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.

Project description:Staphylococcus aureus is one of the most important contagious bacteria causing subclinical bovine mastitis. This bacterial infection is commonly identified by determine the pathogen in bovine milk samples through conventional technique including coagulase test. However, this test has several disadvantages as low sensitivity, risk of biohazard, cost expensive, and limited preparation especially in local area.Aim of this study was to compare and assess the screening method, Mannitol fermentation test (Mannitol salt agar [MSA]), and deoxyribonuclease (DNase) test, for S. aureus identification in milk samples.A total of 224 subclinical bovine mastitis milk samples were collected from four provinces of Thailand and determined S. aureus using conventional method and also subjected to the screening test, MSA and DNase test. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) among both tests were analyzed and compared to the tube coagulase test (TCT), as reference method. Immunological test by latex agglutination and molecular assay by determined spa gene were also used to identify and differentiate S. aureus.A total of 130 staphylococci were isolated by selective media, Gram-stain, and catalase test. The number of S. aureus which identified using TCT, MSA and DNase test were 32, 102, and 74 isolates, respectively. All TCT results were correlated to results of latex agglutination and spa gene which were 32 S. aureus. MSA showed 100% sensitivity, 28.57% specificity, 31.37% PPV, and 100% NPV, whereas DNase showed 53.13% sensitivity, 41.84% specificity, 22.97% PPV, and 73.21% NPV. DNase test showed higher specificity value than MSA but the test presented 26.79% false negative results whereas no false-negative result from MSA when comparing to TCT.MSA had a tendency to be a good preference for screening S. aureus because of its high sensitivity and NPV. The result from this study will improve a choice to use a screening test to diagnose S. aureus of veterinary field for prompt disease controlling and effective treatment.

Dataset Information

2,4-Thiazolidinedione Treatment Improves the Innate Immune Response in Dairy Goats with Induced Subclinical Mastitis.

Publications

2,4-Thiazolidinedione Treatment Improves the Innate Immune Response in Dairy Goats with Induced Subclinical Mastitis.

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