total N and organic C contents, but its ability to predict soil N supply in soils with contrasting physio-chemical properties needs to be evaluated. The objectives of this study were to: (i) evaluate NIRS predictions of measured soil parameters (total N, organic C, and C/N ratio) and as an indirect indicator of soil N supply, namely corn (Zea mays L.) N uptake and (ii) assess the effect of sample set heterogeneity on NIRS predictions. Soil samples (n = 282 in the total set) were collected between 2000 and 2009 from 52 sites across four Canadian provinces and were allocated to fine- (≥ 350 g clay kg-1 n = 101) and coarse-textured subsets (<350 g clay kg-1 n = 181). Prediction models were developed using modified partial least squares regression. Prediction precision was assessed using the coefficient of determination (R²) and ratio of prediction to deviation (RPD = ratio of standard error of prediction corrected for bias to standard deviation of the reference data used in the validation). For the total set, predictions were reliable for total N, organic C and C/N ratio (0.7 ≤ R² ≤ 0.9, 1.75 ≤ RPD ≤ 3), and less reliable for soil N supply (R² < 0.7, RPD < 1.75). Prediction precision for total N, organic C, C/N ratio, and soil N supply increased with set homogeneity (i.e., better in the texture subsets than the total set), resulting in reliable predictions for soil N supply (RPD ≥ 2.00) in the texture subsets. This study demonstrated the possibility of developing reliable NIRS predictive models for total N, organic C, and C/N ratio for soils with contrasting physio- chemical properties, as well as reliable NIRS predictive models for soil N supply within homogeneous texture sets. [ABSTRACT FROM AUTHOR]