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 mecha‑nisms 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 nega‑tively 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 signifi‑cant 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 involve‑ment 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.

Transcriptome‑wide mapping of milk somatic cells upon subclinical mastitis infection in dairy cattle

Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

Month-Wise Prevalence of Subclinical Mastitis in Dairy Cows in Guangdong Province,China

To evaluate the month-wise prevalence of subclinical mastitis （SM） and its relations with climatic temperature and humidity, and to provide references for control and prevention of mastitis in dairy cows in Guangdong, China, California mastitis test method was used to determine the monthly prevalence of SM （MPSM, %） of lactating Holstein cows from 11 dairy farms in Guangdong Province, China, including about 516 cows per monthly examination. The average MPSM on a cow and a quarter basis were 30 and 13% respectively, and there was a positive linear correlation between monthly mean air temperature （MT, °C） and MPSM on a cow basis （r=0.763, P=0.004） or a quarter basis （r=0.577, P=0.049）, but there were no correlations between MT and MPSM on a cow or a quarter basis （P〉0.05）. There was a shift in MPSM trend that the highest MPSM （38.4%） was not for the hottest July with MT at 30.8°C, but for the later September at 27.9°C. The farms need to develop new housing conditions to control cowshed temperatures in the hot season to reduce the mastitis prevalences in the post-hot months.

Changes of Cytokines in Serum and Whey of Dairy Cows with Subclinical Mastitis

In order to study the correlation of different degrees of subclinical mastitis and cytokines,the changes of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8)and tumor necrosis factor-α(TNF-α)in blood and milk and whether the changes of these indexes were caused by mastitis were analyzed.The 40 Holstein cows in lactation period were tested and grouped by somatic cell count(SCC)method in milk:Group C(SCC<5×10~5 cells·mL^(-1)),Group I(5×10~5 cells·mL^(-1)5×10~6 cells·mL^(-1)).The contents of IL-1β,IL-6,IL-8 and TNF-αwere analyzed by radioimmunoassay.The results showed that the contents of IL-6,IL-8 and TNF-αin the blood and milk of different degrees of subclinical mastitis increased with the severity of mastitis,and were higher than those in normal group.The contents of IL-6,IL-8 and TNF-αin serum and whey of dairy cows affected by different degrees of subclinical mastitis were higher than those in normal group(Group C).The content of IL-1βincreased and decreased with the increase of inflammation,and it did not have the value of evaluating mastitis.In conclusion,the severities of cow subclinical mastitis and the contents of cytokines were positive correlations.These indexes could be used as the basis for judging mammary gland injury.

Effects of Xiaoruling on Subclinical Mastitis in Dairy Cows

[Objective] TO investigate the effects of traditional Chinese medicine （TCM） preparation ＂Xiaoruling＂ on the subclinical mastitis in dairy cows. [Method] A total of 28 dairy cows suffering from subclinical mastitis were randomly divided into experimental group （n =20） and control group （ n =8）. The dairy cows in the experimental group were treated with Xiaoruling for 2 weeks. [ Rsults] The cure rate of subclinical mastitis in dairy cows was up to 89.47% and milk yield increased greatly （ P 〈 0.05）. Meanwhile, the milk somatic cell count （SCC） was greatly decreased （ P 〈 0.01 ）. SOD and GSH-Px activities in plasma were increased greatly （ P 〈 0.01 ）. [ Conclusion] Xiaoruling has an effective treatment on subclinical mastitis in dairy cows.

Multiple PCR for Detection of Subclinical Mastitis in Dairy Cow

[ Objective] To develop a rapid, sensitive, specific and accurate diagnostic method for subclinical mastitis in dairy cow. [ Method] A total of 70 samples of suspected clinical mastitis cows were detected in gene level by multiple PCR. [Result] The 58 samples were positive with the developed multiple PCR method and the positive rate was 82.5% （58/70）. The 53 samples were positive with the traditional biochemical method and the positive rate was 75.5% （53/70）. As a result, the coincidence rate of both methods was 92%. [ Conclusion] The multiple PCR method is rapid and specific for the detection of pathogenic subclinical mastitis in dairy cow.

Isolation and Identification of Pathogenic Bacteria Causing Bovine Subclinical Mastitis in Eastern Hebei Province

In this study, the milk samples of 1 021 cows in eight dairy farms in Eastern Hebei Province were collected and detected with LMT reagent and somatic cell count for subclinical mastitis. Pathogenic bacteria in subclinical mastitis positive milk samples were isolated and identified.The results showed that 60.63%（619/1 021） of the sampled cows were diagnosed with subclinical mastitis, and mixed infections accounted for 88.21%（546/619） of the cases. In addition, 82 strains of 14 species were isolated from the subclinical mastitis positive milk samples, including 36 strains of Staphylococcus（43.90%）, 33 strains of Streptococcus（40.24%）, 8 strains of Enterobacteriaceae（9.76%） and 5 strains of Corynebacterium（6.10%）, respectively. The results proved that Staphylococcus aureus and Streptococcus agalactiae are the main pathogenic bacteria causing bovine subclinical mastitis in Eastern Hebei Province.