Isolated perfused udder model for transcriptome analysis in response to Streptococcus agalactiae.

This study aimed to evaluate the transcriptional changes occurring in isolated perfused mammary alveolar tissue in response to inoculation with S. agalactiae and to identify the most affected biological functions and pathways after 3 h. Four udders taken at slaughter from cows with healthy mammary gland were perfused ex situ with warmed and gassed Tyrode's solution. Mammary alveolar tissue samples were taken from the left fore and rear quarters (IQ-inoculated quarters) before inoculation (hour 0) and at 3 h post inoculation (hpi) and at the same times from control right fore and rear quarters (not inoculated: NIQ). A total of 1756 differentially expressed genes (DEGs) were identified between IQ and NIQ at 3 hpi using edgeR package. Within this set of DEGs, 952 were up regulated and mainly involved with innate immune response and inflammatory response, e.g., CD14, CCL5, TLR2, IL-8, SAA3, as well as in transcriptional regulation such as FOS, STAT3 and NFKBIA. Genes down-regulated (804) included those involved with lipid synthesis e.g., APOC2, SCD, FABP3 and FABP4. The most affected pathways were chemokine signaling, Wnt signaling and complement and coagulation cascades, which likely reflects the early stage response of mammary tissue to S. agalactiae infection. No significant gene expression changes were detected by RNA-Seq in the others contrasts. Real time-PCR confirmed the increase in mRNA abundance of immune-related genes: TLR2, TLR4, IL-1ß, and IL-10 at 3 hpi between IQ and NIQ. The expression profiles of Casp1 and Bax for any contrasts were unaffected whereas Bcl2 was increased in IQ, which suggests no induction of apoptosis during the first hours after infection. Results provided novel information regarding the early functional pathways and gene network that orchestrate innate immune responses to S. agalactiae infection. This knowledge could contribute to new strategies to enhance resistance to this disease, such as genomic selection.

Efficacy of a Ruminal Bacteriocin Against Pure and Mixed Cultures of Bovine Mastitis Pathogens.

Bacteriocins have been suggested as an alternative to conventional antibiotics for the prevention and treatment of mastitis infections. Predominant bacteria associated with bovine mastitis (n = 276 isolates) were evaluated for their susceptibility to bovicin HC5, a ruminal bacteriocin produced by Streptococcus equinus HC5. Bovicin HC5 inhibited most (> 80%) of the streptococcal and staphylococcal strains tested, but showed no effect against Escherichia coli strains. Susceptibility and resistance testing indicated that approximately 95% of the S. aureus strains were inhibited by concentrations of bovicin HC5 varying from 40 to 2560 AU ml-1. Bovicin HC5 (62.50 AU ml-1) also inhibited the growth of aerobic and anaerobic mixed cultures of S. aureus and S. agalactiae, but the combination with 0.25 mmol l-1 of EDTA showed even greater bactericidal activity. These results demonstrate that bovicin HC5 is effective against the most prevalent pathogens found in contagious udder infections and could complement the use antibiotics in mastitis prophylaxis and therapy.

Systemic inflammation in early neonatal mice induces transient and lasting neurodegenerative effects.

BACKGROUND: The inflammatory mediator lipopolysaccharide (LPS) has been shown to induce acute gliosis in neonatal mice. However, the progressive effects on the murine neurodevelopmental program over the week that follows systemic inflammation are not known. Thus, we investigated the effects of repeated LPS administration in the first postnatal week in mice, a condition mimicking sepsis in late preterm infants, on the developing central nervous system (CNS). METHODS: Systemic inflammation was induced by daily intraperitoneal administration (i.p.) of LPS (6 mg/kg) in newborn mice from postnatal day (PND) 4 to PND6. The effects on neurodevelopment were examined by staining the white matter and neurons with Luxol Fast Blue and Cresyl Violet, respectively. The inflammatory response was assessed by quantifying the expression/activity of matrix metalloproteinases (MMP), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1, and autotaxin (ATX). In addition, B6 CX3CR1(gfp/+) mice combined with cryo-immunofluorescence were used to determine the acute, delayed, and lasting effects on myelination, microglia, and astrocytes. RESULTS: LPS administration led to acute body and brain weight loss as well as overt structural changes in the brain such as cerebellar hypoplasia, neuronal loss/shrinkage, and delayed myelination. The impaired myelination was associated with alterations in the proliferation and differentiation of NG2 progenitor cells early after LPS administration, rather than with excessive phagocytosis by CNS myeloid cells. In addition to disruptions in brain architecture, a robust inflammatory response to LPS was observed. Quantification of inflammatory biomarkers revealed decreased expression of ATX with concurrent increases in HMGB1, TLR-4, and MMP-9 expression levels. Acute astrogliosis (GFAP(+) cells) in the brain parenchyma and at the microvasculature interface together with parenchymal microgliosis (CX3CR1(+) cells) were also observed. These changes preceded the migration/proliferation of CX3CR1(+) cells around the vessels at later time points and the subsequent loss of GFAP(+) astrocytes. CONCLUSION: Collectively, our study has uncovered a complex innate inflammatory reaction and associated structural changes in the brains of neonatal mice challenged peripherally with LPS. These findings may explain some of the neurobehavioral abnormalities that develop following neonatal sepsis.

Selective vulnerability of rat brain regions to unconjugated bilirubin.

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin (UCB) encephalopathy, although cerebellum also shows selective yellow staining in kernicterus. We previously demonstrated that UCB induces oxidative stress in cortical neurons, disruption of neuronal network dynamics, either in developing cortical or hippocampal neurons, and that immature cortical neurons are more prone to UCB-induced injury. Here, we studied if immature rat neurons isolated from cortex, cerebellum and hippocampus present distinct features of oxidative stress and cell dysfunction upon UCB exposure. We also explored whether oxidative damage and its regulation contribute to neuronal dysfunction induced by hyperbilirubinemia, considering neurite extension and ramification, as well as cell death. Our results show that UCB induces nitric oxide synthase expression, as well as production of nitrites and cyclic guanosine monophosphate in immature neurons, mainly in those from hippocampus. After exposure to UCB, hippocampal neurons presented the highest content of reactive oxygen species, disruption of glutathione redox status and cell death, when compared to neurons from cortex or cerebellum. In particular, the results indicate that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein implicated in the maintenance of cellular oxidation status. However, longer neuronal exposure to UCB caused down-regulation of DJ-1 expression, especially in hippocampal neurons. In addition, a greater impairment in neurite outgrowth and branching following UCB treatment was also noticed in immature neurons from hippocampus. Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis, protected neurons from UCB-induced oxidative stress and necrotic cell death, preventing DJ-1 down-regulation and neuritic impairment. Taken together, these data point to oxidative injury and disruption of neuritic network as hallmarks in hippocampal susceptibility to UCB. Most importantly, they also suggest that local differences in glutathione content may account to the different susceptibility between brain regions exposed to UCB.

Evidence of tricellulin expression by immune cells, particularly microglia.

Tight junctions (TJs) are elaborate structures located on the apical region of epithelial cells that limit paracellular permeability. Tricellulin is a recently discovered TJ protein, which is concentrated at the structurally specialized tricellular TJs but also present at bicellular contacts between epithelial cells, namely in the stomach. Interestingly, several TJ proteins have been found in other than epithelial cells, as astrocytes, and tricellulin mRNA expression was reported in mature dendritic cells. These findings prompted us to look for tricellulin expression in both epithelial and immune cells in the stomach, as well as in microglia, the brain resident immunocompetent cells. Immunohistochemical analysis of human stomach tissue sections revealed peroxidase staining at three-corner contact sites, as well as at the contact between two adjacent epithelial cells, thus evidencing the expression of tricellulin not only at tricellullar but at bicellular junctions as well. Such analysis, further revealed tricellulin immunostaining in cells of the monocyte/macrophage lineage, scattered throughout the lamina propria. Cultured rat microglia exhibited a notorious tricellulin staining, consistent with an extensive expression of the protein along the cell, which was not absolutely coincident with the lysosomal marker CD68. Detection of mRNA expression by real-time PCR provided supportive evidence for the expression of the TJ protein in microglia. These data demonstrate for the first time that microglia express a TJ protein. Moreover, the expression of tricellulin both in microglia and in the stomach immune cells point to a possible role of this new TJ protein in the immune system.

Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation.

Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity.

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 micromol/L UCB, in the presence of 100 micromol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3',5'-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.

Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil.

This study aimed to evaluate virulence factors and genetic markers of antimicrobial resistance in 400 Staphylococcus aureus strains isolated from bovine mastitis in four Brazilian states, as well as to assess the association between these characteristics and field information. Virulence factors and drug resistance genes were identified by PCR screening. Biofilm-forming and hemolytic phenotype were detected using Congo red Tryptic Soy Broth and defibrinated sheep blood agar, respectively. Of all isolates, 83.5% were biofilm-forming and 98.5% strains exhibited biofilm gene icaAD, and a significant association between phenotype and genotype for biofilm was observed (P = 0.0005). Hemolysin genes were observed in 82.85% (hla+hlb+), 16.5% (hla+) and 0.75% (hlb+) isolates, whereas the hemolytic phenotype exhibited was complete and incomplete hemolysis in 64.25%, complete in 28.25%, incomplete in 4.75%, and negative in 2.75% of the strains. Virulence factors genes luk, seb, sec, sed, and tst were observed in 3.5%, 0.5%, 1%, 0.25%, and 0.74% isolates, respectively. The gene blaZ was detected in 82.03% of penicillin-resistant isolates, whereas tetK and aac(6')-Ie-aph(2')-Ia were observed in 33.87% and 45.15% of the tetracycline and aminoglycosides-resistant isolates, respectively. Fluoroquinolone resistance gene mepA was detected for the first time in S. aureus from bovine mastitis. Resistance genes tetM (3.22%), tetL (1.61%), ermA (14.29%), ermB (14.29%), ermC (33.3%), ermT (9.52%), ermY (4.76%), msrA (9.52%), and mphC (9.52%) were also detected among resistant isolates. No association between virulence factors or antimicrobial-resistant genes and year of isolation, geographic origin, or antimicrobial resistance profile was observed. Our results showed that S. aureus strains isolated from bovine mastitis in the four Brazilian states sampled are mainly biofilm-forming and hemolytic, whereas virulence genes associated with enterotoxins, luk and tst, were less frequently observed. Moreover, a wide variety of resistance genes that confer resistance to almost all classes of antimicrobial agents approved for use in animals and humans were found. Overall, the data point to a great pathogenic potential of S. aureus associated with bovine mastitis and to the non-negligible risks to public health of staphylococcal infections from animal origin.

Genetic diversity and antimicrobial resistance in Staphylococcus aureus and coagulase-negative Staphylococcus isolates from bovine mastitis in Minas Gerais, Brazil.

The aims of this study were to determine the antimicrobial susceptibility profile and genetic diversity of Staphylococcus spp. isolated from dairy cows in Minas Gerais, Brazil, and to assess the relationship among the isolates' susceptibility profiles and pulsed-field gel electrophoresis (PFGE) genotypes. Seventy-nine isolates were used, including S. aureus (n = 71) and coagulase-negative staphylococci (CoNS) (n = 8). Susceptibility to 12 antimicrobial agents was performed. All Staphylococcus spp. were subjected to PFGE. Staphylococcus aureus and CoNS isolates exhibited full susceptibility only to cephalothin. The greatest percentages of resistance among Staphylococcus spp. were observed to penicillins, folate pathway inhibitors, and tetracyclines. Twelve S. aureus and four CoNS were classified as multidrug resistance strains. Percentage of MRSA was also higher among CoNS (75%), compared to S. aureus isolates (2.81%). Adopting 100% of similarity, 34 different genotypes were identified. Association of minimum-spanning tree (MST) analysis with data from municipalities, herds, methicillin-resistant S. aureus (MRSA), and resistance patterns for all isolates did not show any clustering. However, a clustering pattern of bacterial species was observed. Results from this study indicate a high frequency of antimicrobial resistance, especially among CoNS, and a high genetic diversity among Staphylococcus spp. isolated from dairy cows with mastitis in Minas Gerais, Brazil.

Retail survey of Brazilian milk and Minas frescal cheese and a contaminated dairy plant to establish prevalence, relatedness, and sources of Listeria monocytogenes isolates.

A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, "brand F" of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.

Bilirubin injury to neurons: contribution of oxidative stress and rescue by glycoursodeoxycholic acid.

It is well established that high levels of unconjugated bilirubin (UCB) can be toxic to the central nervous system, and oxidative stress is emerging as a relevant event in the mechanisms of UCB encephalopathy. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been reported as a cytoprotective and antioxidant molecule. In this study, we investigated if exposure of rat neurons in primary culture to clinically relevant concentrations of UCB leads to oxidative injury. The contribution of oxidative stress in UCB neurotoxicity was further investigated by examining whether the reduction of NO production by NAME, an inhibitor of nitric oxide synthase, prevents the disruption of the redox status and neuronal damage. Moreover, we evaluated the ability of glycoursodeoxycholic acid (GUDCA), the most relevant conjugated derivative in the serum of patients treated with UDCA, to abrogate the UCB-induced oxidative damage. Cultured rat neurons were incubated with 50 or 100microM UCB in the presence of 100microM human serum albumin, alone or in combination with 100microM NAME or with 50microM GUDCA, for 4h at 37 degrees C. Protein carbonyls, 4-hydroxy-2-nonenal-protein adducts, intracellular glutathione content and cell death were determined. The results obtained showed that UCB induces protein oxidation and lipid peroxidation, while diminishes the thiol antioxidant defences, events that were correlated with the extent of cell death. Moreover, these events were counteracted by NAME and abrogated in the presence of GUDCA. Collectively, this study shows that oxidative stress is one of the pathways associated with neuronal viability impairment by UCB, and that GUDCA significantly prevents such effects from occurring. These findings corroborate the antioxidant properties of the bile acid and point to a new therapeutic approach for UCB-induced neurotoxicity due to oxidative stress.

Glycoursodeoxycholic acid and interleukin-10 modulate the reactivity of rat cortical astrocytes to unconjugated bilirubin.

The pathogenesis of bilirubin encephalopathy seems to result from accumulation of unconjugated bilirubin (UCB) within the brain. We have recently demonstrated that UCB causes astroglial release of proinflammatory cytokines and glutamate, as well as cell death. The bile acid glycoursodeoxycholic acid (GUDCA) and the anti-inflammatory cytokine interleukin (IL)-10 have been reported to modulate inflammation and cell survival. In this study we investigated the effect of these therapeutic agents on the astroglial response to UCB. Only GUDCA prevented UCB-induced astroglial death. The secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta elicited by UCB in astrocytes was reduced in the presence of GUDCA and IL-10, whereas the suppression of IL-6 was only counteracted by GUDCA. Neither GUDCA nor IL-10 modulated the accumulation of extracellular glutamate. Additionally, IL-10 markedly inhibited UCB-induced nuclear factor-kappaB nuclear translocation and cytokine mRNA expression, whereas GUDCA only prevented TNF-alpha mRNA expression. Moreover, GUDCA inhibited TNF-alpha- and IL-1beta-converting enzymes, preventing the maturation of these cytokines and their consequent release. Collectively, this study shows that IL-10 action is restricted to UCB-induced release of TNF-alpha and IL-1beta from the astrocytes, whereas GUDCA presents a more ubiquitous action on the astroglial reactivity to UCB. Hence, GUDCA may have potential benefits over an IL-10 therapeutic approach in reducing UCB-induced astrocyte immunostimulation and death.

Influence of hypoxia and ischemia preconditioning on bilirubin damage to astrocytes.

Hypoxia-ischemia in the perinatal period is a common cause of neurologic disability in children and is often associated with neonatal morbidity and mortality. Another frequent condition of the newborn is hyperbilirubinemia and it is well known that deposition of unconjugated bilirubin (UCB) in the central nervous system can damage nerve cells and cause encephalopathy. Interestingly, some studies report the onset of cerebral hypoxia-ischemia as a risk factor for UCB encephalopathy, since that condition often precedes neonatal hyperbilirubinemia. However, the cellular mechanisms triggered by hypoxia-ischemia that may enforce UCB deleterious effects are not well elucidated. Therefore, we designed this study to investigate whether hypoxia (HP) or combined oxygen-glucose deprivation (OGD) followed by reoxygenation, modifies glial cell susceptibility to UCB injury. Thus, cultured astrocytes were exposed to HP or OGD for 4 h and returned to normoxic conditions for another 12 h prior to incubation with UCB for 4 h. HP and OGD effects in UCB toxicity were compared to normoxic conditions. Our results demonstrate that HP and OGD preconditioning increase the vulnerability of glial cells to UCB damage by enhancing some of the deleterious effects of UCB, namely cell death by both apoptosis and necrosis. This preconditioning also augments the UCB-induced stimulation of an inflammatory response by an effect that involves the activation of the nuclear factor kappaB activation. These findings provide a novel basis for the increased risk of brain damage in jaundiced newborns that were previously exposed to hypoxia or ischemia during the perinatal period, namely during delivery.

Estimate of the economic impact of mastitis: A case study in a Holstein dairy herd under tropical conditions.

The aim of this study was to estimate the economic impact of mastitis at the herd level and the weight (percent) of the components of this impact in a Holstein dairy herd under tropical conditions. Three estimates of the economic impact of mastitis were performed. In estimates 1 and 2 the real production and economic indices from February 2011 to January 2012 were considered. In the estimate 1, indices for mastitis classified as ideal were considered, whereas in the estimate 2, the mastitis indices used were those recorded at the farm and at Holstein Cattle Association of Minas Gerais State database (real indices). Ideal mastitis indices were bulk milk somatic cell counts less than 250,000 cells/mL, incidence of clinical mastitis less than 25 cases/100 cows/year, number of culls due to udder health problems less than 5% and the percentage of cows with somatic cell counts greater than 200,000 cells/mL less than 20%. Considering the ideal indices of mastitis, the economic impact was US$19,132.35. The three main components of the economic impact were culling cows (39.4%) and the reduction in milk production due to subclinical and clinical mastitis (32.3% and 18.2%, respectively). Estimate 2 using real mastitis indices showed an economic impact of US$61,623.13 and the reduction in milk production due to mastitis (77.7%) and milk disposal (14.0%) were the most relevant components. The real impact of culling cows was approximately 16 times less than the weight that was considered ideal, indicating that this procedure could have been more frequently adopted. The reduction in milk production was 27.2% higher than the reduction in Estimate 1, indicating a need to control and prevent mastitis. The estimate 3 considered the same indices as estimate 2, but for the period from February 2012 to January 2013. Its economic impact was US$91,552.69. During this period, 161 treatments of cows with an intramammary antibiotic were performed to eliminate Streptococcus agalactiae, and eight cows chronically infected with Staphylococcus aureus were culled. The reduction in milk production due to mastitis was the main component of the economic impact (54.9%). The culling of cows with chronic infection was associated with an increase in the economic impact of mastitis and a reduction in the average productivity per cow. At the herd level reduction in milk production was the component that presented the largest weight in the economic impact of the disease.

Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms.

Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159 S. aureus isolates investigated harboured the cap gene: 80 % carried the cap5 gene and 20 % carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58 % expressed CP5 and 11 % expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6 % of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5 % were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.

The TNF-α/NF-κB signaling pathway has a key role in methamphetamine-induced blood-brain barrier dysfunction.

Methamphetamine (METH) is a psychostimulant that causes neurologic and psychiatric abnormalities. Recent studies have suggested that its neurotoxicity may also result from its ability to compromise the blood-brain barrier (BBB). Herein, we show that METH rapidly increased the vesicular transport across endothelial cells (ECs), followed by an increase of paracellular transport. Moreover, METH triggered the release of tumor necrosis factor-alpha (TNF-α), and the blockade of this cytokine or the inhibition of nuclear factor-kappa B (NF-κB) pathway prevented endothelial dysfunction. Since astrocytes have a crucial role in modulating BBB function, we further showed that conditioned medium obtained from astrocytes previously exposed to METH had a negative impact on barrier properties also via TNF-α/NF-κB pathway. Animal studies corroborated the in vitro results. Overall, we show that METH directly interferes with EC properties or indirectly via astrocytes through the release of TNF-α and subsequent activation of NF-κB pathway culminating in barrier dysfunction.

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study.

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells.

Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

Cytokine production, glutamate release and cell death in rat cultured astrocytes treated with unconjugated bilirubin and LPS.

In hyperbilirubinemic newborns, sepsis is considered a risk factor for kernicterus. Evidence shows that injury to astrocytes triggers cytokine release. We examined the effects of unconjugated bilirubin (UCB) alone, or in combination with LPS, on the release of glutamate and cytokines from astrocytes in conditions inducing less than 10% of cell death. UCB leads to an increase of extracellular glutamate and highly enhances the release of TNF-alpha and IL-1beta, while inhibiting the production of IL-6. LPS potentiates immunostimulatory properties of UCB. These results point out the role of cytokines and provide a basis for the significance of sepsis in UCB encephalopathy.

Evaluation of antibacterial, antifungal and modulatory activity of methanol and ethanol extracts of Padina sanctae-crucis.

BACKGROUND: Multi-resistantmicroorganisms such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida tropicalis e Candida krusei are the main causes of microbial infections. Padina sanctae-crucis is a seaweed often used to check the contamination of ecosystems by materials such as heavy metals, but studies of the antimicrobial activity of the same seaweed were not found. METHODS: The tests for the minimum inhibitory concentration and modulation of microbial resistance, with the use of ethanolic and methanolic extracts of Padina Sanctae-cruces combined with drugs of the class of aminoglycosides and antifungal were used to evaluate the activity against the cited microorganisms. RESULTS: Was observed a modulation of antibiotic activity between the natural products and the E. coli and S. aureus strains, indicating a synergism and antagonism respectively. CONCLUSIONS: The results showed a moderate modulatory effect against some microorganisms studied.