Project description:Mastitis is a multietiological complex disease, which is defined as inflammation of parenchyma of mammary glands. Bacterial infection is the predominant cause of mastitis, though fungal, viral and mycoplasma infections also have been reported. Based on severity of the disease, mastitis can be classified into- subclinical, clinical and chronic forms. Bacterial pathogens from the fresh cow milk were isolated and classified by standard microbiological tests and multiplex PCR. Epidemiological studies have shown that Escherichia coli is the 2nd largest mastitic pathogen after Staphylococcus aureus in India. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC) -PCR profile and presence of virulence genes, a field isolate of E. coli was used for intramammary inoculation in lactating mice. Histopathological examination of H&E stained sections showed severe infiltration of polymorphonuclear neutrophils (PMNs), mononuclear inflammatory cells in the alveolar lumen and also in interstitial space and necrosis of alveolar epithelial cells after 24h. Western blot and immunohistochemical analysis of the mice mammary tissues have shown significant hyperacetylation at histone H3Lys14 residue of both mammary epithelial cells and migrated inflammatory cells. Quantitative real-time PCR and genome-wide gene expression profile in the E. coli infected mice mammary tissue revealed differential expression of genes related to inflammation, immunity, antimicrobial peptide expression, acute phase response and oxidative stress response. Expression levels of milk proteins were also suppressed. ChIP assay from the paraffinized tissues have shown selective enrichment of acetylated histone H3K14 and H4K8 at the promoters of over expressed genes. These data suggests that E. coli infection in the mice mammary tissue leads to histone hyperacetylation at the immune gene promoters which is pre-requisite for expression of inflammatory genes to mount drastic immune response. Abdominal mammary glands of 7 Day post partum lactating female Swiss albino mice was challenged with a pure culture of E. coli isolated from cow milk. Mammary tissues were harvested after 24h of infection. Total RNA was isolated by Qiagen RNeasy mini columns and used for global gene expression and RT-PCR analysis. For RT-PCR, cDNAs corresponding to total mRNAs were synthesized using Oligo-dT primer. Global gene expression analysis were done by single colour labelling of total RNA and hybridization to Agilent 8x60k mouse Microarray.

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: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.

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Alteration of gene expression in mouse mammary tissue upon E. coli infection

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