Differential effect of plant lectins on mast cells of different origins

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 µg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.

Role of matrix metalloproteinases in the development of airway inflammation and remodeling

Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate extracellular matrix (ECM) turnover and so they have been suggested to be important in the process of lung disease associated with tissue remodeling. This has led to the concept that modulation of airway remodeling including excessive proteolysis damage to the tissue may be of interest for future treatment. Within the MMP family, macrophage elastase (MMP-12) is able to degrade ECM components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease including emphysema. Pulmonary fibrosis has an aggressive course and is usually fatal within an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of ECM components in the lung interstitium. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components could justify anti-protease treatments. Indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-MMP-9/TIMP-1 ratio in bronchoalveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. Finally, these observations emphasize that effective treatment of these disorders must be started early during the natural history of the disease, prior to the development of extensive lung destruction and fibrosis.

Effect of pentoxifylline on lung inflammation and gas exchange in a sepsis-induced acute lung injury model

Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.

Ambient particulate air pollution from vehicles promotes lipid peroxidation and inflammatory responses in rat lung

Oxidative stress plays a major role in the pathogenesis of particle-dependent lung injury. Ambient particle levels from vehicles have not been previously shown to cause oxidative stress to the lungs. The present study was conducted to a) determine whether short-term exposure to ambient levels of particulate air pollution from vehicles elicits inflammatory responses and lipid peroxidation in rat lungs, and b) determine if intermittent short-term exposures (every 4 days) induce some degree of tolerance. Three-month-old male Wistar rats were exposed to ambient particulate matter (PM) from vehicles (N = 30) for 6 or 20 continuous hours, or for intermittent (5 h) periods during 20 h for 4 consecutive days or to filtered air (PM <10 µm; N = 30). Rats continuously breathing polluted air for 20 h (P-20) showed a significant increase in the total number of leukocytes in bronchoalveolar lavage compared to control (C-20: 2.61 x 105 ± 0.51;P-20: 5.01 x 105 ± 0.81; P < 0.05) and in lipid peroxidation ([MDA] nmol/mg protein: C-20: 0.148 ± 0.01; P-20: 0.226 ± 0.02; P < 0.05). Shorter exposure (6 h) and intermittent 5-h exposures over a period of 4 days did not cause significant changes in leukocytes. Lipid damage resulting from 20-h exposure to particulate air pollution did not cause a significant increase in lung water content. These data suggest oxidative stress as one of the mechanisms responsible for the acute adverse respiratory effects of particles, and suggest that short-term inhalation of ambient particulate air pollution from street with high automobile traffic represents a biological hazard.

Pharmacological study of anti-allergic activity of Syzygium cumini (L.) Skeels

Myrtaceae is a plant family widely used in folk medicine and Syzygium and Eugenia are among the most important genera. We investigated the anti-allergic properties of an aqueous leaf extract of Syzygium cumini (L.) Skeels (SC). HPLC analysis revealed that hydrolyzable tannins and flavonoids are the major components of the extract. Oral administration of SC (25-100 mg/kg) in Swiss mice (20-25 g; N = 7/group) inhibited paw edema induced by compound 48/80 (50% inhibition, 100 mg/kg; P <= 0.05) and, to a lesser extent, the allergic paw edema (23% inhibition, 100 mg/kg; P <= 0.05). SC treatment also inhibited the edema induced by histamine (58% inhibition; P <= 0.05) and 5-HT (52% inhibition; P <= 0.05) but had no effect on platelet-aggregating factor-induced paw edema. SC prevented mast cell degranulation and the consequent histamine release in Wistar rat (180-200 g; N = 7/group) peritoneal mast cells (50% inhibition, 1 µg/mL; P <= 0.05) induced by compound 48/80. Pre-treatment of BALB/c mice (18-20 g; N = 7/group) with 100 mg/kg of the extract significantly inhibited eosinophil accumulation in allergic pleurisy (from 7.662 ± 1.524 to 1.89 ± 0.336 x 10(6)/cavity; P <= 0.001). This effect was related to the inhibition of IL-5 (from 70.9 ± 25.2 to 12.05 ± 7.165 pg/mL) and CCL11/eotaxin levels (from 60.4 ± 8.54 to 32.8 ± 8.4 ng/mL) in pleural lavage fluid, using ELISA. These findings demonstrate an anti-allergic effect of SC, and indicate that its anti-edematogenic effect is due to the inhibition of mast cell degranulation and of histamine and serotonin effects, whereas the inhibition of eosinophil accumulation in the allergic pleurisy model is probably due to an impairment of CCL11/eotaxin and IL-5 production.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Propofol exerts anti-inflammatory effects in rats with lipopolysaccharide-induced acute lung injury by inhibition of CD14 and TLR4 expression

We investigated the effect of propofol (Prop) administration (10 mg kg-1 h-1, intravenously) on lipopolysaccharide (LPS)-induced acute lung injury and its effect on cluster of differentiation (CD) 14 and Toll-like receptor (TLR) 4 expression in lung tissue of anesthetized, ventilated rats. Twenty-four male Wistar rats were randomly divided into three groups of 8 rats each: control, LPS, and LPS+Prop. Lung injury was assayed via blood gas analysis and lung histology, and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels were determined in bronchoalveolar lavage fluid using ELISA. Real-time polymerase chain reaction was used to detect CD14 and TLR4 mRNA levels, and CD14 and TLR4 protein expression was determined by Western blot. The pathological scores were 1.2 ± 0.9, 3.3 ± 1.1, and 1.9 ± 1.0 for the control, LPS, and LPS+Prop groups, respectively, with statistically significant differences between control and LPS groups (P < 0.05) and between LPS and LPS+Prop groups (P < 0.05). The administration of LPS resulted in a significant increase in TNF-α and IL-1β levels, 7- and 3.5-fold, respectively (P < 0.05), while treatment with propofol partially blunted the secretion of both cytokines (P < 0.05). CD14 and TLR4 mRNA levels were increased in the LPS group (1.48 ± 0.05 and 1.26 ± 0.03, respectively) compared to the control group (1.00 ± 0.20 and 1.00 ± 0.02, respectively; P < 0.05), while propofol treatment blunted this effect (1.16 ± 0.05 and 1.12 ± 0.05, respectively; P < 0.05). Both CD14 and TLR4 protein levels were elevated in the LPS group compared to the control group (P < 0.05), while propofol treatment partially decreased the expression of CD14 and TLR4 protein versus LPS alone (P < 0.05). Our study indicates that propofol prevents lung injury, most likely by inhibition of CD14 and TLR4 expression.

Propofol pretreatment attenuates lipopolysaccharide-induced acute lung injury in rats by activating the phosphoinositide-3-kinase/Akt pathway

The aim of this study was to investigate the effect of propofol pretreatment on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the role of the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway in this procedure. Survival was determined 48 h after LPS injection. At 1 h after LPS challenge, the lung wet- to dry-weight ratio was examined, and concentrations of protein, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) were determined using the bicinchoninic acid method or ELISA. Lung injury was assayed via lung histological examination. PI3K and p-Akt expression levels in the lung tissue were determined by Western blotting. Propofol pretreatment prolonged survival, decreased the concentrations of protein, TNF-α, and IL-6 in BALF, attenuated ALI, and increased PI3K and p-Akt expression in the lung tissue of LPS-challenged rats, whereas treatment with wortmannin, a PI3K/Akt pathway specific inhibitor, blunted this effect. Our study indicates that propofol pretreatment attenuated LPS-induced ALI, partly by activation of the PI3K/Akt pathway.

Treatment with 1,25(OH)2D3induced HDAC2 expression and reduced NF-κB p65 expression in a rat model of OVA-induced asthma

Recent evidence indicates that a deficiency of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) may influence asthma pathogenesis; however, its roles in regulating specific molecular transcription mechanisms remain unclear. We aimed to investigate the effect of 1,25(OH)2D3 on the expression and enzyme activity of histone deacetylase 2 (HDAC2) and its synergistic effects with dexamethasone (Dx) in the inhibition of inflammatory cytokine secretion in a rat asthma model. Healthy Wistar rats were randomly divided into 6 groups: control, asthma, 1,25(OH)2D3 pretreatment, 1,25(OH)2D3 treatment, Dx treatment, and Dx and 1,25(OH)2D3 treatment. Pulmonary inflammation was induced by ovalbumin (OVA) sensitization and challenge (OVA/OVA). Inflammatory cells and cytokines in the bronchoalveolar lavage (BAL) fluid and histological changes in lung tissue were examined. Nuclear factor kappa B (NF-κB) p65 and HDAC2 expression levels were assessed with Western blot analyses and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Enzyme activity measurements and immunohistochemical detection of HDAC2 were also performed. Our data demonstrated that 1,25(OH)2D3 reduced the airway inflammatory response and the level of inflammatory cytokines in BAL. Although NF-κB p65 expression was attenuated in the pretreatment and treatment groups, the expression and enzyme activity of HDAC2 were increased. In addition, 1,25(OH)2D3 and Dx had synergistic effects on the suppression of total cell infusion, cytokine release, and NF-κB p65 expression, and they also increased HDAC2 expression and activity in OVA/OVA rats. Collectively, our results indicated that 1,25(OH)2D3might be useful as a novel HDAC2 activator in the treatment of asthma.

Transtracheal puncture: a forgotten procedure

Transtracheal puncture has long been known as a safe, low-cost procedure. However, with the advent of bronchoscopy, it has largely been forgotten. Two researchers have suggested the use of α-amylase activity to diagnose salivary aspiration, but the normal values of this enzyme in tracheobronchial secretions are unknown. We aimed to define the normal values of α-amylase activity in tracheobronchial secretions and verify the rate of major complications of transtracheal puncture. From October 2009 to June 2011, we prospectively evaluated 118 patients without clinical or radiological signs of salivary aspiration who underwent transtracheal puncture before bronchoscopy. The patients were sedated with a solution of lidocaine and diazepam until they reached a Ramsay sedation score of 2 or 3. We then cleaned the cervical region and anesthetized the superficial planes with lidocaine. Next, we injected 10 mL of 2% lidocaine into the tracheobronchial tree. Finally, we injected 10 mL of normal saline into the tracheobronchial tree and immediately aspirated the saline with maximum vacuum pressure to collect samples for measurement of the α-amylase level. The α-amylase level mean ± SE, median, and range were 1914 ± 240, 1056, and 24-10,000 IU/L, respectively. No major complications (peripheral desaturation, subcutaneous emphysema, cardiac arrhythmia, or hemoptysis) occurred among 118 patients who underwent this procedure. Transtracheal aspiration is a safe, low-cost procedure. We herein define for the first time the normal α-amylase levels in the tracheobronchial secretions of humans.

Rôle des eicosanoïdes post-greffe : implication dans la bronchiolite oblitérante

Le rejet chronique se manifeste dans le poumon par la bronchiolite oblitérante (BO), une pathologie inflammatoire et fibrotique menant à l’oblitération des bronchioles. L’étiologie exacte de cette maladie demeure inconnue. Certaines études suggèrent qu'un déséquilibre des leucotriènes (LT) sur les prostaglandines (PG) favorise la fibrose pulmonaire. Les taux des LT et des PG dans le poumon humain post-transplantation sont inconnus. Nous proposons qu'un déséquilibre de cystéinyl leucotriènes (CysLT) sur la PGE2 existe dans le poumon transplanté et pourrait être impliqué dans la pathogenèse de la BO. Aussi, les leucotriènes contribueraient à la fibrose par la transition épithélio-mésenchymateuse (TEM). Afin de vérifier ces hypothèses, nous avons déterminé les taux de CysLT et de PGE2 dans le liquide de lavage broncho-alvéolaire (LBA) provenant de poumons transplantés chez l'homme ainsi que leurs corrélations cliniques. Nous avons également déterminé la capacité des CysLT à induire l’expression des marqueurs de la TEM in vitro. Nous avons découvert des taux de CysLT et PGE2 supérieurs à la normale dans les LBA des greffés. Un pic prédominant de CysLT sur PGE2 est observée à 52 semaines postgreffe et deux facteurs de risque de la BO, les infections au CMV et à l’Aspergillus, sont associés au ratio CysLT/PGE2> 1. In vitro, les CysLT induisent une répression des marqueurs épithéliaux mais n’induisent pas l’expression de marqueurs mésenchymateux chez les cellules épithéliales bronchiolaires.

Étude des gènes d'Actinobacillus pleuropneumoniae exprimés en conditions d'infection

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes.

Développement d’une nouvelle méthode d’analyse multi-résidus par LDTD/APCI-MS/MS pour la quantification de pesticides et de produits pharmaceutiques dans les eaux usées

Une nouvelle méthode d'extraction en phase solide (SPE) couplée à une technique d'analyse ultrarapide a été développée pour la détermination simultanée de neuf contaminants émergents (l'atrazine, le déséthylatrazine, le 17(béta)-estradiol, l'éthynylestradiol, la noréthindrone, la caféine, la carbamazépine, le diclofénac et le sulfaméthoxazole) provenant de différentes classes thérapeutiques et présents dans les eaux usées. La pré-concentration et la purification des échantillons a été réalisée avec une cartouche SPE en mode mixte (Strata ABW) ayant à la fois des propriétés échangeuses de cations et d'anions suivie d'une analyse par une désorption thermique par diode laser/ionisation chimique à pression atmosphérique couplée à la spectrométrie de masse en tandem (LDTD-APCI-MS/MS). La LDTD est une nouvelle méthode d'introduction d'échantillon qui réduit le temps total d'analyse à moins de 15 secondes par rapport à plusieurs minutes avec la chromatographie liquide couplée à la spectrométrie de masse en tandem traditionnelle (LC-MS/MS). Plusieurs paramètres SPE ont été évalués dans le but d'optimiser l'efficacité de récupération lors de l'extraction des analytes provenant des eaux usées, tels que la nature de la phase stationnaire, le débit de chargement, le pH d'extraction, le volume et la composition de la solution de lavage et le volume de l'échantillon initial. Cette nouvelle méthode a été appliquée avec succès à de vrais échantillons d'eaux usées provenant d'un réservoir de décantation primaire. Le recouvrement des composés ciblés provenant des eaux usées a été de 78 à 106%, la limite de détection a été de 30 à 122 ng L-1, alors que la limite de quantification a été de 88 à 370 ng L-1. Les courbes d'étalonnage dans les matrices d'eaux usées ont montré une bonne linéarité (R2 > 0,991) pour les analytes cibles ainsi qu’une précision avec un coefficient de variance inférieure à 15%.

Détection et caractérisation génétique de Listeria monocytogenes dans une usine d’abattage/découpe de porcs au Québec

Listeria monocytogenes (L. monocytogenes) est un pathogène majeur en santé publique comme les épisodes de 2008 dans les fromages et les charcuteries l’ont démontré. Au Canada, il n’y a pas de surveillance règlementaire de ce microorganisme dans les étapes précédant la transformation de produits prêts-à-manger. Ainsi, la présence et la circulation de ce microorganisme dans ces environnements est peu documentée. Pour décrire ces phénomènes, nous avons effectué un échantillonnage dans une usine d’abattage et de découpe de porcs au Québec, principalement dans les parcs d’attente, et dans l’environnement de l’abattage et de découpe : les échantillonages ont été effectués après lavage et désinfection sur une période de 2 ans. Un nombre de 874 échantillons a été récoltés. Le protocole de détection utilisé était inspiré de la méthode MFHPB-30 de Santé Canada. Les sérotypes ont été obtenus par PCR et les isolats caractérisés par un génotypage RFLP-PFGE en utilisant les enzymes de restriction Apa1 et Asc1. Nous avons détecté la présence de Listeria monocytogemes dans toutes ces étapes de la production. De ces échantillons positifs, 4 sérotypes (principalement 1/2b) ont émergé. Les patrons PFGE ont démontré la présence d’une variété de génotypes dans les zones d’attente et d’abattage de l’usine et la présence d’un type majeur dans l’environnement de la zone de découpe (le type 1 représentant 96.1% des souches à cette étape). De plus, nous avons démontré des liens entre les souches retrouvés au début de la production, en attente, et les souches retrouvées dans la zone de découpe. Ces résultats suggèrent que Listeria monocytogenes entre dans l’usine avec les animaux, contamine les étapes suivantes de la production et que certaines souches peuvent être sélectionnées et leur croissance favorisé dans l’environnement, devenant majoritaires, persistantes et préoccupantes en regars de la santé publique.