Project description:This SuperSeries is composed of the following subset Series:; GSE15019: Transcriptome analysis of bovine mammary gland tissue treated with E. coli for 6 hours; GSE15020: Transcriptome analysis of bovine mammary gland tissue treated with E. coli for 24 hours; GSE15022: Transcriptome analysis of bovine mammary gland tissue of an udder quarter adjacent to an E.coli treated one for 24 hrs Experiment Overall Design: Refer to individual Series
Project description:Investigation of the bovine mammary gland response to Escherichia coli and Staphylococcus aureus infection. The aims of the study were to examine the dynamics of the mammary gland response to pathogen invasion and identify general and pathogen-specific transcriptional profiles. Individual udder quarters of 12 cows were sequentially inoculated with either E. coli or S. aureus over 24 or 72 hours before culling, whilst one quarter was used as an uninfected control.
Project description:We performed a global gene-expression analysis of mammary gland and liver tissue collected from dairy cows that had been exposed to a controlled E. coli infection. At time = 0, each of the periparturient dairy cows received 20-40 colony-forming units of live E. coli in one front quarter of the udder. Biopsy samples of healthy and infected udder tissue were collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent the acute phase response (APR). A time series of liver biopsies was collected at -144, 12, 24, and 192 h relative to time of inoculation. Hf=right forward teat Vf=left forward teat
Project description:We hypothesized that accumulation of milk would influence gene expression in the mammary gland of lactating dairy cows. To test this hypothesis, we enrolled 4 multiparous Holstein cows (150 ± 10 DIM) in a half-udder milk stasis experiment. On day 1 of the experiment, cows were milked at 0430 h and at 1430 h, only right udder halves were milked. Mammary biopsies were obtained from both udder halves on day 2 of the experiment at 0500 h, immediately after right udder halves were milked and 12 h since left udder halves had last been milked. Using Affymetrix GeneChip® Bovine Genome Arrays, we identified 32 genes that were differentially expressed between left (full) and right (empty) udder halves (fold change > 1.5; P < 0.05). Four of the genes were downregulated in response to milk stasis, whereas 28 were upregulated. Differentially expressed genes were associated with extracellular matrix remodeling, tight junction formation, regulation of blood flow, and apoptosis. In addition, four of the differentially expressed genes had been previously identified as candidates for local regulation of milk production in dairy cows. Expression of two of these candidates, early growth response-1 (EGR-1) and thrombospondin-1 (THBS-1), was validated using real-time quantitative RT-PCR. Consistent with microarray results, both genes were upregulated in response to 24-h of milk stasis (P < 0.03). Immunofluorescence was used to localize expression of EGR-1 protein, which was restricted to epithelia and was uniformly distributed. We conclude that accumulation of milk alters gene expression in the bovine mammary gland. In particular, EGR-1 and THBS-1 have emerged as strong candidates for local regulation of milk production in dairy cows.
Project description:We hypothesized that accumulation of milk would influence gene expression in the mammary gland of lactating dairy cows. To test this hypothesis, we enrolled 4 multiparous Holstein cows (150 M-BM-1 10 DIM) in a half-udder milk stasis experiment. On day 1 of the experiment, cows were milked at 0430 h and at 1430 h, only right udder halves were milked. Mammary biopsies were obtained from both udder halves on day 2 of the experiment at 0500 h, immediately after right udder halves were milked and 12 h since left udder halves had last been milked. Using Affymetrix GeneChipM-BM-. Bovine Genome Arrays, we identified 32 genes that were differentially expressed between left (full) and right (empty) udder halves (fold change > 1.5; P < 0.05). Four of the genes were downregulated in response to milk stasis, whereas 28 were upregulated. Differentially expressed genes were associated with extracellular matrix remodeling, tight junction formation, regulation of blood flow, and apoptosis. In addition, four of the differentially expressed genes had been previously identified as candidates for local regulation of milk production in dairy cows. Expression of two of these candidates, early growth response-1 (EGR-1) and thrombospondin-1 (THBS-1), was validated using real-time quantitative RT-PCR. Consistent with microarray results, both genes were upregulated in response to 24-h of milk stasis (P < 0.03). Immunofluorescence was used to localize expression of EGR-1 protein, which was restricted to epithelia and was uniformly distributed. We conclude that accumulation of milk alters gene expression in the bovine mammary gland. In particular, EGR-1 and THBS-1 have emerged as strong candidates for local regulation of milk production in dairy cows. 8 samples from 4 cows
Project description:The benefit of treatment in mild to moderate cases of E. coli mastitis in dairy cows remains a topic of discussion. We investigated the effect of intramammary treatment with Cefapirin + Prednisolone compared to Cefapirin only on gene expression profiles in experimentally induced E. coli mastitis. Five midlactating Holstein Friesian cows were challenged with 100 CFU in 3 quarters and treated with Cefapirin only in one quarter and the combination of Cefaprirn and Prednisolne in another quarter at 4, 12, 24 and 36h post challenge. After 24h (n=2) or 48h (n=3) post challenge cows were sacrificed and mammary gland tissue was collected from each quarter. From each cow total RNA of one non challenged quarter, one challenged not treated quarter, on Cefapirin treated and one Cefapirin + Prednisolone treated quarter was subjected to microarray analysis. Data were analyzed separately for the two sample time points
Project description:Mastitis remains one of the most prevalent and costly diseases impacting the dairy industry worldwide. Escherichia coli is an environmental bacterium that frequently causes intramammary infections, the outcome of which depends on the capacity of the host to recognize and clear the bacterial pathogen. E. coli intramammary infection elicits localized and systemic responses, some of which have been characterized in mammary secretory tissue. However, nothing is known about early transcriptome-wide responses to infection that occur within regions of the teat and mammary parenchyma. Therefore, the objective of the current study was to use microarray analysis to characterize gene expression patterns and process networks that become activated in different regions of the mammary gland during the acute phase of experimentally induced intramammary infection with E. coli. Tissues evaluated were from Furstenberg’s rosette, teat cistern, gland cistern, and lobulo-alveolar regions of control and infected mammary glands, 12 and 24 h after bacterial (or control) infusions. For selected genes, quantitative RT-PCR was performed to confirm the microarray findings (Toll-like receptors; TLR1, TLR2, TLR4, TLR6) and evaluate the correspondence between mRNA level in tissues and protein level in milk (interleukin 8, tumor necrosis factor-alpha, haptoglobin, lipopolysaccharide-binding protein). The main networks activated by E. coli infection pertained to immune and inflammatory response, with marked induction of genes encoding proteins that function in chemotaxis, leukocyte activation and signaling. Genomic response was greatest in tissues of the teat and gland cisterns at 12 h post-infection, while tissue of the lobulo-alveolar region responded only, and most strongly of the regions, 24 h following the infection. Up regulation of TLR2, TLR4, IL8, TNF-alpha and bactericidal/permeability-increasing protein peaked at 12 h post infection in tissues of the teat and in the gland cistern, while the highest level of lipopolysaccharide-binding protein was reached at 24 h in the lobular alveoli region of infected quarters. Very few genes were down–regulated within the timeframe of this study. Similar genetic networks were impacted in all regions during early phases of E. coli intramammary infection, although regional differences throughout the gland were noted. Importantly, the tissues of the teat, which are first to encounter invading bacteria during a natural infection, responded rapidly and intensely, suggesting an important sentinel function for this region. Both resident mammary cells and infiltrating immune cells likely play an important role in recognition of pathogens and production of inflammatory mediators during mastitis. Three healthy lactating Holstein cows were used in this study. All mammary glands were bacteria-free. Immediately following the morning milking, 4 mL of E. coli suspension (100 colony forming units/mL) were infused into one mammary gland of the udder and the contralateral gland was infused with PBS (control gland). After the PM milking 12 h later, the remaining ipsilateral quarter was infused with E. coli. Cows were euthanized 24 h after initial infusions and tissues were collected from 4 regions of each mammary gland: Furstenburg’s rosette, teat cistern, gland cistern and lobuloalveolar regions. Tissues were snap frozen in liquid nitrogen, stored at -80C until processing.
Project description:Expression and differential expression analysis of milk samples from healthy and diseased diary cows. Diseases were grouped by their occurrence in the mammary gland or extra-mammary. Furthermore, the diseases were classified by their severity. All cows were examined thoroughly by the dairy herd manager, trained staff, or a veterinarian. Expression and differential expression was assessed by using the Affymetrix Bovine Genome Array (GPL2112). Control animals (2-4 years old, 1st to 3th lactation, one animal 4th and one 8th lactation) showed no clinical signs of disease and had no abnormalities in the udder or milk. Their somatic cell count (SCC) was less than 100,000 cells/ml milk. Most of the control samples were taken during early lactation (10-100 days post-partum, dpp). Diseased cows were in their 1st to 8th lactation within 10-220 dpp.
Project description:In this study we sought to establish changes within the mammary glands of lactating dairy cows (n=3-4) in response to a single dose of exogenous DEX by performing serial biopsies of alternating quarters of the udder Transcriptomic changes in the mammary glands in response to DEX were identified by RNA sequencing of mammary gland RNA at 0, 12, 24 and 72h post-DEX injection, followed by differential gene expression analysis Coincident with the milk yield and composition changes was the differential expression of 519 and 320 genes at 12 and 24 h after DEX, respectively, with return of all gene expression to baseline levels by 72 h.
Project description:Establishment of an in vitro system to explore molecular mechanisms of mastitis susceptibility in cattle by comparative expression profiling of Escherichia coli and Staphylococcus aureus inoculated primary cells sampled from cows with different genetic predisposition for somatic cell score Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows that were selected for high and low mastitis susceptibility by applying a marker assisted selection strategy considering QTL and molecular marker information of a repetitively confirmed QTL for SCS in the telomeric region on BTA18 The cells were cultivated and subsequently inoculated with heat inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 hours the cells were harvested and comparatively analyzed using microarray expression chip technology to identify differences in mRNA expression profiles attributed to cultivation, inoculation or to genetic predisposition. Six heifers inheriting the favorable paternal QTL allele and five heifers inheriting the unfavorable QTL allele were selected by applying a marker assisted selection strategy. All heifers were kept under the same environmental conditions, had no clinical mastitis and did not show any indication of bacterial infection at slaughter. Primary bovine mammary gland epithelial cell cultures were established from cells sampled from the udder parenchyma of each cow. The cells were challenged with heat inactivated Escherichia coli and Staphylococcus aureus or with PBS for control. After 1, 6 and 24 hours the cells were harvested and mRNA expression was comparatively analyzed between time points for each treatment and each paternally inherited SCS-BTA18-QTL allele, respectively. In addition, the differences in gene expression at time points 1, 6 and 24h between inoculated and respective uninoculated control cells were investigated using the short time series expression miner STEM for co-expression profiling and GO cattegory enrichment analyses.