An Explainable Deep Learning Classifier of Bovine Mastitis Based on Whole-Genome Sequence Data-Circumventing the p >> n Problem.

The serious drawback underlying the biological annotation of whole-genome sequence data is the p >> n problem, which means that the number of polymorphic variants (p) is much larger than the number of available phenotypic records (n). We propose a way to circumvent the problem by combining a LASSO logistic regression with deep learning to classify cows as susceptible or resistant to mastitis, based on single nucleotide polymorphism (SNP) genotypes. Among several architectures, the one with 204,642 SNPs was selected as the best. This architecture was composed of two layers with, respectively, 7 and 46 units per layer implementing respective drop-out rates of 0.210 and 0.358. The classification of the test data resulted in AUC = 0.750, accuracy = 0.650, sensitivity = 0.600, and specificity = 0.700. Significant SNPs were selected based on the SHapley Additive exPlanation (SHAP). As a final result, one GO term related to the biological process and thirteen GO terms related to molecular function were significantly enriched in the gene set that corresponded to the significant SNPs. Our findings revealed that the optimal approach can correctly predict susceptibility or resistance status for approximately 65% of cows. Genes marked by the most significant SNPs are related to the immune response and protein synthesis.

Considering potential roles of selected MicroRNAs in evaluating subclinical mastitis and Milk quality in California mastitis test (+) and infected bovine milk.

This study investigates the relationships between subclinical mastitis and milk quality with selected microRNAs in cow milk. California Mastitis Test (CMT)-positive (n = 20) and negative (n = 20) samples were compared (Experiment I). Additionally, samples with CMT-positive but microbiological-negative, as well as positive for only Staphylococcus subspecies (Staph spp.) and only Streptococcus subspecies (Strep spp.) were examined (Experiment II). Four groups were formed in Experiment II: Group I (CMT and microbiological-negative) (n = 20), Group II (CMT-positive but microbiological-negative) (n = 10), Group III (Staph spp.) (n = 5), Group IV (Strep spp.) (n = 5). While electrical conductivity, somatic cell count (SCC), malondialdehyde (MDA) increased, miR-27a-3p and miR-223 upregulated and miR-125b downregulated in the CMT-positive group in Experiment I. SCC and MDA were higher in CMT-positive groups. miR-27a-3p and miR-223 upregulated in Groups III and IV. While miR-155 is upregulated, miR-125b downregulated in Group IV. Milk fat is positively correlated with miR-148a and miR-223. As miR-27a-3p positively correlated with SCC and MDA, miR-125b negatively correlated with electrical conductivity and SCC. miR-148a and MDA were positively correlated. miR-155 was correlated with fat-free dry matter, protein, lactose, and freezing point. miR-223 was positively correlated with SCC and miR-148a. Results particularly highlight miR-27a-3p and miR-223 as potential biomarkers in subclinical mastitis, especially those caused by Staph spp. and Strep spp., while miR-148a, miR-155, and miR-223 stand out in determining milk quality.

Long non-coding RNA (CMR) involved in autoprotection in S. aureus mastitis in dairy cows by regulating miR-877/FOXM1.

Long non-coding RNAs (LncRNAs) are dysregulated in a variety of human diseases and are highly involved in the development and progression of tumors. Studies on lncRNAs associated with cow mastitis have been lagging behind compared to humans or model animals, therefore, the aim of this study was to explore the mechanism of LncRNAs (CMR) involved in autoprotection against S. aureus mastitis in Bovine Mammary Epithelial Cells (BMECs). First, qRT-PCR was used to examine the relative expression of CMR in a S. aureus mastitis model of BMECs. Then, cell proliferation and apoptosis were detected by EdU and apoptosis assay. Finally, the targeting relationship between miRNAs and mRNA/LncRNAs was determined by dual luciferase reporter gene, qRT-PCR and western blotting techniques. The results showed that CMR was upregulated in the S. aureus mastitis model of BMECs and promoted the expression of inflammatory factors, and SiRNA-mediated CMR inhibited the proliferation of mammary epithelial cells and induced apoptosis. Mechanistically, CMR acts as a competitive endogenous RNA (ceRNA) sponge miR-877, leading to upregulation of FOXM1, a target of miR-877. Importantly, either miR-877 overexpression or FOXM1 inhibition abrogated CMR knockdown-induced apoptosis promoting cell proliferation and reducing inflammatory factor expression levels. In summary, CMR is involved in the regulation of autoprotection against S. aureus mastitis through the miR-877/FOXM1 axis in BMECs and induces immune responses in mammary tissues and cells of dairy cows, providing an important reference for subsequent prevention and control of cow mastitis and the development of targeted drugs.

Identification of candidate genes associated with milk production and mastitis based on transcriptome-wide association study.

Genetic research for the assessment of mastitis and milk production traits simultaneously has a long history. The main issue that arises in this context is the known existence of a positive correlation between the risk of mastitis and lactation performance due to selection. The transcriptome-wide association study (TWAS) approach endeavors to combine the expression quantitative trait loci and genome-wide association study summary statistics to decode complex traits or diseases. Accordingly, we used the farmgtex project results as a complete bovine database for mastitis and milk production. The results of colocalization and TWAS approaches were used for the detection of functional associated candidate genes with milk production and mastitis traits on multiple tissue-based transcriptome records. Also, we used the david database for gene ontology to identify significant terms and associated genes. For the identification of interaction networks, the genemania and string databases were used. Also, the available z-scores in TWAS results were used for the calculation of the correlation between tissues. Therefore, the present results confirm that LYNX1, DGAT1, C14H8orf33, and LY6E were identified as significant genes associated with milk production in eight, six, five, and five tissues, respectively. Also, FBXL6 was detected as a significant gene associated with mastitis trait. CLN3 and ZNF34 genes emerged via both the colocalization and TWAS approaches as significant genes for milk production trait. It is expected that TWAS and colocalization can improve our perception of the potential health status control mechanism in high-yielding dairy cows.

Transcriptomic Changes and Regulatory Networks Associated with Resistance to Mastitis in Xinjiang Brown Cattle.

Xinjiang brown cattle are highly resistant to disease and tolerant of roughage feeding. The identification of genes regulating mastitis resistance in Xinjiang brown cattle is a novel means of genetic improvement. In this study, the blood levels of IL-1ß, IL-6, IL-10, TNF-α, and TGF-ß in Xinjiang brown cattle with high and low somatic cell counts (SCCs) were investigated, showing that cytokine levels were higher in cattle with high SCCs. The peripheral blood transcriptomic profiles of healthy and mastitis-affected cattle were constructed by RNA-seq. Differential expression analysis identified 1632 differentially expressed mRNAs (DE-mRNAs), 1757 differentially expressed lncRNAs (DE-lncRNAs), and 23 differentially expressed circRNAs (DE-circRNAs), which were found to be enriched in key pathways such as PI3K/Akt, focal adhesion, and ECM-receptor interactions. Finally, ceRNA interaction networks were constructed using the differentially expressed genes and ceRNAs. It was found that keynote genes or mRNAs were also enriched in pathways such as PI3K-Akt, cholinergic synapses, cell adhesion molecules, ion binding, cytokine receptor activity, and peptide receptor activity, suggesting that the key genes and ncRNAs in the network may play an important role in the regulation of bovine mastitis.

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.

Genetic parameters for novel mastitis traits defined by combining test-day somatic cell score and differential somatic cell count in the first lactation of Japanese Holsteins.

In this study, we aimed to improve current udder health genetic evaluations by addressing the limitations of monthly sampled somatic cell score (SCS) for distinguishing cows with robust innate immunity from those susceptible to chronic infections. The objectives were to (1) establish novel somatic cell traits by integrating SCS and the differential somatic cell count (DSCC), which represents the combined proportion of polymorphonuclear leukocytes and lymphocytes in somatic cells and (2) estimate genetic parameters for the new traits, including their daily heritability and genetic correlations with milk production traits and SCS, using a random regression test-day model (RRTDM). We derived 3 traits, termed ML_SCS_DSCC, SCS_4_DSCC_65_binary, and ML_SCS_DSCC_binary, by using milk loss (ML) estimates at corresponding SCS and DSCC levels, thresholds established in previous studies, and a threshold established from milk loss estimates, respectively. Data consisted of test-day records collected during January 2021 through March 2022 from 265 herds in Hokkaido, Japan. From these records, we extracted records between 7 to 305 d in milk (DIM) in the first lactation to fit the RRTDM. The model included the random effect of herd-test-day, the fixed effect of year-month, fixed lactation curves nested with calving age groups, and random regressions with Legendre polynomials of order 3 for additive genetic and permanent environmental effects. The analysis was performed using Gibbs sampling with Gibbsf90+ software. The averages (ranges) of the daily heritability estimates over lactation were 0.086 (0.075-0.095) for SCS, 0.104 (0.073-0.127) for ML_SCS_DSCC, 0.137 (0.014-0.297) for SCS_4_DSCC_65_binary, and 0.138 (0.115-0.185) for ML_SCS_DSCC_binary; the heritability curve for SCS_4_DSCC_65_binary was erratic. Genetic correlations within the trait decreased as the DIM interval widened, especially for those integrating DSCC, indicating that these traits should be analyzed using RRTDM rather than repeatability models. The averages (ranges) of genetic correlations with milk yield over lactation were 0.01 (-0.22 to 0.28) for SCS, -0.05 (-0.40 to 0.13) for ML_SCS_DSCC, -0.08 (-0.17 to 0.09) for SCS_4_DSCC_65_binary, and -0.08 (-0.22 to 0.27) for ML_SCS_DSCC_binary. Compared with SCS, the newly defined traits exhibited slightly stronger negative genetic correlations with milk yield. Especially in late lactation stages, the genetic correlation between ML_SCS_DSCC and milk yield was significantly below zero, with a posterior median of -0.40. Furthermore, the new traits showed positive correlations with SCS, having estimates varying from 0.68 to 0.85 for ML_SCS_DSCC, 0.14 to 0.47 for SCS_4_DSCC_65_binary, and 0.61 to 0.66 for ML_SCS_DSCC_binary, depending on DIM. Considering that ML_SCS_DSCC and ML_SCS_DSCC_binary have relatively high heritability (compared with SCS) and favorable genetic correlations with milk production traits and SCS, their incorporation into breeding programs appears promising. Nevertheless, their genetic relationships with (sub)clinical mastitis require further investigation.

Milk exosomal microRNA profiling identified miR-375 and miR-199-5p for regulation of immune response during subclinical mastitis of crossbred cattle.

BACKGROUND: The dairy industry has experienced significant economic losses as a result of mastitis, an inflammatory disease of cows, including both subclinical and clinical cases. Milk exosome microRNAs have gained attention due to their stable and selective wrapping nature, offering potential for the prognosis and diagnosis of bovine mastitis, the most common pathological condition of the mammary gland. METHODS AND RESULTS:  In the present investigation, the microRNA profile of milk exosomes was explored using high-throughput small RNA sequencing data in sub-clinical mastitic and healthy crossbred Vrindavani cattle. In both groups, 349 microRNAs were identified, with 238 (68.19%) microRNAs co-expressed; however, 35 and 76 distinct microRNAs were found in subclinical mastitic and healthy cattle, respectively. Differential expression analysis revealed 11 microRNAs upregulated, and 18 microRNAs were downregulated in sub-clinical mastitic cattle. The functional annotation of the target genes of differentially expressed known and novel microRNAs including bta-miR-375, bta-miR-199-5p and bta-miR-12030 reveals their involvement in the regulation of immune response and inflammatory mechanisms and could be involved in development of mastitis. CONCLUSIONS: The analysis of milk exosomal miRNAs cargos hold great promise as an approach to study the underlying molecular mechanisms associated with mastitis in high milk producing dairy cattle. Concurrently, the significantly downregulated miR-375 may upregulate key target genes, including CTLA4, IHH, IRF1, and IL7R. These genes are negative regulators of immune response pathways, which could be associated with impaired inflammatory mechanisms in mammary cells. According to the findings, bta-miR-375 could be a promising biomarker for the development of mastitis in dairy cattle.

LncRNA CA12-AS1 targets miR-133a to promote LPS-induced inflammatory response in bovine mammary epithelial cells.

Bovine mastitis seriously affects milk production and quality and causes huge economic losses in the dairy industry. Recent studies have shown that long non-coding RNAs (lncRNAs) may regulate bovine mastitis. In this study, the expression of lncRNA CA12-AS1 was significantly upregulated in LPS-induced bovine mammary epithelial cells (bMECs) but negatively correlated with the expression of miR-133a, suggesting that it may be related to the inflammatory response in bMECs. Dual luciferase reporter gene assay revealed that miR-133a is a downstream target gene of lncRNA CA12-AS1. Furthermore, lncRNA CA12-AS1 silencing negatively regulated the expression of miR-133a inhibited the secretion of inflammatory factors (IL-6, IL-8 and IL-1ß) and decreased the mRNA expression levels of nuclear factor kappa B (NF-κB) (p65/p50) and apoptosis-related genes (BAX, caspase3 and caspase9). LncRNA CA12-AS1 silencing also promoted the mRNA expression levels of the Tight junction (TJ) signaling pathway-related genes (Claudin-1, Occludin and ZO-1), apoptotic gene BCL2, proliferation-related genes (CDK2, CDK4 and PCNA) and the viability of bMECs. However, overexpression of lncRNA CA12-AS1 reversed the above effects. These results revealed that lncRNA CA12-AS1 is a pro-inflammatory regulator, and its silencing can alleviate bovine mastitis by targeting miR-133a, providing a novel strategy for molecular therapy of cow mastitis.

Methylome-wide analysis of milk somatic cells upon subclinical mastitis in dairy cattle.

Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae-infected versus healthy; (2) Prototheca-infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae-infected versus Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae-infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca-infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations.

Transcriptome sequencing analysis of bovine mammary epithelial cells induced by lipopolysaccharide.

Mastitis in cows is caused by the inflammation of the mammary glands due to an infection by external pathogenic bacteria. Mammary gland epithelial cells, which are in direct contact with the external environment, are responsible for the first line of defense of the mammary gland against pathogenic bacteria, playing an essential role in immune defense. To investigate the mechanism of bovine mammary epithelial cells in the inflammatory process, we treated the cells with LPS for 12 hours and analyzed the changes in mRNA by transcriptome sequencing. The results showed that compared to the control group, the LPS treatment group had 121 up-regulated genes and 18 down-regulated genes. GO and KEGG enrichment analysis revealed that these differential genes were mainly enriched in the IL-17 signaling pathway, Legionellosis, Cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, and other signaling pathways. Furthermore, the expression of GRO1 and CXCL3 mRNAs increased significantly after LPS treatment. These findings provide new insights for the treatment of mastitis in cows in the future.

dCas9-guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by using Staphylococcus aureus.

Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.

Inferring Causalities of Environmental and Genetic Factors for Differential Somatic Cell Count and Mastitis Pathogens in Dairy Cows Using Structural Equation Modelling.

The aim of this study was to establish and evaluate a structural equation model to infer causal relationships among environmental and genetic factors on udder health. For this purpose, 537 Holstein Friesian cows were genotyped, and milk samples were analyzed for novel traits including differential somatic cell counts and specific mastitis pathogens. In the structural model, four latent variables (intramammary infection (IMI), production, time and genetics) were defined, which were explained using manifest measurable variables. The measurable variables included udder pathogens and somatic differential cell counts, milk composition, as well as significant SNP markers from previous genome-wide associations for major and minor pathogens. The housing system effect (i.e., compost-bedded pack barns versus cubicle barns) indicated a small influence on IMI with a path coefficient of -0.05. However, housing system significantly affected production (0.37), with ongoing causal effects on IMI (0.17). Thus, indirect associations between housing and udder health could be inferred via structural equation modeling. Furthermore, genotype by environment interactions on IMI can be represented, i.e., the detection of specific latent variables such as significant SNP markers only for specific housing systems. For the latent variable genetics, especially one SNP is of primary interest. This SNP is located in the EVA1A gene, which plays a fundamental role in the MAPK1 signaling pathway. Other identified genes (e.g., CTNNA3 and CHL1) support results from previous studies, and this gene also contributes to mechanisms of the MAPK1 signaling pathway.

Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow's milk.

The current study was designed to analyze nutritional parameters and to characterize carbapenemase producing-Klebsiella pneumoniae isolates from bovine mastitic cow's milk. Out of 700 milk samples K. pneumoniae was identified by phenotypic and molecular techniques along with their antibiogram analysis and nutritional analysis was performed using the procedure of Association of Official Analytical Chemists. Carbapenemase-producing K. pneumoniae was detected by phenotypic CarbaNP test followed by molecular characterization of their associated resistant genes blaVIM, blaKPC, blaOXA-48, blaNDM, and blaIMP along with insertion sequence common region 1 (ISCR1) and integrons (Int1, Int2, and Int3) genes. Among nutritional parameters, fat content was observed (2.99%) followed by protein (2.78%), lactose (4.32%), and total solid (11.34%), respectively. The prevalence of K. pneumoniae among bovine mastitis was found 25.71%. Antibiogram analysis revealed that more effective antibiotics was ceftazidime (80%) followed by amikacin (72%), while highly resistant antibiotics was Fusidic acid (100%). Distribution of carbapenemase producer K. pneumoniae was found 44.4%. Among carbapenem resistant genes blaKPC was found 11.25%, blaVIM 2.75%, blaNDM 17.5%, and blaOXA-48 7.5%, while blaIMP gene was not detected. Furthermore, distribution of ISCR1 was found 40%, while integron 1 was found 61.2% followed by integron 2 (20%), and integron 3 (5%). In conclusion, the recent scenario of carbapenemase resistant K. pneumoniae isolates responsible for mastitis may affect not only the current treatment regime but also possess a serious threat to public health due to its food borne transmission and zoonotic potential.

Multiple roles of LncRNA-BMNCR on cell proliferation and apoptosis by targeting miR-145/CBFB axis in BMECs.

Bovine mastitis is one of the most serious and costly disease affecting dairy cattle production. The present study explored the inflammatory response and autoprotective mechanism of a novel specific high expression BMNCR (bovine mastitis related long non-coding RNA) in S. aureus induced mastitis by miR-145/CBFB axis in dairy cows from the perspective of molecular genetics. In bovine mammary epithelial cells, we preformed loss of function experiments to detect changes in cytokine, proliferation and apoptosis by qRT-PCR, western blot, flow cytometry and EdU staining. The results demonstrated that BMNCR significantly increased cell apoptosis, and inhibited cell proliferation. However, the secretion of IL-1α, IL-2, IL-6, IL-8 and IL-12 were enhanced after knock-down BMNCR. Bioinformatics analysis demonstrated that BMNCR could target 8 miRNAs, in-depth analyses indicated that BMNCR acts as a molecular sponge for bta-miR-145 and CBFB was one of 23 target gene of bta-miR-145 . The results of the present study demonstrated that the role of BMNCR in S. aureus induced mastitis can be mediated by sponge bta-miR-145 activating CBFB expression. BMNCR could be a potential target for mastitis diagnosis and therapy, which may enrich the theoretical research of therapeutic intervention, and further increase milk yield and improve milk quality.

Integrated co-expression analysis of regulatory elements (miRNA, lncRNA, and TFs) in bovine monocytes induced by Str. uberis.

Non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), together with transcription factors, are critical pre-, co-, and post-transcriptional regulators. In addition to their criteria as ideal biomarkers, they have great potential in disease prognosis, diagnosis, and treatment of complex diseases. Investigation of regulatory mechanisms in the context of bovine mastitis, as most common and economic disease in the dairy industry, to identify elements influencing the expression of candidate genes as key regulators of the mammary immune response is not yet fully understood. Transcriptome profiles (50 RNA-Seq and 50 miRNA-Seq samples) of bovine monocytes induced by Str. uberis were used for co-expression module detection and preservation analysis using the weighted gene co-expression network analysis (WGCNA) approach. Assigned mi-, lnc-, and m-modules used to construct the integrated regulatory networks and miRNA-lncRNA-mRNA regulatory sub-networks. Remarkably, we have identified 18 miRNAs, five lncRNAs, and seven TFs as key regulators of str. uberis-induced mastitis. Most of the genes introduced here, mainly involved in immune response, inflammation, and apoptosis, were new to mastitis. These findings may help to further elucidate the underlying mechanisms of bovine mastitis, and the discovered genes may serve as signatures for early diagnosis and treatment of the disease.

Integrated analysis of inflammatory mRNAs, miRNAs, and lncRNAs elucidates the molecular interactome behind bovine mastitis.

Mastitis is known as intramammary inflammation, which has a multifactorial complex phenotype. However, the underlying molecular pathogenesis of mastitis remains poorly understood. In this study, we utilized a combination of RNA-seq and miRNA-seq techniques, along with computational systems biology approaches, to gain a deeper understanding of the molecular interactome involved in mastitis. We retrieved and processed one hundred transcriptomic libraries, consisting of 50 RNA-seq and 50 matched miRNA-seq data, obtained from milk-isolated monocytes of Holstein-Friesian cows, both infected with Streptococcus uberis and non-infected controls. Using the weighted gene co-expression network analysis (WGCNA) approach, we constructed co-expressed RNA-seq-based and miRNA-seq-based modules separately. Module-trait relationship analysis was then performed on the RNA-seq-based modules to identify highly-correlated modules associated with clinical traits of mastitis. Functional enrichment analysis was conducted to understand the functional behavior of these modules. Additionally, we assigned the RNA-seq-based modules to the miRNA-seq-based modules and constructed an integrated regulatory network based on the modules of interest. To enhance the reliability of our findings, we conducted further analyses, including hub RNA detection, protein-protein interaction (PPI) network construction, screening of hub-hub RNAs, and target prediction analysis on the detected modules. We identified a total of 17 RNA-seq-based modules and 3 miRNA-seq-based modules. Among the significant highly-correlated RNA-seq-based modules, six modules showed strong associations with clinical characteristics of mastitis. Functional enrichment analysis revealed that the turquoise module was directly related to inflammation persistence and mastitis development. Furthermore, module assignment analysis demonstrated that the blue miRNA-seq-based module post-transcriptionally regulates the turquoise RNA-seq-based module. We also identified a set of different RNAs, including hub-hub genes, hub-hub TFs (transcription factors), hub-hub lncRNAs (long non-coding RNAs), and hub miRNAs within the modules of interest, indicating their central role in the molecular interactome underlying the pathogenic mechanisms of S. uberis infection. This study provides a comprehensive insight into the molecular crosstalk between immunoregulatory mRNAs, miRNAs, and lncRNAs during S. uberis infection. These findings offer valuable directions for the development of molecular diagnosis and biological therapies for mastitis.

Molecular regulatory mechanism of key LncRNAs in subclinical mastitic cows with folic acid supplementation.

BACKGROUND: Folic acid is a water-soluble B vitamin (B9), which is closely related to the body's immune and other metabolic pathways. The folic acid synthesized by rumen microbes has been unable to meet the needs of high-yielding dairy cows. The incidence rate of subclinical mastitis in dairy herds worldwide ranged between 25%~65% with no obvious symptoms, but it significantly causes a decrease in lactation and milk quality. Therefore, this study aims at exploring the effects of folic acid supplementation on the expression profile of lncRNAs, exploring the molecular mechanism by which lncRNAs regulate immunity in subclinical mastitic dairy cows. RESULTS: The analysis identified a total of 4384 lncRNA transcripts. Subsequently, differentially expressed lncRNAs in the comparison of two groups (SF vs. SC, HF vs. HC) were identified to be 84 and 55 respectively. Furthermore, the weighted gene co-expression network analysis (WGCNA) and the KEGG enrichment analysis result showed that folic acid supplementation affects inflammation and immune response-related pathways. The two groups have few pathways in common. One important lncRNA MSTRG.11108.1 and its target genes (ICAM1, CCL3, CCL4, etc.) were involved in immune-related pathways. Finally, through integrated analysis of lncRNAs with GWAS data and animal QTL database, we found that differential lncRNA and its target genes could be significantly enriched in SNPs and QTLs related to somatic cell count (SCC) and mastitis, such as MSTRG.11108.1 and its target gene ICAM1, CXCL3, GRO1. CONCLUSIONS: For subclinical mastitic cows, folic acid supplementation can significantly affect the expression of immune-related pathway genes such as ICAM1 by regulating lncRNAs MSTRG.11108.1, thereby affecting related immune phenotypes. Our findings laid a ground foundation for theoretical and practical application for feeding folic acid supplementation in subclinical mastitic cows.

Heritability and genetic correlations of total and differential somatic cell count with milk yield and composition traits in Italian Simmental cows.

Costs of production have deeply increased each year in the last decades, breeders are continuously looking for more cost effective and more efficient ways to produce milk. Despite the major signs of progress in productivity, it is fundamental to optimize rather than maximize the performances of the dairy cows. Mastitis is still a highly prevalent disease in the dairy sector which causes several economic losses and environmental effect. Its accurate and early diagnosis is crucial to improve profitability of dairy cows and contribute to a more sustainable dairy industry. Among mastitis reduction strategies, there is the urgent need to implement breeding objectives to select cows displaying mastitis resistance by investigating the genetic mechanisms at the base of the inflammatory response. Therefore, in this study we aimed to further understand the genetic background of the differential somatic cell count (DSCC), which provides thorough insights on the actual inflammatory status of the mammary glands. The objectives of this study were to estimate on a cohort of 20,215 Italian Simmental cows over a 3-yr period: (1) the heritability and repeatability values of somatic cell score (SCS) and DSCC, (2) the genetic and phenotypic correlations between these 2 traits and milk production and milk composition traits, (3) the heritability and repeatability values of SCS and DSCC within class of udder health status. Heritability was low both for SCS (0.06) and DSCC (0.08), whereas the repeatability values for these traits were 0.43 and 0.36, suggesting that the magnitude of cow permanent environmental effect for these traits is remarkable. The genetic and phenotypic correlation of SCS with DSCC was 0.612 and 0.605, respectively. Because both significantly differed from the unit, we must consider those traits as different ones. This latter aspect corroborates the need to consider the DSCC as a further indicator of inflammatory status which might be implemented in the Simmental breed genetic evaluation. It is worthy to mention that heritability estimates for SCS and DSCC were the highest in healthy cows compared with the other udder health classes. This implies that when the udder health status changes, it is most likely due to environmental factors rather than aspects related to the animal's genetics. In contrast, the highest additive genetic variance and heritability found for SCS and DSCC in the healthy group might reveal the potential to further implement breeding strategies to select for healthier animals.

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.