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 regu...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.展开更多
The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution ...The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance-and virulence-related gene patterns.Antimicrobial susceptibility was determined by the E-test.Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR.A total of 27 strains were obtained from 326 mastitis milk samples.Streptococcus parauberis isolates(n=11)showed high resistance to erythromycin(90.9%),followed by tetracycline(45.5%),chloramphenicol(36.4%)and clindamycin(27.3%).Streptococcus uberis isolates(n=16)were highly resistant to tetracycline(81.3%)and clindamycin(62.5%).Both species were susceptible to ampicillin.The most prevalent resistance gene in S.uberis was tetM(80.0%),followed by blaZ(62.5%)and ermB(62.5%).However,tetM,blaZ,and ermB genes were only found in 27.3,45.5,and 27.3%,respectively,of S.parauberis.In addition,all of the isolates carried at least one selected virulence-related gene.The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2%of the strains.One S.uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern.More than 59.3%of analysed strains carried 4 to 7 virulence-related genes.Our findings demonstrated that S.parauberis and S.uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology,and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry.The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens,and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.展开更多
BACKGROUND: Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for...BACKGROUND: Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 isolated from bovine milk. METHODS: The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of 13-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS -PAGE, further confirmed by fibrin zymography. RESULTS: Out of the 3 isolated strains, only one isolate expressed 13-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological, biochemical and molecular characterization, it was identified as Streptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement of Streptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively. CONCLUSION: Thus this study presents its novelty by highlighting the potential of Streptococcus uberis TNA-M1 as a significant source for the production of fibrinolytic enzymes.展开更多
Mastitis is a costly disease which hampers the dairy industry. Inflammation of the mammary gland is commonly caused by bacterial infection, mainly Escherichia coli, Streptococcus uberis and Staphylococcus aureus. As m...Mastitis is a costly disease which hampers the dairy industry. Inflammation of the mammary gland is commonly caused by bacterial infection, mainly Escherichia coli, Streptococcus uberis and Staphylococcus aureus. As more bacteria become multi-drug resistant, one potential approach to reduce the disease incidence rate is to breed selectively for the most appropriate and potentially protective innate immune response. The genetic contribution to effective disease resistance is, however, difficult to identify due to the complex interactions that occur. In the present study two published datasets were searched for common differentially expressed genes (DEGs) with similar changes in expression in mammary tissue following intra-mammary challenge with either E. coli or S. uberis. Additionally, the results of seven published genome-wide association studies (GWAS) on different dairy cow populations were used to compile a list of SNPs associated with somatic cell count. All genes located within 2 Mbp of significant SNPs were retrieved from the Ensembl database, based on the UMD3.1 assembly. A final list of 48 candidate genes with a role in the innate immune response identified from both the DEG and GWAS studies was further analyzed using Ingenuity Pathway Analysis. The main signalling pathways highlighted in the response of the bovine mammary gland to both bacterial infections were 1) granulocyte adhesion and diapedesis, 2) ephrin receptor signalling, 3) RhoA signalling and 4) LPS/IL1 mediated inhibition of RXR function. These pathways comprised a network regulating the activity of leukocytes, especially neutrophils, during mammary gland inflammation. The timely and properly controlled movement of leukocytes to infection loci seems particularly important in achieving a good balance between pathogen elimination and excessive tissue damage. These results suggest that polymorphisms in key genes in these pathways such as SELP, SELL, BCAR1, ACTR3, CXCL2, CXCL6, CXCL8 and FABP may influence the ability of dairy cows to resist mastitis.展开更多
文摘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.
基金This study was supported by the National Key Research and Development Project of China(2017YFD0502200)the National Natural Science Foundation of China(31802232)the International Science and Technology Cooperation Project of Gansu Province,China(17YF1WA169).
文摘The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance-and virulence-related gene patterns.Antimicrobial susceptibility was determined by the E-test.Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR.A total of 27 strains were obtained from 326 mastitis milk samples.Streptococcus parauberis isolates(n=11)showed high resistance to erythromycin(90.9%),followed by tetracycline(45.5%),chloramphenicol(36.4%)and clindamycin(27.3%).Streptococcus uberis isolates(n=16)were highly resistant to tetracycline(81.3%)and clindamycin(62.5%).Both species were susceptible to ampicillin.The most prevalent resistance gene in S.uberis was tetM(80.0%),followed by blaZ(62.5%)and ermB(62.5%).However,tetM,blaZ,and ermB genes were only found in 27.3,45.5,and 27.3%,respectively,of S.parauberis.In addition,all of the isolates carried at least one selected virulence-related gene.The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2%of the strains.One S.uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern.More than 59.3%of analysed strains carried 4 to 7 virulence-related genes.Our findings demonstrated that S.parauberis and S.uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology,and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry.The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens,and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.
文摘BACKGROUND: Screening of isolates for their potency to produce streptokinase was an important criterion of this research. The current study emphasizes the strain improvement, optimization and purification studies for enhanced production of streptokinase from Streptococcus uberis TNA-M1 isolated from bovine milk. METHODS: The study was carried out on samples collected from milk sample. Primary screening and characterization is used as an excellent source for the isolation of 13-hemolytic organisms. Strain improvement was done by both physical & chemical mutagenesis. The enzyme activity was checked by clot lysis assay and confirmed by fibrin plate method. The partially purified and crude enzyme were analysed by high-performance liquid chromatography. Molecular weight & enzyme purity was checked by SDS -PAGE, further confirmed by fibrin zymography. RESULTS: Out of the 3 isolated strains, only one isolate expressed 13-haemolysis with streptokinase (SK) activity. Based on the results of radial caseinolytic assay and blood clot dissolving assay, isolate TNA-M1 demonstrated the highest streptokinase activity. Based on morphological, biochemical and molecular characterization, it was identified as Streptococcus uberis and the strain was named as Streptococcus uberis TNA-M1. The results indicated that ultra-violet (UV) and ethyl methane sulfonate (EMS) were effective mutagenic agents for strain improvement of Streptococcus uberis TNA-M1 and enhanced SK productivity. HPLC analysis was performed in order to confirm the presence of streptokinase with the similar retention time (0.875 min) with its standard (0.854) min. SDS-PAGE of the enzyme showed protein band of approximately 47 kDa and confirmed by fibrin zymography. It exhibited fibrinolytic activity, which was more potent than other fibrinolytic enzymes. Glucose and peptone were recorded to be the optimum carbon and nitrogen sources respectively. CONCLUSION: Thus this study presents its novelty by highlighting the potential of Streptococcus uberis TNA-M1 as a significant source for the production of fibrinolytic enzymes.
文摘Mastitis is a costly disease which hampers the dairy industry. Inflammation of the mammary gland is commonly caused by bacterial infection, mainly Escherichia coli, Streptococcus uberis and Staphylococcus aureus. As more bacteria become multi-drug resistant, one potential approach to reduce the disease incidence rate is to breed selectively for the most appropriate and potentially protective innate immune response. The genetic contribution to effective disease resistance is, however, difficult to identify due to the complex interactions that occur. In the present study two published datasets were searched for common differentially expressed genes (DEGs) with similar changes in expression in mammary tissue following intra-mammary challenge with either E. coli or S. uberis. Additionally, the results of seven published genome-wide association studies (GWAS) on different dairy cow populations were used to compile a list of SNPs associated with somatic cell count. All genes located within 2 Mbp of significant SNPs were retrieved from the Ensembl database, based on the UMD3.1 assembly. A final list of 48 candidate genes with a role in the innate immune response identified from both the DEG and GWAS studies was further analyzed using Ingenuity Pathway Analysis. The main signalling pathways highlighted in the response of the bovine mammary gland to both bacterial infections were 1) granulocyte adhesion and diapedesis, 2) ephrin receptor signalling, 3) RhoA signalling and 4) LPS/IL1 mediated inhibition of RXR function. These pathways comprised a network regulating the activity of leukocytes, especially neutrophils, during mammary gland inflammation. The timely and properly controlled movement of leukocytes to infection loci seems particularly important in achieving a good balance between pathogen elimination and excessive tissue damage. These results suggest that polymorphisms in key genes in these pathways such as SELP, SELL, BCAR1, ACTR3, CXCL2, CXCL6, CXCL8 and FABP may influence the ability of dairy cows to resist mastitis.