Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis

Local host response following an intramammary challenge with Staphylococcus fleurettii and different strains of Staphylococcus chromogenes in dairy heifers

Coagulase-negative staphylococci (CNS) are a common cause of subclinical mastitis in dairy cattle. The CNS inhabit various ecological habitats, ranging between the environment and the host. In order to obtain a better insight into the host response, an experimental infection was carried out in eight healthy heifers in mid-lactation with three different CNS strains: a Staphylococcus fleurettii strain originating from sawdust bedding, an intramammary Staphylococcus chromogenes strain originating from a persistent intramammary infection (S. chromogenes IM) and a S. chromogenes strain isolated from a heifer's teat apex (S. chromogenes TA). Each heifer was inoculated in the mammary gland with 1.0 x 10(6) colony forming units of each bacterial strain (one strain per udder quarter), whereas the remaining quarter was infused with phosphate-buffered saline. Overall, the CNS evoked a mild local host response. The somatic cell count increased in all S. fleurettii-inoculated quarters, although the strain was eliminated within 12 h. The two S. chromogenes strains were shed in larger numbers for a longer period. Bacterial and somatic cell counts, as well as neutrophil responses, were higher after inoculation with S. chromogenes IM than with S. chromogenes TA. In conclusion, these results suggest that S. chromogenes might be better adapted to the mammary gland than S. fleurettii. Furthermore, not all S. chromogenes strains induce the same local host response

Histone Deacetylase Inhibitors Prevent Pulmonary Endothelial Hyperpermeability and Acute Lung Injury By Regulating Heat Shock Protein 90 Function

Transendothelial hyperpermeability caused by numerous agonists is dependent on heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores transendothelial permeability. Hyperacetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating lipopolysaccharide (LPS)-induced transendothelial hyperpermeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced transendothelial hyperpermeability, acetylated and suppressed Hsp90 chaperone function, and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor RGFP-966 or the HDAC6-selective inhibitor tubastatin A provided partial protection against LPS-mediated transendothelial hyperpermeability. Similarly, knock down of HDAC3 and HDAC6 by specific small-interfering RNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacological inhibition of both HDAC3 and -6 attenuated the inflammation, capillary permeability, and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased transendothelial hyperpermeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling

Non-classical ProIL-1beta activation during mammary gland infection is pathogen-dependent but caspase-1 independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1

Acute phase response in two consecutive experimentally induced E. coli intramammary infections in dairy cows

<p>Abstract</p> <p>Background</p> <p>Acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) have suggested to be suitable inflammatory markers for bovine mastitis. The aim of the study was to investigate acute phase markers along with clinical parameters in two consecutive intramammary challenges with <it>Escherichia coli </it>and to evaluate the possible carry-over effect when same animals are used in an experimental model.</p> <p>Methods</p> <p>Mastitis was induced with a dose of 1500 cfu of <it>E. coli </it>in one quarter of six cows and inoculation repeated in another quarter after an interval of 14 days. Concentrations of acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) were determined in serum and milk.</p> <p>Results</p> <p>In both challenges all cows became infected and developed clinical mastitis within 12 hours of inoculation. Clinical disease and acute phase response was generally milder in the second challenge. Concentrations of SAA in milk started to increase 12 hours after inoculation and peaked at 60 hours after the first challenge and at 44 hours after the second challenge. Concentrations of SAA in serum increased more slowly and peaked at the same times as in milk; concentrations in serum were about one third of those in milk. Hp started to increase in milk similarly and peaked at 36–44 hours. In serum, the concentration of Hp peaked at 60–68 hours and was twice as high as in milk. LBP concentrations in milk and serum started to increase after 12 hours and peaked at 36 hours, being higher in milk. The concentrations of acute phase proteins in serum and milk in the <it>E. coli </it>infection model were much higher than those recorded in experiments using Gram-positive pathogens, indicating the severe inflammation induced by <it>E. coli</it>.</p> <p>Conclusion</p> <p>Acute phase proteins would be useful parameters as mastitis indicators and to assess the severity of mastitis. If repeated experimental intramammary induction of the same animals with <it>E. coli </it>is used in cross-over studies, the interval between challenges should be longer than 2 weeks, due to the carry-over effect from the first infection.</p

Proteomic Analyses of Host and Pathogen Responses during Bovine Mastitis

The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced

Treatment of an Intramammary Bacterial Infection with 25-Hydroxyvitamin D3

Deficiency of serum levels of 25-hydroxyvitamin D3 has been correlated with increased risk of infectious diseases such as tuberculosis and influenza. A plausible reason for this association is that expression of genes encoding important antimicrobial proteins depends on concentrations of 1,25-dihydroxyvitamin D3 produced by activated immune cells at sites of infection, and that synthesis of 1,25-dihydroxyvitamin D3 is dependent on the availability of 25-hydroxyvitamin D3. Thus, increasing the availability of 25(OH)D3 for immune cell synthesis of 1,25-dihydroxyvitamin D3 at sites of infection has been hypothesized to aid in clearance of the infection. This report details the treatment of an acute intramammary infection with infusion of 25-hydroxyvitamin D3 to the site of infection. Ten lactating cows were infected with in one quarter of their mammary glands. Half of the animals were treated intramammary with 25-hydroxyvitamin D3. The 25-hydroxyvitamin D3 treated animal showed significantly lower bacterial counts in milk and showed reduced symptomatic affects of the mastitis. It is significant that treatment with 25-hydroxyvitamin D3 reduced the severity of an acute bacterial infection. This finding suggested a significant non-antibiotic complimentary role for 25-hydroxyvitamin D3 in the treatment of infections in compartments naturally low in 25-hydroxyvitamin D3 such as the mammary gland and by extension, possibly upper respiratory tract infections

In Vivo Activation of the Intracrine Vitamin D Pathway in Innate Immune Cells and Mammary Tissue during a Bacterial Infection

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D3 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Subsequently, synthesis of 1,25(OH)2D3 by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14+ cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)2D3, was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)2D3 production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)2D3 for local regulation of vitamin D responsive genes

Pro-inflammatory endothelial cell dysfunction is associated with intersectin-1s down-regulation

<p>Abstract</p> <p>Background</p> <p>The response of lung microvascular endothelial cells (ECs) to lipopolysaccharide (LPS) is central to the pathogenesis of lung injury. It is dual in nature, with one facet that is pro-inflammatory and another that is cyto-protective. In previous work, overexpression of the anti-apoptotic Bcl-X_L rescued ECs from apoptosis triggered by siRNA knockdown of intersectin-1s (ITSN-1s), a pro-survival protein crucial for ECs function. Here we further characterized the cyto-protective EC response to LPS and pro-inflammatory dysfunction.</p> <p>Methods and Results</p> <p>Electron microscopy (EM) analyses of LPS-exposed ECs revealed an activated/dysfunctional phenotype, while a biotin assay for caveolae internalization followed by biochemical quantification indicated that LPS causes a 40% inhibition in biotin uptake compared to controls. Quantitative PCR and Western blotting were used to evaluate the mRNA and protein expression, respectively, for several regulatory proteins of intrinsic apoptosis, including ITSN-1s. The decrease in ITSN-1s mRNA and protein expression were countered by Bcl-X_L and survivin upregulation, as well as Bim downregulation, events thought to protect ECs from impending apoptosis. Absence of apoptosis was confirmed by TUNEL and lack of cytochrome c (cyt c) efflux from mitochondria. Moreover, LPS exposure caused induction and activation of inducible nitric oxide synthase (iNOS) and a mitochondrial variant (mtNOS), as well as augmented mitochondrial NO production as measured by an oxidation oxyhemoglobin (oxyHb) assay applied on mitochondrial-enriched fractions prepared from LPS-exposed ECs. Interestingly, expression of myc-ITSN-1s rescued caveolae endocytosis and reversed induction of iNOS expression.</p> <p>Conclusion</p> <p>Our results suggest that ITSN-1s deficiency is relevant for the pro-inflammatory ECs dysfunction induced by LPS.</p

High susceptibility to lipopolysaccharide-induced lethal shock in encephalomyocarditis virus-infected mice

Secondary bacterial infection in humans is one of the pathological conditions requiring clinical attention. In this study, we examined the effect of lipopolysaccharide (LPS) on encephalomyocarditis virus (EMCV) infected mice. All mice inoculated with EMCV at 5 days before LPS challenge died within 24 h. LPS-induced TNF-α mRNA expression was significantly increased in the brain and heart at 5 days after EMCV infection. CD11b+/TLR4+ cell population in the heart was remarkably elevated at 5 days after EMCV infection, and sorted CD11b+ cells at 5 days after EMCV infection produced a large amount of TNF-α on LPS stimulation in vivo and in vitro. In conclusion, we found that the infiltration of CD11b+ cells into infected organs is involved in the subsequent LPS-induced lethal shock in viral encephalomyocarditis. This new experimental model can help define the mechanism by which secondary bacterial infection causes a lethal shock in viral encephalomyocarditis